(Advances in Parasitology 76) Louis M. Weiss and Herbert B. Tanowitz (Eds.) - Chagas Disease, Part B-Academic Press, Elsevier (2011)

SERIES EDITORS
D. ROLLINSON S. I. HAY
Department of Zoology, Spatial Epidemiology and Ecology Group
The Natural History Museum, Tinbergen Building, Department of Zoology
London, UK University of Oxford, South Parks Road
d.rollinson@nhm.ac.uk Oxford, UK
simon.hay@zoo.ox.ac.uk
EDITORIAL BOARD
M. G. BASÁÑEZ R. E. SINDEN
Reader in Parasite Epidemiology, Immunology and Infection Section,
Department of Infectious Disease Department of Biological Sciences,
Epidemiology, Faculty of Medicine Sir Alexander Fleming Building, Imperial
(St Mary’s campus), Imperial College College of Science, Technology and
London, London, UK Medicine, London, UK
S. BROOKER D. L. SMITH
Wellcome Trust Research Fellow and Johns Hopkins Malaria Research Institute
Reader, London School of Hygiene and & Department of Epidemiology, Johns
Tropical Medicine, Faculty of Infectious Hopkins Bloomberg School of Public
and Tropical, Diseases, London, UK Health, Baltimore, MD, USA
R. B. GASSER R. C. A. THOMPSON
Department of Veterinary Science, Head, WHO Collaborating Centre for
The University of Melbourne, Parkville, the Molecular Epidemiology of Parasitic
Victoria, Australia Infections, Principal Investigator, Envi-
ronmental Biotechnology CRC (EBCRC),
N. HALL School of Veterinary and Biomedical
School of Biological Sciences, Bios- Sciences, Murdoch University, Murdoch,
ciences Building, University of Liverpool, WA, Australia
Liverpool, UK
R. C. OLIVEIRA X. N. ZHOU
Centro de Pesquisas Rene Rachou/ Professor, Director, National Institute
CPqRR - A FIOCRUZ em Minas Gerais, of Parasitic Diseases, Chinese Center
Rene Rachou Research Center/CPqRR - for Disease Control and Prevention,
The Oswaldo Cruz Foundation in the Shanghai, People’s Republic of China
State of Minas Gerais-Brazil, Brazil
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CONTRIBUTORS
Punam Amratia
Malaria Public Health & Epidemiology Group, KEMRI-Wellcome Trust
Collaborative Programme, Nairobi, Kenya
Konstantina Boutsika
Swiss Tropical and Public Health Institute; and University of Basel, Basel,
Switzerland
Caroline W. Kabaria
Collaborative Programme, Nairobi, Kenya
Raúl Manzano-Román
Instituto de Recursos Naturales y Agrobiologı́a de Salamanca (IRNASA),
National Research Council, Salamanca, Spain
Kevin Marsh
Collaborative Programme, Nairobi, Kenya; and Centre for Tropical
Medicine & Vaccinology, Nuffield Department of Medicine, University
of Oxford, Oxford, United Kingdom
Paul Monis
Australian Water Quality Centre, South Australian Water Corporation,
Adelaide, South Australia, Australia
Abdisalan M. Noor
Ana Oleaga
vii
viii Contributors
Ricardo Pérez-Sánchez
Allan Schapira
Swiss Tropical and Public Health Institute; and University of Basel, Basel,
Switzerland
Mar Siles-Lucas
Robert W. Snow
R.C. Andrew Thompson

School of Veterinary and Biomedical Sciences, Murdoch University,
Murdoch, West Australia, Australia
CHAPTER 1
Gene Silencing in Parasites:
Current Status and Future
Prospects
Raúl Manzano-Román, Ana Oleaga, Ricardo
Pérez-Sánchez, and Mar Siles-Lucas
Contents 1.1. Introduction 2

1.2. RNAi Mechanisms and Approaches 3
1.2.1. Short-RNA types and RNAi: Basic principles 3
1.2.2. RNAi machinery in parasites 5
1.3. Delivery Tools and Methods in RNA Silencing 14
1.3.1. Uptake and spreading of dsRNAs 14
1.3.2. dsRNA delivery and stability 15
1.3.3. dsRNA delivery in parasites 17
1.3.4. Additional factors affecting the efficiency of
the RNAi outcome in parasites 20
1.4. Systematic Applications of RNAi Technology
in Parasites 21
1.4.1. Protozoa 21
1.4.2. Helminths 25
1.4.3. Arthropods 32
1.5. Future Prospects 40
Acknowledgements 43
References 43
Abstract Parasitic diseases cause important losses in public and veterinary

health worldwide. Novel drugs, more reliable diagnostic techniques
and vaccine candidates are urgently needed. Due to the complexity
Instituto de Recursos Naturales y Agrobiologı́a de Salamanca (IRNASA), National Research Council,

Salamanca, Spain
Advances in Parasitology, Volume 78 # 2012 Elsevier Ltd.

ISSN 0065-308X, DOI: 10.1016/B978-0-12-394303-3.00002-5 All rights reserved.
1
2 Raúl Manzano-Román et al.
of parasites and the intricate relationship with their hosts, devel-

opment of successful tools to fight parasites has been very limited
to date. The growing information on individual parasite genomes is
now allowing the use of a broader range of potential strategies to
gain deeper insights into the host–parasite relationship and has
increased the possibilities to develop molecular-based tools in the
field of parasitology. Nevertheless, functional studies of respective
genes are still scarce. The RNA interference phenomenon resulting
in the regulation of protein expression through the specific degra-
dation of defined mRNAs, and more specifically the possibility of
artificially induce it, has shown to be a powerful tool for the
investigation of proteins function in many organisms. Recent
advances in the design and delivery of targeting molecules allow
efficient and highly specific gene silencing in different types of
parasites, pointing out this technology as a powerful tool for the
identification of novel vaccine candidates or drug targets at the
high-throughput level in the near future, and could enable research-
ers to functionally annotate parasite genomes. The aim of this
review is to provide a comprehensive overview on the current
advances and pitfalls in gene silencing mechanisms, techniques,
applications and prospects in animal parasites.
1.1. INTRODUCTION
Parasitic diseases have a huge impact on both human and veterinary
health worldwide, frequently aggravated owing to the limited—and occa-
sionally absent—current therapeutics and vaccination alternatives. Due to
the historically underserved track of the parasitology-related research, the
World Health Organization (WHO) has lately encouraged the molecular-
based unravelling of the complex biology of parasites to get a broad
knowledge applicable to new developments in this field.
With the completion of several parasite genomes, research in molecular
parasitology has entered the ‘post-genomic’ era. Accompanied by global
transcriptome and proteome analysis, ample datasets have been generated
adding many novel candidates to the list of drug and vaccine targets. The
validation of these new targets can be reached through a combination of
reverse and forward genetics tools. In this context, functional genomic
approaches and methods for the manipulation of genes are essential tools
for deciphering the roles of genes and to validate new targets in parasites,
among them are those based on RNA interference (RNAi).
RNAi is an evolutionarily conserved eukaryotic gene silencing process
at both the transcriptional and post-transcriptional levels that operates by a
variety of molecular mechanisms and may differ among various kingdoms
and phyla. The RNAi is a gene suppression phenomenon triggered by
Gene Silencing in Parasites: Current Status and Future Prospects 3
transposable elements, sense–antisense RNA pairs, RNA hairpins, viruses

RNA and aberrant transcripts (Sledz and Williams, 2005). This process
begins with the presence inside the cell of double- or single-stranded RNA
species that are ‘diced’ into short-RNA species and associate with defined
proteins. This association leads to specific target recognition and effector
function, ultimately consisting in the decrease (knock-down) of the expres-
sion of defined proteins (Czech and Hannon, 2011).
The revolutionary finding of RNAi resulted from the work of Fire and
co-workers, who demonstrated in 1998 that injection of double-stranded
RNA (dsRNA) into the free-living nematode Caenorhabditis elegans leads
to efficient sequence-specific gene silencing (Fire et al., 1998). Over the
past few years, advances in RNAi technology have also been used to
establish functional links between genes and phenotypes in parasites.
However, it has been shown that the RNAi approach does not result in
gene knockdown in some parasite species. This could be due to either
biological or technical reasons. Thus, the definition of the presence of
RNAi pathways and their characterization in parasites is essential before
respective manipulation. Additionally, the best technical approach (e.g.
delivery method) has to be established for each parasite system. The
growing information about these two general aspects to be found in the
recent literature could also aid in defining gene silencing approaches in
parasites to which this technology has not been applied to date.
1.2. RNAi MECHANISMS AND APPROACHES
1.2.1. Short-RNA types and RNAi: Basic principles

Efforts to clone size-fractionated RNAs from cells have recovered various
classes of natural small (short) RNAs. RNAi can be triggered by dsRNA
species, mainly microRNAs (miRNAs), small interference (si) RNAs and
single-stranded RNAs, for example, the PIWI-interacting (pi) RNAs, among
others. These short RNAs can be differentiated by various features including
the nature of their RNA precursors and targets (Matzke et al., 2004).
The RNAi pathway (Fig. 1.1) is activated by exogenous or endogenous
dsRNAs. These are recognized by a RNase III enzyme family member,
which ‘dice’ dsRNAs molecules into double-stranded small RNAs
of 20–25 nucleotides in length, usually of either the interfering (Dicer,
stimulated by siRNAs from exogenous, long dsRNAs precursors) or the
miRNA (Drosha, triggered by endogenous, stem-loop pre-miRNAs) type,
leaving dinucleotide 30 -overhangs and 50 -phosphate groups in each
strand (reviewed in Jinek and Doudna, 2009). RNAi can also be triggered
by single-stranded RNA species, like the so-called piRNAs involved in
the silencing of transposable elements in germ line cells, which are
A B C
Dicer
R2D2 Dicer R2D2 Dicer
Ago
Ago Ago
Ago
dsRNA
siRNA siRNA
R2D2 Target mRNA AAAA
FIGURE 1.1 An overview of the classical cytoplasmic RNAi-mediated gene silencing

mechanism. Long double-stranded (ds) RNA enters the cell cytoplasm and binds to R2D2.
The dsRNA is then cleaved into small interfering (si) RNAs by an RNAse III enzyme called
Dicer (A). The cleaved siRNA complexed with Dicer and R2D2 binds to an argonaute-type
protein, for example, Ago (B), loosing the passenger strand and keeping the antisense
strand and constituting the RNA-induced silencing complex (RISC). This binds to the
specific complementary mRNA sequence, resulting in the cleavage of mRNA and
consequently in the knock-down of the expression of the corresponding protein (C).
processed by endonucleases different from the RNAse III family

(reviewed in Castañeda et al., 2011). The piRNAs are also distinct from
siRNAs and miRNAs in size (26–31 nucleotides), and in their lack of
sequence conservation and increased complexity.
The diced small RNAs are loaded onto the RNA-induced silencing
complex (RISC), a multiprotein complex in which the argonaute family
proteins (among them also the PIWI proteins) function as the core small
RNA-binding component. Following the removal of the passenger—
sense—strand (complementary to the guide—antisense—strand) of the
small RNA duplex from siRNA, the RISC is activated and uses the
remaining single-stranded antisense small RNA as a guide (guide strand
that provides the specificity in RNA silencing). The activated complex
targets complementary mRNA sequences for degradation, ultimately
resulting in the reduction in the levels of the protein encoded by the
degraded mRNA. The siRNAs usually base-pair perfectly and induce
mRNA cleavage only in a single specific target (Castañeda et al., 2011).
The miRNAs also bind to argonaute proteins, although they differ in their
mechanism of action from siRNAs, since they typically contain mis-
matches to target sequencing and rather than stimulating mRNA degra-
dation the presence of the complex results in the inhibition of translation
of many different mRNAs with similar sequences. Deletion mutants of
the majority of miRNAs have no obvious phenotype in C. elegans, pointing
to redundancy among miRNAs (Miska et al., 2007). The single-stranded
piRNAs also bind to argonaute (PIWI) proteins to exert their effect, pre-
senting a wide variation in sequences, generally representing antisense
complementary sequences from transposable elements. The piRNAs
regulate transposon silencing mainly in embryonic development and
spermatogenesis and seem to be functionally redundant with miRNAs

(Halic and Moazed, 2009).
The RNA-dependent RNA polymerases have also been found to play a
role in RNAi in some organisms (e.g. C. elegans; reviewed in Hannon,
2002). Those enzymes amplify the primary RNAi response synthesizing
secondary siRNAs.
Despite high levels of functional conservation, the complexity of the
RNAi machinery and associated proteins varies greatly between different
organisms, and the processing, loading and effects of regulatory small
RNAs can differ between species (reviewed in Siomi and Siomi, 2009).
Good examples of this diversity are the variable number of Dicer and
argonaute proteins found in different organisms (Carthew and
Sontheimer, 2009). In this respect, both RNAse III proteins and molecules
with similar functions, as well as argonaute proteins, have undergone a
high degree of gene duplication, especially in plants and metazoans,
followed by diversification in their function (Hutvagner and Simard,
2008). It is interesting to mention that argonaute proteins have been
segregated into three paralog groups to date, including the AGO-like
subfamily (similar to Arabidopsis thaliana AGO1), the PIWI-like subfamily
(closely related to Drosophila melanogaster PIWI protein) and the WAGO
subfamily (worm specific argonautes) reported in C. elegans (Faehnle and
Joshua-Tor, 2007). As mentioned, specific argonaute proteins can trans-
port specific classes of small regulatory RNAs to distinct cellular compart-
ments to regulate gene expression and may present redundant effects
between different small RNA species. Those peculiarities are of para-
mount importance when designing a silencing approach in a defined
organism.
In the next section, we give an overview on the RNAi machinery
described to date in the three main groups of parasites (protozoa, hel-
minths and arthropods). Then, we focus on the described methods for
dsRNA external delivery and their application in parasites and review in
detail the successful and failed attempts of RNAi in the three main
parasitic groups of protozoa, helminths and arthropods. Finally, we give
some concluding remarks about the future prospects of RNA silencing in
the field of parasitology.
1.2.2. RNAi machinery in parasites

1.2.2.1. Protozoa
Trypanosomatids represents one of the most ancient eukaryotes in which
RNAi has been experimentally verified (Ngô et al., 1998). The RNAi
pathway in Trypanosoma brucei can be initiated by two distinct Dicer-like
enzymes: TbDCL1 that is mostly found in the cytoplasm and TbDCL2 that
primarily localizes in the nucleus, suggesting operational RNAi pathways
in both subcellular compartments (Patrick et al., 2009). The silencing
action relies in the AGO-tryp argonaute protein (Shi et al., 2009). A second
member of this family, PIWI-tryp, has been identified in both T. brucei and
the related species T. cruzi (Garcia Silva et al., 2010). T. cruzi lacks RNAse
III enzymes and AGO-tryp, which suggests that the RNAi pathway could
be alternatively triggered by small RNA precursors similar to the animal
piRNA pathway (reviewed in Batista and Marques, 2011). Those peculia-
rities in the RNAi mechanism from T. cruzi should result in the lack of
RNA silencing when a double-stranded, external siRNA is introduced in
this parasite.
The related parasites from the genus Leishmania show a similar phe-
nomenon: while some Leishmania lack RNAi activity and argonaute or
Dicer genes, those of the subgenus Viannia show active RNAi machinery,
with related components that are orthologs to those found in T. brucei (Lye
et al., 2010). Whether these differences relate to pathogenic differences
between Viannia and non-Viannia Leishmania parasites remains unclear.
Nevertheless, trypanosomatids offer a good model to study the loss of
some or all of the RNAi components during evolution and its relationship
with different aspects of the host–parasite biology.
In apicomplexan parasites, the situation is similar to that in trypano-
somatids. While several reports had described the use of RNAi for gene
silencing in the blood stages of Plasmodium falciparum and Plasmodium
berghei, comparative genomic and additional RNAi studies have con-
cluded that RNAi-related molecular machinery is absent in malaria para-
sites (Baum et al., 2009). These contradictory results could account for a
specific antisense driver effect distinct from RNAi, which should be
further investigated. In contrast, the apicomplexan Toxoplasma gondii
shows a fully functional RNAi pathway, including argonaute and
RNAse III molecules (reviewed in Batista and Marques, 2011). The small
RNAs found in T. gondii co-purifying with the argonaute protein were of
the miRNA type. Intriguingly, the sequencing of miRNAs in T. gondii
showed that many are complementary to specific mRNAs, a characteristic
usually attributed to siRNAs (Braun et al., 2010).
The RNAi pathway has also been characterized in Entamoeba histolytica
(Abed and Anrik, 2005; Zhang et al., 2011), showing the presence of three
argonaute homologues and one molecule with RNAse III activity,
although with a single conserved RNAse III signature domain instead
the two usually found in higher eukaryotes.
In flagellates, some indications of the presence of a functional RNAi
pathway have been reported for Giardia lamblia and Trichomonas vagina-
lis. The phenomenon of the variability of variant surface proteins (VSP)
in G. lamblia seems to be linked with a mechanism related to RNAi,
since several enzymes from this pathway play a role in differential VSP
silencing (Prucca et al., 2008). The involvement in VSP expression regu-
lation of argonaute and Dicer proteins, together with small nucleolar
(sno)RNAs that are similar to miRNAs in G. lamblia RNAi, including

VSP RNAi, has been recently suggested (Saraiya and Wang, 2008).
Similarly, small non-coding RNAs (snoRNAs) and miRNAs have been
found in T. vaginalis (Chen et al., 2009), and some components of the
silencing machinery have been identified in its draft genome (Carlton
et al., 2007).
The transfer RNAs (tRNAs) are as well susceptible to being diced and
give rise to small RNAs. Interestingly, many of the investigated protozoan
parasites, including G. lamblia, T. cruzi and T. gondii, have shown a high
proportion of tRNAs in their small RNA populations. In G. lamblia, tRNA
is actively cleaved and sitRNAs accumulate in encysting parasites
(Li et al., 2008). The presence of abundant tRNAs could represent an
alternative and novel triggering source for RNAi in some parasites.
Further information about RNAi pathways and mechanisms in proto-
zoan parasites can be found in an exhaustive review recently published
by Kolev et al. (2011).
1.2.2.2. Helminths
In vitro maintenance and manipulation of both round and flatworms are
generally a much more complex task than the cultivation and handling of
parasitic protozoa. This has delayed the progress of post-genomic appli-
cations, including gene knock-down, in most helminths as compared with
the respective advances in unicellular parasites. However, in the past few
years, and for nematodes, the information gained in the free-living hel-
minth model C. elegans and its translation into the parasitic worms has
allowed tangible progress towards the development and use of gene
manipulation in the nematode field.
C. elegans has significantly contributed to our understanding of impor-
tant biological processes through RNAi gene silencing. Key players of the
RNAi and their mechanisms of action and biogenesis pathways in
C. elegans have been reviewed in detail by Boisvert and Simard (2008)
and Fischer (2010). A large number of protein factors are required for
RNAi in C. elegans, and its small RNA pathways are intricately linked by
shared factors acting in multiple pathways. Gene knock-down in C.
elegans has been highly successful although the success of the translation
of RNAi approaches from the C. elegans model to parasitic nematodes has
been rather variable. This could be attributed to the absence of defined
RNAi effectors in specific nematodes (Viney and Thompson, 2008). How-
ever, this explanation has been ruled out following recent comparative
genomic analysis done by Dalzell et al. (2011) that shows a similar cover-
age of RNAi functional protein groups in both parasitic nematodes in
which silencing has been successful and has failed. This supports the
broad applicability of RNAi in nematodes and suggests that variable
results of RNAi approaches among nematodes should be attributed to
other factors, for example, adverse culture conditions or differing cuticle

permeability interfering with the RNAi technology and its effects.
Regarding flatworms, the vast majority of the RNAi approaches have
been applied on trematodes. Protocols for gene silencing in schistosomes
have been described in detail (Ndegwa et al., 2007) and assays using
RNAi directed against several genes in Fasciola hepatica have also been
successful (e.g. McGonigle et al., 2008; Rinaldi et al., 2008). In Schistosoma
mansoni, both Dicer and argonaute proteins have been described in a
landmark report (Verjovski-Almeida et al., 2003) and later shown to be
differentially expressed in various developmental stages, raising the pros-
pect that RNAi technologies might be employed to decipher gene func-
tion in different life stages of this parasite (reviewed in Krautz-Peterson
et al., 2010). Krautz-Peterson et al. (2010) have proposed two RNAi path-
way models for schistosomes: (i) exogenous dsRNA is bound by SmDicer
and diced into siRNAs, and the resulting siRNAs are loaded into a RISC
that includes an Argonaute protein (SmAgo) and homologues of the
RNA-binding protein Fmr1 and the nuclease Tudor-SN, then the siRNAs
drive the identification and cleavage of cognate mRNAs to effect gene
silencing; (ii) miRNA pathway model in which the primary precursor
miRNA transcripts (pri-miRNAs) are processed in the nucleus by
SmDrosha and the resultant pre-miRNAs are exported to the cytoplasm
via Exportin-5, where they bind to SmDicer to generate miRNAs, which
are loaded into the miRNA-induced silencing complex that represses the
cognate mRNA translation.
Recommendations and proposals for RNAi approaches and large-
scale screening have been provided for S. mansoni, showing that not all
genes are susceptible to the same degree to RNAi knock-down regulation
(e.g. Stefanic et al., 2010). Nevertheless, evidences have shown that RNAi
works powerfully in schistosomes and that RNAi-based silencing could
become a high-throughput routine approach to study gene function in
this flatworm.
Among cestodes, RNAi data are scarcer than for trematodes, probably
due to the still limited genetic information currently available for the class
Cestoda. Few examples of cestode gene knock-down can be found in the
literature, although data from other platyhelminthes, like those above-men-
tioned for schistosomes, suggest that this technology should be broadly
applicable in this phylum. Genes from Moniezia expansa and Echinococcus
multilocularis have been successfully silenced by RNAi (Mizukami et al.,
2010; Pierson et al., 2010) demonstrating RNAi functionality, although the
relevant molecular machinery has not been elucidated to date. At least a
gene homology search performed by Spiliotis et al. (2010) on the assembly
version of the E. multilocularis whole genome project verified that this
parasite contains and expresses the components necessary for RNAi.
1.2.2.3. Arthropods
Gene silencing approaches have been regularly applied in the field of
entomology, namely, D. melanogaster, and the in vivo gene function studies
done in this species make it the equivalent model to C. elegans for arthro-
pods. Efforts towards the application of the RNAi technology in ticks and
mosquitoes, due to their relevance as vector of diseases, have also resulted
in a broad number of publications demonstrating its utility.
The siRNA pathway has been best studied in mosquitoes due to its
role in antiviral immunity (Saleh et al., 2009). The pathway is mediated by
Dicer2, R2D2 and Ago2, with orthologs present in almost all mosquito
groups (reviewed in Belles, 2010). To date, RNA gene silencing has been
used for investigating a number of genes in around 30 species of insects
representing a variety of orders which reflects a conserved core of the
RNAi molecular machinery throughout arthropods, although their RNAi
pathways may differ. Differences in sensitivity also highlight specific
regulatory molecules for some mosquito species that could also reflect
their different physiology, for example, differing vector competence.
The two other major RNAi pathways (miRNA and piRNA) have also
been characterized in mosquitoes (Campbell et al., 2008; reviewed in
Belles, 2010).
In ticks, RNAi has been applied successfully in the study of tick gene
function, in the screening of vaccine candidates and in understanding the
tick–pathogen interface. However, only one putative RNAi pathway has
been described for hard ticks so far (de la Fuente et al., 2007; Kurscheid
et al., 2009). The molecules already identified include a tick Dicer, RISC-
associated Ago-2 and FMRp proteins, an RNA-dependent RNA polymer-
ase (EGO-1) and several homologues implicated in dsRNA uptake and
processing. Comprehensive reviews about RNAi mechanisms in ticks
have been done by de la Fuente et al. (2007) and Kurscheid et al. (2009).
Both publications proposed complementary models of dsRNA-mediated
RNAi in ticks, including a potential tick RdRP-based mechanism of
dsRNA amplification and a systemic RNA phenomenon (spreading of
RNAi from cell to cell and thus to subsequent generations through the
germ line), similar to that described in C. elegans but absent in flies and
other animals (Tomoyasu et al., 2008). This implies that tick RNAi path-
ways may differ from those of other arthropods, a difference that war-
rants further investigation. In this respect, it should be mentioned that
recent evidence suggesting a systemic RNAi in mosquitoes (e.g. Zhang
et al., 2010) should be further verified by identifying the RdRP or SID-1
orthologs in mosquitoes.
Similarly, reports of successful gene silencing studies in sand flies,
tsetse flies, flesh and horn flies, bugs and mites have been lately published
(see Table 1.1).
TABLE 1.1 RNAi approaches in parasites
Parasite Stage RNAi source RNAi delivery method
Protozoa
Trypanosoma brucei Bloodstream and procyclic RNAi libraries and vectors, Electroporation
forms dsRNA
Plasmodium falciparum Trophozoites dsRNA Soaking, electroporation
Plasmodium berghei Trophozoites siRNA Host intravenous injection
Leishmania braziliensis Trophozoites dsRNA Transfection
Toxoplasma gondii Tachyzoites RNAi vectors, siRNA, dsRNA Electroporation
Giardia lamblia Trophozoites siRNA, dsRNA, RNAi vectors Electroporation
Entamoeba histolytica Trophozoites siRNA, dsRNA, RNAi vectors, Soaking, electroporation,
shRNA, bacteria expressing feeding
dsRNA
Trichomonas vaginalis Trophozoites siRNA Transfection
Helminths
Nematoda
Nippostrongylus Adult worms dsRNA Soaking
brasiliensis
Brugia malayi Female worms dsRNA Soaking
Onchocerca volvulus Larvae (L3) siRNA Soaking
Litosomoides sigmodontis Adult worms dsRNA Soaking, electroporation
Ascaris suum Larvae (L3) dsRNA Soaking
Trichostrongylus Larvae (L1) siRNA, bacteria expressing Soaking, electroporation,
colubriformis dsRNA feeding
Haemonchus contortus Larvae (L1–L4), adult dsRNA Soaking, electroporation,
worms feeding
Heligmosomoides Larvae (L1), adult worms dsRNA, RNAi vectors, bacteria Soaking, electroporation,
polygyrus expressing dsRNA feeding
Ostertagia ostertagi Larvae (L1, L3) dsRNA Soaking, electroporation
Trematoda
Schistosoma mansoni Cercaria, larvae, sporocysts, dsRNA, shRNA Soaking, electroporation,
miracidia, schistosomula, in vivo injection to host
adult worms, eggs
Schistosoma japonicum Schistosomula siRNA Soaking
Fasciola hepatica Newly excysted juveniles siRNA Soaking, electroporation
Opisthorchis viverrini Adult worms dsRNA, siRNA Electroporation
Cestoda and
monogeneans
Moniezia expansa Adult worms dsRNA Soaking, electroporation,
Echinococcus Primary cells, protoscoleces siRNA Electroporation
multilocularis
Neobenedenia girellae Adult worms dsRNA Soaking
Arthropods
Insects
Mosquitoes
Aedes albopictus C6/36 Cells siRNA Transfection
Aedes aegypti Isolated fat bodies, embryos, Synthetic RNA, transgenes, Soaking, in vivo injection,
larvae, adult females dsRNA, viruses, inverted transgenesis
repeat constructs
Anopheles albimanus Adult females dsRNA In vivo injection
Anopheles gambiae Cells, larvae, adult females dsRNA, siRNA Soaking, in vivo injection,
feeding
Anopheles dirus Adult females dsRNA In vivo injection
(continued)
TABLE 1.1 (continued)
Parasite Stage RNAi source RNAi delivery method
Anopheles stephensi Cells, adult females dsRNA Transfection, in vivo

injection
Armigeres subalbatus Pupae, adult females dsRNA In vivo injection
Culex pipiens Females dsRNA In vivo injection
Flies
Lutzomyia longipalpis Nymphs-4, adult females dsRNA In vivo injection
Glossina morsitans Adults dsRNA Feeding, in vivo injection
Other diptera
Sarcophaga peregrina NIH-Sape-4 cells, larvae dsRNA Soaking, in vivo injection
Lucilia cuprina Embryos dsRNA In vivo injection
Haematobia irritans Adult females dsRNA In vivo injection
Hemiptera
Rhodnius prolixus Nymph-2 and -4 dsRNA Feeding, in vivo injection
Triatoma brasiliensis Nymph-4 dsRNA In vivo injection
Ixodid ticks
Amblyomma americanum Adult females, salivary dsRNA In vivo injection, ex vivo
glands organ soaking
Amblyomma hebraeum Reproductive organs, dsRNA In vivo injection
salivary glands
Dermacentor variabilis Adult males and females dsRNA and siRNA In vivo injection
Dermacentor marginatum Adults dsRNA In vivo injection
Haemaphysalis longicornis Adult females dsRNA In vivo injection
Ixodes scapularis IDE cells, eggs, nymphs, dsRNA Soaking, electroporation,
adult females feeding, in vivo injection
Ixodes ricinus Salivary glands, adult dsRNA, siRNA Soaking, in vivo injection
females
Rhipicephalus evertsi Females dsRNA In vivo injection
evertsi
Rhipicephalus microplus Cells, Adults dsRNA Soaking, in vivo injection
Rhipicephalus sanguineus Adult females dsRNA In vivo injection
Argasid ticks
Ornithodoros moubata Adult females dsRNA In vivo injection
Ornithodoros erraticus
Mites
Varroa destructor Adult females dsRNA Soaking, in vivo injection
Crustacea
Lepeophtheirus salmonis Adults dsRNA In vivo injection
Caligus rogercresseyi Adults dsRNA In vivo injection
In summary, our knowledge on RNAi pathways in arthropods is

rapidly increasing, showing a conserved core of the RNAi molecular
machinery throughout arthropods, as well as specific features for each
group.
1.3. DELIVERY TOOLS AND METHODS IN RNA SILENCING
1.3.1. Uptake and spreading of dsRNAs

The polyanionic character of dsRNA, together with its vulnerability to
nuclease cleavage, makes uptake and duration of dsRNAs inside the cells
manipulated through RNAi technology suboptimal, due to poor cellular
uptake and rapid clearance of externally delivered dsRNA molecules
(reviewed in Wullner et al., 2009). Some other factors may influence
the silencing effect, including the concentration of dsRNA that should
be optimized for every target gene and organism, the nucleotide sequence
and length of the dsRNA fragment that determine off-target effects, the
turnover rate of the target protein influencing the persistence of the
silencing effect and the life stage of the target organisms since younger
stages often show larger silencing effects (reviewed in Huvenne and
Smagghe, 2010). In addition, the application of the RNAi technology in
parasites shows specific drawbacks depending on the parasite characte-
ristics like cell complexity and surface composition.
The mechanisms of dsRNA uptake and spreading have been mainly
described in the model organism C. elegans. Two dsRNA uptake
mechanisms have been shown in this helminth: the transmembrane
channel- and the endocytosis-mediated mechanisms. Regarding the
first mechanism, research with C. elegans systemic RNAi defective
mutants (sid) resulted in the description of two proteins involved in
RNAi (reviewed in Hunter et al., 2006). SID-1 is a multispan transmem-
brane protein that functions in passively transporting dsRNA molecules
into the C. elegans cells. SID-2 is mainly found in the intestine tissue of
the worm and facilitates environmental RNAi. Both proteins could
cooperate through the intervention of SID-2 as a first step to detect
external dsRNA and induce SID-1 activity to internalize it. Little is
known to date about the presence or requirements of those proteins in
parasites: SID-1 has been described in schistosomes (Krautz-Peterson
et al., 2010) and insects (reviewed in Huvenne and Smagghe, 2010),
and SID-2 has never been detected in parasitic organisms, being proba-
bly absent in protozoan parasites. The second dsRNA uptake mecha-
nism based on endocytosis is more likely to be a universal, shared
mechanism among parasites. Many organisms, including parasites,
have exhibited RNAi effects when soaked in medium with dsRNA.
This could be mediated by the internalization of environmental dsRNA

by endocytic-related components, as demonstrated by the lack of silenc-
ing effects after dsRNA treatment of clathrin heavy chain-defective
insects and after treatment with drugs against endosome maturation
(reviewed in Huvenne and Smagghe, 2010). The uptake mechanisms
could be evolutionarily conserved in different organisms, although the
actors of the mechanism could be diverse and should be characterized
in each organism (Saleh et al., 2006).
Another important aspect of this endocytosis-based uptake mecha-
nism is the link between antiviral immunity and RNAi, recently demon-
strated in D. melanogaster (Saleh et al., 2009). In this model, viral dsRNA is
taken up by uninfected cells, which mount an antiviral RNAi response,
limiting virus replication. The finding of viruses in parasites has been
mainly reported in protozoa, but the presence of viral infections in all
parasite taxa is most likely to occur. Thus, similar links between RNAi
pathways and antiviral immunity could be inferred in parasites and
would support the idea that endocytic uptake of dsRNA is a universal
phenomenon.
Besides dsRNA uptake mechanisms, C. elegans has also been the
model to characterize spreading of the RNAi. Exposure of C. elegans to
external dsRNA results in a systemic RNAi effect on neighbouring cells
and can be transmitted to the progeny (Tijsterman et al., 2004), an indica-
tion that dsRNA is taken up by somatic and germ line cells distant from
the point of dsRNA delivery. The nucleotide-binding protein family
called RSD is involved in this systemic effect. A comprehensive revision
of the presence of those molecules in parasitic nematodes has been
recently published (Dalzell et al., 2011). In other parasite taxa, no RSD
proteins have been identified, although alternative nucleotide-binding
proteins could functionally substitute the RSD proteins in some cases.
Without a system for the spreading of dsRNA, the effects of RNAi could,
however, be local and confined to accessible and sensible organs like the
gut in multicellular parasites.
1.3.2. dsRNA delivery and stability

The biggest challenge with RNAi in using dsRNA among non-model
organisms is delivery, since it is imperative that dsRNAs reach the cyto-
plasm of the target cell to become effective and induce silencing. An ideal
dsRNA delivery approach will seek to optimize all the steps in the
delivery process, from the need for efficient uptake and trafficking into
the cytoplasm to the need to maintain the stability and integrity of the
RNAs inside the target cells. So far, there is no universal method for
siRNA delivery as they all present several limitations; however, the
field of dsRNA delivery has advanced rapidly due, in part, to the
utilization of many delivery systems that have previously been shown to

have success in the delivery of nucleic acids for gene therapy.
Artificial dsRNA delivery strategies for RNAi include soaking, injec-
tion, feeding, transfection and electroporation. Nanotechnology is giving
new solutions to the problem of gene silencing delivery methods. Verti-
cally aligned carbon nanofiber arrays (VACNFs) grown on a silicon
substrate can be used as a parallel gene delivery platform in mammalian
cells. In 2008, Mann et al. investigated the application of VACNFs as a
platform for the rapid assay of tetracycline-inducible RNAi-mediated
gene silencing on nanostructured cell cultures, and this technical prece-
dent could help address new types of experimental questions in many
fields of study, including parasitology research (Mann et al., 2008).
The dsRNA is usually accompanied or modified to improve its half-
life and to facilitate its entry inside the target cell. These modified delivery
methods include the use of liposomes or nanoparticles, viral and vector
delivery, bacterial delivery and also chemical modification of siRNA,
either by conjugation with protein components or by modification of the
RNA backbone (Fig. 1.1). It can be difficult to predict the best method for
silencing a given gene target. Each of the delivery methods has particular
advantages and limitations, and when the null phenotype is not known,
all methods should be tried. The relative dosage of dsRNA may account
for some limitations.
Carrier-mediated methods consist in the delivery of dsRNA together
with a lipophilic molecule, and in the use of lipofected or nanoparticle-
conjugated dsRNA. In order to improve cellular uptake, dsRNAs can be
complexed with cationic lipofection reagents that interact with the nega-
tively charged RNA. However, this approach is limited to target sites that
are readily accessible, so siRNAs cannot be delivered to tissues deep
within the body in multicellular organisms. These methods can also
improve RNAi due to the increase in dsRNA stability. The most common
strategies for raising the half-life of dsRNAs are to increase their molecu-
lar weight by complexing the dsRNA into defined lipids or by encapsula-
tion into polymer-based particles.
Deliverable dsRNA can also be integrated in vector-like or viral par-
ticles that could result in nuclear delivery of gene expression cassettes
expressing short- or long-hairpin RNA. Sliva and Schnierle (2010) have
summarized the main practical issues about different virus vectors with
the aim of enhancing therapeutic efficacy. A long-term silencing effect can
be potentially achieved integrating the dsRNA in an expression cassette
that could be stably integrated into the host cell genome or expressed
episomally. Stable integration is mediated by lentivirus-based vectors,
and this has been proved in mammalian cells. In parasites, this has been
applied only recently in schistosomes (Ayuk et al., 2011; Tchoubrieva
et al., 2010) and E. histolytica (Linford et al., 2009), although the relevance
of this technology for its general use in parasites has still to be demon-
strated. More transient expression without genome integration is
provided by adenoviruses which will result in short-term gene silencing.
However, viral siRNA delivery strategies face the same challenges and
problems as other gene therapeutic approaches, for example, insertional
mutagenesis and immunogenicity.
Expression vectors are used as well for dsRNA delivery. Synthetic
libraries consisting of siRNAs or shRNA expression vectors could be
one of the methods of choice for high-throughput knock-down studies
in parasites because of their flexibility and their relatively low price.
RNAi libraries allow performing high-throughput gene knock-down
studies on a genome-wide or pathway-focused basis for the rapid and
effective identification of effective siRNAs to silence any gene of inter-
est, giving a great potential in functional genomics, therapeutics and
generation of genetically modified animal models (Zhao et al., 2005). In
many cases, those libraries are compatible with standard chips to allow
for easy identification of effector sequences. Invading, non-pathogenic
bacteria can be used as ‘carriers’ for dsRNA integrated in DNA plas-
mids, with the advantages of being safer than viral delivery, present
trivial genetic engineering and the ability to control the vector using
antibiotics.
Modification of dsRNA itself can also improve its half-life. Locked
nucleic acid is a family of conformationally locked nucleotide analogs
with unprecedented hybridization affinity towards complementary DNA
and RNA (Mook et al., 2007). Other modifications are also well tolerated
with improving the binding affinity and nuclease resistance (reviewed in
Blidner et al., 2007).
Conjugation of dsRNAs to specific proteins has also been shown to
increase its in vivo half-life, offering, in addition, the chance to target
dsRNAs to specific cells. Recently, several publications have reviewed
the progress with numerous chemical modification strategies that have
been identified allowing the overcome of many obstacles regarding the
inherent properties of dsRNAs, and the factors that must be considered
when assessing the activity of modified dsRNAs (Chernolovskaya and
Zenkova, 2010; Deleavey et al., 2009; Gaglione and Messere, 2010).
1.3.3. dsRNA delivery in parasites

Systematic delivery of dsRNA remains a challenge in translating RNAi to
parasitology research as a conventional option. Many attempts using
several delivery and carrier strategies have been applied in parasites,
although as mentioned, strategies and outcomes vary depending on the
type of parasite to manipulate. In addition, these manipulations are
normally approached in in vitro, axenically maintained parasites.
Although as parasites developing in their natural hosts could show dif-

ferences when compared to in vitro maintained parasites (e.g. transcribe a
different panel of genes), the need to develop and perform host–parasite
RNAi studies either in co-culture for some protozoan parasites or in vivo is
imperative. These types of studies have been shown to be feasible in the
schistosome mouse model, in which the in vivo administration of siRNA
to infected mice is effective in silencing parasite genes (Pereira et al.,
2008). It is worth mentioning that this induction of parasite RNAi silenc-
ing in vivo by injecting siRNAs directly into the host models may present
an attractive alternative to other commonly used therapeutic delivery
strategies.
A summary of the delivery methods successfully assayed to date in
different parasites is shown in Table 1.1. For protozoa, the first attempts
were made by direct incubation of parasites with dsRNA, which usually
resulted in gene silencing. Later, transfection experiments in some proto-
zoa, for example, T. brucei, also resulted in significant transient reduction
of target mRNA levels, although transfection reagents could elicit
biological responses that are artifactual (Bellofatto and Palenchar, 2008).
This organism has been also used to assay high-throughput siRNA deliv-
ery approaches, for example, electroporation of plasmids or libraries (e.g.
Glover and Horn, 2009). Electroporation is the most applied method for
dsRNA delivery in many other RNAi susceptible parasitic protozoa.
Feeding of bacteria expressing dsRNA is also a powerful method for the
study of gene function in phagocytic protozoa, for example, E. histolytica,
which is also technically suitable for the study of a large number of genes
(Solis et al., 2009).
In helminths soaking, electroporation and feeding methods have also
been used for the delivery of dsRNA (Table 1.1). Soaking has been the
method predominantly used, although not always with good results and
reproducibility. Wide variations in the dsRNA uptake have been reported
for different developmental stages in nematodes (reviewed in Geldhof
et al., 2007). Accessibility of target genes has also shown to be an impor-
tant factor in the efficiency of RNAi after dsRNA soaking; genes
expressed in gut and other parasite compartments in close contact with
the environment appear to be more amenable to RNAi knock-down
(Samarasinghe et al., 2011). Electroporation could be an alternative, but
it has also shown variable results, causing mortality in some parasitic
stages. Additionally, it has still to be investigated how deep the electro-
porated dsRNA penetrates and how much it disseminates throughout the
worm tissues.
In arthropods, and more specifically in ticks, several delivery methods
have been attempted for the application of dsRNA to different develop-
mental stages, and their respective advantages and drawbacks, as well
as the detailed experimental designs to perform them, have been
reviewed by de la Fuente et al. (2007) and shown in a highly instructive

video-publication by Kocan et al. (2011). These methods are incubation or
soaking, injection, capillary feeding and virus-transported dsRNA.
It is worth mentioning that injection of dsRNA into adult ticks has
been the most universal method used for in vivo RNAi in ticks, having the
additional possibility of generating high numbers of treated individuals
(eggs and larvae) via inherited RNAi (reviewed in de la Fuente et al.,
2007). Incubation with dsRNA solutions has been used mainly for in vitro
and ex vivo experiments with tick cells and tick organs (salivary glands).
Soaking of live ticks into dsRNA has been proposed as an alternative
method to induce RNAi in a large number of individuals in immature tick
stages with minimal manipulation and trauma (de la Fuente et al., 2007).
Recently, Karim et al. (2010) demonstrated that electroporation of
dsRNA into Ixodes scapularis eggs and nymphs is a simple and efficient
method for specific gene silencing in both developmental stages in large
numbers of individuals, overcoming the tedious and often traumatic
nature of delivering dsRNA to tick nymphs by microinjection or capil-
lary feeding. Delivery of dsRNA and siRNA to ticks by electroporation
could significantly advance RNAi applications over the next few years
leading to discoveries in tick functional genomics and tick-borne disease
research.
Two main in vivo approaches have been established in mosquitoes for
RNAi-mediated gene silencing: transient silencing by direct injection of
dsRNA and in situ expression of dsRNA from a stably integrated trans-
gene designed to contain an inverted repeat of the target sequence. Both
approaches have been described in detail by Catteruccia and Levashina
(2009) providing information on their use and limitations. Direct injection
of dsRNA in females is the most universally used RNAi approach in
mosquitoes, although the generation of transgenic lines expressing stable
RNAi transgenes has the advantage of providing a supply of mutant
mosquitoes for in-depth phenotypical and biochemical analyses, allowing
time and tissue-specific knock-down of the target genes through the use
of appropriate promoters (e.g. Khoo et al., 2010).
Recently, other in vivo approaches have been successfully applied in
mosquitoes; especially promising is larval feeding on chitosan–dsRNA
complexes (Zhang et al., 2010). Additionally, in vitro incubation of mos-
quito cells with dsRNA solutions has being also used (e.g. Peterson and
Luckhart, 2006).
In arthropods other than ticks and mosquitoes, dsRNA injection has
been the most used delivery methods for RNAi. This concept of dsRNA
feeding has also been successfully tested in flies and reduvids, although
its efficiency was lower than that of dsRNA injection and depended on
the developmental stage and tissue where the target gene was expressed.
In some cases, soaking in dsRNA solutions turned out to be as effective as
dsRNA injection, while less labour intensive and traumatic, thus allowing
processing a larger number of individuals with significantly higher
survival rates (Campbell et al., 2010a).
1.3.4. Additional factors affecting the efficiency of the

RNAi outcome in parasites
The efficiency of RNAi in parasites depends on several factors besides
the dsRNA or siRNA delivery method, including the targeted gene, the
off-site effects, the time at which the knock-down level and associated
phenotypes are analyzed and the parasite culture conditions. The best-
studied parasite to illustrate those drawbacks is S. mansoni. Regarding
the targeted gene, it has been shown that not all consistency, specificity
and degree of knock-down RNAi effects depend on the gene to be
silenced. Several authors have found different RNAi outcomes depend-
ing on the target gene sequence (Mourão et al., 2009; Stefanic et al.,
2010), the tissue in which the gene is expressed and the secondary
structure of its mRNA. Atkinson et al. (2010) found that the trematode
nervous tissue was specially refractory to RNAi, a phenomenon also
observed in C. elegans in which it was related to the lack of the trans-
membrane transporter SID-1 in the neurons (Winston et al., 2002).
Recently, Krautz-Peterson et al. (2010) observed different susceptibility
to RNAi silencing for three genes of S. mansoni and attributed the
refractoriness of one of them (SPRM1hc) to the secondary structure of
its mRNA, which make it less accessible to RISC than other mRNA
targets.
The silencing of some genes or gene segments may produce non-
specific or off-target effects that are not consistent with the predicted
interaction with and/or degradation of specific target transcripts, but
affecting instead phenotype-associated non-target genes (Yoshino et al.,
2010). Off-site effects are more frequent when dsRNA is delivered by the
soaking method (e.g. Ndegwa et al., 2007). Thus, it seems imperative to
include the adequate controls to rule out off-site effects to properly
evaluate each RNAi experiment.
Regarding timing and culture conditions, it has been shown that
consistent RNAi effects in Schistosoma spp. are only detectable after 6
days of in vitro culture, while analysis of parasites, an earlier culture
points, revealed inconsistent effects (Correnti et al., 2005; Geldhof et al.,
2007). Maintenance of the different parasite developmental stages, when
applicable, in optimal conditions is imperative to apply RNAi effectively.
This has been a major drawback of RNAi in schistosomes and other
parasites, which has lately fostered the standardization and optimization
of culture conditions for several parasites.
1.4. SYSTEMATIC APPLICATIONS OF RNAi TECHNOLOGY

IN PARASITES
1.4.1. Protozoa
Protozoan parasites are the cause of more sickness, death, mutilation and
debilitation in the world than any other group of disease-causing orga-
nisms. Both vector-trasmitted and foodborne protozoa have a huge
impact in underdeveloped countries, affecting millions of people and
animals and causing enormous rates of morbidity.
Parasites exercise strict control over the expression of the genes
involved in pathogenicity, differentiation, immune evasion or drug
resistance. However, until now, the mechanisms regulating gene expres-
sion are poorly understood in protozoa. This lack of knowledge is also
due to the fact that protozoan parasites are represented by organisms
with highly divergent genetic backgrounds, and thus with different
regulatory mechanisms among different groups. To date, a large num-
ber of novel gene products involved in processes pertinent to the life
cycles of some parasitic protozoa have emerged through several studies,
but many of such genes cannot be disrupted easily using conventional
approaches. Gene silencing technology could assist to elucidate the
function of many of those newly identified molecules. Unfortunately,
and as mentioned, protozoan parasites are genetically heterogeneous
with respect to RNAi pathways and components, which do not appear
to be present in all protozoan parasites and, when present, many mole-
cules are not conserved among members of the same phylum (Meissner
et al., 2007).
The technology to down-regulate gene expression for the analysis of
gene function was first applied in T. brucei and appeared to be the
technique of choice for down-regulating gene products in African trypa-
nosomes (reviewed in Atayde et al., 2011; Batista and Marques, 2011).
Of critical importance for the trypanosome bloodstream form is a dense
protective layer of a vast repertoire of variant surface glycoproteins
(VSGs). In 2009, Smith et al. demonstrated that blocking the actively
expressed VSG by RNAi in T. brucei resulted in the arrest of cell cycle,
a phenomenon that was reversible when a second VSG was expressed.
This study highlighted novel cell-cycle checkpoints that have been
further characterized by Denninger et al. (2010), also by using RNAi
technology.
High-throughput RNAi experiments in the bloodstream parasite form
have also been performed (Kalidas et al., 2011; Mackey et al., 2011). Those
studies show that a proportion of the expressed trypanosome genome is
required for efficient parasite propagation, thus representing potential drug
targets. Some of them have already been identified in high-throughput
format analysis, among them the kinases CRK12 and ERK8 are essential for
parasite proliferation (Mackey et al., 2011). Approaches conducted to the
massive sequencing of RNAi in bloodstream and promastigote T. brucei
stages have been done as well, resulting in the genetic validation of nume-
rous new potential drug targets (Alsford et al., 2011).
Some other T. brucei molecules have been specifically characterized to
be essential for parasite survival by RNAi technology. Some examples are
the ornithine decarboxylase and the spermidine synthase, implied in
polyamine biosynthesis, which are important for growth arrest and cell
death in trypanosomes (Price et al., 2010; Taylor et al., 2008). In relation to
parasite mitochondrial biology, the proteins called prohibitins have an
essential role for mitochondrial-mediated translation (Tyc et al., 2010) and
the so-called mitochondrial RNA-binding 1 protein complex has been
shown to be essential for mitochondrial functionality and thus for parasite
viability (e.g. Sharma et al., 2010). Related with evasion mechanisms, a
small conserved mitochondrial protein, namely, frataxin, has been linked
through RNAi studies with parasite protection against reactive oxygen
species (Long et al., 2008).
The procyclic forms found in the tsetse fly vector have also been
manipulated by RNAi. These studies have shown that procyclic vacuolar
proteins play an important role in the intracellular iron utilization system,
also related with parasite ‘defences’ as well as in the maintenance of
normal cellular morphology in T. brucei (Lu et al., 2007).
These steps towards large-scale trypanosome applications and initia-
tives related with RNAi studies could link thousands of previously
uncharacterized and ‘hypothetical’ genes from T. brucei to essential func-
tions and could ultimately result in the definition of new control tools
against one of the major pathogens of humans and livestock.
In other members of the same family, specifically T. cruzi, the major
conventional molecules involved in RNAi have not been detected, with
the exception of an AGO/PIWI protein. This, together with the tRNAs-
derived small RNAs actively produced by T. cruzi, could give some
biological significance to the RNAi pathway in this parasite. Nevertheless,
every RNAi trial attempted in T. cruzi to date has failed. The absence of
some RNAi components and gene promoters in the genome of T. cruzi
could account for the presence of alternative epigenetic control mecha-
nisms in this parasite. This control could be related with its relationship
with the respective hosts. In this respect, an outstanding study of a
genome-wide RNAi screen using cellular microarrays of a printed
siRNA library of the human genome has recently reported host factors
required for T. cruzi infection (Genovesio et al., 2011). This investigation
recognized several cellular membrane proteins and others as crucial
players for parasite invasion, revealing new potential targets for antipa-
rasitic therapy.
In Leishmania parasites, a similar phenomenon has been described,

since some species present RNAi molecules and some others do not.
Only one publication has reported a successful silencing experiment in
Leishmania braziliensi (Lye et al., 2010). Gene replacement techniques,
which require the total disruption of the gene of interest generating
null-mutants, are the only resource actually developed for epigenetic
approaches in leishmania (e.g. Balaña-Fouce et al., 2008).
In apicomplexan parasites, RNAi attempts have been done mainly in
P. falciparum and T. gondii. The components of the RNAi machinery have
not been found to date in malaria parasites (Baum et al., 2009), although
intriguingly some RNAi assays have resulted in modified phenotypes
(Kumar et al., 2002; Malhotra et al., 2002; McRobert and McConkey,
2002; Mohmmed et al., 2003; Tuteja and Pradhan, 2010). Unspecific effects
cannot be ruled out, although some examples (additive effect of two
dsRNAs targeting two different genes, negligible effects of control
dsRNAs; Tuteja and Pradhan, 2010) seem to point out that observed
effects could be specific. dsRNA effects on plasmodial parasites could
be attributed to other factors, for example, an antisense effect (Militello
et al., 2005). RNA synthesis is a common transcriptional phenomenon in
P. falciparum and is catalyzed by RNA polymerase II. The antisense loci
found to date in this parasite are not predicted to contain open reading
frames, thus antisense RNA may be a novel regulator of stage-specific
gene expression in this parasite.
RNAi studies have also been applied in mosquito vectors for plasmo-
dial species to investigate the nature of the host–parasite relationship and
to assist in the identification of antiparasitic targets (Brown and
Catteruccia, 2006).
The genome of T. gondii harbours gene candidates with convincing
similarity to ‘classical’ RNAi genes. In addition, the analysis by Braun
et al. (2010) of the repertoire of small RNAs in several T. gondii isolates
represents a milestone towards the understanding of the role of RNAi in
this parasite. This species can be better readily epigenetically manipu-
lated for experimental research compared with other apicomplexan para-
sites. Any essential gene or function identified in this parasite could be
important for understanding other apicomplexan parasites. Nevertheless,
RNAi functional studies in this parasite are still relatively scarce.
Al-Anouti and Ananvoranich (2002) first demonstrate that RNAi could
be a very useful tool for the study of gene expression in T. gondii inte-
rrupting expression of the enzyme uracil phosphoribosyltransferase. The
expression of T. gondii enolase, adenosine kinase and hypoxanthine–
xanthine–guanine phosphoribosyltransferase genes have been success-
fully silenced as well (Holmes et al., 2010; Yu et al., 2008, 2009). Also, a
specific RNAi library to T. gondii glycolytic genes was constructed by
Ananvoranich et al. (2006), demonstrating that dsRNA-induced gene
silencing provides a rapid assessment on the loss-of-function effect and

could be useful for elucidating gene function as a step towards develop-
ment of anti-toxoplasmosis vaccines and therapeutic reagents. Only a
couple of RNAi studies have sought host factors influencing toxoplasmo-
sis progress to date (Ahn et al., 2009; Witola et al., 2011).
The analysis of the G. lamblia genome has revealed the presence of
several RNA silencing genes and snoRNAs as precursors for miRNA-like
molecules. The functional significance of snoRNAs in RNA silencing has
to be established yet, since their mRNA targets are unknown. Additio-
nally, high-throughput approaches have allowed the identification of mi-
RNAs specific to G. lamblia although potential RNA targets have to be
defined as well. Regardless, database mining predicts that G. lamblia has
an RNAi pathway, and it is accepted that control of variant-specific
surface protein (VSP) switching involves components of the RNAi
machinery (reviewed in Atayde et al., 2011).
Gene down-regulation mediated by externally delivered dsRNA in
E. histolytica RNAi methodologies has been shown functional and has
been applied very successfully (reviewed in Atayde et al., 2011; Zhang
et al., 2011). Some examples are the transcriptional silencing of the amoe-
bapore (Ehapa) and the cysteine protease 5 genes, resulting in tropho-
zoites which exhibit attenuated virulence (Bansal et al., 2009; Mirelman
et al., 2008). Transcriptional silencing of multiple genes in trophozoites
could provide virulence-attenuated parasites which may be an important
tool, for example, for vaccine development. In this regard, it has been
recently shown that the tRNA gene-mediated silencing mechanism,
known to be functional only in Saccharomyces cerevisiae, is also used by
E. histolytica for epigenetic transcriptional gene silencing (Irmer et al.,
2010). Thus, the use of E. histolytica tRNA arrays could be potentially
applied to silence genes in a high-throughput way for this parasite.
Investigations directed to study the possibility of using tRNAs for silenc-
ing assays in protozoa parasites in which a high content of tRNAs have
been found should be performed. This could be a major step towards the
implementation of RNA silencing as an essential tool for genome mining,
epigenetic studies in T. cruzi, G. lamblia, T. vaginalis and others.
Major drawbacks found in the generation and use of RNAi-attenuated
E. histolytica parasites, for example, long-term cultivation and selection of
‘RNAi’ negative parasites (MacFarlane and Singh, 2008) could also serve
as a guide to define respective obstacles in the application of the RNAi
technology in similar parasites. This and other related shortcomings still
have to be sorted out to reach an ample and generalized use of the RNAi
technology in protozoa.
Regarding T. vaginalis, both miRNAs and snoRNAs have been char-
acterized (Chen et al., 2009). Nevertheless, RNAi studies in this important
parasite or in similar parasites from the same group with impact in
livestock production (Tritrichomonas foetus) have been anecdotic to

date, leaving an open field for RNAi technology application in the near
future. RNAi, like for other protozoan parasites, has also been applied for
the study of host genes related with trichomonosis progression, for exam-
ple, galectins (Okumura et al., 2008).
1.4.2. Helminths
Parasitic helminths have also a great impact on global health and eco-
nomic development. Helminthosis is responsible for enormous levels of
morbidity and mortality, delays in the physical development of children
and loss of productivity related with disability-adjusted life years. It is
estimated that nearly 1billion people are infected with Ascaris lumbri-
coides, 790 millions with Trichuris trichura, 700 with Necator americanus
and Ancylostoma duodenale and 200 with schistosomes (Feasey et al.,
2010). Helminthosis in livestock production is also a raging health pro-
blem. The control of helminth parasites is still an issue to be solved
through the development of new vaccines and drugs. Identification of
novel parasite genes and gene functions would provide new parasite
targets for control.
As mentioned, since the discovery of the RNAi mechanism in the free-
living nematode C. elegans, the RNAi has been applied as a tool for the
study of gene function in a great variety of animals, including parasitic
worms. Nevertheless, RNAi has proven to be effective only for some
genes and species, generally with inconsistent results.
Besides the RNAi-related methodological particularities for each
group of helminths, two main reasons are behind the slow and disa-
ppointing development of RNAi in parasitic helminths: (i) the apparent
lack of homology between some C. elegans genes and the parasite genes,
especially those involved in the parasitic lifestyle and parasite–host rela-
tionship (Geldhof et al., 2007) and (ii) the complexity of the parasitic life
cycles, together with the difficulties for the in vitro culture of their deve-
lopmental stages and the lack of immortal cell lines.
1.4.2.1. Trematodes
In trematodes, most of the RNAi assays have been performed in S. mansoni
and, to a lesser extent, S. japonicum. Additionally, two RNAi studies on
F. hepatica and one in Opisthorchis viverrini have also been reported. Schisto-
somes are parasitic flatworms that cause schistosomiasis, one of the most
prevalent and serious parasitic diseases of humans in tropical and subtrop-
ical regions (Brindley and Pearce, 2007). Fasciolosis holds a similar status in
ruminants, and recently, it has also emerged as a major zoonosis mainly in
rural areas of Central South America, Northern Africa and Central Asia
(Mas-Coma et al., 2009). The sanitary relevance of schistosomes has
stimulated numerous investigations aimed at the sequencing of their tran-

scriptomes and genomes (Berriman et al., 2009). The RNAi technology may
help identify the function of such sequences. In this context, a comprehen-
sive and detailed review about the methodological aspects of RNAi appli-
cation in schistosomes has been recently published (Bhardwaj et al., 2011).
Skelly et al. (2003) demonstrated for the first time the experimental
viability of the RNAi technique in S. mansoni and described a clear knock-
down of the gut-associated cysteine protease, cathepsin B1. After the
former assay by Skelly et al. (2003), a series of RNAi-based experiments
have been made in S. mansoni, which typically consisted in the silencing of
one or two genes in schistosomula and the study of their functions. Some
of these studies have been previously reviewed in Kalinna and Brindley
(2007) and Han et al. (2009). These reports have demonstrated knock-
down of gene expression in several developmental stages of schistosomes
including cultured miracidia, sporocysts, schistosomula, eggs and adult
worms, and have allowed functional analysis of a variety of genes includ-
ing gut proteases, glucose transporters, redox, metabolic and proteolytic
enzymes, signal transduction proteins, tetraspanins, aquaporins and
binding proteins (Table 1.1).
Regarding gut protease function, RNAi has been applied to cathepsin
B1 and cathepsin D (reviewed in Han et al., 2009). Silencing of the cysteine
protease SmCB1, with a central role in haemoglobin digestion in the schis-
tosome gut, has shown that this enzyme is essential for parasite growth.
Digestion of haemoglobin is also accomplished by cathepsin D, and its
silencing results in the lack of black-pigmented heme accumulation in the
schistosome gut, as well as in worm growth retardation. Both proteases
could, therefore, be essential molecules in the mammalian stages of
schistosomes.
Molecules potentially implicated in parasite defence against host
oxidant components have also been functionally studied in S. mansoni
by RNAi. Knock-down of selenium-containing S. mansoni enzyme
thioredoxin glutathione reductase (TGR) expression resulted in the
death of parasites in vitro, indicating that TGR is essential for parasite
survival (Kuntz et al., 2007). On the contrary, silencing of peroxiredoxins
(Prx) 1 and 2 in S. japonicum schistosomula showed that they are not
essential for parasite survival, but that Prx1 may act as a scavenger
against reactive oxygen species (Kumagai et al., 2009). Both could be
excellent drug targets, in spite of their role in parasite survival.
RNAi has also been employed to knock-down a range of signalling
genes from S. mansoni and S. japonicum showing their involvement in
important physiological processes. The silencing of the transforming
growth factor-beta (TGF-b) receptor II gene produced a concomitant
reduction in the expression of gynecophoral canal protein (Osman
et al., 2006). Similarly, knock-down of the S. mansoni inhibin/activin
gene, a novel parasite-encoded TGF-b superfamily member, resulted in

aborted development in eggs, demonstrating that TGF-b signalling plays
a major role in the mating and embryogenesis of schistosomes (Freitas
et al., 2007). In S. japonicum, the Wnt signal transduction pathway was
analyzed showing that a decrease in the transcript levels of the Wnt
canonical pathway genes GSK-3 b and b-catenin through RNAi was
correlated with an increased mortality in schistosomula (Li et al.,
2010). Suppression of S. japonicum calcium-regulated 24kDa heat-stable
protein (CRHSP-24) gene, a major calcineurin phosphoprotein that func-
tions in multiple signal transduction pathways, induced changes in
parasite morphology and caused parasite death as well (Zou et al.,
2011). The potential of those molecules to be used as new drug targets
should be established through further localization/accessibility assays
and studies about their similarities and differences with related host
molecules.
Also related with schistosome mating, suppression of mRNA enco-
ding the gynecophoral canal protein of S. japonicum resulted in the inhibi-
tion of pairing in vivo and in vitro (Cheng et al., 2009), thus making this
molecule a good vaccine target to reduce egg production and act, as the
GST-based vaccine, in reducing the associated pathology. Related with
the potential of reducing egg production, RNAi was recently used for
knock-down of the mago-nashi gene in S. japonicum, which was previ-
ously characterized as an essential product for spermatogenesis and for
regulation of germ line stem cell differentiation in other organisms. The
results showed effects on the formation and maturation of reproductive
organs (reviewed in Han et al., 2009).
Tetraspanins are surface-associated proteins that have shown a good
potential as vaccine-target molecules in schistosomes, The functions of
tetraspanins (TSP-1, TSP-2) in the tegument of S. mansoni adults and
schistosomula were examined by Tran et al. (2010) using RNAi
approaches. The authors found that tetraspanins play important struc-
tural roles impacting development, maturation and stability of the adult
worm tegument. Similarly, Faghiri and Skelly (2009) showed that RNAi
silencing of the aquaporin gene renders the parasites less able to osmo-
regulate and are thus not only less viable but also more resistant to drugs
due to impairment in drug transport.
Some gene functional studies in schistosomal stages related with their
snail hosts have also been approached with the RNAi technology. Gene
knock-down assays have shown that S. mansoni leucine aminopeptidase 1
and 2 are essential for egg hatching and miracidia release (Rinaldi et al.,
2009). Miracidia have been the object for RNAi assays. Application of
RNAi to larval parasites against calmodulin resulted in a ‘stunted growth’
phenotype in sporocysts (Taft and Yoshino, 2011). Early intramolluscan
larval development stages have been used as model for the application of
high-throughput RNAi assays (Mourão et al., 2009). As well as identifying

very valuable methodological criteria for RNAi, this phenotypic screen
pointed to the potential involvement of some of the silenced genes in the
development of S. mansoni inside the snail. Gene knock-down attempts
have also been done in sporocysts glucose transporters, causing morpho-
logical abnormalities and a decreased viability in vivo following infection
of experimental animals (Boyle et al., 2003). This dsRNA treatment was
effective only in the transition from miracidia to sporocysts, but treatment
of fully transformed sporocysts was ineffective, showing the essential role
of this molecule for parasite transformation inside the snail host and thus
for the completion of its life cycle. These studies have opened the way to
define how the described functions could be manipulated to block the
parasite life cycle.
In F. hepatica, RNAi studies are much scarcer than in schistosomes.
Only two RNAi assays have been reported to date although F. hepatica is
an important medical and veterinary parasite whose prevention and
control remain unresolved, and RNAi technology could substantially
help in the discovery of new vaccine and drug targets. Both studies
showed the presence of a viable RNAi pathway in F. hepatica by mani-
pulating newly excysted juveniles with dsRNA to knock-down the leu-
cin aminopeptidase (Rinaldi et al., 2008), and the proteases cathepsin B
and L (McGonigle et al., 2008). Importantly, silencing of either of these
two enzymes significantly reduced penetration of the rat intestinal wall
by newly excysted juveniles in an ex vivo model experiment. A similar
situation is found for Opisthorchis viverrini, in which only one RNAi
approach has been done, resulting in the silencing of cathepsin B
(Sripa et al., 2011).
1.4.2.2. Cestodes and monogeneans

A similar situation regarding the scarcity of RNAi assays can be found for
cestodes and monogenea to date, maybe due to the difficulties inherent in
the in vitro and in vivo maintenance of their life cycles rather than to other
factors. The few publications on this topic are very recent. The first work
reports the successful application of RNAi to a cestode, the ruminant
tapeworm M. expansa, demonstrating that cestodes possess a functional
RNAi (Pierson et al., 2010).
In the same year, two reports on E. multilocularis demonstrated RNAi-
mediated knock-down of target gene expression (Mizukami et al., 2010;
Spiliotis et al., 2010). The first work is based in a masterpiece experiment
in cestodes, which is the set up with in vitro culture of primary cells origina-
ting from axenically cultivated metacestode vesicles (Spiliotis et al., 2008).
More importantly, those cells can be maintained in vitro for prolonged
periods of time and are able to fully regenerate infective metacestode vesi-
cles in culture. This system arises as a unique, highly powerfull tool for the
study of cellular and molecular basis of parasite development and host–

parasite interactions, for example, through functional RNAi assays. Thus,
targeting with dsRNA of three different genes has been demonstrated in
cultured E. multilocularis primary cells: glyceraldehyde-3-phosphate dehy-
drogenase, 14-3-3 and ezrin–radixin–moesin-like protein (elp; Spiliotis et al.,
2010). Effective knock-down of mRNA and protein levels was observed
after dsRNA electroporation, although no obvious phenotype was detected.
Soon after this study, a second approach showed the feasibility of
using RNAi in protoscoleces of E. multilocularis maintained in vitro by
silencing the 14-3-3 and the elp genes (Mizukami et al., 2010). E. multi-
locularis protoscoleces also represent a very attractive material for func-
tional studies, due to culture and manipulation facilities that have been
already established. During their experiments, Mizukami et al. (2010)
could detect a sharp and specific decrease in viability for those protosco-
leces that have been subject to RNAi.
Parasitic monogeneans have also been object of RNAi experiments,
although experiments in this group are still scarce. The species Neobene-
denia girellae, infecting the body surface of many species of marine fish, is
a crucial pathogen of commercial fish due to high mortality rates infected
fish, low host specificity and wide distribution. Knocking-down of vas-
related genes by dsRNA soaking of parasites resulted in gametogenesis
inhibition, arising the RNAi technology as a new control method based on
the spreading of dsRNA-treated and sterilized parasites in fish culture
tanks (Ohashi et al., 2007).
1.4.2.3. Nematodes
In nematodes, the RNAi technology has also been applied, although with
variable results, thus showing to be less robust and reproducible than in,
for example, schistosomes. Difficulties in the application of such technol-
ogy in this group of parasites is also related with the lack of in vivo and
in vitro maintenance and propagation alternatives, difficulties in dsRNA
delivery intrinsically related with nematode outer structures and also
potentially with the lack of the SID-1 molecule—present in C. elegans,
schistosomes and insects, related with dsRNA trasmembrane channel-
mediated uptake; reviewed in Viney and Thompson (2008)—incomplete
knock-down and thus partial or null phenotypes, transiency of pheno-
types, unheritability of the knockdown and other technical drawbacks.
These questions have been excellently reviewed by Aboobaker and
Blaxter (2004), Kalinna and Brindley (2007) and Viney and Thompson
(2008).
A very recent and complete review, including experiments systemati-
cally done in Haemonchus contortus L3 worms by Britton et al. (2011),
suggests that differences in knock-down results depending on the target
gene in parasite nematodes could also be related with limited dsRNA
uptake for defined gene expression sites in parasite nematodes (Britton

et al., 2011). The same authors consider alternative approaches to improve
RNAi to study parasitic nematode gene function.
The first RNAi-based functional study in parasite nematodes was
performed by Hussein et al. (2002) on Nippostrongylus brasiliensis. Soaking
adult worms in dsRNA to knock-down the acetylcholinesterase ACh A
isoform gene resulted in 80–90% reduction of the secretion of ACh A, B
and C isoforms. Since then, the application of RNAi has sought the
targeting of numerous genes in many different nematode parasites.
RNAi has been applied to intestinal nematodes Ascaris suum, H. con-
tortus, Heligmosomoides polygyrus, Ostertagia ostertagi and Trichostrongylus
colubriformis. In additional, RNAi functionality has also been reported in
the filarial nematodes Brugia malayi, Onchocerca volvulus and Litomosoides
sigmodontis. Aboobaker and Blaxter (2004), Geldhof et al. (2007), Kalinna
and Brindley (2007) and Knox et al. (2007) have reviewed most of the
RNAi experiments done in parasitic nematodes. Here, RNAi has been
applied to knock down more than 30 different genes with rather variable
results. One of the main conclusions to be drawn from these studies was
that RNAi has been more effective in the filarial nematodes than in the
intestinal nematodes, where the RNAi outcome has been inconsistent
perhaps as consequence of inadequate technical approaches.
The published RNAi assays done in A. suum have been effective. Tar-
geting the pyrophosphatase (PPi) gene by soaking the L3 larval stage in
dsRNA resulted in the reduction of transcript levels and native protein
expression and inhibition of the moulting process (Islam et al., 2005). These
results were consistent with previous results from the same group that
showed moulting inhibition in L3 treated with PPi-specific inhibitors
(Islam et al., 2003). Interestingly, PPi homologs have been detected in
Trichinella spiralis, A. lumbricoides, Toxocara canis, B. malayi and Loa loa,
opening up the chance to characterize a common factor for L3 moulting
and thus to manipulate it and block development in nematodes.
A similar approach resulted in the ablation of the A. suum enolase gene
expression and the gene corresponding to a specific L3 transcript of
unknown ontology, reducing the in vivo survival rate of treated nema-
todes (Chen et al., 2011; Xu et al., 2010).
RNAi in the blood-feeding nematode H. contortus was first attempted
by Kotze and Bagnall (2006). The knock-down of two b-tubulin genes by
soaking reduced transcript levels in three parasitic life stages (L3, L4 and
adults). In addition, motility reduction in L3, but not in L4 or adults, was
shown. Also in 2006, extensive analyses on the efficacy of RNAi in
H. contortus were done in which different genes were targeted in L1–L3
by feeding, soaking and electroporation of dsRNA (Geldhof et al., 2006).
Here, RNAi by feeding was ineffective, and soaking resulted in the
specific decrease of mRNA levels for only two genes (b-tubulin and
COPII). Following electroporation of dsRNA in L1, significant decreases

were observed for two out of four genes tested. The authors concluded
that a limited number of genes in H. contortus were susceptible to RNAi
approaches, and with weak and difficult to reproduce effects. More
recently, the RNAi technique has shown potential to be useful to define
putative vaccine targets in this nematode. The silencing of the H. contortus
aminopeptidase H11 gene in L3 resulted in a decrease in the number of
worms and eggs recovered from lambs upon infection with the silenced
parasites (Samarasinghe et al., 2011). The same authors evaluated the
influence of the transcript levels and gene expression site in the outcome
of soaking H. contortus L3 in dsRNA, targeting several parasite genes.
They found that genes represented by high ESTs numbers or not
expressed in the intestine, excretory cells or amphids were inconsistently
silenced. Technical drawbacks related with soaking and electroporation
with dsRNA in other nematodes, for example, O. ostertagi, have also been
pointed out (Visser et al., 2006).
The efficacy of RNAi in T. colubriformis was investigated by Issa et al.
(2005) using three different delivery methods, namely, soaking, electro-
poration and feeding on dsRNA-carrying bacteria. Ubiquitin and tropo-
myosin were used as target genes. Ubiquitin siRNA or dsRNA delivery
by soaking or electroporation, but not by feeding, inhibited the develop-
ment of T. colubriformis. However, feeding was successful in silencing the
tropomyosin gene.
Inconsistent results have also been reported for RNAi applied to
filarial nematodes. RNAi was first employed to knock-down RNA poly-
merase II, b-tubulin and the microfilaria sheath protein genes in B. malayi
adult worms (Aboobaker and Blaxter, 2003). RNA polymerase and
b-tubulin knock-down produced a reduction in respective transcript
levels that was later resulting in the death of the adult worms. The
knock-down of microfilaria sheath protein was not lethal to adult
worms but resulted in a reduction in microfilariae release and in morpho-
logical changes in those that were released.
The potential blocking of the parasite life cycle has also been assayed
in RNAi experiments. The injection of dsRNA and siRNA targeting the
cathepsin L-like cysteine protease B. malayi gene in parasitized mosqui-
toes resulted in a reduction of gene transcription and a consequent reduc-
tion in worm motility, preventing the migration of larvae to the mosquito
proboscis (Song et al., 2010).
Cathepsins have been shown to play important roles in filarial
biological processes within the host, such as moulting, cuticle remodelling,
embryogenesis, feeding and immune evasion. This has been functionally
demonstrated in several filarial parasites. In O. volvulus silencing of two
cathepsins (L and Z) and the serine protease inhibitor (OV-SP1) genes by
soaking of L3 stage larvae in dsRNA significantly reduced larval moulting
(Ford et al., 2005; Lustigman et al., 2004). Similarly, the knock-down of B.

malayi cathepsins L and Z genes resulted in decreased numbers of released
microfilariae and the disruption of embryogenesis (Ford et al., 2009).
1.4.3. Arthropods
In contrast to the use of the RNAi technology in parasite groups, RNAi is
already regularly applied in the field of entomology to study the mecha-
nism itself and the function, regulation and expression of arthropod genes.
As mentioned previously, most of the RNAi studies have been done in
insect species and particularly in the model organism D. melanogaster. The
growing availability of insect genomes, as for other parasites, is revealing a
large array of genes with unknown functions, and RNAi is allowing their
rapid and straightforward functional characterization in diverse arthropod
fields including innate immunity, embryogenesis, pattern formation,
reproduction, biosynthesis and behaviour. All of these RNAi-based
approaches have generated a huge amount of publications, some of
which were recently reviewed by several authors in different contexts.
Belles (2010) comprehensively reviewed RNAi-based studies in
arthropods, covering 30 species and nine orders, together with Terenius
et al. (2011), which collected detailed data from more than 150 RNAi
experiments in Lepidoptera, and analyzed the variation of RNAi effi-
ciency as a function of the dsRNA features and delivery method, the
target species, developmental stage and tissue, and the function of the
targeted gene. Huvenne and Smagghe (2010) brought together the current
knowledge on the uptake mechanisms of dsRNA in insects, highlighting
the transmembrane channel- and the endocytosis-mediated mechanisms,
and the information on successful RNAi experiments by autonomous
feeding of the target insect showing the potential of RNAi to control
pest insects. On this track, and focusing on non-drosophilid insects,
Mito et al. (2011) reviewed and discussed on the applications of RNAi
for the development of species-specific insecticides.
Among the parasitic arthropods, RNAi has been mainly applied to
mosquitoes and ticks due to their role as vectors of pathogens affecting
man and animals worldwide (e.g. Alphey, 2009; de la Fuente et al., 2007).
Those approaches have resulted in a better understanding of respective
gene function and thus of the vector–host and vector–pathogen interfaces,
paving the way to use the RNAi technology for the development of pest
control measures and transmission-blocking vaccines.
1.4.3.1. Ticks
Tick genome resource availability is scarce compared to those for some
other insects, which has resulted in more limited RNAi attempts in this
group of arthropods when compared with others.
Aljamali et al. (2002) performed the first RNAi assays in Amblyomma

americanum demonstrating the applicability of RNAi in ticks. The silencing
of the salivary histamine binding protein from female ticks by dsRNA
injection resulted in an aberrant tick feeding pattern. Later, RNAi experi-
ments, many of them reviewed by de la Fuente et al. (2007), suggested that
ticks utilize a dsRNA-mediated RNAi similar to that described in flies and
mosquitoes. Additionally, those experiments have consistently demons-
trated that the application of dsRNA via body cavity injection, feeding or
soaking leads to global and persistent gene silencing in treated ticks and
their progeny.
The publications on ticks RNAi refer to 10 ixodid species from the
genera Amblyomma, Dermacentor, Haemaphysalys, Ixodes and Rhipicephalus
(Table 1.1), but not to soft ticks to date. Most of those studies have
consisted in dsRNA-mediated RNAi of a single gene or a few genes in
adult ticks and the subsequent determination of tick survival, engorge-
ment weight, oviposition and egg hatching to analyze the effect of the
silencing of the target gene on tick feeding, digestion and reproduction.
Since related publications are quite numerous, the following will be
restricted to a selection of illustrative RNAi-based studies covering repre-
sentative tick functions and species.
Tick feeding is a complex process involving host defensive responses
(haemostatic, inflammatory and immune), faced by the parasites with
bioactive lipids and proteins that are secreted into the saliva and then
inoculated into the host to assist blood feeding (Francischetti et al., 2009).
Saliva secretion processes have been characterized by RNAi. Silencing of
the A. americanum synaptobrevin and nSec-1 genes resulted in inhibition
of secretion of anticoagulant proteins and aberrant tick feeding, demons-
trating the role of those proteins in salivary glands vesicle exocytosis
(Karim et al., 2005). Similarly, RNAi of the Haemaphysalis longicornis
valosin-containing protein (Boldbaatar et al., 2007) and I. scapularis
Naþ/Kþ-ATPase (Karim et al., 2008) impaired salivation and decreased
the amount of ingested blood. More recently, Campbell et al. (2010b) used
RNAi to silence the Ixodes ricinus aquaporin-1 (IrAQp1) thereby showing
its important role in blood meal water handling.
Midgut-associated proteolytic enzymes for blood meal digestion have
also been studied in RNAi approaches. Some of these proteases have been
studied, showing (i) the involvement of cubilin-related serine proteases
(HlSP, HlSP2 and HlSP3) in lumen haemolysis (Miyoshi et al., 2004, 2007,
2008); (ii) the role of longpepsin (aspartic proteinase) and legumains
HlLgm1 and HlLgm2 (asparaginyl endopeptidases) in the haemoglobin
digestion cascade (Alim et al., 2009; Boldbaatar et al., 2006) and (iii) the
function of leucine aminopeptidase (HlLAP) in both the blood digestion
in the midgut and the supply of nutrients to developing oocytes in the
ovary (Hatta et al., 2007, 2010).
In ticks, iron homeostasis has to be tightly maintained for its uptake,

utilization, transport and storage. Hajdusek et al. (2009) used RNAi to
silence the ferritin-1, ferritin-2 and iron regulatory protein-1 of I. ricinus
and demonstrate the role of these proteins in the non-heme iron storage in
digestive gut cells, its transport through hemolymph and its utilization by
peripheral tissue cells.
Initiation of vitellogenesis by blood feeding is a key event in the
reproductive cycle of the tick. This process involves the massive synthesis
of vitellogenin (Vg) by the fat body, midgut and ovary (Thompson et al.,
2007), the release of Vg to the hemolymph and its incorporation into
growing oocytes through a receptor (VgR). RNAi has allowed the disrup-
tion of VgR expression in Dermacentor variabilis (Mitchell et al., 2007) and
H. longicornis (Boldbaatar et al., 2008) and thus the accumulation of Vg in
oocytes, resulting in the inhibition of tick oviposition.
Subolesin represents a special example of the use of RNAi to discover
the biological function of a novel tick protein, since its functions are
multiple (reviewed in de la Fuente et al., 2007). RNAi silencing of subo-
lesin was shown to reduce tick survival and reproduction and cause
degeneration of gut, salivary gland, reproductive tissues and embryos,
and sterility in males and, more importantly, decreased tick vector capa-
city (de la Fuente et al., 2007). Later on, it has been shown that subolesin
plays a role in the regulation of gene expression, therefore, affecting
multiple cellular processes (de la Fuente et al., 2008; Galindo et al.,
2009). Accordingly, subolesin is a conserved molecule in ticks, and, as
demonstrated in several tick species, its heterologous silencing resulted in
tick physiology impairment (reviewed in de la Fuente et al., 2007).
In addition, subolesin has been successfully knock-down (up to 90%
reduction in gene expression) in the soft tick species Ornithodoros erraticus
and Ornithodoros moubata by injecting dsRNA into adult ticks. Silenced
females fed normally, but their oviposition was completely inhibited
(Manzano-Román et al., 2011), showing that subolesin is also essential
in this tick group. This is the first report of RNAi silencing in soft ticks,
thereby demonstrating a functional involvement of an RNAi machinery.
Regarding tick defence proteins, RNAi has been successful in defining
molecules with anti-inflammatory, immunosuppressive, wound-healing
and anticoagulant roles (reviewed in de la Fuente et al., 2007; Decrem
et al., 2008; Kotsyfakis et al., 2007; Liao et al., 2009), showing that the
silencing of several of those molecule types also resulted in the reduction
of ingested blood.
RNAi has also been applied to understand the molecular interactions
between pathogens and tick vectors towards the development of novel
control measures. Studies on several tick–pathogen pairs have demons-
trated that tick gene expression is modified in response to pathogen
infection (e.g. Zivkovic et al., 2010).
Very different proteins of five tick species have been characterized that
play a role in the infection and transmission of several pathogens, inclu-
ding Anaplasma marginale, Anaplasma phagocytophilum, Borrelia burgdorferi,
Babesia bovis and Babesia gibsoni. A good example of the ample range of
tick proteins affecting bacterial outcome has been recently published for
Anaplasma (de la Fuente et al., 2010). Some examples are subolesin, diffe-
rent proteases, VgR, several salivary proteins and gut receptors, etc. Some
molecules prevent colonization, proliferation or transmission, having a
deleterious effect on bacteria, while some others result in positive out-
comes for bacteria, for example, protecting them against host defences by
binding on the bacterial surface (e.g. Dai et al., 2010).
The transovarian transmission of some microorganisms in ticks has
also been characterized by RNAi. For example, after silencing the Rhipi-
cephalus microplus gene coding for immunophilin, Bastos et al. (2009)
found increased infection by B. bovis in tick larval progeny, thus demon-
strating that immunophilin controls the transovarial transmission of these
protozoa to eggs and larvae.
These results support the notion that RNAi constitutes an important
tool for the study of the tick–pathogen interface and might contribute to
the rapid identification and characterization of potential pathogen trans-
mission-blocking tick vaccine antigens.
In this respect, RNAi has also been applied to characterize tick protec-
tive antigens. Tick control has been primarily based on the use of acari-
cides, but these chemicals have serious drawbacks, including the selection
of tick resistant strains, environmental pollution and contamination of
food products. Among the alternative approaches for tick control, vaccines
have proven to be a feasible, cost-effective and environmental friendly
method. However, since the first release of commercial recombinant anti-
tick vaccine in 1994, the progress in development of new and more effective
vaccines has been disappointing, with the identification of tick protective
antigens as a major limiting step (de la Fuente et al., 2007).
RNAi has been already used in two tracks leading to the discovery of
tick protective antigens, showing that RNAi can be used as a rapid and
cost-effective tool for discovery of candidate vaccine antigens in ticks.
In the first one, RNAi has been applied for systematic screening of
potential protective antigens among unknown genes. This application
was initially proposed by de la Fuente et al. (2005), who demonstrated
its feasibility on I. scapularis. For this, dsRNAs were generated from pools
of cDNA clones and injected into ticks that were allowed to feed on
animals. The dsRNA pools influencing survival, feeding and fertility of
the ticks were then re-analyzed in subpools until individual functional
clones were obtained. Then, the protective clones were sequenced, iden-
tified and expressed to obtain recombinant antigens. Finally, vaccination
of animals with the recombinants was used to confirm the protective
value of the antigen. This approach is limited by the identification of those

tick proteins whose gene silencing leads to a detectable phenotype.
Another limiting factor could be the accessibility of the corresponding
target to the immunological response of the host, which should be
explored on a case-by-case basis in vaccination trials (Almazan et al.,
2010; Willadsen, 2008).
The second track is based on the use of RNAi to silence already known
genes encoding for proteins putatively essential for tick survival, and the
subsequent application of the corresponding protein for vaccination
trials. Some of these candidates (e.g. the R. microplus ubiquitin, subolesin
and Bm86 antigens) have been evaluated as recombinant proteins, either
alone or in combination, in controlled vaccine trials that confirmed the
RNAi prediction on the protective value (Almazan et al., 2010). When
RNAi treatment of ticks and vaccination of the host with recombinant
protein are combined, control efficacy could increase (Merino et al., 2011).
Together, these studies demonstrate that RNAi is an amenable tool for
the study of tick gene function. It is expected that in the near future, the
increasing tick genome sequence information will allow application of
RNAi for functional genome-wide studies to address important issues on
tick physiology, development and gene regulation.
1.4.3.2. Mosquitoes
Mosquitoes are vectors of serious human diseases such as malaria, dengue
fever and yellow fever, and despite efforts to control them, they remain a
serious problem. Identifying novel mosquito genes involved in olfaction,
blood feeding, digestion, reproduction, immunity, etc., is expected to
provide the bases for the development of novel methods to control mos-
quito populations and mosquito-borne diseases (Chen et al., 2008).
The recent genome sequence information for three major mosquito
vectors, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus
(http://www.vectorbase.org/), has been used for comparative genomics
and transcriptional profiling studies that are allowing the identification of
large arrays of novel mosquito genes. RNAi has rapidly become the tool
of choice for characterizing gene function in diverse fields of mosquito
biology and mosquito–pathogen interactions (e.g. Fragkoudis et al., 2009).
This has resulted in the publication of quite numerous functional RNAi
assays in this organism group, including members of the aedines
(A. aegypti, Armigeres subalbtus and C. pipiens) and anophelines (A. gambiae
and Anopheles stephensi). Most of these studies, however, have focussed on
only two species: the main vector of dengue and yellow fever, A. aegypti,
and the African vector of malaria, A. gambiae (Table 1.1).
In those studies, the typical experimental strategy involved microin-
jection of dsRNA into the thorax of adult mosquitoes followed by feeding,
challenging with pathogens, odorants, insecticides or stressing
conditions, and the subsequent examination of the mosquitoes to analyze

the phenotypical effect of the silencing of the targeted gene on the
physiological process under study, including olfaction, feeding, digestion
and metabolism, stress, detoxification, cuticle formation, reproduction,
immunity and diapause regulation. In the following, we present some
representative studies of RNAi in mosquitoes.
Olfaction mediates a wide range of both adult and larval mosquito
behaviours, including feeding, host preference, mate location/selection
and breeding sites for oviposition. Olfaction involves perception of chemi-
cal stimuli provided by odorant molecules and development of specific
responses to such stimuli. Odorants are captured by odorant-binding
proteins (OBP), which transport them to the odorant receptors (OR) on
the dendritic membranes of olfactory neurons. Recent RNAi targeting of
both OBP and OR genes have provided valuable information on their
function in the olfaction mechanism and specificity (Biessmann et al.,
2010; Liu et al., 2010; Pelletier et al., 2010), opening the opportunity to
modify olfaction perception and thus a range of behaviours that could
result in the prevention of mosquito biting and mating.
In temperate climates, adult female mosquitoes overwinter in dia-
pause, a period of dormancy that is characterized by the absence of
host-seeking behaviour, the accumulation of huge fat reserves and an
arrest of ovarian development. Manipulation of the regulation of this
dormancy episode could result, for example, in the induction of dormant,
non-host-seeking mosquitoes. This has been exemplified in several stu-
dies from Dr. Denlinger’s team. In particular, they have recently used
RNAi to investigate the diapause mechanism in C. pipiens, to evidence the
participation of the insulin/FOXO signalling pathway in the regulation of
diapause (Sim and Denlinger, 2008, 2009a). They have also evidence for
the involvement of some fatty acid synthases in the accumulation of fat
reserves in overwintering females (Sim and Denlinger, 2009b), and for the
role of ribosomal proteins S3 and S2 in follicle development in non-
diapausing females (Kim and Denlinger, 2010; Kim et al., 2010).
Vitellogenesis and reproduction have also been characterized by
RNAi in mosquitoes. Similar to the situation in ticks, anautogenous mos-
quitoes require the intake of vertebrate host blood to initiate a reproduc-
tive cycle involving egg production. The amino acids (AA) derived from
the blood meal are used by the mosquito fat body to synthesize yolk
protein precursors, mainly vitellogenin (Vg), in a process termed vitello-
genesis. Vg is then released to the haemolymph and taken up by the
ovaries and deposited into developing oocytes through a specific VgR.
In the mosquito A. aegypti, Vg gene expression has been intensively
studied by the team of Dr. Raikhel in a long series of elegant experiments,
in which they used RNAi as tool for reverse functional genomics. This
team demonstrated the transcription of Vg gene being tightly regulated by
the combined inputs of several molecules, including the steroid hormone

20-hydroxyecdysone (20E) cascade and the nutritional AA/Target-of-
Rapamycin (TOR) signalling. This serine/threonine kinase is responsible
for transducing the AA signal, activating the phosphorylation of S6 kinase
that is required for activation of translational events (Park et al., 2006; Roy
and Raikhel, 2011). Furthermore, by injecting a specific antagomir,
the same team has produced RNAi-mediated depletion of the miRNAi
miR-27 in order to demonstrate the function of miR-27 as positive regula-
tor in both blood digestion and egg development. In fact, miR-27 per se is
regulated by the 20E and AA/TOR pathways (Bryant et al., 2010).
The innate immune system of mosquito vectors comprises three func-
tional categories of genes involved in pathogen recognition, signalling
pathways mediating signal amplification, modulation and transduction
and effector mechanisms mediating pathogen clearance from the host
(Baton et al., 2008). RNAi is itself a major antiviral immune mechanism
in mosquitoes. In RNA-based antiviral immunity, viral dsRNA is recog-
nized and processed into siRNAs by the mosquito Dicer. After that, these
virus-derived siRNAs guide specific antiviral immunity through RNAi
and related RNA silencing effector mechanisms (Ding, 2010). Numerous
papers report on the use of RNAi to characterize the function of mosquito
immune genes. Studies on general immune mechanisms and effectors
have focused on pattern recognition receptors, antibacterial and antifun-
gal signalling pathway molecules and proteins involved in other cellular
effector mechanisms (reviewed in Dong et al., 2006; Moita et al., 2005; Shin
et al., 2003, 2006; Wang et al., 2006).
RNAi has also been used in the characterization of the mosquito–
pathogen interface, many investigations looking at immune-related
mosquito molecules. Molecular interactions at the mosquito–pathogen
interface ensure survival and development of both the pathogen and the
vector. Therefore, understanding the molecular interactions between
pathogens and their mosquito vectors is fundamental towards the deve-
lopment of novel control measures. In the past decade, an intense research
effort has been deployed towards the identification and functional cha-
racterization of the mosquito genes involved in pathogen-induced immune
responses using a variety of approaches including comparative genomics,
transcriptional profiling and RNAi-based functional analysis (Baton et al.,
2008). These efforts have been focused mainly on two mosquito–pathogen
associations, A. gambiae-Plasmodium sp. and A. aegypti-dengue virus
2 (DENV-2). The mosquito innate immune responses to arbovirus infec-
tion have been reviewed in detail by Fragkoudis et al. (2009), and those to
malaria parasites have been recently reviewed by Brown and Catteruccia
(2006) and Baton et al. (2008), including RNAi approaches.
Most of these RNAi-based studies have targeted genes from the
mosquito immune defence mechanisms rather than genes needed by
pathogens to develop in the mosquito vector. Regarding the mosquito

anti-malarial genes subjected to RNAi knock-down, most of them are
those involved in ookinete killing and melanization during midgut pene-
tration (reviewed in Brown and Catteruccia, 2006; Baton et al., 2008). With
respect to the antiviral mosquito genes subjected to knock-down, some
are components of signalling pathways, while others form part of the
RNAi-based antiviral defence (reviewed in Fragkoudis et al., 2009).
Among the most recent papers, Guo et al. (2010) present a useful
approach to identify novel A. aegypti proteins interacting to DENV-2.
These authors developed the first draft of the mosquito protein interac-
tion network using a computational approach and identified 714 putative
DENV-associated A. aegypti proteins that group into four main functional
categories (replication/transcription/translation, immunity, transport
and metabolism). Ten of these putative DENV-associated proteins were
randomly selected for validation by RNAi-mediated gene silencing, and
dengue viral titre in mosquito midguts was significantly reduced for five
of them.
Collectively, these results support the notion that RNAi could be a
powerful tool for high-throughput characterization of the immune system
of insect disease vectors, thus contributing to the identification and cha-
racterization of potential mosquito targets for the development of novel
methods to control mosquito populations, parasites inside mosquitoes
and mosquito-borne diseases.
After the discovery that RNAi is one of the mosquito’s major defences
against arboviruses, it has been reported that suppression of this pathway
increases viral load in infected mosquitoes (Sanchez-Vargas et al., 2009).
Cirimotich et al. (2009) used a Sindbis virus engineered to express a
protein that binds to dsRNA and presumably protects it from processing
in the RNAi pathway, thereby acting as an RNAi suppressor. This engi-
neered virus produces much more virus particles than normal in infected
mosquitoes and is lethal to a range of mosquito species (A. aegypti, Aedes
albopictus, C. trithaeniorhynchus). This approach is an example of new
genetics-based development, potentially useful in ‘population suppre-
ssion’ strategies for mosquito control (Alphey, 2009).
The opposite goal, that is, the artificial boosting of RNAi-based anti-
viral mosquito immunity to obtain virus-resistant mosquitoes, has also
been developed. Antiviral RNAi has been used to confer resistance to
DENV in transgenic A. aegypti mosquitoes, by expressing a hairpin RNA
corresponding to a fragment of the virus (Franz et al., 2009). Using tissue-
and time-specific promoters, the expression of hairpin RNA can be li-
mited to the midgut—the first cells to be infected— and only after blood
meal, minimizing potential problems for mosquito fitness derived from
the constitutive expression of a long-hairpin RNA. A discussion on the
advantages and problems of using this kind of transgenic resistant
mosquito strains in ‘population replacement’ strategies for mosquito

control can be found in Alphey (2009).
1.4.3.3. RNAi in other parasitic arthropods

Novel RNAi protocols have also been developed for several other para-
sitic arthropods different from ticks and mosquitoes. These include insect
disease vectors such as tsetse flies (Glossina morsitans morsitans), reduviid
bugs (Rhodnius prolixus and Triatoma brasiliensis) and sand flies (Lutzomyia
longipalpis), and other medically and/or veterinary important pests such
as the flesh fly Sarcophaga peregrina, horn fly Haematobia irritans, sheep
blowfly Lucilia cuprina and the honey bee mite Varroa destructor (e.g.
Araujo et al., 2009; Caljon et al., 2010; Campbell et al., 2010b; Concha
and Scott, 2009; Sant’Anna et al., 2009; Torres et al., 2011; Walshe et al.,
2009; Wang et al., 2009; Yang et al., 2010).
Similar to ticks and mosquitoes, RNAi has been successfully used in
these other arthropod groups for (i) the analysis of gene function, that
is, R. prolixus nitrophorins (Araujo et al., 2009); (ii) the analysis of the
vector–pathogen relationship, that is, G. m. morsitans-T. brucei (Wang
et al., 2009) and (iii) the identification of candidate protective antigens
for the development of vaccines again insect pests, that is, H. irritans
(Torres et al., 2011).
Additionally, RNAi protocols have also been successfully applied to
parasite arthropods of fish, and more specifically to copepods of the
family Caligidae. These are major ectoparasites of farmed and wild
Atlantic salmon and thus have a huge economical importance in aquacul-
ture. Intra-haemoceloic injection of dsRNA to Lepeophtheirus salmonis
adults directed to the prostaglandine synthase of this parasite resulted
in persistent knock-down of its expression (Campbell et al., 2009). The
akirin molecule of Caligus rogercresseyi, an ortholog of tick subolesin, has
been also silenced by RNAi, resulting in a lethal knock-down for a
percentage of the dsRNA-treated parasites (Carpio et al., 2011).
1.5. FUTURE PROSPECTS
The RNAi phenomenon was first described only 10 years ago. Since then,
many advances have been made in the use and manipulation of this
pathway in different organisms. The major advances have been accom-
plished in model organisms such as C. elegans and Drosophila. Unfortu-
nately, many of those advances often cannot be directly translated to
related organisms, for example, parasites.
The development of novel tools to fight against parasites is still ham-
pered by the lack of information about parasite biology and the complex
relationship with their hosts. The RNAi phenomenon has opened new
and interesting opportunities to generate information about the largely

uncharacterized gene functions in parasites.
The information regarding the knowledge about RNAi and gene silen-
cing in parasites has also evolved very fast, showing its enormous poten-
tial in the field of parasitology for functional annotation of genes and
genomes. This potential is actually applicable for parasites in which the
RNAi pathway is present and has been characterized, and from which the
genome is already known. For parasites lacking the RNAi pathway, it
should be further investigated if alternative mechanisms similar to that of
RNAi are functional. RNAi could also be approached in defective para-
sites by exploring the artificial transfection of the RNAi components from
a related parasite.
Common efforts towards the elucidation of parasite genomes and RNAi
mechanisms could lead to the application of high-throughput-based RNAi
assays for the integral annotation of parasite genes, and thus to the know-
ledge of parasite physiology and further definition of new targets to fight
against them. For high-throughput screenings in parasites, automated
phenotyping approaches will need to be incorporated.
RNAi is also a promising tool for studying the parasite–host interface
through the identification of pathogenic determinants. This has been shown
to be feasible injecting RNAi targeting either the parasite or the host to in
vivo experimental models, from which the gained information after silenc-
ing would give a more realistic view about functionality of the silenced
molecules than the in vitro assays. In this respect, therapeutic gene silencing
in parasites or infected hosts for the study of the host–parasite relationship
is a virtually unexplored field to be considered in the future.
Nevertheless, there are still some drawbacks that have to be overtaken
to get into a proper and fully satisfactory application of the RNAi tech-
nology in the parasitology field. Those inconveniences were enumerated
already in 2004 by Aboobaker and Blaxter referring to helminth
RNAi applicability—which life cycle stage?, which method of dsRNA
delivery?, which target gene?, duration of the RNAi effect?— and they
are still valid to some extent. This will, of course, depend first in the type
of parasite we want to manipulate and on some of their characteristics
(see Box 1.1), like dsRNA permeability, potential siRNA amplification
and amenability to be manipulated (and so maintained and multiplied/
transformed) in vitro or in vivo. Some parasites could be finally shown to
be resistant to RNAi for any or several of those reasons, and then alter-
natives should be sought, for example, use of models, generation of
parasite cell lines or application of RNAi approaches to the parasite inside
their natural hosts.
In many cases, the development of specific technical tools is going to
be needed to deal best with the intricacies of the parasite and the stage we
intend to manipulate through RNAi.
BOX 1.1
BARRIERS FOR ACHIEVING IN VITRO OR IN VIVO

SILENCING IN PARASITES
Surrounding
tissues, serum
nucleases, acidic
medium
Internalization
and endosomal
entrapment-
Cellular uptake
lysosomal
degradation
Extracellular
Pharmacokinetics
matrix
and
Tegument
biodistribution
Cuticle
Multiple barriers are faced by artificially delivered dsRNA mole-

cules to reach their specific targets inside the parasites’ cells. Some of
those barriers are solely attributable to the parasite itself—external
parasite’s structures and components: the extracellular matrix at the
surface of unicellular parasites and additionally, the tegument in fla-
worms and cuticle in roundworms— and can result in poor dsRNA
penetration. In addition, dsRNA cellular uptake can be more or less
effective depending on the parasite cell type. Some others are due to
the host and should be taken into consideration when performing an
in vivo RNAi approach. The type of tissues surrounding the targeted
parasite, nucleases present in the serum, and potential acidic medium
could result in dsRNA degradation and entrapment. The dsRNA
could also be lost by host cell internalization, entrapment in endo-
somes and lysosomal degradation. Finally, other factors influencing
the quantity of dsRNA reaching its target inside the parasite are shared
by the host and the parasite—pharmacokinetics and biodistribution.
BOX 1.2
Liposomes and Chemical

Nanoparticles Other carriers Conjugation Others
lipoplexes modifications
•Lipid-based •Lipid based •Polymers •Lipids •Ribose 2 ¢-OH •Penetration

transection nanoparticles •Peptides •Aptamers modifications enhancers
•Cationic •Inorganic •Parasite •PEG •Phosphodiester •Protectors
liposomes nanoparticles receptors backbone
•Dinamic
•PEGylated •Antibodies polyconjugates modifications
liposomes •Ribose
•Biodegradable
•Stable nucleic particles modifications
acid-lipid •Polymeric •Base
particles nanocarriers modifications
Improve siRNA nuclease resistance

Improve siRNA potency
Reduce siRNA immunogenicity
Reduce siRNA off-target effects
Improve siRNA pharmacokinetics
Allow in vivo delivery
siRNA formulations for systemic application face a series of hur-

dles before reaching the cytoplasm of the target cell. These drawbacks
have been overcome with the development of better delivery
approaches through the modification of RNA molecules and the use
of alternative vehicles and components to improve stability and deliv-
ery of siRNA to finally promote its trafficking to the cytoplasm and
uptake in the RISC complex.
The recent advances in this field applied to other organisms and

models, for example, new dsRNA delivery methods (see Box 1.2), could
be translated to parasites. This, together with an extensive collaboration in
the parasitology community, could uncover many unknown aspects of
parasites and parasitic diseases.
ACKNOWLEDGEMENTS
Thanks are given to Dr. Bernadette Connolly (University of Aberdeen, UK) and Dr. Norbert
Müller (University of Berne, Switzerland) for critical reading of the chapter.
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CHAPTER 2
Giardia—From Genome to
Proteome
R.C. Andrew Thompson* and Paul Monis†

2.2. Current Status of Genome and Proteome Projects 59
2.3. What is Giardia?—Evolutionary Biology
and Phylogeny 60
2.4. Taxonomy and Nomenclature 62
2.5. The Maintenance of Giardia in Nature 66
2.5.1. Life cycle and development 66
2.5.2. Hosts 68
2.5.3. Transmission 72
2.6. Interaction Between Cycles 74
2.6.1. Zoonotic transmission 75
2.7. Functional Significance of Genetic Variation 79
2.7.1. Developmental biology 79
2.7.2. Pathogenesis, variation in virulence and
polyparasitism 81
2.8. Conclusions 83
References 84
Abstract In this review, the current status of genomic and proteomic

research on Giardia is examined in terms of evolutionary biology,
phylogenetic relationships and taxonomy. The review also describes
how characterising genetic variation in Giardia from numerous
hosts and endemic areas has provided a better understanding of
life cycle patterns, transmission and the epidemiology of Giardia
infections in humans, domestic animals and wildlife. Some progress
* School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, West Australia, Australia
{
Australian Water Quality Centre, South Australian Water Corporation, Adelaide, South Australia, Australia

57
58 R.C. Andrew Thompson and Paul Monis
has been made in relating genomic information to the phenotype

of Giardia, and as a consequence, new information has been
obtained on aspects of developmental biology and the host–
parasite relationship. However, deficiencies remain in our under-
standing of pathogenesis and host specificity, highlighting the
limitations of currently available genomic datasets.
2.1. INTRODUCTION
The taxonomy, life cycle patterns and zoonotic potential of Giardia (Fig. 2.1)
infecting mammals and birds have been poorly understood and contro-
versial for many years (Thompson and Monis, 2004). However, the devel-
opment of molecular tools for characterising isolates of Giardia directly
from faeces or environmental samples has helped to resolve the taxonomy
of the most common forms of Giardia parasitising mammals, and we
use the nomenclature proposed in this review (Table 2.1). In addition,
major advances have been made in understanding the transmission and
epidemiology of Giardia and giardiasis. The availability of full genome
sequences for several species of Giardia now offers the potential to better
understand host specificity and pathogenesis which will require not only
a greater emphasis on bioinformatic analysis but also the application of
proteomic technologies to Giardia in order to fully realise the value of the
available genomic data. In this review, which seeks to ‘update’ our earlier
review on genetic variation in Giardia (Thompson and Monis, 2004), we
discuss how ‘marrying’ available genetic and phenotypic data in the
context of improvements in proteomics will provide important insights
into the host–parasite relationship.
FIGURE 2.1 Scanning electron micrograph of Giardia trophozoites showing ventral

adhesive disc anteriorly and flagella in the trophozoite at top right (courtesy of Dr. Peta
Clode, Centre for Microscopy, Characterisation and Analysis, University of Western
Australia).
Giardia—From Genome to Proteome 59
TABLE 2.1 Species of Giardiaa
Species Assemblage Host(s)
G. duodenalis A Humans and other primates, dogs, cats,

livestock, rodents and other wild mammals
G. enterica B Humans and other primates, dogs, cats and
some species of wild mammals
G. canis C/D Dogs, other canids
G. bovis E Cattle and other hoofed livestock
G. cati F Cats
G. simondi G Rats
G. ?b H Pinnipeds
G. ?b ? Marsupial (Quenda, bandicoot)
G. microti Rodents
G. psittaci Birds
G. ardeae Birds
G. muris Rodents
G. agilis Amphibians
Details in Monis et al. (2009) and Thompson and Monis (2011).

a
Designation based on original taxonomic description.
b
Novel lineages of Giardia distinct from the described species, likely new species but not yet formally
described.
2.2. CURRENT STATUS OF GENOME AND

PROTEOME PROJECTS
Currently, there are genome sequences available from three isolates of

Giardia: WB, GS and P15, representing Giardia duodenalis (syn. Giardia
intestinalis; Giardia lamblia), Giardia enterica and Giardia bovis (Assemblages
A, B and E—Table 2.1), respectively ( Jerlstrom-Hultqvist et al., 2010a).
One of the limitations of the genome data is the quality of the GS and P15
assemblies in GC-rich regions. Different genome sequencing platforms
have different strengths and weaknesses, including how well they deal
with repeat regions or extremes in GC content, and so additional data
from alternative sequencing approaches will likely be beneficial for
improved data for these regions ( Jerlstrom-Hultqvist et al., 2010b).
In addition, genome sequences have been obtained from Spironucleus
salmonicida and Spironucleus barkhanus (Roxstrom-Lindquist et al., 2010),
demonstrating large genome differences between the morphologically
identical species. Comparison of the Giardia genomes has identified a
core set of proteins common to the three assemblages studied, as well as
major differences in particular gene families (such as variant-pecific sur-

face antigens), which could underlie the biological differences (such as
host range, disease type) between assemblages (Franzen et al., 2009;
Jerlstrom-Hultqvist et al., 2010a,b). A useful tool for data mining the
Giardia genome data is GiardiaDB (http://giardiadb.org/giardiadb/),
which provides an integrated and searchable database that combines
genome sequence data and chromosome maps with gene and protein
expression data, including cell localisation of proteins (if known)
(Aurrecoechea et al., 2009).
The Giardia genome data have been used to further elucidate lipid
metabolism (Yichoy et al., 2011), and data mining has provided insight
into the evolution of Giardia and eukaryotic cells (discussed below).
A combination of proteome and genome data has been used to identify
unique basal body proteins (Lauwaet et al., 2011). Proteomic analysis has
also been used to study metabolism in mitosomes ( Jedelsky et al., 2011).
Transcriptomes and proteomics from different growth stages are starting
to be generated and promise to provide further insight into processes
such as excystation and encystation (Birkeland et al., 2010; Kim et al.,
2009). Most recently, proteomic analysis of ventral disc extracts and
comparison with the Giardia genome database has been used to identify
novel proteins associated with the ventral disc and lateral crest (Hagen
et al., 2011). The localisation of these proteins was confirmed by the
expression of green fluorescent protein fusion constructs in trophozoites
(Hagen et al., 2011).
2.3. WHAT IS GIARDIA?—EVOLUTIONARY BIOLOGY

AND PHYLOGENY
Giardia has long been considered to be a primitive, early diverging

eukaryote due to the apparent lack of typical eukaryotic organelles such
as peroxisomes and mitochondria and on the basis of phylogenetic analy-
sis of conserved gene or protein sequences. The early branching of Giardia
(and other diplomonads) was first suggested following the characterisa-
tion of ribosomal RNAs (Edlind and Chakraborty, 1987; Sogin et al., 1989;
van Keulen et al., 1993) and later by analysis of conserved proteins such as
the elongation factors (Hashimoto et al., 1994, 1995). However, on the
basis of morphology and life history, Giardia had previously been sug-
gested to be the most derived member of the order (Brugerolle, 1975). The
status of Giardia as a ‘‘missing link’’ was further challenged by phyloge-
netic analysis of the Diplomonadida using morphological characters,
which also suggested that Giardia was the most highly derived genus in
the order (Siddall et al., 1992). Furthermore, the accuracy of the placement
of Giardia and other diplomonads relative to other eukaryotes has been
questioned because of the observed large differences in GþC composition

that can bias phylogenetic analysis (Leipe et al., 1993) and due to the
possible effects of long branch attraction (Dacks et al., 2002). Lateral gene
transfer has also complicated the elucidation of the evolutionary history
of Giardia, with many genes involved in anaerobic metabolism in Giardia
having been acquired from prokaryotes (Andersson et al., 2003; Nixon
et al., 2002a).
More recent molecular data support Siddall’s original proposal that
Giardia are highly derived, rather than primitive organisms. It is likely
that the divergence of the lineage giving rise to Giardia was subsequent
to the acquisition of introns during eukaryote evolution, following the
detection of a spliceosomal intron and eukaryote-specific spliceosomal
peptides in Giardia (Nixon et al., 2002b). More recent genome sequence
data have added further weight to these findings, identifying additional
cis-spliced introns and also finding novel trans-spliced introns, where
exons are dispersed throughout the genome and a single transcript is
produced by trans-splicing (Kamikawa et al., 2011; Roy et al., 2011).
Analyses of genomic data have also demonstrated the presence of numer-
ous eukaryotic features, such as sequences encoding eukaryotic RNA
processing machinery (Chen et al., 2011), Scaffold/Matrix attachment
regions that are involved in chromatin attachment/DNA organisation in
other eukaryotes (Padmaja et al., 2010), the presence of nucleoli ( Jimenez-
Garcia et al., 2008), meiosis-specific genes (Ramesh et al., 2005) and path-
ways for RNA regulation such as RNA silencing (Ullu et al., 2004) and
microRNAs (Zhang et al., 2009). The presence of mitochondrial remnants,
called mitosomes, has been demonstrated in Giardia (Tovar et al., 2003)
and other amitochondriate protists, as have genes encoding components
of Golgi bodies (Dacks et al., 2003). The sequences involved in protein
targeting for mitosomes have been shown to be conserved and recognised
by the hydrogenosomes of Trichomonas and related to translocases in
mitochondria (Dolezal et al., 2005). Phylogenetic analysis of the genes
encoding type II DNA topoisomerases found that Giardia diverged after
mitochondriate kinetoplastids and that amitochondriate protists were
polyphyletic, adding further evidence that the Giardia lineage diverged
after the acquisition of mitochondria by eukaryotes and that secondary
loss or alternative evolution of organelles has occurred independently
multiple times (He et al., 2005a,b).
A further understanding of Giardia evolution has been gained from
analysis of genome data. While the genome of Giardia is thought to be
compact through the reduction or loss of many metabolic pathways, 40%
of genes (predominantly variant-specific surface proteins) were found to
be duplicates (Sun et al., 2010). Interestingly, phylogenetic analysis of the
duplicated genes suggested that expansion of the variant-specific surface
proteins coincided with the radiation of placental mammals (Sun et al., 2010).
Comparison of predicted small nucleolar RNAs in Giardia demonstrated

similarity with Dictyostelium, Plasmodium, fungi and metazoans, which
were all different to those from Euglenozoa, suggesting that the lineage
containing Giardia diverged later than Trypanosoma and Euglena (Luo et al.,
2009). This observation is concordant with earlier analyses using multi-
gene phylogenies, which separated the excavates into three major
lineages: Diplomonads, Parabasalids and Carpediemonas; Trimastix and
Oxymonads; Euglenozoa, Heterolobosea and Jakobids, with the lineage
including Giardia and other Diplomonads closest to the fungi and animals
(Simpson et al., 2006). However, a more recent study using phylogenomics
suggests that long branch attraction may have affected previous attempts
to elucidate the relationships of the excavate groups, and exclusion of the
most rapidly evolving genes or species from analyses supports the mono-
phyly of the Excavata (Hampl et al., 2009), leaving the placement of the
excavates and the major lineages within it uncertain.
The phylogeny of the Diplomonads has been further elucidated, with
Octomitus being shown to be a sister taxon to Giardia (Keeling and
Brugerolle, 2006) and with Spironucleus, Hexamita and Trepomonas being
shown to be a separate lineage from Giardia/Octomitus (Kolisko et al.,
2008). Interestingly, none of the Enteromonads were basal to the Diplo-
monads (as suggested by Brugerolle, 1975; Siddall et al., 1992) but were
instead polyphyletic within the lineage including Spironucleus, suggesting
either multiple origins of diplokarya or multiple instances of secondary
loss of the duplicated nucleus (Kolisko et al., 2008). A genome comparison
between Spironucleus and Giardia found evidence of lateral gene transfer
from both prokaryotes and eukaryotes, distinct biases in mutations and
polyadenylation signals and differences in codon usage (Andersson et al.,
2007). Of more interest, large genomic differences were found between
the morphologically indistinguishable species S. barkhanus and S. salmo-
nicida, including differences in codon usage, the frequency of allelic
sequence variation and genome size (Roxstrom-Lindquist et al., 2010).
Interestingly, a similar observation has been made for the difference in
allelic sequence variation observed between G. duodenalis and G. enterica
(Assemblages A (WB) and B (GS)) (Roxstrom-Lindquist et al., 2010).
2.4. TAXONOMY AND NOMENCLATURE
The taxonomic placement of Giardia has changed following revisions

to higher order classifications. A key change has been the replacement
of the Diplomonadida with two new orders, the Distomatida Klebs
1892 (Trepomonas, Hexamita, Spironucleus) and the Giardiida Cavalier-
Smith 1996 (Giardia, Octomitus), which are both in the subclass Diplozoa
Dangeard 1910 stat. nov. Cavalier-Smith 1996 (Cavalier-Smith, 2003). As a
consequence, the family Hexamitidae is now in the order Distomatida,

with no apparent families within the Giardiida (Brands, S.J. (comp.) 1989–
present. Systema Naturae 2000. The Taxonomicon. Universal Taxonomic
Services, Zwaag, The Netherlands. [http://taxonomicon.taxonomy.nl/].
Access date: 17-12-2011). The classification suggested by Cavalier-Smith
(2003) still recognised the Enteromonadida, which has more recently been
shown to be polyphyletic within the Distomatida and so should be recon-
sidered (Kolisko et al., 2008). At the higher level, there have been pro-
posed changes to the kingdoms at the base of the eukaryotes, with
Euglenozoa being removed from the infrakingdom Excavata, which still
contains Metamonada (Cavalier-Smith, 2010). Following these revisions,
Giardia belongs to the order Giardiida Cavalier-Smith 1996, subclass
Diplozoa Dangeard, 1910, class Trepomonadea, superclass Eopharyngia,
subphylum Trichozoa (Cavalier-Smith, 2003), phylum Metamonada
Grassé 1952 stat. nov. et emend. Cavalier-Smith, 1981 (Cavalier Smith,
1993). Multigene phylogenetic analyses support previous suggestions that
Giardia is the most highly derived member of the order (Brugerolle, 1975),
but as described above, the molecular data do not support Brugerolle’s
(1975) proposed evolution, which has Giardia and Octomitis being derived
from Trepomonas, Hexamita and Spironucleus.
The morphological descriptions and taxonomic history of Giardia have
been reviewed extensively elsewhere (Monis et al., 2009; Thompson and
Monis, 2004, 2011) and so will not be redescribed here. Despite critical
reviews of the history of Giardia, taxonomic descriptions to clarify time-
lines and the validity of particular names (Monis et al., 2009; Thompson
and Monis, 2004, 2011), there continues to be debate regarding nomencla-
ture, with invalid species names such as G. lamblia still in use by some
investigators. For this review, we will use the nomenclature proposed by
Monis et al. (2009), which allocates previously described species to the
currently recognised assemblages on the basis of apparent host preference
(Table 2.1). This is consistent with the spirit of Filice’s (1952) original
proposal, which was at pains to emphasise that the rationalisation of
Giardia taxonomy was only a temporary solution in the absence of valid
discriminatory criteria for recognising additional species. The nomencla-
ture is also consistent with the relationships inferred by phylogenetic
analysis of alloenzyme electrophoretic data (Monis et al., 2003), illustrated
in Fig. 2.2, and of DNA sequence data from multiple independent loci
(Monis et al., 1999). The sequence-based phylogenies suggest that
G. duodenalis, Giardia cati and G. bovis were clustered together but could
not resolve the branching order, with Giardia canis, G. enterica and Giardia
simondi being external to these three species (Monis et al., 1999). The
available genome data support that G. bovis and G. duodenalis are more
similar to each other than either are to G. enterica ( Jerlstrom-Hultqvist
et al., 2010b).
G. muris (rodent)
G. ardeae (bird)
G. canis (dog)
G. simondi (rat)
(Various, bIII) (Human)

(Mamoset)
(Siamang)
G. enterica
(Dog)
(Human, BIV)
G. cati (Cat)
(Sheep)
(Cattle)
G. bovis
(Pig)
(Guinea pig)
(Various, A?) (Cat)
(Alpaca)
(Cat)
(Cat)
G. duodenalis (Various, AI) (Dog)
(Human)
(Human, AI)
0.1 Roger’s distance
FIGURE 2.2 Diagrammatic representation of the phylogenetic relationships of Giardia

species and the possible infection cycles that occur within these species. The phylogeny
was inferred by Neighbor Joining analysis of Roger’s Distances (modified from Monis
et al. 2003). Host origin for species or lineages is indicated in parentheses.
Acceptance of suggested changes to Giardia nomenclature may have

been hampered through the application of imprecise terminology, for
example, the use of the term ‘genotype’ to describe a group of genetically
diverse organisms such as the Giardia isolates comprising Assemblage B.
A ‘genotype’ reflects the genetic constitution of an organism and can be
broadly interpreted as a group or class of organisms having the same
genetic constitution. The first detailed genetic studies of Giardia identified

a number of genotypes, some of which were shown by clustering analysis
to form distinct groups (Andrews et al., 1989; Meloni et al., 1988). More
detailed analysis of human isolates of Giardia demonstrated quite a large
degree of genetic diversity, but that all the human isolates formed two
broad clusters and that there was some genetic substructuring within
these clusters (Mayrhofer et al., 1995). This study gave rise to the concept
of genetic assemblages of Giardia, with the two assemblages (A and B)
each comprising clusters of related genotypes that were separated from
each other by relatively large genetic distances, of the same order of
magnitude as those separating G. duodenalis from G. muris (Ey et al.,
1997; Mayrhofer et al., 1995). From this, it is evident that it is inappropri-
ate to refer to the assemblages as genotypes and that to do so provides a
false impression of homogeneity within these groups, de-emphasizing the
magnitude of the differences between them, which are sufficient for them
to be recognised as distinct species.
Recent genome sequence data lend support to the distinct species status
of the Giardia assemblages ( Jerlstrom-Hultqvist et al., 2010a). Sequence
data are now available from representative isolates of G. duodenalis (Assem-
blage A) (Morrison et al., 2007; Svard et al., 2003), G. enterica (Assemblage B)
(Franzen et al., 2009) and G. bovis (Assemblage E) ( Jerlstrom-Hultqvist
et al., 2010b). Comparison of the genomes at the amino acid level
( Jerlstrom-Hultqvist et al., 2010a) supports earlier phylogenetic analyses
that suggested that G. duodenalis and G. bovis were more closely related to
each other relative to G. enterica (Monis et al., 1999). The GþC content of
G. enterica and G. bovis was slightly lower than that of G. duodenalis, but the
difference was ascribed to imperfect sequence assemblies of the two newer
genome sequences ( Jerlstrom-Hultqvist et al., 2010b). The three species/
assemblages shared a core set of genes representing 91% of their genomes,
but there were still large differences, particularly in Giardia-specific gene
families and chromosomal rearrangements ( Jerlstrom-Hultqvist et al.,
2010a). Comparison of the genomes of G. duodenalis and G. enterica found
a higher frequency of allelic sequence heterozygosity in G. enterica, but
additional data from other isolates of these two species are required to
determine if this is a characteristic of the species/assemblages or the iso-
lates (WB and GS, respectively) used to obtain the genome sequences
(Franzen et al., 2009). Perhaps the most important indication of the species
status of the assemblages has been demonstrated by a genome-based
comparison of the divergence between the assemblages with divergence
between species within other protozoan genera. The divergence between
G. duodenalis and G. enterica (Assemblages A and B) and between G. enterica
and G. bovis (Assemblages B and E) are similar to those separating Theileria
parva from Theileria annulata, whereas the divergence between G. duodenalis
and G. bovis (Assemblages A and E), while lower, are similar to those
separating Leishmania major and Leishmania infantum ( Jerlstrom-Hultqvist

et al., 2010b).
Previous genetic studies of Giardia isolates have focussed on isolates
from humans and animals in close contact with humans, especially pets
and livestock. More recent studies have examined Giardia isolated from
wildlife, identifying new assemblages or genotypes. A novel species,
based on cyst morphology and phylogenetic analysis of SSU rRNA, and
elongation factor 1 alpha, was isolated from a small marsupial in Austra-
lia (Adams et al., 2004). A survey of marine vertebrates identified a new
lineage, named Assemblage H, which was isolated from grey seal and a
single gull sample (Lasek-Nesselquist et al., 2010). A novel genotype
belonging to Assemblage A has been isolated from a deer (Lalle et al.,
2007), but it is not clear if this is similar to AIII reported from wild-hoofed
mammals and a rabbit (Lebbad et al., 2010) or the novel non-AI, non-AII
genotypes from animal hosts identified by alloenzyme analysis (Monis
et al., 2003). Comparison between studies is made complicated by differ-
ences in terminology, for example, A1, A2, A3, etc., have been used to
arbitrarily describe genotypes or subtypes within Assemblage A (Eligio-
Garcia et al., 2005; Lalle et al., 2005) but without reference to other studies
or earlier descriptions of genetic groups, such as AI and AII, which
represent clusters of genotypes (Read et al., 2004).
2.5. THE MAINTENANCE OF GIARDIA IN NATURE
2.5.1. Life cycle and development

The cyst is the most important transmissible stage in the life cycle of
Giardia. Most authorities consider cysts to be immediately infective
upon being passed in the faeces, but there is evidence that some cysts
undergo a maturation period of up to 7 days before becoming infective
(Caccio and Sprong, 2011; Grant and Woo, 1978; Thompson, 2011).
Between 10 and 25 cysts are considered the minimum dose to initiate an
infection (Caccio and Sprong, 2011; De Carneri et al., 1977; Rendtorff,
1954). Trophozoites are also capable of initiating an infection following
ingestion, as demonstrated experimentally in mice (Thompson, 1999).
However, trophozoites will only have transitory survival in the environ-
ment and initiate infection in circumstances where they are expelled in
large numbers during diarrhoetic episodes leading to contamination of
individuals or surfaces, for example, in day care centres, breeding kennels
and dairies. Cysts can also initiate infections in similar situations and may
lead to longer term contamination of surfaces and the external bodies of
animals, as well as contaminating the environment which may lead to the
establishment of foci supporting a sustained high frequency of
transmission by the faecal–oral route, for example, community situations,

kennels, dairies, etc. (see below).
Following ingestion, excystation takes place shortly after cysts leave
the stomach. The low pH of the stomach environment appears to be the
major factor which initiates the excystation process (Bingham and Meyer,
1979; Boucher and Gillin, 1990; Lauwaet and Gillin, 2009). Excystation
leads to rapid colonisation of the duodenum and jejunum, where the
excysted trophozoites attach to the intestinal mucosa and multiply rap-
idly. Attachment is mediated by Giardia’s unique attachment organelle,
the ventral adhesive disc (Fig. 2.1), and is an essential feature of the
relationship between Giardia and its host and a prerequisite to sustained
infection (Thompson, 2011).
Giardia has long been considered to reproduce asexually by simple
binary fission, but there is increasing evidence from epidemiological and
molecular genetic studies that Giardia is capable of sexual reproduction
(Cooper et al., 2007; Lasek-Nesselquist et al., 2009; Meloni et al., 1995;
Siripattanapipong et al., 2011; and reviewed in Monis et al., 2009) and
genes involved in meiosis have been identified in the Giardia genome
(Ramesh et al., 2005). However, the frequency of recombination is not
known, nor its impact on the epidemiology of giardiasis and the extensive
genetic diversity that characterises the forms of Giardia that infect mam-
mals. The evolutionary advantage of genetic exchange to Giardia would be
the capacity to respond to adversity, for example, selection pressures
imposed by regular exposure to antigiardial drugs or competition with
cohabiting ‘strains’ in circumstances where the likelihood of mixed infec-
tions is common (Hopkins et al., 1999; Monis et al., 2009). As such, it may
be a relatively rare event and further population genetic studies are
required in foci of infection where the frequency of infection is high
(Monis et al., 1999; Thompson and Monis, 2011). The fact that available
data indicate that the genetic assemblages¼species (Table 2.1) of Giardia
are conserved in terms of geographic location and host occurrence sug-
gests that any recombination is not reflected at the species level (Monis
et al., 2009).
The incubation, or pre-patent period, before cysts appear in the faeces,
may be short in both humans and other animals, commencing as early as
3 days postinfection but can range up to 3 weeks depending upon host
species (Flanagan, 1992; Hopkins and Juranek, 1991; Rendtorff, 1954;
Thompson et al., 2008). The duration of infection may vary from a few
days to several months. During the course of infection, trophozoites will
encyst in the posterior small intestine. The trigger for trophozoites to
encyst in vivo is not completely understood but appears to be initiated
by the presence of bile salts and cholesterol (Lauwaet and Gillin, 2009;
Sener et al., 2009). There have been no comparative studies of encystation
in different species of Giardia, with transcriptional analysis of encysting
trophozoites restricted to the WB strain (Morf et al., 2010). Comparison

of different in vitro encystation protocols identified upregulation of a core
set of genes, as well as upregulation (and downregulation) of genes
specific to each protocol (Morf et al., 2010). Comparison of the encysta-
tion-associated genes determined that they all possessed a motif for
binding the Giardia Myb transcription factor (Morf et al., 2010). Further
studies are required to determine what similarities or differences occur
between different species of Giardia and whether differences in regulation
could account for differences in virulence.
The first appearance of any symptoms usually coincides with the onset
of cyst excretion. Cyst excretion is characteristically intermittent in both
humans and other animal species, and cysts are resistant, surviving for at
least 2 months in suitable temperature and moisture conditions (Meyer
and Jarroll, 1980; Thompson, 2011). Encystation must be considered
a major virulence factor as differentiation into a form that can survive in
the environment and infect a new host is vital for transmission and
disease progression.
2.5.2. Hosts
Numerous vertebrate species have been shown to harbour Giardia infec-
tions in nature. To some extent, the current taxonomy reflects the host
range of Giardia (Table 2.1). The majority of species of Giardia appear to
have a relatively restricted host range. However, the two most common
species found in mammals, G. duodenalis and G. enterica, have a low host
specificity and are considered to have zoonotic potential. As a conse-
quence, most data on the distribution and prevalence of Giardia in verte-
brates have come from studies on mammalian hosts, principally domestic
animals. From these studies, three life cycle patterns have been well
defined that maintain Giardia in domestic hosts, including humans
(Monis et al., 2009). Interaction between these cycles occurs in terms of
transmission between hosts (Fig. 2.3). In addition, Giardia cycles of trans-
mission have been identified in numerous species of wildlife, but it is not
clear how the parasite is maintained in nature in wildlife populations, nor
the impact of domestic cycles on the perpetuation of Giardia infections in
wildlife (Fig. 2.3).
2.5.2.1. Humans
Giardia is today well recognised as one of the most prevalent intestinal
infections of humans in both temperate and tropical areas, with preva-
lence rates varying between 2% and 7% in Europe, the United States,
Canada and Australia, to over 40% in developing areas where living
conditions are poor, nutritional levels are often inadequate and concur-
rent infections are common (reviewed in Feng and Xiao, 2011; Thompson,
Dog/cat
cycle
Frequency of Direct (occasional

transmission? waterborne)
Frequency of
transmission?
Human Livestock
cycle cycle
Direct (occasional
waterborne)
f
yo ?
nc
Frequency of Direct (occasional q ue ssion
transmission? waterborne) Fre smi
n
tra na
l
sio
c ca e)
o
t ( rbor n
ec
Dir wate
Wildlife
cycle(s)
FIGURE 2.3 Major cycles of transmission of Giardia in mammalian hosts. Some

assemblages/species are host specific and cycle between their respective hosts, whereas
others have low host specificity and are capable of infecting humans and other animals
(modified from Monis et al., 2009).
2009, 2011). In developed countries, infections with Giardia are most

common in children, especially in day care centres, residents of institu-
tions and travellers. A rising incidence in such settings has led to the
designation of giardiasis as a re-emerging infectious disease in the devel-
oped world (Eckmann, 2003; Savioli et al., 2006; Thompson, 2000, 2004;
Thompson and Monis, 2004). The emerging issue of Giardia infections in
developing regions of the world and the impact on children was a major
factor in the inclusion of Giardia in WHO’s ‘Neglected Diseases Initiative’
(Savioli et al., 2006).
In developing countries, particularly in Asia, Africa and Latin America,
about 200million people have symptomatic giardiasis with some 500,000
new cases reported each year (Savioli et al., 2006; Thompson, 2009; WHO,
1996). Children living in communities in developing countries and among
disadvantaged groups living in isolated communities such as indigenous
Australians are most commonly infected (Al-Mekhlafi et al., 2005; Savioli
et al., 2006; Thompson, 2000, 2009; Thompson and Smith, 2011; Thompson
et al., 2001). These children are most at risk from the chronic consequences
of Giardia infection, as well as the repeated exposure to potentially toxic
drugs in some endemic regions (Thompson et al., 2001).
In developed countries, epidemiological investigations have demon-
strated that travel, swimming in surface water, contact with young chil-
dren and institutional confinement are important risk factors associated
with Giardia infection (Abe and Teramoto, 2012; Hunter and Thompson,
2005; Kettlewell et al., 1998; Stuart et al., 2003; Thompson, 2009). There is
also evidence that contact with farm and companion animals are also risk
factors for infection ( Jagai et al., 2010; Robertson et al., 2010; Warburton
et al., 1994). Infection varies inversely with socio-economic status and is
high in regions where water supplies are poor or non-existent and sanita-
tion and personal hygiene standards are inadequate (Alvarado and
Vásquez, 2006; Balcioglu et al., 2007; Hesham et al., 2005; Hunter and
Thompson, 2005; Savioli et al., 2006; Thompson, 2011). Living in commu-
nity settings with other animals has also been shown to heighten the risk
of infection with Giardia (Inpankaew et al., 2007; Marangi et al., 2010; Salb
et al., 2008; Traub et al., 2003, 2004). Risk factors identified as important in
facilitating emergence of Giardia infection include high environmental
faecal contamination, lack of potable water, inadequate education and
housing, overcrowding and high population density and animal reser-
voirs of infection (reviewed in Thompson, 2011).
2.5.2.2. Dogs and cats

Giardia is a common parasite of dogs and cats globally and is the most
frequently diagnosed enteric parasite of dogs and cats in developed
countries (Ballweber et al., 2010; Scaramozzino et al., 2009; Thompson
et al., 2008; Tangtrongsup et al., 2010). Prevalence rates vary (Ballweber
et al., 2010; Feng and Xiao, 2011) and are influenced by the sampling
strategies and diagnostic methods used (Epe et al., 2010).
Surveys of a variety of canine and feline populations reveal preva-
lences of between 10% in well-cared-for dogs, 36–50% in puppies and
kittens, and up to 100% in breeding establishments and kennels where the
frequency of transmission will be higher (Hahn et al., 1988; Kirkpatrick,
1988; and reviewed in Ballweber et al., 2010; Feng and Xiao, 2011;
Thompson et al., 2008). A recent study in the United States found that
dog park-attending dogs were more likely to be positive for Giardia than
non-dog park-attending dogs (Wang et al., 2011).
Infection with Giardia will result from direct transmission between

animals or from the environment. Dogs and cats may harbour host-
adapted (G canis, Giardia felis) or zoonotic species of Giardia which can
cycle between dogs or cats (Covacin et al., 2011; Thompson et al., 2008;
Upjohn et al., 2010). Host-adapted species, such as G. canis in dogs, is
likely to predominate in breeding establishments and pet shops (Itoh
et al., 2011).
2.5.2.3. Livestock
In livestock, Giardia infections have been reported in cattle, both dairy and
beef, sheep, goats, horses, pigs and cervids (Dixon et al., 2011;
Farzan et al., 2011; Feng and Xiao, 2011; O’Handley and Olson, 2006).
Although all ruminants are likely to be exposed to Giardia shortly after
birth, infections are most common towards the end of the neonatal period
and in calves can be as high as 100% (O’Handley and Olson, 2006; Olson
et al., 2004).
Direct contact between young livestock appears to be the most likely
source of transmission (Becher et al., 2004; Dixon et al., 2011; O’Handley
et al., 1999; St Jean et al., 1987; Wade et al., 2000; Xiao et al., 1993).
Grouping behaviour of calves in pens or paddocks provides ample oppor-
tunities for the transmission of Giardia.
As with dogs and cats, livestock may harbour host-adapted (G. bovis)
or zoonotic species of Giardia, although G. bovis tends to be more prevalent
in cattle (Dixon et al., 2011; Khan et al., 2011). However, G. duodenalis is
most common in young animals (Mark-Carew et al., 2011), and in a recent
survey of pigs in Ontario, Canada, G. enterica was the most common
species found (Farzan et al., 2011).
The role of zoonotic transmission is discussed below, but the introduc-
tion of zoonotic species of Giardia by humans into environments where
cattle are housed may result in infections in cattle which can then be
transmitted between cattle.
2.5.2.4. Wildlife
Although numerous species of wild mammals have been reported to be
infected with Giardia, both in the wild and captivity, the majority of
infections are with zoonotic species (Levecke et al., 2011; Martinez-Diaz
et al., 2011; Siembieda et al., 2011; Soares et al., 2011; and reviewed in
Thompson et al., 2010a). These are considered to have been introduced
into wildlife habitats and once established would appear to be maintained
by direct contact or via the environment even in terrestrial and aquatic
environments presumed to be pristine, for example, muskoxen in the
Arctic and beavers in pristine mountain streams (Thompson et al., 2010a).
Distinct species and genotypes of Giardia have been recovered from
amphibia, reptiles, rodents, bandicoots and birds (Adams et al., 2004;
McRoberts et al., 1996; Monis and Thompson, 2003). Although the ecology
of infections with these host-restricted species of Giardia is not well
understood, it is presumed that infections cycle directly between hosts
and/or the environment. However, there is limited information on the
prevalence of infections in nature. A recent study in Australia found that
infections in bandicoots were not common raising questions about how
the parasite is maintained in nature (Thompson et al., 2010b).
2.5.3. Transmission
2.5.3.1. Faecal–oral transmission
In humans, transmission of Giardia is principally by faecal–oral contami-
nation, which is reflected by higher levels of infection where levels of
hygiene and sanitation are compromised, particularly in tropical and
subtropical environments (Alvarado and Vásquez, 2006; Balcioglu et al.,
2007; Savioli et al., 2006). As such, direct person-to-person transmission is
considered to be more important than waterborne, foodborne or zoonotic
transmission (Hesham et al., 2005; Hunter and Thompson, 2005;
Pawlowski et al., 1987; Schantz, 1991; Thompson, 2004; Thompson and
Smith, 2011). Other environmental factors which will exacerbate the fre-
quency of faecal–oral transmission include day care centres where condi-
tions conducive to faecal–oral contamination are common and high
prevalence rates of Giardia infection have often been observed
(Thompson, 2000, 2011). Indirect transmission, where infection results
through the mechanical transmission of cysts on, for example, flies
(Szostakowska et al., 2004) or other animals such as dogs or livestock,
poses a significant threat particularly in the developing world (Thompson
and Smith, 2011).
In domestic animals, Giardia infections are most common in situations
where the levels of environmental contamination with cysts are high,
such as breeding establishments, kennels, catteries, dog parks, pet
shops, dairies, cattle sheds and in the case of dogs communities with
free roaming dogs (Itoh et al., 2011; Thompson, 2011; Wang et al., 2011).
In addition, direct transmission from the contaminated coats of animals in
breeding and weaning areas will be common.
2.5.3.2. Waterborne transmission

Giardiasis is a frequently diagnosed waterborne disease in developed
countries (Karanis et al., 2007; Levine et al., 1990; Robertson and Lim,
2011; Smith et al., 2007; Thompson, 2004). The consumption of drinking
water other than metropolitan mains, or other filtered supplies, repre-
sents a significant risk for giardiasis (Robertson and Lim, 2011). The
majority of waterborne giardiasis outbreaks in humans have occurred in
unfiltered surface or groundwater systems impacted by surface run off or
sewage discharges ( Jakubowski and Craun, 2002; O’Reilly et al., 2007;

Robertson and Lim, 2011) or systems that have been poorly maintained
(Daly et al., 2010). Irrigation waters used for food crops that are tradition-
ally consumed raw may also represent a high risk as a source of Giardia
(Thurston-Enriquez et al., 2002). Environmental contamination of such
water systems and supplies may result from human, agricultural and
wildlife sources (Heitman et al., 2002).
Waterborne transmission is also a well-documented cause of Giardia
infection in travellers who usually contract infection from drinking local
tap water (Hunter and Thompson, 2005). In the developed world, water-
borne transmission is usually the result of contamination with Giardia of
human origin or a process failure by water utilities, industry or in swim-
ming pools (Dale et al., 2010; Shields et al., 2008; Stuart et al., 2003). Such
contamination may impact negatively on ecosystem health leading to
infections in aquatic wildlife which may then establish reservoirs of
human infection. The role of the beaver as a ‘spill back’ reservoir of
Giardia in North America is the best known example (Thompson et al.,
2009a). Recent studies have also demonstrated that filter-feeding molluscs
and freshwater fish are useful indicators of the presence of waterborne
pathogens, including Giardia of human origin (Lucy et al., 2008; Miller
et al., 2005; Nappier et al., 2010; Thompson and Smith, 2011).
In the developing world, there is a much greater reliance on lakes,
streams and other natural surface water sources for drinking, food prep-
aration, washing clothes and personal hygiene exacerbating the chances
of waterborne infection (Hunter and Thompson, 2005; Thompson and
Smith, 2011). Areas which are prone to flooding face an increased risk of
waterborne infection particularly where basic sewerage systems are used
and containment likely to be compromised (Thompson and Smith, 2011).
Kutz et al. (2009a) also emphasised that climate change has been proposed
to cause increased frequency and magnitude of flooding enhancing trans-
mission of waterborne pathogens such as zoonotic species of Giardia, in
and between terrestrial and marine systems.
2.5.3.3. Foodborne transmission

Giardia is one of the several enteric protozoa that is known to be readily
transmitted on food (Robertson and Lim, 2011; Thompson, 2011), and in
some parts of the world, foodborne transmission may be enhanced
through the use of human waste as fertiliser and inadequate pasteurisa-
tion techniques (Thompson and Smith, 2011). However, most foodborne
transmission is considered to be associated with infected food handlers
and poor hygiene, usually at a local level rather than as the source of
outbreaks of Giardia infection (Barnard and Jackson, 1984; Mintz et al.,
1993; Petersen et al., 1988; Robertson and Lim, 2011; Smith et al., 2007).
Overall, it has been estimated that the number of cases of foodborne
transmission range from 13 to 76 million globally (Thompson and Smith,

2011). Foods associated with cyst contamination have included canned
salmon, salads, sandwiches, raw vegetables and ice (Robertson and Lim,
2011; Smith et al., 2007). The reason there are fewer reported outbreaks
of Giardia infection involving contaminated foods is likely due to the
lack of appropriate tools, or their application, in the past, the sporadic
nature of such outbreaks, lack of awareness and under reporting
(Robertson and Lim, 2011; Thompson and Smith, 2011).
2.6. INTERACTION BETWEEN CYCLES
Although we may have a growing understanding of how Giardia is

maintained in cycles involving domestic animals and wildlife, the ques-
tion of how these cycles may interact, and the host range of the various
genotypes of Giardia involved, is largely unresolved. This is particularly
important with respect to zoonotic and waterborne transmission.
The molecular characterisation of Giardia isolates from different spe-
cies of mammalian hosts throughout the world has confirmed the exis-
tence of host-specific species and two species with broad host ranges
which are zoonotic (Table 2.1). This revised taxonomy largely reflects
the species nomenclature reported by early workers in the field (Monis
et al., 2009; Thompson and Monis, 2011) and helps to better understand
host specificity in terms of the epidemiology of Giardia infections.
The two zoonotic species of Giardia are geographically widespread,
and as more isolates are genotyped, some patterns are emerging on host
occurrence. Overall in humans, the distribution of G. duodenalis and
G. enterica is similar in both developed and developing countries, with
G. enterica more common (58%) in developing than developed countries
(55%) compared to G. duodenalis (37% vs. 40%), but mixed infections are
more common in developing countries (8% vs. 2%) (data from Feng and
Xiao, 2011). In dogs, recent studies have shown that it is not possible to
extrapolate from one geographical region to another in terms of the
species/assemblage composition of Giardia infections in dogs (Ballweber
et al., 2010; Covacin et al., 2011). In Europe, studies had suggested that
Assemblage B has a predominantly human distribution (Sprong et al.,
2009), but a recent study in the United States found a higher frequency of
infections in dogs with G. enterica than G. duodenalis, which has not been
reported elsewhere (Covacin et al., 2011). This suggests that in North
America at least, we cannot assume that G. duodenalis is the most common
of the zoonotic species found in non-human hosts (Covacin et al., 2011).
Indeed, in wildlife, G. enterica often predominates (e.g. Johnston et al.,
2010), whereas in cattle, G. duodenalis is most often reported (Sprong et al.,
2009). However, there is an extensive genetic substructuring within
G. enterica, and it is possible that some subgroups are more commonly

associated with zoonotic infections than others. In humans, there is some
evidence of geographic substructuring (Siripattanapipong et al., 2011;
Wielinga et al., 2011), and G. enterica may be more common in isolated
and/or community settings where the frequency of transmission is high
(Thompson, 2000). Under such circumstances, the parasite is likely to be
exposed to greater selection pressure in terms of exposure to antigiardial
drugs and competitive interactions which might explain why evidence of
recombination in Giardia is mostly confined to G. enterica isolates (Lasek-
Nesselquist et al., 2009; Siripattanapipong et al., 2011; Thompson and
Monis, 2011).
2.6.1. Zoonotic transmission

The application of molecular tools for ‘typing’ isolates of Giardia in faecal
samples from human and non-human mammalian hosts in different parts
of the world has produced a wealth of information on the distribution of
host-specific and zoonotic species of Giardia. From these studies, there is
clear evidence that cysts of zoonotic Giardia do contaminate the environ-
ment in areas where the potential for zoonotic transmission exists. The
epidemiological value of these studies varies with the number of loci used
for genotyping and the number of host species sampled. However, in most
cases, it is possible to extrapolate that a risk of zoonotic transmission exists,
but evidence of how frequently it occurs requires focal studies in defined
endemic areas where transmission dynamics and host range are known.
2.6.1.1. Dogs and cats

The significance of Giardia infection in domestic dogs and cats is consid-
ered to be primarily a public health issue, and the clinical impact on dogs
and cats is generally believed to be minimal (Thompson et al., 2008).
However, Giardia may be associated with gastrointestinal disorders in
dogs (Barutzki et al., 2007; Epe et al., 2010), and more studies are required
to determine whether there are differences in clinical impact between
infections with zoonotic Giardia species and G. canis in dogs, and simi-
larly, whether mixed infections of Giardia species may be clinically more
apparent in dogs.
The zoonotic potential of Giardia infections in dogs and cats was
proposed long before genotyping data was available, but cross-infection
experiments proved difficult to interpret (Thompson and Monis, 2004;
Thompson et al., 1990). Apart from limitations in experimental design,
variability in results will have been affected by the differences in host
specificity of the Giardia isolates used which has now been confirmed
from molecular epidemiological data (Monis et al., 2009).
A number of studies have been undertaken in which domestic dogs,

and to a lesser extent, cats, living in urban areas of developed countries
have been sampled. In the majority of studies, both host-specific, G. canis/
G. felis, and zoonotic species, G. duodenalis and G. enterica, and subgeno-
types have been identified, albeit in varying proportions (Ballweber et al.,
2010; Covacin et al., 2011; Feng and Xiao, 2011; Leonhard et al., 2007;
Suzuki et al., 2011; Volotao et al., 2011). Mixed infections of G. canis and
G. duodenalis or G. enterica have also been reported. As discussed above,
the distribution of zoonotic species varies; for example, in Europe,
G. duodenalis has been reported more commonly than G. enterica which a
recent study found to be the dominant zoonotic species in dogs in the
United States (Covacin et al., 2011). From an epidemiological perspective,
interpretation of the results of these studies demonstrate that a potential
environmental reservoir of Giardia infection exists in urban areas but
without concurrent data from owners or known handlers, information
on the frequency of zoonotic transmission is lacking. However, Bugg et al.
(1999) found that dogs from multi-dog households were more commonly
infected with Giardia than dogs in single-dog households, emphasising
the potential ease with which Giardia can be spread to in-contact animals
and therefore presumably humans (Bugg et al., 1999).
In contrast, a few studies have been undertaken in defined endemic
foci in which both humans and dogs have been sampled and isolates of
Giardia characterised genetically. Results from these molecular epidemio-
logical studies have provided more definitive support for zoonotic trans-
mission but have also highlighted the importance of understanding the
transmission dynamics of Giardia infections.
The first multilocus molecular epidemiological studies to address the
issue of zoonotic transmission were undertaken by Traub et al. (2004) in
tea growing communities in Assam, north-east India, where Giardia
occurs in both humans (up to 21%, depending upon age) and dogs
(20%). Traub and her colleagues found that all infected dogs harboured
zoonotic species of Giardia: G. duodenalis and G. enterica, with some mixed
infections. These studies by Traub et al. (2004) provided the first direct
evidence of zoonotic transmission between dogs and humans, by finding
the same genotype of Giardia in people and dogs, not only in the same
village but also in the same household. Evidence for zoonotic transmis-
sion was supported by strong epidemiological data showing a highly
significant association between the prevalence of Giardia in humans and
the presence of a Giardia positive dog in the same household. A similar
situation was found in Temple communities in Bangkok (Inpankaew
et al., 2007) and northern Canadian indigenous communities (Salb et al.,
2008). Both studies demonstrated zoonotic species of Giardia infecting
dogs and their owners sharing the same living area. In contrast, a molec-
ular epidemiological investigation in remote indigenous communities in
northern Western Australia, which represent highly endemic foci of

Giardia transmission with high rates of infection in children and dogs,
often greater than 50% (Meloni et al., 1993; Thompson, 2000), found that
all but one dog (1/12 dogs) were infected with G. canis (Hopkins et al.,
1997). This result was interpreted as evidence of competitive exclusion,
since the frequency of Giardia transmission is so high in these commu-
nities, with dogs equally likely to be exposed regularly to infection with
G. canis and zoonotic species, mostly G. enterica. Such competitive inter-
actions are likely to ensure that the host-adapted genotypes predominate
in respective host species, as with G. bovis in dairy cattle (Hopkins et al.,
1997, 1999; Thompson and Lymbery, 1996; Thompson and Monis, 2004,
2011; Thompson et al., 1996). Such an interpretation is supported by a
recent study undertaken in a desert community in Peru where 16% of
dogs and 20% of humans were infected with Giardia with all dogs apart
from one infected with G. canis (Cooper et al., 2010). One dog had a mixed
infection with G. canis and G. enterica. In domestic, urban environments,
and in the communities in Assam, Bangkok, northern Canada and Peru,
the frequency of dog-to-dog transmission will be less frequent, and thus
infections acquired with zoonotic species in dogs are likely to persist. It
should be emphasised, however, that the fact that dogs have contact with
young children passing Giardia cysts, as well as discarded nappies/
diapers, means that dogs are likely to act as mechanical transmitters of
zoonotic Giardia since their coats are likely to be contaminated with cysts.
Although competitive interactions between different species of Giardia
have been proposed to explain the predominance of single species infec-
tions in both dogs and cattle (see below), this may reflect the conse-
quences of mixed infections in endemic foci where the frequency of
transmission is very high. In other situations where transmission is spo-
radic, mixed infections may coexist and have been increasingly reported
from multilocus studies in several countries in humans, dogs and cattle
(e.g. Covacin et al., 2011; Dixon et al., 2011; Hussein et al., 2009; Sprong
et al., 2009). The reason why mixed Giardia infections are more common in
domestic dogs in urban areas of developed countries is not clear. Perhaps
it is a reflection of the lower frequency of transmission and/or dietary
differences between well-cared-for dogs living in more affluent environ-
ments and those on a poorer plane of nutrition which do not provide an
intestinal environment supportive of mixed infections.
2.6.1.2. Livestock
Livestock infected with Giardia, particularly cattle, has long been considered
to represent a public health risk as a source of waterborne outbreaks of
giardiasis in humans. This is because livestock is known to be susceptible to
infection with zoonotic species of Giardia as well as G. bovis, and thus the
potential for livestock operations to contaminate ground and surface waters
and considering the large numbers of cysts shed by infected cattle (Donham,
2000). It has been shown that calves infected with Giardia commonly shed
from 105 to 106 cysts per gram of faeces (O’Handley et al., 1999; Xiao, 1994).
However, of the 132 documented waterborne outbreaks (Robertson and
Lim, 2011), there is no evidence incriminating infected cattle in any outbreak
(Hunter and Thompson, 2005; Olson et al., 2004; Thompson, 2004).
Although it would seem likely that runoff and flooding would result in
contamination events, molecular epidemiological data suggest cattle opera-
tions are a minimal risk as a source of environmental contamination with
zoonotic Giardia. Although Giardia is common in both dairy and beef cattle,
it is principally dairy cattle that harbour zoonotic species, usually G. duode-
nalis and less commonly G. enterica (Dixon et al., 2011; Feng and Xiao, 2011),
but only as transitory infections in young animals less than 3 months of age.
Older animals only seem to support infections with G. bovis which may also
be related to competitive exclusion operating in older animals (Thompson
and Monis, 2011). Longitudinal studies in Australia and the United States
(Becher et al., 2004; Mark-Carew et al., 2011) suggest that zoonotic geno-
types may only be present transiently in cattle under conditions where
the frequency of transmission with the livestock species, G. bovis (Assem-
blage E), is high and competition is thus likely to occur (Becher et al., 2004;
Thompson, 2004; Thompson and Monis, 2004, 2011). A recent survey of pigs
on 10 farms in Ontario, Canada, found that over 50% of pigs were infected
on all farms and that 92.1% of isolates were G. enterica, the remainder
being G. bovis (Farzan et al., 2011). These authors considered that there
was potential for zoonotic transmission via cyst-contaminated water.
Animal handlers are at risk from contracting Giardia from dairy cattle
as recently demonstrated in a molecular epidemiological study in India
(Khan et al., 2011). However, reverse zoonotic transmission should be
considered as the possible source of zoonotic Giardia infections in cattle,
particularly in dairy cattle because of more frequent contact with handlers
(Dixon et al., 2011). A molecular epidemiological study in Uganda where
humans appear to have introduced Giardia into a remote national park are
thought to have been the source of Giardia in a small number of cohabiting
dairy cattle (Graczyk et al., 2002).
2.6.1.3. Wildlife
The occurrence of Giardia in wildlife has been the single most important
factor incriminating Giardia as a zoonotic agent. As such, it was the
association between infected animals such as beavers and waterborne out-
breaks in people that led the WHO (1979) to classify Giardia as a zoonotic
parasite. It is therefore surprising that there is so little evidence to support
the role of wildlife as a source of disease in humans, since this has
dominated debate on the zoonotic transmission of Giardia and, in
particular, when water is the vehicle for such transmission (Welch, 2000).
Indeed, there is increasing evidence to suggest that Giardia infections in

wildlife result from environmental contamination from domestic sources,
that is, reverse zoonotic events.
Although wildlife, particularly aquatic mammals, is commonly infected
with Giardia, there is little evidence to implicate such infections as the original
contaminating source in waterborne outbreaks (Appelbee et al., 2005;
Thompson, 2004). It would appear that such animals are more likely to
have become infected from water contaminated with faecal material of
human, or less likely, domestic animal origin (Thompson, 2011). Wildife
may thus serve to amplify the numbers of the originally contaminating isolate
(Bemrick and Erlandsen, 1988; Kutz et al., 2009b; Monzingo and Hibler, 1987;
Thompson, 2004, 2011; Thompson et al., 1990; Thompson et al., 2009a), and
depending upon the nature of the particular ecosystem, a zoonotic reservoir
may be established, as was the case with beavers in North America.
The few studies that have genotyped Giardia of beaver origin, in both
Canada and the United States, have confirmed previous suggestions that
the source of Giardia infection in beavers was likely to be of human origin
(Appelbee et al., 2002, 2005; Sulaiman et al., 2003). The latter authors also
examined Giardia from eight muskrats from the same region and only
three were infected with the expected Giardia microti, and the remaining
five muskrats were infected with zoonotic Giardia, G. enterica.
Several more recent reports have also shown that ‘reverse zoonotic
transmission’ is an important factor that must be considered in understand-
ing the epidemiology of Giardia infections in wildlife. Humans are consid-
ered to be the source of infection in non-human primates and painted dogs
in Africa, marsupials in Australia, coyotes in North America, muskoxen in
the Canadian Arctic, house mice on remote subarctic islands and marine
mammals in various parts of the world (Appelbee et al., 2010; Ash et al.,
2010; Dixon et al., 2008; Graczyk et al., 2002; Johnston et al., 2010; Kutz et al.,
2008; Moro et al., 2003; Teichroeb et al., 2009; Thompson et al., 2009b,
2010b). These reports raise important issues for conservation because we
do not understand the impact Giardia may have on what are possibly naı̈ve
hosts. They may have been exposed to the parasite relatively recently, as a
consequence of habitat disturbance and human encroachment, impairing
health and fitness ( Johnston et al., 2010; Thompson et al., 2010a).
2.7. FUNCTIONAL SIGNIFICANCE OF GENETIC VARIATION
2.7.1. Developmental biology

Widespread differences have been reported between isolates of Giardia
(representing G. duodenalis and G. enterica) in a variety of areas, including
biochemistry, growth rates (in vitro and in vivo), DNA content, drug
sensitivity, site and duration of infection, pH preference, virulence and

susceptibility to infection with a dsRNA virus (Binz, 1996; Binz et al., 1992;
Farbey et al., 1995; Hall et al., 1992; Monis et al., 1996; Reynoldson, 2002;
Thompson et al., 1996). Genome comparative analyses are in their relative
infancy, but they are already revealing some interesting findings. There
is significant variation between the genomes of G. duodenalis, G. bovis and
G. enterica in terms of gene content and polymorphism, chromosome
structure and gene families encoding surface antigens and kinases
( Jerlstrom-Hultqvist et al., 2010b).
G. duodenalis and G. bovis can both be readily cultivated axenically (Ey
et al., 1997), while G. enterica is more difficult to establish in vitro, growing
slower than G. duodenalis in vitro and appearing to grow better than
G. duodenalis in suckling mice (Andrews et al., 1992). At a gross level,
G. duodenalis and G. bovis are overall more similar to each across their
genomes than either are to G. enterica ( Jerlstrom-Hultqvist et al., 2010b).
A comparison of promoter regions for major cyst wall proteins has found
conserved promoters present in both WB and GS isolates of G. duodenalis
and G. enterica, respectively, suggesting regulation of these proteins is
similar in both isolates (Franzen et al., 2009). A similar promoter sequence
has also been found in front of a key regulatory enzyme in WB and the
P15 isolate of G. bovis, but the GS sequence lacks the same promoter
(Franzen et al., 2009; Jerlstrom-Hultqvist et al., 2010b). This variation has
been suggested to cause a difference in the regulation of cyst wall sugar
synthesis in GS and may be the cause of the poor encystation observed
in vitro of GS (Franzen et al., 2009).
The metabolic gene content for GS and WB is the same (Franzen et al.,
2009). Comparison of VSP, NEK kinases and high cysteine membrane
proteins found some that were conserved between WB and GS and
some that were highly divergent (Franzen et al., 2009). The genomic
organisation of the VSP genes has only been analysed in detail for WB,
finding that many genes occur in clusters and that recombination
has occurred between different VSP clusters (Adam et al., 2010). The
regulation of Giardia VSPs is likely to be different to that of other
protozoan parasites, with the WB VSPs predominantly occurring at
internal chromosome locations, whereas subtelomeric location of surface
antigen genes is more common (and in some cases, required for
expression) in trypanosomes or Plasmodium (Adam et al., 2010). The P15
genome appears to be poorer in VSPs compared to WB, although this
could be due to incomplete sequencing of those regions ( Jerlstrom-
Hultqvist et al., 2010b). The differences in some of these gene families
may explain the differences in host range ( Jerlstrom-Hultqvist et al.,
2010b). The genome organisation is different between the three species
(Jerlstrom-Hultqvist et al., 2010b), but the biological significance of this is
not clear.
2.7.2. Pathogenesis, variation in virulence and polyparasitism

A variety of symptoms are associated with Giardia infections. With the
genomic information now available, it should be possible to correlate this
with clinical expression and identify factors associated with virulence.
However, this remains a major challenge, given the variables that need to
be considered.
Most information on the pathogenesis of Giardia infections has been
obtained from studies in rodent models and in vitro culture, which have
shown that Giardia damages brush border microvilli, thus limiting intes-
tinal barrier function resulting in malabsorption and maldigestion
(reviewed in Cotton et al., 2011; Humen et al., 2011; Shukla and Sidhu,
2011). Although such observed changes help in understanding how diar-
rhoeal disease may occur in Giardia infections, it is not clear how the
pathophysiological changes described in rodent models can be extrapo-
lated to humans and other vertebrate hosts, since infection may not result
in overt clinical symptoms. Symptoms are also influenced by species/
breed of host, species/assemblage of Giardia, age, immune competence,
frequency of infection, nutrition and concurrent infections.
In humans, acute and chronic giardiasis present as two very different
diseases. In the former, acute episodes of diarrhoea are most commonly
associated with infection, whereas chronic giardiasis is not characterised by
diarrhoea but is associated with failure to thrive and is often exacerbated by
poor nutrition and polyparasitism (Thompson, 2008; Thompson and Smith,
2011). Furthermore, there is emerging evidence that Giardia infections may
induce post-infectious gastrointestinal symptoms including irritable bowel
syndrome (Hanevik et al., 2009; Kampitak, 2010; Wensaas et al., 2010).
Unfortunately, the impact on health of concurrent/coinfections (poly-
parasitism) has not been adequately taken into account. Giardia com-
monly occurs with other genera of intestinal parasites, particularly in
the developing countries (Thompson and Smith, 2011), and this will
influence the clinical impact of Giardia infections. This makes it difficult
to determine the contribution of each cohabiting pathogen to the clinical
consequences of such mixed infections. For example, the chronicity of
Giardia infections in disadvantaged children whose nutrition may be
suboptimal and who suffer infections with other gastrointestinal parasites
such as Entamoeba, Blastocystis, Hymenolepis and/or hookworm is recog-
nised as an important contributor to poor growth (Sackey et al., 2003;
Thompson, 2000; reviewed in Thompson and Smith, 2011). However, the
situation is further complicated by the fact that mixed infections with
G. duodenalis and G. enterica are also common.
A number of studies have provided data suggesting that acute and
chronic giardiasis may be associated with different species/assemblages
of Giardia (Gelanew et al., 2007; Haque et al., 2005; Homan and Mank, 2001;
Molina et al., 2011; Read et al., 2002; Sahagun et al., 2008). Based on available
data, it had been proposed that G. duodenalis may be more commonly
associated with acute giardiasis and G. enterica with chronic infections
(Thompson and Monis, 2011). In contrast, some recent reports found that
diarrhoea was more common in individuals infected with G. enterica
(Al-Mohammed, 2011; Mahdy et al., 2008, 2009; Pelayo et al., 2008). How-
ever, these reports were from developing and/or rural regions and are
difficult to interpret since Giardia was one of the several other cohabiting
enteric parasites, and in such cases of polyparasitism, it is very difficult to
conclude that non-specific symptoms such as diarrhoea are only due to
Giardia. The clinical impact of enteric protozoan infections is greatest in the
developing world where inadequate sanitation, poor hygiene and proxim-
ity to zoonotic reservoirs, particularly companion animals and livestock, are
greatest. In such circumstances, it is not surprising that infections with more
than one species of enteric protozoan and helminth are common, and in fact,
single infections are rare (Thompson and Smith, 2011). Interpretation of the
results is also complicated by differences in study design and sampling
strategy. From what has been reported in the literature, there is evidence
that infections with G. enterica in humans are more common in rural areas,
particularly in developing countries, and community situations, where the
frequency of transmission is high (Boontanom et al., 2011; Mahdy et al.,
2009; Molina et al., 2011; Yason and Rivera, 2007). This would suggest that
G. enterica is better adapted to such situations which are characterised by
prolonged infections/regular reinfections where acute diarrhoeal episodes
are not in the best interests of the parasite, allowing better survival in mixed
infections. The lack of overt symptoms such as diarrhoea would explain
why infections with G. enterica are more common in such environments
(Molina et al., 2011). Children with such infections are likely not to be
treated, which also raises questions about the long-term consequences of
such chronic infections if they persist and there is no ‘self cure’. This is
thought to be significant in situations where infected children are disadvan-
taged in terms of nutrition and exposure to concurrent enteric infections.
A number of mechanisms have been proposed to explain how Giardia
attaches to intestinal epithelial cells, but most evidence indicates that the
ventral disc plays the major role in attachment and that the cytoskeletal
elements of the disc are the major mediators in this process (Palm and
Svärd, 2009). This is indicated by the fact that microtubule inhibitors,
including known b-tubulin antagonists, have been shown to inhibit
adherence in vitro (Edlind et al., 1990; Magne et al., 1991; Meloni et al.,
1990). It is therefore interesting that a prominent cytoskeletal protein of
the ventral adhesive disc, alpha 2 giardin, which is present in G. duodenalis
(Assemblage A) isolates is absent in G. enterica (Assemblage B) isolates
which may explain the differences emerging in the clinical consequences
of infection with these two species (Steuart et al., 2008).
It is not known whether the complexity of symptomatology that is

seen in humans is seen in other hosts infected with the same species/
assemblage of Giardia. For example, it is not known whether there is any
difference in the clinical outcome in dogs infected with the zoonotic
species or G. canis.
2.8. CONCLUSIONS
The data from Giardia genome sequences (and other related protozoans)
have already improved our understanding of the evolution of Giardia and
eukaryotes in general and have identified some unique strategies that
Giardia has developed during its evolution, such as split introns. The
genome data are also improving our understanding of the metabolism
and cellular processes within Giardia. Comparison of the available Giardia
genomes supports the species status of the currently recognised assem-
blages, suggesting genome-wide differences equivalent to those separat-
ing species in other genera such as Theileria and Leishmania. The
differences that have been identified so far might also explain observed
phenotypic differences, such as differences in encystation caused by
differences in the regulation of key enzymes. These are relatively early
days in the comparative genomics of the different lineages of Giardia, and
more work is required to further compare the regulation of cellular
processes and to determine if there are differences that correlate with
variation in characters such as host range. Importantly, more genome
sequences are required, both from the different species and from multiple
isolates within the same assemblage/species, so that we can determine
the levels of intra- and interspecific differences, and if key differences in
chromosome arrangements or gene family repertoires are conserved
within species. Considering the level of genetic diversity within G. enter-
ica, it will be particularly important to compare the intraspecific variation
since this may underlie differences in host infectivity/disease outcome
among different isolates of G. enterica. The cost of genome sequencing is
continually decreasing, so the challenges to come will be more in the
collection of type material for sequencing, with the largest challenge to
conduct the necessary bioinformatic analysis to make best use of the large
amount of data that can now be readily generated.
There has been a progression in the development of molecular tools
for the identification of Giardia in recent years (Smith and Mank, 2011),
but the challenge for the future is the development of diagnostic assays
that will support clinical management and treatment decisions. For exam-
ple, an ELISA-based assay for use with dogs and cats that will provide not
only sensitive detection of Giardia but also information on species will
support the need for treatment in terms of public health significance and
possibly clinical prognosis. We already have a good stable of drugs with

antigiardial efficacy (Lalle, 2010), but there are limitations due to toxicity,
specificity, dosage, palatability and, possibly, resistance. Mining the
genome and proteome of Giardia will allow the development of new
classes of compounds with improved specificity, thus avoiding any
impact on normal gut microflora as well as improved compliance in
terms of palatability and dosage.
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CHAPTER 3
Malaria Ecotypes and
Stratification
Allan Schapira*,† and Konstantina Boutsika*,†

3.2. Methods 106
3.3. Results 108
3.3.1. Global studies 108
3.3.2. Experiences in different geographical regions 112
3.3.3. Proposed definition, identification and
demarcation of malaria ecotypes and their
implications in five biogeographic regions 137
3.4. Discussion 149
3.4.1. Implications for control programmes 149
3.4.2. Implications for malaria modelling and field
research 153
Acknowledgements 155
References 155
Abstract To deal with the variability of malaria, control programmes need to

stratify their malaria problem into a number of smaller units. Such
stratification may be based on the epidemiology of malaria or on its
determinants such as ecology. An ecotype classification was devel-
oped by the World Health Organization (WHO) around 1990, and it
is time to assess its usefulness for current malaria control as well as
for malaria modelling on the basis of published research. Journal
and grey literature was searched for articles on malaria or Anopheles
combined with ecology or stratification.
* Swiss Tropical and Public Health Institute, Basel, Switzerland

{
University of Basel, Basel, Switzerland

97
98 Allan Schapira and Konstantina Boutsika
It was found that all malaria in the world today could be assigned
to one or more of the following ecotypes: savanna, plains and valleys;
forest and forest fringe; foothill; mountain fringe and northern and
southern fringes; desert fringe; coastal and urban. However, some
areas are in transitional or mixed zones; furthermore, the implications
of any ecotype depend on the biogeographical region, sometimes
subregion, and finally, the knowledge on physiography needs to be
supplemented by local information on natural, anthropic and health
system processes including malaria control.
Ecotyping can therefore not be seen as a shortcut to determine
control interventions, but rather as a framework to supplement avail-
able epidemiological and entomological data so as to assess malaria
situations at the local level, think through the particular risks and
opportunities and reinforce intersectoral action. With these caveats,
it does however emerge that several ecotypic distinctions are well
defined and have relatively constant implications for control within
certain biogeographic regions. Forest environments in the Indo-malay
and the Neotropics are, with a few exceptions, associated with much
higher malaria risk than in adjacent areas; the vectors are difficult to
control, and the anthropic factors also often converge to impose
constraints. Urban malaria in Africa is associated with lower risk than
savanna malaria; larval control may be considered though its role is not
so far well established. In contrast, urban malaria in the Indian subcon-
tinent is associated with higher risks than most adjacent rural areas,
and larval control has a definite, though not exclusive, role.
Simulation modelling of cost-effectiveness of malaria control
strategies in different scenarios should prioritize ecotypes where
malaria control encounters serious technical problems. Further
field research on malaria and ecology should be interdisciplinary,
especially with geography, and pay more attention to juxtapositions
and to anthropic elements, especially migration.
3.1. INTRODUCTION
‘‘Everything about malaria is so moulded and altered by local conditions
that it becomes a thousand different diseases and epidemiological
puzzles. . . While this has provided a fascinating occupation for the epi-
demiologist, it has seemed discouraging enough to the health authorities’’
(Hackett, 1937). To deal with such immense variability—and encourage
the health authorities—some kind of classification is needed. In fact, most
national malaria control programmes stratify their malarial problem into
a number of smaller units, usually geographically defined, where differ-
ent strategies or approaches are applied (Beales and Gilles, 2000; Beales
et al., 1988). Classification of malaria situations should also be useful for
malaria modelling, which is undergoing a renaissance on the background
Malaria Ecotypes and Stratification 99
of increased international funding for malaria control, renewed interest in

elimination and eradication and technical developments, which now
allow for relatively realistic and dynamic assessment of cost-effectiveness
of malaria control in a variety of scenarios (Smith et al., 2008). While it is
possible in modelling to handle any number of permutations of variables,
an evidence-based classification of malaria situations would make it
possible to present model outputs within a humanly manageable number
of scenarios. This would also support communication between modelling
and control programmes.
Over the years, various typologies have been proposed with the aim of
supporting stratification and decision-making in malaria control as well
as the description of malaria and its occurrence. Almost all of them are
based on either the epidemiology of malaria or determinants of the
disease such as ecology or climate.
Among the malariometric classifications, the division based on spleen
or parasite rate into hypo-, meso-, hyper- and holoendemic (Metselaar and
Van Thiel, 1959; WHO, 1951) is well known and easy to understand, but it
has many weaknesses, including the absence of evidence that it has impli-
cations for planning control. An alternative is to classify malaria along the
spectrum of stability, that is, contrasting stable malaria characterized by
highly anthropophilic long-lived vectors in a warm environment, with
unstable malaria, where the characteristics are the opposite. This is more
attuned to decision-making, but the assessment is affected by the scarcity of
data on the longevity of mosquitoes in nature (Kiszewski et al., 2004), and
the utility is limited by the broad intermediate range, where the implica-
tions of Macdonald’s stability index (the number of bites on man taken by
an average mosquito during a normal life-time) (Macdonald, 1957a) are
uncertain. Molineaux (1988) proposed using the basic reproductive rate, as
a refinement of the stability index, incorporating more relevant factors.
However, its estimation is also demanding and hardly possible at a fine
enough scale to deal with important variability for a control programme.
Recently, it has been proposed to base classification and decision-making
on the more readily available measures of parasite prevalence and disease
incidence, especially Plasmodium falciparum prevalence. This may seem a
throwback to the old endemicity classification, but avoids the arbitrary
classes, while proposing further investigation of relationships between
malariometrics and chance of elimination (Hay et al., 2008). However, all
of these quantitative indicators may be poor predictors of resilience to
currently available interventions. For example, stable malaria on the
north coast of South America was easily eliminated by spraying in the
1950s (Giglioli et al., 1976), while unstable urban malaria in India has
withstood multi-pronged attacks (Saxena, 2001).
In classifying malaria situations, it would therefore be desirable to
include elements beyond quantitative epidemiological indicators, for
example, vector bionomics, resistance to biocides and operational determi-

nants of control feasibility. Typologies based on malaria determinants
would be useful if they serve as good proxies for several of such important
elements. The earliest global scheme based on this principle was probably a
climate-based classification (Gill, 1938), but it has never been widely
accepted due to the poor correlation between malaria and climate on a
global scale and in many regions of the world. In contrast, a malaria map
for Africa based on climate suitability has been widely used to illustrate the
presumed distribution of malaria in that continent and has at least an
approximate congruence with epidemiological data (Craig et al., 1999).
Ecological classifications have been promoted, ‘‘because they allow a
classification based on common knowledge about ecological characteris-
tics in a given area without collecting extensive information on vectors,
parasites, meteorology, human characteristics, etc.’’ (Beljaev, 2002). Eco-
logical characterization of local malaria situations was used in the early
twentieth century when anti-larval measures, which require an under-
standing of local physiography and vector bionomics, dominated vector
control options (Bradley, 1994; Takken et al., 1990).
The first global classification incorporating environmental determi-
nants was developed in the 1950s by Macdonald, who, referring to
Wallace’s six zoogeographical regions (Table 3.1), identified the main
vectors and their bionomics in each of 12 zones (Macdonald, 1957b).
TABLE 3.1 Malaria ecotypes and their occurrence in the world according to texts
published 1990–2000
Malaria ecotype Where found
Savanna Sub-Saharan Africa, Southwest Pacific

Plains with traditional South Asia, Central and South America,
agriculture outside Africa China
Highland fringe Africa, Southeast Asia, Southwest
Pacific, South America
Desert fringe and oasis Sahel, southern Africa, West Asia
Forest and forest fringe South and Southeast Asia, South
America
Costal and marshland Africa, South and Southeast Asia, South
and Central America
Urban and peri-urban Africa, South Asia, South America
Agricultural development Superimposed on any of the above
including irrigation ecotypes
War and socio-political
disturbances
This description helped organize the global diversity of anophelines and

provided valuable insights on vector bionomics and control. However, as
a classification scheme, it is a hybrid between a geographical division and
an ecological typology, and it has very limited control implications.
Macdonald’s scheme was criticized by Russian malariologists
as being too top down. As an alternative, they promoted landscape
epidemiology (Pavlovsky, 1966; Sergiev et al., 2007a), seeking to inte-
grate epidemiology with landscape science (Dyakonov, 2007), examin-
ing the interactions between natural and human ecology to convey a
comprehensive local picture of the disease and its determinants and
thereby stimulate thinking about what could be done about it. For
malaria, these principles were applied by Beklemishev from 1940
onwards (Tchesnova, 1998) and further developed by Lysenko, who
recommended the recognition of types of foci within each kind of geo-
graphical area or landscape and zonation based on a combination of
malaria data and ecological type. However, in practice, it was often difficult
to differentiate malaria control in vertical programme strategies according
to this approach (Lysenko, 1960).
Otherwise, during the eradication era from the 1950s to the 1970s,
there was little interest in local assessment and classification, given that
indoor residual spraying (IRS) is much less site specific than larval con-
trol, and those strategies and approaches were highly standardized.
However, in tropical Africa, various classification schemes were devel-
oped, especially by francophone scientists (Carnevale and Mouchet, 2001;
Hamon et al., 1963; Mouchet, 1976). The experiences were crystallized as
an approach consisting of
(A) Identification of the primary faciès épidémiologique as belonging to one
of six major natural regions, namely, (1) equatorial with forest or
savanna and perennial transmission; (2) tropical with humid savanna
and a transmission season exceeding 6 months; (3) Sahelian with dry
savannas or steppes and a transmission season lasting less than
6 months; (4) desert with steppe or desert and short, sometimes
missing transmission season; (5) southern corresponding to the
plateaux of southern Africa with seasonal transmission, which is
interrupted in winter and (6) highland at 1000–2000m altitude,
where transmission is highly variable and limited by temperature
and surface declination.
(B) Identification of secondary factors within each primary faciès: natu-
ral (landform, water bodies, soil characteristics); anthropic factors
(modification of vegetation, water bodies, urbanization, habitat of
humans and cattle) and dynamic factors (natural disasters, climate
change, malaria control, population movement, development of
transport networks) (Mouchet et al., 1993a).
Outside Africa, the failure to eradicate eventually led to renewed

interest in ecology-based classification, aiming to incorporate the lessons
learnt as well as the local and regional experiences and frameworks
referred to above. A global malaria typology was developed, building
on the early ecological characterizations and the Russian and francophone
‘schools’ with environmental characteristics as the primary identifier, but
trying to relate them to the selection of control interventions (Najera, 1989;
Najera et al., 1992), this framework was included in the global malaria
control strategy promoted by World Health Organization (WHO) and
approved by the Ministerial Conference on Malaria in Amsterdam in
1992 (WHO, 1993). The general principle is to link environmental deter-
minants to associated characteristics of malaria epidemiology, vector
bionomics, human ecology and health systems, emphasizing commonal-
ities of particular patterns across the world (Beales and Gilles, 2000;
Najera, 1990). Table 3.1 presents the types defined as these texts con-
verged, with some harmonization of nomenclature.
Around 1990, the term paradigm was frequently used to denote the
need not only for describing particular settings but also to provide exam-
ples of successful control adapted to local determinants. In the following
years, as it was difficult to identify truly paradigmatic control experi-
ences, this term fell into disuse, and the most commonly used term
became ecotypes or eco-epidemiological types. It is proposed here to
use the term (malaria) ecotype, because it parallels the use of ecotype in
biology (Table 3.2), meaning that the malaria system, which is a biological
and social phenomenon, can be studied in the same way as an organism
interacting with an environment.
The latest of these texts is a report issued by WHO in 2006 reviewing
effectiveness and challenges of different vector control measures accord-
ing to ecotype. This publication includes the first seven of the above
ecotypes under ‘steady-state ecosystems’ and the two last as ‘situations
of rapid development change’. It notes that IRS and insecticide-treated
nets (ITNs) are both almost universally effective for malaria control
(though not to the same degree everywhere), and that stratification
according to ecotypes would be useful mainly to identify epidemiological
patterns, local risk factors, risk groups and feasibility of larval control
(WHO, 2006). Given the accessibility of this text, it will not be summar-
ized here, but attention will be given to recent evidence that challenges or
supplements it.
This typology has not been critically addressed in journal literature
and may now be at risk of being perceived as dogma; it is therefore
opportune to revisit it to assess its utility in an era when a wide range of
vector control interventions are being considered on a background of
increased resources and renewed interest in elimination and eradication.
Another reason for revisiting ecology-based malaria classification is that
TABLE 3.2 Terms and acronyms used in this review
Annual parasite index (API) A measure of the number of confirmed malaria cases per thousand people
per year in a defined geographical area
Biogeographic regions or realms/ Major geographic divisions of the biosphere according to distribution of
ecozones/zoo-geographical regions fauna. The original zoogeographical regions of Wallace (1876) have
recently been modified by the World Wildlife Foundation (Olson et al.,
2002) to the following (Fig. 3.1):
1. Palearctic (including most of Eurasia and North Africa)
2. Nearctic (North America)
3. Neotropic (including South and Central America and the Caribbean)
4. Afrotropic (including sub-Saharan Africa, Madagascar and south-western
part of Arabian peninsula)
5. Indo-malay (including Indian subcontinent and Southeast Asia)
6. Australasian (including eastern Indonesia and Southwest Pacific)
7. Oceanic
8. Antarctic
Note: 7 and 8 are malaria free
Cold cloud duration (CCD) Remotely sensed data correlating closely with rainfall (Thomson et al., 1997)
Ecoregion Regions of relative homogeneity in ecological systems or in relationships
among organisms and their environment (Omernik, 1987)
Ecosystem An area of any size with an association of physical and biological
components so organized so that a change in one component may bring
about some corresponding change in other components and in the
operation of the whole system (Bailey, 2009)
Ecotone Transition zone between two communities (Bailey, 2009)
(continued)
Ecotype For malaria: a group of malaria foci, which are similar in terms of physical
and biological environment and most of the following attributes: malaria
epidemiology, vector bionomics, human ecology and health systems
(writers’ proposed definition)
In biology, ecotype refers to species with wide geographic range that develop
locally adapted populations having different limits of tolerance to
environmental factors (Bailey, 2009)
Enhanced vegetation index (EVI) NDVI (see below) corrected for some distortions
Entomological inoculation rate (EIR) The expected number of infectious bites, per person, per unit time
(usually a year)
Geographic information system (GIS) Information system for capturing, storing, analyzing, managing and
presenting data which are spatially referenced (linked to location)
(Bailey, 2009)
Insecticide-treated nets (ITNs) Also including long-lasting insecticidal nets (LLINs)
Indoor residual spraying (IRS) Indoor residual spraying with insecticides. ITN and IRS are the two main
methods of adult vector control in malaria
Malaria focus A defined and circumscribed locality situated in a currently or formerly
malarious area and containing the continuous or intermittent
epidemiological factors necessary for malaria transmission (WHO, 2007)
Normalized difference vegetation index Remotely sensed data based on reflectance factors indicating presence and
(NDVI) density of green vegetation or water (Thomson et al., 1997)
Physiography Landform (including surface geometry and underlying geologic material
(Bailey, 2009))
Receptivity For a malaria-free area: The abundant presence of vector anophelines and the
existence of other ecological and climatic factors favouring malaria
transmission. Receptivity is a reflection of vectorial capacity of local
anophelines during the season most favourable for malaria transmission
(WHO, 1978)
Stratification A process of dividing the malaria problem of a given area, for example, a
country, into a limited number of units, which are sufficient homogenous
internally and sufficiently different from each other that it is rational to
apply different strategies to them
Vectorial capacity The expected number of infectious bites that will arise from all the
mosquitoes that bite a single person in 1 day
Vulnerability For a malaria-free area: Proximity to malarious areas or liability to the
frequent influx of infected individuals or groups and/or of infected
anophelines. The level of awareness of the population concerning malaria,
and the level of sophistication of the health authorities also have an
important bearing (WHO, 1978)
since the early 1990s, the evidence-base has been strengthened by the
availability of geographic information systems (GISs), remote sensing
and spatial analysis (Kitron, 1998).
General criteria for a malaria typology could be presented as follows,
slightly modified from those put forward by Molineaux in 1988 for a
typology based on epidemiological criteria: (a) it does not have too many
types; (b) it provides only one type for every possible malaria situation;
(c) the types are meaningful for control, in terms of what is recommended
and feasible in a situation and of achieving the expected impact and (d) the
diagnosis of situations and the stratification of geographical areas accord-
ing to the types are not too expensive or complicated.
The purpose of this chapter is not to identify the perfect malaria
typology but to assess, review and possibly improve ecological classifica-
tion for malaria control and modelling, keeping in mind the existing
availability of malariometric data. The methodology selected is a qualita-
tive review of published evidence by biogeographic region. This division
of the world has direct implications for mosquito fauna, precedes all
malaria typologies, has stood the test of time and is used by other
disciplines.
3.2. METHODS
Pubmed (http://www.ncbi.nlm.nih.gov/sites/entrez) and ISI Web

of KnowledgeTM(http://apps.isiknowledge.com/UA_GeneralSearch_input.
do? product¼UAandsearch_mode¼GeneralSearch andSID¼Q2A34OLK8A
FDBNLGdOD and preferencesSaved¼) were searched without time limita-
tion, using as search terms ‘malaria’ combined with each of the following:
‘ecology’, ‘ecological’, ‘eco-epidemiological’, ‘ecotype’, ‘geography’ and
‘stratification’. In addition, the search term ‘Anopheles’ was combined with
‘ecology’ or ‘interaction’ for the past 20 years. Major textbooks, monographs,
WHO publications on malaria and websites of some institutions and malaria
control programmes were hand searched for material relevant to the subject.
Recent literature on ecology and disease was examined selectively for
updates on modelling and concepts. The several thousand references found
were scanned by their titles and the number reduced to about 1000. For these,
the abstracts were read, and this led to a selection of the 200 articles and texts
quoted in this review.
The application of ‘eco-epidemiology’ for classifying malaria situa-
tions has been examined by biogeographical region (Table 3.2; Fig. 3.1).
For each biogeographical region, a brief overview of malaria vector bio-
nomics and any general region-wide classification schemes is followed by
a review of research findings related to specified types, with an emphasis
on those of the scheme presented in Table 3.1. This is followed by a review
Palearctic
Nearctic
Oceanic
Afrotropic Indo-malay
Oceanic
Neotropic
Australasian
Antarctic
Biome
TMF: Tropical and subtropical moist broadleaf forests MG: Montane grasslands and shrublands
TDF: Tropical and subtropical dry broadleaf forests T: Tundra
TCF: Tropical and subtropical coniferous forests MF: Mediterranean forests, woodlands and scrub
TeBF: Temperate broadleaf and mixed forests D: Deserts and xeric shrublands
TeCF: Temperate coniferous forests M: Mangroves
BF: Boreal forests/taiga Lakes
TG: Tropical and sub-tropical grasslands, savannas and shrublands Rock and ice
TeG: Temperate grasslands, savannas and shrublands
FG: Flooded grasslands and savannas Biogeographic realm
Conutry
Ecoregions
FIGURE 3.1 The 14 Biomes and Eight Biogeographic Realms of the World as defined by the World Wildlife Foundation. Biomes are coded
in colours. Biogeographic realms are named in the figure. Ecoregions are nested within both biomes and realms. Source: United Nations
Millennium Ecosystem Assessment, Appendix, Fig. 4.3. Permission to reuse is given at www.millenniumassessment.org/en/GraphicResources.
aspx.
of articles relevant to malaria and ecology at the global level. The findings
are summarized in two tables, one describing the general characteristics
of six proposed basic ecotypes at the global level, including their defini-
tions and delimitations (addressing Molineaux’s criterion (a)), and the
other, the variability of those ecotypes according to biogeographic region.
In these two tables, the delimitation of the ecotypes from each other and
their implications for control are specifically addressed, in line with
Molineaux’s criteria (b) and (c).
The key terms used in this review are presented in Table 3.2.
3.3. RESULTS
3.3.1. Global studies

The monumental Biodiversité du paludisme is a thorough global review of
the diversity of malaria epidemiology with emphasis on the vector
aspects (Mouchet et al., 2004a). While referring to previous classification
schemes, this text avoids any attempt at a global typology but does
include a global overview, which can be summarized as follows: The
distribution of malaria follows a gradient: from Afrotropic core areas,
where malaria is endemic and continuous over vast distances, except
where interdicted by climatic factors, with transitional epidemiology in
the ecotones; through tropical Southwest Pacific, then tropical Asia and
South America, where malaria is focal and highly dependent on ecologi-
cal determinants, to subtropical and temperate areas, where malaria is
sporadic and transmission conditional on a convergence of enabling
factors. As may have been noted, the present review draws extensively
on the compilations, reviews and syntheses in Biodiversité. The sequence
in which the world’s biogeographic regions have been presented corre-
sponds to the global gradient just described.
The interactions between agriculture and malaria have been reviewed
by Service (1989), who includes the various possibilities for control and
the circumstances under which they are likely to be feasible and effective.
A more recent and quantitative review found that most dam building and
irrigation in the world takes place in areas with no or little malaria, but
that the risks from environmental change are greatest in areas with
unstable malaria and that remedial measures should be planned at the
design stage (Keiser et al., 2005a).
The effectiveness of environmental management for malaria control
has been reviewed with reference to an ecological typology with four
classes: deep forests, forest fringe and hills; rural malaria attributable to
water resources development and management; rural malaria attribut-
able to wetlands, rivers, streams, coasts and non-agricultural man-made
habitats and urban and peri-urban malaria. Nearly all analyzable studies
showed some effect of environmental measures, but most were con-
founded by concurrent interventions. This review documented that envi-
ronmental management can be highly effective in certain circumstances
and that the practice in the twentieth century, both before and after the
eradication era, had been to select such circumstances, largely excluding
settings (especially savanna and forest malaria), which were or which
were thought to be inappropriate (Keiser et al., 2005a).
Yasuoka and Levins reviewed deforestation worldwide and found that
the effects depended on the type of environmental change and the species
of vector; in particular, sun preference of the vector was associated with
increasing vector density as a result of deforestation. In fact, An. darlingi
prefers breeding sites exposed to the sun or with only partial shade in
contrast to An. dirus in Southeast Asia (Yasuoka and Levins, 2007).
Kiszewski mapped a global malaria stability index in order to describe
the distribution of the global malaria burden, as it would be without
organized malaria control. The index represented the contribution of
regionally dominant vectors to the force of transmission in each geo-
graphic area and incorporated human blood index, daily survival of the
vector, duration of the transmission season and extrinsic incubation
period based on temperature. Vegetation indices from remote sensing
were used to define areas suitable for vectors with ecological require-
ments, such as salt marshes or forests, and altitude limits were used to
define the ranges of vector species. Comparing the resulting map
(Kiszewski et al., 2004) with Fig. 3.1, the congruence between malaria
stability and forests in the Neotropic and Indo-malay is clear.
The Malaria Atlas Project (MAP) has over some years mapped malaria
burdens in the world. An examination comparing several independent
definitions of urban areas with reports on malaria parasite prevalence in
pairs of urban and rural areas found that the Global Rural Urban
Mapping Project (GRUMP) urban extent mask (Center for International
Earth Science Information Network, 2004) proved more accurate than
other delimitations of urban extent to delimit urban areas with lower
malaria burden. However, significantly lower burdens in urban areas
were found only in the Afrotropic (Tatem et al., 2008). The latest iteration
makes use of nearly 8000 geo-referenced prevalence surveys dating since
1985 and model-based geostatistics to create a global map of P. falciparum
endemicity in 2007. Apart from urban and peri-urban areas, it was found
that there was no strong relationship with climate or environmental
covariates, so these were not included in the model (Hay et al., 2009).
Nonetheless, the geographical distribution shows good correspondence
with maps based on other methods including reported incidence maps
and with forest cover in the Indo-malay and Neotropic biogeographic
regions as shown in Fig. 3.2 and Socheat et al. (2003).
Land use in India, 2001
Arable land: yellow
Forests: dark green
Non-agricultural use of land: dark brown
Plantation: light green
Scrub and grass: purple
Unproductive land: Light brown
Source: Environment Atlas of India, Ministry of
Environment and Forest. Map data source Central
Pollution Control Board(CPCB) and National Atlas and
Thematic Mapping Organisation (NATMO)
http://www.soeatlas.org/PDF_Map%20Gallery/Landuse.p
df accessed 16 September 2009
N
W E
N
Jammu & Kashmir S
W E
Jammu & Kashmir
S
Himachal Pradesh
Punjab Himachal Pradesh
Chandigarh
Uttaranchal Punjab
Chandigarh
Haryana Uttaranchal
Delhi Arunachal Pradesh Haryana
Sikkim Delhi Arunachal Pradesh
Uttar Pradesh Sikkim
Rajasthan Assam Uttar Pradesh
Bihar Nagaland
Rajasthan Assam
Nagaland
Meghalaya Bihar
Manipur
Meghalaya
Tripura Manipur
Jharkhand Tripura
Madhya Pradesh West Mizoram Jharkhand
Bengal Madhya Pradesh West Mizoram
Gujarat Bengal
Chhattisgarh Gujarat
Chhattisgarh
Daman & Diu
Orissa Daman & Diu
Dadra & Nagar Haveli Orissa
Maharashtra Dadra & Nagar Haveli
API - 2001 Maharashtra API - 2007
> 10.00 > 10.00
5.01 - 10.00 5.01 - 10.00
Andhra 2.01 - 5.00
Pradesh Andhra 2.01 - 5.00
Goa 1.01 - 2.00 Pradesh 1.01 - 2.00
<= 1.00 Goa < 1.00
Karnataka
Karnataka
Pondicherry Pondicherry
Tamil Tamil
Kerala Nadu Nadu
Kerala
Andaman & Nicobar Islands Andaman & Nicobar Islands
Lakshadweep Lakshadweep
Malaria incidence in India, 2001 and 2007, as indicated by annual parasite index (API).
Source: National Vectorborne Disease Control Programme, India, and WHO
FIGURE 3.2 Comparison of land use and reported malaria incidence in India in 2001 and 2007.
3.3.2. Experiences in different geographical regions

3.3.2.1. Afrotropic region
3.3.2.1.1. General In nearly all of sub-Saharan Africa, malaria transmis-
sion is dominated by a pair of vectors, which are almost ubiquitous and
often transmit malaria in tandem across seasons: An. funestus, which
breeds in streams and shaded water bodies in rural areas, and the highly
ramified and versatile An. gambiae complex, which occupies mainly tem-
porary water bodies, preferably sunlit. An. arabiensis, which belongs to the
An. gambiae complex, is common in relatively arid areas; it is often exo-
philic and zoophilic but is still a very efficient vector. A few vector species
are locally important; they include An. moucheti, which is a main vector in
some forested areas, and An. nili, which is found in various environments,
usually with riverine breeding sites (Carnevale et al., 1992; Mouchet et al.,
2004a; Sinka et al., 2010a).
Exploiting the entomological homogeneity, the MARA/ARMA proj-
ect (http://www.mara.org.za/) mapped malaria in sub-Saharan Africa
based on climate suitability. A combination of rainfall and temperature
was shown to correlate well with the distribution of malaria as shown by
parasitological surveys; in most of the continent, low temperature
correlated closely with altitude leading to unstable malaria in mountain
fringe areas, mainly in Eastern Africa, and low rainfall with desert
fringes in the Sahel and south-western Africa. However, in eastern
South Africa, low winter temperatures limit the distribution of the
malaria vectors, thereby defining a southern fringe related neither to
altitude nor to desert climate (Craig et al., 1999). Epidemiologically, this
setting has practically the same characteristics as mountain fringe further
north on the continent (see below), with risk of epidemics and high
population densities. Further investigations for East Africa including
remote sensing, human settlement and land-use data at high spatial
resolutions found that the best fit was obtained by stratifying the
subcontinent into two ecological zones: one corresponding to highland
and arid ecotones and the other corresponding to other rural areas. In
addition, it was found necessary to distinguish urban areas, where
malaria transmission was always lower than in rural areas with similar
climate (Omumbo et al., 2005). Thus, this classification, which started out
as climate-based, developed into being more physiography-based
because of (a) the close correlation between physiography and climate
and (b) the need to reckon urban areas as a special class, which is not
distinguished by climate.
As mentioned in Section 1.1, a typology for Africa has been proposed
by Mouchet et al. (1993a); it has a slightly greater degree of differentiation,
for example, between an equatorial zone and a tropical zone. The former
would correspond to forest malaria and savanna malaria with perennial
transmission, and the latter to savanna malaria with long seasonal trans-
mission. However, the transition from equatorial to tropical savanna is
gradual, as is also the transition from perennial to seasonal malaria.
In fact, as reported in the same article, the transmission of malaria in the
savanna environment is maintained at a low level during most of the dry
season by An. funestus.
3.3.2.1.2. Savanna In the above analysis, the zone of rural areas with
intense malaria transmission corresponds to savanna malaria. Depending
on geographic region and especially rainfall and vegetation, there may be
up to three extremely efficient vectors in savanna areas: An. gambiae s.s.,
An. arabiensis and An. funestus. Among these, the second is often, and the
first sometimes somewhat exophagic and exophilic. Further investigation
of such areas in western Kenya revealed a fragmented landscape
mainly consisting of agricultural and domestic land uses within which
breeding of malaria vectors was associated with certain land cover
types, of largely agricultural origin, and close to streams (Mutuku et al.,
2009). In arid savanna in Mali, it was found that NDVI correlated well
with malaria incidence (Gaudart et al., 2009). It has been assumed that
larval control has little potential in the African savanna environment,
because the many diverse temporary habitats of An. gambiae are
difficult to cover, while the breeding sites of An. funestus are often difficult
to find and protect. Yet, a recent study in western Kenya found that the
application of bacterial larvicides at a cost of USD 0.9 per inhabitant per
year can lead to an epidemiologically significant reduction in biting
density; however, the site had lower malaria transmission before inter-
vention than is usually found in the savanna environment (Fillinger and
Lindsay, 2006).
3.3.2.1.3. Forest The main vector is An. gambiae s.s., which in some forests
is highly endophilic and therefore easy to control (Carnevale and
Mouchet, 2001), but in others somewhat exophilic. The density is lower
in forests than in savanna areas due to the requirement for sunlight
(Mouchet et al., 2004a). Corresponding to earlier findings, for example,
in Cameroon (Manga et al., 1997), a direct comparison between forested
and deforested adjacent areas in Kenya found that vectorial capacity was
higher in the latter, and this was attributed to higher temperatures and
humidity levels (Afrane et al., 2008). In West Africa, very intense trans-
mission with exacerbation during the rainy season may characterize the
forest-savanna transition zone (Owusu-Agyei et al., 2009).
3.3.2.1.4. Highland fringe The epidemic pattern and temperature and

rainfall influence were shown, for example, in Burundi (Gomez-Elipe
et al., 2007). Altitude variations were shown to be important predictors
of malaria transmission intensity in Zimbabwe (Mabaso et al., 2005).

In Madagascar, the epidemic, highly unstable malaria on the high plateau
at around 1000–1500m above sea level (a.s.l.) is now being controlled by
IRS, but a recent investigation drew attention to the very high burden
affecting all age groups almost equally in the nearby foothill area at
around 900m a.s.l. (Rabarijaona et al., 2009). In Kenyan highland areas,
water bodies identified by remote sensing predicted mosquito breeding
and proximity to high-order streams the distribution of adult mosquitoes
in houses (Li et al., 2008; Mushinzimana et al., 2006). Similarly, proximity
to forest and swamps were both strong predictors of malaria in Kenyan
highlands (Ernst et al., 2009). It was recently found in an area of moderate
transmission intensity in the highlands of western Kenya, where ITNs
were also introduced, that larviciding could reduce risk of malarial infec-
tion in children by 40%, almost the same as the protection afforded by nets
(Fillinger et al., 2009). This is of considerable interest, because larval
control has not in the past been considered to have much potential in
such an environment, where breeding sites are abundant and diverse in
the rainy season.
Ethiopia has large populations without the typical markers of genetic
resistance to malaria found in most other African populations; the
highest population densities are found in highland areas, which are
malaria free, but at risk of malaria epidemics. Following epidemics in
the highland areas in 2003–2004, which, together with those in
Ethiopia in 1959, are the worst malaria epidemics on record anywhere
since 1950 (UNICEF, accessed 12 January 2009 http://www.unicef.org/
ethiopia/malaria.html; Fontaine et al., 1961), there has been increasing
coverage of IRS and ITN and a steady reduction in reported malaria
incidence.
3.3.2.1.5. Desert fringe It is generally assumed that breeding sites are

scarce, dependent on rainfall and/or permanent water bodies, and there-
fore easy to control. In some texts, highland and desert fringe malaria are
treated as one; while the epidemiological pattern is often similar with
unstable malaria and higher age groups being vulnerable in contrast to
the savanna situation, this overlooks important ecological and social
differences: Highlands usually have abundant breeding sites, fertile
soil and high population density enabling a health service infrastruc-
ture. Mosquito nuisance may be very low, and IRS therefore better
accepted than ITNs. In contrast, in thinly populated desert fringe
areas, the people are more often pastoralists constraining the provision
of health care (Sheik-Mohamed and Velema, 1999). The proximity to
cattle may lead to considerable insect nuisance and therefore motivation
for the use of mosquito nets, but this may be constrained by seasonally
or perennially high ambient temperatures, which is one reason for the
preference for insecticide-treated curtains rather than mosquito nets in

trials in Burkina Faso (Procacci et al., 1991). Yet, while it is clear that
the Sahel and Namibia are mainly affected by desert fringe malaria
and Burundi and Rwanda by mountain fringe malaria, it must be
recognized that certain areas of Kenya, Ethiopia and other countries
combine highland and desert fringe characteristics (Noor et al., 2009;
Zhou et al., 2007).
Recent studies in desert fringe areas do not always confirm received
wisdom. In southern Somalia, a clear correlation between malaria and
rainfall was found, but not between malaria and distance to permanent
water bodies; in the country’s north, there were no significant spatial or
climatic covariates, presumably because of data scarcity. Arid areas in
Somalia are among the few in tropical Africa where larvivorous fish have
been tried for malaria control; the intervention led to reduced larval
density, but it was not investigated whether this led to lower malaria
transmission (Mohamed, 2003). Similarly, a more recent controlled study
in Eritrea showed significant reduction in the adult density of An. ara-
biensis (which, typically for an arid area, was the only vector) following
environmental and chemical larval control (Shililu et al., 2007), but epide-
miological impact was not assessed.
The south-western part of the Arabian Peninsula belongs to the Afro-
tropic realm. The main vector is An. arabiensis, and most of the malaria in
that area is highly unstable and rain dependent and can be classified as
desert fringe.
Mouchet et al. (1993a) distinguish Sahélien from savanna malaria by a
duration of the rainy season of less than 5 months and propose a distinc-
tion between desert fringe (Sahélien) and actual desert malaria, where
malaria transmission takes place only in some years. There is merit in
this, but it is difficult to define the demarcation climatically or ecologi-
cally, and it would seem practical to treat desert malaria (which probably
affects very few people) as a subtype under desert fringe malaria.
3.3.2.1.6. Coastal In Africa, the malaria vectors, An. melas and An. merus,
which breed in brackish water (and belong to the An. gambiae complex),
are less efficient than those typically found in the surrounding rural areas.
Incidence may fluctuate widely when seasonal rains reduce salinity,
thereby increasing vectorial capacity (Akogbeto et al., 1992). In Senegal
in a coastal area, the malaria situation was characterized by seasonality,
low level of transmission with all age groups affected and influence of
man-made environmental changes (Diop et al., 2006). In the Senegal river
delta, An. pharoensis, which is otherwise not considered an important
vector outside Egypt, was identified as the main vector, and ITNs were
highly effective there (Carrara et al., 1990).
3.3.2.1.7. Urban Compared to people in savanna areas, urban populations

in Africa, generally, have lower malaria transmission intensity with
higher age maxima for morbidity and mortality. Generally, access to
curative services is better. Using data from a number of studies, it was
shown that there is a rising gradient of entomological inoculation rate
(EIR) when moving from urban areas, where the value is typically 20 or
less infective bites per person per year, over intermediate peri-urban areas
to highly endemic rural areas with EIR values of typically 100–200 (Hay
et al., 2005; Robert et al., 2003). The high level of heterogeneity of malaria
in urban areas has been demonstrated in a number of studies (Machault
et al., 2009) and is easily explained by the scarcity of breeding sites and the
high population density.
In Khartoum, there have been good experiences with larviciding in the
first half of the twentieth century and again in recent years (Elkhalifa
et al., 2008). The effect of larviciding was demonstrated recently in Dar es
Salaam (Geissbuhler et al., 2009), but there are few other demonstrations
of the value of larval control in African cities. In many cases, urban
malaria in Africa is due to interspersion of areas with urban and savanna
characteristics, so breeding may not be technically easier to control than in
rural areas. Recently, it has been observed that increased vector breeding
in polluted water and artificial containers, earlier biting, and increased
exophily could compromise the expected mitigation effect on malaria of
urbanization (Saugeon et al., 2009). A tendency of An. gambiae s.l. in Accra
to breed in domestic containers and polluted water was noted already in
the 1980s, but it was not clear whether this was a genetic adaptation or a
partial replacement of An. gambiae s.s. with An. arabiensis (Chinery, 1984).
There is clearly a need for longitudinal studies on possible vector adapta-
tion to urbanization and on ways to deal with it. The serious mosquito
nuisance in many urban areas (Carnevale and Mouchet, 2001) and
the risks of various arthropod-borne diseases should facilitate effective
promotion of house screening and mosquito nets.
3.3.2.1.8. Agricultural development Wet-rice cultivation is rapidly

increasing in Africa and there has been concern about the potential impact
on malaria. In the savanna zone in Côte d’Ivoire, rice cultivation induced
moderate changes in the seasonality of malaria, but no increase in EIR
or morbidity. In northern Tanzania, rice irrigation was associated with
less malaria than alternative agricultural practices, despite the high vector
productivity in the paddies (Ijumba et al., 2002; Keiser et al., 2002). Even in
the semi-arid sub-Saharan environment in Mali, rice cultivation altered
transmission from seasonal to perennial but reduced annual incidence
more than twofold (Sissoko et al., 2004). In a study investigating the
interactions between environment, vectors, human ecology, health sys-
tem and disease, biannual irrigation rice harvesting, when compared to
annual, was associated with changing gender roles, lesser availability of

cash for women and consequently poorer health seeking behaviour; how-
ever, the situation might look different in a drought year, where the
biannual rice harvest could improve food security (de Plaen et al., 2004;
Henry et al., 2003). In contrast, studies in a highland area in Burundi
showed clearly increased risk (Coosemans, 1991) and in Ethiopian high-
land areas, malaria morbidity was greatly increased near dams (Brewster,
1999). The effect of rice cultivation was also serious in a forest zone, where
the vector density is normally low (Briet et al., 2003). Possibly, the most
serious negative effects of irrigation in Africa were seen in the arid con-
ditions of Gezira, Sudan, where eventually malaria was controlled by IRS.
Subsequently, epidemics followed the relaxation of control and the
development of insecticide resistance related to the use of DDT for cotton
cultivation and subsequently malaria was controlled again by IRS with
newer insecticides; various forms of larval control were attempted but
never proved effective (el Gaddal et al., 1985; WHO, 1985). In urban areas,
rice cultivation is associated with high anopheline densities, but it is not
clear whether it leads to more malaria than there would otherwise be
(Dongus et al., 2009; Matthys et al., 2006).
Thus, in ecosystems with relatively low transmission intensity in
Africa, irrigation usually leads to increased transmission, while in
savanna areas, it has little impact on malaria burden although it may
be associated with increased vector density and a shift towards greater
perennity. This ‘paddies paradox’ is sometimes attributed to conditions
favouring less efficient vectors, but it may also be explained
by increased biting rates motivating people to use protective measures
and by communities near irrigation schemes benefiting from greater
wealth and better access to health care (Ijumba and Lindsay, 2001;
Keiser et al., 2005b). Modification of water management practices in
irrigation schemes may have limited potential in most of Africa, as
water is generally scarce. In Mwea in Kenya, where An. arabiensis is
the main vector, intermittent irrigation at four-day intervals did not have
a significant impact on mosquito densities (Mutero et al., 2000). As
An. arabiensis often emerges as the main vector in rice-field areas, it is
possible that the potential of zooprophylaxis could be better exploited
(Mutero et al., 2004). Generally, the extensive descriptive literature on
malaria and irrigation in Africa contrasts with a paucity of trials of larval
control.
The serious malaria problem at Zambia’s copper mines in the 1920s
shared its main characteristics with malaria in agricultural development
projects. The well-documented success in reducing it mainly through
environmental management 60–80 years ago is a reminder that such
methods may play an important role in some settings dominated by
African malaria vectors (Utzinger et al., 2001).
Madagascar has the same main malaria vectors as continental Africa,

but their bionomics are different. In the highlands, irrigated rice fields are
the main determinant of malaria, with An. funestus as the main vector with
the highest densities, when the rice is close to harvest providing shade
(Mouchet et al., 2004a). In the southern lowlands, irrigated rice fields in
arid zones, which could be classified as desert fringe, also have greatly
increased malaria (Laventure et al., 1996).
3.3.2.1.9. War and socio-political disturbance The particular problems of

malaria related to war and political disturbance are easily understood,
considering the main feature of degradation of health systems. To this
may be added housing problems, environmental damage and migration
of populations with varying malaria exposure. New approaches and tech-
nologies are being developed to deal with these problems (WHO, 2005).
3.3.2.2. Australasian region

This region includes the easternmost part of the Indonesian archipelago,
New Guinea Island, Solomon Islands, Vanuatu, New Caledonia, Australia
and New Zealand. It has a region-specific anopheline fauna in New
Guinea, Solomon Islands, Vanuatu and northern Australia; in contrast,
the malaria vectors in the eastern part of the Indonesian archipelago are
Indo-malay with the exception of Maluku islands (the Molucas) (Mouchet
et al., 2004a; Ndoen et al., 2010). New Caledonia and New Zealand have
always been malaria free, like Oceania, probably because malaria vectors
have never spread there. Malaria has been eliminated from Australia
except for the Torres Strait Islands near Papua New Guinea.
Malaria in New Guinea differs from typical Afrotropic malaria by
generally somewhat lower transmission intensities and the importance
of P. vivax along with P. falciparum. The vector system is quite different
and exceptionally complex. There are three main vectors, all belonging to
the An. punctulatus complex: An. farauti, in itself a species complex, is
responsible for coastal malaria. It is sometimes associated with brackish
water, sometimes with swamps further inland; in fact, some subspecies of
An. farauti and newly recognized species resembling it are important in
highland areas. An. koliensis is highly opportunistic and occupies breed-
ing sites in inland plain areas, which could be considered savannas or
foothills. An. punctulatus is also widespread and considered the main
vector in highlands. Generally, vector density is highly clustered and
correlates with proximity to rivers or swamps and man-made environ-
mental disturbances (Hii et al., 1997). However, the ecology and distribu-
tions of the sibling species are not well understood, and vector systems
may vary considerably from one village to the next and even within
villages (Muller et al., 2003).
Malaria in coastal plains is hyper- to holoendemic with the main

burden concentrated in young children. Transmission becomes unstable
at altitudes of 1300–1600m and ceases above 1700–1800m. Like in Ethio-
pia and Madagascar, a large proportion of the population is concentrated
above the usual limit of transmission of malaria. At altitudes from 200 to
1200m, the population density is lower than in the coastal plain, and this
has been ascribed to the high malaria risk affecting all age groups (Muller
et al., 2003). The emergence of malaria in economic development projects,
mainly mines and plantations, has been described repeatedly in Papua
New Guinea (Pluess et al., 2009; Radford et al., 1976). Usually, these
situations are characterized by population movements, where immune
parasite carriers encounter non-immunes from non-endemic areas com-
bined with the creation of man-made breeding sites.
From New Guinea to Solomon Islands and onwards to Vanuatu,
ecological diversity, vector diversity and transmission intensity decrease.
Most of the malaria in the eastern parts of this region can be described as
coastal, and there are some highly circumscribed examples of environ-
mental management as a supplementary control measure (Schapira,
2002). The particular opportunities and challenges related to malaria
control in estuaries, particularly stream rectification, have been discussed
by Ford (1949).
3.3.2.3. Indo-malay region

3.3.2.3.1. General This realm stretches from Pakistan to the Philippines
and from the Himalayas to Java (Rao, 1984). With subregions separated by
seas and mountain chains and precipitation levels varying from 0 to over
3000mm per year, it harbours immense biodiversity (Trung et al., 2004).
There is a gradient of increasing rainfall from west to east with major
implications for ecosystems, vegetation and malaria vector bionomics.
In most of Pakistan and peninsular India, the main vector is
An. culicifacies. It is mainly found in agricultural areas, stagnant or flowing
water including rice fields, as well as tanks and ponds. In urban areas, it
may be sympatric with An. stephensi. In Sri Lanka, it is more specialized,
preferring ponds along rivers, causing epidemics mainly in the dry sea-
son. It has been considered a zoophilic and inefficient vector, but newer
investigations indicate that one subspecies, E, is anthropophilic and
potent. Subspecies A and C are also vectors, while B is practically refrac-
tory to malaria infection (Barik et al., 2009). A number of species such as
An. aconitus, An. annularis, An. maculatus, An. sinensis and An. superpictus
are primary or secondary vectors in rural areas, often breeding in rice
fields or streams, but rarely pose a major challenge to control efforts
(Beales, 1984).
An. fluviatilis is the main vector in monsoon forest and hills in eastern
India and in the Western Ghats. Its subspecies S is an anthropophilic,
efficient vector, which is endophagic, but not always endophilic (Rao,

1984), while subspecies T and U are weak, zoophilic vectors. The related
An. minimus (Chen et al., 2006) is an important vector in the Indochinese
peninsula, southern China, Bangladesh, Nepal and Northeast India,
where it is abundant in hilly as well as forested areas, being exophilic to
a variable extent. It is sometimes highly zoophilic (especially subspecies
C) and then probably unimportant as a vector (Van Bortel et al., 2004). The
main vector in forested areas in the Philippines and parts of Indonesia,
An. flavirostris, is closely related to An. minimus.
The An. leucosphyrus complex includes several species, which are
potent, anthropophilic and exophilic vectors, found in the rainforests of
Indonesia and Malaysia. The closely related, even more efficient as well as
highly exophilic An. dirus species complex is found in rain forests in the
Indochinese peninsula, Northeast India, eastern Bangladesh, Yunnan
Province in China, but not peninsular India (except subspecies E, which
does not transmit human malaria) (Kalra, 1991; Rao, 1984). It breeds in
shaded stagnant water collections with density increasing after rains.
The An. sundaicus species complex is found in coastal areas from
eastern India over the Indochinese peninsula to Indonesia. The vectorial
status varies from place to place, but the efficiency is never high (Manguin
et al., 2008).
3.3.2.3.2. Plains with traditional agriculture except irrigated rice In rural

areas in India and Pakistan, malaria was historically endemic, but focal
and unstable with wide exacerbations related mainly to climate and
population movements. Nowadays, the recorded malaria incidence in
non-forested rural areas is generally below 2 per 1000 per year. In these
areas, IRS is now mainly used reactively, while various methods for larval
control are used with the intention of minimizing receptivity (Ghosh
et al., 2005; Sharma 1999). The transmission that occurs is often related
to migrant farm labourers from forested areas in eastern India.
In Sri Lanka, rural malaria is related to An. culicifacies breeding in pools
along streams. A recent study indicated that an intermittent flush system
might deal cost-effectively with the vector breeding, though it would be
necessary to be cautious not to create breeding sites for An. varuna, which
prefers flowing water (Konradsen et al., 1998). However, An. culicifacies is
the only widespread vector in Sri Lanka, which, for the second time in its
history, is approaching malaria elimination (WHO, 2008).
In Indonesia, a study in a rural area of Java found a distinct association
between certain land-use classes and the presence of malaria vectors, as
follows: Rice paddy was associated with An. aconitus and An. subpictus,
plantation near human settlement with An. maculatus, bush/shrub with
An. aconitus, An. maculatus and An. sundaicus and bare land and water
body land use on the coast with An. sundaicus (Stoops et al., 2008). All
these vectors are inefficient in Indonesia and nowadays rarely associated
with any transmission. Likewise in the rest of Southeast Asia, malaria is
nowadays rare in undisturbed, socially stable plain areas.
3.3.2.3.3. Forest, forest fringe, deforestation, foothills Tropical rain forest

is mainly found in the Indochinese peninsula including northeast India,
Indonesia, Philippines, southern China and Western Ghats, while mon-
soon forest with wet and dry seasons of almost equal duration predomi-
nates in other parts of India, especially eastern peninsular India.
It has been estimated that out of the national total, the following
percentages of recorded malaria cases were forest related in 1989: Bangla-
desh 87%, India 31%, Indonesia 44%, Nepal 49% and Thailand 42%
(Sharma et al., 1991). While western India has important malaria burdens
in certain rural and especially urban areas (see below), malaria in eastern
and Northeast India and Bangladesh is largely dependent on the presence
of forest and hill (stream-breeding) vectors. The proportion of the malaria
burden including malaria mortality, which is forest related in India, is
likely to have risen considerably since 1989 (Fig. 3.2). Throughout South
and Southeast Asia, forest malaria is determined by the convergence of
several or all of the following factors: High vectorial capacity with vectors
often exhibiting exophily, exophagy and early biting; economic rewards
of forest activities (logging, fuel wood, gem mining), sometimes at night
(smuggling, frog hunting); transmigration, as in eastern Indonesia, where
large populations with no previous malaria exposure are settled near
forests; low population density associated with poor road access and
rudimentary health services. Often the malaria problem is most visible
among migrants and to some extent in forest fringe villages, while it may
be neglected and more severe in ethnic minority groups in villages sur-
rounded by forest (Erhart et al., 2004; Harijani and Arbani, 1991;
Kondrashin et al., 1991; Shrestha et al., 1991). Depending on several
factors, especially patterns of mobility and control measures, malaria
may be transmitted in the forest and in fringe villages (Trung et al.,
2004) or only inside forests (Sanh et al., 2008). The contrast between forest
villages and plain areas in eastern peninsular India was measured in
Sundergarh district, Orissa, where annual malaria incidence in forest
villages was 347.9 per 1000 with mainly children affected against 61.0 in
plain villages with all age groups equally affected (Sharma et al., 2004).
The following subtypes of forest-related (tribal) malaria have been
identified in India:
i. Hilly rain forest and tropical rainforest of North-Eastern States,
where An. dirus is the main vector.
ii. Hilly deforested cultivated areas in the Northeast, where An. mini-
mus and An. dirus supplement each other so that the transmission
may be more intense and prolonged than inside the forest.
iii. Undulating deforested areas with rice cultivation in the Northeast
with lower levels of transmission by An. minimus, An. fluviatilis and
An. nivipes.
iv. Deciduous forest in eastern peninsular India. An. fluviatilis is the
main vector, and the seasonal transmission can be controlled if IRS
can be implemented. In the rain forests of the western sub-Himala-
yan Region in Nepal, the short transmission season makes control
easier.
v. Deforested areas with An. culicifacies and An. fluviatilis. Transmission
is more prolonged and control more difficult, as An. culicifacies is
often insecticide resistant and bites early (Sharma et al., 1996).
Figure 3.2 compares the spatial distribution of malaria and forest cover
in India from 2001–2007. While the correlation is obvious, the exceptions
to the rule are of particular interest. The severe malaria problem in
Rajasthan and to some extent Gujarat in the beginning of the decade in
desert fringe areas is now under a degree of control. In the forest belt
in the Himalayas, the short-season transmission seems to have been con-
trolled. In the easternmost parts of Northeast India, some forested areas are
likewise not highly endemic, because of high altitude, but in others, the
health services are constrained by terrain and unrest so that malaria is more
underreported than elsewhere. That explanation would not be valid for the
forests in Western Ghats in Karnataka and the Tamil Nadu states, which
have relatively strong health systems. Possibly, the health services in those
states have been able to deal effectively with the malaria problem except for
small residual foci; this would give cause for optimism for forest malaria in
eastern India, where the vectors are the same.
In Thailand, Myanmar, eastern Bangladesh, western Cambodia and
southern Laos, more than in India and Vietnam, there is usually a close
association between forests and the An. dirus complex. This vector may
also be present in fruit orchards, but at lower density than in forests
(Obsomer et al., 2007; Oo et al., 2003; Rosenberg and Maheswary, 1982).
While GIS has been used as in Fig. 3.2 to illustrate the overlap between
malaria, forests and ethnic minority groups (Kidson et al., 1999; Mouchet
et al., 2004a), there have been few rigorous spatial studies of malaria and
environment in Southeast Asia. A national malaria survey in Cambodia in
2007 was restricted to populations living in forests and within 5km from
the forest border. Distance to forest as identified on land-use maps
was highly correlated with malaria prevalence, with very low levels of
infection in populations living more than 2km from the forest border.
A similar pattern was found using MODerate-resolution Imaging
Spectroradiometer (MODIS) vegetation index data, especially Enhanced

Vegetation Index (EVI) and Normalized Difference Vegetation Index
(NDVI) to identify forest-covered areas (Cambodia Malaria Survey,
2007). It was found that remote-sensed data may be more useful than
landcover maps, as they are continuously updated, have temporal and
spatial resolution well suited to national level analysis and are free (J. Cox,
personal communication). Also in Cambodia, regression analysis identi-
fied adults and males involved in forest activities as high risk groups,
with additional risks for children in forest-fringe villages. Villages dis-
playing the highest malaria rates were clustered along roads or tracks
penetrating into recently colonized forested areas (Incardona et al., 2007).
Similarly, in Bangladesh in the Chittagong Hill Tract, houses located less
than 3km from forest had higher malaria risk, while the malaria risk was
inversely related to distance to water bodies (Haque et al., 2009).
In Vietnam, an analysis of district level data (which is far from ideal for
such ecological studies, as districts in that country have populations of
about 100,000 and may comprise a variety of landscapes) found signifi-
cant association between malaria, poverty and percent forest cover,
though with substantial residual spatial heterogeneity (Manh et al., 2010).
Other studies have explored the determinants of malaria in or near
forested areas in greater depth and less quantitative precision. In northern
Thailand, land-poor families practice swidden farming inside forests,
where they are infected by An. dirus, and carry parasites to their villages
in the fringe for transmission by An. minimus. Lack of roads limits access
to markets, and rice fields in these upland areas have low productivity, so
people diversify agriculture, increasing their exposure in forests. The
illegal character of some forest activities creates a further obstacle to
access to the health services (Singhanetra-Renard, 1986).
There are very few reports of larval control in areas of forest malaria
(Singh et al., 1989). An elaborate scheme was successfully applied for
protecting a large contingent of soldiers in northeast India during
World War II (Afridi, 1962). A more recent study was done under more
normal conditions in an area with An. fluviatilis and An. culicifacies, and
the environmental control measures were probably effective against the
latter (Singh et al., 1989). It is often stated that IRS is ineffective in forest
malaria because of the exophily, but there are experiences indicating
some effect (Institute for Malariology, Parasitology and Entomology for
Central Vietnam, Qui Nhon, Vietnam, unpublished data), which is not
surprising, since exophily usually does not mean that all mosquitoes
always rest outside houses, and some people living in forests have houses
with sprayable walls. When ITNs were introduced, there were high hopes
that they would solve the problem of forest malaria. However, the forest
malaria problem cannot be reduced to entomology, and even in the best of
circumstances, their effect would be constrained by exophagy and early
biting. A cluster-randomized trial of conventional ITNs in Cambodian

forest villages suggested a reduction of around 30% of malaria incidence
and prevalence (Sochantha et al., 2006), while an entomological trial of
long-lasting insecticidal hammock nets indicated good protection against
the bites of An. minimus, but much less against An. dirus (Sochantha et al.,
2010). In Vietnam, a community-randomized trial in forest and forest
fringe villages found that incidence in villages provided with long-lasting
insecticidal hammock nets was reduced twice as much as in villages
without this intervention (Thang et al., 2009).
3.3.2.3.4. Deforestation Deforestation in South and Southeast Asia seems

to lead to lower malaria risk in most cases. In Orissa, in eastern peninsular
India, the highly anthropophilic and efficient vector, An. fluviatilis S, was
very rare in deforested riverine villages, but common in forested hilly
villages, with higher malaria burden. It was not clear, however, whether
the more hot and dry deforested riverine area would have been more
malarious if forest cover had been maintained (Nanda et al., 2000).
3.3.2.3.5. Foothills In Assam, in Northeast India, in hilly deforested areas,

An. minimus, breeding in perennial seepage streams, may still be an
important vector, responsible for indoor transmission in these areas and
in tea gardens (Dev et al., 2004). A recent study from Quang Tri Province
in Vietnam, where the physiography is more foothill than forest, pre-
sented the health system problems impeding control in a remote thinly
populated ethnic minority area (Morrow et al., 2009). Such findings sug-
gest that the malaria ecotype previously classified as Indochinese hills
malaria (Macdonald, 1956) should be re-instated.
3.3.2.3.6. Highland fringe In much of Southeast Asia, the highest malaria

risk is found at altitudes between 300 and 800m a.s.l., where vectors
belonging to the An. fluviatilis-minimus and/or dirus-leucosphyrus groups
abound depending on the character of the forest. At higher altitudes, the
environment is usually less hospitable with very low population density
so that relatively few people are exposed to epidemic risk; however,
certain areas in Laos and northern Vietnam above 1500m are settled by
the Hmong ethnic group, relatively recent immigrants from China, who
traditionally fear disease at lower altitudes (Lewis, 1951). In the recent
past, forced resettlement of these in endemic foothill areas has led to
malaria epidemics (S. Hoyer, personal communication).
3.3.2.3.7. Desert fringe The north-west of India, especially Rajasthan

State, and parts of Pakistan are semi-desert. These areas have been
known for many years for serious epidemics related to unusually heavy
rainfall and to development projects. A study in 2006 found that not only
was importation of cases the most important determinant of malaria but

also excessive rainfall and low cattle-to-human ratio ( Joshi et al., 2006).
In some areas, An. stephensi is the most important vector, resting mainly in
storage tanks with the implication that IRS may not be ideal, larval control
becoming more important (Sharma et al., 1996). Due to the high summer
temperatures, it may be very difficult to motivate the population to use
mosquito nets (R.K. Das Gupta, personal communication).
3.3.2.3.8. Coast Coastal malaria transmitted by vectors that tolerate

brackish water has been important focally in Indonesia, Malaysia,
Philippines and Viet Nam, and to a lesser extent, Cambodia, Thailand
and India (Poolsuwan, 1995). The main vectors belong to the An. sundaicus
complex including An. sundaicus s.s., An. epiroticus in Vietnam and An.
litoralis in southern Philippines and Sabah, Malaysia. The optimal levels
of salinity corresponding to between 3% and 50% sea water would gener-
ally be found in areas with man-made disturbances of the environment
(Trung et al., 2004).
Coastal malaria in Vietnam is nowadays related to shrimp farming in
areas south of Ho Chi Minh City (Erhart et al., 2004). Over the 10-year
period from 1992 to 2002, there was a dramatic reduction in malaria
transmitted by brackish water breeders in the Vietnamese part of the
Mekong Delta. The reductions could be ascribed to high levels of coverage
with ITNs and widespread availability of treatment with artemisinin
derivatives. Desalination may also have played a role. Between 1992
and 2001, at a cost of 12 billion US dollars, tidal floodgates were installed
on the major rivers and canals and secondary canals were dredged in an
effort to prevent seawater intrusion into the low-lying Ca Mau Peninsula.
The purpose was to improve agricultural productivity (White, 2009); as a
side effect, it may have helped reduce the malaria risk.
Although the threat of coastal malaria was evident in the Andaman
Islands after the tsunami in 2004 (Krishnamoorthy et al., 2005), it has,
generally, lost its former importance in Asia. There may be several expla-
nations for this; coastal vectors are not very efficient and they are largely
endophilic (the vegetation probably does not provide suitable resting
places), so IRS and ITNs should work well; high population density and
economic development allow good access to curative care. A consequence
of the low burden is that environmental management, which celebrated
some of its greatest triumphs in coastal areas in the past (Takken et al.,
1990), may now rarely be cost-effective, except when required for other
purposes by other sectors.
Coastal malaria is still important in the easternmost part of the
Indonesian archipelago, for example, in West Timor, where the vectors
are An. subpictus, and possibly An. barbirostris and An. maculatus (Ndoen
et al., 2010).
3.3.2.3.9. Urban Urban malaria in Asia is practically restricted to the

Indian subcontinent, from Karachi in the west to Kolkata in the east.
It is a consequence of the adaptation of An. stephensi and to some extent
An. culicifacies to breeding in artificial containers. Already by 1903,
malaria was recognized as a serious public health problem in Mumbai;
anti-larval measures were associated with near interruption of transmis-
sion there in the 1940s, but it was in the 1970s that urban malaria emerged
as a major problem in India, the burden often being higher in the cities
than in the surrounding countryside (Rao, 1984). Thus, in the region of
Mewat in Gurgaon district, Haryana state, the burden was highest in the
urban area despite important breeding sites for An. culicifacies in the
surrounding areas classified as irrigation, water catchment, mining and
flood prone (Srivastava et al., 2004).
One of the reasons for the resilience of the problem is that urban
An. stephensi, though mainly endophilic, cannot be controlled by IRS
because of its tendency to rest on the inside walls of wells and containers
and because IRS is not possible in multi-storey buildings with basements,
etc. (Hyma et al., 1983). Control must therefore rely mainly on sanitation
supported by public works and health education, chemical and biological
larviciding and personal protection.
Urban malaria in India is currently considered a serious problem in
131 cities with about 10% of the country’s population. Between 2000 and
2007, the number of reported cases in these cities was reduced from
172,000 to 106,000 and urban malaria now accounts for about 7% of all
recorded cases in the country. None of the affected Indian cities has
become malaria free yet. The challenge is intersectorial and includes
also the control of parasite carriers, who move from poverty-stricken
rural areas to cities in search of employment. It must be addressed
together with other vectorborne diseases and insect nuisance (National
Vectorborne Disease Control Programme, 2009).
There is no urban malaria problem in the Indochinese peninsula (Oo
et al., 2002). The only major environmental difference between Indian and
Southeast Asian cities, is to the writer’s knowledge, the higher air humid-
ity in the latter, but it is not clear whether that can explain the important
difference in anopheline fauna.
3.3.2.3.10. Agricultural development including plantations Two major

types of agricultural development are particularly important: cultivation
of irrigation rice and tree plantations.
The use of river water for irrigation of rice fields goes back at least
3000–5000 years in East, Southeast and South Asia, and should therefore
be considered as traditional agriculture in those regions. In most of
Southeast Asia (Philippines, Vietnam, Cambodia, Laos, Thailand and
Myanmar), malaria is virtually absent in the heavily populated flood
plain areas, where rice is harvested from one to four times a year. Rice
fields in these countries may harbour various anopheline species, often at
high density, but there is hardly any malaria transmission. An. minimus,
an important vector in hilly areas in most of the Indochinese peninsula,
can be found near rice fields in ditches and canals but rarely transmits
malaria in those areas (Meide et al., 2008).
Even close to the forest fringe in Thailand, rice field areas seem almost
free of malaria (Kondrashin et al., 1991). In Bangladesh and West Bengal
in India, land-use changes and increase in population density led to
reduced production of An. philippinensis from ponds, tanks and marsh-
land so that malaria more or less disappeared from the plain areas,
although An. annularis and An. aconitus emerged as rice field breeders,
occasionally causing low-level transmission (Elias, 1996). In Indonesia,
irrigated rice has been associated with malaria transmission, with
An. aconitus as the main vector, for example, in Java and Bali (Harijani
and Arbani, 1991; Konradsen et al., 2004; Worth and Subrahmaniam, 1940),
but this is apparently not a problem at present. In India, An. culicifacies
breeds more in irrigation canals than in the paddy and has, in some cases,
been controlled by flushing (Russell and Knipe, 1942) and intermittent
irrigation. In Central India, An. annularis, breeding in irrigation canals,
may play a role (Singh and Mishra, 2000), and in Sri Lanka the introduction
of irrigation in a dry forest zone led to the emergence of this otherwise
insignificant species as an important vector (Ramasamy et al., 1992).
In most areas of Sri Lanka, rice-field irrigation is no longer an important
source of effective vectors; in contrast, irrigation malaria remains impor-
tant in the arid north-west India (Kondrashin and Kalra, 1989).
It is striking that irrigated rice cultivation is so closely associated with
malaria in the western part of the region and not at all in Southeast Asia.
Some factors in human ecology could play a role, such as the almost
universal use of mosquito nets in Southeast Asia and the traditional
habitation on stilts, but the ethnic Vietnamese (Kinh) build their houses
on the ground. One important factor in arid areas could be the need for
extensive canals, which may be more important as vector breeding sites
than the rice fields per se. Also, anophelines in arid areas are advantaged if
long lived, able to fly far and to aestivate and/or hibernate. The Southeast
Asian counterparts are physiologically adapted to humid conditions hav-
ing wider spiracles. They cannot fly far (Rao, 1984), but they might be
more competitive in the humid ecosystems by investing in large numbers
of offspring to the detriment of longevity, and thereby vectorial capacity
(A. Kiszewski, personal communication).
In hilly areas, rice fields are not always so innocent. Malaria continues
to occur in hilly rice-field areas in Java and West Timor in Indonesia. The
most common vector species in these areas are An. annularis, An. vagus
and An. subpictus, but it is not certain which of them is important in this
environment (Ndoen et al., 2010). In contrast, there is almost no malaria

nowadays in wet-rice areas in hills in Philippines and Vietnam.
A number of studies in areas where malaria is associated with agricul-
tural and other development projects, mainly in western India, indicate
that ‘bio-environmental control’, that is, larval control through the use of
larvivorous fish and/or environmental management, can be very effec-
tive (Dua et al., 1988, 1991, 1997; Ghosh et al., 2005; Konradsen et al., 1998).
However, proof from randomized controlled trials is absent. In fact, that
kind of evaluation is very difficult in such environments, where malaria is
focal with characteristics varying from one site to the next (Sharma and
Sharma, 1989).
3.3.2.3.11. Tea and tree plantations For more than a century, tea gardens
in India have been infamous for malaria, ascribed more to high popula-
tion mobility and poor health care than to high vectorial capacity
(Christophers and Bentley, 1911). In the Indochinese peninsula, rubber,
tree and fruit plantations have often been associated with breeding of
efficient vectors. In Sarawak in Malaysian Borneo, deforestation and
development of an oil palm plantation were associated with a change in
fauna and a major reduction of malaria transmission, but then this study
was not carried through to the full maturation of the oil palms (Chang
et al., 1997).
Thus, tree plantations offer opportunities for breeding of the notorious
Asian forest vectors depending on the extent to which they imitate their
natural environment. Population mobility becomes a major determinant
of the malaria burden. Good health services, which would be expected at
plantations, can mitigate the problem, but the ample availability of cheap
labour is a constraint. One of the writers (AS) was told in a Cambodian
rubber plantation in 1995 that workers often avoided approaching the free
health service for fear of being recorded as sick and losing income. They
preferred to avail themselves of medicine from private pharmacies and
toil on with a fever.
3.3.2.3.12. War and socio-political disturbances Several articles were writ-

ten a few decades ago about the malaria situations among refugees in the
Indochinese peninsula (Baker et al., 1987; Meek, 1988; Meek et al., 1986),
and presently, the worst malaria situation in that subregion is in
Myanmar, which is still affected by chronic conflict in peripheral forested
areas (WHO, 2009). In the past, the arid North-West Frontier Province in
Pakistan had the lowest malaria incidence in the country, but in 2001,
when the area had been affected by warfare and its social and environ-
mental effects for two decades, the highest (Kazmi and Pandit, 2001).
3.3.2.3.13. Stratification in India and Vietnam India, Thailand and Vietnam

are among the countries with the longest continued experience in stratifi-
cation as a tool for national level planning and replanning of control. For
reasons of space, only two of these schemes are summarized here.
A description of the Thai scheme, which has similarities with both of
them, is easily accessible (Malaria Control Programme in Thailand.
Ministry of Public Health of Thailand http://unpan1.un.org/intradoc/
groups/public/documents/APCITY/UNPAN009706.pdf).
Since 1935, malaria researchers in Vietnam have proposed five strati-
fication schemes of the country referring to altitude, forest cover, migra-
tions and influence of brackish water. These schemes have illustrated
malaria and its determinants, but the data to define the exact geographic
confines and the populations of those strata were not available and
there were no major control implications (National Malaria Control
Programme, 2004; Phan, 1998).
In 2003, the national malaria control programme established a classifi-
cation as follows:
1. Without malaria transmission.
2. Risk of malaria resurgence: Former endemic area without local trans-
mission during past 5 years.
3. Low malaria endemic: Malaria morbidity rate 1–5 per 1000 persons
per year.
4. Moderate malaria endemic area: Malaria morbidity rate 5–10 per 1000
persons per year, P. falciparum proportion <70%.
5. High malaria endemic area: Malaria morbidity >10 per 1000 persons
per year and P. falciparum proportion >70%.
Morbidity rates include confirmed and unconfirmed malaria, where
the proportion of confirmed has been increasing in recent years. Each of
these classes is further characterized by landscape, altitude, population
movement and vector species, and they have clear implications for
interventions and surveillance. In category 3, for example, there is no
vector control except for promotion of mosquito nets, which are to be
insecticide treated, if and only if the area borders an area of higher risk.
With morbidity data, the 10 529 communes of the country were classi-
fied by this scheme in 2003 allowing a quantification of target popula-
tions for interventions, although it was recognized that the size of
migrant populations at risk remained uncertain. The framework is
meant to be flexible in such a way that the quantitative epidemiological
criteria for decision-making are modulated locally by the mentioned
contextual factors.
In India, the control experiences of the early twentieth century were
formulated as typologies during the years of reorientation from eradica-
tion to control: urban, irrigation, rural, tea gardens, railway and coal fields
(Pattanayak et al., 1994), later distilled as: tribal, rural, urban, industrial
and border. Responding to an external evaluation in 1985, it was
attempted to use 14 variables to divide the country in to seven strata,
but it was impossible to collect the data at a fine enough scale, and this
approach was abandoned (Sharma et al., 1996). In the 1990s, as the
programme experienced a number of setbacks, it was proposed to go
back to a simple typology of epidemic-prone, tribal, project and urban
and within each of these define epidemiological criteria (annual parasite
incidence (API), slide positivity rate, slide P. falciparum rate, epidemics),
for control measures, and provide some indication of subtypes and their
control implications.
For current national- and state-level malaria control planning in India,
stratification is in practice based on API, supplemented by some other
criteria (slide positivity rate replacing API if the blood examination rate in
a given area is low, high P. falciparum proportion, worsening malaria
situation and extensive population movement). As a general rule, areas
with API above 2 per 1000 are classified as high risk and therefore eligible
for full coverage with IRS or (recently) ITNs (Directorate of National
Vector Borne Disease Control Programme, 2009).
3.3.2.4. Neotropic and Nearctic regions

3.3.2.4.1. General Malaria is no longer endemic in the Nearctic except for
some areas in northern Mexico, where vectors and ecology are no differ-
ent from further south within the Neotropic realm; there is therefore no
separate description of the latter.
The most important vectors and their characteristics can be summar-
ized as follows: An. darlingi is efficient, anthropophilic, endophagic and
widespread in tropical lowland areas in South America and parts of
continental Mesoamerica. It breeds mainly near rivers and is sometimes
described as preferring shaded conditions, sometimes as sun loving.
In open plain areas, it is mainly endophilic, but in forest areas, highly
exophilic. Recently, An. marajoara has emerged as an important lowland
vector associated with wetlands, secondary forests and human interven-
tion (Sinka et al., 2010b). An. nuneztovari is also a widespread lowland
vector, which is less closely associated with forests. It can be anthropo-
philic or zoophilic and its importance as a vector is variable. An. albimanus
and An. aquasalis are exophilic, zoophilic and relatively ineffecient vec-
tors, which may attain very high densities in coastal plains, the former
near the Pacific, the latter mainly by the Atlantic and the Caribbean.
An. pseudopunctipennis prefers stagnant, sun-exposed water, especially
ponds along rivers with filamentous algae and occurs from low to very
high altitude. The subgenus Kerteszia includes a number of vectors asso-
ciated with bromeliads (see forest malaria below) (Mouchet et al., 2004b;
Sinka et al., 2010b).
Rubio-Palis and Zimmerman, using mainly South American data,

explored climatic factors, vegetation, elevation and landform in accordance
with Bailey’s ecosystem geography (Bailey, 1996) to describe five malaria
vector ecoregions, where homogeneity of these factors could be related to
vector density and distribution: coastal, piedmont, savanna, interior low-
land forest and high valleys (Rubio-Palis and Zimmerman, 1997). The
following review of Neotropic malaria follows this typology (Fig. 3.3).
No transmission
API <10
API 10-49
API>=50
FIGURE 3.3 Malaria risk as measured by annual parasite index (API) by municı́pio in
Amazonia legal, 2007 (Source: Ministry of Health) http://portal.saude.gov.br/portal/
saude/profissional/area.cfm?id_area¼1526) and vegetation and deforestation in the
Brazilian Amazon, 2002 (Source: Human Pressure on the Brazilian Amazon Forests
March 2006. World Resources Institute/Imazon http://www.globalforestwatch.org/
common/pdf/Human_Pressure_Final_English.pdf).
3.3.2.4.2. Savanna Savanna landscapes with seasonal and variable rainfall

are found at elevations from less than 100 up to 1500m with variable rainfall,
but always a dry season lasting at least 5 months. Malaria is or was localized
to the more humid areas and transmitted by An. darlingi or An. nuneztovari.
It has now largely been eliminated by IRS from this ecoregion.
3.3.2.4.3. Interior Lowland Forest Forest malaria in South America was

aptly described by Giglioli, bringing out the convergence of determinants,
which makes this malaria system so resilient:
‘‘. . .The reasons for this failure of our eradication campaign in the
remote interior. . .can be easily summarized. 1. The difficulty of
locating all camps and habitations in the forest. 2. The habitual
and frequent displacements of the Indian population from their
permanent settlements to temporary shelters and camps on their
farms in the forest. 3. The exophily of An. darlingi and its persistence
in semi-inhabited forest. 4. The lack of adequate sprayable surfaces
in rudimentary house structures used on the farms and in the
bush camps. 5. The difficulty of access, particularly in the wet
season, when flooding interrupts lines of communication, and in
the dry season, when the upper reaches of many rivers become
unnavigable. . .’’ (Giglioli, 1963).
An. darlingi is the main vector in nearly all forest areas in Central and
South America. It tends to be exophilic in this environment, especially
where human dwellings do not have complete walls, as in traditional
Amerindian villages (Girod et al., 2008). In Venezuela, the main problem
in some areas was exophily and to some extent exophagy of An. nunezto-
vari (Rubio-Palis and Curtis, 1992). In contrast, in Northeast Brazil, malaria
in deforested areas was mainly transmitted by An.marajoara, a vector
previously associated mainly with marshes and assumed to play a mini-
mal role (Conn et al., 2002). It is estimated that at least 70% of P. falciparum
in the Americas now occurs in the Amazon and Orinoco basins, where it is
mainly transmitted by An. darlingi (J. Najera, personal communication).
A multifactorial, spatial analysis with remote sensing data found that, in
the short term, deforestation in the Amazon forest fringe leads to increased
breeding of An. darlingi (de Castro et al., 2006). After a few years of coloni-
zation, agriculture reduces breeding sites, housing improves, and the
residual malaria problem is related to incursions into the forests (Takken
et al., 2005). Similar conclusions were reached by several other groups,
applying different methods in this region (Rubio-Palis and Zimmerman,
1997; Vittor et al., 2009). In the Peruvian Amazon, highly focal malaria
transmission in forest communities was demonstrated; despite the high
levels of precipitation, proximity to rivers was an important determinant
(Bautista et al., 2006). Ecological classification was carried to a finer scale in
the state of Roraima in northern Brazil. Applying different analytical stra-

tegies to an ecological niche model based on landscape elements, human
occupancy and vector distribution, eight ecoregions of importance for
malaria were identified, including five types of dense rainforest and three
types of savanna. Unfortunately, this work does not examine how the
ecoregions correlate with the spatial distribution of malaria and does not
indicate control implications (Rosa-Freitas et al., 2007).
Forest malaria emerged as a major concern in Brazil from the 1970s
with rapidly increasing deforestation, agricultural colonization and
mining in the Amazon (Wallace and Webb, 2007) and especially, when
drug resistance spread to the extent that miners could no longer be
protected by chemoprophylaxis. Over time, malaria spread to the Amer-
indians living inside the forests (Mouchet et al., 2004b). Malaria in Brazil
is now only endemic in Amazonia legal, that is, those states with 12% of the
nation’s population that include areas of the geographic Amazon region.
In 2007, 457,831 malaria cases were recorded in the country, of which only
172 (0.04%) were outside that region. However, 24% of cases were found
in urban areas located near forest or with encroaching forest (Ministério
da Saúde, 2008). Comparing the distribution of malaria with the distribu-
tion of forest cover, it is easy to see the almost perfect congruence between
malaria and forest/forest fringe areas including those, which have
been deforested recently (Fig. 3.3). On the background of a paucity of
research evidence of control methods—contrasting with the rich literature
on spatial description—it is encouraging that a controlled trial among
forest dwellers in the Amazon Region found that insecticide treatment of
hammock nets prevents 56% of new malaria cases, and non-treated nets
presumably have no effects (Magris et al., 2007).
In the Atlantic rainforests of South America and the Caribbean, espe-
cially Brazil, the main vectors, An. (Kerteszia) bellator and An. (Kerteszia)
cruzii, breed in the leaf axils of epiphytic bromeliads in the tree canopy.
They have been difficult to control because of exophily, early biting and
the peculiar breeding site (Gadelha, 1994). However, they are short lived,
and more than 90% of the Atlantic rain forest has disappeared, so brome-
liad malaria is now a sporadic phenomenon, but one that could increase
as a result of reforestation. In the past, bromeliad malaria affected urban
areas and cacao plantations in Trinidad (Marrelli et al., 2007) and coffee
plantations in Brazil (Mouchet et al., 2004b).
Another variant of forest malaria is found in the Lacandon forest in
Belize and south-eastern Mexico, where An. vestitipennis seems to be the
main vector (Arredondo-Jimenez et al., 1998).
3.3.2.4.4. Piedmont This ecoregion comprises foothills between 200 and

1500m altitude in Mexico and Central America and on the west and east
sides of the Andes. The main vectors are the widespread An. nuneztovari
and An. pseudopunctipennis, with An. albimanus being found especially in

the western Andes and An. darlingi in humid lowland areas. P. vivax is
predominant, but P. falciparum closely associated with An. darlingi
occurs focally. The transmission is only of low to moderate intensity.
In western Venezuela, an exophilic An. nuneztovari continues to
challenge control and elimination in the Andean foothills (Mouchet
et al., 2004b).
In Oaxaca, the state in Mexico, which currently has the highest malaria
burden after Chiapas, an ecological study found that transmission was
associated with elevation between 200 and 500m, temporary streams and
larger population size of rural localities. Land use seemed to be of little
importance (Hernandez-Avila et al., 2006); An. darlingi does not inhabit
this part of Mexico, where the remaining forest is located at high altitudes.
The malaria in the state is thus partially coastal, but mainly of piedmont
type, with An. pseudopunctipennis as the main vector; removal of algae
from breeding sites has been identified as one element in the control
strategy (Case Study Mexico, 2009)
3.3.2.4.5. High valley In Andean valleys above 1000m, P. vivax may be

transmitted seasonally by An. pseudopunctipennis. Epidemic P. vivax and
sometimes P. malariae persist in the sparsely populated, underserved
villages in highland areas in Bolivia, Peru and Colombia. In contrast,
malaria has largely been sprayed away in Venezuelan highlands
(Giglioli, 1963). This epidemic-prone ecoregion clearly corresponds to
highland fringe malaria in other parts of the world.
3.3.2.4.6. Coastal The coastal zone as identified by Rubio-Palis and

Zimmerman (1997) includes not only areas under salt water influence
but also plains up to 550m altitude or about 100km from the ocean. By this
definition, the demarcation from piedmont becomes somewhat arbitrary
and related to the landform. In the Caribbean, malaria was always found
only in coastal areas, transmitted by An. albimanus, which is today the
main vector in the island of Hispaniola as well as in the lowlands of
central America, both Atlantic and Pacific. In Pacific coastal areas of
South America, An. aquasalis is the main vector, but generally, malaria is
now a minor problem there. The Pacific coastal environment in Chiapas,
Mexico, where An. albimanus is the main vector, was studied by remote
sensing; it was found that certain land cover types, salt marsh, mudflat,
savanna/woodland and open water, were more productive of this vector
than others (Rojas et al., 1992). It was suggested that this would help
target malaria control efforts, but it was not clear, if this approach
would improve on targeting by conventional epidemiological methods.
In the South American coastal plains facing the Caribbean, An. darlingi,
breeding in irrigation canals, rice fields and cane fields, was an important
endophilic vector. IRS interrupted transmission in these coastal areas, but

in some places, the disease returned in the 1980s because of neglect of
vector control (Pope et al., 1994).
3.3.2.4.7. Urban Urban malaria in South America is, generally, an exten-

sion of surrounding rural malaria. As noted above, malaria is now a
serious problem in cities in the Amazon region of Brazil, where it should
perhaps be considered ‘urban’ from an administrative viewpoint and
‘forest’ entomologically, as it is transmitted by An. darlingi, breeding in
man-made sun-exposed water bodies, but not in artificial containers
(Cabral et al., 2010). Control by IRS or ITN is difficult due to the exophily,
but much could probably be achieved with environmental management
(Gil et al., 2007; Gonçalves and Alecrim, 2004; Olano et al., 1997).
Similarly, certain cities in Colombia have urban malaria transmitted by
An. albimanus with less malaria in the cities than in surrounding
countryside.
3.3.2.4.8. Agricultural development In many areas, agricultural develop-

ment, especially in the savanna environment, has been associated with the
disappearance of malaria. Yet, there are a number of examples of specific
problems arising as a result of agricultural development. Rice-field
malaria was a serious problem in Puerto Rico, in Central America, in
the piedmont of Venezuela and in dry zones of Peru but has largely been
eliminated by IRS (Mouchet et al., 2004b). In Guyana, the elimination of
buffaloes in the Demerara river estuary resulting from mechanization led
to a resurgence of malaria caused by the zoophilic An. aquasalis (Giglioli,
1963). In Central America, resistance of An. albimanus to several classes of
insecticide has been notorious in cotton plantation areas (Georghiou,
1972), but nonetheless, there has been a continued reduction in malaria
over the past 30 years (Pan American Health Organization, 2002). When,
at the start of the twenty-first century, malaria returned to The Dominican
Republic in sugar cane plantations which recruited workers from Haiti,
environmental management helped deal with this problem (WHO, 2008).
3.3.2.4.9. Warfare and social instability The importance of long-term con-

flict is evident especially in Colombia and Peru, where chronic insecurity
in highland areas impede the development of services, which could, in
principle, easily achieve elimination of the unstable malaria transmitted
by the endophilic An. pseudopunctipennis. Conflict may also contribute to
the maintenance of high incidence rates in forested areas of countries like
Honduras and Guatemala (Pan American Health Organization, 2002).
3.3.2.5. Palearctic
3.3.2.5.1. General Malaria in the Palearctic occurs only focally. Like the
Indo-malay, this region encompasses enormous ecological and climatic
variability. Its demarcation from that region is fuzzy, both east and west
of the Himalaya. The malaria situations are best described according to
geographic location.
3.3.2.5.2. Arabian peninsula Malaria in the south-western part of the pen-

insula belongs to the Afrotropic region, as noted above. In the eastern part
of the peninsula, small foci now remain in Oman, where the vectors are
the same as those in the western Indo-malay. The most intense malaria
transmission has been in foothill areas, with An. culicifacies breeding
seasonally in streams (Delfini, 1987).
3.3.2.5.3. Caucasus, Iraq and Turkey Several Caucasian nations experi-

enced a re-emergence of vivax malaria in the 1990s, with An. sacharovi
as the main vector. The valleys and foothills provided a suitable environ-
ment for breeding of this species; in some areas, it was enhanced by dams
and irrigation, and the situation was aggravated by a degradation of
health services following the breakdown of the Soviet Union and popula-
tion movements related to the armed conflict between Armenia and
Azerbaijan (Sergiev et al., 2007b). In Iraq, malaria now remains in Kurdi-
stan, where it is mainly transmitted by An. sacharovi and to some extent
An. superpictus and An. maculipennis. In this area, the ecology could
partially be classified as foothills, partially as irrigation (Mouchet et al.,
2004a). The current malaria focus in eastern Turkey is associated with
irrigated agriculture, which started in the 1970s in the Çukorova plains,
and has led to massive increased breeding of An. sacharovi as well as
attraction of large numbers of migrant workers (Sergiev et al., 2007a).
Social unrest in this area may also have played a role.
3.3.2.5.4. Afghanistan, Central Asia, Iran and Russia The vectors include
the Indo-malay An. culicifacies, An. stephensi and An. fluviatilis, and the
Palearctic An. pulcherrimus, An. hyrcanus and An. superpictus. Despite
considerable progress in the 1960s, malaria was never eliminated in
Afghanistan and serious resurgences occurred following conflicts in the
1980s. In Tajikistan, Uzbekistan and Turkmenistan, malaria had been
practically eliminated but was reignited by movements of infected people
and vectors across the borders from Afghanistan, and the situation fur-
ther worsened when control capacity was affected by the dissolution of
the Soviet Union.
In all these countries, the ecological background situation is valleys
and foothills with traditional agriculture, on which, in many areas,
irrigation is superimposed, greatly increasing vector density. Nomadism

is a factor hampering control efforts, but the main problem has been the
continued violence and instability in Afghanistan (Sergiev et al., 2007b).
The main focus of malaria in Russia in recent years has been in and
around Moscow over the years 2000–2007, where carriers of P. vivax of
diverse origins have encountered high seasonal densities of several Pale-
arctic vectors breeding mainly in water reservoirs (Gordeev et al., 2005).
The focus has now been eliminated as a result of better case management,
increased surveillance and awareness, larviciding and diminishing pop-
ulation movement (Ivanova et al., 2009). Together with a smaller outbreak
in Tashkumyr in Kyrgysztan in 2002 (Usenbaev et al., 2008), this is the first
published example of urban malaria in the Palearctic for several decades.
It is possible that climate change contributed to the outbreak (Mironova
and Ivanova, 2006).
3.3.2.5.5. Central China and the Korean peninsula Vivax malaria continues
to be transmitted in a number of foci in central China and the Korean
peninsula, all of them related to irrigated rice cultivation (Beales, 1984;
Schapira, 2002; Somboon et al., 1994). The main vectors are An. sinensis, a
rice-field breeder with zoophilic and exophilic tendencies, and in hilly
areas of China, the much more efficient, anthropophilic An. lesteri (for-
merly considered as An. anthropophagus) (Qunhua et al., 2004). As one
would expect, various larval control methods may play a role in the
control of the former, while ITN or IRS works well where the latter
dominates (Xu et al., 1998). In contrast to other areas of endemic malaria
in the Palearctic, conflict is not a determinant of malaria in these foci
(which are gradually being eliminated); however, it has been hypothe-
sized that loss of cattle in the 1990s may have increased the vectorial
capacity in North Korea.
3.3.3. Proposed definition, identification and demarcation of

malaria ecotypes and their implications in five
biogeographic regions
Across biogeographic regions, there are striking commonalities between the
characteristics of malaria associated with particular ecological back-
grounds, for example, for forest malaria in the Indo-malay and the Neo-
tropic, and also striking differences, for example, between savanna malaria
in Africa and malaria in plains and valleys with traditional agriculture
(which include savannas) in nearly all other biogeographic regions. Within
biogeographic regions, there are also striking differences between subre-
gions, for example, as regards irrigated rice fields or urban malaria in
different parts of the Indo-malay. In any biogeographic region, major envi-
ronmental differences generally do translate to differences in malaria
epidemiology, but in many cases, the differences are quantitative and

gradual, and they do not always have implications for control. This is
particularly so in the Afrotropic, where the main vectors are ubiquitous
and versatile.
The only global ecological typology attempting to classify all malaria
situations in the world remains the one proposed by WHO (Table 3.1);
when considering the six steady-state types, it has the great advantage of
parsimony. Only two alternative systematically developed typologies have
been published, one for the Neotropic and the other for the Afrotropical.
Both of these overlap with WHO’s, but there are interesting differences.
The approach of Rubio-Palis and Zimmermann (Rubio-Palis and
Zimmerman, 1997), aligning malaria typology with mainstream ecological
stratification in the Neotropic, has the advantage that a malaria situation,
wherever it occurs, can be assigned to a physiographic background. This
may be particularly useful, as malaria has, since 1990, re-emerged in
ecoregions from where it was thought to have been eliminated, especially
in the Palearctic. One ecoregion included in this typology is foothills
(piedmont), which is not specified in the classical typology; in fact, the
findings in the Afrotropical (Madagascar) (Rabarijaona et al., 2009),
Australasian (Muller et al., 2003), Indo-malay (Dev et al., 2004), Neotropic
and Palearctic (Sergiev et al., 2007a,b) regions concur in suggesting that
foothills merit to delimit an ecotype of malaria, where the defining charac-
teristic would be that the terrain often provides niches for vectors which
prefer fast-flowing water, while the temperature is still (almost) as favour-
able to malaria transmission as that in adjoining plains. At its upper
altitude limit, foothills merge into mountain fringes with unstable, temper-
ature-dependent malaria. In many texts, malaria in foothills has implicitly
been clubbed with forest malaria; in fact, although the transition may
be gradual, there are often important differences in predominant vector
species and bionomics, social conditions and amenability to control.
The typology for Africa developed by Mouchet et al. starts with major
faciès épidémiologiques, which are similar to those of Table 3.1, but with
some finer gradations. These gradations have not been shown to be rele-
vant in a programme perspective, and they would not be easily applicable
in a global typology. Yet, this typology has great merit in the separation
between major ecoregions ( faciès) as primary factors and a variety of
secondary factors or processes, which may be natural or anthropic. This
is very much akin to landscape epidemiology and addresses a limitation of
the classical typology, which has only two situations of rapid development
change: agricultural development including irrigation and war and socio-
political disturbances, which seems unnecessarily restrictive. The conver-
gence of physiographic changes and population movement, which often is
associated with malaria risk in agricultural development projects, may also
occur, in relation to, for example, dam building, road- and railway
building, mining, etc. Similarly, the effects of natural disasters may be not
very different from those of wars.
This additional level of analysis can be considered an alternative to
structuring a typology with sub- and sub-subtypes. Such may be justified
at times in national programmes (viz. Indian forest malaria strata). How-
ever, fine subdivisions are difficult to memorize, do not capture juxtapo-
sitions and may encourage control managers to straitjacket all malaria
situations to fit with defined types, instead of locally examining the
implications of interactions between climate, biology, physiography and
human ecology. The weakness of an approach with several levels of
analysis is that the typology abdicates from almost any prescription,
becoming rather a framework. Given that published typologies anyway
tend to avoid prescriptiveness and that decision-making on control must
consider epidemiological data, available resources (in the broadest sense)
and technologies, this is probably rational.
From these observations, it is proposed that a global typology can be
based on a small number of environmental classes, which are represented
in nearly all biogeographic regions and would cover all areas, where
malaria transmission is not interdicted by climate or biogeography:
Savanna, plains and valleys
Forest, forest fringe
Foothill
Mountain fringe and northern and southern fringes
Desert fringe
Coastal
Urban
Despite the profound differences in malaria transmission, African
savanna is grouped together with savannas, plains and valleys outside
Africa for the sake of consistency, and because in any country or region,
savanna, plains and valleys are natural candidates for the role of default
ecotype. Also, there are other ecotypes that differ greatly, even within
biogeographic regions, for example, urban.
In addition, it is proposed that the classical typology is modified
towards a scheme with three levels:
1. Identification of the biogeographical region, or subregion, which
determines, so to say, the menu of vector species from which the
physiography will choose.
2. Classification of the physical environment into one of the above
classes, recognizing that even a small district may have several envi-
ronmental types, where sometimes the juxtaposition of certain envir-
onments may create particular dynamics. Some situations may be
characterized as transitions (ecotones); others rather as mixed
(mosaic), like forests encroaching on Amazonian cities or combined,

as some fringe areas in East Africa combining desert and highland
characteristics.
3. Identification of secondary factors, which can be stationary or
dynamic, is classified as follows:
a. Natural (landform, water bodies, soil characteristics, disasters,
climate change);
b. Anthropic (habitat and habits, population movement, agriculture
(traditional or based on major investments), animal husbandry,
other economic activities, deforestation, modification of water
bodies, transport infrastructure, war and unrest);
c. Health system including malaria control. (This is, of course,
anthropic, but by its intentionality so specific that it appears rea-
sonable to consider it separately).
Thus, a given malaria situation would, for example, be classified as
Afrotropic in a highland fringe environment and with irrigation rice
cultivation as the main anthropic process. Another as localized in the
Indo-malay region, with physiography being a mixture of plains and
monsoon forest with interspersed urban areas and mines attracting
large numbers of migrants, as well as some insurgency causing additional
unpredictable population movement.
The defining characteristics of the seven environmental classes as well
as their demarcations are presented in Table 3.3A, and the major varia-
tions in relation to malaria epidemiology and control in Table 3.3B. The
most useful findings could be summarized as follows.
As has been known for a few decades, forest cover is a very strong
determinant of malaria risk in the Indo-malay and the Neotropic, except
in certain islands, most notably Sri Lanka, where forest vectors have not
arrived, and in areas where the temperatures are too low for malaria
transmission. Forest-related malaria can be delimited. In Cambodia
(Cambodia Malaria Survey, 2007) and Bangladesh (Haque et al., 2009),
malaria risk was greatly increased within 2, respectively, 3km from the
forest border, which can be identified by land-use data or by EVI or
NDVI. Probably, the demarcation is more complex and fuzzy in the
Neotropic, given the propensity of An. darling for deforested areas. It is
important to note that forest-related malaria in those two biogeographic
regions covers a wide spectrum from the viewpoints of vector bionomics,
human ecology and control (Table 3.3B).
In tropical Africa, the reductions in malaria transmission associated
with highland and southern fringes are well known, as are those asso-
ciated with desert fringes. Outside Africa, in non-forested areas, the low
background risk in rural areas may and may not be increased in areas of
agricultural development; the risk is higher, the more arid the area.
TABLE 3.3 Malaria ecotypes as based on physiography, with variations in different biogeographic regions
A. General characteristics of the ecotypes Malaria implications

Physiography Definition and delimitation Demography, human Vector bionomics Morbidity and Control
ecology and health and transmission mortality
system
Savanna, Default: Not belonging to any Population density Vectors usually Concentrated in Anti-adult methods
plains and other ecotype and health service endophilic, but in youngest age constitute primary
valleys coverage variable, certain areas and groups in Africa line of vector
but usually far seasons exophilic. including adverse control
from universal Often zoophilic. If effects on
malaria is pregnancy.
endemic, Elsewhere, pattern
transmission is is variable,
usually seasonal, sometimes
lasting >5 months concentrated in
(Mouchet et al., occupational
1993a) groups
Forest EVI or NDVI consistent with Low population Some, but variable Ranging from Limited effect of anti-
forest. Forest fringe may density; weak degree of exophily African savanna adult methods in
extend up to 2–3km from infrastructure; and exophagy; pattern with most subregions of
forest margin in Indo-malay, population groups high degree of mainly young Indo-malay and
possibly longer in Neotropic, with different anthropophily; children affected Neotropical; anti-
especially if deforestation habitats and variable to only certain larval methods
habits; various heliophily. occupational rarely feasible.
kinds of Transmission only groups Curative service
population in tropical areas provision often
movement very difficult and
must be tailored to
local conditions.
(continued)
Foothill An altitude belt, where Variable population The inclination may Transmission Usually, excellent
transmission is not density; service favour vectors that moderate and focal effect of anti-adult
significantly constrained by access often find niches in may have intense methods if
low temperatures, thus constrained by running water. seasonal operational
depending on latitude. 200– terrain. Vectorial capacity variations. constraints can be
1500m a.s.l. in the Andes Agricultural may be high Usually, all age overcome. Larval
(Rubio-Palis and development may focally groups affected, control may be
Zimmerman, 1997), 200–1200 increase often with peak feasible in specific
m in Papua New Guinea population disease incidence circumstances
(Muller et al., 2003), 200–800 movement, in older children
m in Indochinese peninsula increase or (Rabarijaona et al.,
(writer’s (AS) obs.) decrease 2009)
transmission, and
provide control
opportunities
Highland Malaria unstable depends on Population density Vectors usually Highly unstable, Usually good effect
fringe and temperature variations, varies from high to endophilic, often epidemic. All of anti-adult
northern secondarily also rainfall, very low; sometimes age groups are methods, when
and environmental disturbances sometimes zoophilic affected. With high feasible, but
southern and population movement. nomadism and population acceptability of
fringes Lower altitude limit defined transhumance; density, mortality ITN may be
locally, corresponds to upper infrastructure and in epidemics may constrained by low
altitude of foothill, for heath system be enormous insect nuisance
example, 800–1200m a.s.l. in highly variable. and small
most malaria risk areas. Dams and dwellings with
Upper limit depends on irrigation may open fires. Larval
latitude and local factors greatly increase control may be
affecting outdoor and indoor transmission feasible in some
microclimate, 2800m for circumstances as
vivax in Bolivia (Rubio-Palis supplement
and Zimmerman, 1997), 2000
m in Africa near equator
(Mouchet et al., 1993a), 1600
m in Madagascar (Mouchet
et al., 1993b), 1700–1800m in
Papua New Guinea (Muller
et al., 2003), 1500m in
Indochinese peninsula
(writer’s obs.)
Desert fringe As highland fringe, but rainfall Population density Vectors usually Usually, all age Usually, good effect
is main determinant, while in low; often endophilic, often groups are affected of anti-adult
some areas, temperatures nomadism or zoophilic methods;
may be so high as to limit transhumance. feasibility of ITN
transmission. Duration of Health system may be better for
rainy season up to 5 months, often very weak. mobile
transmission season <5 Development populations
months (Mouchet et al., projects are rare. though sometimes
1993a), may overlap with Irrigation may constrained by
highland fringe, as in East greatly increase high temperatures.
Africa transmission Larval control may
be feasible, in
specific
circumstances as
supplement
Coastal For a clear distinction, coastal Population density Vectors associated Usually, all age Environmental
malaria should be defined as usually high with with brackish groups are management is
malaria in areas, where the relatively good water with varying affected, possible in some
vectors need salinity. infrastructure and degrees of salinity, sometimes circumstances, but
However, coastal vectors in health services depending on occupational nowadays plays a
the Neotropic are catholic in species; anthropic exposure limited role as part
(continued)
this respect, so coastal disturbances often of intentional

malaria there has been essential for malaria control, as
defined as malaria associated breeding sites anti-adult methods
with plains stretching far (Poolsuwan, 1995); are usually
inland (Rubio-Palis and vectors usually effective
Zimmerman, 1997) endophilic
transmission low
to moderate
Urban In most cases, urban malaria is Population density Transmission rarely Usually, all age Anti-adult methods,
caused by the encroachment high, health intense but often groups are affected especially IRS,
of rural/forest landscapes services accessible, highly variable may be difficult to
into urban areas. Urban but dominated by over short implement in truly
malaria sensu stricto is caused private providers distances urbanized areas.
by vectors, which prefer Anti-larval
man-made containers. methods not only
Delimitation: GRUMP urban feasible in
extent in Afrotropic principle but also
constrained by
operational
problems. House
screening, cross-
disease
collaboration and
intersectoral work
merit more
attention
B. Variations of main characteristics by biogeographic region
Afrotropic Australasian Indo-malay Neotropic Palearctic
Savanna, Intense transmission with Intermediate Malaria is unusual, highly focal and unstable and largely limited to
plains and seasonal variation leading to between areas with anthropic environmental modifications. Irrigated rice
valleys stable malaria with burden Afrotropic and the fields are associated with increased malaria risk in the Neotropic
with concentrated in young other regions. and Palearctic regions and in the western, relatively arid part of
traditional children, pregnant women Malaria is the Indo-malay region, but usually not in Southeast Asia.
agriculture and travellers. Control may relatively stable, Man-made rural malaria outside Africa has been seen as one of the
be difficult in West African but man-made main opportunities for environmental management or other
arid savanna, where intense environmental forms of larval control. There are examples of this, mainly in older
transmission is compounded disturbances literature; nowadays, larvivorous fish are used widely in India
by exophily (Molineaux and greatly increase and some other countries. In most of these settings, the vectors are
Gramiccia, 1980). the risk very amenable to adult control. Planning and implementing IRS
Environmental change rarely or ITN are so easy that little attention is paid to targeting or
leads to important alteration evaluation of larval control
in malaria epidemiology, but
population movements may
be important. Recent studies
suggest that both IRS and
chemical larviciding can
have additional effect when
combined with ITN (Fillinger
and Lindsay, 2006;
Kleinschmidt et al., 2007)
Forest Forest malaria manifests as a Seems to have same Forest-related malaria has emerged as the Forest malaria plays
variant of savanna malaria, characteristics as dominant residual malaria problem. At no role
often with less intense savanna malaria, least in Indo-malay, there is evidence for
transmission because of but the diversity of delimitation of forest-related malaria. In
lower vector density. In most, vectors and Cambodia (Cambodia Malaria Survey,
but not all cases, African limited 2007) and Bangladesh (Haque et al., 2009),
(continued)
forest vectors are relatively information makes malaria risk was greatly increased within
endophilic and amenable to it difficult to 2, respectively, 3km from the forest
adult control generalize the role border, which can be identified by land-
of forest use data or by EVI or NDVI. It has serious
environment consequences for the directly affected
often underserved populations; it seeds
malaria foci in environments with lower
vectorial capacity and is a source of multi-
drug resistant falciparum malaria for the
world. The effects of deforestation depend
on the type of environmental change and
the local forest vectors’ heliophily.
Variations especially within Indo-malay
region are important: The highly exophilic
An.dirus may be confined to continental
Southeast Asia; related forest vectors in
Indonesia and Malaysia are less exophilic
and those in peninsular India even less.
There are no true forest vectors in Sri
Lanka and those in the Philippines (except
Palawan) are inefficient and relatively
easy to control. In the Neotropic, the risk is
generally greater in the deforested fringe
than inside the forest. South American
forest malaria has a tendency to invade
cities, while southeast Asian forest vectors
are likely to colonize plantations. Control
must be multi-pronged, deal with
population movements and overcome
geographic, social and societal constraints
Foothill Foothill malaria usually has Mining and Foothill areas often have specific vectors with The residual or re-
similar characteristics as infrastructure breeding sites related to streams; the emerging malaria
savanna malaria, but recent development in vectorial capacity is often higher than in problems often
studies in Madagascar New Guinea have adjacent plain areas, and the malaria, result from
suggest that the disease greatly increased though unstable, is typically not epidemic convergence of
burden extending to all age malaria problems like in the highland fringe, while population
groups merit more attention transmission is less intense and more movement,
controllable than in forest areas. Habit of agricultural
using mosquito nets is becoming development and
increasingly generalized in Southeast degradation of
Asia. Generally, foothill vectors are less services on a
exophilic than those in deep forest; as foothill landscape
found in Mexico, there may be background
opportunities for larval control.
Cultivation of foothills, even rice-field
terraces, usually changes the landscape in
a way that reduces malaria transmission
or risk
Highland Epidemic malaria in highlands related to Occurs sporadically; the human populations Hardly of any
fringe and meteorological variations is a highly significant affected are small. Evidence is scanty in importance, except
southern problem. Because of the limited mosquito nuisance, South and Southeast Asia, better in South as northern fringe
and IRS may be more effective than ITN. Recently, larval America in Central Asia
northern control has shown potential in Kenyan highlands
climate (Fillinger et al., 2009)
fringes
Desert fringe Important in western Africa, the Does not occur Important in Does not occur Of minimal
Horn and south-western restricted importance.
Africa. Much more neglected populations Resurgences in
than highland malaria. Both inhabiting large past 20 years have
major anti-adult methods areas in Pakistan generally not
(continued)
should be highly effective, and North-west occurred in desert

but both meet different India. Control fringe areas
operational challenges. Like challenges similar
in forest areas, there is a to Africa
scope for adapting personal
protection methods to local
culture and human ecology
Coastal A variant of savanna malaria Remains dominant, Much less common Was in most areas Does not occur
with less intense, sometimes especially in than a century ago, wiped out by IRS nowadays
more seasonal transmission. Solomon Islands nowadays only except for a few
Environmental management and Vanuatu, important in parts areas, where it has
is likely to be feasible and where the effect of of Indonesia and persisted or re-
cost-effective only in very adult control is southern Vietnam. emerged
specific situations somewhat The elaborate
constrained by environmental
early biting and management
exophily schemes of the past
have almost
become history
Urban Increasingly important. Recent Similar to African Urban malaria is a Has again become Has re-emerged in a
results with larval control in situation, but problem only in important in South few foci posing;
Dar es Salaam are generally, so far, a the Indian America in areas of controlled by
encouraging but need to be minor problem subcontinent, but forest combined
subjected to cost- there it has encroachment; no measures aiming
effectiveness studies presented serious good examples of at mosquito
(Geissbuhler et al., 2009). challenges to control published breeding and
More attention to house control (Gil et al., 2007) human carriers
screening is needed (Ivanova et al.,
2009)
Note that there are frequent exceptions to the characteristics as described here; that the transitions between ecotypes are gradual (though less so around urban and forest) and that the
proposed delimitations are based on expert opinion, not hard evidence. The recommendations about control measures must be considered tentative; in fact, emphasis has been placed on
the potential implications of recent research, which challenges older assumptions.
Foothills are generally associated with higher malaria risk than non-
forested plains in all biogeographic regions outside Africa. Much of the
recent, highly focal, malaria occurrences in the Palearctic have been
associated with foothills and/or agricultural development projects.
The urban environment is associated with increased risk in most of the
Indian subcontinent, especially the western, arid part; urban malaria is
present in the Neotropic when forests encroach on cities and in a few
exceptional circumstances urban malaria has re-emerged in the Palearctic.
The findings of Tatem et al. (2006) indicate that urban extent as defined by
GRUMP, based on night-time light emissions, is adequate to demarcate
urban areas with relatively low malaria transmission in Africa. Whether
this is also the case outside Africa remains to be further investigated, but
there is no a priori reason to assume that this criterion would work less well
there. In most of Southeast Asia, there is usually no or very little malaria in
either urban areas or adjacent countryside, unless forested.
Thus, there is some evidence for geographic demarcation of forest
malaria and urban malaria in the biogeographic regions, where these
malaria ecotypes are important. For most other types, the transitions
between them are gradual, determined by changing altitude, latitude,
rainfall and salt-water infiltration. The resulting modification in malaria
epidemiology is likewise gradual. The delimitations of these ecotypes
presented in the tables are largely based on published expert opinion
and in some cases, the writers’ field observations.
3.4. DISCUSSION
3.4.1. Implications for control programmes
Knowledge about ecological determinants of malaria could be useful for
control programme stratification in a number of ways: firstly, by improv-
ing the delimitation of areas and populations at various levels of malaria
risk; secondly, by indicating which vector control interventions are most
likely to be effective and cost-effective; thirdly, by indicating any other
special measures and fourthly, by indicating what effects can be expected
from given interventions.
3.4.1.1. Stratification and delimitation of malaria risk

The first step in stratification is normally to delimit populations living in
areas where malaria cannot be transmitted (no risk), areas without
endemic malaria but a risk of outbreaks (epidemic risk) and areas with
endemic areas (WHO, 2009). In theory, ecological determinants should be
useful mainly for distinguishing between the two former categories
because malaria cases are absent from both. There are examples of failures
of ecological prediction of risk, as in the case of unforeseen convergence of
determinants of urban malaria in Russia (see Section 1.3.2.5.4, p. 56).
In contrast, it worked in Southeast Asia, where tree plantations have led
to the return of forest vectors and transmission (see Section 1.3.2.3.11,
p. 43). Thus, for this distinction, the careful use of ecological, climatic and
other determinants together with historical malaria data is probably the
best that can be done.
For delimitation of endemic areas, ecological determinants may be
useful as supplement to epidemiological data. As malaria surveillance is
still usually not case-based, data are referenced at best to health facilities,
and it is assumed that these have coverage areas corresponding to
administrative units. As epidemiological data are usually not available
at a fine enough level to allow vector control targeting by village, local
planners may need to take decisions based on soft knowledge. This soft
knowledge may be expressed by local health service providers, who may
have experience that most malaria cases come from given localities, or it
may be ecological. Overlaying malaria epidemiological data with popu-
lation data and physiographic data makes it possible to identify under-
served populations, which may be assumed to live with endemic
malaria. This is in fact done in mature control programmes (see
Section 1.3.2.3.13, p. 44), but in practice, the only ecological malaria
determinant that is trusted at face value is probably the forest environ-
ment in eastern India, most of Southeast Asia and South America, which
has a very strong correlation with malaria risk (see Section 1.3.2.3.3, p. 31
and Section 1.3.2.4.3, p. 48).
More generally, the certainty and the degree with which a given
ecotype will modify malaria risk are highly variable, depending on the
biogeographic region, the ecotype in question and the associated pro-
cesses. Yet, the forest environment in the Indo-malay and Neotropic
realms stands out because of its relatively clear delimitation and almost
invariably strong association with heightened malaria risk. In tropical
Africa, the implications of the various fringes and urbanization are well
documented, but the delimitations are not sharp. This has implications for
surveillance and assessment of impact rather than for interventions. In
contrast, the knowledge that rice irrigation has different effects according
to background ecology is relatively new, though not a great surprise (see
Section 1.3.2.1.8, p. 24), and this has control implications. As the wide-
spread application of control interventions in Africa changes the epide-
miological pattern, these distinctions are becoming blurred, but it remains
to be seen to what extent ecological and climatic determinants will be
associated with rebounds. As for the other ecotypes, considering the
findings in this review, they should normally be used only as indications
of what must be looked out for rather than for what must be there.
3.4.1.2. Implications for vector control

A major reason for ecological assessment of local malaria problems has
been to determine whether anti-larval methods could be effective to con-
trol malaria. Keiser et al. (2005a) have found that in recent times, inten-
tional methods for larval control remain neglected in favour of anti-adult
methods. Coastal malaria, which in the past was found highly suitable for
larval control in the Indo-malay region, appears to be receding there,
probably as a result of general development (better access to health ser-
vices, higher population density, improved housing and improved envi-
ronmental hygiene) and the availability of anti-adult methods. Similarly,
malaria related to rural development projects is often managed with anti-
adult methods, because of the ease of planning and implementing them. In
contrast, in urban areas, especially in India, anti-adult methods encounter
serious constraints, so that larval control has an important role, although
that role is in need of stronger, updated evidence and should be enhanced
through more research and more advanced intersectoral collaboration.
Larvivorous fish and sometimes source reduction (bio-environmental
control) are widely promoted and applied in some countries in Asia,
especially India, but with little attention to criteria for applying various
methods and evaluation (Dev et al., 2008; Sharma, 1999).
Generally, it seems that the simplicity of planning and implementing
anti-adult methods leaves little room for serious application of larval
control in endemic areas of Asia and the Americas. Those areas, in
which anti-adult methods are most constrained, are (a) urban malaria in
the Indian subcontinent, where larval control can be highly effective and
(b) forest-related malaria, where larval control is usually inapplicable
(except possibly in deforested and semi-urbanized areas in South Amer-
ica). Anti-adult methods in non-forested areas in India are running into
serious problems of multi-insecticide resistance (K. Raghavendra, per-
sonal communication); it is therefore important to revisit the range of
larval control methods, including insect growth regulators, and conduct
well-designed controlled trials guided by competent entomologists and
epidemiologists in defined ecological settings. The problems of vector
control in forest areas with exophilic vectors have not been solved.
There was hope some years ago that insecticide-treated mosquito nets
including hammock nets would provide the solution, but the results have
so far suggested an effect, which is modest—though present and possibly
better in the Amazon than in the most difficult areas of continental
Southeast Asia (Magris et al., 2007; Sochantha et al., 2006; Thang et al.,
2009). Although the problems of control of forest-related malaria can in no
way be reduced to the entomological dimension, there are good reasons to
invest in research and development.
Recent investigations in Africa also suggest a broader role for larvicid-
ing as an adjunct to effective anti-adult intervention. The studies are few
and may have been done in selected areas with propitious ecological and
health system conditions. Nonetheless, they are potentially very impor-
tant. In savanna malaria settings in Africa, anti-adult methods have good
effect (Lim et al., 2011), but this is limited and subject to decay because of
waning population immunity, insecticide resistance and operational fac-
tors (Trape et al., 2011). ITNs are widely accepted as the basic vector
control method. It remains to be seen, whether and where IRS with
alternative insecticides or larval control will be the better primary supple-
ment. In summary, current evidence suggests that only two ecotypes have
reasonably clear implications for larval control. In urban malaria, espe-
cially the Indian type, larval control should be considered. For forest
malaria, there is so far no evidence that larval control is potentially useful.
3.4.1.3. Implications for other control interventions

Local assessments of ecotypes with their anthropic and natural processes
can lead to the identification of special risk groups and risk areas, for
which particular strategies and approaches are needed. It is usually not so
much a question of what to do as of how to do it, for example, finding
ways of providing case management services to populations at high risk,
who may be cut of part of the year, engaging with clandestine work
forces, mining companies, private pharmacies in urban areas, etc.
3.4.1.4. Implications for expectations of impact

Malaria control planning usually mobilizes investments on the basis of
expected reductions of morbidity and mortality. With the currently avail-
able menu of interventions, it can be expected that the outcomes will be
less impressive and more difficult to sustain in the most difficult areas:
those where the transmission (indicated by, e.g. parasite rate) is intense
and those where technical and operational constraints converge, like
forest-related malaria in certain biogeographic regions and subregions,
and to some extent, urban malaria in India and desert fringe areas, with
major operational challenges. Although it is more and more the accumu-
lating experience at the local level, which informs the assessment of
expected impact, stratification based on ecotypes should help generate
an overview, stimulate critical thinking and help communicate to other
sectors why the problem seems to be resilient in certain areas.
3.4.1.5. General
Landscape epidemiology is not as an alternative to, but a supplement to
standard epidemiological information. In many countries, its utility in
national level planning is limited, while in local, for example, district
level planning, it has greater potential, because it is rarely possible to
triangulate all the important information for larger geographical units.
Even if it is not always possible to pay attention to larval control at the
national level, it should be possible at the local level to detect, for example,
a company that can be advised on how to reduce breeding sites according
to what is known about local vector bionomics, or to avoid wasting efforts
on trying to find breeding sites to be seeded with larvivorous fish inside a
forest area. As noted in Mexico, an ‘ecohealth approach’ is a means of
engaging other sectors. Thus, malaria ecotypes could be useful in training
local staff to plan beyond the distribution of commodities, to assist in
targeting, to adapt interventions to local conditions and to engage commu-
nities and other sectors.
3.4.2. Implications for malaria modelling and field research

3.4.2.1. Mapping
Hay et al. found that there are no globally valid environmental covariates
for mapping of the prevalence of falciparum malaria (Hay et al., 2009).
The findings in this review are consistent with this: the geographic varia-
bility in vector bionomics is so great that covariates could only be useful at
the level of biogeographical regions or subregions. However, within
certain regions, certain covariates are extremely important. Thus, future
iterations of global malaria mapping should take into consideration the
strong correlation found by various methods in the Indo-malay and
Neotropic regions between malaria and forest cover. Other findings,
which could perhaps be useful for mapping, are that in the Indian sub-
continent, from Kolkata and westwards, malaria transmission is generally
more intense in urban areas than in the surrounding rural areas, as long as
the latter are not forested. And, in areas dominated by river-irrigated rice-
field cultivation in continental Southeast Asia (from Myanmar to Vietnam
and from Yunnan Province to Malaysia) and in the Philippines, malaria is
virtually absent. This can have important implications for burden estima-
tion, as the populations of these areas are large.
3.4.2.2. Simulation modelling of effects of malaria control strategies

To aid decision-making, models can be structured so that inputs and
outputs are relevant to the needs of the users. One finding of this review
is that ecotyping does not provide a classification of all malaria situations
into a ‘humanly manageable’ number of categories for modelling.
Although there are arguments for working with only six ecotypes, these
have, to a large extent, different implications in the five biogeographical
regions, which have endemic malaria today; sometimes, there are impor-
tant differences between subregions, and to this comes a broad spectrum
of anthropic processes and effects of health systems and malaria control
with profound implications including insecticide and drug resistance and
changed vector behaviour.
Another finding is that a number of ecoregions are affected by malaria,

which in most areas is controllable from a technical viewpoint; in most of
these areas, malaria transmission persists because of health system pro-
blems. This is the case for practically all malaria in the Palearctic and in
plains and valleys outside the Afrotropical and Australasian regions.
Although malaria control strategies with their operational and systemic
constraints can be modelled in these scenarios, it would probably be more
rational to prioritize the ecoregions which are posing serious technical
problems, namely:
All malaria ecoregions in the Afrotropical, with their variability in
intensity of transmission, exophily, seasonality, heterogeneity (espe-
cially urban malaria) and health system performance; most malaria in
Papua New Guinea can probably be covered by applying the same
variations as in Africa although the presence of P. vivax is an added
complication.
Forest malaria in the Indo-malay and the Neotropic with the variability
of intensity of transmission, exophily, length of transmission season
and health system performance.
Indian urban malaria with its limited applicability of anti-adult inter-
ventions, at least IRS.
South American forest-related urban malaria.
Coastal malaria in the Australasian and the Neotropic with the poten-
tial (at least in principle) for source reduction.
Within each of these ecoregions, there is considerable variability
related to anthropic, natural and health system factors. It will probably
not be fruitful to address all of these in modelling. What can be done is to
address the defining variables noted above under each ecotype. There is
an increased interest on malaria bionomic databases which compile this
information from published literature (Diboulo, 2010).
3.4.2.3. Field research

Researchers need to integrate many scientific disciplines if they are to
contribute to the understanding of malaria in its broad social and ecologi-
cal context. Some studies which have combined observation with systems
thinking are the reviews of Palearctic malaria, which integrate evolution-
ary biology, palaeontology, history and epidemiology (Bruce-Chwatt and
de Zulueta, 1980; de Zulueta, 1994), the investigations on human geogra-
phy and malaria in Thailand (Singhanetra-Renard, 1986) and investiga-
tions of West African rice-field malaria (de Plaen et al., 2004). The need for
better understanding of factors related to human ecology may be even
greater. While it is relatively easy to characterize the physiography of an
area, it is difficult to obtain data pertaining to human ecological determi-
nants and to formally describe these in a way that can be used for
modelling. So far, quantitative techniques for studying human migration

are in their infancy. Interesting studies on population movement and
malaria were published in the 1960s, and fortunately, this thread is now
being resumed (Prothero, 1961; Stoddard et al., 2009).
In principle, therefore, the geographical classification of malaria
requires an ongoing multidisciplinary collaboration between epidemiol-
ogists, statisticians, geographers and computing and database specialists
to construct geographically explicit models of both local malaria risk and
ecology. Ecologic niche modelling has been promoted to address the
limitations of empirical spatial statistical analyses (Peterson, 2006), but it
is not certain whether it is realistic to try to assemble all the data needed to
calibrate such models when the disease is determined not only by its
vectors and parasites but also by a myriad of host factors including the
health system, which even has important effects on vector and parasite
biology. The interactions that result from spatial juxtaposition of differing
landscape elements in patterns that vary from place to place are only
beginning to be explored (Lambin et al., 2010; McCallum, 2008).
ACKNOWLEDGEMENTS
This work was supported by the Malaria Modelling Project #39777.01 funded by the Bill and
Melinda Gates Foundation, which also supports KB. We are grateful to Dr. Tom Burkot
(CDC-Atlanta), Dr. José Najera (Crans-sur-Céligny), Dr. Amanda Ross (Swiss TPH) and
Professor Tom Smith (Swiss TPH) for their valuable and constructive comments on earlier
drafts.
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CHAPTER 4
The Changing Limits and
Incidence of Malaria in Africa:
1939–2009
Robert W. Snow,*,† Punam Amratia,* Caroline W.
Kabaria,* Abdisalan M. Noor,*,† and Kevin Marsh*,†

4.2. A Brief History of Malaria Control in Africa 171
4.2.1. Pre-second world war 171
4.2.2. 1948–1960: The global malaria eradication
programme (GMEP) in Africa 172
4.2.3. 1960–1999: Post GMEP 173
4.2.4. 2000–2010: Roll Back Malaria 174
4.3. Defining the Absence of Malaria Risk 175
4.3.1. Excluding malaria risk based on reported
absence and population density 175
4.3.2. The transmission limiting effects of
temperature and aridity 178
4.3.3. Defining transmission stability within the
spatial margins of risk in relation to control
and elimination 179
4.4. The Changing Margins of Malaria Transmission in
Africa 181
4.4.1. Changing boundaries and incidence of
malaria in North Africa and Djibouti 181
4.4.2. Changing boundaries and incidence of
malaria on the islands of Africa 190
* Malaria Public Health & Epidemiology Group, KEMRI-Wellcome Trust Collaborative Programme, Nairobi,
Kenya
{
Centre for Tropical Medicine & Vaccinology, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom

169
170 Robert W. Snow et al.
4.4.3. Changing boundaries of stable malaria risk

and disease incidence in Southern Africa 206
4.4.4. Malaria control in Middle Africa: From GMEP
pilots to RBM 217
4.5. Summary and Discussion 226
4.5.1. Changing limits in North Africa 226
4.5.2. The successes and failures of malaria
elimination on Africa’s islands 233
4.5.3. Elimination and control efforts in Southern
Africa 235
4.5.4. The double dip recession 237
4.5.5. The future 239
Acknowledgements 239
References 240
Abstract Understanding the historical, temporal changes of malaria risk

following control efforts in Africa provides a unique insight into
what has been and might be archived towards a long-term ambition
of elimination on the continent. Here, we use archived published
and unpublished material combined with biological constraints on
transmission accompanied by a narrative on malaria control to
document the changing incidence of malaria in Africa since earliest
reports pre-second World War. One result is a more informed
mapped definition of the changing margins of transmission in
1939, 1959, 1979, 1999 and 2009.
4.1. INTRODUCTION
Africa is often called the ‘‘heartland’’ of malaria. Certainly, malaria has
played a major role in shaping human evolution in Africa and remains a
major public health threat and impediment to economic development.
Although malaria in Africa is often spoken of as if it were a single well-
characterized situation, in fact, the epidemiology and ecology of malaria
are extremely heterogeneous. Over recent years, an increasingly accurate
picture of the scale and heterogeneity of malaria in Africa has emerged.
At the same time, there has been an increasing appreciation that the
malaria situation is changing in many areas, with reports of falling trans-
mission and disease burden in some but by no means all parts of the
continent. It is assumed that many of these changes are related to deliber-
ate intervention, and certainly, there has been a massive increase in
investment in malaria control over the past 10 years, but it should not
be forgotten that the ecology of malaria is shaped by many factors includ-
ing climate, human settlement, human behaviours and factors that may
The Changing Limits and Incidence of Malaria in Africa: 1939–2009 171
affect vector populations, all of which are subject to changes for a multi-
tude of reasons.
Today, there is an increasing emphasis on the concept of ‘‘shrinking
the map’’ of malaria with the initial aim of local elimination and the long-
term aim of global eradication. To shrink a map, one has to begin by
knowing the map accurately and how it may have changed in the past.
Several attempts have been made over the last 60 years to define the
limits of malaria transmission using a variety of climate-driven con-
straints on parasite and vector survival and reported case incidence
(Boyd, 1930; Craig et al., 1999; Dutta and Dutt, 1978; Guerra et al., 2006,
2008; Hay et al., 2009; Kiszewski et al., 2004; Le Lannou, 1936; Lysenko
and Semashko, 1968; Macdonald, 1957; Manguin et al., 2008; Pampana
and Russell, 1955; US War Department, 1944). These mapped products
have been difficult to use sequentially to understand the changing mar-
gins and intensity of risk as each has used different methodologies and
input data. We aim here to define the boundaries of malaria risk in Africa
by reviewing available documented case data together with the applica-
tion of biological and human settlement criteria to define malaria risk at
its natural extent and record how this has changed over the last century.
In doing this, we have brought together for the first time data relating to
past attempts to control and eliminate malaria in different parts of Africa.
4.2. A BRIEF HISTORY OF MALARIA CONTROL IN AFRICA
4.2.1. Pre-second world war

Following Sir Ronald Ross’s discovery of the role played by the mosquito
in the transmission of malaria in 1897, he travelled widely, including
Africa (Egypt, Mauritius, Nigeria, Ghana, Sierra Leone and Zimbabwe),
to promote environmental sanitation using "mosquito brigades" (Nye and
Gibson, 1997; Ross, 1902). Reference to Ross’s recommendations appear
in many Colonial Administration Medical Department annual reports
from 1900 and the reduction of larval breeding sites became a public
health priority after the First World War for many of the rapidly expand-
ing urban centres in Africa. The discovery by Alphonse Laveran of the
blood stages of the malaria parasite in French Foreign Legion troops
stationed in Algeria in 1880 (Bruce-Chwatt, 1981) and the effects of qui-
nine as a therapeutic agent served as the second major approach to
malaria prevention among Europeans in Africa, starting before the First
World War (Shah, 2010). ‘‘Quininisation’’ was practiced as a means of
personal prophylaxis or through mass drug administration, for example,
in Dar es Salaam (Orenstein, 1914), the large towns of the Belgian Congo
(Henrard and Van Hoof, 1933; Van den Branden and Van Hoof, 1923),
Sudan (Henderson, 1934), Tunisia (Husson and Nicolle, 1907) and Algeria
(Sergent and Sergent, 1928). The clinical and epidemiological link
between sustained use of quinine, malaria and blackwater fever became
a major cause for concern early in the twentieth century, and its use as a
means of malaria prevention slowly declined through the 1950s (Foy and
Kondi, 1950; Graham, 1912; Shah, 2010).
Despite an early recognition of the economic impact malaria had on
productivity in the European colonies (League of Nations, 1933), a com-
mon epidemiological portrayal of malaria at the time was that "Africans"
were immune, asymptomatic carriers of infection (Bagster-Wilson, 1939;
Bagster-Wilson and Wilson, 1937; Christophers, 1924; Garnham, 1949;
James, 1929) and that this posed "threats" to the transmission of the
parasite to Europeans. Emphasis was on protecting European settlers
and prevention recommendations included the spatial distances neces-
sary for separate African housing to limit risks to Europeans in Sierra
Leone (Christophers and Stephens, 1900) and Kenya (Paterson, 1928).
4.2.2. 1948–1960: The global malaria eradication programme

(GMEP) in Africa
The Second World War marked a new era in drug discovery and the
development of residual insecticides notably the 8-aminoquinolines such
as chloroquine (Sweeney, 2000) and dichlorodiphenyltrichloroethane
(DDT) (Russell, 1951). These new tools signalled a moment of great
opportunity to tackle the public health burden posed by malaria, and
importantly for the time, the economic growth of colonial Africa
(Colbourne, 1966; Macdonald, 1950; WHO, 1948). A report presented to
the World Health Organization (WHO) in 1948 states: ‘‘It is not enough to
quote that about 3,000,000 deaths are caused yearly by malaria in the
world, or that every year about 300,000,000 cases of malaria occur. . .. . .
that malaria is prevalent in tropical and subtropical areas where food
production and agricultural resources are potentially very high, and that,
by affecting the mass of rural workers, it decreases their vitality and
reduces their working capacity and thus hampers the exploitation of the
natural resources of the country. At a time when the world is poor, it
seems that control of malaria should be the first aim to achieve in order to
increase agricultural output’’ (WHO, 1948).
Two years later at a conference in Kampala, the WHO recommended
‘‘to governments responsible for the administration of African territories
that malaria should be controlled by modern methods as soon as feasible,
whatever the degree of endemicity, and without awaiting the outcome of
further experiments’’ (Dobson et al., 2000; Najera et al., 2011; WHO, 1950).
Immediately after the Second World War, almost every country in Africa
began using chloroquine and DDT. This varied in application and
coverage but had become universal policy very quickly and adapted in
different settings to achieve national ambitions of elimination or sub-
national pilot elimination projects. However, not long after the launch of
the Global Malaria Eradication Programme (GMEP), it was decided that
sub-Saharan Africa was not ready for elimination: ‘‘the prolonged period
of the transmission season and the extremely high degree of malaria
endemicity in the region. . .’’ combined with weak infrastructure ‘‘. . .are
likely to form an effective barrier to a large-scale eradication programme’’
(WHO, 1954).
4.2.3. 1960–1999: Post GMEP

Across Africa, malaria programmes gradually returned to an objective of
controlling, rather than eliminating risk and the GMEP defined control as
‘‘the reduction of the disease to a prevalence where it is no longer a major
public health problem; the concept carries the implication that the
programme will be unending, control having to be maintained by contin-
uous active work’’ sometimes referred to as "pre-elimination" (WHO,
1957, 1961). Despite this conclusion, several countries maintained elimi-
nation ambitions through to the 1970s through the use chemoprophylaxis
(Charmot, 1969; Hamon et al., 1963; Kouznetsov, 1979) and indoor resid-
ual house spraying (IRS) with sustained use of DDT and to a lesser degree
other organochlorides such as benzene hexachloride (BHC), hexachloro-
cyclohexane (HCH), Gammexane and dieldrin, organophosphates
(including malathion and fenitrothion) and carbamates (including pro-
poxur) despite mounting threats of vector resistance to these insecticide
classes (Hamon et al., 1963; Kouznetsov, 1976, 1977). Countries maintain-
ing elimination strategies tended to be located at the margins of stable,
endemic transmission in the northern and southern latitudes of Africa
and the islands off the continental coast line. For the rest of central Africa,
sustained control largely meant the treatment of febrile illness.
From the mid-1980s, trials began in Africa of a new approach to vector
control based on the personal protection afforded by insecticide-treated
bed nets (ITNs) (Lines et al., 1985; Ranque et al., 1984; Snow et al., 1987;
1988). By the mid-1990s, further large-scale trials across Africa had shown
that ITN provided significant, cost-effective protection against child mor-
tality (Lengeler, 2004). However, the community coverage of ITN by 2004
was minimal (Noor et al., 2009). By the late 1990s, the continent was
gripped by a spiralling decline in chloroquine efficacy, leading to wide-
spread treatment failures, evidence of increasing mortality (Snow et al.,
2001; Trape, 2001) and hailed as a public health disaster (White et al.,
1999). This promoted the accelerated development, registration and
deployment of fast-acting artemisinin-based combination therapy (ACT)
(White, 1999). However, in contrast to the rapid adoption after the Second
World War of chloroquine, a drug that would be difficult to register with

regulatory authorities today, protracted policy dialogue (Attaran et al.,
2004, 2006), difficulties in manufacture and distribution and national
procurement and regulation of ACTs have meant that these new medi-
cines reached only a few people who needed them by 2009 (RBM, 2011).
4.2.4. 2000–2010: Roll Back Malaria

Following the recognition that malaria in Africa could not effectively be
addressed by the GMEP, 40 years lapsed before malaria control in Africa
became a significant part of international public health dialogue. A series of
international meetings and declarations during the late 1990s (Greenwood
et al., 2008; Kidson, 1992) led to launch of the Roll Back Malaria (RBM)
movement in 1998 (Nabarro and Taylor, 1998). In April 2000, African
leaders, meeting in Abuja, signed a declaration that said they would
‘‘Halve the malaria mortality for Africa’s people by 2010, through imple-
menting the strategies and actions for Roll Back Malaria’’ (WHO, 2000). This
was to be achieved by ensuring that at least 60% of at-risk populations were
protected or treated with appropriate methods (WHO, 2000); subsequently
redefined to 80% coverage by 2010 (WHO, 2005) and the bar raised even
higher with the launch of the Global Malaria Action Plan (GMAP) in 2008
that called for universal coverage with some form of vector control (RBM,
2008). Where DDT and chloroquine were seen as the magic bullets for
malaria elimination during the era of the GMEP, ITNs, ACTs and new
rapid diagnostic tests were the exciting new tools during the RBM era.
From its inception, RBM concentrated on ‘‘high-burden countries’’, the
result of which was that Africa was for the first time in malaria control
history centre stage of an international effort to tackle malaria.
Underpinning the recent wave of international interest in malaria
control has been a concerted effort to articulate the economic burden and
inequities posed by malaria, creating a poverty trap (Gallup and Sachs,
2001; Sachs and Malaney, 2002; Sachs and McArthur, 2005). This evidence
base increased the profile of malaria as a broad development issue, effec-
tively levered support from key international partners (World Bank, 1993)
and put malaria on the global development map articulated in the Millen-
nium Development Goals (MDGs) (Sachs and McArthur, 2005). Despite
the unquestionable health burden posed by malaria, making an economic
argument for its control has been necessary during each wave of interna-
tional interest in funding its control and elimination since the 1930s.
The Global Fund to fight AIDS, Tuberculosis and Malaria (GFATM)
was established in 2002 to make available large-scale funding to help
achieve health-related MDGs (Feachem and Sabot, 2006). In 1998, spend-
ing on malaria control globally was around 100 million USD (Narasimhan
and Attaran, 2003). Between 2002 and 2009, the Global Fund had approved
5.6 billion USD for malaria grants to African countries. This has been
accompanied by a significant increase in direct bilateral support for
malaria (Snow et al., 2010a). The launch of the President’s Malaria Initia-
tive (PMI) in 2006 massively changed the funding landscape in Africa
(PMI, 2009). By 2009, 21 African countries had sufficient combined per
capita annual donor assistance to meet the targets established at Abuja in
2000 (Snow et al., 2010a). In 2007, a commitment to a global eradication
strategy re-emerged (BMGF, 2007; Feachem and Sabot, 2008; Roberts and
Enserink, 2007) and the GMAP, launched in 2008 by the RBM partnership,
reflected this renewed ambition—a malaria free world (RBM, 2008).
4.3. DEFINING THE ABSENCE OF MALARIA RISK

The territories and boundaries of nation states across Africa have changed
considerably over the past 100 years through colonization by the Otto-
mans and Europeans, wars and struggles for independence. Throughout
our descriptions of risk, we have regarded as separate nation states those
that exist today (Fig. 4.1), but in reviewing reported intervention cover-
age, clinical evidence and changing risk, it is important to recognize the
changing governance boundaries over the past century and where appro-
priate these are defined throughout.
4.3.1. Excluding malaria risk based on reported absence and

population density
Plasmodium falciparum transmission probably reached its natural extent in
Africa around 1900 (Carter and Mendis, 2002), and few African countries
have been completely free from malaria transmission over the last 100
years. The Kingdom of Lesotho (Basutoland pre-1966) is the highest
country in the world with 80% of the population living higher than
1800 m above sea level and has always been regarded as malaria free
(Russell, 1956). The Islands of the Seychelles archipelago, Tromelin, Car-
gados Carajos, Agalega and Rodriguez, Saint Brandon and Chagos in the
Mascarene archipelago were documented in the 1950s as being unable to
support malaria transmission. Similarly, the island of St. Helena, in the
Atlantic Ocean, regarded by the UN as part of Africa, has not supported
malaria transmission (Russell, 1956). The Western Sahara is a barren, arid
area that in 1956 was reported by the Spanish governing authorities to be
completely free from transmission (WHO-Spanish Morocco, 1955).
A careful assembly of historical evidence of risk in the Union of South
Africa pre-1940s suggests that malaria was absent from large parts of the
western part of the country (Sharp and Le Sueur, 1996) and the bordering
southern areas of Namibia and Botswana (De Meillon, 1951; Franco de
FIGURE 4.1 The margins of stable P. falciparum transmission at its presumed natural extent (pre-1939). Dark grey representing no malaria risk;
light grey biologically suitable transmission but population density less than 0.01 people per km2; green represents areas of unstable transmission;
dark pink areas show stable transmission. Although the Western Sahara remains unrecognized by the UN, we consider here as an independent
territory within Africa. The Federation of Rhodesia and Nyasaland formed a semi-autonomous state between 1954 and 1963 before it became three
British governed countries of Southern Rhodesia, Northern Rhodesia and Nyasaland but throughout regarded as independent Zimbabwe, Zambia
and Malawi, respectively; Eritrea and Ethiopia were officially recognized as separate nations in 1993; more recently, South Sudan and The Republic of
Sudan separated in July 2011. Two small territories within the Kingdom of Morocco continue to be occupied by Spain in the North West (Ceuta) and
North-East (Melilla) on the Mediterranean coastline to this day; however, we considered part of the Kingdom approximating to its Ottoman extent
before 1912. The United Arab Republic of Egypt lost the Sinai Peninsula to Israeli forces during the 6-day war of 1967. This disputed territory was not
fully restored to Egypt until after the partial reoccupation by President Sadat as part of the October War in 1973 and negotiated return to Egypt in
1979. We consider the Sinai Peninsula as part of UAR Egypt, and hence the African continent, throughout the entire period under review. In Libya, the
Eastern coastal cities have always been regarded of unstable transmission and this intelligence has been used to indicate this area as unstable (light
pink). The area of present day Namibia became a German Imperial Protectorate in 1884 and was made a South African Mandated Territory after the
First World War. Until independence Namibia was referred to as German South-West Africa. The war for independence from South Africa intensified
from 1973 with the international recognition of the South-West Africa People’s Organization (SWAPO) that eventually resulted in an end to South
African rule in 1988 and Namibian independence in March 1990. The small territory of Walvis Bay, mid-way along the Namibian coast, was part of the
Union and Republic of South Africa up to 1994, but we treat as part of the Republic of Namibia throughout all presentations of risk. AfriPOP
population surfaces for each country were downloaded from www.afripop.org and re-sampled from 100 m resolution to 5 km in ArcGIS 10 (ESRI,
Redlands, CA, USA). These were then reclassed to identify 5 5 km grid squares that reported less than 1 person per 100 km2 (population
density < 0.01 persons per km2). Areas that are biologically suitable for transmission but where populations are less than 0.01 km2 within a
contiguous area are shown in light grey in the figure. These often represent game reserves, conservation areas or deserts. Unsuitable areas for P.
falciparum transmission are shown in dark grey, and based on (a), medical intelligence from Djibouti, South Africa, Namibia and Botswana has been
used to define no risk (see text for details). The Kingdom of Morocco’s range of transmission intensity and risk mapped extensively by Hoeul and
Donadille (1953) and Houel (1954) that mapped pre-elimination extents and the progress of elimination since 1948. These mapped ranges have been
combined with the aridity mask to identify the natural extent of malaria in the Kingdom; (b) Temperature Suitability Indices generated at 1 1 km
resolutions as described by Gething et al. (2011), and constructed using monthly synoptic mean, maximum, and minimum temperature records
obtained from 30-arcsec ( 1 1 km) spatial resolution climate surfaces (Hijmans et al., 2005) converted using spline interpolation to a continuous
time series representing a mean temperature profile across an average year incorporated into a biological model of sporogyny. TSI values above zero
for P. vivax are more ubiquitous across Africa suggesting transmission at higher altitudinal and wider latitudinal limits, including the highlands of
Kenya, Réunion, Madagascar and Ethiopia (Gething et al., 2011), but these are harder to interpret given the absence of reported transmission in these
areas even when P. vivax is prevalent in these countries and (c) areas of extreme aridity (Guerra et al., 2008) based on the enhanced vegetation index
(EVI) derived from the MODerate-resolution Imaging Spectroradiometer (MODIS) sensor imagery, available at approximately 1 1 km spatial
resolution (Guerra et al., 2008; Scharlemann et al., 2008). Temporal Fourier-processed, monthly EVI surfaces were used to develop 12 monthly
surfaces that reclassified EVI 0.1 (arid) and > 0.1 (non-arid) (Guerra et al., 2008). All rivers and lakes shown from single source: Global Lakes and
Wetlands Database (GLWD) (Lehner and Döll, 2004).
et al., 1984a; Ministry of Health, 2001; MoHSS, 1996). These sub-national

limits of risk based on medical intelligence in Southern Africa have been
digitized and excluded as part of the historical range of malaria transmis-
sion in Africa.
The presence of human hosts is clearly necessary to perpetuate trans-
mission of the four malaria parasites that affect man in Africa. Earlier
descriptions of malaria risk have applied the crude limits of unpopulated,
barren areas across the Sahara desert and other low population density
desert areas in southern Africa (Boyd, 1930; Lysenko and Semashko, 1968;
Manguin et al., 2008; Pampana and Russell, 1955). More informed
approaches to excluding human infection risks based on population
density ( 1 person per km2) were implemented by Guerra and colleagues
using global population surfaces developed by the Global Rural Urban
Mapping Project (GRUMP) (Balk et al., 2006; Guerra et al., 2006). These
masks were subsequently felt to be too imprecise due to the resolution
and quality of the population input data used by GRUMP to describe the
distribution of human settlement in Africa (Hay et al., 2009). A new
human population settlement map has recently been developed employ-
ing considerably more input data at higher spatial and temporal resolu-
tions that has substantially improved the modelled spatial predictions at
0.1 0.1 km resolutions of population density in Africa (Afripop, 2011).
Here, we have used these spatial data, re-sampled to 5 5 km, to quanti-
tatively define the spatial limits of parasite transmission based on a
conservative definition 0.01 people per km2 (Fig. 4.1). This mask serves
as a visual guide to the spatial limits of human malaria transmission and
presumes that extremely sparsely populated areas of Africa today corre-
spond to similar settlement patterns over the last century where transmis-
sion is biologically suitable.
4.3.2. The transmission limiting effects of temperature and

aridity
Both altitude (a proxy for low ambient temperature) and deserts have
been used to define the absence of malaria transmission in most previous
iterations of global malaria maps (Boyd, 1930; Dutta and Dutt, 1978).
Temperature plays a key role in determining the transmission of human
malaria based on its relationship with the duration of sporogony and is
particularly relevant to Plasmodium vivax and P. falciparum (Nikolaev,
1935). To provide a plausible mask to eliminate the possibility of trans-
mission across Africa, we have used a recently developed temperature
suitability index (TSI) (Gething et al., 2011). The TSI model uses a
biological framework based on survival of vectors and the fluctuating
monthly ambient temperature effects on the duration of sporogony that
must be completed within the lifetime of a single generation of
Anophelines. This was used to generate at each 1 1 km pixel periods of

an average year when a vector’s lifespan would exceed the time required
for sporogony, and hence when transmission was not precluded by
temperature. If this time exceeded the maximum feasible vector lifespan,
then the cohort was deemed unable to support transmission and the area
classified as being at zero risk (Gething et al., 2011). Here, we have used a
TSI value of zero for P. falciparum to represent no transmission and TSI
values above zero as areas able to sustain some parasite transmission. The
P. falciparum temperature mask highlights the highland areas and moun-
tains of East Africa, the southern mountains of Tanzania, the mountains at
the junction of Democratic Republic of Congo, Rwanda and Burundi, the
highlands in Ethiopia, Mount Cameroun, the Shimbiris mountains in
Somaliland, the Nyika Plateau in Malawi and Mount Nyangani in Eastern
Zimbabwe (Fig. 4.1).
The second important environmental constraint on transmission is
the effect of arid conditions on anopheline development and survival
(Shililu et al., 2004). Limited surface water reduces the availability of
sites suitable for oviposition and reduces the survival of vectors at all
stages of their development through the process of desiccation (Gray
and Bradley, 2005). The ability of adult vectors to survive long enough
to contribute to parasite transmission and of pre-adult stages to ensure
minimum population abundance thus depends on the levels of aridity
and species-specific resilience to arid conditions. We have defined
extreme aridity using the enhanced vegetation index (EVI) and used
data from 12 monthly surfaces to classify into areas likely to support
transmission, defined by an EVI of greater than 0.1 for any two consec-
utive months and areas without two or more consecutive months of an
EVI > 0.1 as unable to support transmission (Guerra et al., 2006, 2008).
This aridity mask identifies small foci of risk across the Sahara that are
likely to support transmission because of their proximity to oases and
seasonal rivers while retaining a plausible mask of virtual zero trans-
mission across the Sahara, in extremely arid areas that make up large
areas of the Horn of Africa and in southern Africa through the aridity
limiting effects of the Kalahari, the Sossusvlei and the Skeleton Coast
(Fig. 4.1).
4.3.3. Defining transmission stability within the spatial margins

of risk in relation to control and elimination
The stable–unstable classification was first introduced into malariology
by Sir Ronald Ross (Ross, 1916) and adapted by George Macdonald for the
measurement of malaria endemicity where stability was defined quanti-
tatively by the average number of feeds that a mosquito takes on man
during its life (Macdonald, 1952, 1957). The measurement of Macdonald’s
stability index demands detailed entomological data that are rarely avail-
able. Qualitatively, stable malaria refers to situations that are relatively
insensitive to natural and man-made changes and unstable malaria
includes areas very sensitive to climatic aberrations and very amenable
to control with ranges of intermediate stability between these extremes.
These qualitative concepts of stability are still in use today.
Critical to the planning of malaria elimination during the GMEP was a
quantitative description of risk for planning control and monitoring prog-
ress. During the preparatory phase, large-scale parasite prevalence surveys
were undertaken to examine feasibility of elimination. During the attack
phase, the aim was to reduce prevalence and incidence to interrupt trans-
mission within 12–18 months and then remove the last reservoir of infec-
tions within a further 24–30 months. Towards the end of attack phase,
parasite prevalence was deemed impractical to monitor effectively and
malaria incidence became the key monitoring metric. It was suggested
that when infection prevalence fell below 2%, national programmes
should invest in combinations of passive, active and mass-blood survey
surveillance of new infections, expressed as an annual parasite incidence
(API) per 1000 people resident in a reporting administrative area. Addi-
tional measures have been variously included but not as regularly
reported including average blood slide examination rates and slide posi-
tivity rates (Pampana, 1969; Pull, 1972; Ray and Beljaev, 1984; Yekutiel,
1960). When the API was less than 1 per 10,000, the consolidation phase
started and comprehensive use of prevention was in theory stopped. API
was originally set at 5 per 10,000, but experience showed that national
programmes often overestimated the coverage and completeness of their
surveillance. The consolidation phase maintained a targeted control com-
ponent, guided by active case detection to eliminate residual foci of
parasite reservoirs. The duration of the consolidation phase was highly
variable (Russell, 1956), but migration to the maintenance phase was usu-
ally initiated after 3 years without local transmission. Theoretically, the
maintenance phase included the introduction of measures to prevent the
reintroduction of malaria.
Several authors have recently revisited the epidemiological definitions
used to signal transitional points from sustained malaria control and a
pathway towards elimination (Cohen et al., 2010; Feachem et al., 2010a,b;
Hay et al., 2008, 2009). In practical terms, it has been generally considered
that a parasite prevalence of less than 1% during peak transmission in a
representative sample of the country, or lower administrative area, with
prevalence in sub-populations of less than 5% (allowing for over-disper-
sion of risk) would constitute a situation referred to as low-stable endemic-
ity and governments may elect to hold this line for disease control (Cohen
et al., 2010). Conditions based on parasite prevalence lower than 1%
become very difficult to measure and qualitatively represent unstable
conditions. Hay and colleagues regard unstable transmission as repre-

sented by an API of less than 1 per 10,000, and this approach is used in
current mapping of malaria risk worldwide (Guerra et al., 2008; Hay et al.,
2009). There is also a growing recognition that zero transmission is both
impossible to measure and too strict a definition in areas where vectors
persist and immigration of infected hosts is high, especially in areas
where the environmental criteria necessary to sustain further transmis-
sion exist. For example, the United States of America has experienced
multiple autochthonous transmission events since it was declared malaria
free in 1956 (Mali et al., 2009). As such elimination is presently regarded
as a state where interventions have interrupted endemic transmission and
limited onward transmission from imported infections below a threshold
at which risk of reestablishment is minimized (Cohen et al., 2010).
Throughout our current description of risk, we have used API as a
measure of stability and reported documented presence and absence of
transmission to define the margins of risk.
4.4. THE CHANGING MARGINS OF MALARIA TRANSMISSION

IN AFRICA
The fixed long-term average climatic conditions together with reported
absence of transmission provide a natural maximal extent of possible
malaria transmission in Africa (Fig. 4.1). However, these margins have
changed over the past 100 years through systematic control, elimination
and prevention of resurgent risks. We review the effects of scaled inter-
ventions that were mounted since the first reported efforts of aggressive
control in North Africa, including the aberrant changes in the Republic
of Djibouti, the islands of Africa in the Atlantic and Indian Oceans
and countries in Southern Africa (South Africa, Botswana, Namibia,
Zimbabwe and Swaziland). These countries represent the historical
margins of Africa’s stable and unstable transmission, and it is important
to define how these limits have contracted and expanded since 1900.
4.4.1. Changing boundaries and incidence of malaria in North

Africa and Djibouti
4.4.1.1. Morocco
Following the first world war, focal attempts at using biological control, a
protracted period of quinine prophylaxis from 1929, followed by the use of
atebrine þ praequine (chloroquine-like drugs) in late 1930s and limited use
of pyrethrum insecticides deployed in areas of agricultural significance
were variously promoted to control malaria across the country (Gaud and
Sicault, 1938; Vialatte, 1923). After the Second World War, Hoeul and
Donadille (1953) mapped the extents of highest transmission along the coast
from Tanger at the point of the Mediterranean to Casablanca further south
on the Atlantic coast stretching inland along rivers and irrigation areas but
declining in intensity towards the Atlas mountains and the desert fringe
areas where foci were identified around oases. The main vectors were An.
labrachiae in the north and central parts of Morocco, a vector refractory to
P. falciparum and supports only P. vivax transmission (De Zulueta et al.,
1975), and An. sergentii perpetuating both P. vivax and P. falciparum across
the entire country (Guy and Holstein, 1968). In 1948, DDT had been intro-
duced for IRS to supplement radical case treatment and control in 33 peri-
urban areas and 28 rural zones augmenting special engineering projects
combined with larviciding in irrigation areas. The case incidence declined
significantly by the late 1950s; from this point, the Gharb region contributed
more than a third of all cases; overall transmission had been reduced to only
nine mapped focal areas (Houel, 1954; Hoeul and Donadille, 1953). By the
early 1960s, 70% of clinical infections were caused by P. vivax (Guy, 1963).
From 1968, a renewed effort was launched to eliminate malaria from the
remaining foci which succeeded in reducing case incidence until a resurgent
risk of malaria in the 1980s. At this time, all new cases were reported as
vivax, and by 1974, it was assumed that the Kingdom of Morocco was
falciparum free. Foci of vivax transmission continued to exist through the
1990s to 2000 in Al Hoecima, Chefchaouen, Taounate and Khouribga pro-
vinces. Chefchaouen, in the rice growing in the North West, 85 km south
east of Tanger remained the last focus of P. vivax transmission by 2000
principally transmitted by An. labranchiae (Faraj et al., 2003, 2008, 2009).
In 2004, the last case of locally acquired P. vivax infections was reported
from this area and the Kingdom was certified malaria free in 2010. The long-
term multiparasite case incidence data have been assembled from multiple
sources and shown in Fig. 4.2.
4.4.1.2. Algeria
In 1904, the Antimalaria Department was established under the direction
of the Institute Pasteur and headed by Etienne Sergeant (Dedet, 2008).
Leading up to the First World War, environmental management domi-
nated approaches to prevention around settler’s farms on the Mitidja
plain and the railway. Between the World Wars, quinine prophylaxis
was promoted for French settler populations and their work force with
continued experimentation with environmental control (drainage, canali-
zation, bush clearing and removal of permanent swamps) (Ciavaldini,
1917; Foley, 1923; Sergent and Sergent, 1928). These activities systemati-
cally expanded across the three Departments of Oran, Constantine and
Algiers until the end of the Second World War. Between 1948 and 1953, an
average of 5300 cases of malaria per year were reported in Algeria (WHO-
Algeria, 1956). In 1948, DDT was introduced for IRS and became the
600 6
Annual malaria incidence (per 100,000 population)

500 5
400 4
300 3
Morocco
200 2 declared
malaria free
in 2010
100 1
0 0
1963
1948
1938
1943
1958
1928
1953
1979
1968
1974
1984
1973
1994
1999
1933
2004
2009
1989
FIGURE 4.2 Kingdom of Morocco. Annual malaria case incidence (both species) per
10,000 per annum 1928–1973 (left hand panel) and slide-confirmed P. vivax malaria 1974–
2010 per 100,000 population (right hand panel). Last confirmed P. falciparum case
detected in 1979. Note case incidence in 1973 ¼ 1.03 per 10,000 population, 3 vivax
cases detected in 2000 and 19 case in 2002, no cases detected in 2001 and 2003 and one
case notified in 2004. Case data derived for 1930–1933 (Gaud, 1947); 1934–1945 (Hoeul
and Donadille, 1953); 1946–1962 (Guy, 1963); 1963 and 1964 (El Aouad, 2009); 1965, 1978
and 1979 (WHO, 1992); 1966–1977, 1980–1981 and 1998 (El Aouad, 2009); 1982–1997 (WHO,
1999); 2002–2010 (WHO-Morocco, 2010). Population has been sourced for 1925–1955
(Goldewijk and Batthes, 1997); 1960–2010 (H-C au Plan, Royaume du Maroc, 2011).
Intercensal growth rates used to compute non-census year population size.
mainstay of control with supporting larval control and use of atebrine and
plasmochine as mass drug administration and prophylaxis (Parrot et al.,
1946). The focus continued to be on the reduction of transmission in Oran,
Constantine and Algiers to protect areas widely settled by French immi-
grants since the 1830s who were able to lobby political support through
direct government representation in Paris (Guy and Gassabi, 1967). The
bloody Algeria war ended 132 years of French rule in 1962 but delayed a
declaration of malaria elimination ambitions until 1968 when there were
over 95,000 cases reported per year (Fig. 4.3). The eradication programme
in the newly independent Algeria was rapidly successful; by 1978, only 30
locally acquired cases of P. vivax were reported in foci in the middle of
Algeria (Benzerrough and Janssens, 1985; Hammadi et al., 2009). Here, we
assume that by 1978 P. falciparum and P. vivax had been eliminated in the
northern territories, focal transmission occurred in the middle of the
country and both P. falciparum and P. vivax remained through 1980 in
the southern-most regions. In 1981, Khemis el Kechna represented nearly
all of the autochthonous cases detected in Algeria that year (51 cases) and
all were P. vivax (Benzeroug and Wery, 1985; Benzerrough, 1990).
Between 1980 and 2007, only 300 confirmed, locally acquired cases were
reported (Fig. 4.3). Importantly between 1985 and 2007, all cases were
16 0.5

14
0.4
12
10
0.3
0.2
6
4
0.1
2
0 0.0
1948
1954
1977
1982
1987
1992
1997
2002
2007
FIGURE 4.3 Algeria: Annual malaria incidence per 10,000 population 1948–1954 (left
hand side) and per 100,000 population 1977–2009 (right hand side). Annual malaria case
data sourced from multiple sources: 1948–1953 (WHO-Algeria, 1956); 1954 (WHO, 1957);
1977–1984 (Benzerrough and Janssens, 1985); 1985–2007 (Hammadi et al., 2009);
2008–2009 (Richard Cibulskis, Personal Communication). Case data converted to annual
incidence between 1948 and 1960 (Goldewijk and Batthes, 1997); 1969–1984 (CICRED,
1974) and census data for the years 1998 and 2008 from ONS, Algeria (2011). Between
census years intercensal growth rates computed to estimate populations. Note no case
data available for review for period 1955–1976; zero cases reported in years 1985, 1989
and 2009; Annual incidence in 2005 and 2006 was 0.003 cases per 100,000 population.
reported from the southern region among an average annual population

of 100,000 residents and represented an average annualized incidence of
less than 1 locally acquired P. falciparum case per 10,000 population at risk
(Boubidi et al., 2010; Hammadi et al., 2009). Small residual foci of
P. falciparum and P. vivax transmission continued to be reported at Tin-
zaouatine in the south between 2003 and 2007, thought to be a result of
suitable local conditions for the vector An. sergentii, and the area is located
on the trans-Saharan highway connecting Algeria to Mali and Niger
(Boubidi et al., 2010). There were no locally acquired cases in 2009 and
2010 (Richard Cibulskis and Ryan O’Neil, Personal Communication).
4.4.1.3. Tunisia
Prior to the First World War larval control, environmental management
and ‘‘quininization’’ were focused in areas of European settlement
(Husson and Nicolle, 1907; Sergent and Sergent, 1906). Epidemics in
1911 and 1933 in Tunisia served as incentives for government responses
and public health action. The epidemic of 1932–1933 doubled the case
incidence in all provinces compared to 1927–1931 (Chadli et al., 1985) and
resulted in 10,000 deaths in the lakeside area of Khelbia (WHO-Tunisia,
1956). During the years 1934–1944, similar approaches to malaria control

to those designed by Algeria were implemented including the use of
larviciding and the mass chemoprophylaxis in the regions of Cap Bon
and Gabès with prémaline (properties of primaquine/chloroquine)
(Decourt et al., 1936; Wassilieff, 1938; WHO-Tunisia, 1956). Over
11 years after the Second World War, 1944–1954, an average of 6500
cases per year were reported in Tunisia among an average population
of 3.8 million people, approximating to 17 cases per 10,000 population at
risk (WHO-Tunisia, 1956; Fig. 4.4). By 1955, amodiaquine was the pre-
ferred drug for prophylaxis. The Tunisian Republic gained independence
from France in 1957; between 1961 and 1966, an aggressive approach to
malaria control was mounted using DDT and a malaria elimination cam-
paign was announced by the Government of Tunisia in 1967. All of the
Northern provinces, where the dominant vectors are An. labranchiae and
An. multicolor, were malaria free by 1968 (Ambroise-Thomas et al., 1976).
Between 1968 and 1977 activities included nationwide active case detec-
tion and radical treatment alongside focal IRS with DDT and larviciding.
By 1972, Tunisia had entered the consolidation phase of elimination and
the foci of remaining transmission were located in most southerly part
of Sfax Governorate, and the three southern Governorates of Gafsa,
Gabes and Medenine where transmission was predominantly by An.
sergentii. The last three autochthonous P. vivax cases of malaria were
officially recorded in 1979. A large-scale school-based serological survey
was conducted between 1990 and 1991 across 20 Governorates including
70 3
60
Annual malaria incidence
Annual malaria incidence
(per 100,000 population)
2
(per 10,000 population)
50
40 2
30 1
20
Last autochthonous
1 case declared in
10
1979
No data
0 0
1949
1970
1975
1980
1985
1990
1995
1934
1939
1944
1954
1959
1969
1964
FIGURE 4.4 Tunisia. Annual malaria case incidence per 10,000 1934–1969 (left hand
panel) and slide confirmed, locally acquired case incidence per 100,000 1970–1995 (right
hand panel). Case data from 1935–1938 to 1955–1978 (Chadli et al., 1985); 1944–1954
(WHO- Tunisia, 1956); 1980–1995 (Mondher, 2010); No data available for review for the
periods 1939–1943. Population data for whole country used to reflect national changes in
incidence from 1925 to 1955 (Goldewijk and Batthes, 1997); 1966, 1975, 1984, 1994 and 2004
(National Institute of Statistics, Tunisia, 2011). Non-census years computed using annual
intercensal growth rates.
approximately 38,000 children none of whom were seropositive for

P. falciparum or P. vivax. The 10 years after 1979 covered a maintenance
phase that included active case detection in the ‘‘hot-spot’’ areas of
southern Tunisia, nationwide passive case detection accompanied by
health worker awareness and active follow-up of infected travellers.
4.4.1.4. Libya
The Kingdom of Libya was historically characterized by very focal trans-
mission around oases and settled farmlands in the southern region of
Fezzan sustained by An. sergentii and An. multicolor (Ramsdale, 1990) and in
the less arid areas to the West in Tripolitania maintained predominantly by
An. multicolor. An. labranchaie is limited in its extent to a small coastal strip
west of Tripoli (Manguin et al., 2008). Following the Italian occupation of
Libya, between 82 and 300 cases of P. vivax were reported from Tripolitania
(Anon, 1944-1950). In the south, it was presumed that P. falciparum was
more significant compared to vivax (Gebreel, 1982). The densely populated
Mediterranean coastal cities towards the East were not thought to sustain
significant transmission (Gebreel, 1982). In 1954, the health and sanitation
division of the United States Operation Mission (USOM) initiated a malaria
control programme (Anon, 1957). The first campaign, using DDT and mass
drug administration with Resochin (chloroquine), began in August 1955
covering 31 localities and reaching 51 localities by 1957 protecting approxi-
mately 23,300 people across the Fezzan Oases. In 1957, this was extended
further to the Taourga Oases. The WHO then began a partnership with the
Kingdom of Libya to launch a campaign of nationwide malaria elimination.
Following on from the USOM collaboration, the renewed elimination cam-
paign achieved rapid success with only 28 cases being reported by 1963
(Gebreel et al., 1985). No locally acquired P. falciparum or P. vivax cases were
reported in the Eastern region of Cyrenaica or Tripolitania from 1963. Cases
continued to be reported from Fezzan in the West including a resurgence of
falciparum malaria between 1964–65 through to 1968 when King Idiris I
was overthrown and the Libyan Arab Jamahiriya was established. Between
1968 and 1973, only 14 vivax autochthonous cases were documented in
Fezzan (Gebreel et al., 1985). There were no locally acquired cases reported
after 1973, and while the country was declared malaria free, in September
1980, an outbreak of vivax malaria, involving 18 subjects, occurred in
Zuara, a coastal town surrounded by marshland 70 km east of the Tunisian
border 120 km west of Tripoli and thought to have been introduced by
migrant workers (Gebreel et al., 1985).
4.4.1.5. Egypt
Across Egypt, both the extent and intensity of malaria risk have changed
over the past 150 years. The building of the Suez Canal under French
contract in 1869, the rapid irrigation of the Nile for agriculture including
lucrative cotton farming during the 1870 s under Ismail Pasha’s rule to
accelerate ‘‘modernization’’ and the building of the Aswan dam changed
the ecology of malaria transmission in Egypt. Perhaps most notable was a
rapidly changing epidemiology in the upper Nile region of Nubia where
An. gambiae s.l. ‘‘invaded’’ in 1942 from North Sudan (Shousha, 1948).
Malaria control began as early as 1900 when Ronald Ross recom-
mended environmental control methods at Ismailia near the recently
completed Suez Canal where in that year 2234 malaria cases were
reported, representing one-third of the town’s population (Bey and
Hussein, 1928; Halawani and Shawarby, 1957). In 1916, the High Malaria
Commission was established to develop a nation-wide malaria control
effort and led to the establishment of the Malaria Control Centre at
Khanka, north-west of Cairo. Between the two World Wars, activities
focused on attacking breeding sites in major towns and oases in the
Western Desert (Bey and Hussein, 1928). By the 1930s, An. pharoensis
was thought to be the predominant vector across much of Egypt
(Kirkpatrick, 1925). During the 1950s, An. pharoensis remained dominant
in irrigated areas and banks of the River Nile while An. sergentii and
An. multicolor were implicated as important vectors elsewhere (Kenawy,
1990; Madwar, 1938). The 1940s epidemic began in the south and eventu-
ally led to almost 38,000 cases reported during 1944 compared to an
average of 15,000 during the 5 years 1939–1943 (WHO-Egypt, 1956;
Fig. 4.5). The cause was the introduction of An. gambiae s.l. from Sudan.
An aggressive gambiae elimination programme successfully eliminated
the vector by 1948 (Shousha, 1948). This success encouraged further
focal eradication projects at Kharga and Dhakla Oases south west of the
Nile valley (Madwar and Shawarby, 1950). Prior to 1945, the
principal vector control methods included larviciding using oiling, Malar-
iol and Paris Green. From 1946, DDT was introduced first at the oases of
Kharkla, Dhakla and Siwa with increased frequency and coverage
through to 1952 and improved control with higher coverage by 1954 in
Fayoum Governorate. Gammaxene and Octa-Klor were used as
adjunct insecticides from late 1950s (Sobky, 1957). In 1940, approximately
50% of all malaria cases were due to P. falciparum in Lower Egypt
and Fayoum Governorate and over 70% in the Oases; by 1953, only
6% of all clinical infections were due to P. falciparum and the main
parasite had become P. vivax (Halawani and Shawarby, 1957). This
change in species dominance coincided with a dramatic decline in
incidence as defined by the slide positivity rates reported by
endemic disease hospitals in Upper and Lower Egypt that declined
from 31% in 1940 to 5.5% in Lower Egypt and 1.8% in Upper Egypt by
1953; with no cases or smear positives being recorded in the canal zone,
Assiut, Girga, Kom Ombo, Aswan and Nubia regions (Halawani and
Shawarby, 1957).
25 1.4

1.2
20
1.0
15
0.8
0.6
10
0.4
5
Last autochthonous
0.2 case declared in
1998
0 0.0
1939
1944
1949
1953
1979
1984
1989
1994
1999
2004
FIGURE 4.5 United Arab Republic of Egypt reported malaria case incidence 1939–1953
per 10,000 (left hand side) and 1979–2004 per 100,000 (right hand side). Annual reported
malaria cases sourced for 1939–1953 (WHO-Egypt, 1956); 1979 (Anon, 1981); 1986 and 1987
(WHO, 1989); 1980–1985 (EMRO-WHO, 1987); 1988 and 1991–1997 (WHO, 1999); 1989 and
1990; 1999–2002 (WHO-EMRO, 2011) and 2003 and 2004 (WHO-Egypt, 2010). National
population used throughout to highlight overall changing incidence 1927, 1937, 1947, 1960,
1966, 1976, 1986, 1996, 2006 from CAPMAS, Egypt (2011). Non-census years computed
using annual intercensal growth rates.
The 1970s witnessed a series of epidemics across the country; how-

ever, from 1979, national case incidence had fallen to below 1 case per
10,000 population, and by 1987, it was reported that there were only 22
locally acquired cases with transmission predominantly in El Fayoum
Governorate. Between 1982 and 1991, malaria cases were reported in
seven governorates: Port Said, Suez, Shakira, Menofia, Beni Suef, Aswan
and Fayoum; however, the cases in all governorates except Fayoum were
very few (Hassan et al., 2003). It seems reasonable therefore to assume
that P. falciparum and P. vivax incidence was unstable for six governorates
between 1980 and 1990 and free of malaria from 1990; however, Fayoum
Governorate remained a stable endemic focus of P. falciparum malaria
through the 1980s to the 1990s with epidemics in 1989 and 1994–1995.
Fayoum is 1800 Km2 and has a unique ecology situated in an irrigated
area fed by the Bahr Youssef tributary of the Nile that ends in the Kaun
Lake and the area lies 20 m below sea level which combined provides
very suitable conditions for An. sergentii (Kenawy et al., 1990; Morsy et al.,
1995). Between 1991 and 1997, all locally acquired cases in Egypt came
from Fayoum including an epidemic of 495 and 313 cases in 1994 and
1995, respectively. Since 1998, there have been no officially reported

autochthonous cases in this governorate or elsewhere in Egypt (Fig. 4.5).
A serological screen of 2800 children aged 1–5 years living in 12 villages in
Fayoum for the detection of specific IgG antibody against pan P. falci-
parum, P. vivax, P. malariae and P. ovale resulted in a seroprevalence of
0.7% but might have been due to cross-reactivity with non-malaria anti-
gens (El Mohamady, 2010), and positives were later confirmed as sero-
negative in another laboratory (Hoda Atta, personal communication). We
therefore assume that the United Arab Republic of Egypt had focal
P. falciparum and P. vivax risks between 1980 and 1999 but that the entire
country was malaria free from 1998 (Fig. 4.5) despite a high malariogenic
potential in Fayoum and Aswan.
4.4.1.6. Djibouti
The French governed territory of the Issa’s and Afar’s (French Somali-
land) is likely to have experienced endemic transmission around Ambouli
before 1910 (Bouffard, 1905); however, the entire territory was regarded
as malaria free from 1910 up to 1973, 4 years before independence in 1977
(Carteron et al., 1978; Mohamed, 1990; Rodier et al., 1995; WHO-Djibouti,
1956). This small country borders the Danakil depression, one of the
hottest places on earth, and large parts of the country are barren rocky
deserts with erratic rainfall averaging 130 mm per year. Anopheles d’thali
was thought to be the historical, potential vector; however during the
early 1970s, an extensive entomological survey across the country could
not identify any malaria vectors (Courtois and Mouchet, 1970). Sixty
percent of the population of the Republic live in Djibouti ville, connected
to Ethiopia by the Addis Abba–Dire Dawa–Djibouti Railway that during
the 1970s served as a route for large refugee populations that expanded
the outskirts of the city and led to urban informal agriculture.
From 1988, malaria epidemics from imported infections began to
appear and led to onward transmission among local resident commu-
nities (Louis and Albert, 1988; Manguin et al., 2008; Rodier et al., 1995).
An. arabiensis is now accepted as the dominant vector of P. falciparum
around Djibouti city particularly among the wadis, agricultural areas
and watering holes around the Ambouli region. Some have argued that
both An. arabiensis and P. falciparum arrived by train from Ethiopia (Fox
et al., 1991; Rogier et al., 2005). From all available evidence, the Republic
of Djibouti was probably malaria free up to 1980; between 1988 and 2007,
reported case incidence ranged between 60 and 120 cases per 10,000
population per year (Osman, 2008; PNLP-Djibouti, 2006, 2011). Since
2008, case incidence has begun to decline to levels of less than 1 case per
10,000 population in 2010 (Hawa Guessod, Personal Communication).
This recent change is reflected in declining slide positivity at two hospi-
tals in Djibouti ville (Ollivier et al., 2011). A seroprevalence survey in 2009
among 4687 people across Djibouti found 1.6% seropositives to P. falci-

parum AMA-1 and MSP16 antigens and not related to recent travel his-
tories (Noor et al., 2011; PNLP-Djibouti, 2009) confirming an unstable
endemicity where transmission is possible.
4.4.2. Changing boundaries and incidence of malaria on the

islands of Africa
4.4.2.1. Cape Verde
The Republic of Cape Verde is an archipelago of 10 (only 9 populated)
volcanic islands in the Atlantic Ocean off the Coast of Senegal. The islands
were uninhabited until used by Portuguese slavers in the fifteenth cen-
tury. The Creole populations across the islands vary considerably in
population density; 25% of the Republic’s population today live in the
city of Praia on Santiago Island. The islands are grouped according to
their windward position: the Barlavento Islands (Santo Antao, São Vicente,
Sta Luzia, São Nicolau, Sal and Boavista) and the Sotavento Islands
(Maio, Santiago Fogo and Brava). Independence from Portugal was
achieved in 1975. Interest in the epidemiology and elimination of malaria
by Portuguese malariologists dated back to the 1930s when extensive
surveys of infection and disease prevalence were undertaken by members
of the Permanent Mission in Cape Verde from the Instituto de Medicina
Tropical, Lisbon (Cambournac and De Meira, 1952; De Meira 1954, 1964;
Monteiro, 1952). Between 1938 and 1954, a total of 201,682 malaria cases
were documented representing an average case incidence of 800 per
10,000 population (Fig. 4.6). Cases were both falciparum and vivax
although predominantly falciparum and were reported from all of the
inhabited Islands (WHO-Cape Verde, 1955). An. pretoriensis is a disputed
vector on the islands (Joana Alves, personal communication) while An.
arabiensis is the widely accepted vector with some doubt over its presence
on São Nicolau (Cambournac et al., 1984; Ferriera, 1945; Joana Alves,
personal communication).
In 1948, a malaria elimination campaign was launched starting on the
island of Sal using DDT, oiling of larval breeding sites and more latterly
with the introduction of Gambusia affinis predatory fish. The campaign
extended to other Islands throughout the 1950s. The campaign was suc-
cessful and malaria was felt to have been eliminated through the removal
of the vector in Sal (1950), São Vicente (1954), Boavista and Maio (1962)
and Santiago (1968) (Cambournac et al., 1984; De Meira, 1963). Although
claimed, malaria-free Santiago still had cases in 1973. Frequent popula-
tion movements between the islands, mainland Africa and Brazil with
increasing air travel always presented a threat to reintroduction of both
vectors and parasites (Cambournac et al., 1984). With the exception of
Santiago, no autochthonous cases were detected for many years on any of
1000 35

900
30
800
700 25
600
20
500
15
400
300 10
200
5
100
1.69
0.04
0.04
0.05
0.04
No data
0 0
1934
1939
1944
1949
1954
1959
1963
1964
1669
1974
1979
1984
1989
1994
1999
2004
2009
FIGURE 4.6 Cape Verde: Annual slide-confirmed malaria case incidence per 10,000 population 1934–1963 (left hand side) and annual,
locally acquired, slide-confirmed case incidence per 100,000 population 1964–2010. Data sources used include 1934–1952 (De Meira, 1954);
1960–1983 (Cambournac et al., 1984); 1984–1985 and 1987–2006 (PNLP-Cape Verde, 2009); 2007–2010 (Joana Alves, personal communication).
No reports available for review for the period 1953–1960. Case incidence computed for entire country per year to highlight changing
national incidence and not per remaining islands at risk, denominators derived for census years 1940, 1950, 1960, 1970, 1980, 1990, 2000 and
projections 2001–2010 (INE Cape Verde, 2011) and non-census years computed using intercensal growth rates. The years 1968–1972 and
1983–1986 no locally acquired cases reported.
the islands since they were declared malaria free, despite imported cases
being detected in almost all islands. In 1973 on the island of Santiago, 148
cases were reported leading to onward transmission of both P. vivax and
P. falciparum (Fig. 4.6) and served as a stimulus to renewed application of
DDT, use of Gambusia fish to supplement chemical larviciding and the use
of chloroquine chemoprophylaxis under a new directorate, the Brigada de
Luta contra o Paludismo in 1977.
In 1979, a further national elimination programme was launched and
the focus was on Santiago with renewed efforts targeting the vector with
DDT and larvicides (temephos). The entire archipelago was returned to
zero incidence between 1983 and 1986. The following year transmission
re-established itself on Santiago and heralded a period of annual cases
being detected despite increased vigilance (Alves, 1994) through to 1995–
1996 when an epidemic occurred in St. Catarina district on Santiago
originating from sub-patent and chloroquine resistance asymptomatic
carriers (Alves et al., 2006, 2009). Current approaches to eliminate malaria
on Santiago include active case detection and case investigation, the use of
artemether–lumefantrine for treatment (since 2008), mefloquine for pro-
phylaxis for travellers, temephos for larviciding and very limited use of
IRS (deltamethrin) for epidemic containment and ITN. Currently, locally
acquired case incidence is below 1.0 per 10,000 on Santiago. On Boavista,
four possible autochthonous cases were detected in 2003, the first since
1962, 10 cases in 2009 and three in 2010. The long-term case incidence data
are shown in Fig. 4.6.
4.4.2.2. São Tomé and Prı́ncipe

The Democratic Republic of São Tomé and Prı́ncipe is made up of two
volcanic islands 140 km apart in the Gulf of Guinea, 250 km from Gabon
on mainland Africa. Like the Cape Verdean islands, they were uninhab-
ited before the Portuguese occupied them for trade in the 1470s. The
volcano topography and plantation agricultural economy define the
transmission of malaria on the two islands (Ceita, 1981). Sao Tomeans
achieved independence from Portugal in 1975. Over 96% of the present
population, of 162,000 people, lives on São Tomé.
Between 1942 and 1944, approximately 5000 cases were documented
on São Tomé ( Joaquim and de Mesquila, 1946); over the period 1946 and
1953 on both islands, an average of 10,000 cases were reported per year
among a population of only 60,000 people, and 25–37% of slide examina-
tions at dispensaries were positive for P. falciparum (WHO-São Tomé and
Prı́ncipe, 1955). In 1955, IRS using DDT and gammexane was limited to
major settled, urban and peri-urban areas and larviciding was addition-
ally used in the town of São Tomé. Over 20,000 people were protected
with mass drug administration/intermittent treatment with chloroquine,
atebrin, paludrine and camoquine (WHO-São Tomé and Prı́ncipe, 1955).
During the late 1970s, a proposal for malaria elimination was redeve-
loped involving epidemiological surveillance with active and passive
screening, radical treatment with chloroquine and primaquine recogniz-
ing the presence of P. vivax on the islands (Pinto et al., 2000a,b), weekly
prophylaxis with chloroquine among selected groups, special screening
at airports and the use of DDT for IRS (Ceita, 1981). By 1980, parasite
prevalence on both Islands had declined to less than 5% (Ceita, 1986).
Owing to a lack of financial support, the programme became less vigilant,
chloroquine resistance emerged and doubts were raised about the sus-
ceptibility of the dominant vector An. gambiae s.s. to DDT (Ribeiro et al.,
1988, 1992).
From 2004, a renewed effort at country-wide IRS using alphacyperme-
thrin was implemented, managed by the Centro National de Endemias,
augmented with the use of LLIN from 2005 and application of Bacillus
thuringiensis israelensis (BTI) following larval mapping exercises and mass
screening and treatment and use of artesunate–amodiaquine for treat-
ment (CNE, 2006). On the smaller island of Prı́ncipe, cases among a
population of approximately 6500 declined from 2537 in 2003 to 51 in
2009 (75 per 10,000 population) (Lee et al., 2010). These successes were
repeated with similar approaches on the island of São Tomé which
achieved almost 100% coverage of the population with LLIN and IRS
(Teklehaimanot et al., 2009; Tseng et al., 2008). On São Tomé, parasite
prevalence declined from 30% to 2.1% by 2007 (Teklehaimanot et al.,
2009), and by 2009, case incidence was 247 per 10,000 population at risk
(WHO, 2010). Impressive reductions in infection prevalence, disease and
mortality incidence have resulted from aggressive and comprehensive
combinations of vector control, screening and treatment. The declining
malaria mortality rates since 2000 are particularly impressive, yet it is
notable that malaria mortality on the islands was probably at its peak
during the early 2000s when compared to previous pre-elimination his-
torical periods (Fig. 4.7). The recent scaled efforts and reductions in
disease incidence are further notable as they have occurred during diffi-
cult periods in the islands’ history with two attempted military coups in
2003 and 2009. On both islands, malaria incidence reflects a stable trans-
mission state by 2009 similar to the late 1970s, neither Island has ever
reached a malaria free or unstable endemic status but the future cycle of
investment in elimination may transform these islands to unstable or
malaria-free conditions.
4.4.2.3. Zanzibar
Zanzibar is composed of two large islands, Unguja (Zanzibar Island) and
Pemba (40 km North-East of Zanzibar) and several smaller islands. The
islands are only 25–50 km from mainland Tanzania. The islands were
governed as part of the Omani Sultanate and as a British Protectorate
250
Malaria mortality rates (per 100,000 population)
200
150
100
50
0 No data
1948
1954
1972
1976
1979
2000
2005
2009
FIGURE 4.7 São Tomé and Prı́ncipe. Annual malaria-specific mortality per 100,000
population. Mortality data sourced from several publications: 1948–1954 (WHO São
Tomé and Prı́ncipe, 1955); 1972–1979 (Ceita, 1981); No data available for review for 1977;
2000–2009 (Teklehaimanot et al., 2009). Population data used for 1955 (WHO São
Tomé and Prı́ncipe, 1955) and 1981–2006 (Instituto Nacional de Estatistica, ST&P 2006).
Non-census years computed using intercensal growth rates.
(1890) until a brief independent Sultanate in 1963 followed by civil war

and the overthrow of the Sultan in 1964. Zanzibar then became part of the
United Republic of Tanzania while retaining its own parliamentary and
governance system under the Revolutionary Government of Zanzibar. In
terms of malaria control, it has always operated independent of mainland
Tanzania, and therefore, we consider a separate territory. Between 1923
and 1933, an average of 6800 malaria cases were recorded per year across
a combined Zanzibar and Pemba population of approximately 280,000
residents and accounted for over 25% of all clinic consultations (Zanzibar
Protectorate, 1923-1966). A larval survey of the island of Zanzibar in 1919
identified An. gambiae and An. funestus as principal vectors (Mansfield-
Aders, 1920), subsequent investigations have found An. merus on Pemba
but not on Unguja (Schwartz et al., 1997). A detailed parasitological
survey among children aged 1–6 years at 26 locations of the island of
Zanzibar, including Tumbatu Island in the north, found an overall preva-
lence of 67% and noted the presence of both P. falciparum and P. vivax
between 1923 and 1926 (Mansfield-Aders, 1927). Spleen rates among
school children remained in excess of 50% on both Pemba and Zanzibar
between 1930 and 1966 (Zanzibar Protectorate, 1923-1966). By 1953, only
limited control was mounted involving larviciding of swamps with oil
and use of Paris Green in ‘‘crab holes’’. DDT was only used in private
residences at a fee and free of charge at all government employees houses
in Zanzibar town (WHO-Zanzibar, 1955).
During the 1960s, Zanzibar mounted a campaign of biannual cycles of

IRS using DDT followed by mass drug administration with amodiaquine
and primaquine and a combination of chloroquine and pyrimethamine
(Delfini, 1969; Dola, 1974; ZMCP, 2009) with a view to interrupting trans-
mission. The programme was successful, reducing parasite prevalence to
6.8% on Zanzibar and 0.8% on Pemba by 1967 (Delfini, 1969). Vigilance and
interest in the final effort to eliminate transmission waned as malaria was
perceived to no longer be a major public health burden (Schwartz et al.,
1997). A second attempt to control, rather than eliminate, malaria was
mounted by the Zanzibar Malaria Control Project (ZMCP) with funding
from the United States in 1984 using two rounds of DDT house spraying
each year by mobile malaria teams and improved use of chloroquine at
dispensaries. However, by 1983, chloroquine resistance had begun to esca-
late (Schwartz et al., 1983), and between 1981 and 1987, mean mortalities of
exposed An. gambiae s.l to DDT were less than 50% (Schwartz et al., 1997).
The programme was abandoned in 1989 after failing to show any percepti-
ble changes in parasite rates at clinics (Schwartz et al., 1997).
In 2001, the Ministry of Health and Social Welfare decided to adopt
ACT, making it one of the first countries to do so in Africa and since 2002
secured substantial funding from the GFATM and US PMI to improve
case management and expand coverage of ITN and IRS using lambda-
cyhalothrin. This programme did not anticipate elimination but followed
international recommendations to halve the malaria burden. Coverage of
vector control remained low by 2004. From 2005 onwards, this began to
change with more than 70% of under-fives and pregnant women sleeping
under an ITN and 96% of houses were covered with IRS by 2008. Parasite
prevalence in young children sampled in the community in 2002 was 47%
and declined to 0.9% by 2008 (ZMCP, 2009). From 2004, Zanzibar began to
witness a precipitous decline in malaria incidence, hospitalizations and
blood transfusions (Aregawi et al., 2011; Bhattarai et al., 2007; ZMCP,
2009). Between 1999 and 2003, there were between 15,000 and 18,500
confirmed cases of malaria each year; in 2005, this declined to 7600
cases. By 2010, 5000 parasitologically confirmed cases were identified
through enhanced surveillance, and in two sentinel areas, community-
based parasite prevalence remained below 1% (Abdullah Ali, personal
communication). Using case incidence and parasite prevalence data, it is
most reasonable to assume that the Zanzibari islands are in a state of low-
stable endemic control and that at no time in the history of elimination
efforts on the islands had they reached unstable conditions.
4.4.2.4. Réunion
The island of Réunion is 200 km from Mauritius and 700 km from Mada-
gascar in the Indian Ocean. This small island is only 63 by 45 km and is
dominated by the Piton de la Fournaise (2631 m above sea level) and Piton
des Neiges (3070 m above sea level) volcanoes. Réunion was colonized by
the French in the 1600s and remains to this day an overseas department of
France. Over the past two centuries, there have been large in-migrations
from Africa, China, Malaysia, Vietnam and India. The island was thought
to have been malaria free before a large epidemic, probably from
imported infections from mainland Africa in 1868 that set in motion a
cycle of frequent, high-burden epidemics ( Julvez et al., 1990a). In 1949,
malaria parasite rates in school children suggested a hypoendemic state
(parasite prevalence < 10%) across the island with transmission of both
P. falciparum (28% of all infections) and P. vivax (66%) (Hamon and
Dufour, 1954). Nevertheless malaria was a significant cause of morbidity
and mortality: 17,459 clinical cases were confirmed in 1946 and 1779
deaths from malaria were recorded by the authorities in 1948 (WHO-
Réunion, 1955). The mortality rate on the island among all age groups,
7.35 per 1000, was equivalent to the presumed malaria mortality in young
children in Africa under stable, hyper-to holoendemic conditions (Rowe
et al., 2006; Snow et al., 1999). Before 1949, larviciding and the presump-
tive treatment of school children using chloroquine were the only meth-
ods used to control malaria.
In 1949, an elimination strategy was launched (Hamon and Dufour,
1954). Following a detailed housing structure and breeding site census of
the island, two divisions were created to stagger DDT house spraying that
began in October 1949 in the first sectors (Sous-le-vent). A year later, it
expanded to all areas on the island and continued annually through to
1953 accompanied by sustained use of chloroquine presumptive treat-
ment to school attending children. Overall parasite prevalence declined
from 2.9% in 1949 to 0.2% in 1952, and malaria mortality declined from 5.6
to 0.6 per 1000 population over the same period (Hamon and Dufour,
1954). After this initial attack phase, a period of consolidation of elimina-
tion efforts were mounted through larviciding of mapped breeding sites,
restricted use of DDT in focal transmission areas and active case and
entomological surveillance. Twenty-six locally acquired infections were
identified between 1956 and 1967 (Denys and Isautier, 1991; Riff and
Isautier, 1995). A mass screen of over 62,000 residents in 1966/1967
identified six possible autochthonous cases in the Mafate area and sur-
veillance identified five possible cases in Saint-Paul in 1971 (Picot, 1976;
Riff and Isautier, 1995). The WHO concluded that transmission had been
interrupted in 1973 and certified Réunion malaria free in March 1979.
Active surveillance since 1965 has included screening of immigrants and
air travellers (Guihard, 2006), and there are on average 150 imported cases
of malaria each year notably from neighbouring islands of Madagascar,
Comoros and Mayotte. The dominant vector, An. arabiensis, remains wide
spread and has not been eliminated (Girod et al., 1999; Morlais et al.,
2005), and the 810,000 residents of the country remain vulnerable to
imported malaria risks (D’Ortenzio et al., 2009; Denys and Isautier, 1991;
Girod et al., 1995; Guihard, 2006; Julvez et al., 1982; Lassalle et al., 2000;
Sissoko et al., 2006).
4.4.2.5. Mauritius
The Republic of Mauritius includes the islands of Mauritius, Cargados
Carajos, Rodrigues and Agalega. The archipelago is located in the south
western part of the Indian Ocean 900 km east of Madagascar. Only the
island of Mauritius has been identified as supporting malaria transmis-
sion. Mauritius was occupied first by the Dutch and French, who found
the islands uninhabited. As with Réunion, it is likely that malaria was
introduced onto the island of Mauritius in the mid-1860s by immigrant
labour (Ross, 1908) and led to a large epidemic in 1867 (Balfour-Kirk, 1934;
CDCU, MoH&QL, 2008). Ronald Ross completed an island-wide investi-
gation of spleen rates in 1906 and found an overall rate of enlarged
spleens of 48% and made recommendations for immediate sanitation to
reduce vector breeding sites (Ross, 1908). In 1910, Smith, reporting to the
Colonial Development Fund, estimated malaria death rates on the island
to be in excess of 12 per 1000 population per year (Smith, 1911).
Before the Second World War, there was very little active prevention
despite some reports of drainage of swamps and wide-spread use of
quinine. Between 1942 and 1943, P. falciparum infection prevalence
among children was 42%, P. vivax prevalence was 22% (Sippe and
Twining, 1946) and An. funestus and An. gambiae s.l. were implicated as
the sole vectors (Colony of Mauritius, 1950). Archived hospital and dis-
pensary returns and census interpolations suggest that there were large
between year variations in the annual incidence of malaria between 1930
and 1948, but most years showed more than 10% of the population
suffering from a clinical attack (Fig. 4.8); the average malaria-specific
mortality was 3.63 per 1000 per year among the entire population during
this period (Colony of Mauritius, 1928–1972).
Immediately after the Second World War, the Ministry of Health
began to implement some of the recommendations made by Ross
40 years earlier with major environmental works (canalization and clean-
ing of streams, drainage of marshes) and oiling of breeding sites. These
efforts concentrated on the Central Plateau, the town of Port Louis and the
drainage of two extensive marshes in Pamplemousses district. In 1948, to
tackle the high incidence on the rest of the island, the Colonial Insecticide
Committee proposed in conjunction with the Government of Mauritius a
Malaria Eradication Scheme (Colony of Mauritius, 1950; Dowling, 1951a,
b, 1952). In November 1948, a detailed housing census led to the creation
of three zones for the attack phase of elimination: Zone 1 using DDT (80%
pp in Kerosene); Zone 2 using DDT 50% Wettable Powder and Zone 3
using Gammexane 50% Wettable Powder. The first round of spraying
2500 70

60
2000
50
1500
40
30
1000
20
Last indigenous
500
case of P. vivax
10
malaria in 1997
0.14
1968 0.13
0 0
1927
1932
1937
1942
1947
1952
1957
1962
1963
1973
1978
1983
1988
1993
1998
2003
2008
FIGURE 4.8 Mauritius. Annual malaria incidence per 10,000 population 1927–1962 (left
hand side) and vivax incidence per 100,000 population 1963–2008 (right hand side).
Annual malaria cases sourced from 1927–1971 (Colony of Mauritius, 1931–1972); 1940–
1953 (WHO Mauritius, 1955); 1961 (WHO, 1967); 1970 and 1971 (WHO, 1971) and 1980–2008
(Communicable Disease Control Unit Mauritius, 2008). Population derived from 1927–
1960 (Colony of Mauritius, 1928–1972); 1961–2008 (CSO Mauritius, 2011) and intercensal
growth rates computed for non-census years to predict population between censuses.
Zero indigenous cases recorded in 1966, 1967, 1969–1972, 1990, 1991, 1993–1995, 1998–
2010. Last indigenous case of P. vivax malaria recorded in 1997 (Tatarsky et al., 2011).
began in January 1949. During the second spray round, the central area
was extended and the ‘‘barrier’’ technique was adopted by spraying of the
outskirts of the town of Port Louis and Mahebourg. The third spray round
began in 1950 and covered over 720,000 rooms providing protection for
over 614,000 people (Colony of Mauritius, 1950). Parasite prevalence
surveys in school children showed a drop from 9.5% infection rates in
1948 to 0.4% in 1950 (Colony of Mauritius, 1950), and the effects on case
incidence was immediate and dramatic (Fig. 4.8).
Between 1953 and 1956, case incidence was below 1 per 10,000 popu-
lation per year. By the end of the attack phase, An. funestus was virtually
extinct (Bryan and Gebert, 1976) while An. gambiae s.l. proved harder to
control notably in the area of Flacq. This led to a more aggressive phase of
breeding site identification and larval control. Between 1960 through to
the early 1970s, mass IRS was replaced with targeted use of DDT accom-
panied by active surveillance to identify residual foci using mobile teams
and screening of immigrants at ports. Apart from an excess of cases
identified in 1960, malaria incidence continued to decline and it was
assumed that local transmission had been interrupted in 1969, the year
after independence from Britain (Fig. 4.8). In 1972, a serological survey
among children living in Black River, high foci of previous transmission,
showed that immunoflourescent antibodies to P. falciparum and P. vivax
were present in less than 0.6% of children aged less than 5 years (Bruce-
Chwatt et al., 1973). The WHO certified Mauritius malaria-free in 1973
which prompted what turned out to be a rather premature article hailing

malaria as ‘‘dead as a dodo’’ (Bruce-Chwatt and Bruce-Chwatt, 1974). At
this point, surveillance vigilance declined as the responsibility for malaria
was absorbed into the wider health system (Tatarsky et al., 2011). In 1975,
P. vivax transmission established itself in village close to Port Louis likely
to have been imported from India. This initial importation event led to an
increasing vivax transmission across the island peaking in the mid-1980s
with over 500 cases each year (Fig. 4.8). Through the use of focal IRS
(DDT), widespread larviciding (temephos), passenger screening and an
up-regulated active case detection system, transmission was contained by
1990. With small vivax outbreaks in 1992 and 1996 (Fig. 4.8), the last
indigenous case recorded in 1997. Since 1998, Mauritius has maintained
the absence of local transmission. Mauritius therefore was able to elimi-
nate P. falciparum and P. vivax transmission in 1969, witnessed re-emer-
gence of P. vivax transmission in 1975 and achieved a second elimination
in 1998.
4.4.2.6. Comoros
Three islands formed the Federal Islamic Republic of Comoros at inde-
pendence from France in 1975, Grand Comore (1024 km2, rising to 2361 m
above sea level with the volcano of Karthala), Anjouan (424 km2 rising to
1578 m above sea level) and the lower altitude Mohéli island (374 km2) in
the Comorian Archipelago. In 1997, Anjouan and Mohéli unsuccessfully
sought independence from the union with Grand Comore. Under a new
constitution in 2001, the islands form an unstable Union of the Comoros
with each island having some political autonomy. The people of this
archipelago, including Mayotte, have been part of the evolving Swahili
corridor since the tenth century and comprise a mixture of Arab and
Bantu people. Altitude, settlement patterns and agriculture determine
the malaria risks across the three islands including malaria-free areas at
high altitudes on Grand Comore.
The first recorded severe epidemics occurred in 1920 (Raynal, 1928a).
An. gambiae and An. funestus are the dominant malaria vectors (Brunhes,
1977) of P. falciparum and the less commonly prevalent P. vivax (< 1%
parasite prevalence) (Blanchy et al., 1987, 1990). Between 1940 and 1943,
reported case incidence was approximately 1555 per 10,000 population
per year (WHO-Comoros, 1955). In June 1953, limited use of DDT was
applied on the islands of Grand Comore and Mohéli, and there is a
suggested use of chloroquine chemoprophylaxis in the 1950s (WHO-
Comoros, 1955). No significant malaria prevention seems to have been
reported up to the 1980s and transmission remained intense and stable.
During 1987, 3370 clinical cases were detected on Grand Comores (popu-
lation 223,600), 1788 on Anjouan (population 163,900) and 1294 on Mohéli
(population 20,400); parasite prevalence among children 2–9 years during
the same year was 51.4%, 23.3% and 44.6% on each of the islands, respec-
tively (Blanchy et al., 1987). In January 1987, a campaign to control malaria
and filariasis was mounted although details of precise activities and
approaches are difficult to establish. In 1988, the Programme National
de Lutte Contre le Paludisme (PNLP) was established. Between 1999 and
2001, case incidence remained high on all islands (Tchen et al., 2006), and
in 2006, malaria accounted for 36% of all clinic consultations (PNLP-
Comoros, 2009).
In 2007, a national plan of action was launched with the aim of
preparing the Comoros for pre-elimination in 2014 and eventual Inter-
ruption of transmission. The new strategy focuses on the wide-scale
distribution of ITN, IRS in selected areas with lambda-cyhalothrin, larval
control with predatory guppies, intermittent presumptive treatment of
pregnant women and enhanced clinical management using artemether–
lumefantrine all implemented with funding from the Global Fund and
some bilateral agency support (PNLP-Comoros, 2009). On the island of
Mohéli, in collaboration with scientists from China, mass treatment of
communities with artemisinin monotherapy (Artequick) and primaquine
as a follow-up treatment began in October 2007 reducing infection preva-
lence from 23% in September 2007 to 1.4% by January 2008 and a further
reduction to 0.4% by June 2009 (Anon, 2007; Bacar, 2010). Whether this
was continued and scaled as an intervention to Grand Comore and
Anjouan, despite WHO recommendations not to use artemisinin mono-
therapy (WHA, 2007), is unclear. By 2009, the PNLP had distributed
almost 170,000 ITN across the three islands by 2009 (WHO, 2010), and
during a mass-free distribution, campaign between November 2010 and
January 2011 on Grand Comore and Anjouan distributed a further 255,000
ITN. Among the 640,000 residents in 2009, over 51,000 presumed cases of
malaria were reported, of which only 10% were confirmed cases (WHO,
2010). Following the reduction of transmission on Mohéli as a result of
mass drug administration, it is not possible to estimate the stability of
endemicity due to the lack of corresponding case incidence data. For
Grand Comore and Anjouan, clinical incidence has probably remained
intense and stable over the past 100 years.
4.4.2.7. Mayotte
The two islands that comprise Mayotte, Mahoré (352 km2) and Pamanzi
(17 km2), are located within the Comorian Archipelago 320 km from
Madagascar and 70 km from the Comorian island of Anjouan. The islands
have been governed by France since 1841, and when the Federal Islamic
Republic of Comoros secured independence from France in 1975, Mayotte
elected to remain a French Territory Overseas. The majority of the popu-
lation live in approximately 70 villages that surround the coastline of the
island of Mahoré. Malaria has been intense and stable on the islands for
many years, and the only parasite identified among clinical cases had
been P. falciparum (Ali Halidi, 1995; Galtier and Blanchy, 1982); however,
cases of vivax have more recently been identified (Loos et al., 2006; Solet
et al., 2007). An. gambiae s.l. and An. funestus maintain transmission,
although An. funestus plays a lesser role (Brunhes, 1977). Some limited
use of dieldrin for IRS was applied in 1954, but there are few other records
suggesting much aggressive control until the 1970s. Parasite prevalence
was 36.5% among children living in villages on Pamanzi in 1972 (Galtier
and Blanchy, 1982). A programme of chloroquine prophylaxis among
school children was started on both islands in 1972 ( Julvez et al., 1990b).
A joint effort to eliminate two, high morbidity burden vector-borne
diseases, malaria and filariasis, was initiated in 1976. The malaria compo-
nent included chloroquine prophylaxis to school children and preschool
children attending dispensaries, IRS using DDT and malathion (subse-
quently, only malathion as culex vectors of filariasis was shown to be
resistant to DDT) and larviciding with temephos (Galtier and Blanchy,
1982). By 1981, coverage was high with 91% of households sprayed and
60% of school children reached with chemoprophylaxis. Among sentinel
villages, the overall parasite rate in all age groups was 25.5% in 1976 but
declined to 0.9% by 1980 (Galtier and Blanchy, 1982). Between 1981 and
1983, it is likely that malaria transmission on the islands was unstable;
however in 1984, early signs of reduced chloroquine efficacy were
observed from Comorian immigrants, and in this year, there was an
epidemic with 64 cases in May ( Julvez et al., 1987) and 394 throughout
1984 ( Julvez et al., 1990b). Parasite prevalence rose to 2.5%, and this
prompted an emergency intervention with IRS using quarterly rounds
of fenitrothion spraying, use of temephos and predator guppy fish
(Lebistes reticulatus) in mapped larval areas and increased active and
passive surveillance including serial, annual serological surveys ( Julvez
et al., 1986, 1987, 1990a,b). The use of chloroquine for chemoprophylaxis
was stopped except for pregnant women. By 1985, parasite prevalence
had declined to 0.3% and 75 clinical cases were reported for the year
( Julvez et al., 1987, 1990b). For the three years 1986, 1987 and 1988, only
8, 44 and 8 cases, respectively, were detected ( Julvez et al., 1990b), and it
is reasonable to assume that the islands had returned to an unstable
transmission state. Resurgent waves of transmission continued through
the early 1990s as identified from age profiles of serological detection of
falciparum-specific antibodies ( Julvez, 1993). A large epidemic occurred
in 1991 with 1724 cases detected through the active and passive surveil-
lance system and parasite prevalence had increased to 1.3% (Receveur
et al., 2004).
By 2001, malaria was the cause of over 1000 clinic presentations, 250
hospital admissions each year (Receveur et al., 2004; Tchen et al., 2006)
and resistance to chloroquine and sulphadoxine–pyrimethamine had
escalated (Roussin et al., 2002). In an attempt to tackle the high disease

burden malaria control in the 2000s focussed on the distribution of ITN to
pregnant women and newborn children, IRS using deltamethrin and the
distribution of rapid diagnostic tests to all clinics (Receveur et al., 2004).
Artesunate–mefloquine was recommended as first-line therapy in 2005
following documented high levels of resistance to chloroquine, pyrimeth-
amine, amodiaquine and quinine (Pettinelli et al., 2004). By 2003, malaria
incidence began to decline with a 25–40% reduction in cases detected
compared to 1999–2002 (Tchen et al., 2006). Cases are more concentrated
in the northern districts of the main island of Mahoré, most notably at
Bandraboua (Solet et al., 2007) where re-emergence of An. funestus has
been documented (Elissa and Karch, 2005). The complete interruption of
transmission on the islands of Mayotte has never been achieved and the
difficulties associated with elimination have been outlined by Receveur
et al. (2004). Brief periods of unstable transmission have been experienced
on the islands since 1939, and the changing status of risk since 1983 where
data are available is shown in Fig. 4.9.
200
180
160
140
120
100
80
60
40
No data
20
0
1983
1988
1993
1998
2003
2008
2010
FIGURE 4.9 Mayotte malaria case incidence per 10,000 population 1983–2010. Annual
malaria case data derived for period 1984–1988 ( Julvez et al., 1990a); 1983, 1989–1994 (Ali
Halidi, 1995); 1995–2004 (Tchen et al., 2006); 2005 and 2006 (Solet et al., 2007) and 2007–
2010 (Jean-Loius Solet, personal communication). No data available for review for the
year 1997. Population for Mayotte derived from Institut National de la Statistique et des
Etudes Economiques (INSEE) for the French Overseas Department, reviewed between
1985 and 1993 (INSEE, 2011a,b) and non-census years using intercensal growth rates.
4.4.2.8. Madagascar
The Republic of Madagascar is the fourth largest island in the world and
includes smaller islands located off its coastline including Nosy Be and
Sainte-Marie. The central highland plateau rises to 1341 m above sea level,
and this densely populated area is characterized by terraced, rice-grow-
ing valleys. It is likely that the first inhabitants arrived from Indonesia
between 300 and 500 years BC followed in the first millennia by Bantu
migrants crossing the Mozambique Channel. Immigrants from Arabia,
India, China, East Africa and Europe have led to a diverse population.
The island gained independence from France in 1960.
P. vivax has probably existed on the island for several centuries; how-
ever, it has been argued that P. falciparum was first introduced by the
French Foreign Legion during the war with the Kingdom in 1878 leading
to severe epidemics (Blanchy et al., 1993). For the past 100 years, the
distribution and intensity of malaria have been governed by the diversity
of ecology across the island, altitude, agriculture and changing human
settlement patterns and population growth (Mouchet et al., 1993). In 1923,
parasite prevalences in the northern part of Madagascar, Diego Suarez
(Antsiranana), were in excess of 64% (Raynal, 1928b) and the spleen rate
among children in the highlands, at Antananarivo, was over 77% in 1927
(Legendre, 1930). P. vivax was recorded in 20% of all malaria infections in
1927 (Legendre, 1930), but vivax now accounts for 6% of all infections and
is concentrated in highlands and the western coastline (PNLP-
Madagascar, 2007). An. gambiae s.s., An. arabiensis and An. funestus are
reported as the most important vectors (Ayala et al., 2006; Bernard, 1954;
Mouchet and Blanchy, 1995); however, their distribution and dominance
in transmission have changed with time (Curtis, 2002; Joncour, 1956).
The antimalaria service of Madagascar was reorganized in 1927
(Legendre, 1930). Between the two world wars, control focused on limited
drug prophylaxis, larval control using ‘‘stoxal’’, Paris Green, Gambusia
fish and drainage works (Bernard, 1950; Legrende, 1930). In 1948, DDT
house spraying began and by 1949 covered almost 25,000 houses in
Tananarive Province. This expanded in 1950 to approximately 46,000
houses in Tananarive (Antananarivo), Tamatave (Toamasina), Antsirabe,
Diego Suarez (Antsiranana) and the island of Santa Marie (Bernard, 1950).
By 1952, it was estimated that over 3 million people were protected
through the spraying of 680,000 households (Bernard, 1954). In addition
to IRS with DDT, the campaign included routine chemoprophylaxis with
chloroquine administered to school children and younger children at
dispensaries at a total of 4924 distribution sites (Bernard, 1954). Supple-
mentary activities included larval control notably in rice irrigation areas
including the use of Gambussia fish. Spleen rates declined from 40% in
1948 to 0.2% by 1953, and by 1952, parasite prevalence among 39,000
sampled children was 0.01% (Bernard, 1954). Crude mortality dropped
by a half between 1948 and 1952 in the town of Tananarive from 21 per
1000 residents to 12.8 and malaria mortality declined from 6 per 1000
population at risk to 0.4 per 1000 over the same period with only 3.7% of
all deaths attributed to malaria by 1952 (Bernard, 1954). IRS, chemopro-
phylaxis and larviciding continued through to 1955 when 50 of the 80
districts in Madagascar had become hypoendemic (< 10% spleen rates in
children aged 2–9 years) and 30 districts located largely on the West of the
Island were mesoendemic, with spleen rates of 10–49% ( Joncour, 1956;
WHO-Madagascar, 1955). Transmission in the highland plateau districts
was extremely low, An. funestus had largely disappeared and in Fianaran-
tosa district zero infection prevalence was recorded in 1955. By 1957, the
highland plateau was regarded as malaria free (Blanchy et al., 1993).
Continued efforts to maintain spraying were largely successful in
maintaining low levels of case incidence through to 1975 in the highland
plateau (Fig. 4.10; Blanchy et al., 1993; Bouma, 2003; Tchen et al., 2006).
Chloroquine prophylaxis (Nivaquinization) was maintained reaching
1400
1200
1000
800
600
400
No data
200
0
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
FIGURE 4.10 Antananarivo Province malaria case incidence per 10,000 population
1972–1989. Annual malaria case data derived for period between 1972–1974 and 1982–
1983 where data presented only as incidence (Bouma, 2003); 1975–1989(Blanchy et al.,
1993) and 1981 (Tchen et al., 2006). No data available for review for the year 1983.
Population for Antananarivo province derived from census bureau review of province
1975 and 1993 (Razafimanjato et al., 1997) and non-census years using intercensal growth
rates. Note in 1979, chloroquine chemoprophylaxis stopped (Blanchy et al., 1993) and
following rise in late 1980s DDT reintroduced in 1993. No data available for review for the
province after this date.
35% of young children and school children between 1977 and 1978 (Laing,
1984) but ended in 1979 (Randrianarivelojosia et al., 2009). About this
time, An. funestus reappeared in the highlands as a result of expanding
rice cultivation (Blanchy et al., 1993). Reduced sensitivity to chloroquine
was documented in 1981 (Le Bras et al., 1982) and became more wide-
spread by 1983 (Deloron et al., 1984). Epidemics occurred in the highland
plateau between 1985 and 1988 (Fig. 4.10). These epidemics had a devas-
tating public health impact and are thought to have doubled malaria-
specific mortality increasing during the late 1980s to over 1.9 per 1000
population (Mouchet and Baudon, 1988; Mouchet et al., 1998) and
prompted the return to routine DDT use in 1993 (Blanchy et al., 1993;
Jambou et al., 1998; Mouchet and Blanchy, 1995) accompanied by
enhanced surveillance (Albonico et al., 1999; Romi et al., 2002). Despite
focused intervention in the highlands, by 1999, the number of presumed
malaria cases in Madagascar exceeded 1.4 million (Tchen et al., 2006).
From 1998, the national malaria programme was reconfigured and
began the promotion of ITN and continued house spraying with combi-
nations of DDT and pyrethroids according to epidemiological stratifica-
tion of the island. Malaria in the highlands once again began to decline
( Jambou et al., 2001; Rabarijaona et al., 2006).
In 2004, it became policy to offer ITN free-of-charge across the island.
Despite day 28 failure rates of over 50% to chloroquine by 2004 (Menard
et al., 2008), home-based management of fevers was promoted using
socially marketed pre-packaged chloroquine. In December 2005, the Min-
istry of Health adopted amodiaquine–artesunate as first-line treatment.
In 2007, the Ministry launched a malaria elimination strategy that
included a preparatory phase and attack phase by 2012, a consolidation
phase to be completed by 2017 and maintenance malaria free-phase from
2018 (PNLP-Madagascar, 2007). Using funds from the GFATM, US PMI
and other bilateral agencies, 6.2 million LLINs were distributed between
2007 and 2009, covering an estimated 57% of the population at risk and
IRS protected 6.9 million people at risk in 2009. According to the WHO,
from 2006 malaria admissions to hospitals declined rapidly through to
2009 and there were only 173 reported malaria deaths in 2009 (WHO,
2010). However, it is hard to interpret these data without knowing the
location of the hospitals or the reliability of mortality reporting during the
year when a coup d’état led to major civil disruption.
Despite remarkable, rapid achievements in reducing transmission in
the Highland Plateau during the first malaria elimination campaign of
1948–1955, it is not clear from available evidence whether transmission
had been interrupted, but it seems reasonable to assume that the area was
rendered unstable through to 1980. The 1980s through to 2005 were
periods when stable transmission and high disease burdens were
reported in the Highland Plateau. P. falciparum risks were country wide,
while evidence suggests that P. vivax transmission is concentrated in the

Highland Plateau and Western districts (PNLP-Madagascar, 2007). Other
than the small area of temperature limiting transmission in Antsirabe,
Antanifotsy and Ambatolampy districts of Vakinankaratra province, at
no time has any other location in Madagascar been strictly malaria free.
4.4.3. Changing boundaries of stable malaria risk and disease

incidence in Southern Africa
4.4.3.1. South Africa
Assembled historical data from a variety of government and research
reports held at the Tzaneen National Institute of Tropical Diseases and
the National Archives in Pretoria were used by various authors to define
the limits of malaria transmission in 1938. Risks before expanded national
control extended to within Durban’s city limits along the Indian Ocean,
included Pretoria in the north and reached the railway crossing point at
Ramatlabama on the Botswana border (Le Sueur et al., 1993; Sharp and Le
Sueur, 1996; Strebel et al., 1988). Since the First World War, it is likely that
malaria transmission has been concentrated in the extended Transvaal
areas of the North-East and the wider Natal region in the South East.
Malaria impeded agricultural development from the turn of the last
century in Northern and Eastern Transvaal. Anti-larval measures started
in 1924 at irrigation sites south of the Hartbeespoort dam west of Pretoria.
Epidemics in 1928 across the Transvaal prompted investigations
(Swellengrebel, 1932) that led to the establishment of the Tzaneen Malaria
Centre and extensive work on breeding site identification and reduction,
education of farmers on personal protection, engaging the national rail-
ways to control vector breeding around stations and the promotion of the
use of quinine through 200 ‘‘quinine distributors’’. In 1944, a trial of
larviciding combined with pyrethrum house spraying was undertaken
at Springbok Flats. Only after the Second World War was progress made
in shrinking the 1938 margins of transmission in the Transvaal using a
strategy of focal elimination employing DDT IRS, continued targeted
larviciding and expanded use of quinine treatment. In the Transvaal,
4439 malaria cases were detected in 1939–1940 and this declined to only
128 by 1949–1950, located along the river tributaries of the Limpopo and
bordering the Kruger Game Reserve (Annecke, 1950). Attack and consoli-
dation phases continued from Western to Central Transvaal through to
the Lowveld from the 1950s and included a period from 1950 to 1969
when BHC was used in preference to DDT (Brink, 1958; Hansford, 1974,
1987). During the 1970s, annual and biannual rounds of DDT house
spraying and active house-to-house surveillance in the Transvaal region
focused on high-risk areas around the Limpopo, White and Crocodile
River valleys, Bushbuckridge, Letaba valley up to Nelspruit (Hansford,
1972, 1974). A WHO sponsored study of intensive active surveillance and

IRS was mounted in 1974 at Makonde that reduced autochthonous cases
from 42 to 10 by 1976 (Smith et al., 1977). Progress across the province
during the 1970s varied depending largely on aberrant rainfall patterns
that led to epidemics. The apartheid era was a period when South Africa’s
borders were rigorously policed, and very few imported cross-border
infections were detected relative to locally acquired cases and imported
infections came from bordering areas of Swaziland, Zimbabwe and
Mozambique. Transvaal was divided in 1994 to Mpumalanga, Limpopo,
Gauteng and North Western provinces. By the late 1980s, all local trans-
mission was restricted to defined areas of Limpopo and Mpumalanga
provinces. Malaria incidence began to rise during the late 1980s and early
1990s (Gerritsen et al., 2008). This increasing clinical burden was coinci-
dental with rapidly changing cross-border human population movement
from Zimbabwe and Mozambique, 5–10% of refugees from the civil war
in Mozambique being asymptomatic carriers of infection in 1985 (Frank
Hansford, personal communication), and emerging chloroquine resis-
tance (Bac et al., 1985; Philip Kruger, personal communication).
Epidemics were common in KwaZulu-Natal at the turn of the last
century. Hill and Haydon (1905) refer to the epidemic that caused 4177
clinical cases and 42 deaths in Durban in 1905. From 1910 screening of
dwellings, use of bed nets and personal protection including the prophy-
lactic use of quinine were recommended (Le Sueur et al., 1993). Severe
epidemics occurred in 1929 and 1932 and over 22,000 malaria deaths were
recorded by magistrates in 1932 (Le Sueur et al., 1993). Malaria Commit-
tees were formed from 1933 among sugar farmers who promoted larval
control, environmental management and the planting of eucalyptus (De
Meillon, 1936). In 1941/1942, experimental use of pyrethroids was used
for weekly house spraying (Hansford, 1987). From 1945, DDT replaced
pyrethrum and by 1956 had extended as far north as Ubombo and Ingwa-
vuma districts. Malaria Committees began to be disbanded from 1952; by
1965, only 36 autochthonous were detected across the province and rou-
tine DDT spraying was discontinued. Case incidence and spatial extents
continued to decline through the 1970s, although they varied depending
on rainfall (Sharp et al., 1988). By the late 1980s, over 90% of cases were
reported from the northern most districts of Ingwavuma and Ubombo
(Craig et al., 2004; Kleinschmidt et al., 2001; Mnzava et al., 1998). Cases in
KwaZulu-Natal started to increase in 1986–1987 and then began a dra-
matic rise from 1991 until over 40,000 cases were reported in 2000
(Fig. 4.11; Craig et al., 2004). The rise in case incidence followed the
replacement of DDT with Deltamethrin for IRS, increasing clinical failures
to chloroquine and rising malaria incidence in southern Mozambique.
DDT was re-instated as the preferred insecticide for IRS in 2000 as
resistance to pyrethroids was documented in KwaZulu-Natal among
50
45
Annual malaria incidence (per 10,000 KwaZulu
40
35
30
population)
25
20
15
10
5
2 1
0
1974
1979
1984
1989
1994
1999
2004
2009
FIGURE 4.11 Annual malaria case incidence in KwaZulu-Natal Province per 10,000
population 1974–2009. Annual Malaria Cases for KwaZulu-Natal 1974–2005 (Craig et al.,
2004; Marlies Craig, unpublished data); 2006 and 2007 (DoH South Africa, 2008); 2008
and 2009 (Rajendra Maharaj, unpublished data). Population has been estimated using the
1996 population census, 20.7% of South Africa’s population lived in KwaZulu-Natal
Province and intercensal growth rates between 1974 and 1991. Provincial population data
for period post-1991 sourced from STATSA (2011).
An. arabiensis close to Mozambique border (Maharaj et al., 2005). First-line

treatment policy was changed in KwaZulu-Natal to ACT in 2001 (Barnes
et al., 2005). Drug policy changes to ACT followed in 2002 in Mpumalanga
and 2004 in Limpopo.
By 2009/2010, less than 6000 cases were detected across South Africa
reflecting a decline since 2000 but not a return to case incidence rates that
prevailed in the 1970s (Fig. 4.12). The largest declines were witnessed in
KwaZulu-Natal Province and less dramatic declines recorded in Limpopo
and Mpumalanga Provinces. By 2010, case incidence was focal and unsta-
ble along a restricted margin from Zimbabwe running south through to
the eastern river valleys in Inkumanze district in Mpumalanga (Aaron
Mabuza, personal communication) and the districts Ingwavuma and
Ubombo in KwaZulu-Natal. There remain practical difficulties in defin-
ing locally acquired versus imported cases across South Africa. Over the
past 5 years, there have been increases in cross-border movement among
the Gaza communities from Mozambique to Gaza settlements in South
Africa across the Kruger National Park; increasing migration from Zim-
babwe to Mutale sub-district in Limpopo and a more significant threat is
posed by the massive immigration that occurs at Beitridge, Limpopo that
has processed, without malaria screening, up to 300,000 economic and
Annual malaria incidence (per 10,000 resident Kwazulu-Natal, 40
35
Mpumalanga, Limpopo population)
30
25
20
15
10
0
1970/1971
1975/1976
1980/1981
1985/1986
1990/1991
1995/1996
2000/2001
2005/2006
2008/2009
FIGURE 4.12 South Africa. Annual malaria case incidence per 10,000 population 1970/
1971–2008/2009. Annual malaria incidence in 1970/1971 was 0.12 per 10,000 popula-
tion—not visible on the graph. Malaria case data provided for period 1970/1971–1980/
1981 (DoH South Africa, 2008); 1981/1982–1995/1996 (WHO, 1999); 1996/1997–2008/
2009 (WHO, 2010). Note cases reported in South Africa for periods July–June and graph
shows starting July 1972 and ending June 2009; it has not been possible to define locally
acquired infections from imported infections from the data available, but from 1999, the
more imported infections were likely than locally acquired. No national data were
available for review for the reporting year July 2009–June 2010. To compute incidence
resident populations in Kwazulu-Natal, Mpumalanga and Limpopo provinces have been
used (STATSA, 2011). Estimates prior to 1991 assume that 39.8% of South Africa’s total
population resides in these three provinces.
political migrants from central and horn of Africa in recent years (Philip
Kruger, personal communication). Many of these migrants move rapidly
to non-receptive areas of Gauteng Province but some remain in the more
malaria receptive areas of Limpopo and Mpumalanga.
4.4.3.2. Namibia
De Meillon conducted an opportunistic survey of communities across
South-West Africa in 1950 and used information on vectors, spleen
rates, parasite rates and reports from local school, railways and mission
authorities to define four zones of transmission (De Meillon, 1951). Areas
in the north including the Ovamboland, Bushmanland and Caprivi were
regarded as intense, stable transmission, while the most southerly areas
from Grootfontein and Franzfontein to the Orange River were likely to be
free of transmission or very focal pockets of occasional transmission
(De Meillon, 1951). The Ministry of Health and Social Services has always
regarded the southern provinces of Karas and Hardap as malaria free
(MoHSS, 1995, 1996) and supported De Meillon’s observations in the
1950s (De Meillon, 1951). It was not until 1965 that a campaign of IRS
was launched using DDT and bendiocarb in urban residential houses in
the North. A malaria public health specialist was provided by South
Africa to establish a network of malaria health inspectors in the areas of
Ovambo and Kavango along the Angolan border in the mid-1960s and
this led to the rapid expansion of DDT house spraying across these areas
with almost 1 million houses sprayed each year by 1970. This programme
was managed from Windhoek with regional officers at Oshakati and
Runtu; however, due to accessibility, the Caprivi area was managed
directly from Pretoria and IRS was less complete in this region (Frank
Hansford, personal communication; Hansford, 1990). Annual mass-blood
surveys and treatment with Darachlor began in 1969; slides were read at
Tzaneen in South Africa and results returned to guide the mapping of
high-risk areas for the next annual spray rounds. Despite the war for
independence mounted by SWAPO in the northern territories, which
led to regular movement across Angola’s borders and periodic disruption
of basic services, IRS control continued although costs and supply began
to impact on coverage by the early 1990s. All Northern provinces have
continued to support stable P. falciparum transmission since 1950, and
following wide-scale use of DDT for IRS almost exclusively maintained
by An. arabiensis. IRS was never mounted in the more southerly districts as
parasite prevalence was intrinsically low.
Independence in 1990 unfortunately coincided with a large malaria
epidemic. In 1991, the national malaria control programme was launched
as part of the National Vector Diseases Control Programme. In 2004,
chloroquine was replaced with artemether–lumefantrine following
increasing chloroquine treatment failures and deltamethrin replaced
bendiocarb for spraying of modern structures. With support from the
Global Fund, distribution of ITNs began in 2000; by 2009, 22% of the
population in the Northern provinces were sleeping under a treated net
and 22% of households had been sprayed within the past year (MoHSS,
2010). Reliable health information on malaria diagnoses is not available
for the years during German occupation or during subsequent Union of
South Africa rule. A concerted effort to improve parasitologically diag-
nosed cases was mounted in 2004 and recent data are hard to interpret
against changing diagnostic practices.
4.4.3.3. Botswana
Malaria risk in the Republic of Botswana, formerly the British Protectorate
of Bechuanaland until independence in 1966, is constrained by latitude
and the Kalahari Desert that makes up 70% of the country’s land mass. In
1958, 98% of all malaria cases were reported from two districts, Ngami-
land and Chobe in the North in areas surrounding the Okavango and
Chobe swamps fed by the Zambezi River (Bechuanaland Protectorate,
1928-1963). Commenting on the combined parasitological data and clinic
returns for the year 1960, the medical department regarded the southern-
most districts of Tsabong through to Gaborone as malaria free but subject
to introduced risks from neighbouring Transvaal (Bechuanaland
Protectorate, 1928-1963). During a national parasitological survey in
1961–1962, no sampled infants were found to harbour infections in
Tsha, Loda, Gaborone, Kanye, Moduchi, Tuli and Ghanzi areas (Franco
de et al., 1984a).
Between 1958 and 1962, few cases were reported along the Limpopo
River, and in 1959, discussions began with the Transvaal Medical Depart-
ment of the Union of South Africa to start cross-border activities in
support of malaria elimination. Malaria control focussed on larval reduc-
tion strategies and the use of DDT for house spraying in major towns
before 1955 and was regarded as successful in reducing the case incidence
in major towns such as Maun, Francistown, Mhalapye and Serowe by
1956 (WHO-Bechuanaland, 1955). The medical department of the Bechua-
naland Protectorate undertook extensive reconnaissance of malaria risks
through school-based parasitological surveys from 1959 to 1962 (Bechua-
naland Protectorate, 1959-62). These mapped data were used to prepare a
malaria elimination strategy with the WHO in January 1961. The use of
DDT for IRS was irregular and incomplete between the 1950s and 1971,
focussed largely in Ngamiland, Chobe and Francistown (Franco de et al.,
1984a). In 1971/1972, fenitrothion was used briefly before being aban-
doned the following year (Franco de et al., 1984a; Mabaso et al., 2004). The
Botswana National Malaria Control Programme was reorganized in 1980
with headquarters at Maun. Improved biannual IRS using DDT use was
employed in the most malarious districts of Ngamiland, Chobe and
Francistown (North-East) throughout the 1980s. There is reference made
to weekly chloroquine prophylaxis for pregnant women and children
below the age of 5 years in the mid-1980s (Franco de et al., 1984a).
Between 1982 and 1984, over 94% of all cases were reported from Maun,
Chobe and Tutume regions (Franco de et al., 1984a). Shortages of DDT in
1987 led to a failure to spray large parts of the endemic regions of Ngami-
land and Tutume (Benthein, 1989).
In 1998, Botswana stopped using DDT and switched to the use of
deltamethrin and lambda-cyhalothrin (MoH, 1999). ITN distribution
began in 1997 but was only made free of charge through vaccine and
antenatal clinics in the northern districts in 2008. Over 250,000 people
were protected by IRS in 2009 and approximately 69,000 LLIN had been
distributed since 2008. Following escalating treatment failures with chlo-
roquine and sulphadoxine–pyrimethamine, Botswana switched to
200
180
160
140
120
100
80
60
40
No data
No data
No data
No data
20
No data
0
1928
1933
1938
1943
1948
1953
1958
1963
1968
1973
1978
1983
1988
1993
1998
2003
2008
FIGURE 4.13 Botswana annual slide-confirmed malaria case incidence 1928–2010 per
10,000 population. Data sources used include 1928–1938 (Bechuanaland Protectorate,
1928–1938); 1945–1953 (WHO-Bechuanaland, 1955); 1954–1960 (Bechuanaland Protector-
ate, 1954–1960); 1963–1973 (WHO, 2002); 1974–1984 (RBM, Southern Africa, 2002); 1985–
2009 (NMCP Botswana, unpublished data, 2009). No data available for review for the
years 1933, 1936, 1939–1944, 1948 and 1971. Reported cases converted to annual incidence
using annual population, to reflect overall changing population sizes with time rather
than population residing in risk areas. Population data from actual census years derived
from MoH reports and National Census Office (Bechuanaland Protectorate, 1934 and
1963; Botswana CSO, 2002–2004; Botswana CSO, 2005) and intercensal growth rates
used to compute non-census years. Annual malaria Incidence in 1928 and 1934 was 280
and 312 per 10,000, respectively, but attenuated on graph.
artemether–lumefantrine in 2007. Botswana experienced malaria epi-

demics in 1988, 1993, 1996 and 1997, but these may have occurred against
a background of rising disease risk through the late 1980s to 2000 where
after case incidence has declined (Fig. 4.13). In 2000, the National Malaria
Control Programme assembled parasitological survey data from the 1990s
and case-reporting data from the national health information system to
confirm that the districts of Kgalagadi, Kweneng, Kgatleng, Gaborone,
Southern (including Good Hope) and South East were for practical pur-
poses malaria free but could be subject to localized epidemics following
imported infections (MoH, 2001). Since 1990, case incidence, while proba-
bly focal in its extent and magnitude, remains above 1 per 10,000 popula-
tion at risk in areas where transmission has been reported between 1990
and 2010 (Fig. 4.13). In September 2010, Botswana launched an elimina-
tion strategy with a renewed emphasis on the use of scaled annual
spraying between October and December with DDT.
4.4.3.4. Zimbabwe
Malaria risks in Zimbabwe are determined by altitude and proximity to
the river valleys of the Zambezi and Limpopo. During the 1920s, Thom-
son remarked on the high risks associated with low-lying areas in the
river valleys of Shamva, Bindura, Sinoia, Gatooma and Victoria but

describes Salisbury (Harare) and Bulawayo as urban centres ostensibly
free from malaria (Thomson, 1929). It is often stated that the central ridge
of mountains that bisects the country from Mutare to Bulawayo is largely
free from malaria above 1200 m (Taylor, 1985; Taylor and Mutambu, 1986)
although others have used 1500 m as the divide (Crees and Mhlanga,
1985). The medical department records of malaria start in the late 1800s
but focus almost exclusively on the morbidity and mortality experiences
of European settlers. What is clear is that annual hospital returns suggest
some malaria risk across the entire country since the turn of the nine-
teenth century when the country was first colonized.
Malaria control began in earnest in 1949 in Mazoe Valley using HCH
for IRS covering over 200,000 people (Alves, 1951; Alves and Blair, 1953;
Blair, 1950) and increased across the lowveld areas through the early
1950s (Alves and Blair, 1955). In 1953, the programme expanded further
to include areas of higher altitude to serve as a ‘‘buffer’’ for European
communities, and by 1955, both lowveld and middleveld areas were
‘‘under control’’ (Alves and Blair, 1955). Between 1957 and 1991, DDT
was the preferred residual insecticide. A number of experimental projects
were also launched in reserve areas using enhanced surveillance (Wolfe,
1964) and mass drug administration with chloroquine among children or
with amodiaquine and primaquine for immigrant labour (Alves, 1958;
Reid, 1962). In 1942, malaria accounted for 10% of all hospitalizations, but
by 1962, this had declined to only 0.3% of admissions (Taylor and
Mutambu, 1986). The city limits of Bulawayo and Harare (previously
Salisbury) were confirmed as malaria free from late 1970s by national
malaria control agencies (NMCP Zimbabwe, 2008) and international
travel advisories (IAMAT, 2004). The spatially restricted campaigns
since the late 1940s were successful in reducing parasite prevalence and
case incidence to a state of unstable transmission by 1959 and through to
the late 1970s. It was hoped that malaria might be eliminated in the
southern provinces during the 1960s although Rhodesia was never sup-
ported by the WHO beyond pre-eradication. Spraying activities
continued throughout the civil war for independence during the 1970s
although disruptions were inevitable.
Since independence in 1980, malaria control was re-energized, and in
1988, deltamethrin replaced DDT for IRS. The country witnessed a num-
ber of severe epidemics of increasing frequency from the mid-1980s with
the most widespread and severe epidemics in 1988 and 1993 (Freeman,
1995; Fig. 4.14). From the first reported evaluation and documentation of
chloroquine resistance in 1984, this spread across the country over the
next 10 years (Makono and Sibanda, 1999). Zimbabwe changed its first-
line treatment policy from chloroquine to a combination of chloroquine–
sulphadoxine/pyrimethamine in 2004; by 2006, artemether-lumefantrine
1800
1600
1400
1200
1000
800
600
400
200
No data
0
1990
2005
2009
1980
1985
1995
FIGURE 4.14 Zimbabwe: Annual malaria case incidence per 10,000 population 1980– 2000
2009. All case data combinations of slide confirmed and presumed cases. No data
available for review for the years 2001–2003. Data 1980–1989 from Freeman (1995);
1990–2009 (WHO, 2010); 2000 extracted from WHO (2002). Population data used to
compute incidence derived from the World Bank database (2011).
had become the recommended first-line treatment. Despite the continued

disruption within the health sector wrought by political unrest (Tren
et al., 2007), progress had been made in increasing coverage of ITN with
42% of children reported sleeping under a treated net the night before in a
national sample survey of over 6000 households in 2008 (NMCP
Zimbabwe, 2010). Coverage with DDT IRS, reintroduced in 2004
(NMCP Zimbabwe, 2008), was considerably lower in 2008 with only
15.5% of households reporting spraying in the last 12 months (NMCP
Zimbabwe, 2010).
The various efforts to control and eradicate malaria over the years
probably led to constrained areas of unstable transmission in the 1950s,
and by 1979, the central districts were regarded as malaria free (Global
Fund—Zimbabwe, 2010). Transmission today is largely supported only
by An. arabiensis which has replaced the previously widespread presence
of An. funestus reported in the 1950s (Reid and Woods, 1957). Epidemics
continue to be common, but by 2009, there were 14 districts that were
malaria free and part of elimination consolidation efforts (Fig. 4.20). The
rise and fall of malaria between 1980 and 2009 is shown in Fig. 4.14, and it
is important to recognize that Zimbabwe is yet to re-establish disease
control to rates described in the early 1980s.
4.4.3.5. Swaziland
The Kingdom of Swaziland is a landlocked country only 200 km by
130 km sharing borders with South Africa and Mozambique. In common
with Zimbabwe, the ecology of malaria is divided along altitudinal lines
with the lowveld (Bushveld) high-risk areas to the East, midveld and the
highveld lower-risk areas to the mountainous regions in the West
(Mastbaum, 1957a). Malaria epidemics in 1937 and 1945 highlight the
severity of malaria in Swaziland; in 1937, hundreds of Swazis died of
malaria (Packard, 1986); in 1945/1946, 6850 cases were reported (Fig. 4.15;
Mastbaum, 1954). The only form of prevention prior to the end of the
Second World War included very limited larval control measures as
recommended by control agencies in the Union of South Africa. The
first malaria control unit was established in 1945 and limited HCH
house spraying began in 1949 (Mastbaum, 1954) which expanded through
the lowveld during the 1950s and a subsequent switch to DDT until 1951
when BHC was used as a cheaper residual insecticide until 1961 (Mabaso
et al., 2004). Dieldrin was also used experimentally in 1955–1956 in some
areas and larviciding was maintained in Bremersdorp (Manzini) and
200
180
Annual malaria incidence (per 10,000
160
140
population)
120
100
80
60
40
20
No data
0
1929
1934
1939
1944
1949
1954
1959
1964
1969
1974
1979
1984
1989
1994
1999
2004
2009
FIGURE 4.15 Kingdom of Swaziland. Annual malaria case incidence 1928/1929–2009/

2010 per 10,000 populations. Total population of Swaziland used throughout to highlight
changing national case incidence despite changing margins of risk. Case data derived
for period 1929–1938 (Ministry of Health, Swaziland, 1930–1938); 1946–1973 (Ministry of
Health, Swaziland, 1974); 1974–1982 (MoH Swaziland, 1983); other years and most recent
years provided by Simon Kunene and Joe Novotny. Annual malaria incidence in 1946 was
370 per 10,000 populations. No data available for review for the years 1939–1945.
Population has been sourced from several sites: 1929–1938 (Ministry of Health, Swaziland,
1930–1939); 1946, 1956 census years (Ministry of Health, Swaziland, 1948 and 1957); 1966,
1976, 1986, 1997 and 2007 (CSO, Kingdom of Swaziland, 2011). Non-census years
computed using annual intercensal growth rates. Annual case reporting is July–June;
therefore, graph starts July 1929 and ends June 2010.
Stegi (Siteki) (Mastbaum, 1957b). By 1955, all rural areas, sugar farms and
irrigation schemes across the Kingdom were protected by IRS. Parasito-
logical surveys were undertaken annually to monitor the impact of the
control programme; in 1945/1946, parasite rates among infants were 37%,
declining to 6% by 1952/1953, 1.2% by 1954/1955 and 0% by 1956
(Mastbaum, 1955, 1957a; WHO-Swaziland, 1955). In concert with Zim-
babwe, Swaziland switched to a system of barrier control in 1958 with the
highveld areas and an intensified buffer of 15 km from the Mozambique
border in the Hhohho and Lubombo regions. An. funestus reduced signif-
icantly in numbers following the scaled IRS campaigns (Mastbaum,
1957b) and An. arabiensis predominates to this day. Spraying operations
were systematically withdrawn from areas that reported no cases within a
2-year period and mass IRS was stopped in 1959. Between 1961 and 1967,
focal IRS was maintained using both BHC and DDT. Between 1968 and
2000, DDT was used for rural IRS and cyfluthrin in houses with painted
walls (Hansford, personal communication; Mabaso et al., 2004). One
important threat to the success of control during the 1950s and 1960s was
the rapid introduction of irrigation and imported labour for the Colonial
Development Cooperation programme to stimulate sugar cane farming
(Packard, 1986). This changed the landscape and risks of malaria including
epidemics in 1966 and 1971 (Fig. 4.15). Between 1956 and 1975, malaria case
incidence was less than 5 per 10,000 population per year with the excep-
tions of the epidemics in 1966 and 1971. By 1970, it is stated that the only
cases were those imported from outside the country (MoHSW, 1999). At
this point, malaria operations were drastically scaled down, funding with-
drawn and the malaria department reduced from 36 staff to 7.
During the early 1980s, large-scale population movements occurred as a
result of refugees fleeing the civil war in Mozambique, for example, 24,000
were settled in Malindza and Ndzevane in 1983 alone (Hansford, 1994). In
1986/1987, spraying ceased due to lack of funding and declining govern-
ment priority. This was followed by a resurgence of malaria risk until
funding from South Africa restored control operations and led to a tempo-
rary decline, but malaria case incidence followed a pattern seen elsewhere
in Southern Africa rising through to a peak in the late 1990s including a
serious epidemic in 1996 that led to 125 malaria deaths (MoHSW, 1999).
Between 1994 and 1999, 70% of cases came from Lubombo on the border
with Mozambique (MoHSW, 1999). In 1999, Swaziland joined forces with
KwaZulu-Natal Province in South Africa and Southern Mozambique to
form the Lubombo Spatial Development Initiative (LSDI) to aggressively
reduce transmission across borders (LSDI, 2007; Sharp et al., 2007). Global
Fund external support increased the national capacity to fund IRS, ITN
distribution, drugs and diagnostics and surveillance in 2003 and 2008. Up
until 2009, the first-line treatment for malaria was chloroquine, and the
Swazi Ministry of Health was the last to change to ACT in Africa in 2010.
Case incidence began to decline from 2000, and for the three consecutive
years 2006–2008, incidence was below 1 per 10,000 population (Fig. 4.15)
and these areas of unstable risk are located in the Eastern regions of
Lubombo and Hhohho. In 2008, the Kingdom of Swaziland launched a
malaria elimination strategy (MoHSW, 2010).
Swaziland has probably witnessed several periods where it
approached the elimination of P. falciparum resulting in unstable case
incidence (late 1950s, early 1970s and late 2000s). These short-lived suc-
cesses do not constitute sustained maintenance of unstable transmission.
The recent declines in case incidence between 2006 and 2008 have resulted
in less than 100 confirmed cases reported each year largely located in the
Eastern regions; therefore, we have treated the mapped extent of the cases
in the East of the country as unstable and the remaining areas are malaria
free. In 2010, the number of reported confirmed cases increased to 253
(Simon Kunene, personal communication) highlighting the need for
vigilance, cooperation with neighbouring Mozambique that provides
seasonal labour and more aggressive containment of transmission if
Swaziland aims to eliminate all local transmission.
4.4.4. Malaria control in Middle Africa: From GMEP pilots

to RBM
4.4.4.1. Before the Second World War
Before, and during, the Second World War, the control of malaria was
largely focused on protecting Europeans settling in central African terri-
tories, military personnel or short-stay colonial administrators. Conse-
quently, control was limited to urban administrative centres, ports and
economic concessions such as mines and farming areas. Prior to the
Second World War, attempts to reduce vector breeding sites were under-
taken in a number of urban and economically important areas in the
highly endemic countries of middle Africa under the colonial administra-
tion of Britain, France, Portugal, Belgium and Germany. Reference is
made to environmental mapping of larval breeding sites and control,
including in some cases penalties for infringement of ‘‘malaria legisla-
tion’’, in Conakry, Guinea (Le Moal, 1906), Dakar, Senegal (Heckenroth,
1922), the ‘‘Dutton Scheme’’ in Bathurst (Banjul), The Gambia (Colony of
The Gambia, 1917), Leopoldville (Kinshasa), Democratic Republic of
Congo (Colonie du Congo Belge, 1931), Khartoum, Sudan (Balfour,
1913), Dar es Salaam (Colonial Development Fund, 1935; Mackay, 1938),
Nairobi, Kisumu and Mombasa, Kenya (De Boer, 1930) and the use of
oiling of breeding sites in large towns in Nigeria (Colony of Nigeria,
1927). Few data exist on the overall impact of these approaches; however,
several examples are worth highlighting.
Nairobi was established as the administrative capital of Kenya in 1905,

and although it is located at 1795 m above sea level, malaria was a
significant problem for residents from 1911. Over 14,000 malaria cases
were recorded in Nairobi in 1913, and malaria cases fluctuated between
2500 and 3600 per year between 1917 and 1919 (Symes, 1940). Three major
epidemics occurred in 1926 (De Mello, 1947; Symes, 1940), 1935 and 1940
(De Mello, 1947; Haynes 1940). Following the 1926 epidemic, malaria was
made a notifiable disease and renewed efforts were established, sup-
ported by legislation, to improve drainage and environmental manage-
ment to reduce the larval breeding sites across the expanding city (De
Mello, 1947; Nairobi Municipality, 1930-1969; Symes, 1940). Notifications
showed a significant decrease of autochthonous malaria cases from an
annual average of 1182 cases in the 1930s, to 317 cases in the 1940s to 250
in the 1950s and finally 49 cases in the 1960s during a period when the
numbers of Nairobi residents had increased 35 times since the 1930s
(Fig. 4.16; Mudhune et al., 2011). Attribution of declining risk to specific
intervention approaches is difficult, but the data shown in Fig. 4.16 sug-
gest that urban malaria control was successful in reducing vector breed-
ing and locally acquired disease incidence before the Second World War.
During the 1920s in Sierra Leone, extensive drainage of wells and
‘‘canalization’’ were undertaken by the local colonial government’s
Annual malaria incidence per 10,000
2000
1800
1600
Nairobi population
1400
1200
1000
800
600
No data
400
200
No data
0
1916
1921
1926
1931
1936
1941
1946
1951
1956
1961
1966
1969
FIGURE 4.16 Nairobi city malaria incidence per 10,000 population 1916–1969 (adapted
from Mudhune et al., 2011). Annual malaria incidence in 1926 was 3649 per 10,000
populations and attenuated in graph. No data were reported in 1921–1925 and 1945. Case
incidence between 1952–1964 was less than 5 per 10,000 and between 1965 and 1969 was
less than 1 per 10,000. Annual malaria incidence has been sourced from several pub-
lications: 1916–1920, 1926, 1928 and 1929 (Symes, 1940); 1930–1939, 1944–1949 (Nairobi
Municipality, 1930–1939 and 1946–1949); 1940–1943 (De Mello, 1947); 1950–1969 (Nairobi
Municipality, 1950–1969). No data available for review for years 1921–1925. Population
between 1916 and 1925 is estimated from historical prediction in 1926 (Symes, 1940) and
1928 (Mitullah, 2003); data on censused population 1929–1949 (Nairobi Municipality,
1930–1949) and 1950–1969 (Nairobi Municipality, 1950–1969). Note that malaria was a
notifiable disease after 1930 through to 1969.
Medical and Public Works Department to improve the malaria situation

in the towns of Freetown, Kissy and Aberdeen and led to significant
reductions in house resting An. gambiae by the early 1940s (Tredre, 1943;
Turner and Walton, 1946). Detailed reconnaissance of local vector breed-
ing and control continued throughout the Second World War as the port
of Freetown was extensively used by the army (Tredre, 1943). In Northern
Zambia, between 1929 and 1949, a comprehensive programme of vegeta-
tion clearance and drainage was mounted around the Roan Antelope
copper mines, accompanied by provision of quinine and promotion of
mosquito nets. Malaria mortality was reduced by 90% among European
employees within 5 years of the programme starting (Utzinger et al.,
2002). At Lagos in Nigeria, drainage of the swamps and provision of
tide gates for the creeks during the Second World War were used to
reduce malaria risks for the British Air Force who had built a base at
Apapa and was thought to have been directly responsible for a reduction
of malaria attack rates from 100 per 1000 to approximately 30 per 1000
stationed soldiers per year (Gilroy and Bruce-Chwatt, 1945). Throughout
the twentieth century, urbanization has led to systematic declines in
malaria risk across many parts of middle Africa. The changing epidemi-
ology of malaria in rapidly growing urban centres in Africa is complex
(Hay et al., 2005; Keiser et al., 2004; Robert et al., 2003); however, the
effects of public heath engineering projects before the Second World War
cannot be underestimated (Keiser et al., 2005; Utzinger et al., 2001, 2002).
4.4.4.2. Vector control and pilot elimination projects post-Second

World War
The 1948 WHO malaria meeting (WHO, 1948) sought to maximize the
advances made in chemical discoveries for antimalarials and insecticides
during the Second World War. Attempts to eliminate malaria in Africa
were predominately located at the margins of stable transmission in the
northern and southern latitudes or on islands. Far fewer national-level
elimination efforts were reported in the countries and territories governed
by colonial powers in Middle Africa. The coverage of malaria prevention
in countries located in this subregion is best summarized from a review of
reports presented to WHO regional meetings in 1955 and 1956 that
brought together national malaria control programmes to review current
progress toward elimination. The meetings were held in Lagos in August
1955 (WHO, 1955) covering most of the Middle African countries and in
Athens in June 1956 where Sudan reported (WHO, 1956). The national
summaries provided at these meetings allow some insight into the scope,
scale, costs and impact of malaria control activities across the continent
for the approximate reporting year of 1953. Across the Middle African
countries, the reported information varied between countries in detail,
completeness and the sources of data provided; three countries did not
provide any information (Ethiopia, Italian Somalia and the British Camer-
oons). Nevertheless, the data generated for the year 1953 provide some
estimate of IRS and chemoprophylaxis coverage. Most countries reported
using some form of IRS with the exception of Guinea-Bissau and Uganda.
The most widely reported insecticide used was DDT; however, countries
also reported using in addition gammexane, BHC, dieldrin, malariol or
hexastan. Overall among the 32 reporting countries, representing approx-
imately 122.5 million people at risk, only 4.9% of the population was
protected by preventative measures and most of the areas protected
were either special projects or urban settings. In 1955, Russell estimated
that in the combined territories of West, Central and Eastern Africa only
8.5% of people at risk of malaria were protected against infection (Russell,
1956). While it is hard to distinguish what constitutes middle, southern
and northern Africa, it was estimated that by 1968 of the 214 million
people living in the entire Africa region exposed to malaria, only 1.03
million (0.5%) were living in areas that had mounted consolidation or
maintenance phases of elimination (Brown et al., 1976). By 1974, among
the 240 million Africans living in potentially malarious areas, only 2.3%
were protected under elimination campaigns, 5.9% were protected by
vector control measures and 3.2% were protected by chemoprophylaxis;
89% remained unprotected by any form of vector control or chemopro-
phylaxis (Brown et al., 1976).
Pilot control and elimination projects across West, Central and Eastern
Africa were in some cases highlighted in the WHO conferences in 1955 and
1956 others began after 1955. These were significant trials covering
thousands of people. The trials provided important information on the
impact on transmission and mortality of house spraying and drug-based
regular prophylaxis or mass treatment. Between 1945 and 1979, IRS pilot
projects were undertaken in Senegal (Locan and Michel, 1962), Sierra Leone
(Davidson, 1947; Walton, 1947, 1949), Liberia (Guttuso, 1967), Ghana
(Eddey, 1944), Nigeria (Bruce-Chwatt et al., 1955, 1957; Foll and Pant,
1966), Cameroon (Chastang, 1959), Togo (Bakri and Noguer, 1977), Demo-
cratic Republic of Congo (Davidson, 1950; Vincke, 1950), Rwanda-Burundi
( Jadin et al., 1953), Tanzania (Draper and Smith, 1960; Smith, 1962; Smith
and Draper, 1959), Kenya (Fontaine et al., 1975; Payne et al., 1976), Ethiopia
(Chand, 1965), Republic of Sudan (BNHP, 1981; El Gaddal et al., 1985;
Mirghani et al., 2010) and Mozambique (Soeiro, 1952, 1956); trials of com-
bined IRS with mass drug administration or chemoprophylaxis in Nigeria
(Molineaux and Gramiccia, 1980; Nájera et al., 1973), Cameroon (Cavalie
and Mouchet, 1961), Burkina Faso (Escudie et al., 1961; Ricosse et al., 1959),
Democratic Republic of Congo (Feuillat et al., 1954; Vincke, 1954), Kenya
(Roberts, 1956, 1964a,b; Strangeways-Dixon, 1950) and Uganda (De Zulueta
et al., 1964) and trials of drug-based control without IRS in Tanzania
(Clyde, 1966, 1967), Ghana (Charles et al., 1962), Kenya (Avery-Jones,
1958), Uganda (Hall and Wilks, 1967) and Sudan (Omer, 1978). What is
clear is that the escalation of IRS or mass drug administration across middle
Africa failed and in most instances did not go beyond pilot projects. High
costs of insecticides, fears of rapid escalation of vector resistance to insecti-
cides and mixed results from malaria elimination pilot projects all contrib-
uted to a failure to expand vector control in Africa (Kouznetsov, 1977;
Nájera, 1999; Nájera et al., 2011). Requirements for successful elimination
programmes highlighted the need for strong and effective health systems
and much of Africa neither had the resources nor was deemed prepared for
the scaling up of attack phases (Cambournac, 1966; Gramiccia, 1966;
Nájera, 1999; WHO AFRO, 1962). By the 1970s, malaria was seen as a health
system problem for much of Africa and its control was integrated into
strategies for the management of illness within the framework of Primary
Health Care (Nájera, 1999).
The mounting fears of resistance to insecticides (notably at first dieldrin)
highlighted the need to rapidly reduce transmission in order to mitigate the
expected lost potency of insecticides in use (Bruce-Chwatt, 1956). This
prompted early investigations into the combined effects of chemoprophy-
laxis in combination with IRS to escalate transmission reduction in highly
endemic areas (Bruce-Chwatt, 1956; D’Alessandro and Buttiens, 2001; Dola,
1974; Kouznetsov, 1979). National programmes of chemoprophylaxis were
beginning to be cited at the WHO Lagos conference in Kenya, Tanzania,
Somaliland, Mozambique, Malawi and Angola; however, the details sur-
rounding these programmes were limited. At the WHO regional confer-
ence in Yaoundé in 1962, it was stated that ‘‘The problem of collective drug
administration for malaria control is of increased interest and importance
in Africa. In a number of African countries where a malaria eradication
programme cannot be put into immediate effect because of technical,
administrative or financial obstacles, the responsible authorities are inter-
ested in the possibilities of malaria control through a large-scale adminis-
tration of antimalarials either to the whole population or to selected and
particularly vulnerable groups’’ (WHO AFRO, 1962).
From as early as the 1960s, chloroquine was widely available in clinics,
shops and private pharmacies across Africa. Sixteen percent of children
presenting to a clinic in Ibadan in 1959 had had some form of anti-malarial
treatment at home before attending the clinic (Onuigbo, 1961). Through-
out the 1960s and 1970s, there were reports of the use of chloroquine and
pyrimethamine as a means of control as Mass Drug Administration in
Middle Africa (von Seidlein and Greenwood, 2003), including school-
based programmes referred to as the ‘‘Daraprim Parade’’ in Eastern
Nigeria (Arthur, 1965), Western Nigeria (Fasan, 1971), Gabon (AFRO-
WHO, 1962), Tanzania (Clyde, 1967) and Kenya (John Ouma, personal
communication). The steady growth in the wide-spread use of chloro-
quine led to a situation following the end of the GMEP activities in Middle
Africa, whereby all fevers were routinely treated with branded forms of
chloroquine (AFRO WHO, 1962). At Saradidi in Western Kenya during
the early 1980s, it was estimated that every person received on average
1.24 chloroquine exposures per year, and 13.4% of the population
received five or more treatments per year (Spencer et al., 1987). With the
scaled introduction of Primary Health Care and expanded availability of
retail drugs (Foster, 1995; McCombie, 1996) during the 1970s and 1980s,
the presumptive treatment of all fevers as malaria with chloroquine was
widespread. The first confirmed case of chloroquine resistant malaria was
reported in Kenya and Tanzania in the late 1970s (Campbell et al., 1979;
Fogh et al., 1979) and spread westwards reaching a presumed complete
incursion across all of Africa by 1989 (Bloland et al., 1993; D’Alessandro
and Buttiens, 2001; Talisuna et al., 2004).
There are very few long time-series data on malaria incidence from
Middle Africa, and this limits our ability to fully understand the changing
clinical epidemiology of malaria in this region between 1950 and the 1990s.
What has been suggested from the examination of cause-specific demo-
graphic surveillance studies across Middle Africa is that malaria-specific
mortality in childhood reduced significantly following independence from
colonial rule and remained at a lower incidence through to the 1990s where
after it rose significantly as a cause of death against a continuing decline in
all-cause mortality (Fig. 4.17; Snow et al., 2001). The rise in malaria
20
18
Malaria mortality per 1000 children p.a.
15
13
10
0
N= 8 20 11
Before 1960 1960–1989 After 1989
FIGURE 4.17 Annualized malaria-specific mortality in children aged 0–4 years old
pre-1960; 1960–1989 and 1990–1999. Box plot showing median (central lines), 25%, 75%
quartile ranges around the median (box width) and upper and lower limits (T) mortality
estimates per 1000 children aged 0–4 years per annum (reproduced from Snow et al.,
2001).
Malaria-specific mortality per 1000 children aged 18
16
14
12
0–4 years p.a.
10
0
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
FIGURE 4.18 Niakhar, Senegal: Malaria-specific mortality per 1000 children 0–4 years
1984–2010 (adapted from Munier et al., 2009; Trape et al., 2012). Malaria defined in
demographic surveillance of Naikhar population using verbal autopsies. In 1992,
chloroquine resistance established; by 2000, sulphadoxine–pyrimethamine (SP) used for
second-line rescue therapy; 2003 amodiaquine (AQ) þ SP became first-line treatment
until replaced by AQ-Artesunate in 2006; in 2008, ITN distribution went to scale.
mortality witnessed at surveillance sites during the 1990s coincided with

established high levels of chloroquine resistance (Snow et al., 2001) and
more temporally associated with documented drug resistance at Niakhar
in Senegal (Fig. 4.18; Munier et al., 2009; Trape et al., 2012). These observa-
tions are further supported by longer-term data on malaria admissions at a
Tea Estate population in Kenya which showed low incidence during the
1960s to early 1980s followed by a rise in malaria reaching peaks in the
1990s, of note in this series is the subsequent decline through to 2009
(Fig. 4.19; Shanks et al., 2002; Stern et al., 2011).
The peak of malaria incidence since the end of the GMEP in Africa was
probably somewhere between the early 1990s and early 2000s in many
sites of Africa where first-line drugs were failing and the prevention of
infection with vector control was minimal. This period coincides with
resurgent risks described earlier for the Malagasy highlands (Fig. 4.10),
malaria mortality in São Tomé and Prı́ncipe (Fig. 4.7), Kingdom of Swazi-
land (Fig. 4.15), South Africa (Fig. 4.12) and Botswana (Fig. 4.13).
4.4.4.3. The RBM era in middle Africa

The RBM initiative and the supporting financial structures provided by
the Global Fund emerged at a time when Africa was facing a rapidly
rising malaria disease burden. Both initiatives were slow to impact on the
1200
Annual malaria admissions
1000
800
600
400
200
0
1966
1971
1976
1981
1986
1991
1996
2001
2006
2009
FIGURE 4.19 Annual malaria admissions in Kericho Tea Estate population, Kenya 1966–
2009 (adapted from Shanks et al., 2002; Stern et al., 2011).
poor coverage of new efficacious tools such as ITN (Noor et al., 2009),
removing failing monotherapies and supporting policy change in favour
of ACTs (Attaran et al., 2006) and the funding necessary to implement
aggressive control started reaching high-burden countries slowly
(Narasimhan and Attaran, 2003; Teklehaimanot and Snow, 2002). The
Scale-Up for Impact initiative was conceived to rapidly change the land-
scape of poor coverage across Africa and achieve near universal access
and use of prevention and clinical care (Campbell and Steketee, 2011). By
2005, new international funding was translating into effective coverage of
prevention (ITN, IRS and intermittent presumptive treatment of malaria
in pregnancy) across middle Africa. Between 2008 and 2010, a total of
about 254 million nets were supplied and delivered to sub-Saharan
Africa, and approximately 34% of young children were sleeping under
an ITN by 2010 (RBM, 2011). About 10% of Africans at risk of malaria
were protected by IRS by 2010 (RBM, 2011) including more recent IRS
policies and implementation in The Gambia, Senegal, Mali, Liberia,
Ghana, Benin, Nigeria, Gabon, Angola, Democratic Republic of Congo,
Zambia, Mozambique, Malawi, Uganda, Kenya, Tanzania, Rwanda, Bur-
undi, Ethiopia and Eritrea. Although coverage was deliberately patchy,
four countries achieved household coverage greater than 50% (RBM,
2011). Overall, IRS coverage estimates are considerably higher in 2010
than those reported during the 1950s and 1960s for Middle Africa. DDT is
used for malaria control in 13 African countries.
Following growing concerns about chloroquine and sulphadoxine–
pyrimethamine resistance and the lack of an international response
(Attaran et al., 2006), remarkably rapid concerted action led to the policy
changes to support novel ACTs as first-line therapies across Africa. In
2003, only four countries in Africa had adopted ACTs as their first-line
therapy (Bosman and Mendis, 2007); by 2010, they were first-line treat-
ment in every malaria endemic country in Africa. Despite rapid policy
change, making sure clinical cases are treated with an ACT has so far
proven to be the most elusive milestone of RBM success nationally and
regionally. These drugs still reach only a fraction of people who need
them. Most countries in Middle Africa, for which data are available,
report that less than 20% of febrile children access an ACT (RBM, 2011).
Not all fevers are malaria and the big-push is to now scale up parasitolog-
ical diagnosis of malaria to improve case-management practices
(D’Acremont et al., 2009).
RBM, the Global Fund and bilateral agencies supporting malaria con-
trol in Africa have all improved how we assess the impact of financial
investments to support disease control and elimination efforts. However,
while there has been a significant improvement in how partners measure
financial investment and coverage of malaria control activities, far less
attention has been given to the documented impact on disease incidence
and death from malaria. Modelled expected impacts of reported interven-
tion coverage form the main evidence base by which partners estimate
deaths averted in Africa since 2000 (Eisele et al., 2009; 2010; Komatsu et al.,
2010; RBM, 2011). These models predict that approximately 0.8 to 1.1
million deaths have been averted since the launch of RBM. Our only
empirical evidence in Middle Africa comes from short-term temporal
coincidence between increased access to effective interventions and the
changing patterns of paediatric hospitalization with severe malaria since
1999 in Eritrea (Nyarango et al., 2006), Ethiopia (Graves et al., 2008; Otten
et al., 2009), The Gambia (Ceesay et al., 2008; 2010); Gabon (Bouyou-Akotet
et al., 2009), Rwanda (Otten et al., 2009; Sievers et al., 2008), Kenya
(O’Meara et al., 2008; Okech et al., 2008; Okiro et al., 2007, 2009), Guinea-
Bissau (Rodrigues et al., 2008), Senegal (Brasseur et al., 2011; Sarrassat et al.,
2008), Tanzania (Mmbando et al., 2010) and Zambia (Chizema-Kawesha
et al., 2010). These reports suggest a wide-spread effect of scaled interven-
tion across middle Africa since 1999 and are consistent with declines seen
in southern Africa and those island states pursuing elimination.
There is little doubt that the epidemiology of malaria is in transition
across Africa, yet there are several important aspects of this change that
need highlighting. Firstly, all is not equal and there are reports from some
high transmission settings in Africa including Western Kenya, (Okiro
et al., 2009), Uganda (Okiro et al., 2011) and Malawi (Roca-Feltrer et al.,
2012); the clinical burden presenting to hospitals has increased since 1999.
Most reports of declining malaria burdens are from settings where the
initial transmission intensity was low to moderate (O’Meara et al., 2010).
Secondly, progress in ensuring that the most vulnerable communities are
protected across Middle Africa has been varied with some countries
achieving more than others with similar levels of donor support
(Flaxman et al., 2010; Hill and Kazembe, 2006; Noor et al., 2009; RBM,
2011; Van Eijk et al., 2010; WHO, 2010). There are very few published
time-series data since 2000 from countries that have been slow to scale
intervention coverage. Thirdly, the temporal association between scaled
coverage of ITN, changing therapeutic policies and declining disease
incidence is not always congruent. At several sites, malaria hospital
admissions began to decline prior to significant coverage of prevention
with ITN, IRS and effective access to ACT. Finally, where declining
incidence of malaria has been documented, the decline has been dramatic;
however, these declines were all reported from a baseline period towards
the end of the 1990s and early 2000s when the malaria burden was at its
recent peak.
4.5. SUMMARY AND DISCUSSION
4.5.1. Changing limits in North Africa

Using the narratives from published reports, mapped extents and descrip-
tions on the locations and species of locally acquired infections, it is possi-
ble to combine historical medical intelligence with biological masks of
temperature and aridity suitability for transmission and human population
density to provide a sequence of spatial risks from 1939 (presumed natural
extent; Fig. 4.1), 1959 (Fig. 4.20A), 1979 (Fig. 4.20B), 1999 (Fig. 4.20C) to
2009/2010 (Fig. 4.20D). The areas of biological suitability coincident with
population densities greater than 0.01 per km2 are often oasis settlements
across the Sahara. The focus here is on P. falciparum risks only, recognizing
there were foci of P. vivax transmission in the Kingdom of Morocco after
1974 in Al Hoceima, Chefchaouen, Taounate and Khouribga provinces and
from 2000 in Chefchaouen province until eliminated, and there was an
outbreak of vivax malaria in 1981 Khemis el Khechna in the north of Algeria.
In 1939, most of the populated areas of North Africa were exposed to stable
transmission of both P. falciparum and P. vivax with the likely exception of
the eastern coastal towns of Libya. By 1969, this spatial extent and the likely
clinical incidence had reduced substantially as part of attack phases of
national, post-independence elimination campaigns.
At the launch of the RBM initiative in 1999, almost all of the North
African territories were P. falciparum free with the exception of the border
area of Tinzaouatine in southern Algeria and Fayoum in the UAR Egypt.
While difficult to establish with certainty, we have left the residual foci as
unstable in southern Algeria by 2009, representing the last area of possible
P. falciparum transmission. Figs. 4.2–4.4 demonstrate the rapid decline
associated with aggressive adult vector control, reconnaissance of larval
breeding sites and active case detection during the initial attack phases of
FIGURE 4.20 The changing margins and stability of P. falciparum transmission (A) 1959, (B) 1979, (C) 1999 and (D) 2009. Dark grey representing
no malaria risk; light grey biologically suitable transmission but population density less than 0.01 person per km2; light green unstable
transmission and dark pink stable transmission. (A) 1959: The remaining focal areas of P. falciparum risk in Kingdom of Morocco as reported and
mapped by Hoeul and Donadille (1953). The regions of Oran, Constantine and Algers in Algeria were under aggressive control from the mid-1940s
that transitioned this area to unstable conditions by 1959 (Benzerrough and Janssens, 1985; Hammadi et al., 2009; Parrot et al., 1946). Elimination
campaigns systematically reduced the margins of malaria risk in Libya with a remaining area of unstable risk in Fezzan region by 1959 (Gebreel
et al., 1985). In Egypt, by 1953, no cases were recorded in the Canal Zone, Assiut, Girga, Kom Ombo, Asswan and Nubia regions (Halawani and
Shawarby, 1957). The Republic of Djibouti was malaria free. The islands of Réunion and Mauritius had substantially reduced malaria incidence to
render each island unstable by 1959. Use of IRS and chemoprophylaxis in Madagascar led to effective control in the highland plateau districts by
1959 (Bernard, 1954) and resulted in exceptionally low transmission and disease incidence. For South Africa, the map produced by Brink (1958)
and narrative provided by Hansford (1974) have been used to constrain the margins of risk in the Transvaal area by 1959 resulting from aggressive
use of DDT and providing evidence of unstable risk in the lower margins. It was felt that these control efforts were mirrored by a changing risk
along the lower margins of Botswana along the Limpopo River (Franco de et al., 1984a). In Zimbabwe, similar attack phases of elimination were
able to reduce case incidence in unstable transmission and provided as mapped extents by Alves and Blair (1955). Case incidence declined
rapidly in the Kingdom of Swaziland though the use of DDT and stable transmission was constrained to Lubombo and Hhohho regions until 1999
(MoHSW, 1999; Simon Kunene, personal communication); the highveld was regarded as malaria free (Fontaine, 1987), and this was digitized using
ARCGIS and regarded as malaria free through to 2009. (B) 1979: The Kingdom of Morocco was free from P. falciparum by 1974. By 1979, falciparum
transmission had been eliminated in the northern territories of Algeria and focal risks persisted in the southern provinces with increasing
stability with increasing latitude (Benzeroug and Wery, 1985). All of the Northern provinces of Tunisia were malaria free by 1968, and by 1979, all
districts were falciparum free (Ambroise-Thomas et al., 1976). Libya was declared malaria free in 1973. Stable transmission in Fayoum region.
Unstable transmission was likely in some parts of Egypt in 1979 at malaria suitable areas but national case incidence dropped to less than 1 in
10,000 (Hassan et al., 2003). 1979 was probably the last period when The Republic of Djibouti was regarded as malaria free. Réunion and
Mauritius were declared malaria free by 1979. Elimination efforts on the islands of Cape Verde had reduced case incidence to zero in all but
Santiago by the late 1960s. It is also likely that combined disease control on Mayotte resulted in a case incidence that would be regarded as
unstable by 1979. Drug-based and IRS control in the highlands of Madagascar sustained unstable control through to 1979. Continued efforts to
eliminate malaria in the Transvaal and KwaZulu-Natal provinces rendered increasing areas unstable and reduced the spatial extent of risk in
South Africa (Craig et al., 2004; Hansford, 1974; Kleinschmidt et al., 2001). In Namibia, combined medical intelligence based on case data
generated by the Ministry of Health and Social Services since the 1980s shows the regions of Khomas and Erango to have conditions that are
borderline malaria free and unstable transmission with consistently low case incidence (MoHSS, 1996). The southern-most risk districts in
Namibia had very few clinical cases during the early 1980s and regarded here as unstable by 1979 (MoHSS, 1996). These qualitative observations
were more systematically quantified using reporting from mapped facilities over the period 2008–2009 (Snow et al., 2010b). In Zimbabwe,
evidence suggests that the areas under control in 1959 remained under control rendering them unstable transmission; the cities of Harare and
Bulawayo were malaria free as were highland districts (NMCP Zimbabwe, 2008). In Swaziland, case incidence data were mapped in 1983 to show
that stable risks were constrained to only the areas located on the east of the country (Franco de et al., 1984b); these cases were digitized and
enveloped using ARCGIS. (C) 1999: A foci of risk in Algeria on the border with Mali at Tinzaouatine continued through to 2009 (Boubidi et al.,
2010). El Fayoum Governorate in Egypt remained a focal area of unstable transmission in 1999 (Hassan et al., 2003) with no autochthonous cases
elsewhere in Egypt. The Republic of Djibouti witnessed a sequence of epidemics from 1988 and in areas where transmission was biologically
suitable leading to the establishment of stable endemicity. Madagascar, Cape Verde (Santiago) and Mayotte witnessed resurgent risks of malaria
during the late 1980s and early 1990s that returned previously unstable areas to stable transmission and high disease burden. Risks in South
Africa were constrained by 1999 to areas located along the Kruger national park and borders with Zimbabwe in the Limpopo and Mpumalanga
Provinces (Philip Kruger and Aaron Mbuza, personal communication) and the two northerly districts of Ingwavuma and Ubombo in KwaZulu-
Natal Province (Craig et al., 2004; Kleinschmidt et al., 2001). The subregional rise in malaria risks affected Zimbabwe against a background of
political crisis and it is assumed that lowveld areas previously under control in Zimbabwe returned to stable transmission by 1999 with the
exceptions of malaria-free situations in Bulawayo, Harare and central highlands. (D) 2009: Since 1998, no locally acquired case has been reported
from Fayoum in Egypt and now the country is malaria free although not certified about. Following efforts to control malaria in the Republic of
Djibouti from 2008, case incidence was unstable (Hawa Guessod, personal communication). In Algeria, Tinzaouatine remains the only area of
unstable risk by 2010. Locally acquired cases have been reported on the Cape Verdean islands of Santiago (mainly Saint Caterina and Santa Cruz)
and Boa Vista in recent years but represent an unstable situation. Cases are concentrated in the northern districts of the main island of Mahoré,
in Mayotte (Solet et al., 2007). Botswana reported no cases in most areas previously free of malaria by 2010; however, locally acquired cases
were detected in Kweneng West and East districts between 2006 and 2008 rendering this area unstable (Ministry of Health, 2009). By 2009,
malaria-free areas extended to include 14 districts in central highlands in Zimbabwe and under consolidation phase of elimination (Global Fund
Zimbabwe, 2010). By 2009, case incidence in South Africa and Swaziland had dropped dramatically and case incidence by district has been used
to delineate unstable and stable areas risks in Limpopo and Mpumalanga (Philip Kruger and Aaron Mbuza, personal communication; Ngomane
and de Jager, 2012), unstable risks in Ingwavuma, KwaZulu-Natal (Marlies Craig and Rajendra Maharaj, personal communication) and unstable
risks within the districts of Hhohho and Lubombo, with one stable endemic district of Mhlangatane, in the Kingdom of Swaziland (Kunene et al.,
2011).
elimination programmes in North Africa. It is, however, important to

recognize that malaria control had a long history in North Africa dating
back over many years prior to the GMEP, and the effects of larval reduc-
tion and mass drug administration on transmission in Morocco, Algeria,
Tunisia and UAR Egypt are likely to have substantially reduced the
endemicity prior to the launch of elimination campaigns. The natural
barrier provided by the vast Saharan desert serves as a protection from
the highly endemic regions of sub-Saharan Africa, thus reducing the risks
of imported, cross-border malaria. Nevertheless, North Africa attracts
many economic migrants from the south. Since 2002, in the Kingdom of
Morocco, over 700 imported malaria cases have been detected (WHO-
Morocco, 2010); between 1980 and 2009, 981 and 2466 imported cases have
been detected in Tunisia (WHO-Tunisia, 2010) and Libya (WHO-Libya,
2010), respectively. Demonstrations in Tunisia in 2010 set off a wave of
political unrest across North Africa and Arabian Peninsula known as the
Arab Spring. It remains uncertain how the building and restructuring of
Algeria, Tunisia, Libya and Egypt will affect the immediate vigilance
required to maintain active detection of imported infections and the
efforts required to contain onward transmission where vectors continue
to provide areas of receptive risk.
4.5.2. The successes and failures of malaria elimination on

Africa’s islands
Small island states are thought to represent unique opportunities to
eliminate malaria (Kaneko et al., 2000), having identifiable vector ecolo-
gies and accessible populations isolated from neighbours harbouring
continued transmission. All the self-governed African islands have
attempted malaria elimination at some stage over the past 70 years.
These islands share several common properties that distinguish them
from mainland Africa. Human settlement was more recent and involved
an ad-mixture of people from Asia and Africa resulting in significant
proportions of the population having duffy-positive red cells and, com-
bined with a history of trade outside Africa, receptive to the establishment
of P. vivax transmission on all the islands in the Atlantic and Indian
Oceans. P. vivax is usually the ‘‘last parasite standing’’ during elimination
campaigns (Baird, 2010) and harder to prevent from reintroduction as
witnessed in Mauritius. All the small islands have distinct agriculture-
based ecologies and human settlement patterns allowing the relatively
easy mapping of vector breeding sites, human risk and stratified spatial
control. With the exception of Zanzibar, migration between mainland
Africa and the islands is quantitatively limited through a sea buffer rather
than a desert buffer for North Africa, with the high transmission countries
of the continent. Increasing air travel has, however, transformed risks of
imported malaria, and special screening and containment programmes at

airports during consolidation phases of elimination in Cape Verde,
Réunion, Mauritius and São Tomé and Prı́ncipe have at some stage been
implemented. Interestingly, the islands of Madagascar and the Union of
Comoros pose the largest threats to the re-establishment of malaria in
Réunion (Denys and Isautier, 1991). Although physically separated from
one another, the islands of the Indian Ocean, therefore, require a subre-
gional effort to reduce the risks of re-establishing transmission in island
states with high malariogenic potential.
Only Réunion (1979) and Mauritius (1973 and 1998) have achieved
malaria elimination since the launch of the GMEP. The Cape Verdean
islands reduced the spatial extent considerably and much earlier, but local
transmission continues on the islands of Santiago and Boavista. Zanzibar
(three attempts), Mayotte (two attempts) and Madagascar (three attempts)
have enjoyed varying degrees of success towards elimination over the past
50 years, often reducing transmission and disease incidence to extremely
low levels but never interrupting transmission. On the islands of São Tomé
and Prı́ncipe and the Union of Comoros, far less was achieved before the
launch of the RBM initiative. An important component of previous elimina-
tion efforts on the islands has been the combination of IRS with wide-scale
use of anti-malarial drugs through mass drug administration, prophylaxis
or screening and treatment. During periods when parasites were sensitive
to chloroquine, this approach would have had a dramatic effect on the
parasite reservoir. Resurgent interest in this approach has been adopted in
Comoros; however, there remain concerns over the use of artemisinin
monotherapy as resistance emerging to this important therapeutic agent
would be a disaster on far more than a local scale.
A consistent theme throughout the combined histories of malaria
elimination attempts across Africa’s islands is the impact of waning
political support and financial commitments to maintaining prevention
and surveillance when disease burdens are reduced to very low levels.
Sustaining the malaria-fee status in Mauritius and the progressive effects
of early elimination attempts in São Tomé and Prı́ncipe, Zanzibar and
Madagascar were all jeopardised by weakened enthusiasm and commit-
ment to programme efforts. Failures were also attributed to emerging
drug resistance (Cape Verde, Mayotte, Zanzibar and Madagascar), chang-
ing patterns of land use (Madagascar) and imported infections accompa-
nied by declining surveillance efforts (Mauritius and Cape Verde).
Anticipating a long game, demanding constant vigilance rather than a
short-term win, is critical to sustaining success towards elimination.
All islands that have yet to interrupt transmission have witnessed
dramatic reductions in the incidence and public health burden posed by
malaria since 2005. This is coincident with the scaling up of replacement
ACT first-line treatments, provision of free ITN and targeted IRS made
possible following a massive increase in financial resources provided by

the Global Fund and other international agencies. In addition to the
independently governed islands, Bioko, one of the islands of Equatorial
Guinea, saw a huge reduction in child mortality and malaria incidence
following scaled IRS and ITN coverage since 2005 (Kleinschmidt et al.,
2009). The disease reduction successes across high-burden islands have
encouraged a renewed wave of enthusiasm for elimination: the Union of
Comoros, Madagascar and São Tomé and Prı́ncipe have all explicitly
developed elimination attack, maintenance and consolidation strategies
to achieve malaria-free status before 2020.
The Revolutionary Government of Zanzibar and its 1.2 million residents
now face a difficult decision to either maintain aggressive control to sustain
a very low prevalence and incidence of disease (low-stable endemic control)
or embark on a pathway to elimination. An elimination feasibility study
reviewed the risks posed by imported infections from travellers each year
(between 10,000 and 25,000 air travellers per month), its close connectivity
by ferry and boats to mainland Tanzania and the economic costs (ZMCP,
2009). The report concluded that the vulnerability posed by imported infec-
tions, high receptivity on the islands and the costs (US $1.88 per capita for
sustained control versus US $2.87 per capita for elimination over 25 years)
argued in favour of sustaining low-stable endemic control (ZMCP, 2009).
4.5.3. Elimination and control efforts in Southern Africa

Malaria control activities began as national campaigns from 1948 in South
Africa, the Kingdom of Swaziland and Zimbabwe and the 1960s in
Botswana and Namibia. Prior to 1948, malaria prevention was not wide-
spread and tended to focus on the use of quinine prophylaxis among
European settlers and limited vector control notably efforts to improve
environmental sanitation, oiling and use of Paris Green. From the late
1940s, the wide-scale use of IRS programmes with a variety of residual
insecticides, but mostly DDT, across many areas of Southern Africa was
able to achieve rapid and substantial reductions in transmission and inci-
dent cases. None of the southern African countries have managed to sub-
stantially reduce the margins of transmission that prevailed in 1939
(Fig. 4.1), or completely interrupt transmission within the margins, but
have enjoyed periods of low case incidence that would qualify as unstable
transmission between the 1959 and 1979 (Fig. 4.20A and B) coincidental with
periods of aggressive IRS campaigns (Mabaso et al., 2004; Mastbaum, 1965).
Resurgent risks began to emerge in the 1980s (Figs. 4.11–4.15) and have
been variously attributed to large-scale population movements during the
1970s and 1980s due to regional conflicts, waning political commitment
and funding, periodic interruption of IRS, emerging drug resistance and
the HIV epidemic. The renewed political commitment to malaria control
and elimination in the 2000s served to galvanize efforts in Southern Africa

and have led to recent successes in reducing the burden of malaria in every
country from its second peak. However, it is important to recognize that
during the period when the international development lens was focussed
elsewhere and malaria in Africa was not a priority, much of Southern
Africa experienced a lower malaria incidence than they do presently
following substantial investment and a renewed political interest.
There are a number of reasons why none of the Southern African
countries have eliminated malaria. The dominant vectors, An. arabiensis
and An. funestus, are considerably more efficient than their counterparts
in North Africa and breeding sites harder to map than on islands. Insecti-
cide resistance, behavioural adaptation and changing species dominance
have posed challenges to IRS campaigns across the subregion (Enayati
and Hemingway, 2010). Compared to North Africa, southern African
countries have been considerably poorer and have realized independent
governance representing the majority of the population much later. The
continued presence of asymptomatic carriage among semi-immune resi-
dents and the constant introduction of new migrant infection render
elimination particularly difficult even with the most aggressive combi-
nations of active and passive surveillance. Towards the end of the attack
phase of elimination, mass screening and active surveillance of popula-
tions who are likely to harbour infections asymptomatically through
acquired anti-parasitic immunity is necessary. With the exception of
South Africa, all other southern African countries have not mounted
active surveillance since the 1960s, and the slide examination rates for
presumed clinical cases have been poor. During a pilot approach to
active screening in the southern part of Zimbabwe in the early 1960s, it
was recognized that this is an expensive element of the attack phase of
elimination, demanding skilled human resources and a carefully sensi-
tized population (Wolfe, 1964). Prolific cross-border seasonal migration
from neighbouring highly endemic countries such as Angola and
Mozambique continues to pose a larger threat to interrupting transmis-
sion in Namibia and Swaziland and South Africa. In recognition of the
subregional threats, initiatives have started across borders including the
LSDI (Mozambique, Swaziland, South Africa) (LSDI, 2007; Sharp et al.,
2007), Trans-Zambezi Malaria Initiative and the Trans-Kunene Initiative
(SARN, 2011).
Recent progress in reducing case incidence has prompted the Ministers
of Health in the subregion to launch the Africa Malaria Elimination Cam-
paign supported by the African Union (AU) and the Southern Africa Devel-
opment Community (SADC). The concept of the Malaria Elimination 8 (E8)
was proposed and signed as the E8 Windhoek Agreement in 2009. The
countries on mainland Africa that constitute the E8 include those regarded
as having the greatest potential to eliminate malaria by 2015: Botswana,
Namibia, South Africa and The Kingdom of Swaziland and second line
neighbours Angola, Mozambique, Zambia and Zimbabwe (E8, 2010).
4.5.4. The double dip recession

Accounts of current emerging changes in the epidemiology of malaria
sometimes give the impression that GMEP was an irrelevance to Africa
and that the malaria situation was unchanged from the 1950s until the
past few years. In fact, this was not the case; the 1950s, 1960s and 1970s
saw dramatic successes in reducing the burden of malaria. These were
most obvious at the limits of the malaria map, but declining child mortal-
ity (albeit from extremely high levels) and malaria-specific mortality in
sites across sub-Saharan Africa in the post-colonial era suggests that a
significant degree of control was achieved elsewhere.
The GMEP was a moment of tremendous expectation and brought into
sharp relief the burden posed by malaria across Africa. Rapid adoption of
IRS led to impressive declines in disease incidence in almost every area
where this control approach was taken to scale in the North, the South
and the islands of Africa. In addition to vector control, the wide-scale
availability of and use of chloroquine were probably of major importance.
The same was also true at sites where IRS and prophylaxis were intro-
duced under pilot schemes in sub-Saharan Africa. What emerged was
that despite huge reductions in disease burden, transmission in most
settings was not interrupted within the few years that the GMEP had
hoped to eliminate malaria. Against a waning enthusiasm for elimination
in Africa, countries located at the margins, nonetheless, continued to
pursue carefully coordinated elimination strategies after independence
from colonial rule.
By 1979, no part of North Africa was considered to be subject to
stable P. falciparum transmission, Réunion was certified malaria free in
1975, Mauritius was P. falciparum free (despite reintroduced P. vivax),
malaria risks were exceptionally low on one remaining island of Cape
Verde, Madagascar had achieved near interruption of transmission
across the highland provinces and the spatial margins of stable risk
had reduced significantly in Zimbabwe, South Africa and the Kingdom
of Swaziland by 1979 (Fig. 4.20B). However around the late 1980s, things
began to unravel. The years leading up to 1999 saw a precipitous rise in
disease incidence in Southern Africa and the islands where P. falciparum
transmission had been reduced to barely detectable levels in 1979 had
returned to stable endemic levels (Fig. 4.20C). Because of pre-emptive,
earlier elimination achievements, North Africa was largely protected
from the re-expansion of stable transmission, although it is notable
that the Kingdom of Morocco and Egypt witnessed resurgent P. vivax
risks during the same period. Thus, rather than being the baseline
against which we should measure what is happening now, the 1990s

should probably be regarded as an exceptional period in which malaria
was on the rise following a period of control, albeit limited, in many
parts of Africa.
The reasons for this are probably multifactorial: because interest in
malaria control had fallen off the international agenda since the late 1970s,
effective new tools such as impregnated bed nets failed to be taken up. In
areas that had enjoyed protracted periods of effective control populations
were naive to the clinical consequences of infection having failed to
develop collective immunity. In many areas, especially those where vec-
tor control had never been widely applied, the widespread use of chloro-
quine had probably played the major role in controlling morbidity and
mortality which began to be lost with increasing drug failure. The result,
beginning in the late 1980s and early 1990s, was a wave of increasing
malaria incidence and deaths in many countries, including those located
at the margins of stable transmission. A rise in incidence was seen across
large parts of Southern Africa and the highland fringes of East Africa and
Madagascar, and there was a stalling of progress towards elimination in
North African countries yet to achieve a malaria-free status (Morocco,
Egypt, Algeria). At the same time, malaria mortality was rising across
many parts of sub-Saharan Africa and in some areas may have doubled.
By 1999, the international community had recognized the need for global
action with a focus on Africa, new funding was made available and the
subsequent 10 years led to the re-establishment of effective control opera-
tions in Southern Africa and the African islands leading to a renewed
contraction of stable endemicity by 2009 (Fig. 4.20D).
Thus, it seems plausible that the public health burden of malaria
across much of Africa south of the Sahara witnessed a substantive decline
following the Second World War. These achievements were probably
sustained through the 1970s and early 1980s, and at some point towards
the end of the 1980s into the 1990s, malaria incidence began to rise reach-
ing, in some areas, pre-1940s levels by the late 1990s. Since 2000, evidence
exists of a declining incidence of malaria in many (though by no means
all) parts of Africa. It is reasonable to assume that the first ‘‘dip’’ in
malaria in Africa was largely related to deliberate attempts at control;
certainly, this was the case at the limits of transmission. Similarly, the
massively increased investment in malaria control must be playing an
important role in the second or ‘‘double dip’’ in malaria. However, it is
also important to recognize that there may be other factors at play; in
several areas, it is clear that the beginnings of the current decline in
transmission considerably preceded the widespread application of new
investments in control and that these are insufficient to explain the timing
and degree of the changes. Many factors, including climatic, socioeco-
nomic, and biological factors, could potentially be lending a hand to the
undoubted effects of vector control and availability of effective drugs.

Understanding these factors is important because whilst they seem to be
moving in the right direction at the moment, there is no guarantee that
this will always be the case.
4.5.5. The future

The past few years have seen renewed international commitment and
investment in global malaria control. Its successes have led to a new
optimism and a refocussing of the world’s attention on the importance
of eradication as the long-term goal of our efforts. At the same time,
there has been concern that Africa may once again be neglected and
financial resources for a global programme diverted from high-burden
countries to support shrinking the malaria map at the low-risk margins
of the world outside Africa. Against this back ground, there are cer-
tainly many lessons to be drawn from the long experience in Africa of
attempts at malaria control and elimination, and we have attempted to
bring together for the first time in this review this accumulated experi-
ence in some depth. Although it is clear that the final steps to elimina-
tion, even in apparently favourable circumstances are difficult,
prolonged and susceptible to setback from many causes, perhaps the
most important point for the future is that reducing malaria to a minor
problem in terms of disease or deaths is an inescapable point on the way
to elimination. Here, the repeated lesson from control programmes
around Africa is that this can be achieved remarkably quickly, and
this should be the unremitting focus of African and international efforts
until it is achieved.
ACKNOWLEDGEMENTS
This chapter is the result of funding provided by the Wellcome Trust, UK as part of
fellowship support to RWS (079080) and AMN (095127) and the Wellcome Trust Core
Grant to the Kenyan Major Overseas Programme (092654)
This review has only been possible with the gracious help and assistance provided by
librarians and archivists in Europe and Africa particularly the library staff at The Wellcome
Institute, London; the Institute Pasteur, Paris (Agés Raymond-Denise, Catherine Cecilio,
Daniel Demellier and Dominique Dupenne); the Institute of Tropical Medicine, Antwerp
(Dirk Schoonbaert); Sapienza—Università di Roma, Rome (Gilberto Corbellini, Mauro
Capocci); Instituto Higiene Medicina Tropical, Project RIDES CPLP, Lisbon (Virgı́lio do
Rosário, Susana Nery); the World Health Organization library in Geneva (Marie Sarah
Villemin Partow), Sudan Civilization Institute, Khartoum (Jaffar Mirghani, Alaa Moawia);
Wellcome Library, National Public Health Laboratory Service, Nairobi (Anne Mbeche);
National Institutes for Health archives, Amani (William Kisinza, Jumanne Gwau, Japhet
Kimbesa). Of additional note for acknowledgement are the invaluable on-line library
resources provided by Armed Forces Pest Management Board Defense Pest Management
Information Analysis Centre Literature Retrieval System—AFMIC Library: http://lrs.afpmb.
org; the World Health Organizations malaria and country report repositories: http://whqlib-
doc.who.int/malaria/; Inter-university health library, Paris, France: http://www.biusante.
parisdescartes.fr/debut.htm; South African Medical journal archives: http://archive.samj.
org.za/index.php and the Institute of Tropical Medicine, Antwerp, Belgium http://lib.itg.be.
The authors are also indebted to malariologists, surveillance officers and malaria control
programme managers from across Africa including; Joana Alves (Cape Verde), Rajae El
Aouad (Morocco), Richard Kamwi and Benson Ntomwa (Namibia); Simon Kunene and
Joseph Novotny (Swaziland); Philip Kruger, Aaron Mabuza, Marlies Craig, Rajendra
Maharaj and Karen Barnes (South Africa); Abdulla Ali and Justin Cohen (Zanzibar); Jean-
Francois Trape (Senegal); Richard Cibulskis and Ryan O’Neil (Algeria and Botswana); Hawa
Guessod (Djibouti); Milijaona Randrianarivelojosia (Madagascar), Jean-Louis Solet (Mayotte
and Réunion); Ghasem Zamani and Hoda Atta (Morocco and Egypt) and especially our
gratitude to Frank Hansford for his detailed descriptions of malaria and its control in
Namibia, Botswana, Swaziland and South Africa. Finally, we are grateful for the assistance
provided by Clara Mundia for help with proof reading.
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INDEX
Note: Page numbers followed by ‘‘f’’ indicate figures, and ‘‘t’’ indicate tables.
A B
Afrotropic geographical region Biodiversité du paludisme, 108
agricultural development Biogeographical regions, malaria ecotypes
ecosystems, 117 and stratification
environmental management, 117 agricultural development, 140–149
Madagascar, 118 ecological typology, 138
vector density, 116–117 physiographic changes, 138–139
coastal, 115 physiography, with variations, 131f, 140
desert fringe plains and valleys, 139
demarcation, 115 population movement, 140
health service infrastructure, structuring, typology, 139
114–115 temperature-dependent, 138
malaria control, 115 urban environment, 149
forest, 113
highland fringe C
distribution, adult mosquitoes,
Cestodes and monogeneans
113–114
E. multilocularis protoscoleces, 29
Ethiopia, 114
marine fish, 29
malaria transmission, 112
metacestode vesicles, 28–29
savanna, 113
protoscoleces, 29
urban, 116
war and socio-political disturbance, 118
D
Annual parasite incidence (API)
malaria control planning, 130 ‘‘Daraprim Parade’’, 221–222
malaria risk, 131, 131f Djibouti, malaria transmission, 189–190
API. See Annual parasite incidence (API)
Arthropods E
RNAi machinery
E8. See Malaria Elimination 8 (E8)
antiviral immunity, 9
Enhanced vegetation index (EVI)
gene silencing approaches, 9
extreme aridity, 179
RNAi approaches, 9, 10t
forest-covered areas, 122–123
tick gene function, 9
land-use data, 140
systematic applications, RNAi technology
EVI. See Enhanced vegetation index (EVI)
developmental stage and tissue, 32
embryogenesis, 32
G
entomology, 32
mosquitoes, 36–40 Gene silencing, parasites
parasitic, 40 barriers, in vitro/in vivo silencing,
ticks, 32–36 41, 42
transmission-blocking vaccines, 32 development, novel tools, 40–41
263
264 Index
Gene silencing, parasites (cont.) Afghanistan, central Asia, Iran and

global transcriptome and proteome Russia, 136–137
analysis, 2 Arabian peninsula, 136
in vitro assays, 41 Caucasus, Iraq and Turkey, 136
molecular parasitology, 2 Central China and Korean peninsula,
RNAi mechanism and approaches 137
machinery, 5–14 ecological and climatic variability, 136
short-RNA types, 3–5 Giardia
sequence-specific, 3 evolutionary biology and phylogeny
siRNA amplification, 41 Diplomonads, 62
systematic applications, RNAi technology gene transfer, 60–61
arthropods, 32–40 mitochondrial remnants, 61
helminths, 25–32 multigene phylogenies, 61–62
protozoa, 21–25 trans-splicing, 61
technical approach, 3 genetic variation
technical tools, 41 developmental biology, 79–80
tools and methods pathogenesis, 81–83
dsRNA delivery, 17–20 genome and proteome projects
factors affecting efficiency, 20 GiardiaDB, 59–60
stability, dsRNA, 15–17 GS and P15 assemblies, 59–60
uptake and spreading, dsRNAs, 14–15 proteomic analysis, 60
transcriptional and post-transcriptional hosts
levels, 2–3 dogs and cats, 70–71
Genetic variation, Giardia humans, 68–70
developmental biology livestock, 71
G. duodenalis and G. bovis, 80 wildlife, 71–72
genome comparative analyses, 79–80 interaction, cycles
metabolic gene content, 80 G. duodenalis and G. enterica, 74–75
pathogenesis zoonotic transmission, 75–79
enteric protozoan infection, 81–82 life cycle and development
gastrointestinal parasites, 81 cysts, 66–67
G. enterica, 81–82 excretion, cyst, 68
giardiasis, acute and chronic, 81–82 excystation, 67
intestinal epithelial cells, 82 in vitro encystation, 67–68
Geographical regions reproduction, 67
afrotropic scanning electron micrograph,
agricultural development, 116–118 trophozoites, 58f
coastal, 115 species, 59t
desert fringe, 114–115 taxonomy and nomenclature
forest, 113 Diplomonadida replacement, 62–63
highland fringe, 113–114 multigene phylogenetic analyses, 62–63
malaria transmission, 112 phylogenetic relationships, 64f
savanna, 113 transmission
urban, 116 faecal–oral, 72
war and socio-political disturbance, 118 foodborne, 73–74
Australasian region waterborne, 72–73
anopheline fauna, 118 Giardiasis
population density, 119 acute and chronic, 81–82
stream rectification, 119 description, 72–73
vector system, 118 G. duodenalis, 81–82
neotropic and nearctic regions, 130–135 Global Malaria Action Plan (GMAP),
palearctic 174–175
Index 265
Global Malaria Eradication Programme optimal levels, salinity, 125

(GMEP) shrimp farming, 125
Africa, 1948–1960, 172–173 deforestation, 124
post, 1960–1999 desert fringe, 124–125
defined, control, 173 foothills, 124
ITNs, 173–174 forest fringe, deforestation and foothills
‘‘pre-elimination’’, 173 EVI, 122–123
Second World War, before Indochinese peninsula, 121
breeding sites, 217 larval control, 123–124
Nairobi city, 218, 218f national malaria survey, 2007, 122–123
vegetation clearance and drainage, NDVI, 122–123
218–219 subtypes, 121–122
vector control, 219–223 vectorial capacity, 121
Global rural urban mapping project highland fringe, 124
(GRUMP), 109, 178 plains, traditional agriculture, 120–121
GMAP. See Global Malaria Action Plan stratification, India and Vietnam
(GMAP) API, 129–130
GMEP. See Global Malaria Eradication morbidity rates, 129
Programme (GMEP) stratification schemes, 129
GRUMP. See Global rural urban mapping Thai scheme, 129
project (GRUMP) tea and tree plantations, 128
urban
H flood prone, 126
intersectorial, 126
Helminths
war and socio-political disturbances, 128
RNAi machinery, parasites
Indoor residual spraying (IRS)
C. elegans, 7–8
Algeria, 182–184
cestodes, 8
Botswana, 211–212
gene silencing, schistosomes, 8
forest malaria, 123–124
in vitro maintenance, 7
larval control, 101
knock-down regulation, 8
Mayotte, 201–202
Morocco, 181–182
cestodes and monogeneans, 28–29
São Tomé and Prı́ncipe
developmental stages, 25
alphacypermethrin, 193
gene function, 25
DDT and gammexane, 192
global health and economic
Swaziland, 215–216
development, 25
Tunisia, 184–186
livestock production, 25
vector control, 134–135
nematodes, 29–32
Zanzibar island, 195
trematodes, 25–28
Insecticide-treated nets (ITNs), 102
I IRS. See Indoor residual spraying (IRS)
Islands of Africa, malaria transmission
Indo-Malay region Cape Verde
agricultural development An. pretoriensis, 190
bio-environmental control, 128 incidence, 191f
rice cultivation, 127 national eradication programme, 192
types, 126 Comoros
An. fluviatilis, 119 altitude, settlement patterns and
anthropophilic and exophilic vectors, 120 agriculture, 199
biodiversity, 119 DDT, 199–200
coast ITN and IRS, 200
environmental management, 125 PNLP, 199–200
266 Index
Islands of Africa, malaria transmission control programmes

(cont.) anthropic and natural processes, 152
Madagascar expectations, impact, 152
amodiaquine–artesunate, 205 landscape epidemiology, 152–153
chloroquine prophylaxis, 203–205 stratification and delimitation, risk,
incidence, 204f 149–150
ITN and continued house spraying, vector control, 151–152
204–205 ecological classifications, 100
P. falciparum and P. vivax, 205–206 ecology-based classification, 102
P. vivax, 203 epidemiology, 99
spleen rates, 203–204 geographical regions
Mauritius afrotropic, 112–118
incidence, 197, 198f Australasian region, 118–119
Malaria Eradication Scheme, 197–198 Indo-malay region, 119–130
P. vivax transmission, 198–199 neotropic and nearctic regions, 130–135
spleen rates, 197 palearctic, 136–137
Mayotte global studies
artesunate–mefloquine, 201–202 and agriculture interactions, 108
chloroquine prophylaxis, 201 biomes and biogeographic realms, 107f,
incidence, 202f 109
ITN and IRS, 201–202 classes, ecological typology, 108–109
Réunion, 195–197 deforestation, 109
São Tomé and Prı́ncipe diversity, epidemiology, 108
IRS, DDT and gammexane, 192 land use and incidence, comparison,
LLIN, 193 109, 111f
mortality, 194f MAP and GRUMP, 109
volcano topography and plantation stability and vegetation index, 109
agricultural economy, 192 immense variability, 98–99
Zanzibar IRS, 101
artemisinin-based combination ITNs, 102
therapy (ACT), 195 journal literature, 102–106
DDT, 193–194, 195 Macdonald’s scheme, 101
larval survey, 193–194 malaria modelling and field, 153–155
ZMCP, 195 malariometric classifications, 99
ITNs. See Insecticide-treated nets (ITNs) malariometric data, 106
methods
M
biomes and biogeographic realms,
Malaria Atlas project (MAP), 109 106–108, 107f
Malaria control, Africa eco-epidemiology, 106–108
GMEP Pubmed, 106
1948–1960, 172–173 terms and acronyms, 102, 103t
post GMEP, 1960–1999, 173–174 vector bionomics, 99–100
middle Africa Wallace’s zoogeographical regions,
RBM, 223–226 100–101, 100t
Second World War, before, 217–219 Malaria elimination
vector control, 219–223 E8, 236–237
pre-second world war, 171–172 Islands of Africa
RBM, 174–175 ACT, 234–235
Malaria ecotypes and stratification infections, travellers, 235
biogeographical regions. See IRS and anti-malarial drug
(Biogeographical regions, malaria combination, 234
ecotypes and stratification) ITN and IRS, 234–235
Index 267
Southern Africa olfaction, 37

E8 Windhoek Agreement, 236–237 phenotypical effect, 36–37
IRS programmes, 235 RNAi pathway, 39
prolific cross-border seasonal transcriptional profiling, 38
migration, 236 vitellogenesis and reproduction, 37
Malaria Elimination 8 (E8), 236–237
Malaria in Africa N
changing limits, North Africa
Arab Spring, 226–233 NDVI. See Normalized difference vegetation
P. falciparum transmission, 226–233 index (NDVI)
spatial risks, 226, 230f Nematodes
control. See (Malaria control, Africa) cathepsins, 31–32
double dip recession gene transcription, 31
chloroquine, 238 moulting process, 30
GMEP, 237 nematode gene function, 29–30
P. falciparum transmission, 237–238 parasitic life stages, 30–31
elimination RNAi technology, 29
islands, 233–235 technical approaches, 30
Southern Africa, 235–237 ubiquitin and tropomyosin, 31
risk exclusion Neotropic and Nearctic regions
GRUMP, 178 agricultural development, 135
Plasmodium falciparum transmission, API, 131, 131f
175–178, 176f characteristics, 130
transmission climatic factors, 131
islands, 190–206 coastal, 134–135
middle Africa, 217–226 high valley, 134
North Africa and Djibouti, 181–190 interior lowland forest
Southern Africa, 206–217 agricultural colonization, 133
temperature and aridity, 178–179 ecological classification, 132–133
transmission stability epiphytic bromeliads, 133
low-stable endemic situation, 180–181 malaria system, 132
multiple autochthonous transmission multifactorial, spatial analysis, 132–133
events, 180–181 piedmont, 133–134
phases, 180 savanna, 132
stable–unstable classification, 179–180 urban, 135
Malaria modelling and field warfare and social instability, 135
field research Normalized difference vegetation index
geographical classification, 155 (NDVI)
human ecology, 154–155 land-use data, 140
mapping, 153 malaria incidence, 113
simulation modelling, effects North Africa, malaria transmission
drug resistance, 153 Algeria
ecoregions, 154 annual incidence, 183f, 184f
health system factors, 154 P. falciparum and P. vivax, 182–184
MAP. See Malaria Atlas project (MAP) quinine prophylaxis, 182–184
Mosquitoes Egypt
antiviral mosquito immunity, 38–39 An. gambiae s.l, 186–187
human diseases, vectors, 36 Fayoum, 188–189
immune defence mechanisms, 38–39 incidence, 188, 188f
induction, dormant, 37 Libya, 186
innate immune system, 38 Morocco, 181–182
malaria. See (Malaria in Africa) Tunisia, 184–186
268 Index
P RNA silencing
dsRNA delivery and stability
Parasites. See Gene silencing, parasites
carrier-mediated methods, 16
PMI. See President’s malaria initiative (PMI)
gene therapy, 15–16
PNLP. See Programme National de Lutte
hybridization affinity, 17
Contre le Paludisme (PNLP)
in vivo half-life, 17
President’s malaria initiative (PMI),
lentivirus-based vectors, 16–17
174–175
locked nucleic acid, 17
Programme National de Lutte Contre le
synthetic libraries, 17
Paludisme (PNLP), 199–200
VACNFs, 16
Protozoa
dsRNA delivery, parasites
RNAi machinery, parasites
developmental stage and tissue,
apicomplexan parasites, 6
19–20
draft genome, 6–7
electroporation and feeding
Leishmania, 6
methods, 18
pathway, 6
experimental designs, 18–19
transfer RNAs (tRNAs), 7
parasitology research, 17–18
trypanosomatids, 5–6
phenotypical and biochemical
analyses, 19
antisense effect, 23
transient reduction, mRNA levels, 18
anti-toxoplasmosis vaccines, 23–24
factors affecting RNAi efficiency
epigenetic control mechanisms, 22
off-target effects, 20
gene down-regulation, 24
parasite developmental stages, 20
gene function, 21
trematode nervous tissue, 20
gene replacement techniques, 23
uptake and spreading, dsRNAs
immune evasion/drug resistance, 21
RSD, systemic effect, 15
livestock production, 24–25
SID-1, transmembrane protein, 14–15
massive sequencing, 21–22
surface composition, 14
plasmodial species, 23
Roll back malaria (RBM)
polyamine biosynthesis, 22
DDT and chloroquine, 174
procyclic vacuolar proteins, 22
GFATM, 174–175
trypanosome applications, 22
PMI, 174–175
vector-trasmitted and foodborne
RBM era, middle Africa
protozoa, 21
ACTs, 224–225
VSP, 24
chloroquine and
sulphadoxine–pyrimethamine
R
resistance, 224–225
RBM. See Roll back malaria (RBM) GFATM, 225
RNAi. See RNA interference (RNAi) ITN, 223–224, 225–226
RNA interference (RNAi)
S
machinery, parasites
arthropods, 9–14 Southern Africa, malaria transmission
helminths, 7–8 Botswana
protozoa, 5–7 DDT, 211–212
parasitic arthropods deltamethrin and lambda-cyhalothrin,
caligidae, 40 211–212
insect disease vectors, 40 incidence, 212f
short-RNA types National Malaria Control Programme,
argonaute proteins, 5 211–212
cytoplasmic gene silencing mechanism, school-based parasitological survey,
3–4, 4f 211
RISC, 4–5 Namibia, 209–210
Index 269
South Africa physiological processes, 26–27

anti-larval measures, 206–207 RNAi assays, 25–26
DDT, 207–208 schistosome mating, 27
incidence rates, 208–209, 209f silencing, cathepsin B, 28
KwaZulu-Natal, 207–209, 208f sporocysts glucose transporters, 27–28
pyrethroids, 207–208 tetraspanins, 27
Swaziland TGR expression, 26
control programmes, 215–216 TSI. See Temperature suitability index (TSI)
incidence, 215–216, 215f
LSDI, 216–217 V
P. falciparum elimination, 217
VACNFs. See Vertically aligned carbon
Zimbabwe
nanofiber arrays (VACNFs)
An. arabiensis and An. funestus, 214
Variant surface proteins (VSP)
control programmes, 213
expression regulation, 6–7
DDT, 213–214
RNAi machinery, 24
incidence, 214f
Vertically aligned carbon nanofiber arrays
risks, 212–213
(VACNFs), 73–74
T VSP. See Variant surface proteins (VSP)
Temperature suitability index (TSI),

Z
178–179
Ticks Zoonotic transmission, Giardia
anticoagulant roles, 34 dogs and cats
bioactive lipids and proteins, 33 cross-infection experiments, 75
gene silencing, 33 gastrointestinal disorders, 75
genome resource availability, 32 mixed infections, 76, 77
immunophilin, 35 molecular epidemiological studies,
iron homeostasis, 34 76–77
novel control measures, 34 livestock
proteolytic enzymes, 33 animal handlers, 78
recombinant proteins, 36 pigs, 78
vaccines, 35 waterborne outbreaks, 77–78
vitellogenesis, 34 wildlife
Trematodes aquatic mammals, 79
developmental stages, schistosomes, 26 beavers, 79
gut protease function, 26 ‘reverse zoonotic transmission’, 79
CONTENTS OF VOLUMES
IN THIS SERIES
Volume 41 M. Albonico, D.W.T. Cromption, and

L. Savioli
Drug Resistance in Malaria Parasites of
Animals and Man DNA Vaocines: Technology and
W. Peters Applications as Anti-parasite and
Anti-microbial Agents
Molecular Pathobiology and Antigenic J.B. Alarcon, G.W. Wainem and
Variation of Pneumocystis carinii D.P. McManus
Y. Nakamura and M. Wada
Ascariasis in China
P. Weidono, Z. Xianmin and Volume 43
D.W.T. Crompton
Genetic Exchange in the
The Generation and Expression of Trypanosomatidae
Immunity to Trichinella spiralis in W. Gibson and J. Stevens
Laboratory Rodents
R.G. Bell The Host-Parasite Relationship in
Neosporosis
Population Biology of Parasitic A. Hemphill
Nematodes: Application of
Genetic Markers Proteases of Protozoan Parasites
T.J.C. Anderson, M.S. Blouin and P.J. Rosenthal
R.M. Brech Proteinases and Associated Genes of
Schistosomiasis in Cattle Parasitic Helminths
J. De Bont and J. Vercruysse J. Tort, P.J. Brindley, D. Knox, K.H. Wolfe,
and J.P. Dalton
Parasitic Fungi and their
Volume 42 Interaction with the Insect
Immune System
The Southern Cone Initiative Against A. Vilcinskas and P. Götz
Chagas Disease
C.J. Schofield and J.C.P. Dias
Phytomonas and Other Trypanosomatid
Parasites of Plants and Fruit Volume 44
E.P. Camargo
Cell Biology of Leishmania
Paragonimiasis and the Genus B. Handman
Paragonimus
Immunity and Vaccine Development in
D. Blair, Z.-B. Xu, and T. Agatsuma
the Bovine Theilerioses
Immunology and Biochemistry of N. Boulter and R. Hall
Hymenolepis diminuta
The Distribution of Schistosoma bovis
J. Anreassen, E.M. Bennet-Jenkins, and
Sonaino, 1876 in Relation to
C. Bryant
Intermediate Host Mollusc-Parasite
Control Strategies for Human Intestinal Relationships
Nematode Infections H. Moné, G. Mouahid, and S. Morand
271
272 Contents of Volumes in This Series
The Larvae of Monogenea Satellites, Space, Time and the African

(Platyhelminthes) Trypanosomiases
I.D. Whittington, L.A. Chisholm, and D.J. Rogers
K. Rohde Earth Observation, Geographic
Sealice on Salmonids: Their Biology Information Systems and
and Control Plasmodium falciparum Malaria in
A.W. Pike and S.L. Wadsworth Sub-Saharan Africa
S.I. Hay, J. Omumbo, M. Craig, and
R.W. Snow
Volume 45
Ticks and Tick-borne Disease Systems in
The Biology of some Intraerythrocytic Space and from Space
Parasites of Fishes, Amphibia S.E. Randolph
and Reptiles
The Potential of Geographical
A.J. Davies and M.R.L. Johnston
Information Systems (GIS) and
The Range and Biological Activity of FMR Remote Sensing in the Epidemiology
Famide-related Peptides and and Control of Human Helminth
Classical Neurotransmitters Infections
in Nematodes S. Brooker and E. Michael
D. Brownlee, L. Holden-Dye, and R.
Advances in Satellite Remote Sensing of
Walker
Environmental Variables for
The Immunobiology of Gastrointestinal Epidemiological Applications
Nematode Infections in Ruminants S.J. Goetz, S.D. Prince, and J. Small
A. Balic, V.M. Bowles, and E.N.T.
Forecasting Diseases Risk for Increased
Meeusen
Epidemic Preparedness in Public
Health
Volume 46 M.F. Myers, D.J. Rogers, J. Cox, A.
Flauhalt, and S.I. Hay
Host-Parasite Interactions in
Acanthocephala: A Morphological Education, Outreach and the Future of
Approach Remote Sensing in Human Health
H. Taraschewski B.L. Woods, L.R. Beck, B.M. Lobitz, and
M.R. Bobo
Eicosanoids in Parasites and Parasitic
Infections
A. Daugschies and A. Joachim
Volume 48
The Molecular Evolution of
Volume 47 Trypanosomatidae
J.R. Stevens, H.A. Noyes, C.J. Schofield,
An Overview of Remote Sensing and and W. Gibson
Geodesy for Epidemiology and
Public Health Application Transovarial Transmission in the
S.I. Hay Microsporidia
A.M. Dunn, R.S. Terry, and J.E. Smith
Linking Remote Sensing, Land Cover
and Disease Adhesive Secretions in the
P.J. Curran, P.M. Atkinson, G.M. Foody, Platyhelminthes
and E.J. Milton I.D. Whittington and B.W. Cribb
Spatial Statistics and Geographic The Use of Ultrasound in Schistosomiasis

C.F.R. Hatz
Information Systems in
Epidemiology and Public Health Ascaris and Ascariasis
T.P. Robinson D.W.T. Crompton
Contents of Volumes in This Series 273
Volume 49 Volume 52
Antigenic Variation in Trypanosomes: The Ecology of Fish Parasites with
Enhanced Phenotypic Variation in a Particular Reference to
Eukaryotic Parasite Helminth Parasites and their
H.D. Barry and R. McCulloch Salmonid Fish Hosts in Welsh
Rivers: A Review of Some of the
The Epidemiology and Control of Human
Central Questions
African Trypanosomiasis
J.D. Thomas
J. Pépin and H.A. Méda
Biology of the Schistosome Genus
Apoptosis and Parasitism: from the
Trichobilharzia
Parasite to the Host Immune
P. Horák, L. Kolárová, and C.M. Adema
Response
G.A. DosReis and M.A. Barcinski The Consequences of Reducing
Transmission of Plasmodium
Biology of Echinostomes Except
falciparum in Africa
Echinostoma
R.W. Snow and K. Marsh
B. Fried
Cytokine-Mediated Host Responses
during Schistosome Infections:
Volume 50 Walking the Fine Line Between
The Malaria-Infected Red Blood Cell: Immunological Control and
Structural and Functional Changes Immunopathology
B.M. Cooke, N. Mohandas, and R.L. K.F. Hoffmann, T.A. Wynn, and D.W.
Coppel Dunne
Schistosomiasis in the Mekong Region:

Epidemiology and Phytogeography
S.W. Attwood Volume 53
Molecular Aspects of Sexual Interactions between Tsetse
Development and Reproduction in and Trypanosomes with
Nematodes and Schistosomes Implications for the Control of
P.R. Boag, S.E. Newton, and R.B. Gasser Trypanosomiasis
S. Aksoy, W.C. Gibson, and M.J. Lehane
Antiparasitic Properties of Medicinal
Plants and Other Naturally Enzymes Involved in the Biogenesis of
Occurring Products the Nematode Cuticle
S. Tagboto and S. Townson A.P. Page and A.D. Winter
Diagnosis of Human Filariases (Except
Volume 51 Onchocerciasis)
M. Walther and R. Muller
Aspects of Human Parasites in which
Surgical Intervention May Be
Important
D.A. Meyer and B. Fried Volume 54
Electron-transfer Complexes in Ascaris Introduction – Phylogenies,
Mitochondria Phylogenetics, Parasites and the
K. Kita and S. Takamiya Evolution of Parasitism
D.T.J. Littlewood
Cestode Parasites: Application of In Vivo
and In Vitro Models for Studies of the Cryptic Organelles in Parasitic Protists
Host-Parasite Relationship and Fungi
M. Siles-Lucas and A. Hemphill B.A.P. Williams and P.J. Keeling
Phylogenetic Insights into the Evolution The Mitochondrial Genomics of Parasitic

of Parasitism in Hymenoptera Nematodes of Socio-Economic
J.B. Whitfield Importance: Recent Progress, and
Implications for Population Genetics
Nematoda: Genes, Genomes and the
and Systematics
Evolution of Parasitism
M. Hu, N.B. Chilton, and R.B. Gasser
M.L. Blaxter
The Cytoskeleton and Motility in
Life Cycle Evolution in the Digenea: A
Apicomplexan Invasion
New Perspective from Phylogeny
R.E. Fowler, G. Margos, and G.H. Mitchell
T.H. Cribb, R.A. Bray, P.D. Olson, and
D.T.J. Littlewood
Progress in Malaria Research: The Case Volume 57
for Phylogenetics Canine Leishmaniasis
S.M. Rich and F.J. Ayala J. Alvar, C. Cañavate, R. Molina, J.
Phylogenies, the Comparative Moreno, and J. Nieto
Method and Parasite Evolutionary Sexual Biology of Schistosomes
Ecology H. Moné and J. Boissier
S. Morand and R. Poulin
Review of the Trematode Genus Ribeiroia
Recent Results in Cophylogeny Mapping (Psilostomidae): Ecology, Life
M.A. Charleston History, and Pathogenesis with
Inference of Viral Evolutionary Rates Special Emphasis on the Amphibian
from Molecular Sequences Malformation Problem
A. Drummond, O.G. Pybus, and A. P.T.J. Johnson, D.R. Sutherland,
Rambaut J.M. Kinsella and K.B. Lunde
Detecting Adaptive Molecular Evolution: The Trichuris muris System: A Paradigm

Additional Tools for the of Resistance and Susceptibility to
Parasitologist Intestinal Nematode Infection
J.O. McInerney, D.T.J. Littlewood, and L.J. Cliffe and R.K. Grencis
C.J. Creevey Scabies: New Future for a Neglected
Disease
Volume 55 S.F. Walton, D.C. Holt, B.J. Currie, and
D.J. Kemp
Contents of Volumes 28–52
Cumulative Subject Indexes for Volumes
28–52
Contributors to Volumes 28–52
Volume 58
Leishmania spp.: On the Interactions they
Establish with Antigen-Presenting
Volume 56 Cells of their Mammalian Hosts
J.-C. Antoine, E. Prina, N. Courret, and
Glycoinositolphospholipid from
T. Lang
Trypanosoma cruzi: Structure,
Biosynthesis and Immunobiology Variation in Giardia: Implications
J.O. Previato, R. Wait, C. Jones, for Taxonomy and Epidemiology
G.A. DosReis, A.R. Todeschini, N. R.C.A. Thompson and P.T. Monis
Heise and L.M. Previata
Recent Advances in the Biology of
Biodiversity and Evolution of the Echinostoma species in the
Myxozoa ‘‘revolutum’’ Group
E.U. Canning and B. Okamura B. Fried and T.K. Graczyk
Human Hookworm Infection in the Volume 61

21st Century
S. Brooker, J. Bethony, and P.J. Hotez Control of Human Parasitic
Diseases: Context and Overview
The Curious Life-Style of the David H. Molyneux
Parasitic Stages of Gnathiid Isopods
N.J. Smit and A.J. Davies Malaria Chemotherapy
Peter Winstanley and Stephen Ward
Insecticide-Treated Nets
Volume 59 Jenny Hill, Jo Lines, and Mark Rowland
Genes and Susceptibility to Control of Chagas Disease
Leishmaniasis Yoichi Yamagata and
Emanuela Handman, Colleen Elso, and Jun Nakagawa
Simon Foote
Human African Trypanosomiasis:
Cryptosporidium and Cryptosporidiosis Epidemiology and Control
R.C.A. Thompson, M.E. Olson, G. Zhu, E.M. Fèvre, K. Picozzi, J. Jannin,
S. Enomoto, Mitchell S. Abrahamsen S.C. Welburn and I. Maudlin
and N.S. Hijjawi
Chemotherapy in the Treatment and
Ichthyophthirius multifiliis Fouquet and Control of Leishmaniasis
Ichthyophthiriosis in Freshwater Jorge Alvar, Simon Croft, and
Teleosts Piero Olliaro
R.A. Matthews
Dracunculiasis (Guinea Worm Disease)
Biology of the Phylum Nematomorpha Eradication
B. Hanelt, F. Thomas, and A. Schmidt- Ernesto Ruiz-Tiben and Donald
Rhaesa R. Hopkins
Intervention for the Control of Soil-
Volume 60 Transmitted Helminthiasis in the
Community
Sulfur-Containing Amino Acid
Marco Albonico, Antonio Montresor, D.W.
Metabolism in Parasitic Protozoa T. Crompton, and Lorenzo Savioli
Tomoyoshi Nozaki, Vahab Ali, and
Masaharu Tokoro Control of Onchocerciasis
Boakye A. Boatin and Frank O. Richards,
The Use and Implications of Ribosomal Jr.
DNA Sequencing for the
Discrimination of Digenean Species Lymphatic Filariasis: Treatment, Control
Matthew J. Nolan and Thomas H. Cribb and Elimination
Eric A. Ottesen
Advances and Trends in the Molecular
Systematics of the Parasitic Control of Cystic Echinococcosis/
Platyhelminthes Hydatidosis: 1863–2002
Peter D. Olson and Vasyl V. Tkach P.S. Craig and E. Larrieu
Wolbachia Bacterial Endosymbionts of Control of Taenia solium Cysticercosis/
Filarial Nematodes Taeniosis
Mark J. Taylor, Claudio Bandi, and Achim Arve Lee Willingham III and
Hoerauf Dirk Engels
The Biology of Avian Eimeria with an Implementation of Human
Emphasis on their Control by Schistosomiasis Control: Challenges
Vaccination and Prospects
Martin W. Shirley, Adrian L. Smith, and Alan Fenwick, David Rollinson, and
Fiona M. Tomley Vaughan Southgate
Volume 62 Targeting of Toxic Compounds to the

Trypanosome’s Interior
Models for Vectors and Vector-Borne Michael P. Barrett and Ian H. Gilbert
Diseases
D.J. Rogers Making Sense of the Schistosome
Surface
Global Environmental Data for Patrick J. Skelly and R. Alan Wilson
Mapping Infectious Disease
Distribution Immunology and Pathology of
S.I. Hay, A.J. Tatem, A.J. Graham, Intestinal Trematodes in Their
S.J. Goetz, and D.J. Rogers Definitive Hosts
Rafael Toledo, José-Guillermo Esteban, and
Issues of Scale and Uncertainty in Bernard Fried
the Global Remote Sensing of
Disease Systematics and Epidemiology of
P.M. Atkinson and A.J. Graham Trichinella
Edoardo Pozio and K. Darwin Murrell
Determining Global Population
Distribution: Methods, Applications
and Data
D.L. Balk, U. Deichmann, G. Yetman,
Volume 64
F. Pozzi, S.I. Hay, Leishmania and the Leishmaniases:
and A. Nelson A Parasite Genetic Update and
Defining the Global Spatial Limits of Advances in Taxonomy,
Malaria Transmission in 2005 Epidemiology and Pathogenicity
C.A. Guerra, R.W. Snow and S.I. Hay in Humans
Anne-Laure Bañuls, Mallorie Hide and
The Global Distribution of Yellow Fever Franck Prugnolle
and Dengue
D.J. Rogers, A.J. Wilson, S.I. Hay, and Human Waterborne Trematode and
A.J. Graham Protozoan Infections
Thaddeus K. Graczyk and Bernard Fried
Global Epidemiology, Ecology and
Control of Soil-Transmitted Helminth The Biology of Gyrodctylid
Infections Monogeneans: The ‘‘Russian-Doll
S. Brooker, A.C.A. Clements and Killers’’
D.A.P. Bundy T.A. Bakke, J. Cable, and P.D. Harris
Tick-borne Disease Systems: Mapping Human Genetic Diversity and the

Geographic and Phylogenetic Space Epidemiology of Parasitic
S.E. Randolph and D.J. Rogers and Other Transmissible Diseases
Michel Tibayrenc
Global Transport Networks and
Infectious Disease Spread
A.J. Tatem, D.J. Rogers and S.I. Hay Volume 65
Climate Change and Vector-Borne ABO Blood Group Phenotypes and
Diseases Plasmodium falciparum Malaria:
D.J. Rogers and S.E. Randolph Unlocking a Pivotal Mechanism
Marı́a-Paz Loscertales, Stephen Owens,
James O’Donnell, James Bunn, Xavier
Bosch-Capblanch, and Bernard J. Brabin
Volume 63
Structure and Content of the Entamoeba
Phylogenetic Analyses of Parasites in the histolytica Genome
New Millennium C. G. Clark, U. C. M. Alsmark, M.
David A. Morrison Tazreiter, Y. Saito-Nakano, V. Ali,
S. Marion, C. Weber, C. Mukherjee, Volume 67

I. Bruchhaus, E. Tannich, M. Leippe,
T. Sicheritz-Ponten, P. G. Foster, Introduction
J. Samuelson, C. J. Noël, R. P. Hirt, Irwin W. Sherman
T. M. Embley, C. A. Gilchrist, An Introduction to Malaria Parasites
B. J. Mann, U. Singh, J. P. Ackers, Irwin W. Sherman
S. Bhattacharya, A. Bhattacharya,
A. Lohia, N. Guillén, M. Duchêne, The Early Years
T. Nozaki, and N. Hall Irwin W. Sherman
Epidemiological Modelling for Show Me the Money

Monitoring and Evaluation of Irwin W. Sherman
Lymphatic Filariasis Control In Vivo and In Vitro Models
Edwin Michael, Mwele N. Malecela- Irwin W. Sherman
Lazaro, and James W. Kazura
Malaria Pigment
The Role of Helminth Infections in Irwin W. Sherman
Carcinogenesis
David A. Mayer and Bernard Fried Chloroquine and Hemozoin
Irwin W. Sherman
A Review of the Biology of the
Parasitic Copepod Lernaeocera Isoenzymes
branchialis (L., 1767)(Copepoda: Irwin W. Sherman
Pennellidae
The Road to the Plasmodium falciparum
Adam J. Brooker, Andrew P. Shinn, and
Genome
James E. Bron
Irwin W. Sherman
Carbohydrate Metabolism
Irwin W. Sherman
Volume 66 Pyrimidines and the Mitochondrion
Strain Theory of Malaria: The First Irwin W. Sherman
50 Years The Road to Atovaquone
F. Ellis McKenzie,* David L. Smith, Irwin W. Sherman
Wendy P. O’Meara, and Eleanor
M. Riley The Ring Road to the Apicoplast
Irwin W. Sherman
Advances and Trends in the Molecular
Systematics of Anisakid Nematodes, Ribosomes and Ribosomal Ribonucleic
with Implications for their Acid Synthesis
Evolutionary Ecology and Irwin W. Sherman
Host–Parasite Co-evolutionary
De Novo Synthesis of Pyrimidines
Processes
and Folates
Simonetta Mattiucci and Giuseppe
Irwin W. Sherman
Nascetti
Salvage of Purines
Atopic Disorders and Parasitic Infections
Aditya Reddy and Bernard Fried Irwin W. Sherman
Heartworm Disease in Animals and Polyamines

Humans Irwin W. Sherman
John W. McCall, Claudio Genchi, Laura H. New Permeability Pathways
Kramer, Jorge Guerrero, and and Transport
Luigi Venco Irwin W. Sherman
Hemoglobinases Tracking Transmission of the Zoonosis

Irwin W. Sherman Toxoplasma gondii
Judith E. Smith
Erythrocyte Surface Membrane Proteins
Irwin W. Sherman Parasites and Biological Invasions
Alison M. Dunn
Trafficking
Irwin W. Sherman Zoonoses in Wildlife: Integrating Ecology
into Management
Erythrocyte Membrane Lipids Fiona Mathews
Irwin W. Sherman
Understanding the Interaction
Invasion of Erythrocytes Between an Obligate Hyperparasitic
Irwin W. Sherman Bacterium, Pasteuria penetrans
Vitamins and Anti-Oxidant Defenses and its Obligate Plant-Parasitic
Irwin W. Sherman Nematode Host, Meloidogyne spp.
Keith G. Davies
Shocks and Clocks
Irwin W. Sherman Host–Parasite Relations and Implications
for Control
Transcriptomes, Proteomes Alan Fenwick
and Data Mining
Irwin W. Sherman Onchocerca–Simulium Interactions and the
Population and Evolutionary Biology
Mosquito Interactions of Onchocerca volvulus
Irwin W. Sherman Marı́a-Gloria Basáñez, Thomas
S. Churcher, and Marı́a-Eugenia Grillet
Volume 68 Microsporidians as Evolution-Proof
Agents of Malaria Control?
HLA-Mediated Control of HIV and HIV
Jacob C. Koella, Lena Lorenz, and Irka
Adaptation to HLA
Bargielowski
Rebecca P. Payne, Philippa C. Matthews,
Julia G. Prado, and Philip J. R. Goulder
An Evolutionary Perspective on
Volume 69
Parasitism as a Cause of Cancer The Biology of the Caecal Trematode
Paul W. Ewald Zygocotyle lunata
Bernard Fried, Jane E. Huffman, Shamus
Invasion of the Body Snatchers:
Keeler, and Robert C. Peoples
The Diversity and Evolution of
Manipulative Strategies in Fasciola, Lymnaeids and Human
Host–Parasite Interactions Fascioliasis, with a Global
Thierry Lefévre, Shelley A. Adamo, David Overview on Disease Transmission,
G. Biron, Dorothée Missé, David Epidemiology, Evolutionary
Hughes, and Frédéric Thomas Genetics, Molecular Epidemiology
and Control
Evolutionary Drivers of Parasite-Induced
Santiago Mas-Coma, Marı́a Adela Valero,
Changes in Insect Life-History Traits:
and Marı́a Dolores Bargues
From Theory to Underlying
Mechanisms Recent Advances in the Biology of
Hilary Hurd Echinostomes
Rafael Toledo, José-Guillermo Esteban, and
Ecological Immunology of a Tapeworms’
Bernard Fried
Interaction with its Two Consecutive
Hosts Peptidases of Trematodes
Katrin Hammerschmidt and Martin Kašný, Libor Mikeš, Vladimı́r
Joachim Kurtz Hampl, Jan Dvořák,
Conor R. Caffrey, John P. Dalton, and Components of Asobara Venoms and their
Petr Horák Effects on Hosts
Sébastien J.M. Moreau, Sophie Vinchon,
Potential Contribution of
Anas Cherqui, and Geneviève Prévost
Sero-Epidemiological Analysis
for Monitoring Malaria Strategies of Avoidance of Host Immune
Control and Elimination: Defenses in Asobara Species
Historical and Current Geneviève Prévost, Géraldine Doury,
Perspectives Alix D.N. Mabiala-Moundoungou,
Chris Drakeley and Jackie Cook Anas Cherqui, and Patrice Eslin
Evolution of Host Resistance and
Volume 70 Parasitoid Counter-Resistance
Alex R. Kraaijeveld and H. Charles
Ecology and Life History Evolution of J. Godfray
Frugivorous Drosophila Parasitoids Local, Geographic and Phylogenetic
Frédéric Fleury, Patricia Gibert, Scales of Coevolution in Drosophila–
Nicolas Ris, and Roland Allemand Parasitoid Interactions
Decision-Making Dynamics in S. Dupas, A. Dubuffet, Y. Carton, and
Parasitoids of Drosophila M. Poirié
Andra Thiel and Thomas S. Hoffmeister Drosophila–Parasitoid Communities as
Dynamic Use of Fruit Odours to Locate Model Systems for Host–Wolbachia
Host Larvae: Individual Learning, Interactions
Physiological State and Genetic Fabrice Vavre, Laurence Mouton, and
Variability as Adaptive Bart A. Pannebakker
Mechanisms A Virus-Shaping Reproductive Strategy
Laure Kaiser, Aude Couty, and in a Drosophila Parasitoid
Raquel Perez-Maluf Julien Varaldi, Sabine Patot,
The Role of Melanization and Cytotoxic Maxime Nardin, and Sylvain Gandon
By-Products in the Cellular Immune
Responses of Drosophila Against
Parasitic Wasps
A. Nappi, M. Poirié, and Y. Carton
Volume 71
Cryptosporidiosis in Southeast
Virulence Factors and Strategies of
Asia: What’s out There?
Leptopilina spp.: Selective Responses
Yvonne A.L. Lim, Aaron R. Jex,
in Drosophila Hosts
Huw V. Smith, and Robin B. Gasser
Mark J. Lee, Marta E. Kalamarz,
Indira Paddibhatla, Chiyedza Small, Human Schistosomiasis in the Economic
Roma Rajwani, and Shubha Govind Community of West African States:
Epidemiology and Control
Variation of Leptopilina boulardi Success in
Héléne Moné, Moudachirou Ibikounlé,
Drosophila Hosts: What is Inside the
Achille Massougbodji, and Gabriel
Black Box?
Mouahid
A. Dubuffet, D. Colinet, C. Anselme,
S. Dupas, Y. Carton, and M. Poirié The Rise and Fall of Human
Oesophagostomiasis
Immune Resistance of Drosophila Hosts
A.M. Polderman, M. Eberhard, S. Baeta,
Against Asobara Parasitoids: Cellular
Robin B. Gasser, L. van Lieshout,
Aspects
P. Magnussen, A. Olsen, N.
Patrice Eslin, Geneviève Prévost,
Spannbrucker, J. Ziem,
Sébastien Havard, and Géraldine Doury
and J. Horton
Volume 72 Combating Taenia solium Cysticercosis

in Southeast Asia: An Opportunity
Important Helminth Infections in for Improving Human Health and
Southeast Asia: Diversity, Potential Livestock Production Links
for Control and Prospects for A. Lee Willingham III, Hai-Wei Wu, James
Elimination Conlan, and Fadjar Satrija
Jürg Utzinger, Robert Bergquist, Remigio
Olveda, and Xiao-Nong Zhou Echinococcosis with Particular Reference
to Southeast Asia
Escalating the Global Fight Against Donald P. McManus
Neglected Tropical Diseases Through
Interventions in the Asia Pacific Food-Borne Trematodiases in Southeast
Region Asia: Epidemiology, Pathology,
Peter J. Hotez and John P. Ehrenberg Clinical Manifestation and Control
Banchob Sripa, Sasithorn Kaewkes,
Coordinating Research on Neglected Pewpan M. Intapan, Wanchai
Parasitic Diseases in Southeast Asia Maleewong, and Paul J. Brindley
Through Networking
Remi Olveda, Lydia Leonardo, Feng Zheng, Helminth Infections of the Central
Banchob Sripa, Robert Bergquist, and Nervous System Occurring in
Xiao-Nong Zhou Southeast Asia and the Far East
Shan Lv, Yi Zhang, Peter Steinmann,
Neglected Diseases and Ethnic Minorities Xiao-Nong Zhou, and Jürg Utzinger
in the Western Pacific Region:
Exploring the Links Less Common Parasitic Infections in
Alexander Schratz, Martha Fernanda Southeast Asia that can Produce
Pineda, Liberty G. Reforma, Nicole M. Outbreaks
Fox, Tuan Le Anh, L. Tommaso Peter Odermatt, Shan Lv, and Somphou
Cavalli-Sforza, Mackenzie K. Sayasone
Henderson, Raymond Mendoza, Jürg
Utzinger, John P. Ehrenberg, and Ah
Sian Tee Volume 73
Controlling Schistosomiasis in Southeast Concepts in Research Capabilities
Asia: A Tale of Two Countries Strengthening: Positive Experiences
Robert Bergquist and Marcel Tanner of Network Approaches by TDR in
the People’s Republic of China and
Schistosomiasis Japonica: Control and Eastern Asia
Research Needs Xiao-Nong Zhou, Steven Wayling, and
Xiao-Nong Zhou, Robert Bergquist, Robert Bergquist
Lydia Leonardo, Guo-Jing Yang,
Kun Yang, M. Sudomo, and Multiparasitism: A Neglected Reality on
Remigio Olveda Global, Regional and Local Scale
Peter Steinmann, Jürg Utzinger, Zun-Wei
Schistosoma mekongi in Cambodia and Du, and Xiao-Nong Zhou
Lao People’s Democratic Republic
Sinuon Muth, Somphou Sayasone, Health Metrics for Helminthic Infections
Sophie Odermatt-Biays, Samlane Charles H. King
Phompida, Socheat Duong, and Implementing a Geospatial Health Data
Peter Odermatt Infrastructure for Control of Asian
Elimination of Lymphatic Filariasis in Schistosomiasis in the People’s
Southeast Asia Republic of China and the
Mohammad Sudomo, Sombat Chayabejara, Philippines
Duong Socheat, Leda Hernandez, John B. Malone, Guo-Jing Yang, Lydia
Wei-Ping Wu, and Robert Bergquist Leonardo, and Xiao-Nong Zhou
The Regional Network for Asian Studies on the Parasitology,

Schistosomiasis and Other Phylogeography and the Evolution of
Helminth Zoonoses (RNASþ ): Host–Parasite Interactions for the
Target Diseases in Face of Snail Intermediate Hosts of Medically
Climate Change Important Trematode Genera in
Guo-Jing Yang, Jürg Utzinger, Shan Lv, Southeast Asia
Ying-Jun Qian, Shi-Zhu Li, Qiang Stephen W. Attwood
Wang, Robert Bergquist, Penelope
Vounatsou, Wei Li, Kun Yang, and
Xiao-Nong Zhou
Volume 74
Social Science Implications for Control of
Helminth Infections in Southeast The Many Roads to Parasitism: A Tale of
Asia Convergence
Lisa M. Vandemark, Tie-Wu Jia, and Robert Poulin
Xiao-Nong Zhou Malaria Distribution, Prevalence, Drug
Towards Improved Diagnosis of Zoonotic Resistance and Control in Indonesia
Trematode Infections in Southeast Iqbal R.F. Elyazar, Simon I. Hay, and
Asia J. Kevin Baird
Maria Vang Johansen, Paiboon Cytogenetics and Chromosomes of
Sithithaworn, Robert Bergquist, and Tapeworms (Platyhelminthes,
Jürg Utzinger Cestoda)
The Drugs We Have and the Drugs We Marta Špakulová, Martina Orosová, and
Need Against Major Helminth John S. Mackiewicz
Infections Soil-Transmitted Helminths of Humans
Jennifer Keiser and Jürg Utzinger in Southeast Asia—Towards
Research and Development of Integrated Control
Antischistosomal Drugs in the Aaron R. Jex, Yvonne A.L. Lim, Jeffrey
People’s Republic of China: Bethony, Peter J. Hotez, Neil D. Young,
A 60-Year Review and Robin B. Gasser
Shu-Hua Xiao, Jennifer Keiser, Ming-Gang The Applications of Model-Based
Chen, Marcel Tanner, and Jürg Geostatistics in Helminth
Utzinger Epidemiology and Control
Control of Important Helminthic Ricardo J. Soares Magalhães, Archie C.A.
Infections: Vaccine Development as Clements, Anand P. Patil, Peter W.
Part of the Solution Gething, and Simon Brooker
Robert Bergquist and Sara Lustigman
Our Wormy World: Genomics,
Proteomics and Transcriptomics in Volume 75
East and Southeast Asia Epidemiology of American
Jun Chuan, Zheng Feng, Paul J. Trypanosomiasis (Chagas Disease)
Brindley, Donald P. McManus, Louis V. Kirchhoff
Zeguang Han, Peng Jianxin,
and Wei Hu Acute and Congenital Chagas Disease
Caryn Bern, Diana L. Martin, and
Advances in Metabolic Profiling of Robert H. Gilman
Experimental Nematode and
Trematode Infections Cell-Based Therapy in Chagas Disease
Yulan Wang, Jia V. Li, Jasmina Saric, Antonio C. Campos de Carvalho,
Jennifer Keiser, Junfang Wu, Jürg Adriana B. Carvalho, and
Utzinger, and Elaine Holmes Regina C.S. Goldenberg
Targeting Trypanosoma cruzi Sterol Volume 76

14a-Demethylase (CYP51)
Galina I. Lepesheva, Fernando Villalta, Bioactive Lipids in Trypanosoma cruzi
and Michael R. Waterman Infection
Fabiana S. Machado, Shankar Mukherjee,
Experimental Chemotherapy and Louis M. Weiss, Herbert B. Tanowitz,
Approaches to Drug Discovery for and Anthony W. Ashton
Trypanosoma cruzi Infection
Frederick S. Buckner Mechanisms of Host Cell Invasion by
Trypanosoma cruzi
Vaccine Development Against Kacey L. Caradonna and Barbara A.
Trypanosoma cruzi and Chagas Burleigh
Disease
Juan C. Vázquez-Chagoyán, Gap Junctions and Chagas Disease
Shivali Gupta, and Daniel Adesse, Regina Coeli Goldenberg,
Nisha Jain Garg Fabio S. Fortes, Jasmin, Dumitru
A. Iacobas, Sanda Iacobas,
Genetic Epidemiology of Antonio Carlos Campos de
Chagas Disease Carvalho, Maria de Narareth
Sarah Williams-Blangero, Meirelles, Huan Huang, Milena B.
John L. VandeBerg, John Blangero, Soares, Herbert B. Tanowitz,
and Rodrigo Corrêa-Oliveira Luciana Ribeiro Garzoni, and
Kissing Bugs. The Vectors of Chagas David C. Spray
Lori Stevens, Patricia L. Dorn, The Vasculature in Chagas Disease
Justin O. Schmidt, John H. Klotz, Cibele M. Prado, Linda A. Jelicks,
David Lucero, and Stephen A. Klotz Louis M. Weiss, Stephen M.
Advances in Imaging of Animal Models Factor, Herbert B. Tanowitz, and
of Chagas Disease Marcos A. Rossi
Linda A. Jelicks and Herbert B. Tanowitz Infection-Associated Vasculopathy in
The Genome and Its Implications Experimental Chagas Disease:
Santuza M. Teixeira, Najib M. El-Sayed, Pathogenic Roles of Endothelin and
and Patrı́cia R. Araújo Kinin Pathways
Julio Scharfstein and Daniele Andrade
Genetic Techniques in Trypanosoma cruzi
Martin C. Taylor, Huan Huang, and Autoimmunity
John M. Kelly Edecio Cunha-Neto, Priscila Camillo
Teixeira, Luciana Gabriel Nogueira,
Nuclear Structure of Trypanosoma cruzi and Jorge Kalil
Sergio Schenkman, Bruno dos Santos
Pascoalino, and Sheila C. Nardelli ROS Signalling of Inflammatory
Cytokines During Trypanosoma cruzi
Aspects of Trypanosoma cruzi Stage Infection
Differentiation Shivali Gupta, Monisha Dhiman, Jian-jun
Samuel Goldenberg and Andrea Rodrigues Wen, and Nisha Jain Garg
Ávila
Inflammation and Chagas Disease: Some
The Role of Acidocalcisomes in the Stress Mechanisms and Relevance
Response of Trypanosoma cruzi André Talvani and Mauro M. Teixeira
Roberto Docampo, Veronica Jimenez,
Sharon King-Keller, Zhu-hong Li, and Neurodegeneration and
Silvia N.J. Moreno Neuroregeneration in Chagas
Disease
Signal Transduction in Trypanosoma cruzi Marina V. Chuenkova and Mercio
Huan Huang PereiraPerrin
Adipose Tissue, Diabetes and Chagas Robert P. Hirt, Natalia de Miguel, Sirintra
Disease Nakjang, Daniele Dessi, Yuk-Chien Liu,
Herbert B. Tanowitz, Linda A. Jelicks, Nicia Diaz, Paola Rappelli, Alvaro
Fabiana S. Machado, Lisia Esper, Acosta-Serrano, Pier-Luigi Fiori, and
Xiaohua Qi, Mahalia S. Desruisseaux, Jeremy C. Mottram
Streamson C. Chua, Philipp E. Scherer,
Cryptic Parasite Revealed: Improved
and Fnu Nagajyothi
Prospects for Treatment and Control
of Human Cryptosporidiosis
Through Advanced Technologies
Volume 77 Aaron R. Jex, Huw V. Smith, Matthew J.
Nolan, Bronwyn E. Campbell, Neil D.
Coinfection of Schistosoma (Trematoda) Young, Cinzia Cantacessi, and Robin B.
with Bacteria, Protozoa and Gasser
Helminths
Amy Abruzzi and Bernard Fried Assessment and Monitoring of
Onchocerciasis in Latin America
Trichomonas vaginalis Pathobiology: New Mario A. Rodrı́guez-Pérez, Thomas R.
Insights from the Genome Sequence Unnasch, and Olga Real-Najarro

(Advances in Parasitology 76) Louis M. Weiss and Herbert B. Tanowitz (Eds.) - Chagas Disease, Part B-Academic Press, Elsevier (2011)

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(Advances in Parasitology 76) Louis M. Weiss and Herbert B. Tanowitz (Eds.) - Chagas Disease, Part B-Academic Press, Elsevier (2011)

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SERIES EDITORS

First edition 2012

For information on all Academic Press publications

Printed and bound in UK

R.C. Andrew Thompson

Contents 1.1. Introduction 2

Abstract Parasitic diseases cause important losses in public and veterinary

Instituto de Recursos Naturales y Agrobiologı́a de Salamanca (IRNASA), National Research Council,

Advances in Parasitology, Volume 78 # 2012 Elsevier Ltd.

of parasites and the intricate relationship with their hosts, devel-

transposable elements, sense–antisense RNA pairs, RNA hairpins, viruses

1.2. RNAi MECHANISMS AND APPROACHES

1.2.1. Short-RNA types and RNAi: Basic principles

R2D2 Dicer R2D2 Dicer

FIGURE 1.1 An overview of the classical cytoplasmic RNAi-mediated gene silencing

processed by endonucleases different from the RNAse III family

spermatogenesis and seem to be functionally redundant with miRNAs

1.2.2. RNAi machinery in parasites

(sno)RNAs that are similar to miRNAs in G. lamblia RNAi, including

other factors, for example, adverse culture conditions or differing cuticle

Parasite Stage RNAi source RNAi delivery method

Parasite Stage RNAi source RNAi delivery method

Anopheles stephensi Cells, adult females dsRNA Transfection, in vivo

In summary, our knowledge on RNAi pathways in arthropods is

1.3. DELIVERY TOOLS AND METHODS IN RNA SILENCING

1.3.1. Uptake and spreading of dsRNAs

This could be mediated by the internalization of environmental dsRNA

1.3.2. dsRNA delivery and stability

utilization of many delivery systems that have previously been shown to

1.3.3. dsRNA delivery in parasites

Although as parasites developing in their natural hosts could show dif-

reviewed by de la Fuente et al. (2007) and shown in a highly instructive

1.3.4. Additional factors affecting the efficiency of the

1.4. SYSTEMATIC APPLICATIONS OF RNAi TECHNOLOGY

In Leishmania parasites, a similar phenomenon has been described,

silencing provides a rapid assessment on the loss-of-function effect and

livestock production (Tritrichomonas foetus) have been anecdotic to

stimulated numerous investigations aimed at the sequencing of their tran-

gene, a novel parasite-encoded TGF-b superfamily member, resulted in

high-throughput RNAi assays (Mourão et al., 2009). As well as identifying

1.4.2.2. Cestodes and monogeneans

study of cellular and molecular basis of parasite development and host–

uptake for defined gene expression sites in parasite nematodes (Britton

COPII). Following electroporation of dsRNA in L1, significant decreases

(Ford et al., 2005; Lustigman et al., 2004). Similarly, the knock-down of B.

Aljamali et al. (2002) performed the first RNAi assays in Amblyomma

In ticks, iron homeostasis has to be tightly maintained for its uptake,

value of the antigen. This approach is limited by the identification of those

conditions, and the subsequent examination of the mosquitoes to analyze

the combined inputs of several molecules, including the steroid hormone

pathogens to develop in the mosquito vector. Regarding the mosquito

mosquito strains in ‘population replacement’ strategies for mosquito

1.4.3.3. RNAi in other parasitic arthropods

1.5. FUTURE PROSPECTS

and interesting opportunities to generate information about the largely

BARRIERS FOR ACHIEVING IN VITRO OR IN VIVO

Multiple barriers are faced by artificially delivered dsRNA mole-

Liposomes and Chemical

•Lipid-based •Lipid based •Polymers •Lipids •Ribose 2 ¢-OH •Penetration

Improve siRNA nuclease resistance

siRNA formulations for systemic application face a series of hur-

The recent advances in this field applied to other organisms and

Contents 2.1. Introduction 58