Disponible en ligne sur

www.sciencedirect.com

Médecine et maladies infectieuses 43 (2013) 1–9

General review

Diagnosis and epidemiology of leptospirosis

Diagnostic et épidémiologie de la leptospirose
M. Picardeau
Institut Pasteur, unité de biologie des spirochètes, centre national de référence et centre collaborateur de l’OMS de la leptospirose, 28, rue du Docteur-Roux, 75724
Paris cedex 15, France
Received 26 October 2012; accepted 27 November 2012
Available online 18 January 2013

Abstract
Leptospirosis is a zoonosis found worldwide, the main reservoir of which is the rat. Human infection generally results from exposure to contam-
inated river or lake water or animals. Around 600 cases are diagnosed per year in France. Half of these cases occur in French overseas territories,
where the incidence can be more than 100 times higher than in mainland France. Leptospirosis has been under-diagnosed because of non-specific
symptoms, inadequate surveillance system, and lack of readily available quick and simple diagnostic tests. Most cases of leptospirosis are currently
detected by PCR amplification of bacterial DNA from the blood during the first week after the onset of symptoms, or by detection of antibodies
during the second week of the disease. More than 300 serovars have been identified among leptospires, including serovar Icterohaemorrhagiae, the
most frequent in human infections. Leptospirosis remains a major public health issue in many developing countries, one century after discovering
the causative agent. Leptospirosis is expected to become more important due to a rapid urbanization in developing countries (slums), global
warming, and extreme climatic events (floods).
© 2013 Published by Elsevier Masson SAS.

Keywords: Leptospira; Leptospirosis; Spirochetes; Diagnosis; Serological tests

Résumé
La leptospirose est une zoonose de répartition mondiale dont le rat constitue le principal réservoir. La leptospirose est généralement liée aux
contacts avec l’eau douce ou les animaux. En France, quelques 600 cas annuels sont diagnostiqués, dont la moitié provient des départements et
territoires d’Outre-Mer où l’incidence peut être 100 fois plus élevée qu’en Métropole. La leptospirose reste cependant sous-estimée du fait de
l’absence de symptômes spécifiques, d’un système de surveillance défaillant et d’un manque de tests de diagnostic rapides et simples à réaliser.
Le diagnostic s’effectue principalement par la détection de l’ADN bactérien dans le sang par PCR lors de la première semaine de la maladie
ou par la recherche des anticorps à partir de la deuxième semaine. Plus de 300 sérovars sont retrouvés chez les leptospires, parmi lesquels le
sérovar Icterohaemorrhagiae est l’un des plus fréquemment rencontrés en clinique humaine. Près d’un siècle après la découverte de l’agent causal
de la leptospirose, cette zoonose reste un problème de santé publique majeur dans de nombreux pays en voie de développement, notamment à
cause de l’urbanisation grandissante (bidonvilles), le réchauffement climatique et l’apparition plus fréquente de phénomènes climatiques extrêmes
(inondations).
© 2013 Publié par Elsevier Masson SAS.

Mots clés : Leptospira ; Leptospirose ; Spirochètes ; Diagnostic ; Sérologie

1. Introduction reservoir includes mostly rodents; they excrete leptospires in

Pathogenic leptospires are responsible for a worldwide
zoonosis, leptospirosis, in which humans are occasional hosts
in a cycle involving wild and domestic animals. The animal
E-mail addresses: mathieu.picardeau@pasteur.fr, mpicard@pasteur.fr
their urine and thus contaminate hydric environment, transmit-
ting the disease to other animals or to humans [1–3].
Leptospires belong to the phylum of spirochetes that presents
unique morphological characteristics in the bacterial world.
Leptospires are helix shaped and have an internal locomotor
organ, the endoflagellum, which gives them a great mobility,
even in the most viscous media (Fig. 1). The genus Leptospira

0399-077X/$ – see front matter © 2013 Published by Elsevier Masson SAS.

http://dx.doi.org/10.1016/j.medmal.2012.11.005
2 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9

and saprophytes, according to their rRNA 16S sequence, and for

which virulence has not been demonstrated experimentally.
Leptospirosis is a major public health issue in many countries,
especially in Latin America and in South-East Asia. The num-
ber of severe leptospirosis is estimated at more than one million
per year, with a case fatality rate ranging around 10% [5].
The reported yearly incidence usually ranges from 0.1 to 1 per
100,000 inhabitants in temperate climates and is higher than 10
per 100,000 inhabitants in tropical regions. In France, around
600 cases are diagnosed every year, half of which in over-
seas territories where the incidence can be 100 times higher
than in continental France. Most of the leptospirosis cases are
diagnosed by serological tests; but antibodies are detected in
blood more than one week after onset of symptoms. The bac-
Fig. 1. Electron micrographs of leptospires. Leptospires are thin and helical teriological diagnosis is difficult because it requires a specific
bacteria with a diameter of 0.15 to 0.3 ␮m and a length ranging from 6 to 20 culture medium and the time for the culture of leptospires is
␮m. especially long, thus inducing a late result (several weeks). The
Vue en microscope électronique de leptospires. Le diamètre cellulaire est de 0,15
␮m pour une longueur de 6–20 ␮m. Ces bactéries ont une forme hélicoïdale
sero-diagnostic technique has been used at the Pasteur Insti-
avec des extrémités caractéristiques en crochets. tute since 1917 [6]. Nevertheless, PCR bacterial DNA detection
made on early biological samplings tends to replace serological
tests.
Table 1
Referent strains for the 20 species described in the genus Leptospira.
Souches de référence des 20 espèces aujourd’hui décrites dans le genre Lep- 2. Diagnosis
tospira.
Species Serogroup Serovar Strain 2.1. What kind of sample, what test, and when to test?
Pathogenic
L. interrogans Icterohaemorrhagiae Copenhageni Fiocruz LI-130 Infection by pathogenic leptospires may be divided in two
L. kirschneri Grippotyphosa Grippotyphosa Moskva V stages (Fig. 2). The first stage of leptospirosis is septicemia or
L. noguchii Panama Panama CZ 214 K the acute stage, which lasts from 3 to 10 days, with headaches
L. borgpetersenii Sejroe Sejroe M84 and myalgia. During that stage, leptospires are found in blood in
L. weilii Celledoni Celledoni Celledoni
L. santarosai Tarassovi Atlantae LT81
a decreasing number until 15 days after onset of symptoms [1,7].
L. alexanderi Manhao Manhao 3 L 60 Samples must be collected up to 2 days after initiating antibio-
L. alstonii ND Sichuan 79,601 therapy to detect leptospires. The second stage, or immune stage,
L. kmetyi ND ND Bejo-Iso 9 usually occurs during the second week after onset of symptoms,
Intermediate and lasts from 4 to 30 days. During this stage, the increased
L. wolffii ND ND Korat-H2
L. licerasiae ND Varillal VAR010
L. inadai Tarassovi Kaup LT64-68
L. fainei Hurstbridge Hurstbridge BUT6
L. broomii Undesignated ND 5399
Saprophytes
L. wolbachii Codice Codice CDC
L. meyeri Semaranga Semaranga Veldrat
L. biflexa Semaranga Patoc Patoc 1
L. vanthielii Holland Holland WaZ Holland
L. terpstrae ND ND LT 11-33
L. yanagawae Semaranga Saopaulo Sao Paulo

ND: not determined.

includes 20 Leptospira species (Table 1) and more than 300

serovars, grouped in 20 serogroups, have been described. The
lipopolysaccharide (LPS) structure is the main serovar determi-
nant. Leptospires are classified in three groups according to their
phylogenicity and pathogenicity [4]. There are six saprophytic
species (environmental non-pathogenic strains), nine pathogenic
species (strains isolated from humans or animals) and five so-
called “intermediate” species, which are distinct from pathogens
Fig. 2. Kinetics of leptospiral infection in blood. Infection produces lep-
tospiremia in the first few days after exposure, which is rapidly followed by
migration of leptospires to target organs. Antileptospiral IgM are detectable
before the appearance of agglutinating and IgG antibodies.
Cinétique de l’infection dans le sang. L’infection entraîne une bactériémie
durant les premiers jours après l’exposition qui est rapidement suivie par une
migration des leptospires vers les organes cibles. Les IgM antileptospires sont
détectables avant l’apparition des anticorps agglutinants et des IgG.
 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9
antibody titer is correlated to elimination of leptospires from
blood [2].
Leptospires antigens or DNA may not be detected in blood,
in some cases of leptospirosis, maybe because of a weak or
short leptospiremia during the acute stage, or because of a
late sampling, or because of antibiotic administration which
rapidly eliminates leptospires from blood. Sampling prior to
sero-conversion during the acute stage of the disease may give
false negative results in antibody detection.
Coagulated blood (containing serum and blood clot) or non-
coagulated blood (containing plasma, red and white blood cells,
and platelets) will be collected depending on the test to be made.
Several authors have reported that EDTA plasma gives the best
results for the gene amplification; conversely, serum or hep-
arinized plasma are less sensitive than other blood fractions
[8–12]. Using serum or plasma for the serological tests gives
equivalent results but serum should be used preferentially [13].
Leptospires may also be detected in urine or cerebrospinal fluid
(CSF), 10 days after onset of symptoms [2].
Several marketed kits are available for the rapid isolation
of DNA in blood or urine, for diagnostic tests relying on the
purification of nucleic acids [12]. Using magnetic beads may
allow concentrating nucleic acids or antigens in samples [14,15].
2.2. Direct examination
Leptospires are 6 to 20 ␮m long and 0.15 ␮m in diameter.
Dark field microscopy is required for their observation because
of their size: 102 to 106 leptospires/mL of blood may be observed
during the acute stage of leptospirosis [7]. The threshold of
detection is close to 104 leptospires/mL of blood on direct exam-
ination. Theoretically, leptospirosis may be diagnosed by direct
blood examination during the first week after onset of symptoms.
This method is cheap (but a dark field microscope is required),
the risk of false positives due to cell debris and other artifacts is
important [16].
2.3. Gene amplification
2.3.1. PCR
PCR has in recent years been increasingly used for the diag-
nosis of leptospirosis, and even tends to replace serological
methods in endemic zones because of its sensitivity and capacity
to give an early diagnosis. Real-time PCR (SYBR Green or Taq-
man technology) is faster than regular PCR and less sensitive to
contaminations. Several regular or real-time PCR methods have
been developed for the detection of pathogenic leptospires but
only a few have been clinically validated [17–20]. The detec-
tion threshold is usually 10 to 100 leptospires/mL of blood or
urine [8,11,12,21]. The bacterial load may be obtained if a panel
of standard DNA (standard curve) is included. Nevertheless, no
correlation has currently been found between the bacterial load
detected in blood and the patient’s prognosis [7,22,23]. A pos-
itive PCR reveals the presence of pathogenic leptospires in the
sample (if the target is well specific of the pathogenic species) but
in no case does it allow to directly identify the serovar. Analyzing
the amplification products by melting curves [24] or sequencing
3
[25,26] may allow identifying the species and, in some cases,
the serovar or the genotype.
2.3.2. Isothermal methods
In recent years, a growing number of isothermal amplification
techniques have been developed, some of which were applied
to leptospires [27–29]. Isothermal amplification is an attractive
alternative to PCR. This method requires just a heating unit,
and no thermal cycler, to maintain a constant temperature of 60
to 65 ◦ C, making it the best method for developing countries.
An effective and specific amplification can be performed in
1 hour with polymerase DNA and six primers in isothermal con-
ditions. The amplified DNA can then be detected by simple
eye-observation of fluorescence or of turbidity, without using
electrophoresis gels [30]. Loop-mediated isothermal amplifica-
tion type (LAMP) methods targeting genes lipL41 or rrs have
recently been developed for the quick detection of pathogenic
leptospires [27–29]. The specificity of these methods is rela-
tively weak and the detection threshold ranges between two and
100 leptospires per reactive mixture [27–29]. The usefulness of
LAMP for the diagnosis of leptospirosis should be assessed in
endemic zones.
2.4. Serological tests
2.4.1. The microscopic agglutination test (MAT)
The microscopic agglutination test (MAT) or Martin and
Pettit test was developed almost one century ago at the Pas-
teur Institute [6]. This test remains the reference test today
and consists in dark field microscopy assessment of the agglu-
tination degree of live leptospires cultures by the patient’s
serum. The MAT requires maintaining a collection of living
strains, which will be used as antigens. The antigen collec-
tion includes strains representative of the main serogroups. The
MAT is thus a specific serogroup method (and not serovar). At
the Leptospirosis National Reference Center (French acronym
CNRL) (Pasteur Institute), 24 strains are used (including non-
pathogenic strain Leptospira biflexa strain Patoc 1 which has for
particularity to cross-react with several antigens of pathogenic
serogroups) to which may be added local strains from some over-
seas regions for example. A smaller panel of antigens (according
to French guidelines for biological procedures, the test should be
performed with a minimum of nine antigens) may lead to non-
detection of serogroups not presents in the panel. The MAT’s
principle consists in incubating serial dilutions of the patient’s
serum with various leptospires strains. A serum is considered
as positive, at a given dilution and for the tested antigen, if at
least 50% of leptospires are agglutinated compared to a con-
trol antigen without serum. The threshold titer is set at 1/100
for mainland France and 1/400 for endemic zones (French
Polynesia, Guadeloupe, Martinique, Reunion, Mayotte, New
Caledonia, Futuna). The MAT is a subjective technique, expen-
sive and difficult to implement, requiring great expertise both to
perform and analyze it. The MAT becomes positive between D8
and D10, with a usually higher titer for antigen L. biflexa (strain
Patoc) with sometimes numerous co-agglutinins for several
serogroups. The antibody kinetics varies from one individual to
4 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9
the next. Only a late serum test (> D20) may allow identifying
the serogroup. Nevertheless, the MAT has limitations as an epi-
demiological indicator and lacks precision. Indeed, there is not
always a correlation between the serogroup identified by MAT
and the one obtained by identification after isolating the strain
[31,32]. The rate of antibodies decreases during several months
after the infection and may persist at residual levels for several
years. It is thus very important to have clinical and chronolog-
ical documentation (onset of the disease and date of sampling)
for a reliable analysis. A titer of 1/100 or 1/200 may corre-
spond to the beginning of leptospirosis, to a previous infection,
or to vaccinal antibodies. Comparing MAT with IgM enzyme-
linked immunosorbent assay (Elisa) is important in this case,
so as to draw conclusions. Finally, an early antibiotherapy (first
days after onset of symptoms) may eliminate leptospires rapidly
enough to decrease the antibody determined later by the MAT
[33]. Confirming a case of leptospirosis by the MAT requires
two samplings made 2 weeks apart, with sero-conversion or
significant increase of the antibody titers.
2.4.2. IgM enzyme-linked immunosorbent assay (Elisa)
Conventional serological methods such as enzyme-linked
immunosorbent assay (Elisa) are widely used for the diagnosis
of leptospirosis. Several IgM Elisa are available on the mar-
ket, based on the detection of antibodies against a total extract
of leptospires; usually the saprophytic strain L. biflexa, which
shares several surface antigens with pathogenic strains. Some
“homemade” IgM Elisa containing a total cellular extract of
the local epidemic strain or of recombinant leptospires pro-
teins are also used [2,3]. The specificity and the sensitivity
of these Elisa are quite variable. For example, Panbio’s IgM
Elisa has a sensitivity and specificity of 76% and 82% respec-
tively in Thailand [34], 35% and 98% in Hawaii [35], and 61%
and 66% in Laos [36]. Theses reported variations may be due
to differences in the studied population (previous exposure to
pathogenic or environmental leptospires). They may also be due
to the definition of confirmed cases; the MAT is most often
used as reference test [37]. Despite the weak performances of
Elisa, several authors have reported that antileptospires antibod-
ies could be detected earlier with this test than with the MAT
[2,37–41]. Antileptospires IgM may be detected 4 to 5 days after
the onset of symptoms, before detection of IgG and of aggluti-
nating antibodies, and persist at least 5 months in patients [42]
(Fig. 2). A positive Elisa gives no indication on the infecting
serovar/serogroup and is not sufficient to diagnose a case of
leptospirosis; it must be confirmed by MAT (see above), PCR,
or culture.
2.4.3. Other serological methods
Several other tests may be used to screen for antibodies
including macro-agglutination, complement fixation reaction,
indirect immunofluorescence, hemagglutination, and latex bead
agglutination tests [2,3]. Nevertheless, these tests, even if
some are marketed, are rarely used and lack specificity or
sensitivity. In France, a 2005 ministry decree published in
the Official Journal specifies that the thermo-resistant (TR)
antigen macro-agglutination test must be used as screening
test and that the MAT should be used only if the screening
test is positive. But this test which consists in watching the
degree of TR antigen agglutination (a heated formaldehyde
treated suspension of the saprophytic strain L. biflexa) and of
the tested serum on a glass slide does not allow screening
a significant number of positive cases [43]. More recently,
immuno-chromatographic strip tests “Lateral flow assays” have
been developed in various laboratories; these can be used at the
patient’s bedside with a drop of blood, and results are avail-
able in 10 minutes [44–46]. These tests use a membrane coated
with total cellular extract or with a protein used to capture anti-
bodies targeting leptospires when a drop of sampled blood is
deposited. The antibody capture is visualized by a reaction with
a colorimetric detection agent (a colloidal gold conjugate of
protein-A) after migration of antigen–antibody complexes by
capillarity.
2.4.4. Conclusions
Currently, no diagnostic technique is completely satisfactory.
The MAT and PCR are the two techniques allowing to confirm
the diagnosis of leptospirosis (culture requires a one month incu-
bation and does not allow making a quick diagnosis), but the
MAT is used only by a few reference laboratories and detects
antileptospires antibodies late (furthermore, a second sampling
is advised to confirm leptospirosis) and PCR on a blood sample
is possible only for a few days during the first week of the dis-
ease. Thus, there is an urgent need to develop new techniques
for an easy to use and quick detection of antibodies or antigens
at the acute stage of the disease.
3. Epidemiology
3.1. Bacterial identification
Leptospires may be isolated from blood of patients pre-
senting with leptospirosis during the first week after onset
of symptoms, but also later, from urine or CSF. Sampling
must be performed in strictly sterile conditions; and seeding
in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium
must be done as soon as possible after the sampling: 5-fluoro-
uracil (100 ␮g/mL) may be added to the medium to prevent
contamination, or in presence of contaminants, the culture
should be strained on a 0.22 ␮m filter. The cultures are incu-
bated at 28 to 30 ◦ C and observed every week by dark field
microscopy, for 2 months. Positive cultures should be sent to
the CNRL for identification. Serogrouping may then be per-
formed and various molecular biology techniques can be used
such as pulsed-field gel electrophoresis (PFGE) [47,48], Multi
Locus Sequence Typing [MLST] [49], Multiple Loci Variable
Number of Tandem Repeats (VNTR) Analysis (MLVA) [50,51],
or analysis in MALDI-TOF mass spectrometry [52]. Neverthe-
less, it should be kept in mind that isolation of strains is rare, and
that in mainland France, for example, less than five strains are
isolated per year. Thus, there is a great lack of data on circulating
strains as well in humans as in animals.
 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9
3.2. Reservoirs and transmission
Leptospirosis is found worldwide, in rural and urban sur-
roundings in temperate and tropical climates. The reservoir
is mostly animal. In the rat, the main reservoir, the infection
presents as an asymptomatic chronic disease with colonization
of leptospires in renal tubules and excretion in the environment
via urine. All other mammal species, wild and domestic, may
be reservoirs. The infection may induce reproduction disorders
(bovines, pigs, goats, sheep), or uveitis (horses), or more severe
presentations close to human leptospirosis (dogs). The serovars
are usually associated to a specific animal reservoir (which is
why it is useful to try to isolate strains for a better comprehension
of epidemiology): rats usually host serovars of the Icterohaem-
orrhagiae serogroup; the serovar Canicola is associated to dogs,
etc.
Leptospires are very sensitive to drying and to acid pH but
some pathogenic species are able to survive in water for sev-
eral weeks [53]. Bacterial transmission to man occurs by direct
contact with urine, blood, or animal tissue of an infected ani-
mal, or, most often, by exposure to contaminated environment.
Leptospires penetrate the human body through skin lesions or
mucosa of the eyes, mouth, or nose after contact with contam-
inated water. Leptospirosis is usually due to contact with fresh
water or animals.
3.3. Incidence and distribution of serogroups
Leptospirosis is a public health issue in several countries of
Latin America and of South-East Asia. Outbreaks of leptospiro-
sis occur regularly during the rainy season in slums or “favelas”
of Brazilian megapoles, for example. Currently, one billion peo-
ple live in slums throughout the world and this population should
increase two-fold in the next 20 years [54]. The living condi-
tions in these slums, especially in tropical regions, are favorable
for the transmission of leptospirosis by rats [55,56]. The risk
for leptospirosis is mainly occupational (individuals working in
rice fields and sugar cane fields, etc.) except in case of natural
catastrophes (cyclones, floods, etc.). In developed countries of
temperate zones, leptospirosis is a disease, which affects prefer-
entially some exposed occupational categories (farmers, animal
raisers, slaughterhouse personnel, sewage workers, veterinar-
ians, etc.) and outdoor sportsmen (fishing, water sports) by
contact fresh water soiled by the urine of infected animals.
There are great regional differences in the incidence, related
to climatic conditions and also to the heterogeneity in access to
diagnosis, the decreased activity of a local referent laboratory,
etc.
Icterohaemorrhagiae is the most often implicated infectious
serogroup; this is the case in mainland France and French
Atlantic and Pacific territories. The rat is a traveller; it has spread
its serogroup worldwide.
3.4. Mainland France
In France, the incidence ranges at 0.5 cases/100,000 inhabi-
tants (around 300 cases notified every year) but it is 10 to 100
5
times higher in overseas French territories (Fig. 3). France has
one of the highest incidences among developed countries [57].
The greatest number of cases in mainland France occurs dur-
ing the summer/fall period (mainly from August to October)
(Fig. 4). In 2011, the serogroup Icterohaemorrhagiae was pre-
dominant (36% of cases were due to this serogroup), followed
by serogroups Grippotyphosa (21%), Australis (8%), and Cani-
cola (7%) (Fig. 5). Nevertheless, the serogroup could not be
determined in a significant number of cases (13%) because of
cross-reactions among several serogroups in the MAT. The main
risk factors identified by the authors of a case-control study made
in mainland France, from 1999 to 2000, were injuries, practicing
canoe/kayak, contact with water, and contact with wild rodents
[58]. A significant number of cases were imported; thus, in 2011,
despite the small number of documented cases, 10% of cases
were imported after travelling to an endemic zone (Latin Amer-
ica, West Indies, South-East Asia, North Africa). A report made
by the French Food Safety Agency (French acronym AFSSA,
now called ANSES) mentioned leptospirosis as one of the human
diseases the incidence of which could be modified by climate
change in mainland France [59].
3.5. Overseas French territories
The regional incidence of leptospirosis is quite variable in
overseas French territories located in tropical zones (from five
cases per 100,000 habitants in Guyana to more than 1000
cases/100,0000 habitants in Futuna). Unusual meteorological
phenomena, local specificities, lack of surveillance, and/or diag-
nostic difficulties are the reasons for these variations. It should
be noted that in these regions, the diagnosis is mostly made
with PCR and isolation of strains is rare. Thus, it is difficult to
know more about circulating strains. Nevertheless, there is a pre-
dominance of serogroup Icterohaemorrhagiae (> 40%) among
serogroups identified by the MAT, in most regions, except for
Mayotte where this serogroup has not been identified in the last
decade [60].
3.6. In the French West Indies and Guyana zone
Globally, the importance of leptospirosis is probably under-
estimated in the French West Indies and Guyana. Most cases in
this region are diagnosed in Guadeloupe and Martinique.
The average incidence is 17 cases/100,000 habitants in
Guadeloupe and Martinique. The number of cases is constantly
increasing with 142 cases in Martinique and 165 cases in Guade-
loupe in 2011, and an incidence superior to 35 cases per 100,000
inhabitants. The most frequent serogroups are Icterohaemorrha-
giae (40–41%), Canicola (12–14%) and Sejroe (10–12%), in
Guadeloupe and Martinique; whereas the serogroups Ballum
(15%) and Cynopteri (11%) are mainly reported in Guadeloupe,
and the serogroup Pyrogenes (10%) in Martinique. The increas-
ing number of cases is probably due to the implementation of a
surveillance project in this region, to assess more precisely the
importance of leptospirosis in these territories. Most cases occur
between August and December during the rainy season.
6 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9

Fig. 3. Number of cases of leptospirosis in Mainland France and French overseas territories.
Nombre de cas de leptospirose en France métropolitaine et en Outre-Mer (Données Leptospirosis National Reference Center [CNRL]).
Data from Leptospirosis National Reference Center.

Fig. 4. Number of cases of leptospirosis per months in Mainland France.

Variation saisonnières de la leptospirose humaine en France métropolitaine (Données Leptospirosis National Reference Center [CNRL]).
Data from Leptospirosis National Reference Center.

Fig. 5. Serogroup distribution of cases of leptospirosis in Mainland France. AUS: Australis; CAN: Canicola; GT: Grippotyphosa; IH: Icterohaemorrhagiae; SEJ:
Sejroe.
Répartition des principaux sérogroupes identifiés par MAT parmi les cas positifs en France métropolitaine (Données Leptospirosis National Reference Center
[CNRL]).
Data from Leptospirosis National Reference Center.
 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9
In Guyana, the MAT is not used systematically and the
surveillance network is not well developed. The average inci-
dence is five cases per 100,000 inhabitants. There also, the
serogroup Icterohaemorrhagiae is predominant (42%), followed
by Canicola (23%).
3.7. In the Indian Ocean zone
Leptospirosis is endemic all over the Indian Ocean zone. The
average incidence is nine cases per 100,000 inhabitants in the
Reunion island. The incidence has been constantly increasing
since 2007, probably because a better surveillance network was
implemented. The distribution of cases follows the usual season-
ability of the disease with a majority of cases during the rainy
season from December to April. The serogroups Icterohaem-
orrhagiae (65%) and Canicola (18%) are the most frequently
implicated in the cases diagnosed by the MAT.
In Mayotte, the average incidence is 25 cases/100,000
inhabitants. Since 2007, the epidemiological surveillance was
strengthened by using PCR diagnosis, and by isolation of strains
(Mayotte Hospital Center). In 2011, a record number of 171
cases were diagnosed by PCR at the Mayotte Hospital Center.
Most of the isolated strains (70%) belonged to the serogroup
Mini, which confirms a specific epidemiology, different from
neighboring islands such as the Reunion and Seychelles. The
serogroup Icterohaemorrhagiae was not isolated on the island,
whereas it was predominant in the Reunion and Seychelles.
3.8. In the Pacific zone
It is in this region that leptospirosis incidence is the highest.
Even without taking into account the specific situation of Futuna,
the incidence in New Caledonia and in French Polynesia puts
these regions among the most at risk worldwide. Early real time
PCR diagnosis, ELISA, as well as confirmation with the MAT
(only for New-Caledonia) are used routinely.
In French Polynesia, the average incidence is 39
cases/100,000 inhabitants. Cases are diagnosed all year round
with a marked decrease during the dry season from August
to November. During the first trimester of 2010 marked by
cyclone depressions bringing strong rainfall in several Polyne-
sian islands, the total number of cases was twice higher than the
average of the previous 4 years. The two dominant serogroups
were Icterohaemorrhagiae (50%) and Australis (32%).
In New Caledonia, the average incidence is 45 cases/100,000
inhabitants. The phenomenon “the Niña” was responsible for
strong rainfall in 2008, 2009, and 2011, epidemic years marked
by a flare of leptospirosis. On the contrary, an “el Niño” stage
associated with a high water deficit (2010) may have contributed
to a sharp decrease in the number of cases. The serogroup
Icterohaemorrhagiae (63%) was the most often identified in
cases diagnosed with the MAT, followed by serogroups Australis
(13%), and Pyrogenes (11%).
In Wallis-and-Futuna, the average incidence was
1060 cases/100,000 inhabitants between 2007 and 2009
in Futuna, followed by a significant decrease in the number
of cases in 2010 (240 cases/100,000 inhabitants). This record
7
incidence seemed related to the traditional lifestyle of inhabi-
tants (great promiscuity between humans, rats, and pigs) in a
region with strong rainfalls. The serogroups determined by the
MAT belonged to Icterohaemorrhagiae (55%) and Australis
(44%).
4. Conclusions
Several WHO reports mention the emergent aspect of lepto-
spirosis. This is mainly due to climate change (global warming
and more frequent extreme climate phenomena) and demo-
graphic variations (constant increase of the population living
in slums). But, epidemiological data, both for humans and for
animals is quite limited. Thus, we have little information on
circulating strains. This information is crucial for a better under-
standing of epidemiology, but also to assess diagnostic tests and
to develop new vaccines.
Disclosure of interest
The author declares that he has no conflicts of interest con-
cerning this article.
Acknowledgements
I would like to thank the Leptospirosis National Refer-
ence Center team (P. Bourhy, A. Landier, F. Zinini, S. Bremont,
S. Etiemble and S. Murguet) for its contribution to the diagnosis
and surveillance of leptospirosis.
References
[1] Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA,
et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect
Dis 2003;3:757–71.
[2] Levett PN. Leptospirosis. Clin Microbiol Rev 2001;14:296–326.
[3] McBride AJ, Athanazio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin
Infect Dis 2005;18:376–86.
[4] Cerqueira GM, Picardeau M. A century of Leptospira strain typing. Infect
Genet Evol 2009;9:760–8.
[5] Abela-Ridder B, Sikkema R, Hartskeerl RA. Estimating the burden of
human leptospirosis. Int J Antimicrob Agents 2010;36:S5–7.
[6] Martin L, Pettit A. Sero-diagnostic de la spirochaetose icterohaemorrhag-
ique. Bull Mem Soc Med Hop Paris 1918;42:672–5.
[7] Agampodi SB, Matthias MA, Moreno AC, Vinetz JM. Utility of quantitative
polymerase chain reaction in leptospirosis diagnosis: association of level
of leptospiremia and clinical manifestations in Sri Lanka. Clin Infect Dis
2012;54:1249–55.
[8] Smythe LD, Smith IL, Smith GA, Dohnt MF, Symonds ML, Barnett LJ,
et al. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.
BMC Infect Dis 2002;8:13.
[9] Levett PN, Morey RE, Galloway RL, Turner DE, Steigerwalt AG, Mayer
LW. Detection of pathogenic leptospires by real-time quantitative PCR. J
Med Microbiol 2005;54:45–9.
[10] Kositanont U, Rugsasuk S, Leelaporn A, Phulsuksombati D, Tantitanawat
S, Naigowit P. Detection and differentiation between pathogenic and sapro-
phytic Leptospira spp. by multiplex polymerase chain reaction. Diagn
Microbiol Infect Dis 2007;57:117–22.
[11] Stoddard RA, Gee JE, Wilkins PP, McCaustland K, Hoffmaster AR.
Detection of pathogenic Leptospira spp. through TaqMan polymerase
chain reaction targeting the LipL32 gene. Diagn Microbiol Infect Dis
2009;64:247–55.
8 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9
[12] Bourhy P, Bremont S, Zinini F, Giry C, Picardeau M. Comparison of real-
time PCR assays for detection of pathogenic Leptospira spp. in blood
and identification of variations in target sequences. J Clin Microbiol
2011;49:2154–60.
[13] Goris MGA, Leeflang MMG, Boer KR, Goeijenbier M, van Gorp ECM,
Wagenaar JFP, et al. Establishment of valid laboratory case definition for
human leptospirosis. J Bacteriol Parasitol 2012;3:132.
[14] Taylor MJ, Ellis WA, Montgomery JM, Yan KT, McDowell SW. Magnetic
immuno-capture PCR assay (MIPA): detection of Leptospira borgpetersenii
serovar hardjo. Vet Microbiol 1997;56:135–45.
[15] Schreier S, Doungchawee G, Triampo D, Wangroongsarb P, Hartskeerl RA,
Triampo W. Development of a magnetic bead fluorescence microscopy
immunoassay to detect and quantify Leptospira in environmental water
samples. Acta Trop 2012;122:119–25.
[16] Vijayachari P, Sugunan AP, Umapathi T, Sehgal SC. Evaluation of dark-
ground microscopy as a rapid diagnostic procedure in leptospirosis. Indian
J Med Res 2001;114:54–8.
[17] Ahmed A, Engelberts MF, Boer KR, Ahmed N, Hartskeerl RA. Devel-
opment and validation of a real-time PCR for detection of pathogenic
leptospira species in clinical materials. PLoS One 2009;4:e7093.
[18] Villumsen S, Pedersen R, Borre MB, Ahrens P, Jensen JS, Krogfelt KA.
Novel TaqMan® PCR for detection of Leptospira species in urine and
blood: Pit-falls of in silico validation. J Microbiol Methods 2012;91:
184–90.
[19] Thaipadungpanit J, Chierakul W, Wuthiekanun V, Limmathurotsakul D,
Amornchai P, Boonslip S, et al. Diagnostic accuracy of real-time PCR
assays targeting 16S rRNA and lipL32 genes for human leptospirosis in
Thailand: a case-control study. PLoS One 2011;6:e16236.
[20] Slack A, Symonds M, Dohnt M, Harris C, Brookes D, Smythe L. Evaluation
of a modified Taqman assay detecting pathogenic Leptospira spp. against
culture and Leptospira-specific IgM enzyme-linked immunosorbent assay
in a clinical environment. Diagn Microbiol Infect Dis 2007;57:361–6.
[21] Slack AT, Symonds ML, Dohnt MF, Smythe LD. Identification of
pathogenic Leptospira species by conventional or real-time PCR and
sequencing of the DNA gyrase subunit B encoding gene. BMC Microbiol
2006;6:95.
[22] Segura ER, Ganoza CA, Campos K, Ricaldi JN, Torres S, Silva H, et al.
Clinical spectrum of pulmonary involvement in leptospirosis in a region
of endemicity, with quantification of leptospiral burden. Clin Infect Dis
2005;40:343–51.
[23] Truccolo J, Serais O, Merien F, Perolat P. Following the course of human
leptospirosis: evidence of a critical threshold for the vital prognosis using
a quantitative PCR assay. FEMS Microbiol Lett 2001;204:17–321.
[24] Merien F, Portnoi D, Bourhy P, Charavay F, Berlioz-Arthaud A, Baranton
G. A rapid and quantitative method for the detection of Leptospira species
in human leptospirosis. FEMS Microbiol Lett 2005;249:139–47.
[25] Perez J, Goarant C. Rapid Leptospira identification by direct sequenc-
ing of the diagnostic PCR products in New Caledonia. BMC Microbiol
2010;10:325.
[26] Cerqueira GM, McBride AJ, Hartskeerl RA, Ahmed N, Dellagostin OA,
Eslabão MR, et al. Bioinformatics describes novel Loci for high-resolution
discrimination of leptospira isolates. PLoS One 2010;5:e15335.
[27] Sonthayanon P, Chierakul W, Wuthiekanun V, Thaipadungpanit J, Kalam-
baheti T, Boonsilp S, et al. Accuracy of loop-mediated isothermal
amplification for diagnosis of human leptospirosis in Thailand. Am J Trop
Med Hyg 2011;84:614–20.
[28] Koizumi N, Nakajima C, Harunari T, Tanikawa T, Tokiwa T, Uchimura
E, et al. A new loop-mediated isothermal amplification method for rapid,
simple, and sensitive detection of Leptospira spp. in urine. J Clin Microbiol
2012;50:2072–4.
[29] Lin X, Chen Y, Lu Y, Yan J, Yan J. Application of a loop-mediated isother-
mal amplification method for the detection of pathogenic Leptospira. Diagn
Microbiol Infect Dis 2009;63:237–42.
[30] Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a
rapid, accurate, and cost-effective diagnostic method for infectious dis-
eases. J Infect Chemother 2009;15:62–9.
[31] Kusum M, Boonsarthorn N, Biaklang M, Sina U, Sawanpanyalert
P, Naigowit P. Comparison of leptospiral serovars identification by
serology and cultivation in north-eastern region, Thailand. J Med Assoc
Thai 2005;88:1098–102.
[32] Smythe LD, Wuthiekanun V, Chierakul W, Suputtamongkol Y, Tiengrim S,
Dohnt MF, et al. The microscopic agglutination test (MAT) is an unreliable
predictor of infecting Leptospira serovar in Thailand. Am J Trop Med Hyg
2009;81:695–7.
[33] Faine SB, Adler B, Bolin C, Perolat P. Leptospira and leptospirosis. 2nd
ed. Melbourne, Australia: MediSci; 1999.
[34] Desakorn V, Wuthiekanun V, Thanachartwet V, Sahassananda D, Chier-
akul W, Apiwattanaporn A, et al. Accuracy of a commercial IgM Elisa for
the diagnosis of human leptospirosis in Thailand. Am J Trop Med Hyg
2012;86:524–7.
[35] Effler PV, Bogard AK, Domen HY, Katz AR, Higa HY, Sasaki DM. Evalu-
ation of eight rapid screening tests for acute leptospirosis in Hawaii. J Clin
Microbiol 2002;40:1464–9.
[36] Blacksell SD, Smythe L, Phetsouvanh R, Dohnt M, Hartskeerl R, Symonds
M, et al. Limited diagnostic capacities of two commercial assays for the
detection of Leptospira immunoglobulin M antibodies in Laos. Clin Vac-
cine Immunol 2006;13:1166–9.
[37] Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J,
Suputtamongkol Y, Chierakul W, et al. Fool’s gold: why imperfect reference
tests are undermining the evaluation of novel diagnostics: a re-evaluation
of 5 diagnostic tests for leptospirosis. Clin Infect Dis 2012;55:322–31.
[38] Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA,
et al. Evaluation of four commercially available rapid serologic tests for
diagnosis of leptospirosis. J Clin Microbiol 2003;41:803–9.
[39] Doungchawee G, Kositanont U, Niwetpathomwat A, Inwisai T, Sagarasaer-
anee P, Haake DA. Early diagnosis of leptospirosis by IgM immunoblot
testing. Clin Vaccine Immunol 2008;15:492–8.
[40] Aviat F, Rochereau-Roulet S, Branger C, Estavoyer JM, Chatrenet B,
Orsonneau JL, et al. Synthetic peptide issued from Hap1/LipL32 for new
early serodiagnosis of human leptospirosis. Comp Immunol Microbiol
Infect Dis 2010;33:375–87.
[41] Cumberland P, Everard CO, Levett PN. Assessment of the efficacy of an
IgM Elisa and microscopic agglutination test (MAT) in the diagnosis of
acute leptospirosis. Am J Trop Med Hyg 1999;61:731–4.
[42] Silva MV, Camargo ED, Batista L, Vaz AJ, Brandão AP, Nakamura PM,
et al. Behaviour of specific IgM, IgG and IgA class antibodies in human
leptospirosis during the acute phase of the disease and during convales-
cence. J Trop Med Hyg 1995;98:268–72.
[43] Picardeau M, Cornet M, Morel V, Sertour N, Chaumet D, Brachet E, et al.
Impact du changement de nomenclature médicale sur le diagnostic et la
surveillance de la leptospirose en France. BEH 2008;37:329–31.
[44] Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C,
et al. Lateral flow assay for rapid serodiagnosis of human leptospirosis.
Clin Diagn Lab Immunol 2001;8:166–9.
[45] Sehgal SC, Vijayachari P, Sugunan AP, Umapathi T. Field application of
Lepto lateral flow for rapid diagnosis of leptospirosis. J Med Microbiol
2003;52:897–901.
[46] Ribeiro GS, Nabity SA, Medeiros MA, Takahashi D, Aquino AL, Damião
AO, et al.Accuracy of the Dual Path Platform (DPP) assay for the rapid
diagnosis of leptospirosis. 2011.
[47] Galloway RL, Levett PN. Application and validation of PFGE for serovar
identification of leptospira clinical isolates. PLos Negl Trop Dis 2010;e824.
[48] Herrmann JL, Bellenger E, Perolat P, Baranton G, Saint Girons I. Pulsed-
field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid
method of serovar identification. J Clin Microbiol 1992;30:1696–702.
[49] Ahmed N, Devi SM, Valverde Mde L, Vijayachari P, Machang’u RS, Ellis
WA, et al. Multilocus sequence typing method for identification and geno-
typic classification of pathogenic Leptospira species. Ann Clin Microbiol
Antimicrob 2006;5:28.
[50] Salaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application
of multilocus variable number tandem repeat analysis for molecular typing
of the agent of leptospirosis. J Clin Microbiol 2006;44:3954–62.
[51] Nalam K, Ahmed A, Devi SM, Francalacci P, Baig M, Sechi LA, et al.
Genetic affinities within a large global collection of pathogenic Leptospira:
implications for strain identification and molecular epidemiology. PLoS
One 2010;5:e12637.
 M. Picardeau / Médecine et maladies infectieuses 43 (2013) 1–9
[52] Rettinger A, Krupka I, Grünwald K, Dyachenko V, Fingerle V, Konrad
R, et al. Leptospira spp. strain identification by MALDI-TOF MS is an
equivalent tool to 16S rRNA gene sequencing and multi locus sequence
typing (MLST). BMC Microbiol 2012;12:185.
[53] Trueba G, Zapata S, Madrid K, Cullen P, Haake D. Cell aggregation: a
mechanism of pathogenic Leptospira to survive in fresh water. Int Microbiol
2004;7:35–40.
[54] Lau CL, Smythe LD, Craig SB, Weinstein P. Climate change, flooding,
urbanisation and leptospirosis: fuelling the fire? Trans R Soc Trop Med
Hyg 2010;104:631–8.
[55] Riley LW, Ko AI, Unger A, Reis MG. Slum health: diseases of neglected
populations. BMC Int Health Hum Rights 2007;7:2.
[56] Reis RB, Ribeiro GS, Felzemburgh RD, Santana FS, Mohr S, Melendez
AX, et al. Impact of environment and social gradient on leptospira infection
in urban slums. PLoS Negl Trop Dis 2008;2:e228.
9
[57] Baranton G, Postic D. Trends in leptospirosis epidemiology in France.
Sixty-six years of passive serological surveillance from 1920 to 2003. Int
J Infect Dis 2006;10:162–70.
[58] Nardone A, Capek I, Baranton G, Campèse C, Postic D, Vaillant V,
et al. Risk factors for leptospirosis in metropolitan France: results of
a national case-control study, 1999–2000. Clin Infect Dis 2004;39:
751–3.
[59] Dufour B, Moutou F, Hattenberger AM, Rodhain F. Global change: impact,
management, risk approach and health measures – the case of Europe. Rev
Sci Tech 2008;27:529–50.
[60] Bourhy P, Collet L, Lernout T, Zinini F, Hartskeerl RA, van der Lin-
den H, et al. Human leptospira isolates circulating in Mayotte (Indian
Ocean) have unique serological and molecular feature. J Clin Microbiol
2012;50:307–11.