Laboratory Diagnostic Methods

Suitable for diagnosis of Malaria

in Zambia.

By Alick Mwambungu

Email. mwambungup@yahoo.com
 Introduction
• Malaria refers to the disease process resulting from human infection
of parasites belonging to the genus plasmodia.
• Reported cases each year number between 300 and 500 million
worldwide.
• These results in over 1 million deaths annually

Malaria Distribution
 Malaria situation in Zambia
450 From 1976 to 1999 there was a high
No of cases/1000 population

400
350
increase in malaria incidence rates.
300 In 1976 the incidence rate was 121.5
250 Incidence
200 rate/1000 cases per 1000 population(I case/8
150
100
persons).
50
0
By 1999 the incidence rate rose to
1970 1980 1990 2000 2010 304.4 cases per 1000 population(I
Year
case for every 3 persons.

• First line drug of choice used to be Chloroquine but due to increase

in plasmodial resistance,MOH changed to artemether Lumefantrine.
• The cost of these drugs is high hence need for accurate malaria
diagnosis.
• Due to an increase in malaria cases coupled with the shortfall in the
lab personnel, the MOH is encouraging the use of RDTS.
• Various RDTS are being marketed in Zambia and hence the need to
evaluate them.
 Aims and objectives

• Main aim
• To study the lab diagnostic methods suitable for malaria
diagnosis in Zambia.
• Specific aims
• To examine the sensitivity and specificity of the RDTs
• To assess post-treatment HRP2/pLDH antigen persistent

• To evaluate relationship between antigen persistence

and gametocyteamia in P.falciparum monoinfection.
• To assess the prevalence of malaria in blood donations
during the rainy season in Zambia.
• To assess the cost of RDTs against microscopy testing
of malaria.
 Experimental Work

• Study was conducted in Ndola District at Chipulukusu

clinic in Zambia.
Methods
• Fresh capillary blood was collected by finger prick
• Thick and thin films were prepared and stained to
identify and quantify parasites.
• Filter paper spotted and allowed to dry and stored for
PCR.
• Rapid Diagnostic Tests(RDTS) carried
out(ICT,HRP2,Carestart pLDH and Carestart combo).
General principle of RDTs
 Interpretation of RDTS test Reaction
ICT

• Left: A positive result

for P.falciparum.
• Right: Negative test
Result.
• Test was invalid if no
control line was seen.
 Interpretation of test reaction(HRP2)

A
A) Positive reaction:

B.
B) Negative reaction

C.

C) Invalid reaction:
 Interpretation of reactions-Carestart
combo.
A) P.falciparum or A
mixed infection
B
B) P.falciparum
reaction C
C) P.vivax,ovale or
malariae reaction D

D) Negative reaction
E) Invalid reaction E(i)

E(ii)
 Evaluation of sensitivity and
specificity

• 240 patients at Chipulukusu clinic, OPD

were tested by:
– Microscopy
– 4 Rapid Diagnostic test kits(RDTS)
 Results-Evaluation of RDTS

Results of 240 patients screened by Microscopy and RDTS

Graphical representation of the results obtained

Positive Negative Total
from the 240 patients.

No.of Positive/Negative samples

180
Microscopy 134 106 240
160
140
120
100 Positive
HRP2 145 95 240 80 Negative
60
40
20
0

T
H
H
P2
HRP2/pLDH 157 83 240

py

IC
D
LD
co

HR

pL
/p
os

P2
icr

HR
M

pLDH 137 103 240 Diagnostic test

ICT 148 92 240

Comparison of RDTS against microscopy
diagnosis of 240 patients
HRP2 pLDH ICT HRP2-pLDH
Combo

Negative Positive Negative Positive Negative Positive Negative Positive

Microscopy 88 21 99 10 84 15 83 26
Negative (TN) (FP) (TN) (FP) (TN) (FP) (TN) (FP)

Microscopy 7 124 4 127 18 123 0 131

Positive (FN) (TP) (FN) (TP) (FN) (TP) (FN) (TP)

Total 95 145 103 137 102 138 83 157

TN: True Negatives, FP: False Positives, FN: False Negatives, TP: True Positives
 Sensitivity,Specificity,PPV and NPV
HRP2 pLDH HRP2-pLDH ICT
• Calculated using the Epi-table combo
combo
software. Sensitivity (%) 95 97 100 87

Specificity (%) 81 91 76 85

• Specificity= TN/(TN +FP) PPV (%) 86 93 83 89

• PPV = TP/(TP +FP) NPV (%) 93 96 100 82

• NPV = TN/(TN+FN)

sensitivity,specificity,PPV and NPV

120

• Sensitivity =TP/(TP + FN) 100

80 Sensitivity (%)
Specificity (%)
60
PPV (%)
40 NPV (%)

20

0
HRP2 pLDH HRP2- ICT
pLDH
Test kit
 Evaluation of Post-treatment antigen
persistence

• 32 of the 240 patients with parasiteamia of

800-20,000/µl re-examined for malaria on
day 7 and 14 after treatment.
• Thick blood film made and stained
• RDTS performed along with microscopy.
 Evaluation of post-treatment antigen
persistence
35
No. of cases with persistent

30 Day 0 Day 7 Day 14

HRP2/pLDH antigens

25 Mic

20 hrp2
pldh Microscopy 32 2 2
15
combo
10 ict

5 HRP2 32 21 17
0
day 0 day7 day 14
Days pLDH 32 6 2

HRP2-pLDH 32 20 17

ICT 32 20 17
 Association between antigen
persistence and gametocyteamia

• All patients negative for asexual-stage of

P.falciparum on microscopy but positive
with gametocytes were enrolled for this
study.
• Patients were re-examined on day 7 and
14 using microscopy and the four RDTS.
Association between Gametocyteamia
and HRP2/pLDH persistence

Day 0 Day 7 Day 14

ICT

HRP2-pLDH
Day 14 Microscopy 15 7 4
Day 7 (Gametocytes)
pLDH
Day 0
HRP2 12 6 3
HRP2

pLDH 3 0 0
Micro(GM)

HRP2-pLDH combo 13 8 4
0 5 10 15 20
No. of Gametocytes or HRP2/pLDH positives ICT 12 7 3
Presence of Malaria parasites in Blood
donations

• Thin and thick blood films prepared from

200 fresh EDTA blood samples from
donors at regional blood Bank.
• Samples also screened with two RDTS
HRP2 and Carestart pLDH.
Prevalence of malarial parasites in blood
donations
Microscopy HRP2 pLDH

Positive 13(6.5%) 23(11.5%) 13(6.5%)

• A total of 200 blood

Negative 187 176 187
samples tested for the
presence of malarial Total 200 200 200

parasites.
 Discussion
Choice of RDT

• Carestart pLDH had the highest specificity(91%).

• Positive Predictive value of 93%.

• Sensitivity of 97% indicates that only 3% of the cases

were missed.
Evaluation of post-treatment antigenemia

• Persistent antigenemia was observed to

be lower in Carestart pLDH based test
assay than the HRP2.
• pLDH can be used to monitor treatment
outcome.
 Association between persistent HRP2/pLDH
antigens and gametocyteamia

• HRP2 method was better at detecting

blood samples which contained
gametocytes but negative for asexual
parasites.
• But in clinical settings without microscopy,
the kit can’t distinguish between
P.falciparum asexual and sexual infection.
Prevalence of malaria parasites in Blood
donations.
• Human plasmodia remain infectious in blood for
1-12 days.
• Positivity was detected in 6.5% of 200 blood
donor samples.
• This result is high.
• Need to introduce cost-effective screening of
blood donors in Zambia.
• Carestart pLDH detected all the cases, hence
can be an ideal test kit.
 Cost Comparison

• Microscopy still remains the cheaper form

of malaria diagnosis(20 cents per test).
• The cost of Carestart pLDH was 70 cents
per test.
• Reduced expenditure on treatment for
negative patients could balance the extra
costs of using RDTS.
 Conclusion

• pLDH based test assays are better than HRP2

based techniques in detecting asexual P.falciparum
infections in clinical cases and for monitoring
response to treatment,
• Though the kits are more expensive than the HRP2
based assays, could reduce the effect of drug
resistance and promote rational drug use.
• Microscopy still remains best tool for parasite
quantification and mixed infection detection.
• Need to start blood donor screening for malaria
infection