Malaria Diagnosis In Zambia

Laboratory Diagnostic Methods Suitable for diagnosis of Malaria in Zambia. By Alick Mwambungu Email. [email protected]

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

No of cases/1000 population

Malaria situation in Zambia 450 400 350 300 250

Incidence rate/1000

200 150 100 50 0 1970 1980 1990 2000 2010 Year

• • • •

From 1976 to 1999 there was a high increase in malaria incidence rates. In 1976 the incidence rate was 121.5 cases per 1000 population(I case/8 persons). By 1999 the incidence rate rose to 304.4 cases per 1000 population(I 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) Negative reaction

B.

C.

C) Invalid reaction:

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

A B C D

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

HRP2/pLDH

157

83

240

pLDH

137

103

240

ICT

148

92

240

M

Negative

Diagnostic test

IC T

240

H

95

Positive

pL D

145

icr

HRP2

180 160 140 120 100 80 60 40 20 0 P2 /p LD H

240

P2

106

HR

134

Graphical representation of the results obtained from the 240 patients.

HR

Total

os co py

Negative

No.of Positive/Negative samples

Microscopy

Positive

Comparison of RDTS against microscopy diagnosis of 240 patients HRP2

pLDH

ICT

HRP2-pLDH Combo

Negative

Positive

Negative

Positive

Negative

Positive

Negative

Microscopy Negative

88 (TN)

21 (FP)

99 (TN)

10 (FP)

84 (TN)

15 (FP)

83 (TN)

Microscopy Positive

7 (FN)

124 (TP)

4 (FN)

127 (TP)

18 (FN)

123 (TP)

0 (FN)

131 (TP)

Total

95

145

103

137

102

138

83

157

TN: True Negatives, FP: False Positives, FN: False Negatives, TP: True Positives

Positive

26 (FP)

Sensitivity,Specificity,PPV and NPV HRP2-pLDH combo combo

ICT

Sensitivity (%)

95

97

100

87

Specificity (%)

81

91

76

85

PPV (%)

86

93

83

89

93

96

100

82

Calculated using the Epi-table software.

• • • •

pLDH

Specificity= TN/(TN +FP) PPV = TP/(TP +FP) NPV = TN/(TN+FN) Sensitivity =TP/(TP + FN)

NPV (%) sensitivity,specificity,PPV and NPV

•

HRP2

120 100 80

Sensitivity (%) Specificity (%)

60 40 20 0 HRP2

pLDH

HRP2pLDH

Test kit

ICT

PPV

(%)

NPV

(%)

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 No. of cases with persistent HRP2/pLDH antigens

35

Day 0

Day 7

Day 14

Microscopy

32

2

2

HRP2

32

21

17

pLDH

32

6

2

HRP2-pLDH

32

20

17

ICT

32

20

17

30 25

Mic

20

hrp2 pldh

15

combo

10

ict

5 0 day 0

day7 Days

day 14

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

Microscopy (Gametocytes)

15

7

4

HRP2

12

6

3

pLDH

3

0

0

HRP2-pLDH combo

13

8

4

ICT

12

7

3

ICT HRP2-pLDH

Day 14 Day 7

pLDH

Day 0

HRP2 Micro(GM) 0

5

10

15

20

No. of Gametocytes or HRP2/pLDH positives

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

• A total of 200 blood samples tested for the presence of malarial parasites.

HRP2

23(11.5%)

pLDH

Positive

13(6.5%)

13(6.5%)

Negative

187

176

187

Total

200

200

200

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