Phenotyping Plasmodium vivax in Indonesia using Standard Chloroquine Therapy. Puji Budi Setia Asih. Eijkman Institute for Molecular Biology

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1 Phenotyping Plasmodium vivax in Indonesia using Standard Chloroquine Therapy Puji Budi Setia Asih Eijkman Institute for Molecular Biology

2 Background Plasmodium vivax threatens 2.6 billion people with a debilitating and potentially lethal infection Price et al, 2008 Approximately millions vivax cases each year, mostly in Asia and South AmericaBonila et al, 2006 In Indonesia 3 million malaria cases were reported annually, half of which are vivax malariadepart. Of Health, 2007 Chloroquine resistance in P. falciparum was first reported in the 1959, but In P. Vivax, reported approx. 40 years later Price et al, 2001

3 Chloroquine Treatment Failure Reported in 2004 Report from northeastern Papua showed the prevalence of CQ resistant P. vivax in 2003 was approximately 84% Sumawinata et al, 2003

4 Plasmodium Life Cycle P.vivax P.ovale

5 Vivax Malaria: A Neglected Infection Research on P. vivax has lagged markedly behind P. falciparum (P. vivax can not be continuously grown in vitro) Front-line drugs : Chloroquine and Primaquine have been more than 50 years in service, their mechanisms of activity againts the parasite are not well understood Standardized means of determining parasite resistance to either drug do not exist despite almost 20 years of mounting evidence of resistance (Baird 2009) An in vivo test procedure was described over a decade ago and has been applied, at least partially, in some to determine resistance. However, it should be noticed that unlike P.falciparum, P.vivax sporozoites could stay dormant and initiate a new wave of asexual blood stage and may confound the phenotyping

6 Antimalarial drugs for Vivax Malaria 1 st Line: 4-aminoquinolines = chloroquine 8-aminoquinolines = primaquine (Standard Chloroquine Therapy = 25 mg/kg BW, with 10 mg/kb BW for D0 and D1, and 5 mg/kb BW for D2. + primaquine for 14 days (0.25 mg/kg/day). 2 nd Line: ACT = Artemisinin-based Combination Therapy + Primaquine (for 14 days) (1 st antimalarial drug for vivax malaria mid-2009, WHO 2009)

7 Vivax Treatment Failure (Baird 2009) Classification 1. Direct Treatment Falure: If subjects consistently show parasitemia until the end of treatment at day Early Treatment Failure: Subjects show parasitemia between Days 3-7, with or without fever. 3. Recurrent Treatment Failure: Asexual parasitemia once become undetectable but reappears at any time between Day 8-28 with or without fever. 4. Adequate Clinical Parasitological Response: Subjects had no recurrent parasitemia diagnosed microscopically by Day 28

8 The present study aims to rigorously phenotype P.vivax isolates through in vivo drug sensitivity test using standard chloroquine therapy

9 Materials and Methods (1) : Origin of P. vivax Isolates in this study SentaniPapua West Sumba Drawn not to scale

10 Materials and Methods (2) : Treatment 1. Subjects were given a supervised treatment of total 25 mg/kg body weight of CQ over a three- day period, with 10 mg/kb body weight for D0 and D1, and 5 mg/kb body weight for D2. 2. Primaquine therapy against relapse was not provided until discontinuation from the study, i.e., day of recurrence or day 29.

11 Materials and Methods (3): Follow up: 1. Days 1, 2, 3, 7, 14, 21 and 28, or at any time they complained of illness. 2. Blood sample collected by finger prick for microscopic, molecular genotyping using Pvmsp1 and Pvcsp genes, and CQ+DCQ blood levels. Whole blood CQ+DCQ levels : Blood levels of CQ and its major metabolite DCQ, were measured using high-performance liquid chromatography (HPLC). An approximately 100 µl whole blood was spotted on to filter paper on day of enrollment (D0), at the time of recurrence of parasitemia and at day 28

12 RESULTS

13 Vivax Phenotyping Result from West Sumba and Papua Study Sites Total Sample Treatment Outcome (%) DTF ETF RTF ACPR Sumba Papua 73 (1/18) (2/18) (15/18) 100 (67/67*) 73 (46/73) * Number in bracket indicates samples examined; DTF = Direct Treatment Failure; ETF = Early Treatment Failure; RTF = Recurrence Treatment Failure Asih et al, manuscript in preparation

14 HPLC analysis of the CQ plus DCQ blood level on recrudescent days among the subjects with treatment failure Subject s Code Drug Level (ng/ml of blood) D0 DR Phenotype* CRPV , ,476 Resistant CRPV-007 ND 231,376 Resistant CRPV-008 ND 181,125 Resistant CRPV , ,879 Resistant CRPV-012 ND 423,673 Resistant CRPV , ,986 Resistant CRPV , ,559 Resistant CRPV , ,695 Resistant CRPV-033 ND ND NA CRPV , ,109 Resistant CRPV-041 ND 147,022 Resistant CRPV ,917 ND NA CRPV-045 ND 43,9121 NA CRPV , ,000 Resistant CRPV ,781 ND NA CRPV ,094 ND NA CRPV ,706 24,151 NA CRPV-064 ND ND NA ND; not determined; NA, not applicable; DR, day recrudescent; *MEC Minimum Effective concentration = 100 ng/ml of Blood Asih et al, manuscript in preparation

Genotyping of Plasmodium vivax Subject s PvMSP1 PvCSP Day of Phenotype Code D0 DR DO DR Recurrence CRPV-005 1 1 a a Day 17 Resistant CRPV-007 1 1 b b Day 15 Resistant CRPV-008 1 1 b b Day 15

15 Genotyping of Plasmodium vivax Subject s PvMSP1 PvCSP Day of Phenotype Code D0 DR DO DR Recurrence CRPV a a Day 17 Resistant CRPV b b Day 15 Resistant CRPV b b Day 15 Resistant CRPV a a Day 3 Resistant CRPV b b Day 14 Resistant CRPV b a Day 5 Resistant CRPV b a Day 7 Resistant CRPV a b + c Day 14 Resistant CRPV b a + b Day 33 NA CRPV a b Day 14 Resistant CRPV B B Day 14 Resistant CRPV A A Day 14 NA CRPV a a Day 20 NA CRPV a a Day 21 Resistant CRPV a a Day 21 NA CRPV a a Day 21 NA CRPV a b Day 18 NA CRPV a b Day 11 NA Genetic variation of the P.vivax isolates at day 0 and days of recurrence based on PvMSP-1 and PvCSP genes PvMSP1 = P.vivax merozoite surface protein-1: PvCSP = P.vivax circumsporozoite: D0 = day 0: DR = day of recurrence: NA: not applicable because no CQ blood level: 1 = 350 bps: 2 = 400 bps: a = 680 bps: b = 710 bps: c = 740 bps: Asih et al, manuscript in preparation

16 Conclusion 1. This study has successfully identified isolates of P. vivax that are clearly resistant to standard chloroquine therapy following a rigorous phenotyping procedures. 2. This result corroborates previous reports that indicate the wide distribution of CQ-resistant P. vivax in this region of Indonesia. This study is part of PRIOR Project 11 and Funded through A Grant-In-Aid from The Netherlands Foundation for the Advancement of Tropical Research, The Netherlands Foundation for Health Research and Development.

17 Acknowledgment We thank the community and District Health Department in West Sumba and West Papua, Indonesia, for their cooperation. Budhi Leksana and Amir Faisal NIHRD/NAMRU-2, Department of Health, Jakarta for his assistance during HPLC analysis. Sumba Papua

Antimalarial drug resistance

Antimalarial drug resistance Md Mushfiqur Rahman*, Leonard Ortega**, R M Rastogi* and Krongthong Thimasarn* Abstract Antimalarial drug resistance is of great concern in the WHO South-East Asia (SEA) Region.