Antimalarial drug efficacy and resistance in malaria-endemic countries in HANMAT-PIAM_net countries of the Eastern Mediterranean Region 2016-2020: Clinical and genetic studies.

INTRODUCTION: The World Health Organization recommends regular monitoring of the efficacy of nationally recommended antimalarial drugs. We present the results of studies on the efficacy of recommended antimalarials and molecular markers of artemisinin and partner resistance in Afghanistan, Pakistan, Somalia, Sudan and Yemen. METHODS: Single-arm prospective studies were conducted to evaluate the efficacy of artesunate-sulfadoxine-pyrimethamine (ASSP) in Afghanistan and Pakistan, artemether-lumefantrine (AL) in all countries, or dihydroartemisinin-piperaquine (DP) in Sudan for the treatment of Plasmodium falciparum. The efficacy of chloroquine (CQ) and AL for the treatment of Plasmodium vivax was evaluated in Afghanistan and Somalia, respectively. Patients were treated and monitored for 28 (CQ, ASSP and AL) or 42 (DP) days. Polymerase chain reaction (PCR)-corrected cure rate and parasite positivity rate at Day 3 were estimated. Mutations in the P. falciparum kelch 13 (Pfk13) gene and amplifications of plasmepsin (Pfpm2) and multidrug resistance-1 (Pfmdr-1) genes were also studied. RESULTS: A total of 1680 (249 for ASSP, 1079 for AL and 352 for DP) falciparum cases were successfully assessed. A PCR-adjusted ASSP cure rate of 100% was observed in Afghanistan and Pakistan. For AL, the cure rate was 100% in all but four sites in Sudan, where cure rates ranged from 92.1% to 98.8%. All but one patient were parasite-free at Day 3. For P. vivax, cure rates were 98.2% for CQ and 100% for AL. None of the samples from Afghanistan, Pakistan and Yemen had a Pfk13 mutation known to be associated with artemisinin resistance. In Sudan, the validated Pfk13 R622I mutation accounted for 53.8% (14/26) of the detected non-synonymous Pfk13 mutations, most of which were repeatedly detected in Gadaref. A prevalence of 2.7% and 9.3% of Pfmdr1 amplification was observed in Pakistan and Yemen, respectively. CONCLUSION: High efficacy of ASSP, AL and DP in the treatment of uncomplicated falciparum infection and of CQ and AL in the treatment of P. vivax was observed in the respective countries. The repeated detection of a relatively high rate of Pfk13 R622I mutation in Sudan underscores the need for close monitoring of the efficacy of recommended ACTs, parasite clearance rates and Pfk13 mutations in Sudan and beyond. Registration numbers of the trials: ACTRN12622000944730 and ACTRN12622000873729 for Afghanistan, ACTRN12620000426987 and ACTRN12617001025325 for Pakistan, ACTRN12618001224213 for Somalia, ACTRN12617000276358, ACTRN12622000930785 and ACTRN12618001800213 for Sudan and ACTRN12617000283370 for Yemen.

Efficacy of artemisinin-based combination therapies for the treatment of falciparum malaria in Pakistan (2007-2015): In vivo response and dhfr and dhps mutations.

Artesunate+sulfadoxine-pyrimethamine (AS+SP) and artemether+lumefantrine (AL) are the first- and second line treatments, respectively, for the treatment of falciparum infections and dihydroartemsinin+piperaquine (DHA+PPQ) is a potential candidate in case AS+SP or AL fails in Pakistan. The therapeutic efficacies of AS+SP (5 sites in 2007, 2 sites in 2011 and 2 sites in 2012), AL (2 sites in 2012) and DHA+PPQ (2 sites in 2015) were evaluated in seven sentinel sites. Clinical and parasitological outcomes were evaluated among eligible patients. Mutations of the P. falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes were investigated. After PCR correction, a 98.5-100% adequate clinical and parasitological response (ACPR) for AS+SP and a 98.8-100% ACPR for AL were observed by day 28, as well as a 100% ACPR by day 42 for DHA+PPQ. The prevalences of mutants dhfr S108N (100%) and C59R (98%-100%) reached or were near fixation. The double dhfr (C59R/S108N) mutant was dominant (96%-100%) at all sites. The triple dhfr (N51I/C59R/S108N) mutant was rare (1.1%-2.3%). The prevalence of dhps A437G varied between 38% and 70%. A combination of triple dhfr/dhps (C59R/S108N+A437G or N51I/S108N+A437G) mutants was observed (38%-69%). A quadruple dhfr/dhps (N51I/C59R/S108N+A437G) mutation was very rare and no quintuple (N51I/C59R/S108N+A437G/K540E) mutations were detected. AS+SP remains highly effective in Pakistan. However, molecular data indicate that SP resistance is being established, although mutations that confer a high risk of SP treatment failure are rare or non-existent. This underscores the need for close monitoring of both in vivo AS+SP efficacy and dhfr and dhps mutations to inform national treatment policy. Trial registration numbers: ISRCTN21926128 (2007), ACTRN12611001244998 (2011), ACTRN12612001090808 (2012), ACTRN12615001248550 (2015).

Plasmodium vivax: prevalence of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax clinical isolates from Pakistan.

The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR-RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I(13)P(33)F(57)S(58)T(61)S(117)I(173)/A(383)A(553) (43.9%), I(13)P(33)F(57)S(58)T(61)N(117)I(173)/A(383)A(553) (33.6%) and I(13)P(33)F(57)R(58)T(61)N(117)I(173)/A(383)A(553) (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.

Detection of mixed Plasmodium falciparum & P. vivax infections by nested-PCR in Pakistan, Iran & Afghanistan.

BACKGROUND & OBJECTIVES: Species identification and information on transmission pattern of malaria parasite in any malaria endemic area is key to success for a malaria control programme. In this investigation, malaria diagnosis using molecular method was used to assess the transmission pattern of malaria parasite in three malaria endemic regions: Afghanistan, Iran and Pakistan. METHODS: Blood samples were collected from the patients presenting with vivax malaria from Afghanistan (n=108), Iran (n=200) and Pakistan (n=199). Malaria parasite detection was made by the gold standard (microscopy) and also nested-PCR assay, using 18S small sub-unit ribosomal RNA (ssrRNA) gene. RESULTS: Based on microscopy method, the level of mixed infection was zero to 2.5 per cent; however, nested-PCR assay detected 6.5, 22 and 23.5 per cent mixed infections in samples collected from Afghanistan, Iran and Pakistan, respectively. The present results showed that the co-infection of P. vivax with P. falciparum was frequent in malaria endemic regions of Iran and Pakistan. INTERPRETATION & CONCLUSION: The present data suggest the need for improving microscopy diagnosis method and the clinician should also have careful clinical observation, along with the reports on Giemsa- stained thick blood films, particularly in summer time when P. vivax is predominant. Also sharing information on transmission pattern of mixed infection among these countries may help in designing better control strategies for malaria.

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3alpha and pvcsp genes as molecular markers.

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3alpha (msp-1 and msp-3alpha) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n=187) and Iran (n=150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3alpha, three major types: type A (1800bp), type B (1500bp) and type C (1200bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3alpha with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.