Sustained efficacy of artesunate-sulfadoxine-pyrimethamine against Plasmodium falciparum in Yemen and a renewed call for an adjunct single dose primaquine to clear gametocytes

Abstract

BackgroundIn Yemen, artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been used as first-line treatment for uncomplicated falciparum malaria, which accounts for about 99 % of malaria cases. There is evidence that resistance to SP is increasing, with potential negative impact on efficacy, and in particular on curbing transmission. This study aims: (a) to evaluate the therapeutic efficacy of AS + SP treatment for uncomplicated falciparum malaria in Yemen; (b) to investigate the frequency of mutations in Plasmodium falciparum genes associated with resistance to AS (Kelch 13 propeller domain, pfK13) and SP (dihydrofolate reductase, pfdhfr, and dihydropteroate synthase, pfdhps); and (c) to assess the adequacy of this ACT to clear gametocytes.MethodsA 28-day in vivo evaluation of the clinical and parasitological response to three-day course of AS + SP was carried out in two areas of high endemicity (Hodeidah and Al-Mahwit provinces, Tehama region) in Yemen according to standard WHO protocol 2009. Clinical and parasitological indices were monitored over a 28-day follow-up, and the outcome was PCR-corrected. The frequencies of mutations in the pfdhfr, pfdhps, and pfK13 genes were obtained by sequencing following amplification.ResultsEighty-six patients completed the study, with a cure rate of 96.5 % (94.2 % PCR-uncorrected). Whereas four (4.7 %) patients still showed parasitaemia on day 2 post-treatment, all were found negative for asexual malaria stages on days 3 and 7. The efficacy of gametocyte clearance was poor (14.5, 42.5 and 86.0 % on days 7, 14 and 28, respectively), with gametocytes persisting throughout the study in some patients. All the isolates sequenced had the pfk13 propeller domain wild-type allele, and mutations associated with SP failure were observed only for pfdhfr with the double mutation (S108N + N51I) found in 65.4 % of the isolates sequenced.ConclusionIn Yemen, AS + SP therapy remains effective for the treatment of uncomplicated falciparum malaria. Mutations were not detected in pfk13 or pfdhps, though double mutations were observed for pfdhfr. The observed persistent gametocytaemia re-enforces calls to add a single dose primaquine to this ACT in order to minimizes the potential for transmission and enhance regional efforts to eliminate malaria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1344-0) contains supplementary material, which is available to authorized users.

Increased prevalence of pfdhfr and pfdhps mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium falciparum isolates from Jazan Region, Southwestern Saudi Arabia: important implications for malaria treatment policy

“…This finding is consistent with the only previous study on potential mutations in pfkelch13 in isolates, which was conducted in the Taif region of Saudi Arabia [23]. Similar findings have also been reported in other countries either in the Arabian Peninsula or in the East Mediterranean Region including Yemen, Sudan and Somalia [27,35,36]. On the other hand, few non-synonymous pfkelch13 mutations were identified in P. falciparum isolates from other neighbouring countries including Qatar, Iran and Ethiopia; however, none of those mutations were associated with artemisinin resistance [36][37][38].…”

“…The thin and thick blood films of patients who tested positive for malaria by the rapid diagnostic test were stained with diluted Giemsa and then examined microscopically for confirmation and parasitaemia estimation. The parasite species was identified and parasitaemia was estimated by counting the asexual stages against 300 leukocytes and then multiplying by 25, under the assumption of a mean total white blood cell count of 7500 cells per μL of blood [ 27 ]. The level of parasitaemia (parasite density) was categorized into three levels: low (< 1000 parasites/μL of blood), moderate (1000–9999 parasites/μL of blood), and high (≥ 10,000 parasites/μL of blood).…”

“…no. 69506, Germany) following the manufacturer’s instructions and the procedures described in a previous study [ 27 ]. Before any molecular analysis for resistant markers was undertaken, all P. falciparum positive samples were confirmed by using a PCR assay that targeted the small subunit ribosomal RNA genes, according to the Singh protocol [ 28 ].…”

“…The mutations were identified by direct sequencing. Details of the primer sequences used for the pfkelch13 , pfdhfr and pfdhps genes have been described previously [ 27 ].…”

“…A single-run PCR was performed using specific primers designed to obtain an amplicon corresponding to nucleotides 1094–2127 (codons 364–709) [ 27 ]. A 50-µL reaction consist of 20 µL of ExPrime Tag Premix (GENET BIO, Korea), 200 nM of each primer and 2 µL of parasite genomic DNA template.…”