Khalid B. Beshir

Residual Plasmodium falciparum Parasitemia in Kenyan Children After Artemisinin-Combination Therapy Is Associated With Increased Transmission to Mosquitoes and Parasite Recurrence

Background. Parasite clearance time after artemisinin-based combination therapy (ACT) may be increasing in Asian and African settings. The association between parasite clearance following ACT and transmissibility is currently unknown. Methods. We determined parasite clearance dynamics by duplex quantitative polymerase chain reaction (qPCR) in samples collected in the first 3 days after treatment of uncomplicated malaria with ACT. Gametocyte carriage was determined by Pfs25 quantitative nucleic acid sequence–based amplification assays; infectiousness to mosquitoes by membrane-feeding assays on day 7 after treatment. Results. Residual parasitemia was detected by qPCR in 31.8% (95% confidence interval [CI], 24.6–39.8) of the children on day 3 after initiation of treatment. Residual parasitemia was associated with a 2-fold longer duration of gametocyte carriage (P = .0007), a higher likelihood of infecting mosquitoes (relative risk, 1.95; 95% CI, 1.17–3.24; P = .015), and a higher parasite burden in mosquitoes (incidence rate ratio, 2.92; 95% CI, 1.61–5.31; P < .001). Children with residual parasitemia were also significantly more likely to experience microscopically detectable parasitemia during follow-up (relative risk, 11.25; 95% CI, 4.08–31.01; P < .001). Conclusions. Residual submicroscopic parasitemia is common after ACT and is associated with a higher transmission potential. Residual parasitemia may also have consequences for individual patients because of its higher risk of recurrent parasitemia.

Malaria Transmission After Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine: A Randomized Trial

BACKGROUND Artemisinin-based combination therapy (ACT) reduces the potential for malaria transmission, compared with non-ACTs. It is unclear whether this effect differs between ACTs. METHODS A total of 298 children (age, 6 months to 10 years) with uncomplicated falciparum malaria were randomized to artemether-lumefantrine (AL; n = 153) or dihydroartemisinin-piperaquine (DP; n = 145) in Mbita, a community in western Kenya. Gametocyte carriage was determined by molecular methods on days 0, 1, 2, 3, 7, 14, 28, and 42 after treatment initiation. The gametocyte infectiousness to mosquitoes was determined by mosquito-feeding assays on day 7 after beginning therapy. RESULTS The cumulative risk of recurrent parasitemia on day 42 after initiation of treatment, unadjusted by polymerase chain reaction findings, was 20.7% (95% confidence interval [CI], 14.4-28.2) for AL, compared with 3.7% (95% CI, 1.2-8.5) for DP (P < .001). The mean duration of gametocyte carriage was 5.5 days (95% CI, 3.6-8.5) for AL and 15.3 days (95% CI, 9.7-24.2) for DP (P = .001). The proportion of mosquitoes that became infected after feeding on blood from AL-treated children was 1.88% (43 of 2293), compared with 3.50% (83 of 2371) for those that fed on blood from DP-treated children (P = .06); the oocyst burden among mosquitoes was lower among those that fed on blood from AL-treated children (P = .005) CONCLUSIONS: While DP was associated with a longer prophylactic time after treatment, gametocyte carriage and malaria transmission to mosquitoes was lower after AL treatment. CLINICAL TRIALS REGISTRATION NCT00868465.

Amodiaquine Resistance in Plasmodium falciparum Malaria in Afghanistan Is Associated with the pfcrt SVMNT Allele at Codons 72 to 76

ABSTRACT Mutations in the Plasmodium falciparum genes pfcrt and pfmdr1 are selected by amodiaquine treatment in Africa. To examine the importance of these mutations in amodiaquine-treated Asian parasites, we determined pre- and posttreatment genotypes for amodiaquine treatment failures from a clinical trial in Afghanistan. The pfcrt codon 72 to 76 haplotype SVMNT was present in all samples tested, both before and after treatment. Amodiaquine did not clearly select for any pfmdr1 genotype, but a novel mutation, pfmdr1 N86F, was detected in four samples. We provide in vivo data to support the in vitro correlation between pfcrt SVMNT and increased resistance to the metabolite of amodiaquine.

Measuring the efficacy of anti-malarial drugs in vivo: quantitative PCR measurement of parasite clearance

BackgroundArtemisinin-based combination therapy, currently considered the therapy of choice for uncomplicated Plasmodium falciparum malaria in endemic countries, may be under threat from newly emerging parasite resistance to the artemisinin family of drugs. Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy. This phenotype is likely to become a more important trial endpoint in studies of anti-malarial drug efficacy, but currently requires frequent, closely spaced blood sampling in hospitalized study participants, followed by quantitation of parasite density by microscopy.MethodsA simple duplex quantitative PCR method was developed in which distinct fluorescent signals are generated from the human and parasite DNA components in each blood sample. The human amplification target in this assay is the β tubulin gene, and the parasite target is the unique methionine tRNA gene (pgmet), which exhibits perfect sequence identity in all six Plasmodium species that naturally infect humans. In a small series of malaria cases treated as hospital in-patients, the abundance of pgmet DNA was estimated relative to the human DNA target in daily peripheral blood samples, and parasite clearance times calculated.ResultsThe qPCR assay was reproducibly able to replicate parasite density estimates derived from microscopy, but provided additional data by quantification of parasite density 24 hours after the last positive blood film. Robust estimates of parasite clearance times were produced for a series of patients with clinical malaria.ConclusionsLarge studies, particularly in Africa where children represent a major proportion of treated cases, will require a simpler blood sample collection regime, and a method capable of high throughput. The duplex qPCR method tested may fulfil these criteria, and should now be evaluated in such field studies.

Directional selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu loci of Plasmodium falciparum in Kenyan children treated with ACT.

Background The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42. Methods DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure. Results Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72–76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia. Conclusions Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence.

Markers of anti-malarial drug resistance in Plasmodium falciparum isolates from Swaziland: identification of pfmdr1-86F in natural parasite isolates

BackgroundThe development of Plasmodium falciparum resistance to chloroquine (CQ) has limited its use in many malaria endemic areas of the world. However, despite recent drug policy changes to adopt the more effective artemisinin-based combination (ACT) in Africa and in the Southern African region, in 2007 Swaziland still relied on CQ as first-line anti-malarial drug.MethodsParasite DNA was amplified from P. falciparum isolates from Swaziland collected in 1999 (thick smear blood slides) and 2007 (filter paper blood spots). Markers of CQ and sulphadoxine-pyrimethamine (SP) resistance were identified by probe-based qPCR and DNA sequencing.ResultsRetrospective microscopy, confirmed by PCR amplification, found that only six of 252 patients treated for uncomplicated malaria in 2007 carried detectable P. falciparum. The pfcrt haplotype 72C/73V/74I/75E/76T occurred at a prevalence of 70% (n = 64) in 1999 and 83% (n = 6) in 2007. Prevalence of the pfmdr1-86N allele was 24% in 1999 and 67% in 2007. A novel substitution of phenylalanine for asparagine at codon 86 of pfmdr1 (N86F) occurred in two of 51 isolates successfully amplified from 1999. The pfmdr1-1246Y allele was common in 1999, with a prevalence of 49%, but was absent among isolates collected in 2007. The 86N/184F/1246D pfmdr1 haplotype, associated with enhanced parasite survival in patients treated with artemether-lumefantrine, comprised 8% of 1999 isolates, and 67% among 2007 isolates. The pfdhfr triple-mutant 16C/51I/59R/108N/164I haplotype associated with pyrimethamine resistance was common in both 1999 (82%, n = 34) and 2007 (50%, n = 6), as was the wild-type 431I/436S/437A/540K/581A/613A haplotype of pfdhps (100% and 93% respectively in 1999 and 2007). The quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA, associated with high-level resistance to SP, was rare (9%) among 1999 isolates and absent among 2007 isolates.ConclusionsThe prevalence of pfcrt and pfmdr1 alleles reported in this study is consistent with a parasite population under sustained CQ drug pressure. The low prevalence of dhps-437G and dhps-540E mutations (ISGEAA) and the rarity of quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA suggest that SP retains some efficacy in Swaziland. Anti-malarial policy changes in neighbouring countries may have had an impact on the prevalence of molecular markers of anti-malarial resistance in Swaziland, and it is hoped that this new information will add to understanding of the regional anti-malarial resistance map.

Contribution of Plasmodium knowlesi to multi-species human malaria infections in North Sumatera, Indonesia

Summary Indonesia is aiming for malaria elimination by 2030. A parasitological survey of 3731 individuals in North Sumatera province used polymerase chain reaction to identify 1169 with parasitemia and highly specific amplification of sicavar genes to identify Plasmodium knowlesi infection in 377.

Lack of K13 mutations in Plasmodium falciparum persisting after artemisinin combination therapy treatment of Kenyan children

AbstractBackgroundStudies in Southeast Asia reported a strong relationship between polymorphisms at the propeller domain of the Kelch 13 (K13) protein encoded by the Plasmodiumfalciparumk13(pfk13) gene and delayed parasite clearance after artemisinin treatment. In Africa, P. falciparum remains susceptible and combination therapy regimens which include an artemisinin component display good efficacy. Using quantitative real-time PCR (qPCR), sub-microscopic persistence of P. falciparum has previously been reported in one-third of children treated with artemisinin combination therapy (ACT) in western Kenya. In this study, further investigation was made to evaluate whether these sub-microscopic residual parasites also harbour mutations at the propeller region of pfk13 and whether the mutations, if any, affect treatment outcome.MethodsThe pfk13 propeller domain was genotyped in DNA samples obtained in 2009 from Kenyan children treated with artemether–lumefantrine (AL) and dihydroartemisinin–piperaquine (DP). Paired samples at pre-treatment (day 0) and day of treatment failure (day 28 or 42) for 32 patients with documented recurrent parasitaemia were available for genotyping. Additional day 3 DNA samples were available for 10 patients.ResultsNo mutation associated with artemisinin resistance in Southeast Asia was observed. Only one DP-treated patient harboured a non-synonymous mutation at codon 578 (A578S) of pfk13-propeller gene in the day 0 sample, but this allele was replaced by the wild-type (A578) form on day 3 and on the day of recurrent parasitaemia. The mutation at amino acid codon 578 showed no association with any phenotype. Polymorphisms in pfk13 were not responsible for parasite persistence and gametocyte carriage in the children treated with ACT.ConclusionThis study contributes to the ongoing surveillance of suspected artemisinin resistance parasites in Africa by providing baseline prevalence of k13-propeller mutations in western Kenya with samples collected from a longitudinal study. Clinical Trials Registration NCT00868465.

Clinical trial of extended-dose chloroquine for treatment of resistant falciparum malaria among Afghan refugees in Pakistan

BackgroundFalciparum malaria is a significant problem for Afghan refugees in Pakistan. Refugee treatment guidelines recommended standard three-day chloroquine treatment (25 mg/kg) for first episodes and extended five-day treatment (40 mg/kg) for recrudescent infections, based on the assumption that a five-day course would more likely achieve a cure. An in-vivo randomized controlled trial was conducted among refugees with uncomplicated falciparum malaria to determine whether five-day treatment (CQ40) was more effective than standard treatment (CQ25).Methods142 falciparum patients were recruited into CQ25 or CQ40 treatment arms and followed up to 60 days with regular blood smears. The primary outcome was parasitological cure without recrudescence. Treatment failures were retreated with CQ40. PCR genotyping of 270 samples, from the same and nearby sites, was used to support interpretation of outcomes.Results84% of CQ25 versus 51% of CQ40 patients experienced parasite recrudescence during follow-up (adjusted odds ratio 0.17, 95%CI 0.08-0.38). Cure rates were significantly improved with CQ40, particularly among adults. Fever clearance time, parasite clearance time, and proportions gametocytaemic post-treatment were similar between treatment groups. Second-line CQ40 treatment resulted in higher failure rates than first-line CQ40 treatment. CQ-resistance marker pfcrt 76T was found in all isolates analysed, while pfmdr1 86Y and 184Y were found in 18% and 37% of isolates respectively.ConclusionsCQ is not suitable for first-line falciparum treatment in Afghan refugee communities. The extended-dose CQ regimen can overcome 39% of resistant infections that would recrudesce under the standard regimen, but the high failure rate after directly observed treatment demonstrates its use is inappropriate.

Defining Plasmodium falciparum treatment in South West Asia: a randomized trial comparing artesunate or primaquine combined with chloroquine or SP.

Introduction Antimalarial resistance has led to a global policy of artemisinin-based combination therapy. Despite growing resistance chloroquine (CQ) remained until recently the official first-line treatment for falciparum malaria in Pakistan, with sulfadoxine-pyrimethamine (SP) second-line. Co-treatment with the gametocytocidal primaquine (PQ) is recommended for transmission control in South Asia. The relative effect of artesunate (AS) or primaquine, as partner drugs, on clinical outcomes and gametocyte carriage in this setting were unknown. Methods A single-blinded, randomized trial among Afghan refugees in Pakistan compared six treatment arms: CQ; CQ+(single-dose)PQ; CQ+(3 d)AS; SP; SP+(single-dose)PQ, and SP+(3 d)AS. The objectives were to compare treatment failure rates and effect on gametocyte carriage, of CQ or SP monotherapy against the respective combinations (PQ or AS). Outcomes included trophozoite and gametocyte clearance (read by light microscopy), and clinical and parasitological failure. Findings A total of 308 (87%) patients completed the trial. Failure rates by day 28 were: CQ 55/68 (81%); CQ+AS 19/67 (28%), SP 4/41 (9.8%), SP+AS 1/41 (2.4%). The addition of PQ to CQ or SP did not affect failure rates (CQ+PQ 49/67 (73%) failed; SP+PQ 5/33 (16%) failed). AS was superior to PQ at clearing gametocytes; gametocytes were seen on d7 in 85% of CQ, 40% of CQ+PQ, 21% of CQ+AS, 91% of SP, 76% of SP+PQ and 23% of SP+AS treated patients. PQ was more effective at clearing older gametocyte infections whereas AS was more effective at preventing emergence of mature gametocytes, except in cases that recrudesced. Conclusions CQ is no longer appropriate by itself or in combination. These findings influenced the replacement of CQ with SP+AS for first-line treatment of uncomplicated falciparum malaria in the WHO Eastern Mediterranean Region. The threat of SP resistance remains as SP monotherapy is still common. Three day AS was superior to single-dose PQ for reducing gametocyte carriage. Trial Registration ClinicalTrials.gov bold>