Reply to Gonçalves et al

TO THE EDITOR—We agree with Goncalves et al that our study was restricted to treat-ment-seeking persons with symptomatic falciparum malaria. As such, the study did not assess the relative infectiousness of what may well be a larger, asymptom-atic, and often submicroscopic malaria transmission reservoir. The absence of studies on the infectiousness of asym-ptomatic persons with submicroscopic malariainlow-transmissionsettings yields little evidence for or against tar-geting these populations as a strategy to accelerate malaria elimination; indeed, further evaluation is warranted

Longitudinal Pooled Deep Sequencing of the Plasmodium vivax K12 Kelch Gene in Cambodia Reveals a Lack of Selection by Artemisinin

The emergence of artemisinin resistance among Plasmodium falciparum in the Greater Mekong subregion threatens malaria control interventions and is associated with multiple unique mutations in K13 (PF3D7_1343700). The aim of this study was to survey Cambodian Plasmodium vivax for mutations in the K13 ortholog (K12, PVX_083080) that might similarly confer artemisinin resistance. Extracted DNA from Cambodian isolates collected between 2009 and 2012 was pooled by province and year and submitted for next-generation sequencing. Single-nucleotide polymorphisms (SNPs) were identified using a pile-up approach that detected minority SNPs. Among the 14 pools, we found six unique SNPs, including three nonsynonymous SNPs, across six codons in K12. However, none of the SNPs were orthologous to artemisinin resistance–conferring mutations in PF3D7_1343700, and nonsynonymous changes did not persist through time within populations. These results suggest a lack of selection in the P. vivax population in Cambodia due to artemisinin drug pressure

Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia

BACKGROUND: Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of “immediate ex vivo” (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission. METHODS: Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC(50)) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC(50) values from the two methods was made using Wilcoxon matched pair tests and Pearson’s correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples. RESULTS: IC(50) values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC(50) value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC(50) best-fit sigmoidal curves), relative to only a 40% success rate for the MSF assay, when evaluating ex vivo Cambodian isolates. Reduced sensitivity of the MSF assay is likely due to an interference of WBCs in clinical samples. CONCLUSIONS: For clinical samples not depleted of WBCs, HRP-2 ELISA is superior to the MSF assay at evaluating fresh P. falciparum field isolates with low parasitaemia (<0.2%) generally observed in Southeast Asia

A deep sequencing tool for partitioning clearance rates following antimalarial treatment in polyclonal infections

BACKGROUND AND OBJECTIVES: Current tools struggle to detect drug-resistant malaria parasites when infections contain multiple parasite clones, which is the norm in high transmission settings in Africa. Our aim was to develop and apply an approach for detecting resistance that overcomes the challenges of polyclonal infections without requiring a genetic marker for resistance. METHODOLOGY: Clinical samples from patients treated with artemisinin combination therapy were collected from Tanzania and Cambodia. By deeply sequencing a hypervariable locus, we quantified the relative abundance of parasite subpopulations (defined by haplotypes of that locus) within infections and revealed evolutionary dynamics during treatment. Slow clearance is a phenotypic, clinical marker of artemisinin resistance; we analyzed variation in clearance rates within infections by fitting parasite clearance curves to subpopulation data. RESULTS: In Tanzania, we found substantial variation in clearance rates within individual patients. Some parasite subpopulations cleared as slowly as resistant parasites observed in Cambodia. We evaluated possible explanations for these data, including resistance to drugs. Assuming slow clearance was a stable phenotype of subpopulations, simulations predicted that modest increases in their frequency could substantially increase time to cure. CONCLUSIONS AND IMPLICATIONS: By characterizing parasite subpopulations within patients, our method can detect rare, slow clearing parasites in vivo whose phenotypic effects would otherwise be masked. Since our approach can be applied to polyclonal infections even when the genetics underlying resistance are unknown, it could aid in monitoring the emergence of artemisinin resistance. Our application to Tanzanian samples uncovers rare subpopulations with worrying phenotypes for closer examination

Lon et al. Malaria Journal 2014, 13:96

Blackwater fever in an uncomplicated Plasmodium falciparum patient treated with dihydroartemisinin-piperaquin

Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens

Polymorphisms within Plasmodium falciparum vaccine candidate antigens have the potential to compromise vaccine efficacy. Understanding the allele frequencies of polymorphisms in critical binding regions of antigens can help in the designing of strain-transcendent vaccines. Here, we adopt a pooled deep-sequencing approach, originally designed to study P. falciparum drug resistance mutations, to study the diversity of two leading transmission-blocking vaccine candidates, Pfs25 and Pfs48/45. We sequenced 329 P. falciparum field isolates from six different geographic regions. Pfs25 showed little diversity, with only one known polymorphism identified in the region associated with binding of transmission-blocking antibodies among our isolates. However, we identified four new mutations among eight non-synonymous mutations within the presumed antibody-binding region of Pfs48/45. Pooled deep sequencing provides a scalable and cost-effective approach for the targeted study of allele frequencies of P. falciparum candidate vaccine antigens

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

In Cambodia, where Plasmodium vivax and Plasmodium falciparum are coendemic and intense multimodal malaria-control interventions have reduced malaria incidence, P. vivax malaria has proven relatively resistant to such measures. We performed comparative genomic analyses of 150 P. vivax and P. falciparum isolates to determine whether different evolutionary strategies might underlie this species-specific resilience. Demographic modeling and tests of selection show that, in contrast to P. falciparum, P. vivax has experienced uninterrupted growth and positive selection at multiple loci encoding transcriptional regulators. In particular, a strong selective sweep involving an AP2 transcription factor suggests that P. vivax may use nuanced transcriptional approaches to population maintenance. Better understanding of P. vivax transcriptional regulation may lead to improved tools to achieve elimination

Partner-Drug Resistance and Population Substructuring of Artemisinin-Resistant Plasmodium falciparum in Cambodia

Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance

Capacity-building efforts by the AFHSC-GEIS program

Capacity-building initiatives related to public health are defined as developing laboratory infrastructure, strengthening host-country disease surveillance initiatives, transferring technical expertise and training personnel. These initiatives represented a major piece of the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) contributions to worldwide emerging infectious disease (EID) surveillance and response. Capacity-building initiatives were undertaken with over 80 local and regional Ministries of Health, Agriculture and Defense, as well as other government entities and institutions worldwide. The efforts supported at least 52 national influenza centers and other country-specific influenza, regional and U.S.-based EID reference laboratories (44 civilian, eight military) in 46 countries worldwide. Equally important, reference testing, laboratory infrastructure and equipment support was provided to over 500 field sites in 74 countries worldwide from October 2008 to September 2009. These activities allowed countries to better meet the milestones of implementation of the 2005 International Health Regulations and complemented many initiatives undertaken by other U.S. government agencies, such as the U.S. Department of Health and Human Services, the U.S. Agency for International Development and the U.S. Department of State

Microscopic Plasmodium falciparum Gametocytemia and Infectivity to Mosquitoes in Cambodia

Although gametocytes are essential for malaria transmission, in Africa many falciparum-infected persons without smear-detectable gametocytes still infect mosquitoes. To see whether the same is true in Southeast Asia, we determined the infectiousness of 119 falciparum-infected Cambodian adults to Anopheles dirus mosquitoes by membrane feeding. Just 5.9% of subjects infected mosquitoes. The 8.4% of patients with smear-detectable gametocytes were >20 times more likely to infect mosquitoes than those without and were the source of 96% of all mosquito infections. In low-transmission settings, targeting transmission-blocking interventions to those with microscopic gametocytemia may have an outsized effect on malaria control and elimination