Low-technology cooling box for storage of malaria RDTs and other medical supplies in remote areas

<p>Abstract</p> <p>Background</p> <p>With the increase in use of point-of-care diagnostic tests for malaria and other diseases comes the necessity of storing the diagnostic kits and the drugs required for subsequent management, in remote areas, where temperatures are high and electricity supply is unreliable or unavailable.</p> <p>Methods</p> <p>To address the lack of temperature-controlled storage during the introduction of community-based malaria management in Cambodia, the Cambodian National Centre for Parasitology, Entomology and Malaria Control (CNM) developed prototype evaporative cooling boxes (Cambodian Cooler Boxes - CCBs) for storage of perishable medical commodities in remote clinics. The performance of these CCBs for maintaining suitable storage temperatures was evaluated over two phases in 2005 and 2006-7, comparing conditions in CCBs using water as designed, CCBs with no water for evaporation, and ambient storage room temperatures. Temperature and humidity was monitored, together with the capacity of the RDTs recommended for storage between 2 to 30 degree Celsius to detect low-density malaria parasite samples after storage under these conditions.</p> <p>Results</p> <p>Significant differences were recorded between the proportion of temperatures within the recommended RDT storage conditions in the CCBs with water and the temperatures in the storage room (p < 0.001) and maximum temperatures were lower. RDTs stored at ambient temperatures were negative when tested with parasitized blood (2,000 parasites per micro litre) at 210 days, while the field RDTs kept in CCBs with water gave positive results until 360 days.</p> <p>Discussion and Conclusions</p> <p>The CCB was an effective tool for storage of RDTs at optimal conditions, and extended the effective life-span of the tests. The concept of evaporative cooling has potential to greatly enhance access to perishable diagnostics and medicines in remote communities, as it allows prolonged storage at low cost using locally-available materials, in the absence of electricity.</p

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

Lon et al. Malaria Journal 2014, 13:96

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

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

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

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

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

Circulation and characterization of seasonal influenza viruses in Cambodia, 2012‐2015

Background: Influenza virus circulation is monitored through the Cambodian influenza‐like illness (ILI) sentinel surveillance system and isolates are characterized by the National Influenza Centre (NIC). Seasonal influenza circulation has previously been characterized by year‐round activity and a peak during the rainy season (June‐November). Objectives: We documented the circulation of seasonal influenza in Cambodia for 2012‐2015 and investigated genetic, antigenic, and antiviral resistance characteristics of influenza isolates. Patients/Methods Respiratory samples were collected from patients presenting with influenza‐like illness (ILI) at 11 hospitals throughout Cambodia. First‐line screening was conducted by the National Institute of Public Health and the Armed Forces Research Institute of Medical Sciences. Confirmation of testing and genetic, antigenic and antiviral resistance characterization was conducted by Institute Pasteur in Cambodia, the NIC. Additional virus characterization was conducted by the WHO Collaborating Centre for Reference and Research on Influenza (Melbourne, Australia). Results: Between 2012 and 2015, 1,238 influenza‐positive samples were submitted to the NIC. Influenza A(H3N2) (55.3%) was the dominant subtype, followed by influenza B (30.9%; predominantly B/Yamagata‐lineage) and A(H1N1)pdm09 (13.9%). Circulation of influenza viruses began earlier in 2014 and 2015 than previously described, coincident with the emergence of A(H3N2) clades 3C.2a and 3C.3a, respectively. There was high diversity in the antigenicity of A(H3N2) viruses, and to a smaller extent influenza B viruses, during this period, with some mismatches with the northern and southern hemisphere vaccine formulations. All isolates tested were susceptible to the influenza antiviral drugs oseltamivir and zanamivir. Conclusions: Seasonal and year‐round co‐circulation of multiple influenza types/subtypes were detected in Cambodia during 2012‐2015