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Abstract Background Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013–2019) was characterized. Methods Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13—a molecular marker of artemisinin resistance. Result The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p

Abstract Background Malaria remains a major public health threat and tools sensitive to detect infections in low malaria transmission areas are needed to progress elimination efforts. Pregnant women are particularly vulnerable to malaria infections. Throughout pregnancy they access routine antenatal care, presenting a unique sentinel population to apply novel sero-surveillance tools to measure malaria transmission. The aim of this study was to quantify the dynamic antibody responses to multiple antigens during pregnancy so as to identify a single or multiple antibody response of exposure to malaria in pregnancy. Methods This study involved a secondary analysis of antibody responses to six parasite antigens [five commonly studied merozoite antigens and the variant surface antigen 2-chondroitin sulphate A (VAR2CSA), a pregnancy-specific erythrocytic antigen] measured by enzyme-linked immunosorbent assay (ELISA) over the gestation period until delivery (median of 7 measurements/woman) in 250 pregnant women who attended antenatal clinics located at the Thai-Myanmar border. A multivariate mixture linear mixed model was used to cluster the pregnant women into groups that have similar longitudinal antibody responses to all six antigens over the gestational period using a Bayesian approach. The variable-specific entropy was calculated to identify the antibody responses that have the highest influence on the classification of the women into clusters, and subsequent agreement with grouping of women based on exposure to malaria during pregnancy. Results Of the 250 pregnant women, 135 had a Plasmodium infection detected by light microscopy during pregnancy (39% Plasmodium falciparum only, 33% Plasmodium vivax only and 28% mixed/other species), defined as cases. The antibody responses to all six antigens accurately identified the women who did not have a malaria infection detected during pregnancy (93%, 107/115 controls). Antibody responses to P. falciparum merozoite surface protein 3 (PfMSP3) and P. vivax apical membrane antigen 1 (PvAMA1) were the least dynamic. Antibody responses to the antigens P. falciparum apical membrane antigen 1 (PfAMA1) and PfVAR2CSA were able to identify the majority of the cases more accurately (63%, 85/135). Conclusion These findings suggest that the combination of antibodies, PfAMA1 and PfVAR2CSA, may be useful for sero-surveillance of malaria infections in pregnant women, particularly in low malaria transmission settings. Further investigation of other antibody markers is warranted considering these antibodies combined only detected 63% of the malaria infections during pregnancy.

Abstract Background Plasmodium malariae is a widely spread but neglected human malaria parasite, which causes chronic infections. Studies on genetic polymorphisms of anti-malarial drug target genes in P. malariae are limited. Previous reports have shown polymorphisms in the P. malariae dihydrofolate reductase gene associated with pyrimethamine resistance and linked to pyrimethamine drug pressure. This study investigated polymorphisms of the P. malariae homologous genes, chloroquine resistant transporter and multidrug resistant 1, associated with chloroquine and mefloquine resistance in Plasmodium falciparum. Methods The orthologous P. malariae crt and mdr1 genes were studied in 95 patients with P. malariae infection between 2002 and 2016 from Thailand (N = 51) and Myanmar (N = 44). Gene sequences were analysed using BioEdit, MEGA7, and DnaSP programs. Mutations and gene amplifications were compared with P. falciparum and Plasmodium vivax orthologous genes. Protein topology models derived from the observed pmcrt and pmmdr1 haplotypes were constructed and analysed using Phyre2, SWISS MODEL and Discovery Studio Visualization V 17.2. Results Two non-synonymous mutations were observed in exon 2 (H53P, 40%) and exon 8 (E278D, 44%) of pmcrt. The topology model indicated that H53P and E278D were located outside of the transmembrane domain and were unlikely to affect protein function. Pmmdr1 was more diverse than pmcrt, with 10 non-synonymous and 3 synonymous mutations observed. Non-synonymous mutations were located in the parasite cytoplasmic site, transmembrane 11 and nucleotide binding domains 1 and 2. Polymorphisms conferring amino acid changes in the transmembrane and nucleotide binding domains were predicted to have some effect on PmMDR1 conformation, but were unlikely to affect protein function. All P. malariae parasites in this study contained a single copy of the mdr1 gene. Conclusions The observed polymorphisms in pmcrt and pmmdr1 genes are unlikely to affect protein function and unlikely related to chloroquine drug pressure. Similarly, the absence of pmmdr1 copy number variation suggests limited mefloquine drug pressure on the P. malariae parasite population, despite its long time use in Thailand for the treatment of falciparum malaria.

Abstract Background Rapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated. Methods Positive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January 2016. The RDT samples were stored for 6 months, 9 months, 20 months, 21 months, and 32 months before DNA extraction and subsequent molecular analysis of P. falciparum kelch 13 (pfkelch13) mutations, P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum plasmepsin 2 (pfplasmepsin2) gene amplification. In addition, performance of four whole genome amplification (WGA) kits were compared, including REPLI-g®, MALBACTM, PicoPLEX®, and GenomePlex®, for which DNA quantity and quality were compared between original DNA and post-WGA products. Results The proportion of successful amplification of the different molecular markers was similar between blood spots analysed from RDTs stored for 6, 9, 20, 21, or 32 months. Successful amplification was dependent on the molecular markers fragment length (p value

Abstract Background Mutations in Pfkelch13 and Pfplasmepsin2/3 gene amplification are well-established markers for artemisinin and piperaquine resistance in Plasmodium falciparum, a widespread problem in the Greater Mekong Subregion (GMS). The Plasmodium vivax parasite population has experienced varying drug pressure dependent on local drug policies. We investigated the correlation between drug pressure from artemisinins and piperaquine and mutations in the P. vivax orthologous genes Pvkelch12 and Pvplasmepsin4 (Pvpm4), as candidate resistance markers. Methods Blood samples from 734 P. vivax patients were obtained from Thailand (n = 399), Lao PDR (n = 296) and Cambodia (n = 39) between 2007 and 2017. Pvkelch12 and Pvpm4 was amplified and sequenced to assess gene mutations. To assess PvPM4 gene amplification, a Taqman® Real-Time PCR method was developed and validated. Selection of non-synonymous mutations was assessed by its ratio with synonymous mutations (Ka/Ks ratios). Mutation rates were compared to the estimated local drug pressure. Results Polymorphisms in Pvkelch12 were rare. Pvkelch12 mutations V552I, K151Q and M124I were observed in 1.0% (7/734) of P. vivax samples. V552I was the most common mutation with a frequency of 0.7% (5/734), most of which (4/5) observed in Ubon Ratchathani, Thailand. Polymorphisms in Pvpm4 were more common, with a frequency of 40.3% (123/305) in 305 samples from Thailand, Lao PDR and Cambodia, but this was not related to the estimated piperaquine drug pressure in these areas (Pearson’s χ2 test, p = 0.50). Pvpm4 mutation V165I was most frequent in Tak, Thailand (40.2%, 43/107) followed by Pailin, Cambodia (43.5%, 37/85), Champasak, Lao PDR (40.4%, 23/57) and Ubon Ratchathani, Thailand (35.7%, 20/56). Pvpm4 amplification was not observed in 141 samples from Thailand and Cambodia. For both Pvkelch12 and Pvpm4, in all areas and at all time points, the Ka/Ks values were

Abstract Background Plasmodium vivax malaria elimination can only be achieved by the deployment of 8-aminoquinolines (primaquine and tafenoquine) in combination with ACT to kill both blood and liver-stage parasites. However, primaquine and the other 8-aminoquinolines cause dose-dependent haemolysis in subjects with G6PD deficiency, an X-linked disorder of red blood cells that is very common in populations living in tropical and subtropical areas. In order to inform safer use of 8-aminoquinolines in the Greater Mekong Subregion, a multi-centre study was carried out to assess the prevalence of G6PD deficiency and to identify the main G6PD variants in samples collected in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. Methods Blood samples were collected in the five countries during National Malaria Surveys or during Population Surveys. During Population Surveys samples were characterized for G6PD phenotype using the Fluorescent Spot Test. Samples were then genotyped for a panel of G6PD mutations. Results G6PD deficiency was found to be common in the region with an overall mean prevalence of deficient or mutated hemizygous males of 14.0%, ranging from a mean 7.3% in Thailand, 8.1% in Lao PDR, 8.9% in Vietnam, 15.8% in Myanmar and 18.8% in Cambodia. Mahidol and Viangchan mutations were the most common and widespread variants found among the nine investigated. Conclusions Owing to the high prevalence of G6PD deficiency in the Greater Mekong Subregion, strategies for vivax malaria elimination should include point-of-care G6PD testing (both qualitative and quantitative) to allow safe and wide treatment with 8-aminoquinolines.

Abstract Background Plasmodium vivax malaria is characterized by relapses arising from the hypnozoite stages in the liver. The only currently registered drug for radical treatment to prevent relapse is primaquine. Primaquine, a prodrug, requires metabolism through the liver cytochrome CYP2D6 isoenzyme to its active metabolite. Mutations in the CYP2D6 gene may thus affect primaquine efficacy. A SNPs genotyping technique was developed to characterize the CYP2D6 genetic variants and tested this in the patients with Plasmodium vivax infection collected in a Karen population on the Thailand–Myanmar border, where P. vivax malaria is endemic. Methods Direct sequencing of PCR-reamplified products (DSP) was used to uncover exonic CYP2D6 sequence variations. Subsequently, an allele-specific oligonucleotide probe real-time SNPs genotyping (ASO) assay was developed for rapid detection of the four clinically relevant CYP2D6 variants occurring in this population. These two in-house developed assays were used to genotype CYP2D6 mutations in blood samples obtained from 70 Karen adults. Results Results showed a high degree of concordance between the DSP and ASO methods. Six CYP2D6 point mutations were identified within the Karen population: C100T, C1039T, G1661C, G1846A, C2850T and G4180C, at frequencies of 0.43, 0.43, 0.76, 0.02, 0.32 and 0.76, respectively. The CYP2D6*2, *4, *5, *10 and *36 allelic frequencies were 0.33, 0.02, 0.03, 0.40 and 0.01, respectively. Alleles conferring an intermediate CYP2D6 metabolizer phenotype comprised 46% of the total number of alleles. Conclusion The newly developed ASO assay is a reliable and rapid tool for large-scale CYP2D6 genotyping. The high frequency of the CYP2D6*10 allele in the Karen population warrants further assessment of its association with the radical curative efficacy of primaquine.

Abstract Background The detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda. Results The HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z′-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4–100) diagnostic sensitivity and 97.9% (95% CI 94.8–99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4–100) and 88.9% (95% CI 70.8–97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96–100%) and 97.3% (CI 95% 94–99%). Conclusions The HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.

Abstract Background Plasmodium malariae is characterized by its long asymptomatic persistence in the human host. The epidemiology of P. malariae is incompletely understood and is hampered by the limited knowledge of genetic polymorphisms. Previous reports from Africa have shown heterogeneity within the P. malariae circumsporozoite protein (pmcsp) gene. However, comparative studies from Asian countries are lacking. Here, the genetic polymorphisms in pmcsp of Asian isolates have been characterized. Methods Blood samples from 89 symptomatic P. malariae-infected patients were collected, from Thailand (n = 43), Myanmar (n = 40), Lao PDR (n = 5), and Bangladesh (n = 1). pmcsp was amplified using semi-nested PCR before sequencing. The resulting 89 pmcsp sequences were analysed together with 58 previously published pmcsp sequences representing African countries using BioEdit, MEGA6, and DnaSP. Results Polymorphisms identified in pmcsp were grouped into 3 populations: Thailand, Myanmar, and Kenya. The nucleotide diversity and the ratio of nonsynonymous to synonymous substitutions (dN/dS) in Thailand and Myanmar were higher compared with that in Kenya. Phylogenetic analysis showed clustering of pmcsp sequences according to the origin of isolates (Asia vs. Africa). High genetic differentiation (Fst = 0.404) was observed between P. malariae isolates from Asian and African countries. Sequence analysis of pmcsp showed the presence of tetrapeptide repeat units of NAAG, NDAG, and NAPG in the central repeat region of the gene. Plasmodium malariae isolates from Asian countries carried fewer copies of NAAG compared with that from African countries. The NAPG repeat was only observed in Asian isolates. Additional analysis of 2 T-cell epitopes, Th2R and Th3R, showed limited heterogeneity in P. malariae populations. Conclusions This study provides valuable information on the genetic polymorphisms in pmcsp isolates from Asia and advances our understanding of P. malariae population in Asia and Africa. Polymorphisms in the central repeat region of pmcsp showed association with the geographical origin of P. malariae isolates and can be potentially used as a marker for genetic epidemiology of P. malariae population.

Abstract Background Genetic diversity of the three important antigenic proteins, namely thrombospondin-related anonymous protein (TRAP), apical membrane antigen 1 (AMA1), and 6-cysteine protein (P48/45), all of which are found in various developmental stages of Plasmodium parasites is crucial for targeted vaccine development. While studies related to the genetic diversity of these proteins are available for Plasmodium falciparum and Plasmodium vivax, barely enough information exists regarding Plasmodium malariae. The present study aims to demonstrate the genetic variations existing among these three genes in P. malariae by analysing their diversity at nucleotide and protein levels. Methods Three surface protein genes were isolated from 45 samples collected in Thailand (N = 33), Myanmar (N = 8), and Lao PDR (N = 4), using conventional polymerase chain reaction (PCR) assay. Then, the PCR products were sequenced and analysed using BioEdit, MEGA6, and DnaSP programs. Results The average pairwise nucleotide diversities (π) of P. malariae trap, ama1, and p48/45 were 0.00169, 0.00413, and 0.00029, respectively. The haplotype diversities (Hd) of P. malariae trap, ama1, and p48/45 were 0.919, 0.946, and 0.130, respectively. Most of the nucleotide substitutions were non-synonymous, which indicated that the genetic variations of these genes were maintained by positive diversifying selection, thus, suggesting their role as a potential target of protective immune response. Amino acid substitutions of P. malariae TRAP, AMA1, and P48/45 could be categorized to 17, 20, and 2 unique amino-acid variants, respectively. For further vaccine development, carboxyl terminal of P48/45 would be a good candidate according to conserved amino acid at low genetic diversity (π = 0.2–0.3). Conclusions High mutational diversity was observed in P. malariae trap and ama1 as compared to p48/45 in P. malariae samples isolated from Thailand, Myanmar, and Lao PDR. Taken together, these results suggest that P48/45 might be a good vaccine candidate against P. malariae infection because of its sufficiently low genetic diversity and highly conserved amino acids especially on the carboxyl end.

Abstract Background Malaria has declined dramatically along the Thai–Myanmar border in recent years due to malaria control and elimination programmes. However, at the same time, artemisinin resistance has spread, raising concerns about the efficacy of parenteral artesunate for the treatment of severe malaria. Case presentation In November 2015 and April 2017, two patients were treated for severe malaria with parenteral artesunate. Quinine was added within 24 h due to an initial poor response to treatment. The first patient died within 24 h of starting treatment and the second did not clear his peripheral parasitaemia until 11 days later. Genotyping revealed artemisinin resistance Kelch-13 markers. Conclusions Reliable efficacy of artesunate for the treatment of severe malaria may no longer be assured in areas where artemisinin resistance has emerged. Empirical addition of parenteral quinine to artesunate for treatment is recommended as a precautionary measure.

Abstract Background Artemisinin resistance, linked to polymorphisms in the Kelch gene on chromosome 13 of Plasmodium falciparum (k13), has outpaced containment efforts in South East Asia. For national malaria control programmes in the region, it is important to establish a surveillance system which includes monitoring for k13 polymorphisms associated with the clinical phenotype. Methods Between February and December 2013, parasite clearance was assessed in 35 patients with uncomplicated P. falciparum treated with artesunate monotherapy followed by 3-day ACT in an isolated area on the Myanmar–Thai border with relatively low artemisinin drug pressure. Molecular testing for k13 mutations was performed on dry blood spots collected on admission. Results The proportion of k13 mutations in these patients was 41.7%, and only 5 alleles were detected: C580Y, I205T, M476I, R561H, and F446I. Of these, F446I was the most common, and was associated with a longer parasite clearance half-life (median) 4.1 (min–max 2.3–6.7) hours compared to 2.5 (min–max 1.6–8.7) in wildtype (p = 0·01). The prevalence of k13 mutant parasites was much lower than the proportion of k13 mutants detected 200 km south in a much less remote setting where the prevalence of k13 mutants was 84% with 15 distinct alleles in 2013 of which C580Y predominated. Conclusions This study provides evidence of artemisinin resistance in a remote part of eastern Myanmar. The prevalence of k13 mutations as well as allele diversity varies considerably across short distances, presumably because of historical patterns of artemisinin use and population movements.

Abstract Background Singapore has been certified malaria-free by the World Health Organization since November 1982. However, sporadic autochthonous malaria outbreaks do occur. In one of the most recent outbreaks of vivax malaria, an entomological investigation identified Anopheles sinensis as the most probable vector. As metaphase karyotype studies divided An. sinensis into two forms, A and B, with different vector competence: the investigation of vector competence of An. sinensis found in Singapore was thus pursued using Plasmodium vivax field isolates from the Thailand–Myanmar border. Methods Adults and larvae An. sinensis were collected from Singapore from 14 different locations, using various trapping and collection methods between September 2013 and January 2016. Molecular identification of An. sinensis species were conducted by amplifying the ITS2 and CO1 region using PCR. Experimental infections of An. sinensis using blood from seven patients infected with P. vivax from the Thailand–Myanmar border were conducted with Anopheles cracens (An. dirus B) as control. Results Phylogenetic analysis showed that An. sinensis (F22, F2 and collected from outbreak areas) found in Singapore was entirely Form A, and closely related to An. sinensis Form A from Thailand. Artificial infection of these Singapore strain An. sinensis Form A resulted in the development of oocysts in four experiments, with the number of sporozoites produced by one An. sinensis ranging from 4301 to 14,538. Conclusions Infection experiments showed that An. sinensis Form A from Singapore was susceptible to Thai–Myanmar P. vivax strain, suggesting a potential role as a malaria vector in Singapore.

Abstract Background Polyclonal blood-stage infections of Plasmodium vivax are frequent even in low transmission settings, allowing meiotic recombination between heterologous parasites. Empirical data on meiotic products are however lacking. This study examined microsatellites in oocysts derived by membrane feeding of mosquitoes from blood-stage P. vivax infections at the Thai–Myanmar border. Methods Blood samples from patients presenting with vivax malaria were fed to Anopheles cracens by membrane feeding and individual oocysts from midguts were obtained by dissection after 7 days. DNA was extracted from oocysts and parental blood samples and tested by microsatellite analysis. Results A focused study of eight microsatellite markers was undertaken for nine blood stage infections from 2013, for which derived oocysts were studied in six cases. One or more alleles were successfully amplified for 131 oocysts, revealing high levels of allelic diversity in both blood and oocyst stages. Based on standard criteria for defining minor alleles, there was evidence of clear deviation from random mating (inbreeding) with relatively few heterozygous oocysts compared to variance across the entire oocyst population (FIT = 0.89). The main explanation appeared to be natural compartmentalisation at mosquito (FSC = 0.27) and human stages (FCT = 0.68). One single human case produced a total of 431 successfully amplified loci (across 70 oocysts) that were homozygous and identical to parental alleles at all markers, indicating clonal infection and transmission. Heterozygous oocyst alleles were found at 15/176 (8.5%) successfully amplified loci in the other five cases. There was apparently reduced oocyst heterozygosity in individual oocysts compared to diversity within individual mosquitoes (FIS = 0.55), but this may simply reflect the difficulty of detecting minor alleles in oocysts, given the high rate of amplification failure. Inclusion of minor allele peaks (irrespective of height) when matching peaks were found in related blood or oocyst samples, added 11 minor alleles for 9 oocysts, increasing the number of heterozygous loci to 26/176 (14.8%; p = 0.096). Conclusion There was an apparently low level of heterozygous oocysts but this can be explained by a combination of factors: relatively low complexity of parental infection, natural compartmentalisation in humans and mosquitoes, and the methodological challenge of detecting minor alleles.

Abstract Background A targeted malaria elimination project, including mass drug administrations (MDA) of dihydroartemisinin/piperaquine plus a single low dose primaquine is underway in villages along the Thailand Myanmar border. The intervention has multiple components but the success of the project will depend on the participation of the entire communities. Quantitative surveys were conducted to study reasons for participation or non-participation in the campaign with the aim to identify factors associated with the acceptance and participation in the mass drug administrations. Methods The household heads in four study villages in which MDAs had taken place previously were interviewed between January 2014 and July 2015. Results 174/378 respondents (46 %) completed three rounds of three drug doses each, 313/378 (83 %) took at least three consecutive doses and 56/378 (15 %) did not participate at all in the MDA. The respondents from the two villages (KNH and TPN) were much more likely to participate in the MDA than respondents from the other two villages (HKT and TOT). The more compliant villages KNH and TPN had both an appearance of cohesive communities with similar demographic and ethnic backgrounds. By contrast the villages with low participation were unique. One village was fragmented following years of armed conflict and many respondents gave little inclination to cooperate with outsiders. The other village with low MDA coverage was characterised by a high percentage of short-term residents with little interest in community interventions. A universal reason for non-participation in the MDA applicable to all villages was an inadequate understanding of the intervention. Conclusions It is unlikely that community engagement can unite fragmented communities in participating in an intervention, which benefits the community. Understanding the purpose and the reasons underlying the intervention is an important pre-condition for participation. In the absence of direct benefits and a complete understanding of the indirect benefits trust in the investigators is critical for participation.

Abstract Microscopy performed on stained films of peripheral blood for detection, identification and quantification of malaria parasites is an essential reference standard for clinical trials of drugs, vaccines and diagnostic tests for malaria. The value of data from such research is greatly enhanced if this reference standard is consistent across time and geography. Adherence to common standards and practices is a prerequisite to achieve this. The rationale for proposed research standards and procedures for the preparation, staining and microscopic examination of blood films for malaria parasites is presented here with the aim of improving the consistency and reliability of malaria microscopy performed in such studies. These standards constitute the core of a quality management system for clinical research studies employing microscopy as a reference standard. They can be used as the basis for the design of training and proficiency testing programmes as well as for procedures and quality assurance of malaria microscopy in clinical research.

Abstract Background The Sustainable Development Goals (SDG) call for increased gender equity and reduction in malaria-related mortality and morbidity. Plasmodium vivax infections in pregnancy are associated with maternal anaemia and increased adverse perinatal outcomes. Providing radical cure for women with 8-aminoquinolines (e.g., primaquine) is hindered by gender-specific complexities. Case presentation A symptomatic episode of vivax malaria at 18 weeks of gestation in a primigravid woman was associated with maternal anaemia, a recurrent asymptomatic P. vivax episode, severe intra-uterine growth restriction with no other identifiable cause and induction to reduce the risk of stillbirth. At 5 months postpartum a qualitative glucose-6-phosphate dehydrogenase (G6PD) point-of-care test was normal and radical cure with primaquine was prescribed to the mother. A 33% fractional decrease in haematocrit on day 7 of primaquine led to further testing which showed intermediate phenotypic G6PD activity; the G6PD genotype could not be identified. Her infant daughter was well throughout maternal treatment and found to be heterozygous for Mahidol variant. Conclusion Adverse effects of vivax malaria in pregnancy, ineligibility of radical cure for pregnant and postpartum women, and difficulties in diagnosing intermediate levels of G6PD activity multiplied morbidity in this woman. Steps towards meeting the SDG include prevention of malaria in pregnancy, reducing unnecessary exclusion of women from radical cure, and accessible quantitative G6PD screening in P. vivax-endemic settings.

Abstract Background Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. Methods A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l’Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). Results By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. Conclusion The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.

Abstract Oxidative agents can cause acute haemolytic anaemia in persons with G6PD deficiency. Understanding the relationship between G6PD genotype and the phenotypic expression of the enzyme deficiency is necessary so that severe haemolysis can be avoided. The patterns of oxidative haemolysis have been well described in G6PD deficient hemizygous males and homozygous females; and haemolysis in the proportionally more numerous heterozygous females has been documented mainly following consumption of fava beans and more recently dapsone. It has long been known that 8-aminoquinolines, notably primaquine and tafenoquine, cause acute haemolysis in G6PD deficiency. To support wider use of primaquine in Plasmodium vivax elimination, more data are needed on the haemolytic consequences of 8-aminoquinolines in G6PD heterozygous females. Two recent studies (in 2017) have provided precisely such data; and the need has emerged for the development of point of care quantitative testing of G6PD activity. Another priority is exploring alternative 8-aminoquinoline dosing regimens that are practical and improve safety in G6PD deficient individuals.

Abstract Background Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated. Results Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity. Conclusions The MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Malaria elimination will be possible only with serious attempts to address asymptomatic infection and chronic infection by both Plasmodium falciparum and Plasmodium vivax. Currently available drugs that can completely clear a human of P. vivax (known as "radical cure"), and that can reduce transmission of malaria parasites, are those in the 8- aminoquinoline drug family, such as primaquine. Unfortunately, people with glucose-6- phosphate dehydrogenase (G6PD) deficiency risk having severe adverse reactions if exposed to these drugs at certain doses. G6PD deficiency is the most common human enzyme defect, affecting approximately 400 million people worldwide Scaling up radical cure regimens will require testing for G6PD deficiency, in at two levels: 1) individual level to ensure safe case management, and 2) population level to understand the risk in the local population to guide Plasmodium vivax treatment policy. Several technical and operational knowledge gaps must be addressed to expand access to G6PD deficiency testing and to ensure that a patient's G6PD status is known before deciding to administer an 8-aminoquinoline-based drug. In this report from a stakeholder meeting held in Thailand on October 4 and 5, 2012, G6PD testing in support of radical cure is discussed in detail. The focus is on challenges to the development and evaluation of G6PD diagnostic tests, and on challenges related to the operational aspects of implementing G6PD testing in support of radical cure. The report also describes recommendations for evaluation of diagnostic tests for G6PD deficiency in support of radical cure. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax infections in pregnancy are associated with low birth weight and anaemia. This parasites species is also characterised by relapses, erythrocytic infections initiated by the activation of the dormant liver stages, the hypnozoites, to mature. Genotyping of P. vivax using microsatellite markers has opened the way to comparative investigations of parasite populations. The aim of the study was to assess whether there were any differences between the parasites found in pregnant and non-pregnant patients, and/or between the admission infections and recurrent episodes during follow-up. Methods: Blood samples were collected from 18 pregnant and 18 non-pregnant patients, who had at least two recurrent episodes during follow-up, that were recruited in two previous trials on the efficacy of chloroquine treatment of P. vivax infections on the Thai-Myanmar border. DNA was purified and the P. vivax populations genotyped with respect to eight polymorphic microsatellite markers. Analyses of the genetic diversity, multiplicity of infection (MOI), and a comparison of the genotypes in the samples from each patient were conducted. Results: The P. vivax parasites present in the samples exhibited high genetic diversity (6 to 15 distinct allelic variants found for the 8 loci). Similar expected heterozygosity (He) values were obtained for isolates from pregnant (0.837) and non-pregnant patients (0.852). There were modest differences between the MOI values calculated for both admission and recurrence samples from the pregnant patients (2.00 and 2.05, respectively) and the equivalent samples from the non-pregnant patients (1.67 and 1.64, respectively). Furthermore, the mean number of distinct alleles enumerated in the admission samples from the pregnant (6.88) and non-pregnant (7.63) patients were significantly lower than that found in the corresponding recurrent episodes samples (9.25 and 9.63, respectively). Conclusions: The P. vivax populations circulating in inhabitants along the Thai-Myanmar border, an area of low malaria transmission, displayed high genetic diversity. A subtle increase in the multiplicity of P. vivax infections in pregnant patients suggests a higher susceptibility to infection. The higher allelic diversity in the relapse as compared to the admission samples in both patient groups is consistent with the hypothesis that a febrile episode promotes the activation of hypnozoites. [ABSTRACT FROM AUTHOR]

Abstract Background Considering the uncertainty of safety of anti-malarial drugs in pregnancy, efficacy studies are one of the few sources of clinical safety data. Complete safety evaluation is not usually incorporated in efficacy studies due to financial and human resource constraints. This review reports the methods used for the assessment of safety of artemisinin-based and quinine-based treatments in efficacy studies in pregnancy. Methods Methodology of assessment and reporting of safety in efficacy studies of artemisinin-based and quinine-based treatment in pregnancy was reviewed using seven databases and two clinical trial registries. The protocol was registered to PROSPERO (CRD42017054808). Results Of 48 eligible efficacy studies the method of estimation of gestational age was reported in only 32 studies (67%, 32/48) and ultrasound was used in 18 studies (38%, 18/48). Seventeen studies (35%, 17/48) reported parity, 9 (19%, 9/48) reported gravidity and 13 (27%, 13/48) reported both. Thirty-eight studies (79%, 38/48) followed participants through to pregnancy outcome. Fetal loss was assessed in 34 studies (89%, 34/38), but the definition of miscarriage and stillbirth were defined only in 11 (32%, 11/34) and 7 (21%, 7/34) studies, respectively. Preterm birth was assessed in 26 studies (68%, 26/38) but was defined in 16 studies (62%, 16/26). Newborn weight was assessed in 30 studies (79%, 30/38) and length in 10 studies (26%, 10/38). Assessment of birth weight took gestational age into account in four studies (13%, 4/30). Congenital abnormalities were reported in 32 studies (84%, 32/38). Other common risk factors for adverse pregnancy outcomes were not well-reported. Conclusion Incomplete reporting and varied methodological assessment of pregnancy outcomes in anti-malarial drug efficacy studies limits comparison across studies. A standard list of minimal necessary parameters to assess and report the safety component of efficacy studies of anti-malarials in pregnancy is proposed.

Abstract Background There is no agreed standard method to assess the efficacy of anti-malarials for uncomplicated falciparum in pregnancy despite an increased risk of adverse outcomes for the mother and the fetus. The aim of this review is to present the currently available evidence from both observational and interventional cohort studies on anti-malarial efficacy in pregnancy and summarize the variability of assessment and reporting found in the review process. Methods Efficacy methodology and assessment of artemisinin-based treatments (ABT) and quinine-based treatments (QBT) were reviewed systematically using seven databases and two clinical trial registries (protocol registration—PROSPERO: CRD42017054808). Pregnant women in all trimesters with parasitologically confirmed uncomplicated falciparum malaria were included irrespective of symptoms. This review attempted to re-calculate proportions of treatment success applying the same definition as the standard WHO methodology for non-pregnant populations. Aggregated data meta-analyses using data from randomized control trials (RCTs) comparing different treatments were performed by random effects model. Results A total of 48 eligible efficacy studies were identified including 7279 treated Plasmodium falciparum episodes. While polymerase chain reaction (PCR) was used in 24 studies for differentiating recurrence, the assessment and reporting of treatment efficacy was heterogeneous. When the same definition could be applied, PCR-corrected treatment failure of ≥ 10% at any time points was observed in 3/30 ABT and 3/7 QBT arms. Ten RCTs compared different combinations of ABT but there was a maximum of two published RCTs with PCR-corrected outcomes for each comparison. Five RCTs compared ABT and QBT. Overall, the risk of treatment failure was significantly lower in ABT than in QBT (risk ratio 0.22, 95% confidence interval 0.07–0.63), although the actual drug combinations and outcome endpoints were different. First trimester women were included in 12 studies none of which were RCTs of ABT. Conclusions Efficacy studies in pregnancy are not only limited in number but use varied methodological assessments. In five RCTs with comparable methodology, ABT resulted in higher efficacy than QBT in the second and third trimester of pregnancy. Individual patient data meta-analysis can include data from observational cohort studies and could overcome some of the limitations of the current assessment given the paucity of data in this vulnerable group.