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A single co-administered dose of a triple-drug regimen (ivermectin, diethylcarbamazine, and albendazole) has been shown to be safe and more efficacious for clearing Wuchereria bancrofti microfilariae than the standard two-drug regimen of diethylcarbamazine plus albendazole in clinical trials. However, the effectiveness of mass drug administration with the triple-drug regimen compared with the two-drug regimen is unknown. We compared the effectiveness of mass drug administration with the triple-drug and two-drug regimens for reducing microfilariae prevalence to less than 1% and circulating filarial antigen prevalence to less than 2%, levels that are unlikely to sustain transmission of lymphatic filariasis, in Papua New Guinea.This open-label, cluster-randomised study was done in 24 villages in a district endemic for lymphatic filariasis in Papua New Guinea. Villages paired by population size were randomly assigned to receive mass drug administration with a single dose of the triple-drug oral regimen of ivermectin (200 μg per kg of bodyweight) plus diethylcarbamazine (6 mg per kg of bodyweight) plus albendazole (400 mg) or a single dose of the two-drug oral regimen of diethylcarbamazine (6 mg per kg of bodyweight) plus albendazole (400 mg). This is a follow-on study of a previously reported safety study (ClinicalTrials.govNCT02899936). All residents aged 5 years or older and non-pregnant women were asked to participate. After cross-sectional night blood microfilariae and circulating filarial antigen surveys, mass drug administration was provided at baseline and repeated 12 months later. The primary outcomes were mean prevalence of microfilariae and circulating filarial antigen at 12 months and 24 months, assessed in all residents willing to participate at each timepoint. This study is registered with ClinicalTrials.gov, NCT03352206.Between Nov 18, 2016, and May 26, 2017, 4563 individuals were enrolled in 24 clusters; 12 clusters (2382 participants) were assigned to the triple-drug regimen and 12 clusters (2181 participants) to the two-drug regimen. Mean drug ingestion rates (of residents aged ≥5 years) were 66·1% at baseline and 63·2% at 12 months in communities assigned to the triple-drug regimen and 65·9% at baseline and 54·9% at 12 months in communities assigned to the two-drug regimen. Microfilariae prevalence in the triple-drug regimen group decreased from 105 (4·4%) of 2382 participants (95% CI 3·6-5·3) at baseline to nine (0·4%) of 2319 (0·1-0·7) at 12 months and four (0·2%) of 2086 (0·1-0·5) at 24 months. In the two-drug regimen group, microfilariae prevalence decreased from 93 (4·3%) of 2181 participants (95% CI 3·5-5·2) at baseline to 29 (1·5%) of 1963 (1·0-2·1) at 12 months and eight (0·4%) of 1844 (0·2-0·9) at 24 months (adjusted estimated risk ratio 4·5, 95% CI 1·4-13·8, p=0·0087, at 12 months; 2·9, 95% CI 1·0-8·8, p=0·058, at 24 months). The prevalence of circulating filarial antigen decreased from 523 (22·0%) of 2382 participants (95% CI 20·3-23·6) at baseline to 378 (16·3%) of 2319 (14·9-17·9) at 12 months and 156 (7·5%) of 2086 (6·4-8·7) at 24 months in the triple-drug regimen group and from 489 (22·6%) of 2168 participants (20·7-24·2) at baseline to 358 (18·2%) of 1963 (16·7-20·1) at 12 months and 184 (10·0%) of 1840 (8·7-11·5) at 24 months in the two-drug regimen group; after adjustment, differences between groups were not significant.Mass administration of the triple-drug regimen was more effective than the two-drug regimen in reducing microfilariae prevalence in communities to less than the target level of 1%, but did not reduce circulating filarial antigen prevalence to less than 2%. These results support the use of mass drug administration with the triple-drug regimen to accelerate elimination of lymphatic filariasis.BillMelinda Gates Foundation.

BackgroundCurrently, the gold standard to assess parasite developmental stages in mosquitoes is light microscopy. Microscopy can miss low-density infections, is time-consuming and not species-specific. Enzyme-linked immunosorbent assay (ELISA) has been the alternative technique to evaluate the infectivity of mosquitoes especially in field studies however it is semi-quantitative. Molecular techniques that have been used to detect the mosquito stages of malaria parasites including P. vivax. Here, we present a quantitative real-time assay (qPCR) that can be used to detect low-density P. vivax oocyst and sporozoite infections while comparing parasites extracted by the conventional DNA extraction and heating methods.MethodsColony reared Anopheles farauti mosquitoes were exposed to blood samples collected from infected individuals using a direct membrane feeding assay. The fully fed mosquitoes were kept for 7 and 14 days post-feed before dissection to confirm presence of oocysts and sporozoites. Infected mosquito guts and the salivary glands (with the head and thorax) were stored and DNA was extracted either by heating or by performing conventional column-based DNA extraction. Following DNA extraction the infected samples were subjected to qPCR to detect P. vivax parasites.ResultsDNA extraction of 1 or more oocysts by heating resulted in an overall sensitivity of 78% (57/73) and single oocysts infections were detected with a sensitivity of 82% (15/17) in the heating arm. We observed a 60% (18/30) sensitivity with sporozoites where DNA was extracted using the conventional DNA extraction method. We show that the heating method significantly improved the detection of oocysts over conventional DNA extraction. There was no significant difference in the DNA copy numbers when comparing the detection of oocysts from the conventional DNA extraction versus heating. However, we observed that the DNA copy numbers of the sporozoites detected in the heating arm was significantly higher than in the conventional DNA extraction arm.ConclusionWe have adapted a qPCR assay which, when coupled with heating to release DNA reduces sample processing time and cost. Direct qPCR after heating will be a useful tool when investigating transmission blocking vaccines or antimalarials or when evaluating field caught mosquitoes for the presence of malaria parasites.

BackgroundLong lasting insecticidal nets (LLIN) are a key vector control tool used for the prevention of malaria. Active ingredient (AI) measurements in LLIN are essential for evaluating their quality and effectiveness. The main aim of the present study was to determine the utility of X-ray fluorescence (XRF) spectroscopy as a suitable in-field tool for total AI quantification in LLINs.MethodsNew and unused LLIN samples containing deltamethrin (PermaNet® 2.0, n = 35) and alpha-cypermethrin (SafeNet®, n = 43) were obtained from batches delivered to PNG for mass distribution. Insecticides were extracted from the LLINs using a simple extraction technique and quantified using liquid chromatography mass spectrometry (LC-MS). The LC-MS results were correlated with in-field XRF spectroscopy measurements on the same nets. Operators were blinded towards the identity of the nets. Bioefficacy of the LLIN samples was tested using WHO cone bioassays and test results were correlated with total AI content.ResultsThe results indicate a close agreement between the quantitative XRF and LC-MS. Interestingly, the total AI content was negatively correlated with bioefficacy in PermaNet® 2.0 (especially, in recently manufactured nets). In contrast, AI content was positively correlated with bioefficacy in SafeNet®. These results indicate that the chemical content analysis in predelivery inspections does not always predict bioefficacy well.ConclusionXRF is a promising in-field method for quantification of both deltamethrin and alpha-cypermethrin coated LLINs. Since total AI content is not always a predictor of the efficacy of LLINs to kill mosquitoes, bioefficacy measurements should be included in predelivery inspections.

BackgroundCurrently, the gold standard to assess parasite developmental stages in mosquitoes is light microscopy. Microscopy can miss low-density infections, is time-consuming and not species-specific. This can place limitations on studies, especially when the infection status of larger mosquito populations is important and studies are done in co-endemic settings with multiple circulating parasite species. Enzyme-linked immunosorbent assay (ELISA) has been the alternative technique to evaluate the infectivity of mosquitoes especially in field studies however it is semi-quantitative. Molecular techniques that have been used to detect the mosquito stages of malaria parasites includingP. vivax. Here, we present a quantitative real-time assay (qPCR) assay that can be used to detect low-densityP. vivaxoocyst and sporozoite infections. Parasite detection via qPCR after performing the conventional DNA extraction versus direct qPCR following heating of the infected mosquito samples was compared.MethodsColony rearedAnopheles farautimosquitoes were exposed to blood samples collected from infected individuals using a direct membrane feeding assay. The fully fed mosquitoes were kept for 7 and 14 days post-feed before dissection to confirm presence of oocysts and sporozoites. Infected mosquito guts and the salivary glands (with the head and thorax) were stored and DNA was extracted either by heating or by performing conventional column-based DNA extraction. Following DNA extraction the infected samples were subjected to qPCR to detectP. vivaxparasites.ResultsDNA extraction of 1 or more oocysts by heating resulted in an overall sensitivity of 78% (57/73) and single oocysts infections were detected with a sensitivity of 82% (15/17) in the heating arm as well. We observed a 60% (18/30) sensitivity with sporozoites where DNA was extracted using the conventional DNA extraction method prior to qPCR diagnosis. We show that the heating method significantly improved the detection of oocysts over conventional DNA extraction. There was no significant difference in the DNA copy numbers when comparing the detection of oocysts from the conventional DNA extraction versus heating. There was also no significant difference in the detection rate of sporozoite samples when comparing the two DNA extraction protocols. However, we observed that the DNA copy numbers of the sporozoites detected in the heating arm was significantly higher than in the conventional DNA extraction arm.ConclusionWe have adapted a qPCR assay which, when coupled with heating to release DNA reduces sample processing time and cost. Direct qPCR after heating will be a useful tool when investigating transmission blocking vaccines or antimalarials or when evaluating field caught mosquitoes for the presence of malaria parasites.

Background Direct membrane feeding assays (DMFA) are an important tool to study parasite transmission to mosquitoes. Mosquito feeding rates in these artificial systems require optimization, as there are a number of factors that potentially influence the feeding rates and there are no standardized methods that apply to all anopheline species. Methods A range of parameters prior to and during direct membrane feeding (DMF) were evaluated for their impact on Anopheles farauti sensu stricto feeding rates, including the starving conditions and duration of starving prior to feeding, membrane type, DMF exposure time, mosquito age, feeding in the light versus the dark, blood volume, mosquito density and temperature of water bath. Results The average successful DMFA feeding rate for An. farauti s.s. colony mosquitoes increased from 50 to 85% when assay parameters were varied. Overnight starvation and Baudruche membrane yielded the highest feeding rates but rates were also affected by blood volume in the feeder and the mosquito density in the feeding cups. Availability of water during the pre-feed starvation period did not significantly impact feeding rates, nor did the exposure duration to blood in membrane feeders, the age of mosquitoes (3, 5 and 7 days post-emergence), feeding in the light versus the dark, or the temperature (34 °C, 38 °C, 42 °C and 46 °C) of the water bath. Conclusion Optimal feeding conditions in An. farauti s.s. DMFA were to offer 50 female mosquitoes in a cup (with a total surface area of ~ 340 cm2 with 1 mosquito/6.8 cm2) that were starved overnight 350–500 µL of blood (collected in heparin-coated Vacutainer tubes) per feeder in feeders with a surface area ~ 5 cm2 (with a maximum capacity of 1.5 mL of blood) via a Baudruche membrane, for at least 10–20 min. Graphical Abstract

Background: Direct membrane feeding assays (DMFA) are an important tool to study parasite transmission to mosquitoes. Mosquito feeding rates in these artificial systems require optimization as there are a number of factors that potentially influence the feeding rates and there are no standardized methods that apply to all Anopheline species.Methods: A range of parameters prior to and during direct membrane feeding (DMF) were evaluated for their impact on Anopheles farauti feeding rates; including the starving conditions and duration of starving prior to feeding, membrane type, DMF exposure time, mosquito age, illumination level, blood volume, mosquito density and temperature of water bath. Results: The average successful DMFA feeding rate for An. farauti colony mosquitoes increased from 50 % to 85 % when assay parameters were varied. Overnight starvation and Baudruche membrane yielded the highest feeding rates but rates were also affected by blood volume in the feeder and the mosquito density in the feeding cups. Availability of water during the pre-feed starvation period did not significantly impact feeding rates, nor did the exposure duration to blood in membrane feeders nor, the age of mosquitoes (3, 5 and 7 days post-emergence), illumination during feeding or the temperature (34 °C, 38 °C, 42 °C and 46 °C) of the water bath.Conclusion: Optimal feeding conditions in An. farauti DMFA were to offer 50 female mosquitoes in a cup (with a total surface area of ~340 cm2 with 1 mosquito / 6.8 cm2) that were starved overnight 350-500 µL of blood (collected in heparin coated vacutainers) per feeder in feeders with a surface area ~5 cm2 (with a maximum capacity of 1.5 mL of blood) via a Baudruche membrane, for at least 10-20 min.

Papua New Guinea (PNG) has the highest malaria transmission outside of Africa. Long-lasting insecticidal nets (LLINs) are believed to have helped to reduce average malaria prevalence in PNG from 16% in 2008 to 1% in 2014. Since 2015 malaria in PNG has resurged significantly. Here, we present observations documenting decreased bioefficacy of unused LLINs with manufacturing dates between 2013 and 2019 collected from villages and LLIN distributors in PNG. Specifically, we show that of n = 167 tested LLINs manufactured after 2013, only 17% are fulfilling the required World Health Organisation bioefficacy standards of ≥ 80% 24 h mortality or ≥ 95% 60 min knockdown in bioassays with pyrethroid susceptible Anopheles farauti mosquitoes. In contrast, all (100%, n = 25) LLINs with manufacturing dates prior to 2013 are meeting these bioefficacy standards. These results suggest that decreased bioefficacy of LLINs is contributing to the malaria resurgence in PNG and increased scrutiny of LLIN quality is warranted., Malaria prevalence in Papua New Guinea has risen in recent years after almost a decade of decline. In this study, the authors demonstrate that long-lasting insecticidal nets used in the country that were manufactured since 2013 have significantly reduced bioefficacy.

Improved methods for malaria diagnosis are urgently needed. Here, we evaluate a novel method named rotating-crystal magneto-optical detection (RMOD) in 956 suspected malaria patients in Papua New Guinea. RMOD tests can be conducted within minutes and at low cost. We systematically evaluate the capability of RMOD to detect infections by directly comparing it with expert light microscopy, rapid diagnostic tests and polymerase chain reaction on capillary blood samples. We show that compared to light microscopy, RMOD exhibits 82% sensitivity and 84% specificity to detect any malaria infection and 87% sensitivity and 88% specificity to detect Plasmodium vivax. This indicates that RMOD could be useful in P. vivax dominated elimination settings. Parasite density correlates well with the quantitative magneto-optical signal. Importantly, residual hemozoin present in malaria-negative patients is also detectable by RMOD, indicating its ability to detect previous infections. This could be exploited to reveal transmission hotspots in low-transmission settings., Here Arndt et al. establish rotating-crystal magneto-optical detection (RMOD) as a near-point-of-care diagnostic tool for malaria detection and report a sensitivity and specificity of 82% and 84%, respectively, as validated by analyzing a clinical population in a high transmission setting in Papua New Guinea.

Improved methods for malaria diagnosis are urgently needed. Here, we tested a novel rotating-crystal magneto-optical diagnostic (RMOD) test in 964 suspected malaria patients in Papua New Guinea. RMOD tests can be obtained within minutes and at low cost. Capillary blood samples were also subjected to rapid diagnostic tests, expert light microscopy and polymerase chain reaction to systematically evaluate the capability of RMOD to detect infections. Compared to light microscopy, as the gold standard, RMOD exhibited 82% sensitivity and 84% specificity to detect any malaria infection. This increased to 87% sensitivity and 88% specificity for Plasmodium vivax, indicating that RMOD could be useful in P. vivax dominated elimination settings. Parasite density correlated well with the quantitative magneto-optical signal. Importantly, residual hemozoin present in malaria negative patients was also detectable by RMOD, indicating its ability to detect previous infections, which can be exploited to reveal transmission hotspots in low-transmission settings.

Continuous malaria control in Papua New Guinea has resulted in a marked decline of Plasmodium falciparum and P. vivax prevalence. Yet, an increasing proportion of submicroscopic infections, many of them carrying gametocytes, demands for novel strategies to target residual transmission., Background The scale-up of effective malaria control in the last decade has resulted in a substantial decline in the incidence of clinical malaria in many countries. The effects on the proportions of asymptomatic and submicroscopic infections and on transmission potential are yet poorly understood. Methods In Papua New Guinea, vector control has been intensified since 2008, and improved diagnosis and treatment was introduced in 2012. Cross-sectional surveys were conducted in Madang Province in 2006 (with 1280 survey participants), 2010 (with 2117 participants), and 2014 (with 2516 participants). Infections were quantified by highly sensitive quantitative polymerase chain reaction (PCR) analysis, and gametocytes were quantified by reverse-transcription qPCR analysis. Results Plasmodium falciparum prevalence determined by qPCR decreased from 42% in 2006 to 9% in 2014. The P. vivax prevalence decreased from 42% in 2006 to 13% in 2010 but then increased to 20% in 2014. Parasite densities decreased 5-fold from 2006 to 2010; 72% of P. falciparum and 87% of P. vivax infections were submicroscopic in 2014. Gametocyte density and positivity correlated closely with parasitemia, and population gametocyte prevalence decreased 3-fold for P. falciparum and 29% for P. vivax from 2010 to 2014. Conclusions Sustained control has resulted in reduced malaria transmission potential, but an increasing proportion of gametocyte carriers are asymptomatic and submicroscopic and represent a challenge to malaria control.

Background Aedes aegypti and Ae. albopictus are important vectors of infectious diseases, especially those caused by arboviruses such as dengue, chikungunya and Zika. Aedes aegypti is very well adapted to urban environments, whereas Ae. albopictus inhabits more rural settings. Pyrethroid resistance is widespread in these vectors, but limited data exist from the Southwest Pacific Region, especially from Melanesia. While Aedes vector ecology is well documented in Australia, where incursion of Ae. albopictus and pyrethroid resistance have so far been prevented, almost nothing is known about Aedes populations in neighbouring Papua New Guinea (PNG). With pyrethroid resistance documented in parts of Indonesia but not in Australia, it is important to determine the distribution of susceptible and resistant Aedes populations in this region. Methods The present study was aimed at assessing Aedes populations for insecticide resistance in Madang and Port Moresby, located on the north and south coasts of PNG, respectively. Mosquitoes were collected using ovitraps and reared in an insectary. Standard WHO bioassays using insecticide-treated filter papers were conducted on a total of 253 Ae. aegypti and 768 Ae. albopictus adult mosquitoes. Subsets of samples from both species (55 Ae. aegypti and 48 Ae. albopictus) were screened for knockdown resistance mutations in the voltage-sensitive sodium channel (Vssc) gene, the target site of pyrethroid insecticides. Results High levels of resistance against pyrethroids were identified in Ae. aegypti from Madang and Port Moresby. Aedes albopictus exhibited susceptibility to pyrethroids, but moderate levels of resistance to DDT. Mutations associated with pyrethroid resistance were detected in all Ae. aegypti samples screened. Some genotypes found in the present study had been observed previously in Indonesia. No Vssc mutations associated with pyrethroid resistance were found in the Ae. albopictus samples. Conclusions To our knowledge, this is the first report of pyrethroid resistance in Ae. aegypti mosquitoes in PNG. Interestingly, usage of insecticides in PNG is low, apart from long-lasting insecticidal nets distributed for malaria control. Further investigations on how these resistant Ae. aegypti mosquito populations arose in PNG and how they are being sustained are warranted. Electronic supplementary material The online version of this article (10.1186/s13071-019-3585-6) contains supplementary material, which is available to authorized users.

Background. Differentiation between gametocyte-producing Plasmodium falciparum clones depends on both high levels of stage-specific transcripts and high genetic diversity of the selected genotyping marker obtained by a high-resolution typing method. By analyzing consecutive samples of one host, the contribution of each infecting clone to transmission and the dynamics of gametocyte production in multiclone infections can be studied. Methods. We have evaluated capillary electrophoresis based differentiation of 6 length-polymorphic gametocyte genes. RNA and DNA of 25 µL whole blood from 46 individuals from Burkina Faso were simultaneously genotyped. Results. Highest discrimination power was achieved by pfs230 with 18 alleles, followed by pfg377 with 15 alleles. When assays were performed in parallel on RNA and DNA, 85.7% of all pfs230 samples and 59.5% of all pfg377 samples contained at least one matching genotype in DNA and RNA. Conclusions. The imperfect detection in both, DNA and RNA, was identified as major limitation for investigating transmission dynamics, owing primarily to the volume of blood processed and the incomplete representation of all clones in the sample tested. Abundant low-density gametocyte carriers impede clone detectability, which may be improved by analyzing larger volumes and detecting initially sequestered gametocyte clones in follow-up samples

This study examined the acceptability, durability and bio-efficacy of pyrethroid-impregnated durable lining (DL) over a three-year period post-installation in residential homes across Papua New Guinea (PNG).; ZeroVector(®) ITPS had previously been installed in 40 homes across four study sites representing a cross section of malaria transmission risk and housing style. Structured questionnaires, DL visual inspections and group interviews (GIs) were completed with household heads at 12- and 36-months post-installation. Three DL samples were collected from all households in which it remained 36-months post-installation to evaluate the bio-efficacy of DL on Anopheles mosquitoes. Bio-efficacy testing followed WHO guidelines for the evaluation of indoor residual spraying.; The DL was still intact in 86 and 39% of study homes at the two time periods, respectively. In homes in which the DL was still intact, 92% of household heads considered the appearance at 12-months post installation to be the same as, or better than, that at installation compared to 59% at 36-months post-installation. GIs at both time points confirmed continuing high acceptance of DL, based in large part of the perceived attractiveness and functionality of the material. However, participants frequently asserted that they, or their family members, had ceased or reduced their use of mosquito nets as a result of the DL installation. A total of 16 houses were sampled for bio-efficacy testing across the 4 study sites at 36-months post-installation. Overall, combining all sites and samples, both knock-down at 30 min and mortality at 24 h were 100%.; The ZeroVector(®) DL installation remained highly acceptable at 36-months post-installation, the material and fixtures proved durable and the efficacy against malaria vectors did not decrease. However, the DL material had been removed from over 50% of the original study homes 3 years post-installation, largely due to deteriorating housing infrastructure. Furthermore, the presence of the DL installation appeared to reduce ITN use among many participating householders. The study findings suggest DL may not be an appropriate vector control method for large-scale use in the contemporary PNG malaria control programme.

Background This study examined the acceptability, durability and bio-efficacy of pyrethroid-impregnated durable lining (DL) over a three-year period post-installation in residential homes across Papua New Guinea (PNG). Methods ZeroVector® ITPS had previously been installed in 40 homes across four study sites representing a cross section of malaria transmission risk and housing style. Structured questionnaires, DL visual inspections and group interviews (GIs) were completed with household heads at 12- and 36-months post-installation. Three DL samples were collected from all households in which it remained 36-months post-installation to evaluate the bio-efficacy of DL on Anopheles mosquitoes. Bio-efficacy testing followed WHO guidelines for the evaluation of indoor residual spraying. Results The DL was still intact in 86 and 39% of study homes at the two time periods, respectively. In homes in which the DL was still intact, 92% of household heads considered the appearance at 12-months post installation to be the same as, or better than, that at installation compared to 59% at 36-months post-installation. GIs at both time points confirmed continuing high acceptance of DL, based in large part of the perceived attractiveness and functionality of the material. However, participants frequently asserted that they, or their family members, had ceased or reduced their use of mosquito nets as a result of the DL installation. A total of 16 houses were sampled for bio-efficacy testing across the 4 study sites at 36-months post-installation. Overall, combining all sites and samples, both knock-down at 30 min and mortality at 24 h were 100%. Conclusions The ZeroVector® DL installation remained highly acceptable at 36-months post-installation, the material and fixtures proved durable and the efficacy against malaria vectors did not decrease. However, the DL material had been removed from over 50% of the original study homes 3 years post-installation, largely due to deteriorating housing infrastructure. Furthermore, the presence of the DL installation appeared to reduce ITN use among many participating householders. The study findings suggest DL may not be an appropriate vector control method for large-scale use in the contemporary PNG malaria control programme.

Papua New Guinea (PNG) is undertaking intensified efforts to control malaria. The National Malaria Control Program aims to reduce the burden of disease by large-scale distribution of insecticide-treated bednets, improved diagnosis and implementation of new treatments. A scientific program monitoring the effect of these interventions, including molecular epidemiology studies, closely accompanies the program. Laboratory assays have been developed in (or transferred to) PNG to measure prevalence of infection and intensity of transmission as well as potential resistance to currently used drugs. These assays help to assess the impact of the National Malaria Control Program, and they reveal a much clearer picture of malaria epidemiology in PNG. In addition, analysis of the geographical clustering of parasites aids in selecting areas where intensified control will be most successful. This paper gives an overview of current research and recently completed studies in the molecular epidemiology of malaria conducted in Papua New Guinea.

Plasmodium vivax has the ability to relapse from dormant parasites in the liver weeks or months after inoculation, causing further blood-stage infection and potential onward transmission. Estimates of the force of blood-stage infections arising from primary infections and relapses are important for designing intervention strategies. However, in endemic settings their relative contributions are unclear. Infections are frequently asymptomatic, many individuals harbor multiple infections, and while high-resolution genotyping of blood samples enables individual infections to be distinguished, primary infections and relapses cannot be identified. We develop a model and fit it to longitudinal genotyping data from children in Papua New Guinea to estimate the incidence and seasonality of P vivax primary infection and relapse. The children, aged one to three years at enrolment, were followed up over 16 months with routine surveys every two months. Blood samples were taken at the routine visits and at other times if the child was ill. Samples positive by microscopy or a molecular method for species detection were genotyped using high-resolution capillary electrophoresis for P vivax MS16 and msp1F3, and P falciparum msp2. The data were summarized as longitudinal patterns of success or failure to detect a genotype at each routine time-point (eg 001000001). We assume that the seasonality of P vivax primary infection is similar to that of P falciparum since they are transmitted by the same vectors and, because P falciparum does not have the ability to relapse, the seasonality can be estimated. Relapses occurring during the study period can be a consequence of infections occurring prior to the study: we assume that the seasonal pattern of primary infections repeats over time. We incorporate information from parasitological and entomology studies to gain leverage for estimating the parameters, and take imperfect detection into account. We estimate the force of P vivax primary infections to be 11.5 (10.5, 12.3) for a three-year old child per year and the mean number of relapses per infection to be 4.3 (4.0, 4.6) over 16 months. The peak incidence of relapses occurred in the two month interval following the peak interval for primary infections: the contribution to the force of blood-stage infection from relapses is between 71% and 90% depending on the season. Our estimates contribute to knowledge of the P vivax epidemiology and have implications for the timing of intervention strategies targeting different stages of the life cycle., Author Summary Plasmodium vivax is the most widespread of the malaria species affecting humans. It has the ability for parasites to lie dormant in liver cells and then to relapse weeks or months later, causing further blood-stage infections and onward transmission. Relapses present a challenge to control and elimination programs. The contribution of relapses to the force of blood-stage infection is not well established. While genotyping can distinguish individual infections, the difficulty lies in the inability to distinguish primary infections (occurring shortly after an infectious mosquito bite) and relapses. This is a gap in the knowledge of the epidemiology of P vivax. We develop a statistical model to tease out and estimate the contributions of primary infections and relapses to the force of blood-stage infection. We use data from a cohort of children in Papua New Guinea with genotyped routine blood samples. The study area has both P vivax and P falciparum malaria: we use the seasonality of P falciparum to estimate the seasonality of P vivax primary infections. We also take into account infections occurring prior to the study period and their subsequent relapses during the study period. We find that approximately 80% of the force of blood-stage infection l is contributed by relapses and that primary infections and relapses have different seasonal patterns. The findings are important to the epidemiology of P vivax and for designing intervention strategies targeting different stages of the parasite life cycle.

BACKGROUND: Drug resistance remains a major obstacle to malaria treatment and control. It can arise and spread rapidly, and vary substantially even at sub-national level. National malaria programmes require cost-effective and timely ways of characterizing drug-resistance at multiple sites within their countries. METHODS: An improved multiplexed post-PCR ligase detection reaction-fluorescent microsphere assay (LDR-FMA) was used to simultaneously determine the presence of mutations in chloroquine resistance transporter (crt), multidrug resistance 1 (mdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes in Plasmodium falciparum (n = 727) and Plasmodium vivax (n = 574) isolates collected in 2006 from cross-sectional community population surveys in two geographically distinct regions (Madang and East Sepik) of Papua New Guinea (PNG) where strong regional differences in in vivo aminoquinoline and antifolate therapeutic efficacy had previously been observed. Data were compared to those of a follow-up survey conducted in 2010. RESULTS: Despite some very low parasite densities, the assay successfully amplified all P. falciparum and P. vivax loci in 77 and 69 % of samples, respectively. In 2006, prevalences of pfdhfr (59R-108 N) double mutation/wild type pfdhps haplotype, pfcrt SVMNT haplotype (72S-76T double mutation), and 86Y pfmdr1 mutation all exceeded 90 %. For P. vivax, 65 % carried at least two pvdhfr mutations, 97 % the 647P pvdhps mutation and 54 % the 976F pvmdr1 mutation. Prevalence of mutant haplotypes was higher in Madang than East Sepik for pfcrt SVMNT (97.4 vs 83.3 %, p = 0.001), pfdhfr (59R-108 N) (100 vs 90.6 %, p = 0.001), pvdhfr haplotypes (75.8 vs 47.6 %, p = 0.001) and pvmdr1 976F (71.2 vs 26.2 %, p < 0.001). Data from a subsequent Madang survey in 2010 showed that the prevalence of pfdhps mutations increased significantly from 30 % (p < 0.001) as did the prevalence of pvdhfr mutant haplotypes (from 75.8 to 97.4 %, p = 0.012). CONCLUSIONS: This LDR-FMA multiplex platform shows feasibility for low-cost, high-throughput, rapid characterization of a broad range of drug-resistance markers in low parasitaemia infections. Significant geographical differences in mutation prevalence correlate with previous genotyping surveys and in vivo trials and may reflect variable drug pressure and differences in health-care access in these two PNG populations.

INTRODUCTION: The importance of Plasmodium vivax in malaria elimination is increasingly being recognized yet little is known about its population size and population genetic structure in the South Pacific an area that is the focus of intensified malaria control. METHODS: We have genotyped 13 microsatellite markers in 295 P. vivax isolates from four geographically distinct sites in Papua New Guinea (PNG) and one site from Solomon Islands representing different transmission intensities. RESULTS: Diversity was very high with expected heterozygosity values ranging from 0.62 to 0.98 for the different markers. Effective population size was high (12'872 to 19'533 per site). In PNG population structuring was limited with moderate levels of genetic differentiation. F ST values (adjusted for high diversity of markers) were 0.14 0.15. Slightly higher levels were observed between PNG populations and Solomon Islands (F ST?=?0.16). CONCLUSIONS: Low levels of population structure despite geographical barriers to transmission are in sharp contrast to results from regions of low P. vivax endemicity. Prior to intensification of malaria control programs in the study area parasite diversity and effective population size remained high.

Background Discrimination of gametocyte-producing P. falciparum clones depends on high expression of one or more polymorphic stage-specific markers and on the genetic diversity of these markers in the study area. Pfs230 and pfg377 are classical length-polymorphic markers for differentiation of gametocytes. Because of variable PCR fragment sizes, these markers are particularly well suited to distinguish gametocytes of multiple P. falciparum clones within a patient. We aimed at improving the resolution of both markers by creating amplicons spanning several polymorphic domains of these genes and by increasing the sizing accuracy by capillary electrophoresis using an automated sequencer. Material and methods We assessed the genetic diversity and the multiplicity of pfs230 and pfg377 in 80 DNA samples from Papua New Guinea by nested-PCR and following sizing by capillary electrophoresis. We also investigated novel size-polymorphic gametocyte markers, such as PF11.1 (PF10_0374), PF11_0214, PFI0205w and PFL0105w ,a s well as SNPbased genotyping approaches. Results We observed high diversity with pfs230 (He=96.3) and pfg377 (He=89.4). 17 and 13 different alleles were found for pfs230 and pfg377, respectively. The multiplicity of infection (MOI) of pfs230 and pfg377 was compared with the asexual MOI by marker msp2 for each sample. Discussion Gametocyte typing of field samples requires RNA sampling and high gametocyte-specific expression of the genotyping marker. A gametocyte trendline was used to evaluate the detection limit of the nested-RT-PCR of pfs230 and pfg377 in comparison to the standard marker for gametocyte detection, pfs25. We discuss the application of high-resolution gametocyte genotyping for studies on malaria transmission.

Background Reports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates. Methods Pvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples. Results Genotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele. Conclusions The LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.