BACKGROUND: Detection of malaria parasitaemia in samples that are negative by rapid diagnostic tests (RDTs) requires resource-intensive molecular tools. While pooled testing using a two-step strategy provides a cost-saving alternative to the gold standard of individual sample testing, statistical adjustments are needed to improve accuracy of prevalence estimates for a single step pooled testing strategy. METHODS: A random sample of 4670 malaria RDT negative dried blood spot samples were selected from a mass testing and treatment trial in Asembo, Gem, and Karemo, western Kenya. Samples were tested for malaria individually and in pools of five, 934 pools, by one-step quantitative polymerase chain reaction (qPCR). Maximum likelihood approaches were used to estimate subpatent parasitaemia (RDT-negative, qPCR-positive) prevalence by pooling, assuming poolwise sensitivity and specificity was either 100% (strategy A) or imperfect (strategy B). To improve and illustrate the practicality of this estimation approach, a validation study was constructed from pools allocated at random into main (734 pools) and validation (200 pools) subsets. Prevalence was estimated using strategies A and B and an inverse-variance weighted estimator and estimates were weighted to account for differential sampling rates by area. RESULTS: The prevalence of subpatent parasitaemia was 14.5% (95% CI 13.6-15.3%) by individual qPCR, 9.5% (95% CI (8.5-10.5%) by strategy A, and 13.9% (95% CI 12.6-15.2%) by strategy B. In the validation study, the prevalence by individual qPCR was 13.5% (95% CI 12.4-14.7%) in the main subset, 8.9% (95% CI 7.9-9.9%) by strategy A, 11.4% (95% CI 9.9-12.9%) by strategy B, and 12.8% (95% CI 11.2-14.3%) using inverse-variance weighted estimator from poolwise validation. Pooling, including a 20% validation subset, reduced costs by 52% compared to individual testing. CONCLUSIONS: Compared to individual testing, a one-step pooled testing strategy with an internal validation subset can provide accurate prevalence estimates of PCR-positivity among RDT-negatives at a lower cost.

BACKGROUND: Over the last two decades, the scale-up of vector control and changes in the first-line anti-malarial, from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemether-lumefantrine (AL), have resulted in significant decreases in malaria burden in western Kenya. This study evaluated the long-term effects of control interventions on molecular markers of Plasmodium falciparum drug resistance using parasites obtained from humans and mosquitoes at discrete time points. METHODS: Dried blood spot samples collected in 2012 and 2017 community surveys in Asembo, Kenya were genotyped by Sanger sequencing for markers associated with resistance to SP (Pfdhfr, Pfdhps), CQ, AQ, lumefantrine (Pfcrt, Pfmdr1) and artemisinin (Pfk13). Temporal trends in the prevalence of these markers, including data from 2012 to 2017 as well as published data from 1996, 2001, 2007 from same area, were analysed. The same markers from mosquito oocysts collected in 2012 were compared with results from human blood samples. RESULTS: The prevalence of SP dhfr/dhps quintuple mutant haplotype C(50)I(51)R(59)N(108)I(164)/S(436)G(437)E(540)A(581)A(613) increased from 19.7% in 1996 to 86.0% in 2012, while an increase in the sextuple mutant haplotype C(50)I(51)R(59)N(108)I(164)/H(436)G(437)E(540)A(581)A(613) containing Pfdhps-436H was found from 10.5% in 2012 to 34.6% in 2017. Resistant Pfcrt-76 T declined from 94.6% in 2007 to 18.3% in 2012 and 0.9% in 2017. Mutant Pfmdr1-86Y decreased across years from 74.8% in 1996 to zero in 2017, mutant Pfmdr1-184F and wild Pfmdr1-D1246 increased from 17.9% to 58.9% in 2007 to 55.9% and 90.1% in 2017, respectively. Pfmdr1 haplotype N(86)F(184)S(1034)N(1042)D(1246) increased from 11.0% in 2007 to 49.6% in 2017. No resistant mutations in Pfk13 were found. Prevalence of Pfdhps-436H was lower while prevalence of Pfcrt-76 T was higher in mosquitoes than in human blood samples. CONCLUSION: This study showed an increased prevalence of dhfr/dhps resistant markers over 20 years with the emergence of Pfdhps-436H mutant a decade ago in Asembo. The reversal of Pfcrt from CQ-resistant to CQ-sensitive genotype occurred following 19 years of CQ withdrawal. No Pfk13 markers associated with artemisinin resistance were detected, but the increased haplotype of Pfmdr1 N(86)F(184)S(1034)N(1042)D(1246) was observed. The differences in prevalence of Pfdhps-436H and Pfcrt-76 T SNPs between two hosts and the role of mosquitoes in the transmission of drug resistant parasites require further investigation.

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas.

We appreciate the thoughtful commentary provided by Hamer and Miller [1] and are pleased that they arrived at many of the same conclusions that we did; however, we would like to clarify a few points. | | First, we wish to correct the statement that, in our trial, mass testing and treatment (MTaT) was only implemented within the core areas of clusters. Rather, MTaT was implemented throughout intervention clusters, which included a core area ranging between 1 and 3 Km in diameter, and a 300-m buffer. As described, to limit contamination, inclusion criteria for the analytic sample required residence within the core area [2, 3].

BACKGROUND: Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. METHODS: Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. RESULTS: The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012. CONCLUSION: The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.

BACKGROUND: Anti-malarial drug resistance remains a major threat to global malaria control efforts. In Africa, Plasmodium falciparum remains susceptible to artemisinin-based combination therapy (ACT), but the emergence of resistant parasites in multiple countries in Southeast Asia and concerns over emergence and/or spread of resistant parasites in Africa warrants continuous monitoring. The World Health Organization recommends that surveillance for molecular markers of resistance be included within therapeutic efficacy studies (TES). The current study assessed molecular markers associated with resistance to Artemether-lumefantrine (AL) and Dihydroartemisinin-piperaquine (DP) from samples collected from children aged 6-59 months enrolled in a TES conducted in Siaya County, western Kenya from 2016 to 2017. METHODS: Three hundred and twenty-three samples collected pre-treatment (day-0) and 110 samples collected at the day of recurrent parasitaemia (up to day 42) were tested for the presence of drug resistance markers in the Pfk13 propeller domain, and the Pfmdr1 and Pfcrt genes by Sanger sequencing. Additionally, the Pfpm2 gene copy number was assessed by real-time polymerase chain reaction. RESULTS: No mutations previously associated with artemisinin resistance were detected in the Pfk13 propeller region. However, other non-synonymous mutations in the Pfk13 propeller region were detected. The most common mutation found on day-0 and at day of recurrence in the Pfmdr1 multidrug resistance marker was at codon 184F. Very few mutations were found in the Pfcrt marker (< 5%). Within the DP arm, all recrudescent cases (8 sample pairs) that were tested for Pfpm2 gene copy number had a single gene copy. None of the associations between observed mutations and treatment outcomes were statistically significant. CONCLUSION: The results indicate absence of Pfk13 mutations associated with parasite resistance to artemisinin in this area and a very high proportion of wild-type parasites for Pfcrt. Although the frequency of Pfmdr1 184F mutations was high in these samples, the association with treatment failure did not reach statistical significance. As the spread of artemisinin-resistant parasites remains a possibility, continued monitoring for molecular markers of ACT resistance is needed to complement clinical data to inform treatment policy in Kenya and other malaria-endemic regions.

Progress with malaria control in western Kenya has stagnated since 2007. Additional interventions to reduce the high burden of malaria in this region are urgently needed. We conducted a two-arm, community-based, cluster-randomized, controlled trial of active case detection and treatment of malaria infections in all residents mass testing and treatment (MTaT) of 10 village clusters (intervention clusters) for two consecutive years to measure differences in the incidence of clinical malaria disease and malaria infections compared with 20 control clusters where MTaT was not implemented. All residents of intervention clusters, irrespective of history of fever or other malaria-related symptoms, were tested three times per year before the peak malaria season using malaria rapid diagnostic tests. All positive cases were treated with dihydroartemisinin-piperaquine. The incidence of clinical malaria was measured through passive surveillance, whereas the cumulative incidence of malaria infection was measured using active surveillance in a cohort comprising randomly selected residents. The incidence of clinical malaria was 0.19 cases/person-year (p-y, 95% CI: 0.13-0.28) in the intervention arm and 0.24 cases/p-y (95% CI: 0.15-0.39) in the control arm (incidence rate ratio [IRR] 0.79, 95% CI: 0.61-1.02). The cumulative incidence of malaria infections was similar between the intervention (2.08 infections/p-y, 95% CI: 1.93-2.26) and control arms (2.19 infections/p-y, 95% CI: 2.02-2.37) with a crude IRR of 0.95 (95% CI: 0.87-1.04). Six rounds of MTaT over 2 years did not have a significant impact on the incidence of clinical malaria or the cumulative incidence of malaria infection in this area of high malaria transmission.

BACKGROUND: Global gains towards malaria elimination have been heterogeneous and have recently stalled. Interventions targeting afebrile malaria infections may be needed to address residual transmission. We studied the efficacy of repeated rounds of community-based mass testing and treatment (MTaT) on malaria infection prevalence in western Kenya. METHODS: Twenty clusters were randomly assigned to three rounds of MTaT per year for two years or control (standard-of-care for testing and treatment at public health facilities along with government sponsored mass long-lasting insecticidal net (LLIN) distributions). During rounds community health volunteers visited all households in intervention clusters and tested all consenting individuals with a rapid diagnostic test. Those positive were treated with dihydroartemisinin-piperaquine. Cross-sectional community infection prevalence surveys were performed in both study arms at baseline and each year after three rounds of MTaT. The primary outcome was the effect size of MTaT on parasite prevalence by microscopy between arms by year adjusted for age, reported LLIN use, enhanced vegetative index, and socio-economic status. RESULTS: Demographic and behavioral characteristics, including LLIN usage, were similar between arms at each survey. MTaT coverage ranged between 75.0-77.5% and 81.9-94.3% between the three rounds in year 1 and year 2, respectively. The adjusted effect size of MTaT on the prevalence of parasitemia between arms was 0.93 (CI: 0.79-1.08) and 0.92 (0.76-1.10) after year 1 and 2, respectively. CONCLUSIONS: MTaT performed three times per year over two years did not reduce malaria parasite prevalence in this high-transmission area.

BACKGROUND: Ethiopia has set a goal for malaria elimination by 2030. Low parasite density infections may go undetected by conventional diagnostic methods (microscopy and rapid diagnostic tests) and their contribution to malaria transmission varies by transmission settings. This study quantified the burden of subpatent infections from samples collected from three regions of northwest Ethiopia. METHODS: Sub-samples of dried blood spots from the Ethiopian Malaria Indicator Survey 2015 (EMIS-2015) were tested and compared using microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (nPCR) to determine the prevalence of subpatent infection. Paired seroprevalence results previously reported along with gender, age, and elevation of residence were explored as risk factors for Plasmodium infection. RESULTS: Of the 2608 samples collected, the highest positive rate for Plasmodium infection was found with nPCR 3.3% (95% CI 2.7-4.1) compared with RDT 2.8% (95% CI 2.2-3.5) and microscopy 1.2% (95% CI 0.8-1.7). Of the nPCR positive cases, Plasmodium falciparum accounted for 3.1% (95% CI 2.5-3.8), Plasmodium vivax 0.4% (95% CI 0.2-0.7), mixed P. falciparum and P. vivax 0.1% (95% CI 0.0-0.4), and mixed P. falciparum and Plasmodium malariae 0.1% (95% CI 0.0-0.3). nPCR detected an additional 30 samples that had not been detected by conventional methods. The majority of the nPCR positive cases (61% (53/87)) were from the Benishangul-Gumuz Region. Malaria seropositivity had significant association with nPCR positivity [adjusted OR 10.0 (95% CI 3.2-29.4), P < 0.001]. CONCLUSION: Using nPCR the detection rate of malaria parasites increased by nearly threefold over rates based on microscopy in samples collected during a national cross-sectional survey in 2015 in Ethiopia. Such subpatent infections might contribute to malaria transmission. In addition to strengthening routine surveillance systems, malaria programmes may need to consider low-density, subpatent infections in order to accelerate malaria elimination efforts.

BACKGROUND: Innovative approaches are needed to limit antimalarial resistance evolution. Understanding the role of intermittent preventive treatment in pregnancy (IPTp) on the selection for resistance and the impact such selection has on pregnancy outcomes can guide future interventions. METHODS: Plasmodium falciparum isolates (n = 914) from 2 randomized clinical trials were screened for pfmdr1 copy number variation and pfcrt, pfmdr1, pfdhfr, and pfdhps resistance markers. The trials were conducted between 2010 and 2013 in Benin, Gabon, Kenya, and Mozambique to establish the efficacy of IPTp-mefloquine (MQ) compared with IPTp-sulphadoxine-pyrimethamine (SP) in human immunodeficiency virus (HIV)-uninfected and to IPTp-placebo in HIV-infected women. RESULTS: In HIV-uninfected women, the prevalence of pfcrt mutants, pfdhfr/pfdhps quintuple mutants, and pfmdr1 copy number was similar between women receiving IPT-SP and IPTp-MQ. However, prevalence of pfmdr1 polymorphism 86Y was lower in the IPTp-MQ group than in the IPTp-SP group, and within the IPTp-MQ group it was lower at delivery compared with recruitment. No effect of IPTp-MQ on resistance markers was observed among HIV-infected women. The carriage of resistance markers was not associated with pregnancy outcomes. CONCLUSIONS: Selection of wild-type pfmdr1 polymorphism N86 by IPTp-MQ highlights the strong selective pressure IPTp can exert and the opportunity for using negative cross-resistance in drug choice for clinical treatment and IPTp.

BACKGROUND: Measures of malaria burden using microscopy and rapid diagnostic tests (RDTs) in cross-sectional household surveys may incompletely describe the burden of malaria in low-transmission settings. This study describes the pattern of malaria transmission in Ethiopia using serological antibody estimates derived from a nationwide household survey completed in 2015. METHODS: Dried blood spot (DBS) samples were collected during the Ethiopian Malaria Indicator Survey in 2015 from malarious areas across Ethiopia. Samples were analysed using bead-based multiplex assays for IgG antibodies for six Plasmodium antigens: four human malaria species-specific merozoite surface protein-1 19kD antigens (MSP-1) and Apical Membrane Antigen-1 (AMA-1) for Plasmodium falciparum and Plasmodium vivax. Seroprevalence was estimated by age, elevation and region. The seroconversion rate was estimated using a reversible catalytic model fitted with maximum likelihood methods. RESULTS: Of the 10,278 DBS samples available, 93.6% (9622/10,278) had valid serological results. The mean age of participants was 15.8 years and 53.3% were female. National seroprevalence for antibodies to P. falciparum was 32.1% (95% confidence interval (CI) 29.8-34.4) and 25.0% (95% CI 22.7-27.3) to P. vivax. Estimated seroprevalences for Plasmodium malariae and Plasmodium ovale were 8.6% (95% CI 7.6-9.7) and 3.1% (95% CI 2.5-3.8), respectively. For P. falciparum seroprevalence estimates were significantly higher at lower elevations (< 2000 m) compared to higher (2000-2500 m) (aOR 4.4; p < 0.01). Among regions, P. falciparum seroprevalence ranged from 11.0% (95% CI 8.8-13.7) in Somali to 65.0% (95% CI 58.0-71.4) in Gambela Region and for P. vivax from 4.0% (95% CI 2.6-6.2) in Somali to 36.7% (95% CI 30.0-44.1) in Amhara Region. Models fitted to measure seroconversion rates showed variation nationally and by elevation, region, antigen type, and within species. CONCLUSION: Using multiplex serology assays, this study explored the cumulative malaria burden and regional dynamics of the four human malarias in Ethiopia. High malaria burden was observed in the northwest compared to the east. High transmission in the Gambela and Benishangul-Gumuz Regions and the neglected presence of P. malariae and P. ovale may require programmatic attention. The use of a multiplex assay for antibody detection in low transmission settings has the potential to act as a more sensitive biomarker.

BACKGROUND: Building on the declining trend of malaria in Ethiopia, the Federal Ministry of Health aims to eliminate malaria by 2030. As Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia, the use of primaquine is indicated for both transmission interruption and radical cure, respectively. However, the limited knowledge of the local prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its associated variants has hindered the use of primaquine. METHODS: Some 11,138 dried blood spot (DBS) samples were collected in 2011 as part of a national, household Malaria Indicator Survey, a multi-stage nationally representative survey of all malaria-endemic areas of Ethiopia. A randomly selected sub-set of 1414 DBS samples was successfully genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Considering the geographical position and ethnic mix of the country, three common variants: G6PD*A (A376G), G6PD*A- (G202A) and Mediterranean (C563T) were investigated. RESULTS: Of the 1998 randomly selected individuals, 1429 (71.5%) DBS samples were genotyped and merged to the database, of which 53.5% were from females. G6PD*A (A376G) was the only genotype detected. No sample was positive for either G6PD*A- (G202A) or Mediterranean (C563T) variants. The prevalence of G6PD*A (A376G) was 8.9% [95% confidence interval (CI) 6.7-11.2] ranging from 12.2% in the Southern Nations, Nationalities and Peoples' (95% CI 5.7-18.7) to none in Dire Dawa/Harari Region. CONCLUSION: The common G6PD*A- (G202A) or Mediterranean (C563T) variants were not observed in this nationwide study. The observed G6PD*A (A376G) mutation has little or no clinical significance. These findings supported the adoption of primaquine for P. falciparum transmission interruption and radical cure of P. vivax in Ethiopia. As the presence of other clinically important, less common variants cannot be ruled out, the implementation of radical cure will be accompanied by active haematological and adverse events monitoring in Ethiopia.

Antimalarial drug resistance is an evolving global health security threat to malaria control. Early detection of Plasmodium falciparum resistance through therapeutic efficacy studies and associated genetic analyses may facilitate timely implementation of intervention strategies. The US President's Malaria Initiative-supported Antimalarial Resistance Monitoring in Africa Network has assisted numerous laboratories in partner countries in acquiring the knowledge and capability to independently monitor for molecular markers of antimalarial drug resistance.

The role of killer cell immunoglobulin-like receptors (KIRs) in the transmission of HIV-1 has not been extensively studied. Here, we investigated the association of KIR gene content polymorphisms with perinatal HIV-1 transmission. The KIR gene family comprising 16 genes was genotyped in 313 HIV-1 positive Kenyan mothers paired with their infants. Gene content polymorphisms were presented as presence of individual KIR genes, haplotypes, genotypes and KIR gene concordance. The genetic data were analyzed for associations with perinatal transmission of HIV. There was no association of infant KIR genes with perinatal HIV-1 transmission. After adjustment for gravidity, viral load, and CD4 cell count, there was evidence of an association between reduction in perinatal HIV-1 transmission and the maternal individual KIR genes KIR2DL2 (adjusted OR = 0.50; 95% CI: 0.24-1.02, P = 0.06), KIR2DL5 (adjusted OR = 0.47; 95% CI: 0.23-0.95, P = 0.04) and KIR2DS5 (adjusted OR = 0.39; 95% CI: 0.18-0.80, P = 0.01). Furthermore, these maternal KIR genes were only significantly associated with reduction in perinatal HIV transmission in women with CD4 cell count >/= 350 cells/ mul and viral load <10000 copies/ml. Concordance analysis showed that when both mother and child had KIR2DS2, there was less likelihood of perinatal HIV-1 transmission (adjusted OR = 0.44; 95% CI: 0.20-0.96, P = 0.039). In conclusion, the maternal KIR genes KIR2DL2, KIR2DL5, KIR2DS5, and KIR2DS2 were associated with reduction of HIV-1 transmission from mother to child. Furthermore, maternal immune status is an important factor in the association of KIR with perinatal HIV transmission.

Most human Plasmodium infections in western Kenya are asymptomatic and are believed to contribute importantly to malaria transmission. Elimination of asymptomatic infections requires active treatment approaches, such as mass testing and treatment (MTaT) or mass drug administration (MDA), as infected persons do not seek care for their infection. Evaluations of community-based approaches that are designed to reduce malaria transmission require careful attention to study design to ensure that important effects can be measured accurately. This manuscript describes the study design and methodology of a cluster-randomized controlled trial to evaluate a MTaT approach for malaria transmission reduction in an area of high malaria transmission. Ten health facilities in western Kenya were purposively selected for inclusion. The communities within 3 km of each health facility were divided into three clusters of approximately equal population size. Two clusters around each health facility were randomly assigned to the control arm, and one to the intervention arm. Three times per year for 2 years, after the long and short rains, and again before the long rains, teams of community health volunteers visited every household within the intervention arm, tested all consenting individuals with malaria rapid diagnostic tests, and treated all positive individuals with an effective anti-malarial. The effect of mass testing and treatment on malaria transmission was measured through population-based longitudinal cohorts, outpatient visits for clinical malaria, periodic population-based cross-sectional surveys, and entomological indices.

BACKGROUND: Recent efforts in malaria control have resulted in great gains in reducing the burden of Plasmodium falciparum, but P. vivax has been more refractory. Its ability to form dormant liver stages confounds control and elimination efforts. To compare the efficacy and safety of primaquine regimens for radical cure, we undertook a randomized controlled trial in Ethiopia. METHODS AND FINDINGS: Patients with normal glucose-6-phosphate dehydrogenase status with symptomatic P. vivax mono-infection were enrolled and randomly assigned to receive either chloroquine (CQ) or artemether-lumefantrine (AL), alone or in combination with 14 d of semi-supervised primaquine (PQ) (3.5 mg/kg total). A total of 398 patients (n = 104 in the CQ arm, n = 100 in the AL arm, n = 102 in the CQ+PQ arm, and n = 92 in the AL+PQ arm) were followed for 1 y, and recurrent episodes were treated with the same treatment allocated at enrolment. The primary endpoints were the risk of P. vivax recurrence at day 28 and at day 42. The risk of recurrent P. vivax infection at day 28 was 4.0% (95% CI 1.5%-10.4%) after CQ treatment and 0% (95% CI 0%-4.0%) after CQ+PQ. The corresponding risks were 12.0% (95% CI 6.8%-20.6%) following AL alone and 2.3% (95% CI 0.6%-9.0%) following AL+PQ. On day 42, the risk was 18.7% (95% CI 12.2%-28.0%) after CQ, 1.2% (95% CI 0.2%-8.0%) after CQ+PQ, 29.9% (95% CI 21.6%-40.5%) after AL, and 5.9% (95% CI 2.4%-13.5%) after AL+PQ (overall p < 0.001). In those not prescribed PQ, the risk of recurrence by day 42 appeared greater following AL treatment than CQ treatment (HR = 1.8 [95% CI 1.0-3.2]; p = 0.059). At the end of follow-up, the incidence rate of P. vivax was 2.2 episodes/person-year for patients treated with CQ compared to 0.4 for patients treated with CQ+PQ (rate ratio: 5.1 [95% CI 2.9-9.1]; p < 0.001) and 2.3 episodes/person-year for AL compared to 0.5 for AL+PQ (rate ratio: 6.4 [95% CI 3.6-11.3]; p < 0.001). There was no difference in the occurrence of adverse events between treatment arms. The main limitations of the study were the early termination of the trial and the omission of haemoglobin measurement after day 42, resulting in an inability to estimate the cumulative risk of anaemia. CONCLUSIONS: Despite evidence of CQ-resistant P. vivax, the risk of recurrence in this study was greater following treatment with AL unless it was combined with a supervised course of PQ. PQ combined with either CQ or AL was well tolerated and reduced recurrence of vivax malaria by 5-fold at 1 y. TRIAL REGISTRATION: ClinicalTrials.gov NCT01680406.

BACKGROUND: Although malaria control intervention has greatly decreased malaria morbidity and mortality in many African countries, further decline in parasite prevalence has stagnated in western Kenya. In order to assess if malaria transmission reservoir is associated with this stagnation, submicroscopic infection and gametocyte carriage was estimated. Risk factors and associations between malaria control interventions and gametocyte carriage were further investigated in this study. METHODS: A total of 996 dried blood spot samples were used from two strata, all smear-positives (516 samples) and randomly selected smear-negatives (480 samples), from a community cross-sectional survey conducted at peak transmission season in 2012 in Siaya County, western Kenya. Plasmodium falciparum parasite presence and density were determined by stained blood smear and by 18S mRNA transcripts using nucleic acid sequence-based amplification assay (NASBA), gametocyte presence and density were determined by blood smear and by Pfs25 mRNA-NASBA, and gametocyte diversity by Pfg377 mRNA RT-PCR and RT-qPCR. RESULTS: Of the randomly selected smear-negative samples, 69.6 % (334/480) were positive by 18S-NASBA while 18S-NASBA detected 99.6 % (514/516) smear positive samples. Overall, 80.2 % of the weighted population was parasite positive by 18S-NASBA vs 30.6 % by smear diagnosis and 44.0 % of the weighted population was gametocyte positive by Pfs25-NASBA vs 2.6 % by smear diagnosis. Children 5-15 years old were more likely to be parasitaemic and gametocytaemic by NASBA than individuals >15 years old or children <5 years old while gametocyte density decreased with age. Anaemia and self-reported fever within the past 24 h were associated with increased odds of gametocytaemia. Fever was also positively associated with parasite density, but not with gametocyte density. Anti-malarial use within the past 2 weeks decreased the odds of gametocytaemia, but not the odds of parasitaemia. In contrast, recent anti-malarial use was associated with lowered parasite density, but not the gametocyte density. Use of ITNs was associated with lower odds for parasitaemia in part of the study area with a longer history of ITN interventions. In the same part of study area, the odds of having multiple gametocyte alleles were also lower in individuals using ITNs than in those not using ITNs and parasite density was positively associated with gametocyte diversity. CONCLUSION: A large proportion of submicroscopic parasites and gametocytes in western Kenya might contribute to the stagnation in malaria prevalence, suggesting that additional interventions targeting the infectious reservoir are needed. As school aged children and persons with anaemia and fever were major sources for gametocyte reservoir, these groups should be targeted for intervention and prevention to reduce malaria transmission. Anti-malarial use was associated with lower parasite density and odds of gametocytaemia, but not the gametocyte density, indicating a limitation of anti-malarial impact on the transmission reservoir. ITN use had a protective role against parasitaemia and gametocyte diversity in western Kenya.

BACKGROUND: An initial study of genetic diversity of Plasmodium falciparum in Asembo, western Kenya showed that the parasite maintained overall genetic stability 5 years after insecticide-treated bed net (ITN) introduction in 1997. This study investigates further the genetic diversity of P. falciparum 10 years after initial ITN introduction in the same study area and compares this with two other neighbouring areas, where ITNs were introduced in 1998 (Gem) and 2004 (Karemo). METHODS: From a cross-sectional survey conducted in 2007, 235 smear-positive blood samples collected from children ≤15-year-old in the original study area and two comparison areas were genotyped employing eight neutral microsatellites. Differences in multiple infections, allele frequency, parasite genetic diversity and parasite population structure between the three areas were assessed. Further, molecular data reported previously (1996 and 2001) were compared to the 2007 results in the original study area Asembo. RESULTS: Overall proportion of multiple infections (MA) declined with time in the original study area Asembo (from 95.9 %-2001 to 87.7 %-2007). In the neighbouring areas, MA was lower in the site where ITNs were introduced in 1998 (Gem 83.7 %) compared to where they were introduced in 2004 (Karemo 96.7 %) in 2007. Overall mean allele count (MAC ~ 2.65) and overall unbiased heterozygosity (H e ~ 0.77) remained unchanged in 1996, 2001 and 2007 in Asembo and was the same level across the two neighbouring areas in 2007. Overall parasite population differentiation remained low over time and in the three areas at FST < 0.04. Both pairwise and multilocus linkage disequilibrium showed limited to no significant association between alleles in Asembo (1996, 2001 and 2007) and between three areas. CONCLUSIONS: This study showed the P. falciparum high genetic diversity and parasite population resilience on samples collected 10 years apart and in different areas in western Kenya. The results highlight the need for long-term molecular monitoring after implementation and use of combined and intensive prevention and intervention measures in the region.

BACKGROUND: Monitoring the effectiveness of intermittent preventive therapy in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is crucial owing to increasing SP resistance in sub-Saharan Africa. METHODS: Between 2009 and 2013, both the efficacy of IPTp-SP at clearing existing peripheral malaria infections and the effectiveness of IPTp-SP at reducing low birthweight (LBW) were assessed among HIV-negative participants in 8 sites in 6 countries. Sites were classified as high, medium or low resistance after measuring mutations conferring SP resistance. An individual-level prospective pooled analysis was conducted. RESULTS: Among 1,222 parasitaemic pregnant women, overall PCR-uncorrected and -corrected failure rates by day 42 were 21.3% and 10.0%, respectively (39.7% and 21.1% in high-resistance areas; 4.9% and 1.1% in low-resistance areas). Median time to recurrence decreased with increasing prevalence of Pfdhps-K540E. Among 6,099 women at delivery, each incremental dose of IPTp-SP was associated with a 22% reduction in the risk of LBW (prevalence ratio [PR]=0.78 [95% CI 0.69-0.88], p<0.001). This association was not modified by insecticide-treated net use or gravidity, and remained significant in areas with SP resistance (PR=0.81 [0.67-0.97], p=0.02). CONCLUSIONS: The efficacy of SP to clear peripheral parasites and prevent new infections during pregnancy is compromised in areas with >90% prevalence of Pfdhps-K540E. Nevertheless, in these high resistance areas, IPTp-SP use remains associated with increases in birthweight and maternal haemoglobin. The effectiveness of IPTp in eastern and southern Africa is threatened by further increases in SP-resistance and reinforces the need to evaluate alternative drugs and strategies for the control of malaria in pregnancy.

Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials including current artemisinin-based combination therapies.An antimalarial drug resistance surveillance study involving 203 P. falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine and artemisinin derivatives (artesunate and dihydroartemisinin), and for drug resistance allele polymorphisms in Pfcrt, Pfmdr-1 and the K13 propeller domain (K13).We observed a significant increase in the proportion of samples with the Pfcrt wild type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (p < 0.0001) and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild type N allele in Pfmdr-1 codon 86 (93.5 %), with only 7 (3.50%) samples with the mutant N86Y allele. Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%) with only 12 (6.38%) specimens with the mutant D1246Y allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean= 0.907+/-0.141). None of the sequenced parasites had mutations in the K13.Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya.

BACKGROUND: The effectiveness of sulphadoxine-pyrimethamine (SP) intermittent preventive treatment of malaria in pregnancy (IPTp) might be compromised by high prevalence of resistance-associated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations. As a proxy for IPTp-SP effectiveness, the in vivo efficacy of SP to clear parasitaemia and prevent reinfection in asymptomatic parasitaemic pregnant women in an area with high SP resistance prevalence was assessed. METHODS: Pregnant women 16-26 weeks' gestation with asymptomatic parasitaemia presenting for antenatal care were given IPTp-SP and followed for 42 days. The primary outcome was polymerase chain reaction (PCR) uncorrected 42-day survival rate; the per cent of patients without recrudescence or reinfection by day 42. PCR was used to distinguish recrudescence from reinfection. DNA was sequenced to detect resistance-associated dhfr and dhps mutations. RESULTS: Of 245 pregnant women included in the intention-to-treat analysis, 93.9% cleared their parasitaemia by day 7. The day 42 PCR-uncorrected survival rate was 58.1% (95% confidence interval (CI) 51.5-65.7) and day 42 PCR-corrected survival was 68.7% (CI 61.4-76.0). Recrudescence was more common among primi- than among multigravid women; recrudescence rate 33.3% (CI 25.1-42.4%) versus 21.4% (CI 15.0-29.0%) (log rank test p-value 0.006). The quintuple mutant was present in nearly all samples (95%), while 2% were sextuple mutants with an additional mutation at dhps A581G. CONCLUSIONS: SP efficacy for acute malaria treatment has been compromised by resistance, but SP retains partial activity among pregnant women with asymptomatic parasitaemia, and thus might be useful for IPTp. Nonetheless, research on non-SP IPTp regimens should continue. TRIAL REGISTRATION: ClinicalTrials.gov NCT01120145 .

The molecular basis of sulfadoxine-pyrimethamine (SP) resistance lies in a combination of single-nucleotide polymorphisms (SNPs) in two genes coding for Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. The continued use of SP for intermittent preventive treatment in pregnant women in many African countries, despite SP's discontinuation as a first-line antimalarial treatment option, due to high levels of drug resistance, may further increase the prevalence of SP-resistant parasites and/or lead to selection of new mutations. An antimalarial drug resistance surveillance study was conducted in western Kenya between 2010 and 2013. A total of 203 clinical samples from children with uncomplicated malaria were genotyped for SNPs associated with SP resistance. The prevalence of the triple mutant Pfdhfr C50 I51R59N: 108I164 and the double mutant Pfdhps S436 G437E540: A581A613 genotypes was high. Two triple mutant Pfdhps genotypes were found: S436 G437E540G: 581A613 and H: 436 G437E540: A581A613, with the latter, thus far, uniquely found in western Kenya. The prevalence of the S436 G: 437 E: 540 G: 581A613 genotype was low. However, a steady increase in the triple Pfdhps H: 436 G: 437 E: 540A581A613 genotype was observed since its appearance in early 2000. These isolates shared substantial microsatellite haplotypes with the most common double mutant allele suggesting that this triple mutant allele may have evolved locally. Overall, these findings show that the triple H: 436 G437E540: A581A613 mutant may be increasing in this population and could compromise the efficacy of SP for IPTp if it increases the resistant threshold further.

BACKGROUND: Current available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. It was proposed that malaria parasite-specific low molecular-weight metabolites could be used as biomarkers for the development of a malaria diagnostic tool aimed to address this diagnostic challenge. In this study, high resolution metabolomics (HRM) was employed to identify malaria parasite-specific metabolites in Plasmodium falciparum in vitro culture samples. METHODS: Supernatants were collected at 12 hours interval from 3% haematocrit in vitro 48-hour time-course asynchronized culture system of P. falciparum. Liquid chromatography coupled with high resolution mass spectrometry was applied to discover potential parasite-specific metabolites in the cell culture supernatant. A metabolome-wide association study was performed to extract metabolites using Manhattan plot with false discovery rate (FDR) and hierarchical cluster analysis. The significant metabolites based on FDR cutoff were annotated using Metlin database. Standard curves were created using corresponding chemical compounds to accurately quantify potential Plasmodium-specific metabolites in culture supernatants. RESULTS: The number of significant metabolite features was 1025 in the supernatant of the Plasmodium infected culture based on Manhattan plot with FDR q=0.05. A two way hierarchical cluster analysis showed a clear segregation of the metabolic profile of parasite infected supernatant from non-infected supernatant at four time points during the 48 hour culture. Among the 1025 annotated metabolites, the intensities of four molecules were significantly increased with culture time suggesting a positive association between the quantity of these molecules and level of parasitaemia: i) 3-methylindole, a mosquito attractant, ii) succinylacetone, a haem biosynthesis inhibitor, iii) S-methyl-L-thiocitrulline, a nitric oxide synthase inhibitor, and iv) O-arachidonoyl glycidol, a fatty acid amide hydrolase inhibitor, The highest concentrations of 3-methylindole and succinylacetone were 178 +/- 18.7 pmoles at 36 hours and 157 +/- 30.5 pmoles at 48 hours respectively in parasite infected supernatant. CONCLUSION: HRM with bioinformatics identified four potential parasite-specific metabolite biomarkers using in vitro culture supernatants. Further study in malaria infected human is needed to determine presence of the molecules and its relationship with parasite densities.

BACKGROUND: Although it is well known that drug pressure selects for drug-resistant parasites, the role of transmission reduction by insecticide-treated bed nets (ITNs) on drug resistance remains unclear. In this study, the drug resistance profile of current and previous first-line anti-malarials in Kenya was assessed within the context of drug policy change and scale-up of ITNs. National first-line treatment changed from chloroquine (CQ) to sulphadoxine-pyrimethamine (SP) in 1998 and to artemether-lumefantrine (AL) in 2004. ITN use was scaled-up in the Asembo, Gem and Karemo areas of western Kenya in 1997, 1999 and 2006, respectively. METHODS: Smear-positive samples (N=253) collected from a 2007 cross-sectional survey among children in Asembo, Gem and Karemo were genotyped for mutations in pfcrt and pfmdr1 (CQ), dhfr and dhps (SP), and at pfmdr-N86 and the gene copy number in pfmdr1 (lumefantrine). Results were compared among the three geographic areas in 2007 and to retrospective molecular data from children in Asembo in 2001. RESULTS: In 2007, 69 and 85% of samples harboured the pfmdr1-86Y mutation and dhfr/dhps quintuple mutant, respectively, with no significant differences by study area. However, the prevalence of the pfcrt-76T mutation differed significantly among areas (p <0.02), between 76 and 94%, with the highest prevalence in Asembo. Several 2007 samples carried mutations at dhfr-164 L, dhps-436A, or dhps-613T. From 2001 to 2007, there were significant increases in the pfcrt-76T mutation from 82 to 94% (p <0.03), dhfr/dhps quintuple mutant from 62 to 82% (p <0.03), and an increase in the septuple CQ and SP combined mutant haplotype, K76Y86I51R59N108G437E540, from 28 to 39%. The prevalence of the pfmdr1-86Y mutation remained unchanged. All samples were single copy for pfmdr1. CONCLUSIONS: Molecular markers associated with lumefantrine resistance were not detected in 2007. More recent samples will be needed to detect any selective effects by AL. The prevalence of CQ and SP resistance markers increased from 2001 to 2007 in the absence of changes in transmission intensity. In 2007, only the prevalence of pfcrt-76T mutation differed among study areas of varying transmission intensity. Resistant parasites were most likely selected by sustained drug pressure from the continued use of CQ, SP, and mechanistically similar drugs, such as amodiaquine and cotrimoxazole. There was no clear evidence that differences in transmission intensity, as a result of ITN scale-up, influenced the prevalence of drug resistance molecular markers.

BACKGROUND: Low malaria parasite densities in pregnancy are a diagnostic challenge. PCR provides high sensitivity and specificity in detecting low density of parasites, but cost and technical requirements limit its application in resources-limited settings. Pooling samples for PCR detection was explored to estimate prevalence of submicroscopic malaria infection in pregnant women at delivery. Previous work uses gold-standard based methods to calculate sensitivity and specificity of tests, creating a challenge when newer methodologies are substantially more sensitive than the gold standard. Thus prevalence was estimated using Bayesian latent class models (LCMs) in this study. METHODS: Nested PCR (nPCR) for the 18S rRNA gene subunit of Plasmodium falciparum was conducted to detect malaria infection in microscopy-negative Malawian women on IPTp. Two-step sample pooling used dried blood spot samples (DBSs) collected from placenta or periphery at delivery. Results from nPCR and histology as well as previously published data were used to construct LCMs to estimate assay sensitivity and specificity. Theoretical confidence intervals for prevalence of infection were calculated for two-step and one-step pooling strategies. RESULTS: Of 617 microscopy-negative Malawian women, 39 (6.3%) were identified as actively infected by histology while 52 (8.4%) were positive by nPCR. One hundred forty (22.7%) individuals had past infection assessed by histology. With histology as a reference, 72% of women in the active infection group, 7.1% in the past infection group and 3.2% in histology-negative group were nPCR positive. Using latent class models without a gold standard, histology had a median sensitivity of 49.7% and specificity of 97.6% for active infection while PCR had a median sensitivity of 96.0% and specificity of 99.1%. The true prevalence of active infection was estimated at 8.0% (CI: 5.8-10.5%) from PCR. PCR also had similar sensitivity for detecting either peripheral or placental malaria for submicroscopic infections. One-step pooling would give similar confidence intervals for pool sizes less than 20 while reducing the number of tests performed. CONCLUSIONS: Pooled nPCR testing was a sensitive and resource-efficient strategy and LCMs provided precise prevalence estimates of submicroscopic infections. Compared to two-step pooling, one-step pooling could provide similar prevalence estimates at population levels with many fewer tests required.

BACKGROUND: The Plasmodium falciparum dihydropteroate synthase (Pfdhps) A581 G: mutation in combination with the dhfr/dhps quintuple mutant (the "sextuple" mutant) has been associated with increased placental inflammation and decreased birthweight among women receiving intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP). METHODS: Between 2009-2011, HIV-uninfected delivering women were enrolled in an observational study of IPTp-SP effectiveness in Malawi. Parasites were detected by polymerase chain reaction (PCR); positive samples were sequenced to genotype at dhfr and dhps loci. Presence of Pfdhps-K540 E: was used as a marker for the quintuple mutant. RESULTS: Samples from 1809 women were analysed by PCR; 220 (12%) were positive for P.falciparum. A total of 202 specimens were genotyped at codon Pfdhps-581; 17 (8.4%) harbored the sextuple mutant. The sextuple mutant was associated with higher risks of patent infection in peripheral (adjusted prevalence ratio [aPR] 2.76; 95% confidence interval 1.82-4.18) and placental blood (aPR 3.28 [1.88-5.78]), and higher parasite densities. Recent SP was not associated with increased parasite densities or placental pathology, overall and among women with dhps-A581 G: mutant parasites. CONCLUSIONS: IPTp-SP failed to inhibit parasite growth but did not exacerbate pathology among women infected with sextuple mutant parasites. New interventions to prevent malaria in pregnancy are needed urgently.

BACKGROUND: Monitoring local malaria transmission intensity is essential for planning evidence-based control strategies and evaluating their impact over time. Anti-malarial antibodies provide information on cumulative exposure and have proven useful, in areas where transmission has dropped to low sustained levels, for retrospectively reconstructing the timing and magnitude of transmission reduction. It is unclear whether serological markers are also informative in high transmission settings, where interventions may reduce transmission, but to a level where considerable exposure continues. METHODS: This study was conducted through ongoing KEMRI and CDC collaboration. Asembo, in Western Kenya, is an area where intense malaria transmission was drastically reduced during a 1997-1999 community-randomized, controlled insecticide-treated net (ITN) trial. Two approaches were taken to reconstruct malaria transmission history during the period from 1994 to 2009. First, point measurements were calculated for seroprevalence, mean antibody titre, and seroconversion rate (SCR) against three Plasmodium falciparum antigens (AMA-1, MSP-119, and CSP) at five time points for comparison against traditional malaria indices (parasite prevalence and entomological inoculation rate). Second, within individual post-ITN years, age-stratified seroprevalence data were analysed retrospectively for an abrupt drop in SCR by fitting alternative reversible catalytic conversion models that allowed for change in SCR. RESULTS: Generally, point measurements of seroprevalence, antibody titres and SCR produced consistent patterns indicating that a gradual but substantial drop in malaria transmission (46-70%) occurred from 1994 to 2007, followed by a marginal increase beginning in 2008 or 2009. In particular, proportionate changes in seroprevalence and SCR point estimates (relative to 1994 baseline values) for AMA-1 and CSP, but not MSP-119, correlated closely with trends in parasite prevalence throughout the entire 15-year study period. However, retrospective analyses using datasets from 2007, 2008 and 2009 failed to detect any abrupt drop in transmission coinciding with the timing of the 1997-1999 ITN trial. CONCLUSIONS: In this highly endemic area, serological markers were useful for generating accurate point estimates of malaria transmission intensity, but not for retrospective analysis of historical changes. Further investigation, including exploration of different malaria antigens and/or alternative models of population seroconversion, may yield serological tools that are more informative in high transmission settings.

Immunoglobulin (Ig) GM and KM allotypes, genetic markers of gamma and kappa chains, are associated with humoral immune responsiveness. Previous studies have shown the relationships between GM6-carrying haplotypes and susceptibility to malaria infection in children and adults; however, the role of the genetic markers in placental malaria (PM) infection and PM with HIV co-infection during pregnancy has not been investigated. We examined the relationship between the gene polymorphisms of Ig GM6 and KM allotypes and the risk of PM infection in pregnant women with known HIV status. DNA samples from 728 pregnant women were genotyped for GM6 and KM alleles using polymerase chain reaction-restriction fragment length polymorphism method. Individual GM6 and KM genotypes and the combined GM6 and KM genotypes were assessed in relation to PM in HIV-1 negative and positive women, respectively. There was no significant effect of individual GM6 and KM genotypes on the risk of PM infection in HIV-1 negative and positive women. However, the combination of homozygosity for GM6(+) and KM3 was associated with decreased risk of PM (adjusted OR, 0.25; 95% CI, 0.08-0.8; P = 0.019) in HIV-1 negative women while in HIV-1 positive women the combination of GM6(+/-) with either KM1-3 or KM1 was associated with increased risk of PM infection (adjusted OR, 2.10; 95% CI, 1.18-3.73; P = 0.011). Hardy-Weinberg Equilibrium (HWE) tests further showed an overall significant positive F(is) (indication of deficit in heterozygotes) for GM6 while there was no deviation for KM genotype frequency from HWE in the same population. These findings suggest that the combination of homozygous GM6(+) and KM3 may protect against PM in HIV-1 negative women while the HIV-1 positive women with heterozygous GM6(+/-) combined with KM1-3 or KM1 may be more susceptible to PM infection. The deficit in heterozygotes for GM6 further suggests that GM6 could be under selection likely by malaria infection.

Pregnant women have abundant natural killer (NK) cells in their placenta, and NK cell function is regulated by polymorphisms of killer cell immunoglobulin-like receptors (KIRs). Previous studies report different roles of NK cells in the immune responses to placental malaria (PM) and human immunodeficiency virus (HIV-1) infections. Given these references, the aim of this study was to determine the association between KIR gene content polymorphism and PM infection in pregnant women of known HIV-1 status. Sixteen genes in the KIR family were analyzed in 688 pregnant Kenyan women. Gene content polymorphisms were assessed in relation to PM in HIV-1 negative and HIV-1 positive women, respectively. Results showed that in HIV-1 negative women, the presence of the individual genes KIR2DL1 and KIR2DL3 increased the odds of having PM, and the KIR2DL2/KIR2DL2 homozygotes were associated with protection from PM. However, the reverse relationship was observed in HIV-1 positive women, where the presence of individual KIR2DL3 was associated with protection from PM, and KIR2DL2/KIR2DL2 homozygotes increased the odds for susceptibility to PM. Further analysis of the HIV-1 positive women stratified by CD4 counts showed that this reverse association between KIR genes and PM remained only in the individuals with high CD4 cell counts but not in those with low CD4 cell counts. Collectively, these results suggest that inhibitory KIR2DL2 and KIR2DL3, which are alleles of the same locus, play a role in the inverse effects on PM and PM/HIV co-infection and the effect of KIR genes on PM in HIV positive women is dependent on high CD4 cell counts. In addition, analysis of linkage disequilibrium (LD) of the PM relevant KIR genes showed strong LD in women without PM regardless of their HIV status while LD was broken in those with PM, indicating possible selection pressure by malaria infection on the KIR genes.

BACKGROUND: Resistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited. METHODS: Temporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed. RESULTS: The prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7 % in the first study (1996-2000) to 88 % in the third study (2008-2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4 % in 1998 to 44.4 % three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996-2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002-2008 and 2008-2009 studies. In addition, in the 2008- 2009 study, 5.3 % of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples. CONCLUSIONS: There was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.