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Background: In India, malaria is not uniformly distributed. Chhattisgarh is a highly malarious state where both Plasmodium falciparum and Plasmodium vivax are prevalent with a preponderance of P. falciparum. Malaria in pregnancy (MIP), especially when caused by P. falciparum, poses substantial risk to the mother and foetus by increasing the risk of foetal death, prematurity, low birth weight (LBW), and maternal anaemia. These risks vary between areas with stable and unstable transmission. The specific objectives of this study were to determine the prevalence of malaria, its association with maternal and birth outcomes, and use of anti-malarial preventive measures for development of evidence based interventions to reduce the burden of MIP. Methods: A cross-sectional study of pregnant women presenting to antenatal clinics (ANC) or delivery units (DU), or hospitalized for non-obstetric illness was conducted over 12 months in high (Bastar) and low (Rajnandgaon) transmission districts in Chhattisgarh state. Intensity of transmission was defined on the basis of slide positivity rates with a high proportion due to P. falciparum. In each district, a rural and an urban health facility was selected. Results: Prevalence of peripheral parasitaemia was low: 1.3% (35/2696) among women at ANCs and 1.9% at DUs (19/1025). Peripheral parasitaemia was significantly more common in Bastar (2.8%) than in Rajnandgaon (0.1%) (p < 0.0001). On multivariate analysis of ANC participants, residence in Bastar district (stable malaria transmission) was strongly associated with peripheral parasitaemia (adjusted OR [aOR] 43.4; 95% CI, 5.6-335.2). Additional covariates associated with parasitaemia were moderate anaemia (aOR 3.7; 95% CI 1.8-7.7), fever within the past week (aOR 3.2; 95% CI 1.2-8.6), and lack of formal education (aOR 4.6; 95% CI 2.0-10.7). Similarly, analysis of DU participants revealed that moderate anaemia (aOR 2.5; 95% CI 1.1-5.4) and fever within the past week (aOR 5.8; 95% CI 2.4-13.9) were strongly associated with peripheral and/or placental parasitaemia. Malaria-related admissions were more frequent among pregnant women in Bastar, the district with greater malaria prevalence (51% vs. 11%, p < 0.0001). Conclusions: Given the overall low prevalence of malaria, a strategy of enhanced anti-vector measures coupled with intermittent screening and targeted treatment during pregnancy should be considered for preventing malaria-associated morbidity in central India. [ABSTRACT FROM AUTHOR]

Background: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021., Methods: A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1)., Results: Sequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%., Conclusion: This is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT., (© 2024. The Author(s).)

Background: Due to low numbers of active infections and persons presenting to health facilities for malaria treatment, case-based surveillance is inefficient for understanding the remaining disease burden in low malaria transmission settings. Serological data through the detection of IgG antibodies from previous malaria parasite exposure can fill this gap by providing a nuanced picture of where sustained transmission remains. Study enrollment at sites of gathering provides a potential approach to spatially estimate malaria exposure and could preclude the need for more intensive community-based sampling., Methods: This study compared spatial estimates of malaria exposure from cross-sectional school- and community-based sampling in Haiti. A total of 52,405 blood samples were collected from 2012 to 2017. Multiplex bead assays (MBAs) tested IgG against P. falciparum liver stage antigen-1 (LSA-1), apical membrane antigen 1 (AMA1), and merozoite surface protein 1 (MSP1). Predictive geospatial models of seropositivity adjusted for environmental covariates, and results were compared using correlations by coordinate points and communes across Haiti., Results: Consistent directional associations were observed between seroprevalence and environmental covariates for elevation (negative), air temperature (negative), and travel time to urban centers (positive). Spearman's rank correlation for predicted seroprevalence at coordinate points was lowest for LSA-1 (ρ = 0.10, 95% CI: 0.09-0.11), but improved for AMA1 (ρ = 0.36, 95% CI: 0.35-0.37) and MSP1 (ρ = 0.48, 95% CI: 0.47-0.49)., Conclusions: In settings approaching P. falciparum elimination, case-based prevalence data does not provide a resolution of ongoing malaria transmission in the population. Immunogenic antigen targets (e.g., AMA1, MSP1) that give higher population rates of seropositivity provide moderate correlation to gold standard community sampling designs and are a feasible approach to discern foci of residual P. falciparum transmission in an area., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Malaria rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen are widely used for detection of active infection with this parasite and are the only practical malaria diagnostic test in some endemic settings. External validation of RDT results from field surveys can confirm appropriate RDT performance., Methods: A community-based cross-sectional survey was conducted between July and November 2017 enrolling participants of all ages in households from 15 villages in four border regions of Tanzania: Geita, Kigoma, Mtwara and Ruvuma. All participants had an RDT performed in the field and provided a blood sample for later laboratory multiplex antigen detection of HRP2. In assessing the continuous HRP2 levels in participant blood versus RDT result, dose-response logistic regression provided quantitative estimates for HRP2 limit of detection (LOD)., Results: From the 15 study villages, 6941 persons were enrolled that had a RDT at time of enrollment and provided a DBS for later laboratory antigen detection. RDT positive prevalence for the HRP2 band by village ranged from 20.0 to 43.6%, but the magnitude of this prevalence did not have an effect on the estimated LOD of RDTs utilized in different villages. Overall, HRP2 single-target tests had a lower LOD at the 95% probability of positive RDT (4.3 ng/mL; 95% CI 3.4-5.4) when compared to pLDH/HRP2 dual target tests (5.4 ng/mL; 4.5-6.3), though this difference was not significant. With the exception of one village, all other 14 villages (93.3%) showed RDT LOD estimates at 90% probability of positive RDT between 0.5 and 12.0 ng/mL., Conclusions: Both HRP2-only and pLDH/HRP2 combo RDTs utilized in a 2017 Tanzania cross-sectional survey of border regions generally performed well, and reliably detected HRP2 antigen in the low ng/mL range. Though single target tests had lower levels of HRP2 detection, both tests were within similar ranges among the 15 villages. Comparison of quantitative HRP2 detection limits among study sites can help interpret RDT testing results when generating population prevalence estimates for malaria infection., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Since 2005, artemisinin-based combination therapy (ACT) has been recommended to treat uncomplicated falciparum malaria in Madagascar. Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the first- and second-line treatments, respectively. A therapeutic efficacy study was conducted to assess ACT efficacy and molecular markers of anti-malarial resistance., Methods: Children aged six months to 14 years with uncomplicated falciparum malaria and a parasitaemia of 1000-100,000 parasites/µl determined by microscopy were enrolled from May-September 2018 in a 28-day in vivo trial using the 2009 World Health Organization protocol for monitoring anti-malarial efficacy. Participants from two communes, Ankazomborona (tropical, northwest) and Matanga (equatorial, southeast), were randomly assigned to ASAQ or AL arms at their respective sites. PCR correction was achieved by genotyping seven neutral microsatellites in paired pre- and post-treatment samples. Genotyping assays for molecular markers of resistance in the pfk13, pfcrt and pfmdr1 genes were conducted., Results: Of 344 patients enrolled, 167/172 (97%) receiving ASAQ and 168/172 (98%) receiving AL completed the study. For ASAQ, the day-28 cumulative PCR-uncorrected efficacy was 100% (95% CI 100-100) and 95% (95% CI 91-100) for Ankazomborona and Matanga, respectively; for AL, it was 99% (95% CI 97-100) in Ankazomborona and 83% (95% CI 76-92) in Matanga. The day-28 cumulative PCR-corrected efficacy for ASAQ was 100% (95% CI 100-100) and 98% (95% CI 95-100) for Ankazomborona and Matanga, respectively; for AL, it was 100% (95% CI 99-100) in Ankazomborona and 95% (95% CI 91-100) in Matanga. Of 83 successfully sequenced samples for pfk13, no mutation associated with artemisinin resistance was observed. A majority of successfully sequenced samples for pfmdr1 carried either the NFD or NYD haplotypes corresponding to codons 86, 184 and 1246. Of 82 successfully sequenced samples for pfcrt, all were wild type at codons 72-76., Conclusion: PCR-corrected analysis indicated that ASAQ and AL have therapeutic efficacies above the 90% WHO acceptable cut-off. No genetic evidence of resistance to artemisinin was observed, which is consistent with the clinical outcome data. However, the most common pfmdr1 haplotypes were NYD and NFD, previously associated with tolerance to lumefantrine., (© 2021. The Author(s).)

Background: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field., Methods: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay., Results: Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL., Conclusions: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs., (© 2021. The Author(s).)

Background: Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated., Methods: Children aged 6-59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing., Results: No pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72-76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons., Conclusion: In 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique., (© 2021. The Author(s).)

Background: The current first-line treatments for uncomplicated malaria recommended by the National Malaria Control Programme in Mali are artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). From 2015 to 2016, an in vivo study was carried out to assess the clinical and parasitological responses to AL and ASAQ in Sélingué, Mali., Methods: Children between 6 and 59 months of age with uncomplicated Plasmodium falciparum infection and 2000-200,000 asexual parasites/μL of blood were enrolled, randomly assigned to either AL or ASAQ, and followed up for 42 days. Uncorrected and PCR-corrected efficacy results at days 28 and 42. were calculated. Known markers of resistance in the Pfk13, Pfmdr1, and Pfcrt genes were assessed using Sanger sequencing., Results: A total of 449 patients were enrolled: 225 in the AL group and 224 in the ASAQ group. Uncorrected efficacy at day 28 was 83.4% (95% CI 78.5-88.4%) in the AL arm and 93.1% (95% CI 89.7-96.5%) in the ASAQ arm. The per protocol PCR-corrected efficacy at day 28 was 91.0% (86.0-95.9%) in the AL arm and 97.1% (93.6-100%) in the ASAQ arm. ASAQ was significantly (p < 0.05) better than AL for each of the aforementioned efficacy outcomes. No mutations associated with artemisinin resistance were identified in the Pfk13 gene. Overall, for Pfmdr1, the N86 allele and the NFD haplotype were the most common. The NFD haplotype was significantly more prevalent in the post-treatment than in the pre-treatment isolates in the AL arm (p < 0.01) but not in the ASAQ arm. For Pfcrt, the CVIET haplotype was the most common., Conclusions: The findings indicate that both AL and ASAQ remain effective for the treatment of uncomplicated malaria in Sélingué, Mali.

Background: As malaria incidence and transmission in a region decreases, it becomes increasingly difficult to identify areas of active transmission. Improved methods for identifying and monitoring foci of active malaria transmission are needed in areas of low parasite prevalence in order to achieve malaria elimination. Serological assays can provide population-level infection history to inform elimination campaigns., Methods: A bead-based multiplex antibody detection assay was used to evaluate a chimeric Plasmodium vivax MSP1 protein (PvRMC-MSP1), designed to be broadly immunogenic for use in vaccine studies, to act as a pan-malaria serological tool based on its ability to capture IgG in plasma samples obtained from naturally exposed individuals. Samples from 236 US travellers with PCR confirmed infection status from all four major Plasmodium species infecting humans, Plasmodium falciparum (n = 181), Plasmodium vivax (n = 38), Plasmodium malariae (n = 4), and Plasmodium ovale (n = 13) were tested for IgG capture using PvRMC-MSP1 as well as the four recombinant MSP1-19 kD isoforms representative of these Plasmodium species., Results: Regardless of infecting Plasmodium species, a large proportion of plasma samples from infected US travellers provided a high assay signal to the PvRMC-MSP1 chimeric protein, with 115 high responders out of 236 samples assessed (48.7%). When grouped by active infection, 38.7% P. falciparum-, 92.1% of P. vivax-, 75.0% P. malariae-, and 53.4% of P. ovale-infected individuals displayed high assay signals in response to PvRMC-MSP1. It was also determined that plasma from P. vivax-infected individuals produced increased assay signals in response to the PvRMC-MSP1 chimera as compared to the recombinant PvMSP1 for 89.5% (34 out of 38) of individuals. PvRMC-MSP1 also showed improved ability to capture IgG antibodies from P. falciparum-infected individuals when compared to the capture by recombinant PvMSP1, with high assay signals observed for 38.7% of P. falciparum-infected travellers in response to PvRMC-MSP1 IgG capture compared to just 1.1% who were high responders to capture by the recombinant PvMSP1 protein., Conclusions: These results support further study of designed antigens as an approach for increasing sensitivity or broadening binding capacity to improve existing serological tools for determining population-level exposure to Plasmodium species. Including both broad-reacting and Plasmodium species-specific antigen-coated beads in an assay panel could provide a nuanced view of population-level exposure histories, an extensive IgG profile, and detailed seroestimates. A more sensitive serological tool for detection of P. vivax exposure would aid malaria elimination campaigns in co-endemic areas and regions where P. vivax is the dominant parasite.

Background: In 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated., Methods: This study was carried out in two sites where the transmission of P. falciparum malaria is hyper-endemic: Klouékanmey and Djougou. Blood samples were collected from 178 febrile children 6-59 months old with confirmed uncomplicated P. falciparum malaria and were genotyped for SNPs associated with SP resistance., Results: The Pfdhfr triple mutant IRN (N51I, C59R, and S108N) was the most prevalent (84.6%) haplotype and was commonly found with the Pfdhps single mutant A437G (50.5%) or with the Pfdhps double mutant S436A and A437G (33.7%). The quintuple mutant, Pfdhfr IRN/Pfdhps GE (A437G and K540E), was rarely observed (0.8%). The A581G and A613S mutant alleles were found in 2.6 and 3.9% of isolates, respectively. Six isolates (3.9%) were shown to harbour a mutation at codon I431V, recently identified in West African parasites., Conclusions: This study showed that Pfdhfr triple IRN mutants are near fixation in this population and that the highly sulfadoxine-resistant Pfdhps alleles are not widespread in Benin. These data support the continued use of SP for chemoprevention in these study sites, which should be complemented by periodic nationwide molecular surveillance to detect emergence of resistant genotypes.

Background: Histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic tests (RDTs) are effective and widely used for the detection of wild-type Plasmodium falciparum infections. Although recent studies have reported false negative HRP2 RDT results due to pfhrp2 and pfhrp3 gene deletions in different countries, there is a paucity of data on the deletions of these genes in Tanzania., Methods: A community-based cross-sectional survey was conducted between July and November 2017 in four regions: Geita, Kigoma, Mtwara and Ruvuma. All participants had microscopy and RDT performed in the field and provided a blood sample for laboratory multiplex antigen detection (for Plasmodium lactate dehydrogenase, aldolase, and P. falciparum HRP2). Samples showing RDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped to detect the presence/absence of pfhrp2/3 genes., Results: Of all samples screened by the multiplex antigen assay (n = 7543), 2417 (32.0%) were positive for any Plasmodium antigens while 5126 (68.0%) were negative for all antigens. The vast majority of the antigen positive samples contained HRP2 (2411, 99.8%), but 6 (0.2%) had only pLDH and/or aldolase without HRP2. Overall, 13 samples had an atypical relationship between a pan-Plasmodium antigen and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 RDT results but P. falciparum positive by microscopy were also chosen for pfhrp2/3 genotyping. The summation of false negative RDT results and laboratory antigen results provided 35 total samples with confirmed P. falciparum DNA for pfhrp2/3 genotyping. Of the 35 samples, 4 (11.4%) failed to consistently amplify positive control genes; pfmsp1 and pfmsp2 and were excluded from the analysis. The pfhrp2 and pfhrp3 genes were successfully amplified in the remaining 31 (88.6%) samples, confirming an absence of deletions in these genes., Conclusions: This study provides evidence that P. falciparum parasites in the study area have no deletions of both pfhrp2 and pfhrp3 genes. Although single gene deletions could have been missed by the multiplex antigen assay, the findings support the continued use of HRP2-based RDTs in Tanzania for routine malaria diagnosis. There is a need for the surveillance to monitor the status of pfhrp2 and/or pfhrp3 deletions in the future.

Background: With increasing interest in eliminating malaria from the Caribbean region, Haiti is one of the two countries on the island of Hispaniola with continued malaria transmission. While the Haitian population remains at risk for malaria, there are a limited number of cases annually, making conventional epidemiological measures such as case incidence and prevalence of potentially limited value for fine-scale resolution of transmission patterns and trends. In this context, genetic signatures may be useful for the identification and characterization of the Plasmodium falciparum parasite population in order to identify foci of transmission, detect outbreaks, and track parasite movement to potentially inform malaria control and elimination strategies., Methods: This study evaluated the genetic signals based on analysis of 21 single-nucleotide polymorphisms (SNPs) from 462 monogenomic (single-genome) P. falciparum DNA samples extracted from dried blood spots collected from malaria-positive patients reporting to health facilities in three southwestern Haitian departments (Nippes, Grand'Anse, and Sud) in 2016., Results: Assessment of the parasite genetic relatedness revealed evidence of clonal expansion within Nippes and the exchange of parasite lineages between Nippes, Sud, and Grand'Anse. Furthermore, 437 of the 462 samples shared high levels of genetic similarity-at least 20 of 21 SNPS-with at least one other sample in the dataset., Conclusions: These results revealed patterns of relatedness suggestive of the repeated recombination of a limited number of founding parasite types without significant outcrossing. These genetic signals offer clues to the underlying relatedness of parasite populations and may be useful for the identification of the foci of transmission and tracking of parasite movement in Haiti for malaria elimination.

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.

Background: Chile is one of the South American countries certified as malaria-free since 1945. However, the recent increase of imported malaria cases and the presence of the vector Anopheles pseudopunctipennis in previously endemic areas in Chile require an active malaria surveillance programme., Methods: Specimens from 268 suspected malaria cases-all imported-collected between 2015 and 2018 at the Public Health Institute of Chile (ISP), were diagnosed by microscopy and positive cases were included for epidemiological analysis. A photo-induced electron transfer fluorogenic primer real-time PCR (PET-PCR) was used to confirm the presence of malaria parasites in available blood samples. Sanger sequencing of drug resistance molecular markers (pfk13, pfcrt and pfmdr1) and microsatellite (MS) analysis were performed in confirmed Plasmodium falciparum samples and results were related to origin of infection., Results: Out of the 268 suspected cases, 65 were Plasmodium spp. positive by microscopy. A total of 63% of the malaria patients were male and 37% were female; 43/65 of the patients acquired infections in South American endemic countries. Species confirmation of available blood samples by PET-PCR revealed that 15 samples were positive for P. falciparum, 27 for Plasmodium vivax and 4 were mixed infections. The P. falciparum samples sequenced contained four mutant pfcrt genotypes (CVMNT, CVMET, CVIET and SVMNT) and three mutant pfmdr1 genotypes (Y184F/S1034C/N1042D/D1246Y, Y184F/N1042D/D1246Y and Y184F). MS analysis confirmed that all P. falciparum samples presented different haplotypes according to the suspected country of origin. Four patients with P. vivax infection returned to the health facilities due to relapses., Conclusion: The timely detection of polymorphisms associated with drug resistance will contribute to understanding if current drug policies in the country are appropriate for treatment of imported malaria cases and provide information about the most frequent resistant genotypes entering Chile.

Background: Serological data indicating the presence and level of antibodies against infectious disease antigens provides indicators of exposure and transmission patterns in a population. Laboratory testing for large-scale serosurveys is often hindered by time-consuming immunoassays that employ multiple tandem steps. Some nations have recently begun using malaria serosurveillance data to make inferences about the malaria exposure in their populations, and serosurveys have grown increasingly larger as more accurate estimates are desired. Presented here is a novel approach of antibody detection using bead-based immunoassay that involves incubating all assay reagents concurrently overnight., Results: A serosurvey in was performed in Haiti in early 2017 with both sera (n = 712) and dried blood spots (DBS, n = 796) collected for the same participants. The Luminex ® multiplex bead-based assay (MBA) was used to detect total IgG against 8 malaria antigens: PfMSP1, PvMSP1, PmMSP1, PfCSP, PfAMA1, PfLSA1, PfGLURP-R0, PfHRP2. All sera and DBS samples were assayed by MBA using a standard immunoassay protocol with multiple steps, as well a protocol where sample and all reagents were incubated together overnight-termed here the OneStep assay. When compared to a standard multi-step assay, this OneStep assay amplified the assay signal for IgG detection for all 8 malaria antigens. The greatest increases in assay signal were seen at the low- and mid-range IgG titers and were indicative of an enhancement in the analyte detection, not simply an increase in the background signal of the assay. Seroprevalence estimates were generally similar for this sample Haitian population for all antigens regardless of serum or DBS sample type or assay protocol used., Conclusions: When using the MBA for IgG detection, overnight incubation for the test sample and all assay reagents greatly minimized hands-on time for laboratory staff. Enhanced IgG signal was observed with the OneStep assay for all 8 malaria antigens employed in this study, and seroprevalence estimates for this sample population were similar regardless of assay protocol used. This overnight incubation protocol has the potential to be deployed for large-scale malaria serosurveys for the high-throughput and timely collection of antibody data, particularly for malaria seroprevalence estimates.

Background: The Plasmodium falciparum parasite is the only human malaria that produces the histidine-rich protein 2 and 3 (HRP2/3) antigens. Currently, HRP2/3 are widely used in malaria rapid diagnostic tests (RDTs), but several global reports have recently emerged showing genetic deletion of one or both of these antigens in parasites. Deletion of these antigens could pose a major concern for P. falciparum diagnosis in Haiti which currently uses RDTs based solely on the detection of the HRP2/3 antigens., Methods: From September 2012 through February 2014, dried blood spots (DBS) were collected in Haiti from 9317 febrile patients presenting to 17 health facilities in 5 departments throughout the country as part of a bed net intervention study. All DBS from RDT positive persons and a random sampling of DBS from RDT negative persons were assayed for P. falciparum DNA by nested and PET-PCR (n = 2695 total). All PCR positive samples (n = 331) and a subset of PCR negative samples (n = 95) were assayed for three malaria antigens by a multiplex bead assay: pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and HRP2/3. Any samples positive for P. falciparum DNA, but negative for HRP2/3 antigens were tested by nested PCR for Pfhrp2 and Pfhrp3 gene deletions., Results: Of 2695 DBS tested for Plasmodium DNA, 345 (12.8%) were originally found to be positive for P. falciparum DNA; 331 of these had DBS available for antigen detection. Of these, 266 (80.4%) were positive for pAldo, 221 (66.8%) positive for pLDH, and 324 (97.9%) were positive for HRP2/3 antigens. Seven samples (2.1%) positive for P. falciparum DNA were not positive for any of the three antigens by the bead assay, and were investigated for potential Pfhrp2/3 gene deletion by PCR. These samples either successfully amplified Pfhrp2/3 genes or were at an estimated parasite density too low for sufficient DNA to perform successful genotyping., Conclusions: Malaria positive samples in multiple Haitian sites were found to contain the HRP2/3 antigens, and no evidence was found of Pfhrp2/3 deletions. Malaria RDTs based on the detection of the HRP2/3 antigens remain a reliable P. falciparum diagnostic tool as Haiti works towards malaria elimination.

Background: The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance., Methods: Eligible patients were enrolled at the four sites, treated with standard doses of AL, and monitored for 28 days with clinical and laboratory assessments. The main outcomes were PCR corrected cure rates, day 3 positivity rates, safety of AL, and prevalence of single nucleotide polymorphisms in Plasmodium falciparum kelch 13 (Pfk13) (codon positions: 440-600) and P. falciparum multi-drug resistance 1 (Pfmdr1) genes (codons: N86Y, Y184F and D1246Y), markers of artemisinin and lumefantrine resistance, respectively., Results: Of 344 patients enrolled, three withdrew, six were lost to follow-up; and results were analysed for 335 (97.4%) patients. Two patients had treatment failure (one early treatment failure and one recrudescent infection) after PCR correction, yielding an adequate clinical and parasitological response of > 98%. Day 3 positivity rates ranged from 0 to 5.7%. Common adverse events included cough, abdominal pain, vomiting, and diarrhoea. Two patients had serious adverse events; one died after the first dose of AL and another required hospitalization after the second dose of AL (on day 0) but recovered completely. Of 344 samples collected at enrolment (day 0), 92.7% and 100% were successfully sequenced for Pfk13 and Pfmdr1 genes, respectively. Six (1.9%) had non-synonymous mutations in Pfk13, none of which had been previously associated with artemisinin resistance. For Pfmdr1, the NFD haplotype (codons N86, 184F and D1246) was detected in 134 (39.0%) samples; ranging from 33.0% in Mlimba to 45.5% at Mkuzi. The difference among the four sites was not significant (p = 0.578). All samples had a single copy of the Pfmdr1 gene., Conclusion: The study indicated high efficacy of AL and the safety profile was consistent with previous reports. There were no known artemisinin-resistance Pfk13 mutations, but there was a high prevalence of a Pfmdr1 haplotype associated with reduced sensitivity to lumefantrine (but no reduced efficacy was observed in the subjects). Continued TES and monitoring of markers of resistance to artemisinin and partner drugs is critical for early detection of resistant parasites and to inform evidence-based malaria treatment policies. Trial Registration ClinicalTrials.gov NCT03387631.

Background: The Angolan government recommends three artemisinin-based combinations for the treatment of uncomplicated Plasmodium falciparum malaria: artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Due to the threat of emerging anti-malarial drug resistance, it is important to periodically monitor the efficacy of artemisinin-based combination therapy (ACT). This study evaluated these medications' therapeutic efficacy in Benguela, Lunda Sul, and Zaire Provinces., Methods: Enrollment occurred between March and July 2017. Study participants were children with P. falciparum monoinfection from each provincial capital. Participants received a 3-day course of a quality-assured artemisinin-based combination and were monitored for 28 (AL and ASAQ arms) or 42 days (DP arm). Each ACT was assessed in two provinces. The primary study endpoints were: (1) follow-up without complications and (2) failure to respond to treatment or development of recurrent P. falciparum infection. Parasites from each patient experiencing recurrent infection were genotyped to differentiate new infection from recrudescence of persistent parasitaemia. These parasites were also analysed for molecular markers associated with ACT resistance., Results: Of 608 children enrolled in the study, 540 (89%) reached a primary study endpoint. Parasitaemia was cleared within 3 days of medication administration in all participants, and no early treatment failures were observed. After exclusion of reinfections, the corrected efficacy of AL was 96% (91-100%, 95% confidence interval) in Zaire and 97% (93-100%) in Lunda Sul. The corrected efficacy of ASAQ was 100% (97-100%) in Benguela and 93% (88-99%) in Zaire. The corrected efficacy of DP was 100% (96-100%) in Benguela and 100% in Lunda Sul. No mutations associated with artemisinin resistance were identified in the pfk13 gene in the 38 cases of recurrent P. falciparum infection. All 33 treatment failures in the AL and ASAQ arms carried pfmdr1 or pfcrt mutations associated with lumefantrine and amodiaquine resistance, respectively, on day of failure., Conclusions: AL, ASAQ, and DP continue to be efficacious against P. falciparum malaria in these provinces of Angola. Rapid parasite clearance and the absence of genetic evidence of artemisinin resistance are consistent with full susceptibility to artemisinin derivatives. Periodic monitoring of in vivo drug efficacy remains a priority routine activity for Angola.

Background: Artemisinin-based combination therapy is the first-line anti-malarial treatment for uncomplicated Plasmodium falciparum infection in Angola. To date, the prevalence of polymorphisms in the pfk13 gene, associated with artemisinin resistance, and pfmdr1, associated with lumefantrine resistance, have not been systematically studied in Angola., Methods: DNA was isolated from pretreatment and late treatment failure dried blood spots collected during the 2015 round of therapeutic efficacy studies in Benguela, Lunda Sul, and Zaire Provinces in Angola. The pfk13 propeller domain and pfmdr1 gene were sequenced and analysed for polymorphisms. Pfmdr1 copy number variation was assessed using a real-time PCR method. The association between pfmdr1 and pfk13 mutations and treatment failure was investigated., Results: The majority of pretreatment (99%, 466/469) and all late treatment failure (100%, 50/50) samples were wild type for pfk13. Three of the pretreatment samples (1%) carried the A578S mutation commonly observed in Africa and not associated with artemisinin resistance. All 543 pretreatment and day of late treatment failure samples successfully analysed for pfmdr1 copy number variation carried one copy of pfmdr1. The NYD haplotype was the predominant pfmdr1 haplotype, present in 63% (308/491) of pretreatment samples, followed by NFD, which was present in 32% (157/491) of pretreatment samples. The pfmdr1 N86 allele was overrepresented in day of late treatment failure samples from participants receiving artemether-lumefantrine (p value 0.03)., Conclusions: The pretreatment parasites in patients participating in therapeutic efficacy studies in 2015 in Angola's three sentinel sites showed genetic evidence of susceptibility to artemisinins, consistent with clinical outcome data showing greater than 99% day 3 clearance rates. The lack of increased pfmdr1 copy number is consistent with previous reports from sub-Saharan Africa. Although pfmdr1 NYD and NFD haplotypes were overrepresented in artemether-lumefantrine late treatment failure samples, their role as markers of resistance was unclear given that these haplotypes were also present in the majority of successfully treated patients in the artemether-lumefantrine treatment arms.

Background: Rapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys., Methods: Individuals' blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey., Results: There was a sigmoidal dose-response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD., Conclusions: Measures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration.

Background: Recent anti-malarial resistance monitoring in Angola has shown efficacy of artemether-lumefantrine (AL) in certain sites approaching the key 90% lower limit of efficacy recommended for artemisinin-based combination therapy. In addition, a controversial case of malaria unresponsive to artemisinins was reported in a patient infected in Lunda Sul Province in 2013., Methods: During January-June 2015, investigators monitored the clinical and parasitological response of children with uncomplicated Plasmodium falciparum infection treated with AL, artesunate-amodiaquine (ASAQ), or dihydroartemisinin-piperaquine (DP). The study comprised two treatment arms in each of three provinces: Benguela (AL, ASAQ), Zaire (AL, DP), and Lunda Sul (ASAQ, DP). Samples from treatment failures were analysed for molecular markers of resistance for artemisinin (K13) and lumefantrine (pfmdr1)., Results: A total of 467 children reached a study endpoint. Fifty-four treatment failures were observed: four early treatment failures, 40 re-infections and ten recrudescences. Excluding re-infections, the 28-day microsatellite-corrected efficacy was 96.3% (95% CI 91-100) for AL in Benguela, 99.9% (95-100) for ASAQ in Benguela, 88.1% (81-95) for AL in Zaire, and 100% for ASAQ in Lunda Sul. For DP, the 42-day corrected efficacy was 98.8% (96-100) in Zaire and 100% in Lunda Sul. All treatment failures were wild type for K13, but all AL treatment failures had pfmdr1 haplotypes associated with decreased lumefantrine susceptibility., Conclusions: No evidence was found to corroborate the specific allegation of artemisinin resistance in Lunda Sul. The efficacy below 90% of AL in Zaire matches findings from 2013 from the same site. Further monitoring, particularly including measurement of lumefantrine blood levels, is recommended.

Background: Plasmodium ovale is rarely described in Senegal. A case of clinical malaria due to P. ovale wallikeri in West Central of Senegal is reported., Case: A 34-year-old male baker in Dakar, with no significant previous medical history, was admitted to a health clinic with fever and vomiting. Fever had been lasting for 4 days with peaks every 48 h. As monospecific Plasmodium falciparum HRP-2 RDT was negative, he was treated with antibiotics. However, owing to persisting symptoms, he was referred to the emergency unit of the Youssou Mbargane Diop Hospital, Dakar, Senegal. Clinical examination found impaired general condition. All other physical examinations were normal. Laboratory tests showed anaemia (haemoglobin 11.4 g/dl), severe thrombocytopaenia (platelets 30 × 10(9)/mm(3)), leukopenia (3650/mm(3)), lymphocytopenia (650/mm(3)). Renal function was normal as indicated by creatininaemia and uraemia (11 mg/l and 0.25 g/l, respectively) and liver enzymes were slightly elevated (aspartate aminotransferase 77 UI/l and alanine aminotransferase 82 UI/l). Blood smear evaluations in Parasitology Laboratory of Aristide Le Dantec Hospital showed malaria parasites of the species P. ovale with a 0.08 % parasitaemia. Molecular confirmation was done by real time PCR targeting the 18S rRNA gene. The P. ovale infection was further analysed to species level targeting the potra gene and was identified as P. ovale wallikeri. According to the hospital's malaria treatment guidelines for severe malaria, treatment consisted of intravenous quinine at hour 0 (start of treatment) and 24 h after initial treatment, followed by artemether-lumefantrine 24 h later. A negative microscopy was noted on day 3 post-treatment and the patient reported no further symptoms., Conclusion: Malaria due to non-falciparum species is probably underestimated in Senegal. RDTs specific to non-falciparum species and/or pan specific RDTs should be included as tools of diagnosis to fight against malaria in Senegal. In addition, a field-deployable molecular tool such as the loop-mediated isothermal amplification can be considered as an additional useful tool to detect low malaria parasite infections and for speciation. In addition, national malaria control policies should consider other non-falciparum species in treatment guidelines, including the provision of primaquine for the treatment of relapsing parasites.

Background: Determining the source of malaria outbreaks in Ecuador and identifying remaining transmission foci will help in malaria elimination efforts. In this study, the genetic signatures of Plasmodium falciparum isolates, obtained from an outbreak that occurred in northwest Ecuador from 2012 to 2013, were characterized., Methods: Molecular investigation of the outbreak was performed using neutral microsatellites, drug resistance markers and pfhrp2 and pfhrp3 genotyping., Results: A majority of parasite isolates (31/32) from this outbreak were of a single clonal type that matched a clonal lineage previously described on the northern coast of Peru and a historical isolate from Ecuador. All but one isolate carried a chloroquine-resistant pfcrt genotype and sulfadoxine- and pyrimethamine-sensitive pfdhps and pfdhfr genotypes. Pfmdr1 mutations were identified in codons 184 and 1042. In addition, most samples (97 %) showed presence of pfhrp2 gene., Conclusions: This study indicates that parasites from a single clonal lineage largely contributed to this outbreak and this lineage was found to be genetically related to a lineage previously reported in the Peruvian coast and historical Ecuadorian parasites.

Background: As a nation reduces the burden of falciparum malaria, identifying areas of transmission becomes increasingly difficult. Over the past decade, the field of utilizing malaria serological assays to measure exposure has grown rapidly, and a variety of serological methods for data acquisition and analysis of human IgG against falciparum antigens are available. Here, different immunoassays and statistical methods are utilized to analyse samples from a low transmission setting and directly compare the estimates generated., Methods: A subset of samples (n = 580) from a 2012 Haitian nationwide malaria survey was employed as sample population of low falciparum endemicity. In addition to the Haitian samples, samples from 247 US residents were used as a reference population of 'true seronegatives'. Data acquisition was performed through standard ELISA and bead-based multiplex assays assaying for IgG antibodies to the Plasmodium falciparum antigens MSP-1p19, MSP-1p42(D), MSP-1p42(F), and AMA-1. Appropriate parametric distributions and seropositivity cutoff values were determined by statistical measures., Results: Data from both assays showed a strong positive skew, and the lognormal distribution was found to be an appropriate statistical fit to the Haitian and American populations. The American samples served as a good serological true negative population for the multiplex assay, but not for ELISA-based data. Mixture model approaches to determine seronegative and seropositive populations from the Haitian data showed a high degree of distribution overlap-likely due to the historical low falciparum transmission in this nation. Different fittings to the reversible catalytic model resulted depending upon the immunoassay utilized and seropositivity cutoff method employed. Data were also analysed through fitting to penalized B-splines, presenting another possible analytical tool for the analysis of malaria serological data., Conclusions: Standardization of serological techniques and analyses may prove difficult as some tools can prove to be more useful depending on the area and parasite in question, making clear interpretation a vital pursuit. The presented analysis in the low-endemic nation of Haiti found malaria-naive US residents to be an appropriate seronegative reference population for the multiplex assay, and this assay providing consistent estimates between MSP-1 and AMA-1 antigens of percent seropositives for this low-endemic population.

Background: The World Health Organization (WHO) recommends parasitologic confirmation of suspected malaria cases before treatment. Due to the limited availability of quality microscopy services, this recommendation has become scalable following increased use of antigen-detecting malaria rapid diagnostic tests (RDTs) in many malaria-endemic countries. This study was carried out to monitor quality of RDT performance in selected health facilities using two quality assurance (QA) methods: reference microscopy and detection of parasite DNA by real-time quantitative polymerase chain reaction (qPCR) on dried blood spots (DBS)., Methods: Blood samples for QA were collected from patients undergoing RDT for diagnostic confirmation of malaria during two to three consecutive days per month in 12 health facilities in rural Tanzania. Stained blood smears (BS) were first examined at the district hospitals (BS1) and then at a reference laboratory (BS2). Discordant BS1 and BS2 results prompted a third examination. Molecular analysis was carried out at the Ifakara Health Institute laboratory in Bagamoyo., Results: Malaria RDTs had a higher positivity rate (6.5%) than qPCR (4.2%) or microscopy (2.9% for BS1 and 2.5% for BS2). Poor correlation was observed between RDT and BS results: BS1 (K = 0.5), BS2 (K = 0.43) and qPCR (K = 0.45), challenging the utility of these tests for RDT QA. In addition, many challenges related to qPCR processing were recorded and long delays in obtaining QA test results for both microscopy and qPCR., Conclusions: Overall there was limited agreement among the three diagnostic approaches and neither microscopy nor qPCR appear to be good QA options for RDTs under field conditions.

Background: Recent studies have demonstrated the deletion of the histidine-rich protein 2 (PfHRP2) gene (pfhrp2) in field isolates of Plasmodium falciparum, which could result in false negative test results when PfHRP2-based rapid diagnostic tests (RDTs) are used for malaria diagnosis. Although primary diagnosis of malaria in Honduras is determined based on microscopy, RDTs may be useful in remote areas. In this study, it was investigated whether there are deletions of the pfhrp2, pfhrp3 and their respective flanking genes in 68 P. falciparum parasite isolates collected from the city of Puerto Lempira, Honduras. In addition, further investigation considered the possible correlation between parasite population structure and the distribution of these gene deletions by genotyping seven neutral microsatellites., Methods: Sixty-eight samples used in this study, which were obtained from a previous chloroquine efficacy study, were utilized in the analysis. All samples were genotyped for pfhrp2, pfhrp3 and flanking genes by PCR. The samples were then genotyped for seven neutral microsatellites in order to determine the parasite population structure in Puerto Lempira at the time of sample collection., Results: It was found that all samples were positive for pfhrp2 and its flanking genes on chromosome 8. However, only 50% of the samples were positive for pfhrp3 and its neighboring genes while the rest were either pfhrp3-negative only or had deleted a combination of pfhrp3 and its neighbouring genes on chromosome 13. Population structure analysis predicted that there are at least two distinct parasite population clusters in this sample population. It was also determined that a greater proportion of parasites with pfhrp3-(and flanking gene) deletions belonged to one cluster compared to the other., Conclusion: The findings indicate that the P. falciparum parasite population in the municipality of Puerto Lempira maintains the pfhrp2 gene and that PfHRP2-based RDTs could be considered for use in this region; however continued monitoring of parasite population will be useful to detect any parasites with deletions of pfhrp2.

Background: Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated., Methods: DNA from the dried blood spots was extracted using QIAGEN methodology. All 2,989 samples were screened using the PET-PCR assay in duplicate. Samples with a cycle threshold (CT) of 40 or less were scored as positive. A subset of the total samples (534) was retested using a nested PCR assay for confirmation. In addition, these same samples were also tested using a TaqMan-based real-time PCR assay., Results: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7). These same samples were also found to be positive by the nested and TaqMan-based methods. The nested PCR detected an additional positive sample in a subset of 534 samples that was not detected by either PET-PCR or TaqMan-based PCR method., Conclusion: While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples. Given the ease of use and lower cost than the nested PCR, the PET-PCR provides an alternative assay for the rapid screening of a large number of samples in laboratory settings.

Background: Few studies have assessed placental malaria infections from low transmission areas by histopathology to define their impact and underlying mechanisms., Methods: Peripheral smears and rapid diagnostic tests (RDTs), placental smears and histological samples, birth weight and gestational age were collected from 2,282 deliveries in three hospitals during a one-year (2006-2007) continuous cross-sectional survey in Madhya Pradesh. Placental histopathology included all 50 cases positive by microscopy or RDT plus 456 randomly selected samples of women negative for malaria by microscopy or RDT. Histological examination included parasites, inflammatory cells, pigment in fibrin, and morphological changes., Results: There were 52 histology-positive cases; 38 (73.1%) active (acute and chronic) and 14 past infections. Intervillous parasitaemia was low (60% had < 1% parasitaemia) and monocytosis mostly mild (63%). Compared with uninfected placentas, acute Plasmodium falciparum infections were associated with stillbirth (RR 3.8, 95% CI 1.2-12.1), lower maternal haemoglobin (mean difference: 1.5 g/dL, 95% CI 0.5-2.5), lower birth weight (mean difference 451 g, 95% CI 169-609) and shorter gestation (mean difference 0.8 weeks, 95% CI 0.2-1.4). Chronic or past infections were not associated with these outcomes. Among the 11 peripheral Plasmodium vivax cases, placental parasites were absent, but they were associated with increased placental polymorphonuclear cells., Conclusions: Malaria associated stillbirth and low birth weight in women with low protective immunity may result, at least in part, from a shortened gestation triggered by acute infection, stressing the importance of early malaria detection.

Background: Plasmodium vivax is a predominant species of malaria in parts of South America and there is increasing resistance to drugs to treat infections by P. vivax. The existence of latent hypnozoites further complicates the ability to classify recurrent infections as treatment failures due to relapse, recrudescence of hyponozoites or re-infections. Antigen loci are putatively under natural selection and may not be an optimal molecular marker to define parasite haplotypes in paired samples. Putatively neutral microsatellite loci, however, offer an assessment of neutral haplotypes. The objective here was to assess the utility of neutral microsatellite loci to reconcile cases of recurrent parasitaemia in Amazonian P. vivax populations in Peru., Methods: Patient blood samples were collected from three locations in or around Iquitos in the Peruvian Amazon. Five putatively neutral microsatellite loci were characterized from 445 samples to ascertain the within and amongst population variation. A total of 30 day 0 and day of recurrent parasitaemia samples were characterized at microsatellite loci and five polymorphic antigen loci for haplotype classification., Results: The genetic diversity at microsatellite loci was consistent with neutral levels of variation measured in other South American P. vivax populations. Results between antigen and microsatellite loci for the 30 day 0 and day of recurrent parasitaemia samples were the same for 80% of the pairs. The majority of non-concordant results were the result of differing alleles at microsatellite loci. This analysis estimates that 90% of the paired samples with the same microsatellite haplotype are unlikely to be due to a new infection., Conclusions: A population-level approach was used to yield a better estimate of the probability of a new infection versus relapse or recrudescence of homologous hypnozoites; hypnozoite activation was common for this cohort. Population studies are critical with the evaluation of genetic markers to assess P. vivax biology and epidemiology. The additional demonstration of microsatellite loci as neutral markers capable of distinguishing the origin of the recurrent parasites (new infection or originating from the patient) lends support to their use in assessment of treatment outcomes.

Background: Accurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country's diagnostic laboratory; and, (ii) determine the assay's sensitivity and specificity compared to a nested 18S rRNA PCR., Methods: Samples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR., Results: Compared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory in Tanzania were in 100% concordance with the results obtained at the reference laboratory in the USA., Conclusion: The PET-PCR is a new molecular diagnostic tool with similar performance characteristics as commonly used PCR methods that is less expensive, easy to use, and amiable to large scale-surveillance studies in developing country settings.

Background: Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy., Methods: A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator., Results: Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC50 value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs., Conclusion: Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC50 value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended.

Background: Malaria elimination is being pursued in five of seven Central American countries. Military personnel returning from peacekeeping missions in sub-Saharan Africa could import chloroquine-resistant Plasmodium falciparum, posing a threat to elimination and to the continued efficacy of first-line chloroquine (CQ) treatment in these countries. This report describes the importation of P. falciparum from among 150 Guatemalan army special forces and support staff who spent ten months on a United Nations' peacekeeping mission in the Democratic Republic of the Congo (DRC) in 2010., Methods: Investigators reviewed patients' medical charts and interviewed members of the contingent to identify malaria cases and risk factors for malaria acquisition. Clinical specimens were tested for malaria; isolated parasites were characterized molecularly for CQ resistance., Results: Investigators identified 12 cases (8%) of laboratory-confirmed P. falciparum infection within the contingent; one case was from a soldier infected with a CQ-resistant pfcrt genotype resulting in his death. None of the contingent used an insecticide-treated bed net (ITN) or completely adhered to malaria chemoprophylaxis while in the DRC., Conclusion: This report highlights the need to promote use of malaria prevention measures, in particular ITNs and chemoprophylaxis, among peacekeepers stationed in malaria-endemic areas. Countries attempting to eliminate malaria should consider appropriate methods to screen peacekeepers returning from endemic areas for malaria infections. Cases of malaria in travellers, immigrants and soldiers returning to Central America from countries with CQ-resistant malaria should be assumed to be carry resistant parasites and receive appropriate anti-malarial therapy to prevent severe disease and death.

Malaria has been part of Peruvian life since at least the 1500s. While Peru gave the world quinine, one of the first treatments for malaria, its history is pockmarked with endemic malaria and occasional epidemics. In this review, major increases in Peruvian malaria incidence over the past hundred years are described, as well as the human factors that have facilitated these events, and concerted private and governmental efforts to control malaria. Political support for malaria control has varied and unexpected events like vector and parasite resistance have adversely impacted morbidity and mortality. Though the ready availability of novel insecticides like DDT and efficacious medications reduced malaria to very low levels for a decade after the post eradication era, malaria reemerged as an important modern day challenge to Peruvian public health. Its reemergence sparked collaboration between domestic and international partners towards the elimination of malaria in Peru.

Background: Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras., Methods: A total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrollment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species., Results: Of the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite., Conclusions: Molecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.

Background: Understanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions., Methods: This study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (dhfr) and dihydropterote synthase (dhps) genes. Microsatellite alleles spanning 138 kb around dhfr and dhps, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized., Results: By 1992, the South-Asian dhfr triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, dhfr triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, dhps double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two dhfr mutant alleles and the possible emergence of a selective sweep of double mutant dhps alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various dhfr and dhps mutants relative to each other based on a theoretical model tailored to P. falciparum. The data indicate that drug selection acted differently on the resistant alleles of dhfr and dhps, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate., Conclusions: The complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on Plasmodium falciparum populations.

Background: The mechanisms underlying the pathogenesis of cerebral malaria (CM) syndrome are not well understood. Previous studies have shown a strong association of inflammatory chemokines, apoptotic markers and angiogenic molecules with CM associated mortality. Recognizing the importance of angiopoietins (ANG) in the pathogenesis of CM, a retrospective investigation was carried out in a hospital cohort of malaria patients with Plasmodium infection in central India to determine if these factors could be suitable markers of CM associated severity., Methods: Patients enrolled in the study were clinically characterized as healthy controls (HC), mild malaria (MM), CM survivors (CMS) and CM non-survivors (CMNS) based on their malaria status and hospital treatment outcome. Plasma ANG-1 and ANG-2 levels were assessed using sandwich ELISA. Receiver operating characteristic (ROC) curve analysis was used to calculate area under the curve (AUC) for each biomarker in order to assess predictive accuracy of individual biomarkers., Results: The plasma levels of ANG-1 were lower in CMS and CMNS compared to control groups (mild malaria and healthy controls) at the time of hospital admission. On the contrary, ANG-2 levels positively correlated with malaria severity and were significantly higher in CMNS. The ratio of ANG-2/ANG-1 was highest in CMNS compared to other groups. Receiver operating characteristic curves revealed that compared to ANG-1 (AUC = 0.35), ANG-2 (AUC = 0.95) and ratio of ANG-2/ANG-1 (AUC = 0.90) were better markers to discriminate CMNS from MM cases. However, they were less specific in predicting fatal outcome amongst CM cases at the time of hospital admission., Conclusion: These results suggest that at the time of admission plasma levels of ANG-2 and ratio of ANG-2/ANG-1 are clinically informative biomarkers to predict fatal CM from MM cases while they have limited usefulness in discriminating fatal CM outcomes in a pool of CM cases in endemic settings of Central India.

Background: Malaria in pregnancy in India, as elsewhere, is responsible for maternal anemia and adverse pregnancy outcomes such as low birth weight and preterm birth.It is not known whether prevention and treatment strategies for malaria in pregnancy (case management, insecticide-treated bednets, intermittent preventive therapy) are widely utilized in India., Methods: This cross-sectional study was conducted during 2006-2008 in two states of India, Jharkhand and Chhattisgarh, at 7 facilities representing a range of rural and urban populations and areas of more versus less stable malaria transmission. 280 antenatal visits (40/site) were observed by study personnel coupled with exit interviews of pregnant women to assess emphasis upon, availability and utilization of malaria prevention practices by health workers and pregnant women. The facilities were assessed for the availability of antimalarials, lab supplies and bednets., Results: All participating facilities were equipped to perform malaria blood smears; none used rapid diagnostic tests. Chloroquine, endorsed for chemoprophylaxis during pregnancy by the government at the time of the study, was stocked regularly at all facilities although the quantity stocked varied. Availability of alternative antimalarials for use in pregnancy was less consistent. In Jharkhand, no health worker recommended bednet use during the antenatal visit yet over 90% of pregnant women had bednets in their household. In Chhattisgarh, bednets were available at all facilities but only 14.4% of health workers recommended their use. 40% of the pregnant women interviewed had bednets in their household. Only 1.4% of all households owned an insecticide-treated bednet; yet 40% of all women reported their households had been sprayed with insecticide. Antimalarial chemoprophylaxis with chloroquine was prescribed in only 2 (0.7%) and intermittent preventive therapy prescribed in only one (0.4%) of the 280 observed visits., Conclusions: A disconnect remains between routine antenatal practices in India and known strategies to prevent and treat malaria in pregnancy. Prevention strategies, in particular the use of insecticide-treated bednets, are underutilized. Gaps highlighted by this study combined with recent estimates of the prevalence of malaria during pregnancy in these areas should be used to revise governmental policy and target increased educational efforts among health care workers and pregnant women.

Background: Mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP). Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases., Methods: This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutations in 31 African countries. Maps were created to summarize the location and prevalence of the highly resistant dhfr triple mutant (N51I, C59R, S108N) genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data., Results: A database consisting of dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa., Conclusion: Increasing prevalence of the dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent. Continued assessment of the effectiveness of SP for the treatment of clinical malaria and intermittent preventive treatment in pregnancy is needed. The creation of a centralized resistance data network, such as the one proposed by the WorldWide Antimalarial Resistance Network (WWARN), will become a valuable resource for planning timely actions to combat drug resistant malaria.

Background: One of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed Plasmodium infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails., Findings: Here we evaluated three PCR assays (nested, semi-nested multiplex, and one-tube multiplex) for the simultaneous detection of human malaria parasites using experimentally mixed cocktails of known quantities of laboratory derived DNA. All three assays detected individual species with high sensitivity and specificity when DNA was from any one single species; however, experimentally mixed DNA cocktails with all four species present were correctly identified most consistently with the nested method. The other two methods failed to consistently identify all four species correctly, especially at lower concentrations of DNA -subclinical levels of malaria (DNA equivalent to or less than 10 parasites per microliter)., Conclusions: The nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections. Further optimization and/or new molecular gene targets may improve the success rate of detecting multiple parasite species simultaneously using traditional PCR assays.

Background: Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined., Methods: Fifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya., Results: An increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21)., Conclusions: This study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.

Background: Past studies in India included only symptomatic pregnant women and thus may have overestimated the proportion of women with malaria. Given the large population at risk, a cross sectional study was conducted in order to better define the burden of malaria in pregnancy in Jharkhand, a malaria-endemic state in central-east India., Methods: Cross-sectional surveys at antenatal clinics and delivery units were performed over a 12-month period at two district hospitals in urban and semi-urban areas, and a rural mission hospital. Malaria was diagnosed by Giemsa-stained blood smear and/or rapid diagnostic test using peripheral or placental blood., Results: 2,386 pregnant women were enrolled at the antenatal clinics and 718 at the delivery units. 1.8% (43/2382) of the antenatal clinic cohort had a positive diagnostic test for malaria (53.5% Plasmodium falciparum, 37.2% Plasmodium vivax, and 9.3% mixed infections). Peripheral parasitaemia was more common in pregnant women attending antenatal clinics in rural sites (adjusted relative risk [aRR] 4.31, 95%CI 1.84-10.11) and in those who were younger than 20 years (aRR 2.68, 95%CI 1.03-6.98). Among delivery unit participants, 1.7% (12/717) had peripheral parasitaemia and 2.4% (17/712) had placental parasitaemia. Women attending delivery units were more likely to be parasitaemic if they were in their first or second pregnancy (aRR 3.17, 95%CI 1.32-7.61), had fever in the last week (aRR 5.34, 95%CI 2.89-9.90), or had rural residence (aRR 3.10, 95%CI 1.66-5.79). Malaria control measures including indoor residual spraying (IRS) and untreated bed nets were common, whereas insecticide-treated bed nets (ITN) and malaria chemoprophylaxis were rarely used., Conclusion: The prevalence of malaria among pregnant women was relatively low. However, given the large at-risk population in this malaria-endemic region of India, there is a need to enhance ITN availability and use for prevention of malaria in pregnancy, and to improve case management of symptomatic pregnant women.

Background: Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine implicated in the pathogenesis of a number of human diseases including inflammatory neurological diseases. Its role in the pathogenesis of cerebral malaria is unknown. Cerebral malaria is a life-threatening complication of falciparum malaria with approximately 20%-30% of patients dying despite appropriate anti-malarial treatment. The reason for this cerebral malaria mortality is still unknown although host proinflammatory factors have been shown to be evidently important. The current study investigated the role of circulating MIF in the pathogenesis and outcomes of cerebral malaria., Findings: Three categories of subjects contributed to this study: healthy controls subjects, mild malaria patients, and cerebral malaria patients. The cerebral malaria patients were further grouped into cerebral malaria survivors and cerebral malaria non-survivors. MIF levels in the peripheral blood plasma, obtained at the time of enrollment, were measured using standard ELISA methods. In logistic regression on cerebral malaria patients, log MIF levels were found to be significantly associated with fatal outcome (odds ratio 4.0; 95%CI 1.6, 9.8; p = 0.003). In multinomial logistic regression log MIF levels were found to be significantly associated with patient category (p = 0.004)., Conclusion: This study suggests that elevated levels of MIF in the peripheral blood of cerebral malaria patients may be associated with fatal outcomes.

Background: Plasmodium falciparum infection causes cerebral malaria (CM) in a subset of patients with anti-malarial treatment protecting only about 70% to 80% of patients. Why a subset of malaria patients develops CM complications, including neurological sequelae or death, is still not well understood. It is believed that host immune factors may modulate CM outcomes and there is substantial evidence that cellular immune factors, such as cytokines, play an important role in this process. In this study, the potential relationship between the antibody responses to the merozoite surface protein (MSP)-1 complex (which consists of four fragments namely: MSP-1(83), MSP-1(30), MSP-1(38) and MSP-1(42)), MSP-6(36) and MSP-7(22) and CM was investigated., Methods: Peripheral blood antibody responses to recombinant antigens of the two major allelic forms of MSP-1 complex, MSP-6(36) and MSP-7(22) were compared between healthy subjects, mild malaria patients (MM) and CM patients residing in a malaria endemic region of central India. Total IgG and IgG subclass antibody responses were determined using ELISA method., Results: The prevalence and levels of IgG and its subclasses in the plasma varied for each antigen. In general, the prevalence of total IgG, IgG1 and IgG3 was higher in the MM patients and lower in CM patients compared to healthy controls. Significantly lower levels of total IgG antibodies to the MSP-1(f38), IgG1 levels to MSP-1(d83), MSP-1(19) and MSP-6(36) and IgG3 levels to MSP-1(f42) and MSP-7(22) were observed in CM patients as compared to MM patients., Conclusion: These results suggest that there may be some dysregulation in the generation of antibody responses to some MSP antigens in CM patients and it is worth investigating further whether perturbations of antibody responses in CM patients contribute to pathogenesis.

Background: Plasmodium falciparum in a subset of patients can lead to cerebral malaria (CM), a major contributor to malaria-associated mortality. Despite treatment, CM mortality can be as high as 30%, while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM is mediated by alterations in cytokine and chemokine homeostasis, inflammation as well as vascular injury and repair processes although their roles are not fully understood. The hypothesis for this study is that CM-induced changes in inflammatory, apoptotic and angiogenic factors mediate severity of CM and that their identification will enable development of new prognostic markers and adjunctive therapies for preventing CM mortalities., Methods: Plasma samples (133) were obtained from healthy controls (HC, 25), mild malaria (MM, 48), cerebral malaria survivors (CMS, 48), and cerebral malaria non-survivors (CMNS, 12) at admission to the hospital in Jabalpur, India. Plasma levels of 30 biomarkers ((IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, G-CSF, GM-CSF, IFN-gamma, IP-10, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, Fas-ligand (Fas-L), soluble Fas (sFas), soluble TNF receptor 1 (sTNF-R1) and soluble TNF receptor 2 (sTNFR-2), PDGF bb and VEGF)) were simultaneously measured in an initial subset of ten samples from each group. Only those biomarkers which showed significant differences in the pilot analysis were chosen for testing on all remaining samples. The results were then compared between the four groups to determine their role in CM severity., Results: IP-10, sTNF-R2 and sFas were independently associated with increased risk of CM associated mortality. CMNS patients had a significantly lower level of the neuroprotective factor VEGF when compared to other groups (P < 0.0045). The ratios of VEGF to IP-10, sTNF-R2, and sFas distinguished CM survivors from non survivors (P < 0.0001)., Conclusion: The results suggest that plasma levels of IP-10, sTNF-R2 and sFas may be potential biomarkers of CM severity and mortality. VEGF was found to be protective against CM associated mortality and may be considered for adjunctive therapy to improve the treatment outcome in CM patients.

Background: Plasmodium falciparum can cause a diffuse encephalopathy known as cerebral malaria (CM), a major contributor to malaria associated mortality. Despite treatment, mortality due to CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM and other forms of severe malaria is multi-factorial and appear to involve cytokine and chemokine homeostasis, inflammation and vascular injury/repair. Identification of prognostic markers that can predict CM severity will enable development of better intervention., Methods: Postmortem serum and cerebrospinal fluid (CSF) samples were obtained within 2-4 hours of death in Ghanaian children dying of CM, severe malarial anemia (SMA), and non-malarial (NM) causes. Serum and CSF levels of 36 different biomarkers (IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, CRP, G-CSF, GM-CSF, IFN-gamma, TNF-alpha, IP-10, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, SDF-1alpha, CXCL11 (I-TAC), Fas-ligand [Fas-L], soluble Fas [sFas], sTNF-R1 (p55), sTNF-R2 (p75), MMP-9, TGF-beta1, PDGF bb and VEGF) were measured and the results compared between the 3 groups., Results: After Bonferroni adjustment for other biomarkers, IP-10 was the only serum biomarker independently associated with CM mortality when compared to SMA and NM deaths. Eight CSF biomarkers (IL-1ra, IL-8, IP-10, PDGFbb, MIP-1beta, Fas-L, sTNF-R1, and sTNF-R2) were significantly elevated in CM mortality group when compared to SMA and NM deaths. Additionally, CSF IP-10/PDGFbb median ratio was statistically significantly higher in the CM group compared to SMA and NM groups., Conclusion: The parasite-induced local cerebral dysregulation in the production of IP-10, 1L-8, MIP-1beta, PDGFbb, IL-1ra, Fas-L, sTNF-R1, and sTNF-R2 may be involved in CM neuropathology, and their immunoassay may have potential utility in predicting mortality in CM.