Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four  Studies relating gametocyte density to parasite density, mosquito feed studies that have shown human to mosquito transmission in low-density infections, as well as modeling suggest that these low chronic infections have a potential to contribute significantly to ongoing transmission in a community.4,7,8 As a result, asymptomatic individuals with chronic infection remain reservoirs that contribute to onward transmission and represent a serious concern for elimination efforts.9–12Nearly half of the world’s population remains at risk for malaria, despite substantial gains in morbidity and mortality from 2001 to 2015.10–13 Although these two tests have been largely successful in control efforts for case management, they have failed to identify malaria infections among significant proportions individuals infected with malaria but presenting with no clinical symptoms. For example, in Zambia, where the P. falciparum prevalence was less than 10% from 2009 to 2012, the sensitivity of RDT compared with nested polymerase chain reaction (PCR) in a reactive case detection strategy was only 17% in individuals with asymptomatic infections.14 Additionally, in western Kenya, when children and adolescents with no clinical symptoms in highland (low-transmission) and lowland (high-transmission) areas were tested for P. falciparum malaria by microscopy and quantitative PCR (qPCR), the average microscopy-based prevalence was 13.3%, whereas the qPCR-based prevalence was 20.9%, an almost two-fold difference.5 Similar variability in malaria prevalence by microscopy and/or RDT versus qPCR has been also reported in South America and southeast Asia, where the rates of asymptomatic infections were four to five times that of symptomatic infections.3,9,15–20 These data suggest that a large proportion of individuals who have no clinical symptoms of malaria, but are infected, have low-density infections. A challenge in interpretation of these ratios is also the variability in the sensitivity of the various PCR methodologies applied. To support ACD strategies that reduce the hidden burden of malaria and make elimination feasible, a more sensitive, field-deployable P. falciparum malaria infection detection test is required.4,12,14Some elimination programs use active case detection (ACD) strategies such as mass or focal test-and-treat following an index case identified at a health clinic. ACD is performed outside of health clinics and is largely dependent on rapid diagnosis by the two most commonly used field tools: microscopy and/or RDTs.P. falciparum biomarkers in asymptomatic subpatent infections in the field is important for establishing the required limit of detection (LOD) for a highly sensitive diagnostic test. One of the most commonly used tools, microscopy, identifies both gametocytes and asexual parasites and can achieve an LOD of 4–20 parasites/µL (p/µL) in laboratories with expert microscopists and approximately 200 p/µL in field conditions with inexperienced microscopists.4,10,13,21 In addition, microscopy lacks reproducibility and has variable sensitivity and a high false-positive rate.22 The recent introduction of RDTs has changed the malaria diagnostics landscape, allowing rapid diagnosis and treatment in both clinical and nonclinical settings. However, it has a detection threshold similar to that of microscopy and therefore is largely useful only in diagnosing symptomatic malaria.4,23 Molecular laboratory-based methods such as qPCR and quantitative reverse-transcription PCR (qRT-PCR) enable detection of low levels of parasite nucleic acids (often with LODs < 1 p/µL), and have helped distinguish and quantify low-density infections missed by microscopy and RDTs.4,5 A recently developed histidine-rich protein II (HRP2) enzyme-linked immunosorbent assay (ELISA) with an established laboratory LOD of 5 picograms/mL (pg/mL) permits the evaluation of new more sensitive HRP2-based RDTs based on HRP2 concentration. Accordingly, measuring P. falciparum biomarkers using current high-performance diagnostics will generate fundamental data about biomarker concentrations in asymptomatic active infections and subsequently inform the diagnostic LOD required to detect the reservoir population.The quantification of ™ Malaria Ag P.f Ultra Sensitive RDT (uRDT) was developed for field use and evaluated for detection of low-density infections. Compared with the current commercial HRP2-based SD BIOLINE Malaria Ag P.f. RDT (RDT), the uRDT uses the same immunochromatographic cassette platform, has the same whole blood volume requirements (5 µL), and takes only 5 minutes longer to result, making this device a promising and highly sensitive field tool. Currently, the best P. falciparum malaria RDTs have a LOD in the range of 600–1,000 pg/mL HRP2.24,25 The uRDT has an order of magnitude improvement in LOD for HRP2 , such that overall, the uRDT may fulfill many of the necessary characteristics for field use, but has not yet been assessed against asymptomatic infections in malaria-endemic areas. Furthermore, the proportion of infections detected by the uRDT compared with RDT during the course of infection has not been explored. Although P. falciparum-induced blood-stage malaria (IBSM) human challenge studies are traditionally performed to measure vaccine or drug efficacy and pharmacodynamics, they can also inform the impact of LOD on early detection of a new infection by observing parasitemia and HRP2 concentrations by highly sensitive qRT-PCR and ELISA methods, respectively.26 For example, in a clinical vaccine trial, PCR with a test sensitivity of approximately 20 p/mL detected malaria infection 5 days earlier than thick smears read by expert microscopists in both naive and previously exposed volunteers.27Although laboratory assays are more sensitive than microscopy and RDTs, they are also more expensive, require highly trained personnel, take longer to produce results, and are more difficult to deploy for large-scale field use. An AlereP. falciparum IBSM challenge studies.In this study, the new uRDT was evaluated for performance using whole blood specimens collected from asymptomatic individuals in Nagongera, Tororo District, Uganda, and a Karen village (TOT), Myanmar, representing high- and low-transmission settings, as well as from naive individuals enrolled in All specimens were collected from consenting study participants or with the assent of children and consenting legal care givers within the context of studies approved by the relevant institutional review boards (IRBs). Specimens from Uganda were collected under a study approved by the University of California San Francisco (UCSF) (IRB No.11-05995), Makerere University (IRB No. 2011-0167), and London School of Hygiene and Tropical Medicine (LSHTM) (IRB No. 5943); those from Myanmar were collected under a study approved by OxTREC (reference no. 1017-13 and 1015-13), by the Tak Community Advisory Board, and by the relevant village committees. The IBSM challenge studies were approved by the Queensland Institute for Medical Research (QIMR) . Specimens were received at PATH as delinked and anonymized for analysis as per study consent forms.P. falciparum studies performed in 2015 .IBSM challenge studies were performed at QIMR, Queensland, Australia. Whole blood specimens were collected from enrolled human volunteers across four IBSM P. falciparum parasitized red blood cells on Day 0. On Days 0 and 4, blood was collected once from each participant, and then collected twice daily at 12-hour intervals from Day 5 to Day 6.5. On Day 7, participants were admitted to the study unit, treated with an antimalarial, and observed for at least 72 hours. If participants were clinically well, then outpatient monitoring occurred to ensure safety and clearance of parasites until discharge from the study.All participants were deemed healthy and had not been previously exposed to malaria. Data are presented on specimens collected Days 0–7. Participants were inoculated intravenously with approximately 1,800–2,800 3D7 P. falciparum IBSM studies were performed in Queensland, Australia. For the Uganda and Myanmar uRDT studies, the inclusion criteria for asymptomatic individuals were no fever history in the previous 7 days, axillary temperature less than 37.5°C, the absence of other clinical signs of malaria, and no malaria treatment within the previous 60 days.Specimens for performance evaluations of the uRDT were collected at two clinical research sites: Nagongera, Uganda, and a Karen village (TOT), Myanmar. In addition, 28 All participants provided informed consent. As part of the ongoing cohort study, study participants were required to visit a health clinic every 3 months for routine visits, at which time blood was collected by venipuncture and microscopy was performed. For this study, samples were taken during 2 consecutive routine visits from May to October 2015.In Nagongera, Tororo District, Uganda, 100 random households were selected and visited to recruit children 6 months to 11 years old, as well as their primary caregivers, into a surveillance cohort as previously described.In TOT, Myanmar, study teams visited households and recruited both children and adults as part of an ongoing study to assess mass drug administration (MDA) for malaria elimination. All participants provided informed consent. Pregnant women and children less than 6 months of age were excluded.At all field sites and the IBSM site, 1–2 mL of whole blood was collected from each study participant by venipuncture into a labeled ethylenediaminetetraacetic acid (EDTA) vacutainer tube. The vacutainer was inverted gently several times to ensure adequate anticoagulation. For the field studies, whole blood specimens were then transported to a local laboratory in insulated containers containing conditioned ice packs or ambient temperature. Upon arrival at the laboratory, blood specimens were processed within 6–24 hours as per external study partners’ instructions for molecular and antigen detection testing. After processing, specimens were immediately stored at −80°C. If specimens could not be processed in the same day, then specimens were stored at 4°C for a maximum of 12 hours.29,30 qRT-PCR was subsequently performed using a multiplex assay for P. falciparum and pan-Plasmodium-specific 18S rRNA amplicons using a SensiFAST™ Probe Lo-ROX One-Step Kit .31 Absolute copy number quantification was performed against an in vitro-transcribed RNA standard curve in a malaria-negative whole blood eluate and copy numbers were converted to estimated p/mL as described30 parasite density estimates are based on the 18S rRNA content of synchronized ring-stage P. falciparum parasites and the 18S rRNA-to-parasite conversion factor may vary for non-P. falciparum species. qRT-PCR-positive specimens were classified as P. falciparum, P. falciparum or mixed, non-P. falciparum, and Plasmodium spp.At each site’s laboratory, whole blood specimens were prepared for qRT-PCR by aliquoting and mixing 50 µL of EDTA-anticoagulated whole blood into each of two tubes containing 2 mL NucliSENS lysis buffer . Fifty microliters of specimen were also added to each of five spots on a standard dried blood spot (DBS) card , then dried, desiccated, and frozen in gas-impermeable bags. The lysis buffer tubes and DBS cards were then frozen onsite and stored at −80°C until shipping. The University of Washington performed RNA nucleic acid extraction as previously described.32,33 For high-volume qPCR-positive blood samples, malaria parasite species were identified using nested PCR specific for P. falciparum (microsatellite marker Pk2), P. vivax (microsatellite marker 3.502), and Plasmodium malariae (18S rRNA) as described previously.32,33The Myanmar specimens were also examined with the high-volume qPCR at Mahidol University as described.P. falciparum HRP2 and human C-reactive protein (CRP) were quantified in collected whole blood specimens using a recently developed Q-plex array ELISA kit . The measurement of HRP2 and CRP used a sandwich ELISA and competitive ELISA methods, respectively. For P. falciparum HRP2 and CRP, the analytical LODs are 5 pg/mL and 25 ng/mL, respectively. Only the P. falciparum-specific HRP2 data are presented herein.At PATH, P. falciparum HRP2 Q-plex ELISA was performed according to the manufacturer’s kit instructions. The competitor for CRP and calibrator for P. falciparum HRP2 were prepared in an 8-point 3-fold standard dilutions with sample diluent containing heterophilic antibodies and rheumatoid factor blockers. Positive and negative controls and a blank were also included on each plate. The positive control was P. falciparum ITG (recombinant HRP2)-spiked negative North American whole blood, the negative was North American whole blood only, and the blank was standard diluent. Each specimen was tested in duplicate. To each plate well, 12.5 µL of whole blood and 37.5 µL of sample diluent were added, to make a total volume of 50 µL. The plate was then sealed, wrapped with foil, and placed on a shaker at 500 rpm for 1 hour at room temperature. After washing the plate three times with 1× wash buffer, 50 µL of detection mix containing biotinylated detector antibodies was added to each well and incubated on a shaker (500 rpm) for 20 minutes. The plate was washed three times followed by the addition of 50 µL of 1× streptavidin–horseradish peroxidase (HRP), incubated again for 20 minutes on the shaker (500 rpm), and then washed six times. Equal volumes of substrate A (hydrogen peroxide) and substrate B  were mixed, of which 50 µL was then added to each well. The plate was then imaged using a Q-view imager . Quality control specified for replicates a < 15% variation, and the blank and negative control wells had a capture image pixel intensity value ≤150. The analytical LOD of P. falciparum HRP2 in the Q-plex array, 5 pg/mL, was used as the positive–negative threshold for all specimens. The upper LOD was 14,600 pg/mL. The Five-Parameter Logistics model with weighting was used to interpolate the data based on the standard curve and determine P. falciparum HRP2 concentration in each specimen.The All whole blood specimens were tested in singlet using the Standard Diagnostics, Inc. (SD) BIOLINE Malaria Ag P.f. RDT  and in duplicate using Alere Malaria Ag P.f RDT ULTRA SENSITIVE  according to the manufacturer’s instructions. Briefly, the RDT and uRDT were stored and used at room temperature, and blood specimens were defrosted and placed on ice prior to use. For both RDTs, 5 µL of whole blood was transferred by calibrated pipette to the round specimen well, followed by the addition of four drops of diluent into the square well. The test results were read at 15 minutes for the RDT and at 20 minutes for the uRDT. The results were interpreted as invalid (no control line), positive (control line and test line present), or negative .P. falciparum IBSM challenge model were produced using Microsoft Excel 2013 . The sensitivities of the RDT and uRDT against qRT-PCR or Q-plex ELISA reference tests were calculated as true positives/, and specificity calculated as true negatives/. The positive and negative predictive values were calculated as true positives/ and true negatives/, respectively. For the IBSM studies, two-tailed Z tests with a significance level of 0.05 were performed to test the null hypothesis that the proportion of uRDT positives was equivalent to that of the RDT positives.The histograms depicting the distributions of parasite density and HRP2 in asymptomatic infections and uRDT detection from Uganda, Myanmar, and P. falciparum IBSM studies, a total of 93 whole blood specimens from Day 4 to Day 7, comprising the time points prior to treatment (Day 7), were collected from 16 human volunteers. In Myanmar, 493 whole blood specimens comprising all individuals residing in the Karen village were collected from April 21 to May 7, 2015. In Uganda, a total of 607 specimens were collected.Across four All parasite density estimates for this study were determined by qRT-PCR and described in p/µL. HRP2 concentration was determined using the Q-plex ELISA, which has a LOD of 5 pg/mL GST-W2 recombinant HRP2 , and described in pg/mL.P. falciparum. Of these, 92/95 (97%) were detected by qRT-PCR and 62/95 (65%) positive by Q-plex ELISA. Of the 92 qRT-PCR-positive specimens, 61 (66%) were also Q-plex ELISA positive. The parasite densities and HRP2 concentrations are shown in R2 = 0.45), but when extended to 2,200 p/µL, the correlation improved (R2 = 0.88 [data not shown]). The distributions of the specimens sorted by either parasite density or HRP2 concentration are shown in From Days 4 to 7, 93 post-challenge specimens from 16 participants were tested by both qRT-PCR and Q-plex ELISA for Plasmodium 18S rRNA consistent with infection by qRT-PCR, of which five (3.9%) specimens were P. falciparum positive only, four (3.1%) were P. falciparum mixed positive, 97 (78.2%) were P. vivax positive, and 18 (14.1%) were Plasmodium spp. positive. Plasmodium vivax positivity was determined from the qRT-PCR pan-Plasmodium-positive samples and the high-volume qPCR followed by nested PCR to specifically identify P. vivax. There were a total of nine (1.8%) P. falciparum-positive specimens  in the overall collection (N = 493). The parasite densities of the nine positive P. falciparum specimens by qRT-PCR ranged from 0.2 to 136.9 p/µL  and a median of 88.4 p/µL. The 96 specimens that were non-P. falciparum by qRT-PCR, and P. vivax positive only by qPCR, had a pan-Plasmodium spp. parasite density range of 0.11–15,697.2 p/µL with a geometric mean of 31.2 p/µL (95% CI: 20.1–48.6) and a median of 23.5 p/µL. The corresponding qPCR parasite densities for the P. vivax-infected specimens ranged from 0.5 to 32,724.4 p/µL with a geometric mean of 42.2 p/µL (95% CI: 27.5–64.7) and median of 37.4 p/µL.A total of 124 specimens were positive for 6.9 p/µL  with a gP. falciparum qRT-PCR  were P. falciparum only positive, 12 (4.1%) were P. falciparum mixed, and 31 (10.6%) were non-P. falciparum. There were a total of 261 (43%) P. falciparum-positive specimens, including the P. falciparum mixed specimens, in the overall collection (N = 607). Quantification of P. falciparum parasite density revealed a wide distribution, 0.01–235,095 p/µL  and a median of 367.2 p/µL. The 96 specimens that were non-P. falciparum by qRT-PCR had pan-Plasmodium spp. parasite density range of 0.09–1,373.5 p/µL with a geometric mean of 17.1 p/µL (95% CI: 7.0–42.0) and a median of 11.3 p/µL.095 p/µL . The geoPlasmodium falciparum HRP2 was quantified in 269 specimens with concentrations ranging from 6.1 to 14,600 pg/mL  (0 pg/mL) . Of the Thirty-five specimens were qRT-PCR positive but Q-plex ELISA negative; all of these had low parasite densities. The overall geometric mean for these specimens was 0.74 p/µL (95% CI: 0.37–1.5) and median was 1 p/µL. The distributions of the specimens sorted by either parasite density or HRP2 concentration are shown in The correlation between HRP2 and parasite density was poor, although the median and mean values tended to increase with parasite density .P. falciparum negative by qRT-PCR . The performance of the uRDT and RDT against confirmed parasite density and HRP2 concentration is shown in Performance of the uRDT and RDT during early infection was evaluated in IBSM studies at the non-endemic Australian site, where preceding infections in study participants could be excluded. The uRDT was able to detect infections as early as 1.5 days sooner than the RDT during early onset infections  uRDT-positive specimens were detected. Almost half  of qRT-PCR and all  of Q-plex ELISA-confirmed P. falciparum-positive specimens were also uRDT positive, and one uRDT-positive specimen was neither qRT-PCR positive nor Q-plex ELISA positive. The details of qRT-PCR specimens in terms of parasite density, HRP2 concentration, and RDT and uRDT performance are shown in P. falciparum specimens, and both tests were equivalent in terms of specificity (The detection of cificity .N = 607) were tested with both the RDT and uRDT. The prevalence of malaria infection by uRDT as compared with RDT was 40.7% (247/607) and 29.5% (179/607), respectively. Most of the positive specimens by uRDT  and by RDT  were also qRT-PCR positive, such that there were 29 uRDT- and 16 RDT-positive results confirmed as non-parasitemic by qRT-PCR, of which 26 uRDT and only four RDT were confirmed HRP2 positive by the Q-plex-ELISA. The details of qRT-PCR specimens in terms of parasite density, HRP2 concentration, and RDT and uRDT performance are shown in All blood specimens from asymptomatic study participants  and 92% (95% CI: 88–94), respectively, in comparison to 62% (95% CI: 56–68) and 95% (95% CI: 92–97), respectively, for the RDT . Using tP. falciparum malaria, the Alere Malaria Ag P.f. uRDT using specimens collected from different epidemiological settings, specifically for detecting asymptomatic infections. The performance was also evaluated in IBSM studies with malaria-naive study participants in the context of early infection HRP2 dynamics and time-to-infection detection in new infections. The performance of the uRDT was compared with that of currently commercially available SD BIOLINE Malaria Ag P.f. RDT . The RDT has an LOD of approximately 800 pg/mL HRP225 as compared with uRDT, which has an LOD of 80 pg/mL .These studies sought to determine the performance of a new highly sensitive RDT for 6,20 In Nagongera (high transmission), several immune mechanisms likely influence these varying levels of parasite densities.13,34 In low-transmission areas such as TOT village, where there have been rapid decreases in malaria prevalence, just two or three bites can form exposure-related immunity and suppress parasite loads.21,34,35 Many of the asymptomatic infections identified in these studies are likely due to previous exposure with parasite densities suppressed to submicroscopic levels.The qRT-PCR data and Q-plex ELISA showed that in a high-transmission setting such as Nagongera, Uganda, there was a wider range of parasite densities and HRP2 concentrations in asymptomatic infections than in the low-transmission TOT village, Myanmar, where parasite biomarker concentrations were relatively low. The results for parasite densities are consistent with previous studies in low- and high-transmission settings.36,37 There are several possible factors that may explain these disparities, including parasite sequestration,HRP2 persistence, clonal variation in HRP2 expression, and prevalence of parasite strains with gene deletions in pfhrp2 or pfhrp3 or both. Sequestration occurs when P. falciparum trophozoites and schizonts adhere to vasculature and effectively disappear from peripheral blood circulation such that parasites cannot be visualized by microscopy.38,39 At the same time, HRP2 released during schizont rupture can remain in circulating blood during sequestration events, making it easier to detect by HRP2-based tests such as ELISAs and RDTs.38 Alternatively, HRP2 can also remain in detectable amounts after parasite clearance, resulting in “false-positives test results” from the perspective of the test result reflecting active infection.10,24,40–43The relationship between parasite density and HRP2 concentration is poor, as demonstrated previously.38,44 The Q-plex HRP2 assay does not cross-react with rheumatoid factor (manuscript in preparation). In the Myanmar study, the Q-plex assay only detected HRP2 in one out of 484 qRT-PCR-negative specimens. In the Uganda study, however, there were a significant number of study participants who had detectable levels of HRP2 by the Q-plex assay but for whom active infection could not be confirmed by qRT-PCR. These HRP2-positive, qRT-PCR-negative samples all had low HRP2 concentrations and may support the presence of residual HRP2 in a high-prevalence setting.An HRP2-based diagnostic test produces false positives with respect to parasite density because either the specimen truly has residual HRP2 but no intact parasites or because the assay is erroneously detecting HRP2 where there is none; for example, cross-reaction with other proteins such as rheumatoid factor.The availability of a highly sensitive ELISA for HRP2, with different capture and detection reagents to the RDTs, as well as qRT-PCR facilitated the comparison of the performance of the uRDT and the RDT against both parasite density and HRP2 concentration in the blood. Notably, in Myanmar, the uRDT only detected one false positive in 484 confirmed negatives by qRT-PCR for a specificity of 99.7%. This specimen was also HRP2 negative by the Q-plex ELISA. In the Uganda study, 26 of the 29 non-parasitemic uRDT-positive tests were confirmed HRP2 positive by the HRP2 Q-plex ELISA, resulting in an improvement in specificity of the test from 92% as confirmed by 18S rRNA qRT-PCR to 99% by confirmed antigenemia (HRP2 Q-plex ELISA). In contrast, only four of 16 non-parasitemic RDT were confirmed HRP2 positive such that the specificity remained essentially the same, 95% and 96% compared with qRT-PCR and Q-plex ELISA reference assays, respectively. Thus, the uRDT is highly specific for individuals with asymptomatic infection or who have recently been infected.P. falciparum prevalence of 43%, the sensitivity by uRDT was greater than that of RDT, 84% and 62%, respectively. In Myanmar, with a P. falciparum prevalence of 1.8%, the improved LOD of the uRDT resulted in the detection of 4/9 confirmed P. falciparum infections as compared with zero cases with the RDT. Both of the tests used in this study are based on HRP2 detection as such they will not detect parasites with pfhrp2 deletions. In the studies described here, there were no obvious high parasite density, negative HRP2 cases observed, that would be indicative of infections consisting of predominantly pfhrp2/pfhrp3 deletions. In locations where there is a confirmed high prevalence of pfhrp2/pfhrp3 deletions, this should be considered.45–47 Further studies are required in other settings with low-to-intermediate malaria transmission to better understand the performance of the uRDT with respect to the asymptomatic reservoir. The ability of the uRDT to detect infections earlier than the RDT, as demonstrated in the P. falciparum IBSM study, also adds further support to the utility of the improved uRDT LOD. The IBSM study showed that as early as 5 days after inoculation with infected erythrocytes, infection could be detected in naive individuals using the uRDT at significantly larger proportions than the RDT. In fact, on Day 6.5, 12 hours before treatment commenced, the RDT still detected no infections as compared with 7/14 infections by the uRDT. The impact of early detection as demonstrated in the IBSM studies on elimination programs in field settings will likely vary depending on transmission dynamics in a population at the time of screening.By using the Q-plex ELISA, this study was able to confirm a more than 10-fold improvement in HRP2 LOD of the uRDT as compared with RDT using clinical specimens from asymptomatic patients. This improvement in LOD of the uRDT led to an increase in detection of asymptomatic cases in all the studies. In Uganda, with a P. falciparum true positives relative to qRT-PCR and Q-plex ELISA results at all parasite LODs is encouraging for its potential use in malaria elimination settings. Low parasite density infections not detected by microscopy are potentially also infectious to mosquitos. Modeling data have suggested that when microscopy is used for malaria diagnosis in the field in a low-prevalence setting (10–20%), approximately 20–50% of asymptomatic individuals harboring parasites contribute to mosquito infection and continued transmission.21 With a diagnostic tool that has an LOD of 200 p/µL, it was shown in Burkina Faso that 55% of the infectious reservoir would be detected, followed by 83% at 20 p/µL and 95% at 2 p/µL.21 Although the studies in Myanmar and Uganda were unable to perform mosquito feeding studies to investigate the transmissibility of low-density infections, the uRDT demonstrated its ability to detect a greater number of individuals with low-density infections, and potentially also infectious individuals as compared with current field-based diagnostic tools such as microscopy and RDT. Future studies investigating infectiousness of the asymptomatic reservoir using mosquito challenge studies should be performed in combination with biomarker quantification as performed in the present study. Additionally, it would be valuable to study gametocyte density and dynamics in low-density infections and its contribution to oocyst and sporozoite development in the mosquito vector. Together, these results would support and improve model predictions of uRDT impact on different epidemiologies and elimination strategies.The ability of the uRDT to detect large proportions of P. falciparum IBSM challenge study, specimens were collected from only 16 people at five time points, which resulted in a relatively small sample size for early infection analyses. Future studies should expand the survey to a larger sample size. Additionally, given that the IBSM study participants had no previous parasite exposure, it would be interesting to investigate and confirm early infection detection by uRDT in similar studies in malaria-endemic areas. In the Uganda study, the asymptomatic infection prevalence was too high to be considered as an elimination setting. Moreover, a majority of the Uganda specimens were from children, which may not be representative of the parasite density profile in asymptomatic infections in this setting. The Nagongera data, however, do provide useful information about biomarker levels and infection detection of asymptomatic subpopulations in high-transmission areas.Although the present study provided a comprehensive characterization of biomarkers and uRDT performance in asymptomatic infections from two regions with distinct endemicity and also in a challenge model for early infection dynamics, there were several limitations. The sample size for the Myanmar survey was too small to provide statistically significant sensitivity values in low-prevalence settings where most asymptomatic infections have very low parasite density. The blood specimens were collected after mass drug administration, which very likely contributed to low malaria prevalence in this area. Likewise, in the The improved performance characteristics of the Alere Malaria Ag P.f. uRDT over the current malaria RDTs in asymptomatic patients from Myanmar and Uganda, as well as naive participants in the IBSM studies, are highly indicative of the utility of this test as a tool for malaria elimination. This performance should be verified under more operational conditions using finger stick blood specimens and performed by programmatic or health-care workers. Further studies and modeling are warranted to assess the impact and cost-benefit of the uRDT over other tests such as PCR in different malaria elimination strategies.


Plasmodium falciparum molecular barcode was used to characterize parasite populations from infected individuals identified through passive and active case detection in an area approaching malaria elimination in southern Zambia.Substantial reductions in the burden of malaria have been documented in parts of sub-Saharan Africa, with elimination strategies and goals being formulated in some regions. Within this context, understanding the epidemiology of low-level malaria transmission is crucial to achieving and sustaining elimination. A 24 single-nucleotide-polymorphism P. falciparum-infected participants enrolled in a serial cross-sectional, community-based cohort study from 2008 to 2013 and from passively detected, P. falciparum-infected individuals enrolled at five rural health centres from 2012 to 2015 were compared. Changes in P. falciparum genetic relatedness, diversity and complexity were analysed as malaria transmission declined.The study was conducted in the catchment area of Macha Hospital in Choma District, Southern Province, Zambia, where the parasite prevalence declined over the past decade, from 9.2% in 2008 to less than 1% in 2013. Parasite haplotypes from actively detected, Actively detected cases identified in the community were most commonly rapid diagnostic test negative, asymptomatic and had submicroscopic parasitaemia. Phylogenetic reconstruction using concatenated 24 SNP barcode revealed a separation of parasite haplotypes from passively and actively detected infections, consistent with two genetically distinct parasite populations. For passively detected infections identified at health centres, the proportion of detectable polyclonal infections was consistently low in all seasons, in contrast with actively detected infections in which the proportion of polyclonal infections was high. The mean genetic divergence for passively detected infections was 34.5% for the 2012–2013 transmission season, 37.8% for the 2013–2014 season, and 30.8% for the 2014–2015 season. The mean genetic divergence for actively detected infections was 22.3% in the 2008 season and 29.0% in the 2008–2009 season and 9.9% across the 2012–2014 seasons.Distinct parasite populations were identified among infected individuals identified through active and passive surveillance, suggesting that infected individuals detected through active surveillance may not have contributed substantially to ongoing transmission. As parasite prevalence and diversity within these individuals declined, resource-intensive efforts to identify the chronically infected reservoir may not be necessary to eliminate malaria in this setting.The online version of this article (doi:10.1186/s12936-017-1810-3) contains supplementary material, which is available to authorized users. Substantial reductions in the burden of malaria have been documented in parts of sub-Saharan Africa  and malaPlasmodium falciparum molecular barcode assay consisting of 24 unlinked, single nucleotide polymorphisms (SNPs) has been used to characterize unique genetic signatures and track circulating P. falciparum parasite populations [A ulations . The barulations –14, and ulations .This study was conducted in an area of southern Zambia that experienced a dramatic decrease in malaria transmission over the past decade . The molAnopheles arabiensis [2 with roughly 30,000 residents consisting primarily of villagers living in small, scattered homesteads. The prevalence of P. falciparum infection declined in this area over the past decade, from 9.2% in 2008 to less than 1% in 2013 [The study was conducted in the catchment area of Macha Hospital in Choma District, Southern Province, Zambia, where there is a single rainy season which lasts from November through April, followed by a cool dry season from April until August and a hot dry season from August through November. Malaria transmission peaks during the rainy season  and the abiensis , 17. The in 2013 . Artemis in 2013  and into in 2013  and more in 2013 .Plasmodium parasites by a nested PCR as described below and molecular barcoding was attempted for all samples positive for Plasmodium parasites by nested PCR .Satellite images were used to develop a sampling frame for the random selection of households to enroll participants in longitudinal and cross-sectional malaria surveys . The ideFourteen rural health centres (RHCs) that serve the catchment population surrounding Macha Hospital sent a weekly text message report of the number of RDTs used, number of positive RDTs, and the number of people treated for malaria to study staff at Macha Research Trust . Five ofThe DBS were stored at −20 °C in individual plastic bags containing desiccant until DNA extraction. DBS collected from February to September 2008 were initially stored at room temperature and subsequently at −20 °C. Parasite DNA was extracted using the Chelex© method from one dried blood spot . BrieflyPlasmodium multi-species nested PCR assay as previously described [cytb). Briefly, in the primary PCR step, 6 μL of DNA extract was added to 0.2 mL tubes containing a 19 μL reaction mix made up of DNase free water and final concentrations of 200 μM dNTPs, 1× ThermoPol PCR buffer , 1 μM forward and 1 μM reverse primers and DNA Taq polymerase  in 25 μL reaction volume. In the nested PCR step, 3 μL of the primary PCR product was added to 0.2 mL PCR tubes containing 22 μL of reaction mix with DNase free water and the final concentrations of dNTPs, Thermopol buffer, forward and reverse primers, and Taq DNA polymerase in a 25 μL reaction mix as described above. No template controls were included in each experiment and reactions were run in a Techne™ TC-412 thermo cycler . Amplified product was detected by electrophoresis on 1% agarose gel and viewed under UV light.Infection was confirmed using a escribed , 23 targThe 24 SNP molecular barcode assays were run using a TaqMan protocol at the Macha Research Trust laboratory in Zambia . DNA wasP. falciparum strains obtained from MR4  with known haplotypes for all 24 SNPs. Typically, 12 SNP assays were run for five samples at a time with controls on a 96-well plate. The assays were run on the Applied Biosystems StepOnePlus™ , and the Roche LightCycler 480™  real time PCR systems.Pre-amplified samples were diluted 1:20 with TE buffer prior to running the 24 SNP molecular barcoding assay as previously described [Parasites sampled from peripheral blood are those in the haploid intra-erythrocytic stage of their lifecycle. . Thus, SHaplotypes, composed of the concatenated 24 SNPs, were aligned using the ClustalW method in MEGA6 software, with mixed calls represented as a third possible allele . PhylogeParasite genetic complexity was determined by the number of mixed calls at each of the 24 SNPs. Samples with four or more mixed calls were categorized as polyclonal infections . TemporaParasite genetic diversity was evaluated by determining divergence from the most common barcode for each transmission season stratified by whether the parasite was identified through passive or active surveillance. The most common barcode for each season was determined by allelic frequency at each SNP to determine the nucleotide diversity. A modified ‘SNP π’, was developed to account for missing data and mixed allele calls, and was used to measure the seasonal parasite genetic divergence . Details2 test. RDT and microscopy results across transmission seasons were compared for actively detected cases using a chi2 test.Data on age and sex analysed for passively detected cases. Age, sex, symptoms of malaria, and RDT and microscopy results were analysed for actively detected cases. Median ages between actively and passively detected infected individuals and across transmission seasons were compared using a Kruskal–Wallis test. Sex differences between groups and across transmission seasons were compared using a chiThe 24 SNP molecular barcode was performed on 72 samples from actively detected malaria cases collected from 2008 to 2013 and 46 samples from passively detected malaria cases collected from 2012 to 2015. Households of actively detected cases and the RHC from which the passively detected cases were identified came from the same underlying source population  infections and populations of parasites infecting different communities . VariatiTypically, as malaria transmission declines, a genetic bottleneck is created, reducing opportunities for outcrossing in the mosquito midgut and leading to reduced parasite diversity among passively detected, symptomatic malaria cases , 28. ThiPassively detected cases also had fewer complex infections, with a low proportion of polyclonal infections, consistent with recent infection with single parasite clones. In contrast, actively detected cases had more complex infections, with a high proportion of polyclonal infections. Between the 2008 and 2009–2010 transmission seasons there was an indication of a non-statistically significant, decreasing trend in the proportion of polyclonal infections; however, this proportion subsequently increased and remained high, indicating that the actively detected population of mostly asymptomatic and sub-patent infections was composed of many different parasite clones. While transmission declined, these individuals may have harboured parasites acquired over time and maintained at low levels of parasitaemia. Chronic infections have been shown to persist for up to a decade –32.Parasite genetic diversity remained relatively high and constant for passively detected cases throughout the observed malaria transmission seasons. This finding, along with the phylogenetic separation from actively detected cases and the paucity of detectable polyclonal infections, suggests these passively detected cases may represented recent infections with parasites imported into the study area. In contrast, parasite genetic diversity was lower among actively detected cases, consistent with a chronically infected population. The decreasing parasite diversity among the actively detected cases over the study period is consistent with loss of parasites from this chronically infected reservoir as malaria transmission declined.P. falciparum infections [Previous studies using this genotyping approach were reported from Senegal and Malawi. The molecular barcode was used in Senegal to determine the clonal and epidemic expansion of passively detected clinical fections . After efections . Betweenfections . In Malafections . Childrefections . To the Analyses of spatial hotspots of asymptomatic parasitemia and clinical malaria cases have been previously published from low and moderate transmission settings in Kenya and Mali –35. In tThere were several major limitations of these analyses. The first was that passive and active case detection did not overlap in time except for one malaria transmission season. However, given the absence of seasonal phylogenetic clustering for both passively and actively detected cases, and because all samples came from the same underlying source population, the combined phylogenetic tree is likely an accurate representation of the phylogenetic relationships between the two parasite populations. Additionally, when constructing the combined phylogenetic tree, samples were indexed by season so within season relationships between the two populations would not be masked. The second limitation was the high number of mixed infections from which single haplotypes were difficult to resolve. To account for this, a third allele was used to indicate mixed infections in calculating the phylogenetic relatedness and mixed infections were accounted for when calculating genetic divergence , 14. TheDistinct parasite populations were found among infected individuals identified through active and passive surveillance in a low transmission setting in southern Zambia. The actively detected population represented a chronically infected, reservoir that may not contribute substantially to ongoing transmission. As parasite prevalence and diversity within the actively detected cases declined over time, resource-intensive efforts to identify and treat the chronically infected reservoir through reactive case detection, or focal or mass drug administration, may not be necessary to eliminate malaria in this setting.Additional file 1. Flow diagram of sample selection.Additional file 2. The modified ‘SNP π’ used to account for missing data and mixed allele calls was calculated as follows.


Plasmodium falciparum malaria before achieving elimination of all forms of malaria, including Plasmodium vivax in 2025. The emergence of artemisinin-resistant P. falciparum in western Cambodia over the last decade has drawn global attention to support the ultimate goal of P. falciparum elimination, whereas the control of P. vivax lags much behind, making the 2025 target gradually less achievable unless greater attention is given to P. vivax elimination in the country. The following review presents in detail the past and current situation regarding P. vivax malaria, activities of the National Malaria Control Program, and interventional measures applied. Constraints and obstacles that can jeopardize our efforts to eliminate this parasite species are discussed.The Cambodian National Strategic Plan for Elimination of Malaria aims to move step by step toward elimination of malaria across Cambodia with an initial focus on  Plasmodium falciparum and Plasmodium vivax coexist. The emergence of artemisinin-resistant P. falciparum in western Cambodia over the last decade has drawn global attention to support its “containment” with an ultimate goal of P. falciparum elimination. However, the control of P. vivax lags much behind.The Kingdom of Cambodia, which lies in the center of the Indochina peninsula in southeastern Asia has a population of 15,840,251 (in 2015) consisting of 2.2 million households, in 13,406 villages in 25 provinces and one municipality (Phnom Penh)  campaign using dichlorodiphenyltrichloroethane, malaria prevalence rates reduced from 60% to 1% by the early 1960s.In 2001, the country introduced artemisinin-based combination therapies (ACT)s (artesunate plus mefloquine) at a national scale.Plasmodium falciparum was the predominant species among confirmed malaria cases until 2010. With the roll out and scale-up of multispecies (Pan) rapid diagnostic tests (RDTs) in 2009 , the proportion of infections due to P. falciparum shifted and for the first time in 2011, the number of P. vivax cases  was higher than the number of P. falciparum cases  registered in the public sector . In 2014, based on malaria RDT detection , non-P. falciparum infections (mostly P. vivax) accounted for 46%  of cases, followed by 32%  of mixed infections of both P. falciparum and non-P. falciparum and 21%  of pure P. falciparum cases  and particularly prevail in Stung Treng Province along the Lao People's Democratic Republic–Cambodia border   confirmed that recurrences occurred significantly later after dihydroartemisinin–piperaquine treatment  than after chloroquine treatment  (P < 0.05) . This may also explain why, despite the introduction of combo RDTs , P. vivax case counts did not significantly increase in Zone 1 the same way it was observed in the surrounding areas (Zones 2 and 3). Moreover, P. vivax prevalence declined slightly in all zones in 2012 after the introduction of dihydroartemisinin–piperaquine treatment in the entire country in 2010 and the mass distribution of LLINs in late 2011 and early 2012, suggesting that a portion of new, mosquito-borne infection P. vivax cases might be preventable by nets.Malaria transmission and  malaria . As showP. falciparum: P. vivax ratio over the past decade along with progressive reduction in malaria cases , it is reasonable to assume that relapses are responsible for a large proportion of P. vivax cases.Overall, there has been a clear trend toward the reversal of the ia cases . As repoP. vivax cases  with a saw-tooth pattern of periodic increases observed in 2006 and 2009 , the age group 15–49 years  continues to predominate among P. vivax and P. falciparum, with huge implications for vivax malaria control, is the occurrence of dormant P. vivax parasites in the liver (hypnozoites). After an infectious mosquito bite, vivax sporozoites migrate to the liver, invade hepatocytes, and then multiply, forming a schizont that eventually bursts, releasing merozoites into the bloodstream; these cells then invade the reticulocytes and enter the erythrocytic cycle. All P. falciparum liver-stage parasites undergo immediate asexual replication, whereas a proportion of P. vivax liver-stage parasites enter a dormant state (hypnozoite). Only a fraction of the initial sporozoites begin the first erythrocytic cycle inducing the primary episode of vivax malaria. The rest form hypnozoites that reactivate several weeks to months (or even years) after primary infection, causing multiple subsequent vivax malaria episodes called relapses. After the initial treatment of vivax malaria cases, recurrences can result from recrudescence , reinfection (new infectious mosquito bite), or relapses (from dormant liver stages).A key biological difference between P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria.Plasmodium vivax recurrences after falciparum malaria have been also observed in western Cambodia. In a combined analysis of 243 patients recruited in two malaria treatment trials, 20/43 (47%) of those with P. falciparum gametocytes on admission developed P. vivax malaria by day 28 of follow-up. The presence of P. falciparum gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia posttreatment.P. vivax parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcription PCR in a subset of patients strengthened the observed association, whereas PCR detection of P. vivax parasitemia at follow-up was similar to microscopy results. It has been suggested first that the presence of P. falciparum gametocytes may indicate increased malaria exposure in general: patients carrying gametocytes may be more likely to have acquired a P. vivax infection in the past and to harbor hypnozoites in their liver when they are infected with P. falciparum or second that the competition from a second malaria species (P. vivax in that case) may boost falciparum gametocytogenesis as an evolutionary adaptation. The authors of this study concluded that in Cambodia, the majority of P. vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites.It is known that when patients with Since 2012, the first-line treatment of uncomplicated malaria  recommended by the National Malaria Program is based on ACT treatment of 3 days (dihydroartemsinin–piperaquine). For vivax malaria, radical cure with primaquine (0.75 mg/kg weekly for 8 weeks or 0.25 mg/kg daily for 14 days) is recommended only if glucose-6-phosphate dehydrogenase (G6PD) deficiency testing is performed and G6PD deficiency is excluded. Practically, no point-of-care to detect G6PD deficiency is available in health facilities in Cambodia, and primaquine is not used routinely.16Plasmodium infections detected by microscopy declined in each successive national survey since 2004. Weighted national prevalence by microscopy was 4.4% in 2004, 2.6% in 2007, and 0.9% in 2010, and had declined to 0.1% in 2013. In 2010 and 2011, a focused screening and treatment (FSAT) using PCR for case detection was carried out in villages of Pailin Province, the artemisinin resistance hotspots, supported this observation.P. falciparum positive and 1.04% P. vivax positive; the FSAT 2011 screened 5,229 people and found only 0.11% P. falciparum–positive cases and 0.52% P. vivax-positive cases. These consecutive surveys confirmed P. vivax to be the more common species in that area, albeit at a very low prevalence. This observation was also confirmed in Preah Vihear Province  in 2013.P. vivax with a parasite density below 100 parasites per milliliter. More recently, Tripura and othersP. falciparum carriers, 48 (3.3%) P. vivax carriers, and four (0.3%) mixed infections carriers. In 142/1,447 (9.8%) cases, they were not able to identify the species. Monthly follow-up without treatment of 24 adult participants with asymptomatic mono or mixed P. falciparum infections revealed that 3/24 (13%) remained parasitemic for 2–4 months, whereas the remaining 21/24 (87%) participants had cleared their parasitemia after 1 month. In contrast, 12/34 (35%) adult participants with P. vivax monoinfection at M0 had malaria parasites (P. vivax or unindentified Plasmodium sp.) during four or more of the following 11-month surveys. Their data showed limited duration of P. falciparum carriage, but prolonged carriage of P. vivax infections, suggesting that radical treatment of P. vivax infections by 8-aminoquinoline regimens may be required to eliminate all malaria from Cambodia.A series of large-scale malaria surveys  conducted in 2004 , 2007 , 2010 , and 2013  conducted by the CNM and Malaria Consortium, further supports the declines in malaria prevalence  detected by both microscopic examination and PCR.P. falciparum vector studies, as very little data are available about P. vivax transmission in Cambodia. The main malaria transmission season in the country typically runs from June to October in parallel with the rainy season. The common vectors prefer forested areas, so the risk of infection is linked to proximity to forests, affecting particularly migrant populations, mobile populations working in or close to forests, and forest goers.Anophelesmaculatus s.l. is present throughout Cambodia, Anopheles minimus s.l. more prevalent in the west, and Anopheles dirus in the northeast. Other vectors such as Anopheles barbirostris, Anopheles philippinensis, Anopheles vagus, and Anopheles hyrcanus are also present in Cambodia.Anopheles dirus is found in the forested mountains and foothills, cultivated forests, and rubber plantations, whereas An. minimus is frequent outside the forests or in areas where the forests have been cleared, breeds along grassy streams in clear, unpolluted water, and its abundance is determined by water velocity and types of vegetation.Anopheles maculatus is found in hilly or mountainous areas and breeds in or near permanent or semipermanent bodies of clean water like streams or rivers. Anopheles epiroticus is able to breed in water with some salinity, and is therefore typically found in Cambodia's coastal areas. These vectors bite during all hours of the evening, but peak biting hours are usually found to be between 8 pm and 12 am.22Most of information described in this paragraph is derived from An. dirus and An. maculatus have been found to adapt to varying environmental conditions despite the preference of natural forest habitats.An. dirus were found in areas where natural forests were replaced with orchards, tea, coffee, and rubber plantations.P. vivax than for P. falciparum, as the extrinsic incubation period of P. vivax is shorter.Malaria incidence in Cambodia is changing rapidly due to environmental and socioeconomic changes coupled with effective interventions.P. vivax–specific vectors in Cambodia. All main malaria vectors mentioned above were known to be more abundant for transmitting P. falciparum and less abundant for P. vivax. A recent study to identify anopheline vectors in relation to malaria transmission and disease risk in Pailin and Pursat, western Cambodia, was conducted in August–September and November–December 2005.N = 16,160) of seven anopheline species obtained by human landing collections were tested by PCR for P. falciparum and P. vivax, it was found that 11 An. dirus of 9,233 Anopheles  were positive for P. falciparum and four An. dirus and one An. minimus were positive for P. vivax.An. dirus, An. minimus, An. maculatus, and An. barbirostris could transmit both P. falciparum and P. vivax. Considering the increasing changes in their habitats , dominant vector species in Cambodia may change, which may or not affect transmission of malaria parasite species.P. falciparum and P. vivax.There is no available entomology data on Plasmodium falciparum was present in 1,443 (59.9%) and P. vivax in 965 (40.1%) patients. Mean G6PD activity was 11.6  U/g Hb. G6PD deficiency was present in 13.9% of all malaria patients , and severe deficiency  was more common in western (9.1%) than eastern (5.1%) Cambodia (P = 0.01). The researchers found that G6PD deficiency (as well as anemia and hemoglobinopathies) were common in malaria patients, and that decision for a large-scale deployment of primaquine in Cambodia should be preceded by primaquine safety studies in parallel with field evaluation of rapid, point-of-care G6PD tests.The only licensed drug available for the elimination of hypnozoites is primaquine. In combination with a drug for the treatment of blood-stage infection, it provides the only treatment strategy for the radical cure of vivax malaria. However, the safety of primaquine use remains a serious concern. When patients with severe  or mild  defects of G6PD activity receive primaquine, this drug may cause dose-dependent hemolysis resulting in life-threatening acute hemolytic anemia requiring careful medical monitoring and possible blood transfusion. G6PD deficiency in Cambodia is predominantly of the Viangchang variant (871G > A) which leads to severe deficiency. Studies have shown 10–15% prevalence of this variant in the Khmer populations, particularly males.atients 3/2,408, aP. vivax, and no attempt is usually made to determine past history of infection, most P. vivax cases naturally develop multiple relapses, and each episode of relapse is recorded as a new case. If primaquine radical treatment was, it would likely lead to a substantial reduction of P. vivax cases. Development of reliable, easy-to-use RDTs to detect G6PD deficiency at point-of-care is essential to deploying primaquine therapies. A new tool, the CareStart™ G6PD deficiency screening test , has been assessed recently in Cambodia by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method (“gold standard”).N = 903) were collected from Cambodian adults living in Pailin Province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median = 12.0 U/g Hg). On the basis of a normal hemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population were 5.5–17.2 U/g Hg (95th percentile). Ninety-seven subjects (10.7%) had 3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotyping was done in 66 G6PD-deficient subjects, and 63 of these exhibited findings consistent with the Viangchang variant (871G > A). The sensitivity of CareStartG6PD deficiency screening test prototype was disappointing (68%). More recently, the third generation of the CareStart G6PD RDT (www.accessbio.net) was found to be more capable and reliably detected moderate and severe G6PD-deficient individuals (enzyme activity < 30%), suggesting that this novel point-of-care is a promising tool for tailoring appropriate primaquine treatment of malaria elimination by excluding individuals with severe G6PDd for primaquine treatment.34In Cambodia, primaquine had not been mentioned in the national malaria treatment guidelines until May 2012. Primaquine is not used in Cambodia because the entire medical community heeds a common wisdom that primaquine use is dangerous if given to severe G6PD-deficient individuals.P. vivax, in addition to P. falciparum.P. falciparum and P. vivax infections in Cambodia, continues to be fully effective against P. vivax, whereas dihydroartemisinin–piperaquine failure for P. falciparum treatment is emerging in some areas in western and northern Cambodia.43Therapeutic efficacy studies (TES) using WHO protocol have been carried out at sentinel sites around Cambodia since 1991.P. vivax were detected between 2003 and 2005 in Sampouvloun  as a part of antimalarial drug efficacy monitoring by the national control program. The last TES conducted at four sites  from 2008 to 2011  showed proportions of treatment failure of 17.4% in Ratanakiri Province, 2010, following a chloroquine regimen, whereas in the same areas, dihydroartemisinin plus piperaquine treatment was 100% effective.P. falciparum, which is more common in the western provinces, P. vivax “suspected” resistance to chloroquine was detected in the eastern part of Cambodia,P. vivax malaria, mainly to simplify the management of the malaria cases, especially at community level.Sporadic and isolated failures of chloroquine against P. vivax monoinfection were randomized (1:1) to receive pyronaridine–artesunate (target dose of 7.2:2.4 to 13.8:4.6 mg/kg) or chloroquine (standard dose) once daily for 3 days. Each treatment group included 228 randomized patients. The study concluded that pyronaridine–artesunate efficacy in acute uncomplicated P. vivax malaria was at least as good as that of chloroquine in Cambodia.In search of an alternative ACT regimen, a phase III randomized, double-blind clinical trial of pyronaridine–artesunate combination was conducted at five centers across Cambodia, Thailand, India, and Indonesia.P. vivax–focused strategy and financing despite the fact that P. vivax is the key species the national control program will face with from now on. Experiences from elsewhere indicated that eliminating P. vivax is more difficult and usually requires a prolonged process. Therefore, efforts are made to mobilize additional resources required to embark on the elimination strategy and ensure further strengthening of partnerships with key donors and stakeholders at different levels.For several years, the CNM has decentralized implementation of malaria control activities to the provinces, operational districts (ODs), and peripheral health facility levels to be in line with the government's decentralization policy. Currently, there is no At present, the National Strategic Plan for Elimination of Malaria proposes a sequential progression toward preelimination and elimination. Among the activities being prioritized in the move toward malaria elimination are: universal LLIN coverage, universal access to free-of-charge antimalarials , case confirmation by slide/RDT or PCR, health and malaria information system strengthening, active case detection, case investigation and response, linkages with the private sector for diagnosis, treatment and reporting, monitoring of drug resistance and parasite genotyping, strong border control and improved screening tools, innovative protection of night-time outdoor workers, targeting mobile populations, and so on.P. vivax such as G6PD testing before administration of primaquine and access to primaquine for radical treatment of P. vivax malaria, but no agenda has been defined. In addition, there has been increased emphasis on surveillance in recent years. Full investigation of every malaria case and all malaria-related deaths is being encouraged in areas that are approaching elimination/preelimination.Some interventions are also planned specifically for P. vivax, it is envisaged that VMWs will play a role in documenting compliance to the 14-day regimen and following up P. vivax cases for 28 days to observe treatment outcomes.Cambodia follows current WHO guidelines on Test, Treat, Track for people living in endemic areas and will expand the efforts to all mobile/migrant populations at risk. VMWs will be scaled up to cover 4,528 villages  by 2020, thereby ensuring universal access to early diagnosis and treatment. When primaquine will be widely deployed for the radical cure of Standard vector control strategy uses LLINs and hammock nets distributed in mass campaigns at 3-year intervals to all people at risk of malaria, defined as people living within 2 km of the forest. The private sector involving rubber plantations, palm oil plantations, fruit orchards, mining, construction, hydroelectric power projects, and so on will be put under expanding bed net coverage . Personal protection has recently been researchedHealth communication is being promoted through behavior change communication and information, education, and communication strategies in addition to standard case management and vector control approaches. Innovative measures to reach mobile, migrant, and other hard-to-reach populations are also used to distribute bed nets, to spread messages on malaria prevention and care through taxi drivers.47P. falciparum patients who are still blood-smear positive 3 days after initiation of treatment . All these data were disaggregated by species thus allowing for the analyses of P. vivax–specific trends.Before 2009, the main source of malaria case data in Cambodia came from the national Health Information System, which provided aggregate data at OD level. Although useful for reporting total cases, these data were not sufficient to support the stratification of malaria risk by villages. Malaria risk stratification of villages had been based on distance from the forest but with outdated maps. In 2009, as a part of artemisinin-resistant containment, a malaria information system (MIS) was put in place for more efficient and timely reporting process and feedback. The system improved the overall efficiency of the program operation and fulfilled the need for 1) demographic data of all at-risk villages for planning interventions, 2) data relating to bed net distribution and treatment, 3) monthly malaria data by species at village level to identify villages with high incidence and possible transmission, 4) data relating to stock-outs of essential malaria drugs and supplies, and 5) real-time data on individual P. falciparum (and ultimately P. vivax) cases, and 3) real-time data about malaria outbreaks in high-transmission settings. The MIS now allows for village-level malaria stratification, which is being used for refining deployment of malaria control and preelimination activities.P. falciparum case (and ultimately every P. vivax case). Currently, no intervention is targeting P. vivax specifically. However, when primaquine radical treatment will be rolled out countrywide in conjunction with point-of-care G6PD testing, adaptation of these systems will be needed to monitor P. vivax trends and impact of the primaquine intervention more closely.In addition, the following data items were identified as being required at a later stage of the program as it moved toward elimination: 1) malaria data relating to private sector outlets, 2) real-time data of all In recent years, procurement delay has largely been responsible for a lag between what is planned under the national control program and what have actually been implemented. For example, delays in the procurement of RDTs and ACTs have resulted in periodic episodes of stock-outs of diagnostics and antimalarials in the country. Disbursement delays, mainly from the GFATM, have also affected implementation at field level, for example, lack of cash flows for payment of VMW incentives prohibited VMW meetings scheduled monthly, when all VMW records were to be verified and RDT/ACT stocks were to be replenished.P. falciparum. In the second phase, however, P. vivax is likely to pose a major challenge. The need for more efforts and longer time to eliminate P. vivax must be planned ahead and budgeted for while priority is set to keep vigilance against potential reemergence of P. falciparum infections including from neighboring countries.Cross-border issues, conflict areas, migrant populations, remote villages, poor drug quality, and counterfeit drugs remain key challenges for malaria control in Cambodia regardless of parasite species. As a consequence of the operation to contain artemisinin resistance, the first phase of malaria elimination in Cambodia aims at eliminating P. vivax elimination: 1) maintaining maximal intervention efforts for an extended period to eliminate indigenous infections, 2) ensuring the safety of primaquine and compliance with its prolonged treatment course, 3) guarding against the risk of reintroduction of P. vivax into areas without transmission, but where competent anopheline vectors remain, through the entry of P. vivax carriers, and 4) strengthening and maintaining adequate malaria surveillance beyond elimination to prevent reintroduction.Special attention will have to be given to a number of implementation challenges specifically relevant to P. vivax compared with P. falciparum. Drug resistance has been the major obstacle for malaria control in Cambodia, often regarded as the epicenter of multidrug P. falciparum–resistant malaria. Rigorous control efforts, primarily targeting artemisinin-resistant P. falciparum from 2009, have made significant impacts to reduce malaria cases and deaths to the lowest levels ever. Like many P. falciparum/P. vivax coendemic countries in Asia and South America, as P. falciparum cases drop, the burden of P. vivax becomes more prominent. The extent of problems associated with P. vivax, including its overall health and socioeconomic impacts, might not have been anticipated before. Eliminating P. vivax is known to require a lengthier process than eliminating P. falciparum. Without strengthening malaria control policy to target P. vivax and to seriously implement specific actions against the parasite, especially maximizing access to radical cure, it would be unlikely for Cambodia to achieve the ambitious goal of malaria elimination by 2025.Cambodia is a good example of malaria-endemic countries with an impressive decline in malaria burden in the past decade, with a slower decline of


Plasmodium are known to infect a variety of vertebrate hosts, including ungulates . A recent study found that up to a quarter of white-tailed deer (Odocoileus virginianus) in North America are infected with the parasite Plasmodium odocoilei. In addition to occupying an important ecological niche, white-tailed deer are popular game animals and deer farming represents a rapidly growing industry. However, the effect of P. odocoilei infection in this ecologically and economically important ungulate species is unknown. Our work is significant because (i) we identified a high prevalence of P. odocoilei in farmed deer and (ii) we found evidence for both cleared and persistent infection, as well as an association with decreased survival of young fawns.Malaria parasites of the genus  Odocoileus virginianus), an ecologically and economically important species, are the most widely distributed large animals in North America. A recent study indicated that up to 25% of all white-tailed deer may be infected with Plasmodium odocoilei, a malaria parasite belonging to the distinct clade of ungulate-infecting Plasmodium spp. Because the clinical impact of P. odocoilei on deer health and survival is unknown, we undertook a retrospective longitudinal study of farmed Floridian O. virginianus fawns. We found that a substantial proportion (21%) of fawns acquire malaria infection during the first 8 months of life. Some animals naturally clear P. odocoilei infection, while other animals remain persistently positive. Importantly, we found that animals that acquire malaria parasites very early in life have poor survival compared to animals that remain uninfected. Our report thus provides the first evidence of a clinically significant impact of malaria infection in young deer.White-tailed deer . A recent study found that up to a quarter of white-tailed deer (Odocoileus virginianus) in North America are infected with the parasite Plasmodium odocoilei. In addition to occupying an important ecological niche, white-tailed deer are popular game animals and deer farming represents a rapidly growing industry. However, the effect of P. odocoilei infection in this ecologically and economically important ungulate species is unknown. Our work is significant because (i) we identified a high prevalence of P. odocoilei in farmed deer and (ii) we found evidence for both cleared and persistent infection, as well as an association with decreased survival of young fawns. Wild ungulates comprise a diverse and highly successful group of hoofed mammals. Their close ecological and phylogenetic association with livestock such as cattle, goats, sheep, and pigs has made them a center of study for emerging infectious diseases at the livestock-wildlife interface for more than a century. More recently, ungulates have been prioritized in many One Health initiatives due in part to the close association of humans and domestic ungulates, which creates a conduit for zoonotic transmission . Nearly Plasmodium species. In 2016, however, molecular characterization of blood-borne parasites of domestic water buffalo and African goats revealed that two ungulate malaria species, Plasmodium bubalis and Plasmodium caprae, are phylogenetically distinct from all other mammalian Plasmodium spp. and group within a single unique clade. Interestingly, the clade of ungulate-infecting Plasmodium spp. groups most closely with hemosporidian species that infect birds, bats, and lizards, rather than grouping with primate-infecting Plasmodium spp. Ungulate malarial species appear to have branched off after Haemoproteus and Parahaemoproteus spp. (which infect birds) and before Polychromophilus spp. (bats) and bird- and lizard-infecting Plasmodium spp. , the most widely distributed large animal in North America. An intraerythrocytic parasite consistent with malaria, described as Plasmodium odocoilei, had been morphologically described decades ago from blood smears of a splenectomized animal  in South America. Sequences from these parasites also fall within the clade of ungulate-infecting Plasmodium and group most closely with P. odocoilei , parasites have not been readily detected microscopically upon examination of blood smears from supposedly infected (nucleic acid-positive) animals  most often leads to minor upper respiratory illness in adults but represents a leading cause of hospitalization of and life-threatening respiratory distress in young infants . This dintensity . However malaria . Given tP. odocoilei infection was obtained through a longitudinal study of farmed O. virginianus fawns in the Panhandle of Florida. In brief, this study was performed in cooperation with the University of Florida (UF) Cervidae Health Research Initiative (CHeRI), which facilitates private industry-academic institution partnerships for deer health research. This pilot study was a retrospective cohort study, taking advantage of blood samples and survival data previously acquired for the study of epizootic hemorrhagic disease virus (EHDV) epidemiology. Blood was sampled from enrolled fawns at 3, 6, and 8 months of life. In addition, outcomes, including survival and release, were recorded for each animal.Information on the natural course of O. virginianus fawns had acquired P. odocoilei infection at least once during the first 8 months of life , while two additional animals were first positive at 6 months of life (January), and a final animal was positive for the first time at 8 months of life (April). Our study results thus indicate that infection with malaria parasites is common in farmed white-tailed deer.We found that a substantial proportion (7/33 fawns = 21%) of  of life . At any P. odocoileicytB amplicons obtained through this study, we found that there are multiple P. odocoilei strains circulating within the Florida sample population. This finding is consistent with the previous study of P. odocoilei sampled in the eastern United States, which identified six different cytB alleles  in the 8-month sample of OV136. As our primer sets were unbiased with respect to Plasmodium spp., our results support previous observations that white-tailed deer appear to be infected only with multiple closely related P. odocoilei strains, because no other Plasmodium spp. were detected.Based on sequence analysis of all  alleles . Many ofasmodium  12). We. WeP. odcytB genetic signatures were detected. For OV137, the 6- and 8-month samples were genetically identical over the 171-bp cytB sequence that was available for all samples, as well as over a longer amplicon (574 bp) that was common to both the 6- and 8-month OV137 samples. This is suggestive of a single continuous infection, although the data are not conclusive. In contrast, for OV115, the cytB sequences from each time point appeared to be different. While these cytB alleles are distinct from each other, they match cytB alleles found in other, independent infections  present in the population.In the two fawns that had sequential positive samples (OV137 and OV115), different fections . HoweverPlasmodium infection in O. virginianus. Because symptoms associated with disease were not recorded, possible outcomes are limited to resolution of infection, persistence of infection, or death. Two infected animals had subsequent samples in which P. odocoilei nucleic acid was not detected, suggesting that these animals either had naturally resolved the infection or were capable of controlling parasite replication at levels below the limit of detection. In contrast, two animals were repeatedly positive over a period of several months, suggesting either that P. odocoilei is capable of persistent parasitemia or that there was sufficient infection pressure for animals to have become reinfected during the course of study.We observed a range of outcomes of P. odocoilei infection in O. virginianus is associated with increased mortality. In humans living in locations of malaria endemicity, malaria infection is most life-threatening during early childhood, with the majority of attributable deaths occurring in children under the age of 5, prior to the acquisition of immunity .Importantly, we examined whether immunity . Similar6 months , with 7%P. odocoilei was not definitively determined. However, malaria in humans has a substantial impact on the risk for and outcome of coinfections (–Plasmodium infection. We found that a higher percentage of Plasmodium-infected fawns than of Plasmodium-negative controls was EHDV coinfected (P = 0.125). This finding suggests that Plasmodium infection may be associated with an increased risk of acquiring EHDV or may alter viral control. Furthermore, the acute clinical decompensation and presence of EHD viral RNA in heart, spleen, and lung tissues at necropsy strongly suggest that EHDV contributed to death in Plasmodium-infected fawns.The cause of death in animals testing positive for fections –21. For infected  , coincident with the probable timing of waning maternal antibody titers , although careful entomological studies have yet to be performed to confirm the vector competence of A. punctipennis  of 3.37%  was found  mosquito biting rate for O. virginianus, but existing evidence suggests the EIR is comparable to that of human malaria in sub-Saharan Africa. Intriguingly, several deer species possess globin polymorphisms that cause erythrocyte sickling, and recent evolutionary studies suggest that these genetic changes may be undergoing positive selection    , many ofnfection , 21. Plaesvirus) , 31 and esvirus) –35. TherP. odocoilei in particular. Key limitations of our study included its small sample size and relatively sparse sampling frequency. Current understanding of ungulate malaria biology is also significantly hampered by the lack of genomic information. The P. odocoilei genome has not been characterized; neither has that of any other representative ungulate malaria parasite species. As such, all aspects of its molecular genetics and gene repertoire are unknown, which hampers serological studies of prevalence and molecular epidemiological investigations. Of particular interest will also be the multigene families (such as the family of var genes) that mediate adhesion, virulence, and immune evasion in other Plasmodium spp. Comparative genomics has also already revealed unexpected and fundamental differences between murine and primate malaria parasite species in metabolic adaptations . All live-born fawns surviving to 3 months of age were enrolled for further study. Diagnostic blood samples were obtained from client-owned, farmed O. virginianus fawns (n = 33), enclosed within a single 500-acre preserve, at 3, 6, and 8 months of life. The outcomes for all study participants, including death, release , and continued enclosure, were recorded.All studies were performed under the approval of the University of Florida IACUC (IACUC Study 201609390). Fawns were enrolled from a privately owned farm in Gadsden County, FL, that is permitted by Florida Department of Agriculture and Florida Fish and Wildlife Commission to keep, breed, and sell white-tailed deer  and at 6 months  and at 8 months of age (concordant with vaccine booster administration). Blood was stored in EDTA at −4°C for no more than 12 h, prior to frozen storage at −80°C. The samples used in this study were archived and have not yet been published. Giemsa staining of blood smears was not available for these samples.Plasmodium odocoilei analyses and by quantitative PCR (qPCR) for epizootic hemorrhage disease virus (EHDV) analyses. For screening of EHDV, total RNA was extracted using a standard magnetic bead protocol . Multiplex quantitative reverse transcription-PCR (qRT-PCR)  according to the manufacturer’s instructions. Extracted DNA was evaluated by nested PCR for qRT-PCR)  was usedescribed , 38. BritB locus , 39 wereequences . Using scytB coding sequences (CDS) from samples described in this study have been deposited in GenBank under the following accession numbers: MG709243, MG709244, MG709245, MG709246, MG709247, MG709248, MG709249, MG709250, MG709251, and MG709252.Nucleic acid sequences of the partial


Pregnancy during the adolescent period is challenging mainly because of the nutritional demands of both the adolescent and pregnancy period. The risk for anaemia increases especially in developing countries such as Ghana where malaria is endemic and the practice of pica is common. In this study, we sought to determine the prevalence of anaemia, pica practice and malaria infection among pregnant adolescent girls and assess the extent to which these factors are associated.Two hundred and sixty five (265) pregnant adolescent girls were recruited from three hospitals in Accra. Haemoglobin levels, malaria infection and the practice of pica were assessed. Pearson's Chi squared tests were used to determine associations and logistic regression analysis was used to determine the odds of being anaemic. Significance was set at p≤0.05.Anaemia prevalence was 76% with severity ranging from mild (47.8%) to severe (0.8%). About 27.5% were moderately anaemic. Pica was practiced in only 9.1% of the girls. Malaria infection was prevalent in 17.7% of the girls. The logistic regression analysis indicated that pregnant girls with malaria infection were 3.56 times more likely to be anaemic when compared to those without malaria. Also, those who practiced pica were 1.23 times more likely to be anaemic when compared to those who did not practice pica.Anaemia is very prevalent in pregnant adolescent girls and is a public health problem. Drastic measures should be taken to reduce the high prevalence. The adolescent period is characterized by intense growth physically, psychosocially and cognitively. The intensity of this growth period necessitates the increase in nutritional needs. During this period there is a high demand for iron, especially in the adolescent girl because of the onset of mensuration. Iron deficiency is a major cause of anaemia. The risk for anaemia increases when the adolescent girl becomes pregnant. There are several factors that interact and contribute negatively to the outcome of pregnancy in the adolescent girl. The presence of anaemia, practice of pica and malaria has all been implicated. The presence of these conditions in pregnant adolescent girls can pose greater challenge because of the obvious burden of the pregnancy on the rapidly growing mother which additionally has to cater for another developing foetus. Anaemia affects about 38% of pregnant women globally. The prevalence is about 44.6% in Africa and 56% in Ghana . The WorMalaria is an endemic infectious disease and a common public health problem in Ghana, affecting pregnant women. It is globally ranked as the second most common cause of deaths related to infectious disease after tuberculosis . AccordiThe study was a cross sectional study carried out among pregnant adolescent girls between the ages of 15-19 years. The girls were attending ante-natal clinics for the first time at three hospital facilities in the Greater Accra Region of Ghana namely the Mamprobi Polyclinic, James Town Maternity Home (Ussher fort Polyclinic) and the La Polyclinics. A total of 265 pregnant adolescent girls were recruited for the study. All pregnant girls who reported for the first time for ante-natal clinic were approached and invited to be part of the study. Those who consented and were apparently healthy were recruited. Pregnant girls who had cases of haemoglobinopathies were excluded from the study. Ethics approval for the research was obtained from the Ethics and Protocol Review Committee of the School of Biomedical and Allied Health Sciences of the University of Ghana, College of Health Sciences. Permission was also sought from the Greater Accra Health Directorate and the health facilities where the study was undertaken.® One Step Malaria Pf/Pv Ag Test kitwas used to determine the presence of malaria infection according to manufacturer's instructions. Data were entered and analysed using SPSS version 17 and summarized using means and percentages. Pearson's Chi squared tests were used to determine associations and logistic regression analysis was used to determine the odds of being anaemic. The level of significance was set at p = 0.05 with a confidence interval of 95%.Semi-structured questionnaires were used to obtain information regarding patient's background including ages, level of education, marital status and practice of pica obtained. Five (5) ml of blood was drawn into an EDTA tube. Full blood count was performed to determine haemoglobin levels. Cut-off values for the determination of anaemia in pregnancy were set based on World Health Organization (WHO) criteria . The fol213.91, p = 0.003, df =3) and anaemia and pica practices . Majority (51.3%) of those who had malaria were moderately anaemic (The background characteristics of the girls are summarized in  anaemic . About 4 anaemic . The per anaemic . The resAnaemia in pregnancy is an obstetric risk which needs to be treated to prevent complications. The present study reports a disturbingly high (76.2%) prevalence of anaemia among pregnant girls. This is higher than the national prevalence of 56% in pregnant women in Ghana and other reported studies in Kenya 61%), India (63%) and Nigeria (40.4%) %, India –15. The Malaria infection was present in about 17.7% of the girls. In similar studies Uddenfeldt et al  and GlovIn conclusion, this study has demonstrated that anaemia prevalence in adolescent pregnant girls is high. Pregnant girls who were anaemic were more likely to be practicing pica and infected with malaria. This report should further strengthen the need to investigate pica practices in pregnant adolescent girls during antenatal clinics and also step up measures to prevent malaria infections.Prevalence of anaemia in all pregnant women in Ghana;Malaria infection among women who are pregnant in Ghana.Prevalence of anaemia in pregnant adolescent girls in Ghana;The prevalence of the practice of pica among pregnant adolescent girls;The contribution of malaria and pica in anaemia among pregnant adolescent girls.


Plasmodium infections in mothers and children in Uganda has revealed there to be both persistent and increasing parasitemia of certain species, which has clinical significance, despite regular access to frontline antimalarial treatments.A longitudinal cohort study of multispecies  Plasmodium infections and the longitudinal dynamics of Plasmodium species in children. Host age, household location, and Plasmodium falciparum infection were strongly associated with nonfalciparum Plasmodium infections, and Plasmodium malariae infection was associated with splenomegaly. Despite regular artemisinin combination therapy treatment, there was a 3-fold rise in P. malariae prevalence, which was not accountable for by increasing age of the child. Worryingly, our findings reveal the consistent emergence of nonfalciparum infections in children, highlighting the complex dynamics underlying multispecies infections here. Given the growing body of evidence that nonfalciparum malaria infections cause significant morbidity, we encourage better surveillance for nonfalciparum Plasmodium infections, particularly in children, with more sensitive DNA detection methods and improved field-based diagnostics.As part of a longitudinal cohort investigation of intestinal schistosomiasis and malaria in Ugandan children and their mothers on the shorelines of Lakes Victoria and Albert, we documented risk factors and morbidity associated with nonfalciparum  Plasmodium falciparum is the leading cause of malaria, other species—namely, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi and Plasmodium ovale wallikeri—circulate concurrently, although Duffy-negative individuals curtail P. vivax distributions  by microscopy; 82.9% [95% CI, 79.8%–85.8 vs 66.0% [95% CI, 61.9%–69.9% by real-time PCR) see . PlasmodP. falciparum, and P. malariae infections increased with increasing age in children; however the fold difference between the youngest and oldest age category was substantially larger for P. malariae infections than P. falciparum infections (15.3-fold vs 1.3-fold). In contrast, there was no significant difference in P. ovale spp. infection prevalence among the different age groups. The prevalence of highly parasitemic infections (≥5000 parasites/µL) peaked in children aged 1–2 years, then declined in older age groups. In mothers, the prevalence of P. malariae, P. ovale spp., and highly parasitemic infections was very low and did not vary among age groups. For slide-positive malaria and P. falciparum infections, there was a general downward trend in prevalence with increasing age group in mothers. The prevalence of P. falciparum infections as detected by real-time PCR was higher than the prevalence of slide-positive malaria, demonstrative of submicroscopic carriage of Plasmodium parasites in the mothers.The relationship between age and malaria infection prevalence was investigated in children and mothers . The preP. falciparum, P. malariae, and P. ovale spp. infections in children were investigated using logistic regression analysis. This analysis was not carried out in mothers due to the low prevalence of nonfalciparum infections. In univariable analysis, infection with 1 malaria species was associated with infection with each of the other 2 species and also with hookworm infection  was associated with reduced odds of infection, as was sleeping under a bednet in the case of P. falciparum. There was also a positive association between P. falciparum infection and living in a household with goats or sheep .The associations observed among the different malaria species infections  suggesteW  =  −0.602; P  = 0.182; N  = 521) or Lake Victoria . In multivariable models, infection with P. falciparum was associated with moderate anemia and splenomegaly. In addition, high P. falciparum infection levels were associated with fever. Infection with P. malariae was associated with an enlarged spleen, and multispecies malaria infections were more strongly associated with spleen enlargement than single-species malaria infections  .At baseline, there was no obvious visual pattern of infections of any P > .05) . HoweverP > .05) , the arePlasmodium infections in children living in lakeshore communities in Uganda. One of the major risk factors identified for P. malariae and mixed-species infections was host age: infection with any Plasmodium species was much more common in children than in their mothers, and older children were more likely to be infected with each of the Plasmodium species than younger children. This age-prevalence pattern is well established for P. falciparum infections, and similar results have been reported for P. malariae and/or mixed-species infections in sub-Saharan Africa and Papua New Guinea [P. ovale spp. infection and age in our study most likely reflects the low prevalence of P. ovale spp. infections in the baseline survey and that our diagnostic approach could not differentiate the 2 subspecies of P. ovale curtisi and P. ovale wallikeri that have been reported sympatric within this part of Uganda [Our analysis of the SIMI dried blood spot archive has provided a much deeper insight into the complex dynamics and significance of multispecies w Guinea , 37, 38,f Uganda .Plasmodium species and was more common in children along Lake Victoria than along Lake Albert. Children in Bukoba were more at risk of mixed-species malaria infections than elsewhere. Similarly, in Malawi, Bruce et al demonstrated variations in prevalences of different Plasmodium species infections among villages [Anopheles being implicated [Infection prevalence by village varied for all villages . All of villages , althougplicated , 42. Altplicated , with poP. falciparum infection and P. malariae and P. ovale species infections, and the vast majority of P. malariae and P. ovale spp. infections existed as coinfections with P. falciparum. Consistent with this, multiple lottery-kind analysis revealed nonrandom distributions of Plasmodium species. Other studies have reported a frequency of P. falciparum and P. malariae coinfections higher than would be expected [Plasmodium species, an obvious example of which could be shared Anopheles vectors. It is not yet known which vectors play a role in natural transmission of P. malariae and P. ovale spp. in Uganda [We found a strong association between expected , 44–47, expected  and in Mexpected ). Our fin Uganda , 4.Plasmodium infections on any of the clinical indicators of malaria, contrasting with other reports [Plasmodium with P. malariae infection and splenomegaly, which, to our knowledge, is the first time this observation has been made and adds to the growing body of evidence supporting the clinical significance in children of nonfalciparum malaria within mixed-species Plasmodium infections.We did not observe any protective effect of mixed-species versus single-species  reports , 24, altP. malariae prevalence seen here is most worrying, notwithstanding an upward trend in P. ovale spp. prevalence and consistently high P. falciparum prevalence. The rise in P. malariae may be partly explained by the increasing age of the children, even though the association between survey time point and mixed P. falciparum/P. malariae infections was maintained upon controlling for child age. A 4-year longitudinal study of Plasmodium infection in children in rural Burkina Faso found a 15-fold increase in P. malariae prevalence and a 4-fold increase in P. ovale spp. prevalence between 2007 and 2010 [N = 248) received 4 ACT treatments, 27% received 3 treatments, and, based on reporting by mothers, 68% of children received further antimalarial treatment between surveys (unpublished data). Consistent with this hypothesis, we have previously demonstrated the persistence of P. malariae infections after ACT treatment in the SIMI cohort, most likely due to recrudescence of parasitemia after treatment rather than relapse per se [Despite repeated artemisinin combination therapy (ACT) treatments, the dramatic rise in and 2010 , an indiP. malariae may have a relapsing, hepatic hypnozoite stage analogous to P. vivax and P. ovale spp. or that there is sequestration of quiescent blood-stage form analogous to an arrested lymphatic stage observed in rodent Plasmodium species [P. malariae infection in China, Sweden, and the United Kingdom [P. malariae prevalence is perhaps a combination of the long-term persistence of P. malariae parasite processes and drug-induced selection, alongside implementation of more sensitive methods of molecular diagnosis that go beyond the detection thresholds of expert microscopy.In certain settings, it has been argued that  species . This ar Kingdom . The latP. malariae  infections in the face of frontline treatment for P. falciparum. With efforts increasingly directed toward elimination of falciparum malaria, we encourage better surveillance of nonfalciparum Plasmodium infections in the future, particularly in children, with more sensitive DNA detection methods and improved field-based diagnostics.In conclusion, our findings highlight the cryptic burden of nonfalciparum malaria infections and indicate that there is a potential for emergence of The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.Supplementary Table 4Click here for additional data file.Supplementary Figure 1Click here for additional data file.