Plasmodium falciparum-specific antibodies of all Ig classes as compared to the Dogon. However, the proportions of B cell subsets in the Fulani and Dogon that may account for differences in the levels of Ig have not been characterized.Several studies indicate that people of the Fulani ethnic group are less susceptible to malaria compared to those of other ethnic groups living sympatrically in Africa, including the Dogon ethnic group. Although the mechanisms of this protection remain unclear, the Fulani are known to have higher levels of P. falciparum-naïve adults in the U.S. (n = 8). At the time of the blood collection, P. falciparum infection was detected by blood-smear in 16% of the Fulani and 36% of the Dogon volunteers. Thawed lymphocytes were analysed by flow cytometry to quantify B cell subsets, including immature and naïve B cells; plasma cells; and classical, activated, and atypical memory B cells (MBCs).In this cross-sectional study, venous blood was collected from asymptomatic Fulani (n = 25) and Dogon (n = 25) adults in Mali during the malaria season, and from Plasmodium falciparum infection was associated with a higher percentage of plasma cells among Fulani , but not Dogon adults.The overall distribution of B cell subsets was similar between Fulani and Dogon adults, although the percentage of activated MBCs was higher in the Fulani group . The percentage of atypical MBCs was similar between Fulani and Dogon adults . P. falciparum infection is associated with a higher percentage of plasma cells in the Fulani compared to the Dogon, findings that may account for the higher levels of P. falciparum antibodies in the Fulani.These data show that the malaria-resistant Fulani have a higher percentage of activated MBCs compared to the Dogon, and that  Plasmodium falciparum parasite densities compared to individuals of other ethnic groups living sympatrically with the Fulani, including the Dogon [P. falciparum antigens expressed at the liver and blood stages [Several studies have demonstrated that individuals of the Fulani ethnic group in West Africa are at lower risk of malaria and tend to have lower he Dogon . Althougd stages -5, and ed stages . The B c+CD27+CD38− and CD19+CD27++CD38+++, respectively  and long-lived plasma cells (LLPCs), defined in humans by the cell surface markers CD19P. falciparum exposure in Malian children and adults, as well as Peruvian adults [− CD19+ CD20+ CD21− CD27−, similar to a MBC subpopulation initially identified in healthy US individuals in mucosal-associated lymphoid tissues by expression of the inhibitory receptor Fc-receptor-like-4 (FCRL4) [et al showed that compared to naïve B cells and classical MBCs, FCRL4+ MBCs proliferated less well in response to BCR-cross-linking and/or to CD40L and Toll-like receptor 9 (TLR9) agonist CpG, and showed a decreased ability to differentiate into antibody secreting cells in response to polyclonal stimulation [+ MBCs in HIV-viremic [P. falciparum-exposed individuals [+ T cells during chronic viral infections [+ MBCs in P. falciparum-exposed individuals is unknown, we refer to this B cell subset in the context of malaria as ‘atypical’ rather than ‘exhausted’.Recently, it was reported that n adults , is asso (FCRL4) . B cells (FCRL4)  and HCV  (FCRL4) . Moir etmulation . FCRL4+ -viremic  and P. fividuals  also expfections . Given tP. falciparum infected and uninfected individuals from both ethnic groups are presented.Here, the proportion of peripheral blood B cells that are immature and naïve B cells; plasma cells; and classical, activated, and atypical MBCs are compared in Fulani and Dogon adults , and furPlasmodium falciparum transmission is seasonal and intense at this site from July through December. The entomological inoculation was approximately 17 infective bites/person/month in September of 2000. This cross-sectional study includes 50 adults enrolled in October 2008. As is typical in Mali, the P. falciparum-infected adults had no signs or symptoms of malaria when the blood smear was prepared and they were not treated with anti-malarial drugs (the blood smear was not read immediately). The Ethics Committee of the Faculty of Medicine, Pharmacy, and Odonto-Stomatology at the University of Bamako, Mali approved this study. De-identified specimens were obtained from this study for immunological analyses. Written, informed consent was obtained from all study participants.This cross-sectional study was done in October 2008 in Mantéourou, Mali, a rural village approximately 850 km north of the capital of Bamako. A detailed description of the study site has been published elsewhere . ParticiP. falciparum exposure is unlikely. Blood samples were obtained for research use after written informed consent was obtained from all study participants enrolled in a protocol approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, NIH (protocol # 99-CC-0168).Peripheral blood mononuclear cells (PBMCs) from eight healthy adult blood bank donors in the U.S. were also analysed. Demographic and travel history data were not available from these anonymous donors but prior Malian blood samples were drawn by venipuncture into sodium citrate-containing cell preparation tubes . PBMCs were isolated according to the manufacturer's instructions and frozen in foetal bovine serum (FBS)  containing 7.5% dimethyl sulfoxide . U.S. blood samples were drawn into heparinized tubes (BD) and PBMC were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation (Amersham Biosciences) according to the manufacturer's instructions, and frozen in same conditions as Malian samples. PBMCs were kept at −80°C for 24 hours using freezing containers  to control the rate of freezing and then transferred to liquid nitrogen. For analysis, PBMCs were rapidly thawed in a 37°C water bath, washed first in PBS with 10% heat-inactivated FBS and then in complete RPMI ).PBMCs were washed in PBS with 4% heat-inactivated FBS and incubated for 30 min at 4°C with mouse monoclonal fluorescently-labeled antibodies specific for CD10 APC (HI10a) and CD20 APC-H7 (L27) (BD Biosciences); CD19 PerCP‒Cy5.5 (SJ25C1) and CD27 PE-Cy7 (O323) (eBioscience) and CD21 FITC (BL13) (Beckman Coulter). Flow cytometry was performed with a BD™ LSR II Table flow cytometer (BD Biosciences) and data were analysed using FlowJo software .Plasmodium falciparum densities were recorded as the number of asexual parasites/μl of whole blood based on an average leukocyte count of 7,500/μl. Each smear was evaluated separately by two expert microscopists. Any discrepancies were resolved by a third expert microscopist.Thick blood smears were stained with Giemsa and counted against 300 leukocytes. Data were analysed using GraphPad Prism 5 for Mac OS X . The non-parametric Mann–Whitney test was used to compare continuous variables between groups, and the Fisher's exact test was used to compare categorical variables. For all tests, two-tailed p values were considered significant if ≤0.05.P. falciparum infection status data are shown in Table P. falciparum by blood-smear. Of those who were infected, the mean parasite densities for the Fulani and Dogon were 2281.25 ± 1285.9 and 2563.5 ± 1652.9 asexual parasites/μl, respectively. All participants were afebrile and asymptomatic at the time of venous blood draw.Twenty-five Fulani adults and 25 Dogon adults living in the same village in Mali were included in this study. Demographic and P. falciparum-naïve U.S. adults (n = 8).The proportion of various B cell subsets in the peripheral blood of Fulani (n = 25) and Dogon (n = 25) adults was compared by flow cytometry analysis of thawed PBMCs, which had been collected at the peak of the malaria season. These data were compared to B cell profiles of +CD19+), naïve B cells (CD10-CD19+CD20+CD21+CD27-), plasma cells/plasma blasts (CD10-CD19+CD20-CD21-CD27+), classical MBCs (CD10-CD19+CD20-CD21+CD27+), activated MBCs (CD10-CD19+CD20+CD21-CD27+) and atypical MBCs (CD10-CD19+CD20+CD21-CD27-). Figure A panel of fluorophore-conjugated antibodies specific for CD10, CD19, CD20, CD21 and CD27 allowed for the quantification of immature B cells (CD10+) was similar in Fulani and Dogon adults (Figure As a percentage of total lymphocytes, the mean percentage of total B cells (CD19Together these data indicate that the overall distribution of B cell subsets is similar among the Fulani and Dogon adults in this study population, with the exception of activated MBCs, which were increased among the Fulani. Activated MBCs are known to promote signaling cascades that induce their differentiation into antibody secreting cells, thus a higher level of activated MBCs among the Fulani is consistent with their propensity toward higher antibody responses and decreased malaria susceptibility ,6.P. falciparum-exposed individuals at separate study sites in Mali [P. falciparum-exposed populations. The specificity and function of atypical MBCs in the context of P. falciparum transmission remains to be determined. It is possible that atypical MBCs contribute to protection against malaria by regulating the host immune response; on the other hand, it is also conceivable that functional exhaustion of P. falciparum-specific MBCs through repeated infections contributes to the inefficient acquisition and relatively rapid loss of P. falciparum-specific MBCs and long-lived antibodies which has been observed in areas of intense P. falciparum transmission [The higher percentage of atypical and activated MBCs in Fulani and Dogon Malians versus U.S. adults is consistent with previous studies of  in Mali , in The  in Mali , and Per in Mali . These dsmission ,17.Also consistent with previous observations ,11,16, iP. falciparum infection was associated with alterations in B cell subsets, Fulani and Dogon adults were stratified by whether or not the thick blood smears were positive for P. falciparum infection at the time of blood collection. Plasmodium falciparum infection status was not associated with significant differences in the percentages of total B cells (Figure P. falciparum infection was associated with a lower percentage of immature B cells among the Fulani (Figure Plasmodium falciparum infection was not associated with significant differences in the percentage of naïve B cells in either ethnic group (Figures P. falciparum infection was associated with a significantly higher percentage of plasma cells/plasma blasts in the Fulani (Figure P. falciparum-infected Fulani adults is consistent with the higher levels of P. falciparum-specific antibodies observed by others in this ethnic group [P. falciparum infection was not associated with significant changes in the percentages of classical, activated or atypical MBCs in either ethnic group (Figures To assess whether concurrent asymptomatic s Figure A. Howeve Figures C, while ic group -4. Asymp Figures E–G.P. falciparum-specific antibody response of the Fulani group is among the more consistent observations, which led us to investigate potential differences in B cell subpopulations. Indeed, the Fulani had a higher percentage of activated MBCs compared to the Dogon, and that P. falciparum infection was associated with higher percentage of antibody-secreting cells among the Fulani but not the Dogon. These results serve as a basis for further studies of the B cell biology that underlies the association between Fulani ethnicity and decreased malaria susceptibility.It is well established that the Fulani ethnic group is less susceptible to malaria compared to other sympatric ethnic groups in West Africa, but the mechanisms underlying this observation remain obscure. The enhanced The authors declare that they have no competing interests.SP designed and carried out the flow cytometry, analysed the data, and drafted the manuscript. DD participated in the design and coordination of the study in Mali and analysed the clinical data. BT, LHM, MTB, OKD and AD conceived of the study and participated in its design and coordination in Mali. SKP and PDC conceived of the study, participated in its design and coordination in Mali, and helped to analyse the data and draft the manuscript. All authors read and approved the final manuscript.


Malaria has a negative effect on the outcome of pregnancy. Pregnant women are at high risk of severe malaria and severe haemolytic anaemia, which contribute 60-70% of foetal and perinatal losses. Peripheral blood smear microscopy under-estimates sequestered placental infections, therefore malaria rapid diagnostic tests (RDTs) detecting histidine rich protein-2 antigen (HRP-2) in peripheral blood are a potential alternative.Plasmodium falciparum malaria (after childbirth) were conducted using Giemsa microscopy and placental histopathology respectively as the reference standard. The study was conducted in Mbale Hospital, using the midwives to perform and interpret the RDT results. Discordant results samples were spot checked using PCR techniques.HRP-2 RDTs accuracy in detecting malaria in pregnancy (MIP >28 weeks gestation) and placental P. falciparum malaria during pregnancy. At delivery, in non-symptomatic women, RDTs had a 80.9% sensitivity (95% CI 57.4-93.7) and a 87.5% specificity (95%CI 80.9-92.1), PPV of 47.2% (95% CI 30.7-64.2) and NPV of 97.1% (95% CI 92.2-99.1) in detecting placental P. falciparum infections among 173 samples. At delivery, 41% of peripheral infections were detected by microscopy without concurrent placental infection. The combination of RDTs and microscopy improved the sensitivity to 90.5% and the specificity to 98.4% for detecting placental malaria infection (McNemar's X 2> 3.84). RDTs were not superior to microscopy in detecting placental infection (McNemar's X 2< 3.84). Presence of malaria in pregnancy and active placental malaria infection were 38% and 12% respectively. Placental infections were associated with poor pregnancy outcome  (aOR = 37.9) and late pregnancy malaria infection (aOR = 20.9). Mosquito net use (aOR 2.1) and increasing parity (aOR 2.7) were associated with lower risk for malaria in pregnancy.Among 433 febrile women tested, RDTs had a sensitivity of 96.8% (95% CI 92-98.8), specificity of 73.5% (95% CI 67.8-78.6), a positive predictive value (PPV) of 68.0% (95% CI 61.4-73.9), and negative predictive value (NPV) of 97.5% (95% CI 94.0-99.0) in detecting peripheral Use of HRP-2 RDTs to detect malaria in pregnancy in symptomatic women was accurate when performed by midwives. A combination of RDTs and microscopy provided the best means of detecting placental malaria. RDTs were not superior to microscopy in detecting placental infection. With a high sensitivity and specificity, RDTs could be a useful tool for assessing malaria in pregnancy, with further (cost-) effectiveness studies. Plasmodium falciparum malaria remains a major contributor of the disease burden in sub-Saharan Africa (SSA). Children under five years of age and pregnant women represent the most vulnerable population. During pregnancy, malaria infection leads to parasite sequestration in the maternal placental vascular space, with the consequence of increased risks of abortion, stillbirth, prematurity, intra-uterine growth retardation (IUGR) and maternal anaemia ) were active infections (16 acute and five chronic) and the rest past infections. RDTs had a sensitivity of 80.9% (95% CI 57.4-93.7), specificity of 87.5% (95% CI 80.9-92.1), PPV of 47.2% (95% CI 30.7-64.2) and NPV of 97.1% (95% CI 92.2-99.1) against the reference standard of placental histology in detecting placental P. falciparum malaria among febrile pregnant women attending ANC at a hyper-endemic region in eastern Uganda. The procedures in this study were performed by nurses and midwives at the hospital ANC clinic. The operational nature of this evaluation allowed the investigators to demonstrate that RDTs are reliable for diagnosis of P. falciparum malaria in pregnancy when performed by midwives. These findings are comparable to previous reports of diagnosed malaria in pregnancy ranging from 9% to 60% in sub-Saharan Africa [This study found a malaria prevalence of 38% using microscopy and 54% with RDTs among febrile pregnant women at ANC. When compared with microscopy, RDTs demonstrated acceptable sensitivity (97%) and specificity (74%) for the diagnosis of n Africa .The high sensitivity of RDTs among symptomatic pregnant mothers is comparable to results from a previous operational study among outpatients in a similar setting . The higThis study found a relatively low specificity but a high negative predictive value of 97.5%, almost certainly excluding malaria infection. This NPV result gives confidence that if RDTs are used to diagnose malaria in pregnancy very few infected women will be missed. Moreover, false negatives were linked to very low parasite densities that are unlikely to be the cause of illness in such an endemic setting. The consequences of a false negative in an endemic area, particularly when related to low parasitaemia, mean that the patient is unlikely to die. The infection may either clear without illness, as people living in endemic areas have partial immunity, or clinical symptoms will recur and they would seek care again.For clinical application, sensitivity and specificity of the RDTs can be combined into a single measure called likelihood ratio (LR). LR provides a summary of how many times more (or less) likely patients with malaria are to have a positive (or negative) result than women without malaria. This study found a LR+ of two which means that a pregnant woman with malaria is about two times more likely to have a positive RDT test than a woman who has not got malaria in pregnancy. It also found a LR- of 0.025 which means that the probability of having a negative test for pregnant women with malaria is 0.025 times of that of those without malaria. Therefore, women without malaria are about 40 times more likely to have negative test than women with malaria. These results imply that RDTs were better at 'ruling out malaria infection' than in confirming active malaria infection. The benefit of likelihood ratios is that they can be used to help the clinician adapt the sensitivity and specificity to tests of individual patients ,39. In sThis study found that there was an association between RDT result and clinical or demographic factors. During routine care, the clinician takes a history and examines the patient to estimate the chance (probability) of malaria prior to performing a test. A patient's probability of having malaria after the test result is known (post-test probability) is what the clinicians are most interested in as it can help in deciding where to confirm a diagnosis or rule it out. The actions taken after receiving the test results are weighed in light of the history and examination obtained prior to testing. The factors that influenced post-test probability in this study are consistent with known risk factors associated with malaria infection, and include parity, low maternal haemoglobin, sleeping under a mosquito net and advanced gestational age. Previous studies have shown that primigravidae are at increased risk for malaria in pregnancy and most cases of malaria in pregnancy happen towards the end of gestation ,6,40,41.In this study, direct examination of the placental tissue showed that 21% of women had placental infection of which 12% were active infections on the day of delivery. The study demonstrated a modest level of accuracy  of RDTs in detecting placental malaria using peripheral blood at time of delivery. The study allowed the investigators to characterize the burden of placental infection among pregnant women with history of febrile illness during the third trimester. The prevalence of placental malaria is similar to that reported from Nigeria . HoweverThis study was integrated into the existing health system and done by the human resources who would be available for eventual scale-up and in close collaboration with National Malaria Control Programme. The study built on previous work published by others by using an implementation/prospective approach and providing an appropriate context in which malaria is diagnosed. This approach in study conduct was important to validate effectiveness of RDTs given this can be context specific . It has P. falciparum ). The inherently higher rate of false positives recorded for this test may explain the poor PPV observed in the study and also account for lower specificity percentages. It is recommended by the WHO that an RDT should have at least 95% sensitivity at 100 p/µl [The main limitations of this study included the low hospital delivery rate of 47%, which made it impossible to analyse the deliveries out of the hospital. However the hospital delivery rate was higher than the national rate of 30% and the authors attribute this to the study mothers being incentivized to deliver in the hospital. Some samples were not analysed due to missing data, an expected challenge when using data from a routine ANC care setting. These limitations are to be expected in most operational studies but this approach allows the team to learn through implementation. Another limitation is that the RDTs (Diagnosticks) selected for the study displayed a very low sensitivity (59%) against low parasitaemia blood samples in an independent evaluation conducted by FIND/WHO . Additio100 p/µl . It is pP. falciparum malaria in pregnancy with further (cost-) effectiveness studies.Prevalence of active placental malaria infection was 12% in a malaria endemic setting in Uganda. RDTs were accurate and comparable to microscopy for diagnosing malaria in pregnancy when performed by midwives during ANC. RDTs were not statistically better than microscopy in detecting placental infection and both methods were less than optimal. Combined use of RDTs and microscopy on peripheral blood improved detection of placental malaria infection. RDTs high sensitivity and specificity of RDTs means they can complement microscopy for diagnosing The author declares that they have no competing interests.DJK participated in the conception, study design, protocol development, training, and was responsible for project management, data analysis and interpretation, and developed the draft manuscript. NM, CR, DN, and MM participated in the development of tools, interpretation of results and writing of the manuscript. LKT and MN participated in the training of health workers and acquisition of data. HC, JP, SM and JKT participated in the conception study, interpretation of results, provided technical over sight and leadership in writing of the manuscript. All authors read and approved the final manuscript.Matched samples table for infected (n = 21) and non malaria infected placental (n = 152) readings comparing RDTs to microscopy reading based on histopathology as a reference standard among delivering mothers in Mbale Hospital between February and October 2010. The table show the contingency table marginal totals used to compute the McNemar X 2 test comparing RDTs to microscopy reading based on histopathology as a reference standard among 173 delivering mothers in Mbale Hospital between February and October 2010. The X 2 values among the placental-infected and non-infected mothers were 0.25 and 1.565 respectively, both less than the 3.84 cut-off for a significant difference at 5%. The comparison in c and d compares the combined results of both RDTs and microscopy to that of RDTs alone (two degrees of freedom). The extended McNemar X 2 confirmed that combined tests of RDTs and microscopy were significantly better than a single test (X 2 of 6.125 for infected and 9.00 none infected both >than 5.00 cut off).Click here for file


Efforts to monitor malaria transmission increasingly use cross-sectional surveys to estimate transmission intensity from seroprevalence data using malarial antibodies. To date, seroconversion rates estimated from cross-sectional surveys have not been compared to rates estimated in prospective cohorts. Our objective was to compare seroconversion rates estimated in a prospective cohort with those from a cross-sectional survey in a low-transmission population.19 using a multiplex bead antigen assay. We classified individuals as positive for malaria using a cutoff derived from the mean plus 3 standard deviations in antibody responses from a negative control set of unexposed individuals. We estimated prospective seroconversion rates from the longitudinal cohort based on 13 incident seroconversions among 646 person-years at risk. We also estimated seroconversion rates from the cross-sectional survey using a reversible catalytic model fit with maximum likelihood. We found the two approaches provided consistent results: the seroconversion rate for ages ≤11 years was 0.020  estimated prospectively versus 0.023  in the cross-sectional survey.The analysis included two studies from Haiti: a prospective cohort of 142 children ages ≤11 years followed for up to 9 years, and a concurrent cross-sectional survey of 383 individuals ages 0–90 years old. From all individuals, we analyzed 1,154 blood spot specimens for the malaria antibody MSP-1The estimation of seroconversion rates using cross-sectional data is a widespread and generalizable problem for many infectious diseases that can be measured using antibody titers. The consistency between these two estimates lends credibility to model-based estimates of malaria seroconversion rates using cross-sectional surveys. This study also demonstrates the utility of including malaria antibody measures in multiplex assays alongside targets for vaccine coverage and other neglected tropical diseases, which together could comprise an integrated, large-scale serological surveillance platform. Efforts to monitor malaria transmission to inform control strategies increasingly use cross-sectional surveys to estimate transmission intensity from seroprevalence data based on malaria antibodies Investigators have estimated malaria transmission intensity from cross-sectional prevalence surveys using seroconversion rates estimated with a reversible catalytic model 19 (MSP-119) from incident seroconversions measured in a longitudinal cohort of Haitian children ages 0–11 years old, and compare it to the rate estimated with a reversible catalytic model fit to a cross-sectional survey of Haitians aged 0–90 years old. Since the longitudinal data provide a direct measure of the seroconversion rate, a comparison of estimates from the two approaches provides an important check of the model's consistency as currently applied in low-transmission settings.The objective of this study was to estimate the malaria seroconversion rate using antibody measures against merozoite surface protein-1Wuchereria bancroftiStudy populations were set up initially to monitor transmission of lymphatic filariasis (LF) in a setting of intense LF transmission. Both longitudinal and cross sectional studies were carried out in the coastal plain near Léogâne, Haiti, where up to half of the population was infected with The protocols for both studies were reviewed and approved by the Centers for Disease Control and Prevention's Institutional Review Board and the Ethical Committee of St. Croix Hospital . After explaining the purpose of the study in Creole, individuals were asked to provide verbal consent to participate in the research. The human subjects review boards approved the verbal consent process due to low literacy rates in the study communities. In cases of longitudinal follow-up, the study team documented consent at each study visit. Mothers provided consent for young children, and children 7 years or older provided assent. Consent forms included specific permission to share specimens and to test the samples for other infectious diseases.19 fusion protein cloned from P. falciparum isolate 3D7  was coupled to SeroMap beads  in phosphate-buffered saline (pH 7.2) as previously described 6 beads. The MSP-119 assay was included as part of a 28-plex panel for the longitudinal survey minus background (MFI-bg) for the duplicate wells. The magnitude of the fluorescent response (reported in MFI – bg units) is proportional to the amount of antigen-specific IgG antibody present in the sample.A recombinant GST/MSP-119 antibody response was calculated from the logged values of the negative control antibody responses. Because samples from the longitudinal study and the cross-sectional study were run with two different bead lots, two different cutoff values were used: a cutoff of 365 MFI-bg units was calculated from 63 negative control sera for the longitudinal study bead set, and a cutoff of 477 MFI-bg units was calculated from 70 negative control samples for the cross-sectional study bead set.We used adult US citizens with no history of foreign travel as an unexposed population for antibody cutoff values to classify individuals as seropositive. The mean +3*SD for the MSP-1Four children <6 months in the longitudinal cohort and one child age 2 months in the cross-sectional study had evidence of maternal antibodies to multiple antigens  in the multiplex panel; we classified them as seronegative in their first year of life for the analysis. For children who were classified as seropositive during follow-up we plotted their antibody responses to identify those who were incident cases versus those who were positive at their first visit.We estimated age-specific seroprevalence in the longitudinal cohort by combining measurements into one-year to three-year age groups that included enough measurements to estimate each prevalence with reasonable precision. We also estimated the seroconversion and seroreversion rates using incident conversions and reversions divided by the person time at risk over follow-up www.R-project.org).With short periods between measurements such as days, the approach to estimating the rates directly is unbiased With cross-sectional data, direct information about seroconversion and seroreversion is unknown since the same individual is not observed at two points in time. Instead, there is only current status information for an individual at one point in time when they are a particular age. The reversible catalytic model for incidence data can be fit to prevalence data with a simplification where exposure time is measured by age in years Among the 142 children enrolled in the longitudinal study in Leogane, each child was followed for an average of 5.1 years . The study included 771 total antibody measurements, and the average number of measurements per child was 5.4 . The 383 individuals enrolled in the cross-sectional survey in Miton ranged in age from two weeks to 90 years old. Antibody response increased with age in both samples .19 antibody levels. Over the 9 years of follow-up, there were 558 periods where children began periods as seronegative corresponding to 646.185 person years; there were 71 periods where children began periods as seropositive corresponding to 71.844 person years. There were 13 incident seroconversions (rate  = 0.020 per year) and 11 incident seroreversions (rate  = 0.153 per year).In the longitudinal study, 25 children were identified as seropositive for malaria during follow-up. Of these, 13 were incident seroconversions and 12 had seroconverted by their first measurement . No chil19 malaria antibody was very similar when estimated from incident seroconversions in a longitudinal cohort and from a model fit to cross-sectional seroprevalence data. This finding is important because it provides a direct validation of the use of cross-sectional malaria seroprevalence data to estimate seroconversion rates in low-transmission settings. Earlier analyses demonstrated that the seroconversion rate measured from cross-sectional surveys was strongly associated with the entomological inoculation rate In this analysis of two separate studies from Haiti we found that the seroconversion rate for the MSP-119, which underscores its utility as a sensitive surveillance tool in low-transmission settings.The estimation of seroconversion rates using cross-sectional data is a widespread and generalizable problem for many infectious diseases. Although the model used to estimate malaria seroconversion rates from cross-sectional surveys is an extreme simplification of a complex immunological process, numerous field studies including the present study have found that the model fits the data well More recently, Bretscher et al. Our results suggest that the simpler calculation of seroconversion rates directly from incident seroconversions is a viable alternative to a more complex, Hidden Markov Model approach. First, as we have demonstrated there is no need to collapse individual information into age categories and group level seroprevalence to estimate seroconversion rates with longitudinal data – rates can be estimated directly from incident seroconversions 19 was included in the multiplex, along with antigens from enteric pathogens and vaccine-preventable diseases to better understand the public health context in these communities and the potential interactions between LF and other infections. The current results illustrate the potential of this approach to capture seroincidence data for infections beyond those that were the initial focus of the study. Less clear at this point is the extent to which reversible catalytic models might be successfully applied to the other infections we monitored. These efforts are the focus on ongoing efforts in our labs. Independent of whether or not reversible catalytic models can be applied generally as measures of transmission intensity, multiplex serologic assays represent a powerful tool for capturing useful public health data with simple, dried blood spot surveys.Serum samples from these communities were tested by multiplex as part of a study of risk factors for acquisition of LF; MSP-1A limitation of the analysis was that only 25 children were classified as seropositive in the longitudinal study, which meant that it was impossible to estimate age-specific seroconversion rates and it was also impossible to estimate the seroreversion rate with precision. The wide confidence intervals and variation in point estimates for the seroreversion rate in analyses limited to children ≤11 years reflect the lack of information needed to accurately estimate that parameter . Althoug19 antibody responses are considered to be sensitive and specific measures of malaria infection, use of multiple malaria antigens would improve the sensitivity of case detection. We estimate that use of a single antigen would capture at least 75% of incident infections , so our estimates of infection prevalence should be reasonably accurate. Inclusion of additional malaria antigens in future studies, a straightforward approach with the multiplex, will add to the value of these analyses.These studies were not designed to follow malaria specifically and we have no parasitologic data that would confirm that children were, in fact, malaria infected. That limitation notwithstanding, not all malaria infections may manifest as patent parasitemia, so serological measures of infection may be a more accurate representation of the true level of transmission in the population – particularly in areas of low transmission Our finding of close agreement between malaria seroconversion rates estimated in a prospective cohort study with those estimated using a reversible catalytic model fit to cross-sectional prevalence data lends additional credibility to the use of cross-sectional, serological surveys to monitor malaria transmission in low-transmission settings. These results demonstrate the utility of including malaria antibody measures in multiplex assays alongside targets for vaccine coverage and other neglected tropical diseases, which together could comprise an integrated, large-scale surveillance platform.Text S1Statistical Details. This supporting information includes details about the statistical models used in the analysis as well as additional seroconversion rate estimates using a two rate model.(PDF)Click here for additional data file.


Plasmodium ovale is responsible for 5% of imported malaria in French travellers. The clinical and biological features of six clustered cases of P. ovale malaria in an army unit of 62 French soldiers returning from the Ivory Coast are reported.3 in half the cases only, and the haemoglobin count was normal for all patients. The diagnosis was made after at least three thick and thin blood smear searches. Parasitaemia was always lower than 0.5%. All rapid diagnostic tests were negative for HRP2 and pLDH antigens.All patients were symptomatic and developed symptoms on average 50 days after their return and 20 days after the end of chemoprophylaxis (doxycycline). Clinical features included fever (6/6), mostly tertian (4/6), aches (6/6), nausea (3/6), abdominal pain (2/6), diarrhoea (2/6), or cough (2/6). Thrombocytopaenia was lower than 100,000/mmPlasmodium ovale malaria is currently a problem to diagnose in travellers, notably in French soldiers returning from the Ivory Coast. Early attempts at diagnosis are difficult due to the lack of specific clinical features, the rarity of biological changes and the poor sensitivity of diagnostic tools to detect low parasitaemia. Thus, the diagnosis is commonly delayed or missed. Physicians should be aware of this diagnostic challenge to avoid relapses and provide prompt and adequate treatment with chloroquine and radical cure with primaquine. Plasmodium falciparum [Plasmodium vivax from French Guiana and Plasmodium ovale from West Africa [P. ovale malaria in French soldiers who were infected in the Ivory Coast, pointing out the difficulties in diagnosing this species.Malaria is a threat to travellers staying in tropical endemic areas, including soldiers under field conditions ,2. Each lciparum . About hlciparum . Continut Africa ,4. This P. ovale malaria were successively diagnosed among French soldiers returning from the Ivory Coast. They all belonged to the same army unit (62 people), which had stayed in Abijdan, Bouake and Yamoussoukro between 01/10/2007 and 15/04/2008. During the mission, this unit suffered 16 cases of fevers of unknown origin (two of them included in the cases reported here), but no malarial attack confirmed by rapid antigenic tests. An epidemiological study with behavioural questionnaire was conducted in the unit among the cases and uninfected soldiers. The incidence rate for malarial infection was 6/62 (10%) over 6.5 months. All the patients admitted having imperfectly used anti-mosquito measures, but all declared having correctly taken doxycycline chemoprophylaxis during the mission and after their return. No behavioural factors were statistically associated with the occurrence of malaria in this small group.Six cases of 3 (3/6), hepatic cytolysis (3/6), hyperbilirubinaemia (1/6), and elevated CRP (6/6). The haemoglobin count was normal for all patients.All infected cases were men . The first symptoms developed on average 50 days after their return range: 41-72) and an average of 20 days after the end of chemoprophylaxis (range: 11-42). The patients consulted on average two days after the onset of symptoms. The most frequent symptoms were fever (6/6), mostly tertian (4/6) with temperatures higher than 38.5°C in half the cases, aches (6/6) and shivers (5/6). Other symptoms included nausea (3/6), abdominal pain (2/6), diarrhoea (2/6), or cough (2/6). No patient had a splenomegaly. Biological changes  and for the pLDH antigen .The diagnosis was established using thick and thin blood smears, after at least three searches for each case (up to seven times for one case), and on average eight days after the onset of symptoms (range: 3-12). Parasitaemia was always lower than 0.5%. Rapid diagnostic tests were all negative both for the HRP2 antigen  or digestive intolerance (one case treated by intravenous quinine). All attacks were cured. One patient experienced a P. ovale relapse 30 days after his first attack. He received a further course of chloroquine. All patients also received a course of primaquine (15 mg/d for 14 days) to avoid malarial relapse, none having a glucose-6-phosphate-dehydrogenase (G6PD) deficiency. No new relapse was reported after a 24-month follow-up.Three patients received chloroquine (25 milligrams/kilo within three days). Three patients received quinine (24 mg/kg/d for seven days) because of suspected co-infection with P. falciparum is the main target of malarial prevention because this species is widely endemic, very common and clinically severe [P. vivax and P. ovale, are also of concern for travellers in endemic areas. In contrast to P. vivax, which is widespread in Asia and Central and Southern America, P. ovale is only endemic in some African countries [P. ovale have been identified in West Africa [Plasmodium ovale is responsible for rare, travel-acquired infection [P. ovale malaria attacks among French soldiers returning from the Ivory Coast between 2002 and 2007, but not among soldiers in the field, while the incidence of Pl. falciparum regularly decreased over the same period [P. ovale than on P. falciparum. Indeed, P. ovale was very uncommon while using the association chloroquine-proguanil in the French Army before 2002. Thus, preventing P. ovale infection with doxycycline seems challenging because of the lack of action on hypnozoites. A similar failure has also been recently reported with atovaquone plus proguanil chemoprophylaxis [For travellers, y severe . Althougountries . Two subt Africa . Plasmodnfection . Howevere period ,4. Thesephylaxis .P. ovale infection in infected patients is a challenge in the field or on return to non-endemic countries [P. ovale attacks are not specific. Nevertheless, a tertian fever-evocative although non-specific - was present in more than half the patients, possibly due to the time lapse to the delayed diagnosis, which had allowed synchronization of the replication cycle. Moreover, digestive or respiratory symptoms, which were present in half cases, can wrongly suggest a viral infection. However, any fever, associated or not, after return from an endemic area, should be considered to be due to malaria and be followed up by immediate thick and thin blood smears. As a result of health education before and during the mission, all the patients promptly consulted a general practitioner and underwent a search for malaria in a nearby laboratory. In contrast to Plasmodium falciparum and Plasmodium vivax malaria, the haemogram appeared not to be very helpful for Plasmodium ovale because anaemia is frequently missing and thrombocytopaenia mild or absent [P. ovale in imported cases is very low [P. falciparum [P. ovale is far lower at the same time after disease onset [P. ovale, while they are highly sensitive to P. falciparum [P. ovale infection when thick films and blood smears are negative. This could be due to the very low level of circulating antigen or to inadequate antigens used for these tests. This could explain some acute unexplained fevers in the French Army in the field in Africa [P. ovale malaria is suspected, routine microscopic searches with thick and thin blood smears should be repeated, up to three times and in an expert laboratory, if possible [P. ovale using PCR, this tool marks real progress in confirming the diagnosis, although it is still not routinely available [Diagnosing ountries . Symptomountries . Shortenountries . This exr absent , especiavery low , as showlciparum , the levse onset . Unfortulciparum -15. Theyn Africa . When thpossible . Considevailable ,16. It cThe treatment for proven attacks is based on chloroquine (25 mg/kg for three days). The treatment against dormant stages in the liver consists of a radical cure with primaquine (0.5 mg/kg/d for 14 days) in patients without G6PD deficiency ,18. FailP. ovale infection in travellers returning from endemic areas is still a challenge for physicians and requires repeat microscopic searches to detect low parasitaemia. As PCR is not a routine tool, there is a real need to improve the sensitivity of rapid diagnostic tests for this plasmodial species.To date, diagnosing FL: participated in the care of the patients, in the epidemiological study and drafted the manuscript; CR: participated in the care of the patients and drafted the manuscript; MO: participated in the care of the patients; VPS: participated in the epidemiological study; AM: participated in the care of the patients; XD: participated in the epidemiological study; FS: participated in the care of the patients and drafted the manuscript.The authors declare that they have no competing interests.All authors read and approved the final manuscript.


Plasmodium falciparum malaria.To determine whether sequestration of parasitized red blood cells differs between children with uncomplicated and severe P. falciparum histidine rich protein 2, in Gambian children with severe (n = 127) and uncomplicated (n = 169) malaria.We quantified circulating-, total- and sequestered-parasite biomass, using a mathematical model based on plasma concentration of Circulating- and total-, but not sequestered-, parasite biomass estimates were significantly greater in children with severe malaria than in those with uncomplicated malaria. Sequestered biomass estimates in children with hyperlactataemia or prostration were similar to those in uncomplicated malaria, whereas sequestered biomass was higher in patients with severe anaemia, and showed a trend to higher values in cerebral malaria and fatal cases. Blood lactate concentration correlated with circulating- and total-, but not sequestered parasite biomass. These findings were robust after controlling for age, prior antimalarial treatment and clonality of infection, and over a realistic range of variation in model parameters.Extensive sequestration is not a uniform requirement for severe paediatric malaria. The pathophysiology of hyperlactataemia and prostration appears to be unrelated to sequestered parasite biomass. Different mechanisms may underlie different severe malaria syndromes, and different therapeutic strategies may be required to improve survival. Malaria causes around 1 million deaths per year globally.Plasmodium falciparum adhere to vascular endothelium, a phenomenon known as sequestration,P. falciparum histidine rich protein 2 (PfHRP2).Plasmodium vivax can cause SM but exhibits little cyto-adherenceP. falciparum CM the degree of sequestration in cerebral vessels and tissues is extremely variableMost deaths from malaria occur in African children.P. falciparum malaria  were eligible for recruitment. Clinical management followed Gambian government guidelines, with SM cases admitted to hospital. Blood cultures were not routinely performed, but children were excluded if the attending clinician suspected concomitant bacterial infection. SM was defined using modified WHO criteriaWith approval from the Gambia Government/MRC Laboratories Joint Ethics Committee, and the Ethics Committee of the London School of Hygiene and Tropical Medicine, all samples were collected with informed consent from the child's parent or guardian and used for an unmatched case-control study nested within a larger prospective cohort, of which methodological details have been published.PfHRP2 was measured in duplicate in plasma by ELISA kit (Cellabs) following the manufacturer's instructions with addition of a standard curve. Laboratory personnel were unaware of the clinical status of subjects. Circulating-, total- (PfHRP2-derived), and sequestered-parasite biomass estimates were calculated using formulas derived by Dondorp et al.P. falciparum infection was assessed as described previously.Clonality of Streptococcus pneumoniae and non-Typhoid Salmonella (NTS) was determined using quantitative PCR on cDNA.Bacteraemia with metabolically active 10 transformed for parametric analyses to achieve normality, except sequestered biomass  which was analyzed with non-parametric methods. Unpaired t-tests and likelihood-ratio tests were used to compare means and medians, respectively, of groups. Confounding by age, prior antimalarial treatment and clonality of infection was assessed by quantile regression : a model including only intercept was compared by means of likelihood-ratio tests to models with any combination of the above covariates or interaction terms. Correlation was assessed using Spearman's rank correlation coefficient. To allow for the multiplicity of tests resulting from multiple responses and multiple comparisons within a response, a false discovery rate (FDR) of 5% was assumed, using the Benjamini and Hochberg approach.X − 1) times the median sequestered biomass was added. Sensitivity analyses assessed the range within which each model parameter could be varied without rejection of the null hypothesis of equal median sequestered biomass among groups. In addition, the effect of joint variation in the parameters was assessed by sampling 10,000 candidate values from uniform distributions within the limits defined for each individual parameter, determining the frequency with which the likelihood-ratio test did not reject the null hypothesis. The effect of parameter variation on the Spearman's rank correlation between lactate and sequestered biomass was assessed identically.Statistical analyses were performed using PASW statistics 18 (SPSS Inc.), GraphPad Prism (GraphPad Software Inc.) and the R-statistical software (R Foundation). Data was logComplete clinical and laboratory data  were avan = 64), was not significantly different to those with UM. In contrast children with CM, SA, and non-survivors, had very high-sequestered biomass, but these subgroups were relatively small, and only in SA cases was the median sequestered biomass significantly higher than that in UM  than children with UM A. Sequesat in UM , Fig. 3Bat in UM , Fig. 3Bat in UM .P. falciparum malaria,r = 0.0315, P = 0.59), whereas lactate correlated equally well with circulating and total parasite biomass estimates (Spearman r = 0.50 (95% CI 0.40–0.58) and 0.44 (95% CI 0.34–0.53) respectively, both P < 0.001).Blood lactate concentration is strongly associated with mortality in P = 0.95, P = 0.11 and P = 0.57 respectively): overall, the basic model explained the data best. Misclassification bias, due to bacterial sepsis causing severe disease manifestations in children with co-incidental parasitaemia, was assessed by PCR to detect NTS or S. pneumoniae bacteraemia in 160 (54.1%) study subjects with suitable samples (85 of 169 (50.3%) UM and 75 of 127 (59.1%) SM cases); none (95% CI 0–2.3%) were positive. Additionally no study subjects received intravenous antibiotics, making significant misclassification unlikely. Sensitivity analysis revealed that the model was highly robust to a realistic range of variation in parameters  except the sequestered biomass were higher in SM compared with UM, suggesting that extensive pRBC sequestration does not uniformly underlie SM. This unexpected finding may be explained by the fact that the most frequent SM syndromes in this study were LA and SP: children with these syndromes had very similar median sequestered biomass to those with UM and blood lactate concentrations correlated significantly with circulating biomass  but not sequestered biomass. On the other hand, children with CM, SA and fatal cases had high sequestered biomass; although the small numbers of subjects in these groups in this study preclude firm conclusions, our data are consistent with a role for parasite sequestration in these syndromes. However, the low sequestered biomass in children with LA, and the lack of a significant correlation between sequestered biomass and blood lactate concentrations, is a particularly important observation because LA (or a large base deficit) is one of the most frequent entities defining SM and risk of death in African children with malaria.16The present study was undertaken to assess the role of one of the most fundamental processes in Our findings – that overall sequestered-parasite biomass differed only 1.2-fold between SM and UM patients – contrast with findings of an average 10-fold difference between Thai adults with SM and UM in a similarly large study.in vivo in African children with SM,in vitro and non-human primate data).Plasmodium berghei ANKA infection in mice suggests that the sequestration rate and the clearance rate of sequestered-parasites may be dynamic throughout the course of an infection,In assessing the validity of our findings we must consider methodological issues which might influence our results. Plasma concentrations of PfHRP2 in our study were lower than those reported in several other studies of SM,P. falciparum antigens.It is also important to consider potential sources of bias in our study which might influence our results. Antibodies against PfHRP2 might cause under-estimation of sequestered biomass in SM relative to UM cases, but young Gambian children (who are most likely to have SM) are the least likely to have antibodies to How should our results be interpreted? Although the number of children with SA was small, the association with high PfHRP2 concentration is consistent with other studies,This work was supported by core funding from the Medical Research Council, UK to the malaria research programme, and a Medical Research Council, UK, clinical research training fellowship [G0701427 to A.J.C.].The authors have no commercial or other association that might pose a conflict of interest.


A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign.During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks.On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children.HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up. Global control efforts have resulted in a reduction in the incidence of malaria and malarias specific mortality rates. Transmission of malaria decreased remarkably in the Republic of São Tomé and Príncipe (STP) since an integrated malaria control programme was scaled up beginning in 2004,2. WhileIn April 2008, a cross-sectional nationwide malaria surveillance in STP by use of histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs) showed a positive rate of 3.5%, which was further confirmed by microscopy and the inner primers FIP and BIP at 1.6 μM, the loop primers LPF and LPB at 0.8 μM and the outer primers F3 and B3 at 0.2 μM. Amplification was performed at 60°C with a heat block  for 100 min and then heated at 90°C with a heat block for 1 min to terminate the reaction. Duplicate negative control (water) and positive control (water spiked with DNA) were included in each run.One LAMP primer sets for is study. The dupThe positive LAMP reaction was detected by observing the fluorescence in the reaction tube via naked eye under a portable UV lamp. In thisAll patients were seen and the assays, except DNA amplification, were performed on site at the health stations. Blood spots on the filter paper for nested PCR and LAMP were transferred to the molecular diagnostic laboratory within 1 hr after collection. Blood samples for nested PCR and LAMP were treated and assayed individually on the collection day rather than batched and performed at a later time. Time to obtain test results varied depending on the different methods, i.e. 20 min for HRP-2-RDTs, 60 min for microscopy, 3 hr for LAMP, and 8 hr for nested PCR. Diagnosis of malaria was made as a febrile child had at least one positive result of the four malaria tests. Treatment was started as soon as possible at the time when the test results were available. Children with uncomplicated malaria were treated with a three-day course of artesunate-amodiaquine at home except for the first dose taken in front of the nurse’s observation. Children with severe malaria were admitted to hospital and treated with quinine intravenously. Treatment was switched to oral administration as soon as the patient was able to tolerate it. An additional course of primaquine was given for vivax malaria. Home treatment was weekly followed up by a mobile team (consisting of a nurse and a technician), which actively visited patients by taking blood samples for HRP-2-RDTs, optical microscopy, nested PCR and LAMP. Treatment failure was considered if parasitaemia persisted (microscopy positive), or either the nested PCR or the LAMP was positive. When treatment failures occurred after one week of initial treatment, artemether-lumefantrine  was given, with a three-week follow-up.The sensitivity and specificity of the four methods were calculated using either microscopy or nested PCR assay as a reference test. The percentage specificity, sensitivity, PPV, and negative predictive value (NPV) were calculated as reported previously. Ninety-Of the 128 febrile children where a complete set of four malaria tests were carried out, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively . Species identification based on nested PCR and LAMP showed that 62 children exhibited single infection with Plasmodium falciparum and two children were mixed infections . Optical microscopy showed that the parasite density ranged widely, 100–100,000/ul for 47 children on day 0, with a narrower range of 100–10,000/ul in seven children on day 7.Among the 58 children with negative results for both HRP-2-RDTs and microscopy, eight were positive for both nested PCR and LAMP, but one with negative nested PCR was positive for LAMP  no matter that microscopy or nested PCR was used as a gold standard Tables  and4. CThis study has shown that LAMP with high PPV and NPV as compared to nested PCR, can be applied for malaria therapeutic follow-up. HRP-2-RDTs have similar sensitivity as microscopy but less specificity, which can either induce to underdiagnosis because of the moderate NPV or over-diagnosis due to the poor PPV.P. falciparum has been compared with other diagnostic tests, but both sensitivity and specificity of LAMP varied in different studies[Methods for the detection of malaria are central to an elimination programme. Current diagnostic methods applied in endemic areas are either time-consuming, or insensitive for use in conditions of low infection,24. Opti studies,27-30. A studies.P. falciparum and P. vivax. Low parasitaemia and mixed infection may limit the detection, even by use of molecular methods, but this cannot be the excuse for the contradiction in results between LAMP and nested PCR.The inconsistent results between nested PCR and LAMP were found in two cases (one with PCR negative but LAMP positive while one with PCR positive but LAMP negative). This discrepancy may be expected when both nested PCR and LAMP are performed by two technicians independently. It has been reported that the sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections. These pIt has been well documented that blood compositions, such as haemoglobin and IgG/IgM, can interfere with the performance of PCR-34. A raMalaria can be over-diagnosed if febrile children had positive HRP-2-RDTs but negative microscopy. One third of children (23/68) with positive HRP-2-RDTs were negative by microscopy and this can be attributed to low parasite density or to the delayed clearance of circulating HRP-2 antigen. Further verification by molecular tests, positive results from nested PCR and LAMP (10 out of 23) provided the evidence for the low parasitaemia while negative results from nested PCR and LAMP (13 out of 23) explained the possibility for the residual circulating HRP-2 antigens. Other causes for false-positive RDT results, such as gametocytaemia and the presence of the serum rheumatoid factor cannot be excluded,35-37. AIt has been well documented that individuals with low parasite density act as reservoirs for transmission,6,8 and In tropical Africa, the differentiation of malaria from other causes of fever in the absence of microscopy is notoriously difficult. Even febrile children were malaria positive by microscopy it is a challenge to distinguish children who really do have severe malaria from those who have severe febrile illness but coincidental parasitaemia, who may have another infection. Although recent studies reported a strong correlation between plasma PfHRP2, disease severity, and outcome, and suggest that plasma PfHRP2 is a prognostic indicator in African children with severe falciparum malaria,39, measFalse-negative malaria RDT results can occur due to a prozone-like effect in high-density infections. In thisArtesunate plus amodiaquine combination is one ACT recommended by the WHO for use in malaria control programmes and a first line treatment for African children with uncomplicated malaria. This reThe efficacy of the six-dose in three-day regimen of artemether/lumefantrine has been confirmed in many different patient populations around the world, cure rates on 7,14 and 28 days exceeding 95% in the evaluable population. The 14-The weakness of this study was that it was not randomized and the sample size was relatively small. In addition, both nested PCR and LAMP detect the amplification of target DNA while RDTs is antigen capture and microscopy is morphological identification. With the different targets detected, comparisons among these four different tests may be limited. Latent class models have been widely used to estimate diagnostic tests performance measures, such as sensitivity and specificity, as well as the diseases prevalence, in the absence of a gold standard or perfect reference test. In spite of being frequently used as a reference technique, microscopy is in fact an imperfect gold standard, especially in low parasitaemia as in this malaria control island.Although this study was carried out during the rainy season with a short period time and only older children were enrolled, malaria positive rate in febrile children was high (1/3 by microscopy or 1/2 by the nested PCR), which strongly indicates a high risk of malaria epidemics in a low transmission area, like São Tomé. After an intensive treatment and follow-up, there was no mortality observed among these febrile children.In developing countries malaria diagnosis mainly depends on microscopy or RDTs. For an elimination programme in malaria-endemic areas, however, current HRP-2-RDTs and optical microscopy are not adequate enough to evaluate the efficacy of treatment or detect low parasitaemia, especially in people with asymptomatic infection. Both sensitivity and specificity of the LAMP assay were similar to those of nested PCR. With high PPV and NPV, LAMP was the best method of the four tests for malaria diagnosis during a follow-up study. LAMP can be further developed as a point-of-care test, which is a valuable surveillance tool for guiding elimination efforts.The authors declare that they have no competing interests.PWL carried out the nested PCR and conducted data analysis; DDJ assisted with the design of the study, supervised LAMP laboratory work; CTL oversaw fieldwork and collected the field data. HSR contributed to the study coordination and organized field work. VEdR helped in the study design and critically reviewed the manuscript. IFL took full responsibility for the integrity of the data and accuracy of the data and its analysis. MFS led the conceptual design, study coordination, supervision, data interpretation and drafted the manuscript. All authors read and approved the final manuscript.


Malaria has been responsible for the highest mortality in most malaria endemic countries. Even after decades of malaria control campaigns, it still persists as a disease of high mortality due to improper diagnosis and rapidly evolving drug resistant malarial parasites. For efficient and economical malaria management, WHO recommends that all malaria suspected patients should receive proper diagnosis before administering drugs. It is thus imperative to develop fast, economical, and accurate techniques for diagnosis of malaria. In this regard an in-depth knowledge on malaria biomarkers is important to identify an appropriate biorecognition element and utilize it prudently to develop a reliable detection technique for diagnosis of the disease. Among the various biomarkers, plasmodial lactate dehydrogenase and histidine-rich protein II (HRP II) have received increasing attention for developing rapid and reliable detection techniques for malaria. The widely used rapid detection tests (RDTs) for malaria succumb to many drawbacks which promotes exploration of more efficient economical detection techniques. This paper provides an overview on the current status of malaria biomarkers, along with their potential utilization for developing different malaria diagnostic techniques and advanced biosensors. Plasmodium. Five species, P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, are known to affect humans. Characteristic symptoms of the disease include episodes of high fever followed by chills and rigors which are repeated every 48 hrs in falciparum, ovale, or vivax, every 72 hrs in malariae infections, and 24 hrs in knowlesi. P. vivax hypnozoites can lie dormant in the liver and may cause recurrence of the disease [The World Malaria Report 2012 released by World Health Organization (WHO) summarises the data received from 104 malaria endemic countries [ disease . WHO rec disease , 6. Plasmodium species. Although rapid detection tests (RDTs) have facilitated considerably diagnosing malaria, the tests are plagued with many limitations such as variability in results, being nonquantitative, and having poor storage stability in tropical regions. In order to develop an efficient test for diagnosis of malaria, a profound understanding on various malaria-related biomarkers is prerequisite. This review outlines the diagnostic tools for malaria with special focus on the potent biomarkers reported to date and their application for developing rapid and reliable detection techniques including biosensors.In spite of the vast array of tests available today to diagnose malaria, we still await a test that is quantitative and specific to distinguish different P. falciparum infected individuals exhibit asymptomatic malaria [Biomarkers are cellular, biochemical, or molecular alterations measurable in biological samples which indicate any biological, pathogenic, or therapeutic responses . There h malaria . The preμmol glucose/24 hrs/109 RBCs. However, Plasmodium is a voracious scavenger of blood glucose that increases the RBC glucose consumption up to 100-fold. In case of P. falciparum about 60–70% of the glucose is converted to lactic acid and excreted; however, this percentage varies amongst different Plasmodium species and in vitro culture conditions [+ is regenerated by conversion of pyruvate to lactate while the mitochondria contribute minimally to the ATP pool [ Plasmodium. The absence of F0 a and b subunits of the mitochondrial F0-F1 ATP synthase further implies the absence of mitochondrial role in energy generation. However, since parts of the genome sequence are still not known, and the genome being A+T rich that causes difficulty in identifying enzymes, the lack of role of mitochondria in energy generation cannot be ascertained [ P. falciparum LDH (PfLDH) RNA expression level gradually increases, with the peak expression being at 24 to 30 hrs in the intraerythrocytic cycle. This expression declines to zero in the schizont stage. A similar profile that slightly lags behind the RNA expression was observed for the enzyme activity as well [ P. falciparum transcription, it was demonstrated that all glycolytic enzymes are upregulated at the early trophozoite stage during the asexual cycle, coinciding with the time of maximal metabolic activity by the parasite [The red blood cell (RBC) is a nonproliferating cell with a modest requirement of around 5 ATP pool . This reertained . Neverthertained . It has  as well . With thparasite . Plasmodium species except P. knowlesi, which lacks His 195. The active site of the enzyme is located between these two domains. Each monomer of the pLDH protein carries one NADH molecule, and each of the four cofactors occupies identical positions in each monomer [The parasite LDH (pLDH) is a tetramer where each monomer consists of two domain LDH folds. The larger domain comprises the Rossmann fold that binds the cofactor NADH, while the catalytic residues  are located in the other domain. These residues are conserved across all P. falciparum [ Plasmodium species and in the LDH I and II of T. gondii [ Cryptosporidium parvum which causes cryptosporidiosis, a parasitic infection of the mammalian intestinal tract.Protozoal LDHs display some major structural and kinetic differences compared to their mammalian counterparts that may be exploited to develop selective drugs and detection systems for malaria. PvLDH and PfLDH share 26% and 29% sequence identity with human LDH-A (hLDH-A), respectively. Structurally the parasite enzyme differs in having a five-residue insertion in its active site loop which closes down over the active site during catalysis  and alsolciparum  were lat. gondii , 22. How+ from the active site due to the formation of a covalent adduct with pyruvate. Reduced substrate inhibition is attributed to a single amino acid substitution in the protozoal enzyme (Ser163Leu). The single amino acid mutation has also been exploited as a general method to reduce substrate inhibition in L-lactate dehydrogenase enzymes [+ [+ (3-acetylpyridine adenine dinucleotide) [+ binding altering the rate of active site movement or a higher oxidation potential of APAD+ than NAD+ leading to faster hydride transfer to APAD+ is ascribed as one of the reasons for this cofactor preference [+ as inhibitors in a forward reaction of pyruvate to lactate direction it was revealed that this enzyme exhibits ordered sequential bi-bi mechanism of binding in which the NADH cofactor binds before the pyruvate substrate is attached.Kinetically, the parasite enzyme differs by not being inhibited by excess of the substrate pyruvate, a feature not shared by mammalian enzymes . In the  enzymes . The sub enzymes . The mecnzyme Ser3Leu. Thezymes [+ . Anotherleotide) , 27. Incnzyme Ser3Leu. Theeference , 29. By α-ketobutyrate. Also the enzyme does not show an activity with phenylpyruvate [ T. gondii. Although LDHs from P. falciparum and T. gondii have very similar structures, the latter has an additional loop insertion of two residues and several changes in its active site that renders the contrast property [ Toxoplasma enzyme a mild malate dehydrogenase activity [Km for substrates is pH dependent, as the substrate pyruvate binds only when the active site histidine is in the protonated state and lactate is in deprotonated state [Another striking difference of pLDH from other dehydrogenases is its substrate specificity due to long substrate specificity loop. The enzyme efficiency decreases with the increase in number of methylene groups in the substrate, as is seen when pyruvate was replaced with pyruvate , a behavproperty , 30. Theactivity ; this fiactivity . A charaed state .http://dx.doi.org/10.1155/2014/852645). From the inhibition studies of various compounds including gossypol and its derivatives against the parasite enzyme pLDH [pLDH is an attractive target for antimalarial drug design because of its three important attributes. (1) It controls the production of plasmodial ATP, (2) it has unique amino acids at the active site compared to its counterparts from other species, and (3) the protein data bank (PDB) contains many X-ray crystallographic structures of pLDH complexed with various compounds which provide ideal targets for modelling of inhibitors , 3 pentapeptides (Ala-His-His-Ala-Ala), and 33 hexapeptides (Ala-His-His-Ala-Ala-Asp). There is about 85–90% homology between the tandem repeat domains and the regions flanking the repeats of hrp II and hrp III genes which imply that both have originated in the duplication of an ancestral sequence [ hrp II and hrp III genes revealed a 5′ UTR intron which has been shown to have high conservation as the coding intron and a 3′ UTR, having a much greater homology than the histidine repeat region of the coding sequences [ hrps II and III share many similarities, but still it is unclear how these genes function and why HRP II is released in large quantities.The genomic sequence ofsequence . Anotherequences .  Figure in vitro [10-helix conformation as soon as heme molecules are titrated in the solution [10-helix conformation is commonly observed in proteins as short sequences of three to four residues, mostly associated with N- or C-termini of α-helices [10-helix is found to be unusually long in case of HRP II. Along with the changes in the secondary structure of the protein, dimerization of HRP II monomers by formation of intermolecular disulfide bonds also takes place as shown in HRP II transports from the parasite, through the host cell cytoplasm, to the culture supernatantin vitro  which alin vitro , 53. It in vitro , digestiin vitro , and memin vitro . In shorsolution . The 310-helices , 58. HowKd values for heme binding were calculated to be 0.34 μM. In another report the number of binding sites in HRP II-heme complex is estimated as 18 with Kd of 0.94 μM [The actual function of the HRP II is not yet adequately known. However, many reports have demonstrated that its main function is heme binding that may link its role in the heme detoxification in malaria parasites. Spectroscopic studies showed that a single HRP II molecule may bind to multiple heme molecules . By usin 0.94 μM , which i 0.94 μM . Hence t 0.94 μM . In vitro heme polymerization assay performed at the optimum pH 4.0 with native and recombinant HRP II promoted the formation of hemozoin. Polymerization increased with time, protein concentration, and initial concentration of heme [HRP II is also known to initiate the formation of hemozoin. of heme . A study of heme . The for2) of host cells. An important role of HRP II as a buffering protein has been suggested as it helps the parasite to stabilize the changes to the cytoskeleton induced by other released parasitic proteins [At an acidic pH electrostatic interaction takes place between the strongly positive charged histidine of HRP II and negative groups on actin and phosphatidylinositol 4,5-biphosphate  were found where aldo-1 sequence was found to be identical to P. falciparum aldolase, whereas aldo-2 had 13% sequence diversity [ P. berghei, P. chabaudi, P. vinckei, and P. yoelii and from the human malaria parasite P. vivax [ P. falciparum and P. vivax aldolase and showed that aldolase is highly conserved, indicating that antigenic diversity is not a cause of variable RDT sensitivity.Aldolase is a key enzyme in the glycolytic pathway of the parasite. It catalyses the cleavage of fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate . The genleotides , 79. In a brucei , Giardia lamblia , and P. lciparum , suggestiversity . In anotP. vivax . Many reP. vivax  studied  P. falciparum express three GDH isozymes. PfGDH1 is a NADP-dependent glutamate dehydrogenase which is a homohexamer with a subunit of Mr 49,500 [ P. falciparum using western blotting [ P. falciparum. This assay showed a sensitivity and specificity of 86.6% and 96.4%, respectively [Glutamate dehydrogenases (GDHs) are ubiquitous enzymes that occupy an important branch point between carbon and nitrogen metabolism. They are generally involved in ammonium assimilation (NADP-dependent GDHs) or glutamate catabolism (NAD-dependent GDHs).r 49,500 . It has r 49,500 . The cryr 49,500  and 3.1 r 49,500 , respectr 49,500 . PfGDH1 blotting . Monocloectively . P. falciparum infection in the absence of other causes of encephalopathy. If left untreated, it is fatal within 24–72 hrs [Cerebral malaria (CM) is a life-threatening complication of malaria and is defined as an unarousable coma with a4–72 hrs . The adv4–72 hrs , 92. It α were present in children infected with CM as compared to healthy controls [Several other serological biomarkers for CM have also been identified. For example, elevated levels of soluble tumor necrosis factor receptor (sTNF-R) and soluble Fas ligand (sFas), a 26 kDa glycoprotein generated from its membrane bound form by a metalloproteinase-like protease, may indicate CM neuropathy . Cerebrocontrols . The levcontrols  as well controls  angiopoicontrols . EPO hascontrols  in additcontrols . Kassa econtrols  have expcontrols , 99. P. falciparum [Microparticles (MPs), also known as microvesicles, are fragments of plasma membrane shed by various cell types under physiological stress conditions and have also been linked with pathophysiology . MPs havlciparum . Mfonkeulciparum  demonstr Toxoplasma gondii [The serological biomarkers, however, are difficult to use reliably in diagnosing malaria. For instance, CXCR3 and its ligands have been implicated in case of several neurological diseases like West Nile virus , 109, Toa gondii , and HIVa gondii . SerologMicroscopy for malaria diagnosis has been used since 1904 when Gustav Giemsa introduced a mixture of methylene blue and eosin to stain the parasite . This meApart from microscopy, nucleic acid techniques such as PCR and nested PCR , 121 and Plasmodium dsDNA in infected erythrocytes; however, the use of Plasmodium dsDNA as a marker in flow cytometry can result in false positives because pathological conditions are characterized by an efflux of normoblasts and erythroblasts in blood [Flow cytometry (FCM) and automated blood cell counting techniques can be used to detect hemozoin orin blood . LDMS isin blood .A unified sandwich ELISA targeting PfLDH and HRP II was reported that allowed concurrent measurement of the biomolecules . ELISA i P. falciparum density of 1000–2000 parasites/μL [RDTs are quick and portable immunochromatographic dipsticks whose sensitivity generally reaches >95% atsites/μL . They resites/μL . P. falciparum) and pLDH. Most commercial RDTs that detect P. falciparum target HRP II and few others target pLDH. To enhance the efficiency of malaria detection, RDTs targeting many biomarkers at the same time are prepared. The combined immunochromographic-malaria dipstick (ICT) targets HRP II and aldolase for the diagnosis of P. falciparum and P. vivax [ P. falciparum (based on HRP II) and pan LDH for non-falciparum infections, but sensitivities for P. ovale and P. malariae were poor [Dipsticks can be divided into two classes based on their target antigenic biomarkers, namely, HRP II . Results are usually obtained in 5 to 15 minutes . In many P. falciparum infections. This RDT was not acceptable for malaria diagnosis under the field conditions in the Peruvian Amazon due to HRP II and HRP III gene deletions in the malaria parasite genome [Variations in RDT performance have been seen with season, year, age of patient, and presence or absence of fever during consultation . The mose genome –144. Sine genome , 145. HRe genome .μL [ P. falciparum infections in the Asia-Pacific region are likely to be detected at densities <250 parasites/μL. HRP III is also suspected to play a role in the performance of HRP II based tests [ P. falciparum infection. Plasma HRP II has prognostic significance and provides a tool to assess the risk of “true” severe malaria as compared to other severe illnesses in parasitemic African children [Genetic polymorphism may lead to false negative results as was observed when a countrywide assessment of polymorphism was carried out for HRP II, pLDH, and aldolase genes in Madagascar. Higher levels of polymorphism were observed for HRP II and HRP III genes predicting that about 9% of Malagasy isolates could not be detected at parasite densities <250 parasites/μL . This geed tests . Howevered tests . In Africhildren . This juchildren  and theichildren . In spitchildren . A compachildren  by devel12 to 1015 combinatorial oligonucleotide libraries over multiple rounds of in vitro selection, the process commonly known as SELEX [A biosensor is a self-contained integrated device that is capable of providing specific quantitative or semiquantitative analytical information using a biological recognition element that is retained in direct spatial contact with a transduction element . The bioas SELEX . Aptameras SELEX . It is d(1) Electrochemical Biosensors. Lee et al. [e et al.  had devee et al.  and labee et al. . The dete et al. . P. falciparum malaria. To construct the immunosensor, disposable screen-printed electrodes (SPEs) were modified with multiwall carbon nanotubes (MWCNTs) and Au nanoparticles as shown in Different electrochemical immunosensors have also been developed using antibody raised against HRP II. The first disposable amperometric HRP II based immunosensor was developed for the detection of−1).An immunosensor based on magnetic micro- and nanoparticles were also developed for targeting HRP II , the con(2) Optical Biosensors. An immunocapture spectrophotometric diagnostic assay based on PfLDH, which showed a detection limit of 50 parasites/μL and sensitivity of 88%, has been developed by Piper et al. [r et al. . A schem in vivo diagnostic method using a magnetooptic fingertip probe was developed and the method was confirmed by a small preliminary clinical trial [μg/mL could be detected.Besides PfLDH and HRP II, hemozoin has also been used as a biomarker for detecting malaria microscopically. In fact, the characteristic morphology of the crystals can also be considered to distinguish different species of parasites . Hemozoial trial , 172. Fo(3) Piezoelectric Biosensors. A piezoelectric immunosensor was developed using mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol, the schematic configuration of which is shown in t1/2) of 14 days. The sensor was tested on clinical human serum samples and the results were compared with the commercially available ICT kit (NOW) which showed good agreement [greement .The malarial biosensor research, as evident from the above references, is more inclined towards electrochemical based biosensors and the reason may be attributed to some advantages of this biosensing platform; for example, label-free, portable, and small sample volume is needed for the analysis. From the sensitivity point of view electrochemical biosensors targeting HRP II and pLDH showed sensitivity even in the femtomolar range as compared to the other biosensors . CompariIn the last two decades, biomarker research on malarial pathogenicity has taken a leap as witnessed from the discovery of many interesting molecules and functional entities which are significantly distinct from the host counterpart and other host pathogens. Among the biomarkers, pLDH, HRP II, aldolase, and hemozoin formation have received increasing attention for their possible utilizations as drug target and for developing malaria detection techniques. The increase in drug resistance of malaria parasites at alarming rates, caused primarily by the indiscriminate use and overprescription of drugs, has made the current treatment protocol inefficient. Therefore, a rapid, selective, sensitive, and less expensive detection technique is a need of the hour for quick parasitological confirmation in the patients before the administration of drugs. Though RDTs are a milestone in malaria detection due to their rapid detection capability and portable nature, these cannot be exclusively relied upon for reproducible and unambiguous results. In this regard, biosensors have emerged as a reliable diagnostic technique for malaria due to many inherent advantages set by their tenet including reusability, quantitative nature, enhanced signals at low sample volume, and better storage and operational stability. As revealed from the studies, every biomarker has some strengths and limitations. In order to develop an efficient biosensor, the strength of a biomarker needs to be exploited for which a profound understanding on the physiology and metabolism is essential, on the basis of which the biomarker is being instituted. Biosensor development and its confluence with biomarker discovery are therefore important to reach the desired goal in diagnosis and treatment of malaria.Table S1 : List of PDB entries for Plasmodium lactate dehydrogenase.Table S2: List of inhibitors developed against lactate dehydrogenase from P.falciparum.Click here for additional data file.