Plasmodium falciparum and non-falciparum malaria  use a three-band P. falciparum/Pan test such as the SD Bioline Malaria Ag P.f/Pan 05FK60 , hereafter referred to as SD 05FK60, as used by the MSF-Holland clinics in Rakhine state, Myanmar. In spite of published reports of generally good test performance, medical and paramedical staff on the ground often doubt the diagnostic accuracy of these RDTs.Malaria rapid diagnostic tests (RDTs) are commonly used in Médecins Sans Frontières (MSF) programmes to detect acute malaria infection. Programmes in regions with both Parallel testing with malaria microscopy and RDT was conducted at two clinics in Rakhine state, Myanmar, for a period of 14 months as a programmatic response due to doubts and concerns of medical and paramedical staff into malaria RDTs.P. falciparum and 587 P. vivax or P. malariae mono-infections. The overall sensitivity for P. falciparum detection by the SD 05FK60 was 90.2% (95% CI: 87.4-92.6) and for P. vivax/P. malariae 79.4% (95% CI: 75.9-82.6). The overall specificity for P. falciparum detection by the SD 05FK60 was 98.5% (95% CI: 97.7-99.1) and for P. vivax/P. malariae 98.7% (95% CI: 97.9-99.2). The sensitivity for P. falciparum was >91% for parasitaemia levels of >100-1,000 parasites/μl and increased for P. vivax/P. malariae with the parasitaemia level but was overall lower than for P. falciparum 25/408 and 13/420 cases, respectively, of P. falciparum and non-falciparum malaria were missed by the RDT.A total of 2,585 blood samples from non-pregnant suspected malaria patients were examined by the SD 05FK60 RDT and microscopy at two clinics in Myanmar from October 2010 to December 2011. The reference standard microscopy diagnosed 531 P. vivax/P. malariae to a lesser extent than P. falciparum infection. Sensitivity improved with increasing parasitaemia level, however even at higher levels some infections were missed. The SD 05FK60 is adequate for use in settings where high quality microscopy is not available.In field conditions in Myanmar, the SD 05FK60 malaria RDT performed consistent with other reports. The test detected malaria caused by  Microscopy is the reference standard for diagnosing malaria but is time consuming and requires considerable training and experience. It is ill suited as a diagnostic tool in settings where the case load is high and resources limited ,2. LaterPlasmodium falciparum-specific antigen histidine-rich protein 2 (HRP2) and the pan-Plasmodium-specific lactate dehydrogenase (pLDH), expressed in the four most common malaria parasite species [P. falciparum, Plasmodium ovale, Plasmodium malariae and Plasmodium vivax[Among the newer generation of malaria RDTs are three-band tests that detect both the ium vivax,12-16.P. falciparum and non-falciparum malaria are present . In Myanmar, which has an enormous malaria burden, accounting for 7% of all malaria cases in Southeast Asia and India [P. vivax is second only to P. falciparum as the cause of malaria, so the test chosen was the SD Bioline 05FK60 Malaria Ag P.f/Pan , hereafter referred to as SD 05FK60.In 2009, Médecins Sans Frontières (MSF) introduced routine use of HRP2/pan-pLDH RDTs into its programmes at sites where both P. falciparum, with varying results for P. vivax detection [The choice was made for the SD 05FK60 because it was one of the best performing assays in the WHO/TDR /FIND (Foundation for Innovative New Diagnostics)/CDC (Centers for Disease Control) evaluation in Round 1 and because continuous supply and importation to Myanmar could be guaranteed. However, since 2007, evaluations of the SD 05FK60 were conducted in returned travellers or as mentioned before by the WHO/TDR/FIND/CDC programme but not on a field site. In these evaluations, the SD 05FK60 showed good performance for detection of malaria caused by etection -22.However, in spite of the generally good performance of malaria RDTs, their use on the ground can be problematic: medical staff frequently mistrust the results and problems with the products  are encountered regularly -25. ThisMSF has been working in Myanmar’s Rakhine State since 1993. Primary health care (PHC), including the diagnosis and treatment of malaria, is provided free of charge at two clinics, in Sittwe and Thetkalpyin, located in the eastern region of Rakhine. All patients presenting at the Sittwe and Thetkalpyin clinics with fever or a history of fever in the prior 24 hours were immediately tested for malaria on capillary blood with the SD 05FK60 RDT. In addition, a slide was prepared from the same capillary sample and examined separately in the laboratory regardless of the SD 05FK60 result. The laboratory technician was not aware of the SD 05FK60 result when examining the smear. Pregnant women screened for malaria as part of antenatal care received the same procedure but were not included in this analysis.This routine parallel testing of the described population was carried out from 25 October, 2010 until 23 December, 2011 with one month of data lost (23 February to 28 March 2011) as a result of security incidents in eastern Rakhine State.Thick and thin blood films were prepared and stained with 10% Giemsa solution (pH 7.2) for 10 min and examined by light microscopy using x1,000 magnification. At least 200 fields had to be examined before designating a sample ‘negative’, or more accurately, ‘no malaria parasites seen’. Positive findings were graded on the thick smear using the ‘plus’ system scale: + (1 to 9 trophozoites in 100 fields); ++ (1 to 10 trophozoites in 10 fields); +++ (1 to 10 trophozoites per field); ++++ (>10 trophozoites per field). These scores were used to estimate parasite densities: + = 10 to 90 parasites/μl; ++ = 100 to 1,000 parasites/μl, +++ = 1,000 to 10,000 parasites/μl; ++++ = >10,000 parasites/μl, assuming a white blood cell count of 8,000/μl. The species was identified using the thin smear.P. falciparum-positive result was indicated by presence of two colour bands, the P. falciparum test line and the ‘C’ control line. The presence of the ‘Pan’ test line and the ‘C’ control line  indicated a P. vivax, P. ovale. or P. malariae-positive result or a mixed non-falciparum infection. The presence of three colour bands, the ‘P. falciparum’ and ‘Pan’ test lines and the ‘C’ control line indicates a P. falciparum-positive result or a mixed infection. In cases where the control line did not appear, the results were interpreted as invalid and the test repeated with a new device.The SD 05FK60 RDT is a three-band lateral-flow immunochromatographic antigen detection test in a cassette format and testing was carried out according to the manufacturer’s instructions. Readings were taken in daylight, assisted by standard electric lighting. A negative result is indicated by the presence of a single line, the ‘C’ control line, in the result window. A P. falciparum and non-falciparum species separately. When calculating the performance parameters for P. falciparum all negative samples plus all P. falciparum positive samples by microscopy contributed to the analysis. Similarly for the non-falciparum analysis all negative samples and all non-falciparum positive samples by microscopy contributed to the analysis. Likelihood ratios were calculated using the following formulae: LR + = sensitivity/(1–specificity) and LR– = (1–specificity)/sensitivity . Samples with pure gametocytaemia were considered negative. Microscopically identified mixed infections were considered separately Table . Sensitity Table .All malaria RDTs purchased by MSF must pass the WHO lot-testing programme before being shipped to field sites . In addiThis manuscript reports a descriptive retrospective analysis of routinely collected data, and therefore no protocol was submitted for ethical review. Data collected contained no personal identifiers.Between 25 October, 2010 and 23 December, 2011, a blood sample was obtained from each of 2,585 non-pregnant individuals attending the Sittwe and Thetkalpyin clinics in Rakhine State and suspected to have malaria infection. Baseline characteristics of the included population are shown in Table P. falciparum, 587/2,585 (22.7%) with P. vivax or P. malariae. and 1,337/2,585 (51.7%) tested negative; 130 (5.0%) mixed infections  were found and excluded from the main analysis, only misclassification was assessedMicroscopy and RDT results are shown in Table P. falciparum detection by the SD 05FK60 was 90.2% (95% CI: 87.4-92.6) and for P. vivax/P. malariae 79.4% (95% CI: 75.9-82.6). The overall specificity for P. falciparum detection by the SD 05FK60 at both clinics was 98.5% (95% CI: 97.7-99.1) and for P. vivax/P. malariae 98.7% (95% CI: 97.9-99.2).Sensitivity, specificity and predictive values of the SD 05FK60 for detection of malaria are shown in Table P. falciparum detection by the SD 05FK60 in both projects was 96.0% (95% CI: 93.9-97.5%) and for P. vivax 96.3% (95% CI: 94.2-97.8%) at prevalence levels of 28% and 31%, respectively. The negative predictive value (NPV) for P. falciparum detection by the SD 05FK60 in both projects was 96.3% (95% CI: 94.9-97.5%) and for P. vivax 92.5% (95% CI: 90.7-94.2%). Test accuracy indices were calculated separately for the two clinics but no significant difference in performance was observed.The positive predictive value (PPV) for P. falciparum infections were wrongly identified by the SD 05FK60 as non-falciparum infections (Table 2.5% 13/531) of the 3/531 of P. falciparum or mixed infection  of the non-falciparum samples were positive on the P. falciparum and pan-band of which seven had a +++ and five ++++ parasitaemia level. Of the 130 mixed infections 122 were correctly identified by the SD 05FK60 with a positive P. falciparum and pan-band. Two samples only had a positive pan-band and six samples were only positive on the P. falciparum band.Of the non-falciparum infections 2.5% 15/587) were misclassified as 5/587 werP. falciparum specimens and between 69.8% and 97.7% with a ++ to ++++ parasitaemia level. The sensitivity increased consistently with increasing parasitaemia for P. vivax/P. malariae (p < 0.005). 25/408 and 13/420 cases with +++ and ++++ parasitaemia levels, respectively, of P. falciparum and non-falciparum malaria were missed by the RDT.Table The quality control team at the Shoklo Malaria Research Unit (SMRU) in Mae Sot, Thailand, cross-checked 450 slides (225 negative and 225 positive) from MSF’s projects in Myanmar from the year 2010, and 250 slides (125 positives and 125 negatives) from MSF’s projects in Eastern Rakhine State in Myanmar from the year 2011.The overall agreement between the positive results had a kappa value of 0.96  for 2010 and 0.91  for 2011, indicating very good agreement. The overall sensitivity was 98.3% (225/229) in 2010 and 98.4% (123/125) in 2011; overall specificity was 100% (211/211) in 2010 and 99.1% (110/111) in 2011.P. falciparum and satisfactory results for non-falciparum infections. The poorer detection and identification of P. vivax/P. malariae than that of P. falciparum is consistent with other evaluations of the SD 05FK60 and pan-pLDH tests in general [Commercially available HRP-2/pLDH tests have been evaluated previously in Myanmar ,30 but e general -22.P. falciparum and 35.0% for P. vivax[. The same product was retested in Round 3 of this evaluation and showed a slightly decreased PDS of 92.9% for P. falciparum along with an improved detection score of 97.1% for P. vivax[in vitro diagnostic product is changed.However, it should be pointed out that in Round 1 of the WHO/TDR/FIND/CDC evaluation, the panel detection score (PDS) of the SD 05FK60 reported at parasitaemia levels of 200 parasites/μl was 96.2% for  P. vivax. The sam P. vivax. It is uP. falciparum infections and 12 P. vivax/P. malariae infections with +++ or ++++ parasitaemia were undetected by the SD 05FK60. The non-detection of P. falciparum infections at high parasitaemia levels could be explained by the prozone effect [Plasmodium cohort in the area to undermine this argument. However, this does not explain the false negative pan-pLDH band.The sensitivity of the SD 05FK60 improves with increasing parasitaemia. This is a well-known limitation of RDTs. Less expected were the findings that 25 e effect -33 or bye effect . It was P. falciparum mono-infections would have been missed by using the RDT without microscopy. Although the specificity has been high for both P. falciparum and non-falciparum infections: 98.5% and 98.7% respectively, thirteen infections were misidentified as P. vivax/P. malariae malaria and as a result would have been treated with chloroquine, a drug to which P. falciparum is unlikely to be susceptible [P. vivax/P. malariae cases, 121 out of 587 (20.6%) individuals would have not received any malaria treatment despite being infected. Only 31 individuals out of a total of 1,337 (2.3%) would have been unnecessarily treated with anti-malarials on the basis of RDT.Translating these results into the impact on patients, 52 (10.2%) of a total 531 ceptible . ConsideAlthough the results of the performance of the SD 05FK60 are satisfactory, MSF decided to opt for microscopy as the first choice for malaria diagnosis in its programmes in Rakhine State, Myanmar. This was a pragmatic decision based on the clinicians’ greater trust in microscopy, particularly in light of the cases with high parasitaemia that would have been missed if microscopy had not been used in addition to the RDT. MSF reserves RDTs for use in decentralized settings and where a high number of patients precludes microscopy. Therefore MSF decided to continue using microscopy in the two clinics in Rakhine State, Myanmar for the routine diagnosis of malaria, although RDTs have been found to be more cost-effective and more feasible in other settings -38.This study has several limitations. The data were collected as part of routine programmatic activities and were unlinked to patient records. Thus, it was impossible to collect clinical information, such as recent intake of anti-malarials or the presence of other factors, which may have influenced the results. Another limitations is that the + system has been used to grade parasitaemia instead of a parasite count. This less accurate estimation may have influenced the results on sensitivity especially in the stratified analysis of parasitaemia levels.It is important to note that the described data were obtained in true field conditions, i e, conditions that were suboptimal in terms of RDT storage and handling and staff expertise, and non-standardized specimens may have been present. It is known that such factors influence RDT performance ,40, but MSF is able to train and maintain good malaria microscopy in its field settings. However, this may not be possible in a wider public health arena. In 2006 the WHO estimated that, if the recommendation to obtain parasitological confirmation of malaria before prescribing treatment were to be followed, screening would need to be scaled up from 122 million to 780 million tests each year in Southeast Asia alone, a 600% increase. Such scaling up would undoubtedly be difficult to achieve without the use of simple, reliable RDTs [The authors declare that they have no competing interests.WTN organized the sample collection and quality control in Rakhine State and performed the data entry. CSK performed the statistical analysis. CSK, WTN, EP and LS analysed and interpreted the results and drafted the manuscript. All authors approved the final manuscript.


Plasmodium falciparum gametocytes in humans, knowledge of which would be useful in the development of anti-gametocyte vaccines, which would have the capability to reduce malaria transmission from humans to mosquitoes. In a study in the Gambia, mature gametocyte-infected erythrocytes of P. falciparum were found to carry antigens  that were recognised by malaria patient’s plasma antibodies. These anti-GSA antibodies, taken at a single timepoint, were weakly associated with lower duration of gametocyte carriage in these treated patients [in vivo.Malaria transmission-reducing interventions are key components of malaria control and elimination . Howeverpatients ,3. We thP./falciparum-positive children attending school in a rainforest region in Ghana. These children were treated with dihydro-artemisinin piperaquine, and followed up weekly for 1 month.Flow cytometry of cultured gametocyte-infected erythrocytes from 3D7 and from two recently adapted gametocyte-producung lines was used to detect and measure plasma antibodies recognising the erythrocyte surface. Plasma was obtained from asymptomatic By microscopy, 8.9% (15/168) of the children enrolled carried gametocytes and a further 20% of them developed gametocytes during subsequent follow-up.  Preliminary results from 113 samples tested in flow cytometry show that more than 50% of those in the sub-group of children with gametocytes at enrolment carry antibodies to GSA, and we expect this proportion to increase as gametocytes are developed during the follow-up. Further longitudinal flow cytometry, and NASBA analyses will enable us to understand the dynamics between immune responses to gametocytes and gametocyte carriage following treatment of asymptomatic malaria.


Plasmodium falciparum and hepatitis A (HAV) infections are common, especially in children. Co-infections with these two pathogens may therefore occur, but it is unknown if temporal clustering exists.In sub-Saharan Africa, P. falciparum malaria and acute HAV in Kenyan children under the age of 5 years in a cohort of children presenting with uncomplicated P. falciparum malaria. HAV status was determined during a 3-month follow-up period.We studied the pattern of co-infection of P. falciparum malaria was 1.7 (95% CI 0.81–3.1) infections/person-year while the cumulative incidence of HAV over the 3-month follow-up period was 0.27 (95% CI 0.14–0.50) infections/person-year. Children with or without HAV co-infections had similar mean P. falciparum asexual parasite densities at presentation , largely exceeding the pyrogenic threshold of 2,500 parasites/µL in this population and minimizing risk of over-diagnosis of malaria as an explanation.Among 222 cases of uncomplicated malaria, 10 patients were anti-HAV IgM positive. The incidence of HAV infections during P. falciparum malaria suggests that co-infections of these two hepatotrophic human pathogens may result from changes in host susceptibility. Testing this hypothesis will require larger prospective studies.The observed temporal association between acute HAV and  Plasmodium falciparum malaria and hepatitis A virus (HAV) infections are common infections, especially in children In Africa, both P. falciparum malaria and hepatitis B (HBV). In a case-control study in The Gambia, the prevalence of HBV was significantly increased amongst children with severe P. falciparum malaria compared to matched controls P. falciparum malaria, which was higher than the estimated prevalence of HBV in that area (10%) Plasmodium had the lowest prevalence of HBV infection, but there was no correlation with parasitemia P. yoeliiP. falciparum affect the natural course of P. falciparum infections.Epidemiologically relevant interactions, though with contradictory findings, have previously been shown for P. falciparum malaria and acute HAV in a cohort of Kenyan children under the age of 5 years presenting with uncomplicated P. falciparum malaria. We focused on HAV since an initial viral screen  of cases with elevated plasma concentrations of alanine aminotransferase  patients were positive for HAV, but not for any other hepatitis viruses.In this study we aimed to determine the temporal pattern of co-infection of A total of 222 children were included in this study. Forty patients (18.0%) had ALT plasma concentrations above the upper limit of normal for children (36 U/L) at one or more measurements at enrolment or during follow-up, and fifteen patients (6.7%) had ALT levels exceeding 100 U/L. All ten cases of HAV occurred in patient with ALT levels above 36 U/L (range 41–1295 U/L): eight patients had ALT levels ≥100 U/L, and two children had maximal ALT levels between 36–100 U/L (41 and 56 U/L). Demographic and laboratory data of HAV positive and negative children are shown in falciparum malaria infections can lead to increased production of unspecific HAV-IgM P. falciparum parasite density at baseline . The geometrical mean parasitemia in this subgroup was 196,000 parasites/µL , and the median ALT level was 28 U/L (range 16–194 U/L). One patient with a parasitemia of 202,000/µL and a maximal ALT level of 41 U/L . These children were matched on treatment, study date, gender and age with the 15 patients having ALT levels ≥100 U/L. None of these patients showed a positive reaction for IgM-HAV. In addition, we tested an alternative hypothesis that P = 0.001). A possible explanation could be the aggregation of measurements from baseline to day 7, because when only data from baseline were used, the difference ceased to be significant , possibly indicating that other factors such as treatment could have caused the additional increase in ALT levels.Importantly, all but one case coincided with malaria  of HAV uninfected patients (p = 0.37). Of the five patients with a malaria recurrence in the HAV positive group, four were found to be HAV infected at baseline; the other patient had an HAV infection at day 42 and experienced a recurrent P. falciparum infection 1 week later. Four patients had a re-infection; the other sample could not be analyzed. Of the 19 HAV negative patients with a malaria recurrence, 17 had a re-infection, 1 had both recrudescent primary and secondary re-infections, and 1 sample could not be analyzed.Fifty percent (5/10) of patients with HAV infection had a P. falciparum malaria and acute HAV in a cohort of pediatric patients. We found that nine out of ten cases of acute HAV infections occurred simultaneously with P. falciparum malaria infections.The present study describes a temporal association between P. falciparum malaria? Firstly, overestimation of the association might have occurred if patients with HAV infections presented primarily for seeking relief for symptoms of HAV infection, and not of malaria. The baseline blood stage density in our study group was high , far exceeding the pyrogenic threshold of 2,500 parasites/µL in this population and thus, minimizing over-diagnosis of malaria as an alternative explanation P. falciparum mono-infections. Secondly, P. falciparum malaria and/or hepatotoxic side effects of antimalarial drugs can also lead to elevated plasma concentrations of liver enzymes, indicating hepatocellular damage What are the alternative explanations that could have lead to a potential overestimation of the temporal association of active HAV infections with uncomplicated P. falciparum malaria and elevated ALT concentrations (>100 U/L) had a 53% (8/15) chance of being co-infected with HAV, indicating that almost 50% of elevations could not be explained by HAV infections. This percentage is high and warrants consideration whenever determining the safety profile of antimalarial drugs. This study did not reveal any adverse clinical consequences of concurrent infections, except for a significant lower hemoglobin level. We have no ready explanation for this difference. Unlike Plasmodium infections HAV is not known to affect hemoglobin levels. One could speculate that Plasmodium infections in co-infected children were protracted and thus caused a slight additional drop in hemoglobin concentrations. Malaria patients co-infected with HAV were as likely as patients without HAV co-infections to clear clinical symptoms and parasites. By extension, this also indicates that most HAV infections were asymptomatic as expected We found that HAV infections were a common cause for elevated plasma concentrations of an established biomarker of liver cell damage (ALT) in these patients. Children from the Kilifi district with P. falciparum liver stage and/or blood stage infections could, mediated by unknown immune mechanisms, promote HAV replication and thus, increase the chance to detect active HAV infections. Alternatively, coincidental HAV infection could facilitate parasite survival at the liver stage leading to increased numbers of infective merozoites and higher likelihood of subsequent pathogenic blood stage infection, in a similar way to the suggested mechanism for the observed association between malaria and HBV. In the study by Thursz, the increased incidence of severe malaria that was observed in chronic HBV carriers was explained by reduced expression of HLA class I molecules by hepatocytes during chronic HBV infection P. falciparum infected hepatocytes by cytotoxic T lymphocytes (CTLs). Thus, the lysing of parasite-infected hepatocytes by cytotoxic T lymphocytes (CTLs) may have been impaired in HBV carriers leading to an increased susceptibility to severe malaria. Whether similar mechanisms play a role in the association of acute HAV and falciparum malaria remains unclear.The biological mechanism, if any, behind the observed temporal association remains elusive. For instance, P. falciparum and HAV infections in children living in an area of high P. falciparum and HAV transmission. Unfortunately, there is very limited information available about baseline HAV rates in Kenya, which is needed to draw stronger conclusions. We did not measure this; nevertheless we observed a temporal association between falciparum malaria and HAV. Prospective and larger studies are required to further elucidate the epidemiological interaction between these two important human pathogens. Our data also highlight the need for more comprehensive analyses of the complex interactions of prevalent infectious diseases in endemic countries that contribute to an unacceptably high disease burden.In conclusion, we provide tentative evidence for a temporal association between P. falciparum transmission rates have been estimated to range between 22 and 53 infective bites per person per year The study was conducted at the Pingilikani study site of the Kenya Medical Research Institute/Centre for Geographic Medicine Research-Coast (CGMR-C). The Pingilikani study site is located 20 km south of Kilifi town in Kilifi district along the Kenyan coast P. falciparum malaria et al (unpublished data). Ethical approval for this study was granted by the Kenya National Ethics Review Committee, the Oxford Tropical Research Ethics Committee and the Ethics Committee of the Heidelberg University School of Medicine. Children aged 6–59 months presenting to the Pingilikani Health Centre with symptoms suggestive of uncomplicated malaria and a documented P. falciparum infection with an asexual parasite density of 2,000–200,000 parasites/µL, were recruited. Written informed consent was obtained from the parents. Patients were monitored daily until parasite and fever clearance was achieved. Active follow-up was on days 3 and 7, then weekly on days 14, 21, 28, 35, 42, 56, and 63 and 84. All subjects were seen on any day between day 0 and 84 upon request. ALT was measured routinely at baseline and at day 3, 7, 28, 42, 84, and at asexual parasite recurrence. Although ALT levels in patients with active HAV infection are typically 4–100 times elevated This study was nested in an ongoing clinical trial to assess the efficacy and safety of dihydroartemisinin-piperaquine in comparison with artemether-lumefantrine in children with uncomplicated Malaria infection was confirmed by Giemsa-stained thick smear P<0.05 was considered statistically significant. Exact binomial confidence intervals were calculated to compare incidence rates.Analysis was carried out using Stata version 9.2 . Data were entered and checked for inconsistencies by two different persons to reduce faulty entries. The Kolmogorov-Smirnov test was used to verify the normality of distribution of continuous variables. Comparisons between groups were performed using the t-test or two-tailed unpaired Mann-Whitney test for continuous variables evaluated as normally distributed and not-normally distributed, respectively. The chi-square test was used for comparisons between categorical variables. Parasitemia results were summarized using geometrical means and 95% confidence intervals. All the remaining continuous variables were reported with arithmetic means and standard deviation, or by the median and IQR.


Plasmodium vivax infection who were treated with chloroquine and primaquine. The major complications were jaundice and severe anemia. No in vivo chloroquine resistance was detected. These data help characterize the clinical profile of severe P. vivax malaria in Latin America.We describe a case series of 17 patients hospitalized in Manaus (western Brazilian Amazon) with PCR-confirmed  Plasmodium vivax parasites. Of the P. vivax malaria cases, 3,833,477 were reported in Brazil, mainly in the Amazon Region  of these cases were caused by P. vivax). Since the mid-1990s, as P. vivax was becoming the predominant malaria species in Brazil , the capital of the state of Amazonas in the western Brazilian Amazon, is clearly part of a new frontier in the economic development of the Amazon. In 2009, a total of 19,698 cases of malaria were reported in Manaus . Other reports from the same reference center in Manaus have been published regarding unusual complications of P. vivax infection, such as severe rhabdomyolysis , representing 65.1% of the total cases from Manaus. During the same period, 358 (3.2%) patients with P. vivax malaria were hospitalized. A retrospective analysis was performed of case-patients who fulfilled the malaria severity criteria of the World Health Organization (WHO)  and PCR, according to the technique described elsewhere . In 2001 and 2002, a total of 13,056 cases of malaria were diagnosed in this institution , according to the Brazilian Ministry of Health guidelines. In 2006, WHO formally recommended the treatment of severe vivax malaria to be the same as that for severe falciparum malaria, because of the risk for an unrecognized mixed infection  developed in patient 11 two days after she received chloroquine, and she died 3 days later. This patient had a negative thick blood smear from day 3 of treatment with choloroquine. The other 16 patients were followed up after discharge until day 28. None had clinical symptoms of malaria, and all thick blood smears were negative at days 7, 14, and 28.Seventeen patients were included in the analysis, and their clinical and laboratory data are shown in P. vivax malaria with ARDS has been reported in travelers who acquired the infection in Manaus . This age range is similar to that seen in other case series from Latin America, such as in hospitalized children from Venezuela with severe anemia that required blood transfusions , which suggests that this hematologic complication may be a surrogate marker of severity.The concomitant diagnosis of hepatitis A virus infection in patient 3  indicateThe wide range of parasitemia found in our patients does not enable us to comment on the value of this variable as a determinant of severity. Sixteen patients recovered without the use of antimicrobial drugs; therefore, it is highly improbable that bacterial sepsis was a factor for severity in our case series. In unstable transmission areas , malaria in older patients may pose an additional problem because chronic diseases  may predispose a patient to clinical decompensation.P. vivax malaria, studies that assess clinical severity and chloroquine resistance would be unethical. However, our findings suggest that chloroquine resistance would be a problem for individual patients and that the determinants of this resistance need to be clarified. Clearly, areas with chloroquine-resistant P. vivax also report severe P. vivax malaria, but we believe that these studies are not able to establish any firm causality. The finding of both phenomena in some areas may simply reflect high transmission of this species.In vivo chloroquine resistance was not detected in any of the cases that were followed up, despite recent confirmation of the phenomenon in this same locality  and India. These severe P. vivax cases contribute to increased public health costs because of increased hospitalization and the need for intensive care and blood transfusions. The major complications in patients who required hospitalization were jaundice and severe anemia, although whether these complications were responsible for deaths is undetermined.Our retrospective review illustrates the spectrum of severe P. vivax malaria. However, WHO criteria formerly defined for P. falciparum malaria seem to be applicable to most of the severe P. vivax malaria cases reported in hospital-based studies in the literature. PCR should be performed to rule out mixed infections and other common infectious diseases so that reports from different parts of the world are comparable. Despite the small number of patients, our data corroborate previous findings of severe disease found in areas where chloroquine-resistant P. vivax is being reported but suggest that establishing direct causality is not straightforward. We urgently need to know which clinical complications in P. vivax malaria are associated with death to validate severity criteria. A valid biomarker for chloroquine resistance would also enable associative studies to determine the association between resistance and severity.No clear severity criteria exist for


Plasmodium falciparum incidence has decreased 99% and prevalence measured by slide-positivity rate was below 1% in 2009 [Malaria control measures have been successful in reducing malaria mortality and morbidity in sub-Saharan Africa in the last decade. In particular, in Principe Island, São Tomé e Príncipe, after 5 years of control measures,  in 2009 .P. falciparum. The effect of interventions and transmission relapse on the dynamics of infection and immunity to markers of transmission intensity, such as the merozoite surface protein-3 (MSP-3) and of protection against disease, like P. falciparum erythrocyte membrane protein 1 (PfEMPI) at the population level are studied. To calibrate the model, sero-epidemiological data collected in 2005 and 2008 in Principe island with parasitemia determined by polymerase chain reaction (PCR) are used.Mathematical models are developed to assess age and time trends on malaria infection and immunity against conserved and variant surface antigens of P.falciparum PCR parasitemia was above 25% and, assuming endemic equilibrium, the basic reproduction number was still above 1. Patterns of seropositivity to conserved and anti-PfEMPI antibodies reflect cumulative exposure with age. Simulations of interventions that reduce the transmission coefficient mirror well the progression of infection and anti-MSP-3 immunity in the population in 2008 and suggest that prevalence of infection decays faster than immunity. However, when transmission relapses occur, the onset of immunity is slower than the increase of infected individuals, posing problems for severity of new infections. The simulated number of variants against PfEMP1 decayed steeply with interventions and the effect on particular variants associated with severe disease could give some clues on future malaria morbidity.In 2005, with two years of control measures in Principe Island, Based on data from a settlement that has been subject to control measures we explore conditions under which the decay on the diversity of the immune repertoire can increase morbidity if transmission relapses and we aim to propose and inspire strategies to undermine the impact of this issue.


Plasmodium falciparum and Plasmodium vivax are the dominant species accounting for roughly 60 and 40% of malaria cases, respectively. Recently a major shift from P. falciparum to P. vivax has been observed in various parts of the country but the epidemiology of the other human malaria species, Plasmodium ovale spp. and Plasmodium malariae remains poorly understood. The aim of this study was to assess P. ovale curtisi and wallikeri infection in north-west Ethiopia by using microscopy and nested PCR.In Ethiopia Plasmodium species. Samples were collected on Whatman 903 filter papers, stored in small plastic bags with desiccant and transported to Vienna (Austria) for molecular analysis. Data from study participants were entered and analysed by SPSS 20 software.A health institution-based survey using non-probability sampling techniques was conducted at Maksegnet, Enfranze and Kola Diba health centres and Metema hospital in North Gondar. Three-hundred patients with signs and symptoms consistent with malaria were included in this study and capillary blood was collected for microscopic examination and molecular analysis of P. falciparum and 51 P. vivax) by microscopy. By species-specific PCR 233 Plasmodium spp (95% CI: 72.6-82) were detected and the majority 155  were P. falciparum followed by P. vivax 69  and 9  samples were positive for P. ovale. Seven of P. ovale parasites were confirmed as P. ovale wallikeri and two were confirmed as P. ovale curtisi. None of the samples tested positive for P. malariae. During microscopic examination there were high (16.3%) false negative reports and all mixed infections and P. ovale cases were missed or misclassified.Out of 300 study participants , 184 samples were classified positive for malaria (133 P. ovale malaria is under-reported in Ethiopia and provides the first known evidence of the sympatric distribution of indigenous P. ovale wallikeri and P. ovale curtisi in Ethiopia. Therefore, further studies assessing the prevalence of the rare species P. ovale and P. malariae are urgently needed to better understand the species distribution and to adapt malaria control strategies.This study indicates that  Plasmodium ovale (first described by Stevens in 1922) [Plasmodium parasite species that causes human malaria [Malaria remains one of the leading causes of illness and death in the world ,2. Plasmin 1922)  is widel malaria ,5. Relat malaria .P. ovale conducted by researchers from the UK Malaria Reference Laboratory, London, polymorphisms in six loci were examined in 55 isolates. Two distinct major haplotypes of each locus were identified and these did not recombine in any of the parasites examined. Accordingly, P. ovale dimorphism was proposed to reflect the existence of two fully distinct ovale malaria species, which were unexpectedly shown to be broadly sympatric, at the country level, in both Africa and Asia. These two proposed species have been named P. ovale curtisi and P. ovale wallikeri[P. ovale species [In a study on wallikeri. Recentl species ,9.Plasmodium falciparum and Plasmodium vivax are the main species accounting for roughly 60 and 40% of malaria cases [P. falciparum to P. vivax in different parts of the country. Despite First report on P. ovale in Ethiopia by Armstrong JC IN 1969 [P. ovale spp. and Plasmodium malariae has never been elucidated and is rather limited in the entire country [Malaria remains the leading communicable disease in Ethiopia, accounting for approximately 30% of the overall disability-adjusted life years lost. It is estimated that about 75% of the total area of the country and 68% of the population is at risk of infection -12. In tia cases  and rece IN 1969 , the kno country -17.P. ovale tends to be mis-and under-diagnosed when using microscopic examination. Difficulties in the diagnosis are partly the result of the low parasite density that is characteristic of malaria caused by ovale malaria parasites [P. ovale malaria tends to be under-reported.The global burden of ovale malaria is considered to be light, potentially also because arasites ,18,19. Sarasites ,21. In dP. ovale species and the ability of microscopists to detect and identify Plasmodium species in north-west Ethiopia.Therefore, new laboratory diagnostic techniques (such as polymerase chain reaction (PCR)) that can provide higher sensitivity and specificity, without subjective variation, are urgently needed to improve the quality of diagnostics and the understanding of malaria epidemiology -24. The P. falciparum. Both P. vivax and P. falciparum are known to co-exist in the area whereas other Plasmodium species have not been reported in the past two decades from any of the four sites. The only reports of P. ovale in Ethiopia date back to the 1960s [.The study was conducted at three health centres  and one hospital  in North Gondar, northwest Ethiopia. These areas are endemic for malaria and the altitude of the districts ranges from 1,750 to 2100 m above sea level. Malaria is the most prevalent seasonal disease in the area, ranking second among all reported diseases seen at the health centres. October to December is the peak transmission season for he 1960s .A health institution-based survey using non-probability sampling techniques was conducted at three health centres and one hospital in North Gondar, north-west Ethiopia. Patients with signs and symptoms consistent with malaria  were included in this study. Males and females, of any age, with signs and symptoms consistent with malaria and willingness to participate and sign the informed consent, were used as inclusion criteria.Convenience sampling technique was used to recruit study participants for this study. A total of 300 suspected malaria patients were included from March 2 to April 26, 2013. Participants were 184 microscopy-confirmed cases and 116 febrile patients suspected of potentially having malaria and who were negative by microscopy. A structured questionnaire was used to obtain sociodemographic information .From each participant a small amount of capillary blood was collected by finger prick after obtaining informed consent. The total amount of blood was less than 1 mL. Peripheral smear examination of well-prepared and well-stained blood films remains the gold standard for confirming the presence of malaria parasites. Thick and thin smears prepared from the peripheral blood were used for this purpose and for each patient two slides were prepared.Before staining the thin blood films were fixed in methanol for 30 sec. Then smears used for study purposes were stained with 3% Giemsa solution for 30 min, whereas the staining of the routine smear followed the standard techniques established at the respective health facility. Examination of the blood films was conducted according to standardized World Health Organization (WHO) protocols. Thick films were used for the detection of the presence of parasites and parasite species identification was done by using thin films under 100 × oil immersion objective.Two blood spots were collected from each participant, transferred onto filter paper (Whatman 903) and labelled with the participant’s study code and date. Each filter paper was dried individually, carefully avoiding any chance of contamination. The samples were then stored in small plastic bags with desiccant and transported to the Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna (MUV), Vienna, Austria for molecular analysis.Plasmodium DNA from the blood spots on filter paper. To ensure comparable quantities of blood, samples of exactly 4 mm diameter were punched out of the blood spots. Blood spots were soaked overnight in a tube at 4°C with 100 μl phosphate-buffered saline (PBS). The tube was centrifuged at 13,000 revolutions per minute (rpm) for 2 min. After discarding the supernatant, 100 μL of PBS was added to the tube and the sample was centrifuged at 13,000 rpm for 2 min to wash the sediment. After discarding the supernatant, 60 μL of InstaGene Matrix was added. The tube was incubated for 8 min at 70°C, vortexed for 15 sec, boiled for 4 min, and vortexed for 15 sec. Finally, the tube was centrifuged at 13,000 rpm for 1 min and the supernatant was aspirated and purified again with InstaGene Matrix as described. The supernatant was then used for the PCR. If the PCR result was negative, the supernatant was purified again for up to three times.A modified chelex-based DNA extraction method using the InstaGene Whole Blood Kit  was used for the extraction and purification of 2, PCR buffer, 125 μM of each dNTP, and 1 unit of GoTaq DNA polymerase . Nest 1 amplification conditions were as follows: initial denaturation 94°C for 4 min; 25 cycles ; and a final extension at 72°C for 4 min; 2.5 μl of the nest 1 amplification product served as the DNA template for each of the 25 μl nest 2 amplifications.Nested PCR assay was performed for all samples as reported previously ,24-26. EP. falciparum, rVIV1 and rVIV2 for P. vivax, rMAL1 and rMAL2 for P. malariae , rOVA1WC and rOVA2WC for both P. ovale species [The concentration of the nest 2 primers and other constituents were identical to nest 1 amplifications. Nest 2 amplification conditions were identical for all species-specific nest 2 PCRs to those of nest 1 except for the genus-specific primers . In all experiments, a negative control of water and known positive controls were run with the samples. In the nested PCR, the genus-specific primers rPLU1 and rPLU5 for the first PCR and rPLU3 and rPLU4 were used for the second PCR. Whenever this genus-specific nested PCR gave positive results, species-specific nest 2 PCRs were performed for species determination using the following internal primer pairs: rFAL1 and rFAL2 for  species ,24-26.P. ovale spp were further analysed to species level using the primers rOVA1/rOVA2 specific for Plasmodium ovale curtisi and rOVA1v/rOVA2v for Plasmodium ovale wallikeri[The oligonucleotides were obtained from Microsynth . All samples giving positve signals for wallikeri,26. The P. ovale-positive samples were further analysed by sequencing for species confirmation. PCR products were purified with ExoSap-IT®  and sequenced using the BigDye Sequencing Kit and an automatic 310 ABI PRISM sequencer .The amplicons of Data were checked for their completeness before entering for analysis; then all the data from study participants were imported into Excel and analysed by SPSS 20 program software. Descriptive statistics was used to assess the distribution of the sociodemographic characteristics and independent variables as well as frequencies and proportion of malaria-positive samples.This study was expected to pose only minimal risks to the participants. The study protocol was reviewed and approved by the Institutional Ethical Review Board of the University of Gondar. Written informed consent in local language was obtained from each participant and/or his/her legal representative. For patients who cannot read or write the consent form was read in the local language and a thumbprint was collected instead of a signature. Patients who were positive for malaria parasites received standard treatment according to national treatment guidelines.Out of a total of 300 study participants, 167 (55.7%) were male and 133 (44.3%) were female. The majority of the respondents (91.7%) were of Amhara ethnicity and orthodox christianity was the predominant religion in the area accounting for 93.7%. In total 37.3% of the study participants were illiterate and the majority (61%) were in the age group 15 to 44 years  samples classified as positive by microscopic examination were confirmed as being positive by PCR. In addition, 49 samples that were classified as negative by microscopy were positive in the genus-specific nested PCR. Only two  samples that were read as positive by microscopy were considered false positive. The result indicates that 16.3% (49/300) of the malaria cases were missed by microscopy. By species-specific PCR, 233 Plasmodium spp (95% CI: 72.6-82) were detected and the majority 155  were P. falciparum followed by P. vivax 69  and nine  samples were positive for P. ovale. Furthermore, nine  samples gave positive results for P. ovale spp, but all examined samples were negative for P. malariae  samples were mixed malaria infections, which indicates that the majority of 218  cases were mono-infections.All positive samples (184) were mono-infections and all mixed Plasmodium species by microscopy. Three P. ovale parasites were reported as P. vivax, two were reported as P. falciparum, eight P. falciparum as P. vivax and four P. vivax as P. falciparum.Table  AdditionP. ovale spp, out of which the majority, seven cases (77.7%) were confirmed as P. ovale wallikeri and only two cases (23.3%) were confirmed as P. ovale curtisi. Four samples positive for P. ovale in PCR were confirmed by DNA sequencing . However, none were diagnosed as P. ovale, as P. ovale was not known to be endemic in Ethiopia, including in the study areas and, therefore, it was not adequately considered by the microscopists  blood samples collected from symptomatic patients presenting febrile illnesses were positive for Plasmodium species, P. falciparum and P. vivax, are the main and dominant species accounting for roughly 60 and 40% of malaria cases, respectively [P. falciparum to P. vivax in different parts of the country. However, the epidemiology of the P. ovale spp remains poorly understood and there are no recent data on the distribution of this pathogen in Ethiopia (including the study area) [Malaria remains a leading cause of infectious disease in Ethiopia, accounting for approximately 30% of the overall disability-adjusted life years lost. It is estimated that about 75% of the total area of the country and 68% of the population is at risk of infection. In the country, the two ectively . Recentldy area) -17.P. falciparum (66.5%) remains the predominant species followed by P. vivax (29.6%) and P. ovale (3.9%) at the study sites. The majority (93.6%) of infections were mono-infections. This result is concordant with previous studies elsewhere in Ethiopia and the report of the Ethiopian Federal Ministry of Health, in which P. falciparum is the predominant malaria circulating in the country followed by P. vivax. On the other hand, this finding contradicts previous results, which indicated the shifting of P. vivax in the study area and different parts of Ethiopia and no report of P. ovale cases recently [The PCR results of this study indicate that recently -17. Howerecently .P. ovale infections among suspected malaria cases is the first report of P. ovale from this part of the country. Until relatively recently the distribution pattern of P. ovale was considered to be limited to tropical regions in sub-Saharan Africa, Indonesia  and the Philippines [P. vivax[P. ovale cases were misclassified as P. vivax and two were reported as P. falciparum, suggesting difficulty in the diagnosis of P. ovale by microscopic examination in the study area where this pathogen was previously not known to be present and most microscopists are neither trained to detect nor aware of the presence of other species [The 3.9% positive rate for lippines , but not[P. vivax. In anal species .P ovale-positive samples were further analysed with specific PCRs and/or sequenced to classify P. ovale species in the study area and the majority (77.7%) were confirmed as P. ovale wallikeri as compared to 23.3% that were identified as P. ovale curtisi. This finding is in line with the previous report by Sutherland et al. [P. ovale into two species, namely P. ovale curtisi (former classic type) and P. ovale wallikeri (former variant type). This finding is the first of its kind in Ethiopia and is in line with previous reports from South Asia in which both P. ovale curtisi and P. ovale wallikeri occur sympatrically in the Chittagong Hill Tracts, Bangladesh [P. ovale species are morphologically identical and cannot be differentiated by microscopy [majority 7.7% werengladesh ,30. Thescroscopy . Even mocroscopy .Plasmodium species. . Although microscopy remains the gold standard for malaria diagnosis in the field, the limits of detection may significantly differ between microscopists and have previously been estimated to range from 50 to 100 parasites/μl under field conditions [The nested PCR analysis suggests a relatively high false negative rate of 16.3% with only 0.7% false positive cases at microscopy as well as misclassification of nditions . MicroscP. ovale parasites while at the same time assessing the ability of microscopists to detect and identify Plasmodium species. In spite of its limitations , this study assessing of P. ovale spp. is the first of its kind in Ethiopia and provides important information regarding the Plasmodium species spectrum and distribution in the country as the basis for larger scale surveys.The consequences of misdiagnosis of malaria are felt not only at health centres, but also at national levels. Individuals wrongly diagnosed with malaria are exposed to unnecessary side-effects of drugs, and the true causes are not recognized or treated. This scenario is likely to lead to prolonged and worsening illness with loss of income or productivity, and repeated visits to health providers . This stP. ovale wallikeri or P. ovale curtisi. On the one hand this study provides P. ovale malaria is under-reported and the first known evidence of the sympatric distribution of indigenous P. ovale wallikeri and P. ovale curtisi in northwest Ethiopia and is meant to provide the Ethiopian government and researchers with guidance regarding the presence of Plasmodium species other than P. falciparum and P. vivax. At the same time it highlights the importance of adequate training for health staff involved in malaria diagnosis and the inherent limitations of microscopic diagnosis.Despite of the high prevalence of malaria in Ethiopia and in the study areas, there had previously been no report of the presence of either The authors declare that they have no competing interests.AA and HN conceived the study. AA and HFP undertook statistical analysis, undertook molecular analysis of the samples and drafted the manuscript. GG and BT contributed in proposal writing, blood sample collection and microscopic diagnosis of malaria slides. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.


Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research  and in the Malaria & Emerging Parasitic Diseases Laboratory  using different nested PCR methods.The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.Microscopic re-examination in the IMR showed characteristics of P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.This is the first report of human Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.Simian The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization. Plasmodium species to humans was, until recently, thought to be rare [Plasmodium knowlesi infection was described [et al. [Plasmodium knowlesi infections, that simian malaria started to be considered as a concern for human public health.It has been asked whether animal malaria could be considered as true zoonoses. It has been known for some time that malaria of some non-human primates may infect man . The inf be rare . In 1965Plasmodium should not discourage consideration and investigation of them as zoonosis [Plasmodium cynomolgi[, Plasmodium brasilianum[Plasmodium eylesi, Plasmodium knowlesi[Plasmodium inui[Plasmodium schwetzi[Plasmodium simium[The absence of confirmed naturally acquired infections of other simian species of zoonosis . Since 1cynomolgi-10, Plasasilianum, Plasmod knowlesi,12, Plasdium inui, Plasmod schwetzi, and Plaum simium.Plasmodium cynomolgi, P. knowlesi, P. inui and P. eylesi are distributed in Asia, P. brasilianum and P. simium in the New World, while P. schwetzi is found in Africa [P. knowlesi, none of the other species has been found to infect humans in nature.n Africa . With thP. knowlesi infection may progress as a self-limiting malaria with spontaneous cure [Natural ous cure , althougous cure ,16-19, rous cure -24. The ous cure . Clinicaous cure .Plasmodium cynomolgi was first described by Mayer in 1907 in Germany in Macaca cynomolgus (= Macaca fascicularis) imported from Java [P. cynomolgi, by microscopy, are almost identical to that of Plasmodium vivax. The host red blood cell enlarges as the trophozoite grows, accompanied by prominence of Schüffner’s stippling and pigmentations in mature trophozoites. The cytoplasm becomes amoeboid and pigments in small granules with yellowish brown in colour are scattered throughout the cytoplasm. The asexual cycle is completed in 48 hours. The prepatent periods range from seven to 16 days with a mean of 9.8 days. Similarly to P. vivax, P. cynomolgi presents hypnozoites, which can initiate relapses [rom Java . The morrelapses .P. cynomolgi malaria in humans is described.In this report, the first known case of naturally acquired The patient was a 39-year-old Malay woman from the east coast of Peninsular Malaysia (Hulu Terengganu) with no previous history of malaria and an uneventful medical history. She lives in a modern housing area and behind her house there is a small, forested area with occasional sightings of long-tailed macaques. She works as a government nurse in a malaria-free area.Plasmodium malariae/P. knowlesi was concluded. The Giemsa-stained thick blood smears revealed 0.024% Plasmodium parasitaemia  with only asexual stages. She was given a three-day course of oral chloroquine , according to the Malaysian National Antibiotic Guidelines on management of malaria. The patient recovered and was discharged one week later . Microscopic examinations of her blood smears were performed weekly for the first month from discharge followed by monthly examinations for one year. All blood smears were negative.She experienced episodes of mild to moderate 24-hour cycles of morning fevers since 10 January, 2011 with chills and rigor, cough and cold. Four days prior, she visited her mother’s house, in a non-endemic malaria area  and did not travel to any other malaria-endemic areas. The symptoms were non-specific and mimicked a flu-like syndrome. She took two days of medical leave  without seeking hospital treatment. She felt more ill and was still febrile after two weeks and sought treatment at the Terengganu State Health Department. She was admitted to hospital on 20 January. A series of blood films for malaria parasites were taken and screened by microscopy and an initial diagnosis of Anopheles cracens was the predominant mosquito which is the vector of P. inui and P. cynomolgi[The Malaysian Vector Borne Disease Control Programme team did a case investigation among residents within the patient’s housing area and surveyed the area for possible vectors. No other persons were infected and anopheline mosquitoes caught were negative for malaria parasites, as determined by mosquito dissection. In this area cynomolgi.P. vivax rather than P. malariae/P. knowlesi, scanty trophozoites, larger than the size of Plasmodium falciparum, were visible in the “ghost” of red blood cells, with large chromatin dot and thick cytoplasm. Molecular species identification was performed by a nested PCR assay based on the small subunit (SSU) rRNA genes with primers specific for P. vivax, P. falciparum, P. malariae, Plasmodium ovale (Snounou-PCR) [P. knowlesi (Singh-PCR) [P. vivax infection yielding the expected size fragment using the specific-primer pair for P. vivax; while the rest of the species-specific nested PCR assays for P. malariae, P. falciparum and the Singh-PCR for P. knowlesi did not produce any amplifications.Patient blood samples were sent to the Institute for Medical Research  for further species confirmation by microscopy and molecular diagnostic methods. Microscopic re-examination of blood film showed characteristics of nou-PCR)  and primngh-PCR) . MoleculPlasmodium genus amplification (NG-PCR). The NM-PCR method is able to identify the four human malaria species  in two consecutive multiplexing amplifications, while the NG-PCR identifies all Plasmodium species. Primer sequences, concentration and annealing temperature to set up these methods are described in Table A DNA sample was sent to the Malaria & Emerging Parasitic Diseases Laboratory  as part of an informal quality control exchange. The sample was analysed by a modification of the nested multiplex malaria PCR (NM-PCR), also based on the small subunit (SSU) rRNA genes ,29, addiIn the MAPELAB molecular diagnosis by NM-PCR was negative for the four human malaria species and positive for NG-PCR showing an amplified fragment of expected size, confirming that the patient indeed had malaria infection. Sequencing of amplified products, using PLF and UNR primers from the NM-PCR assay, were performed after DNA purification  by Cycle Sequencing using Big Dye Terminator v3.1 in an ABI PRISM® 3700 DNA Analyzer. The sequence obtained has been submitted to the GenBank data base with the accession number JQ794445.P. cynomolgi sequences, 99.9% with P. cynomolgi Mulligan strain from Malaysia (Accession number AB287290), followed by similarities between 99.6 and 99.4% with three other P. cynomolgi SSU rRNA sequences transcribed during the asexual stages , thereby confirming that the patient was infected with P. cynomolgi. Furthermore, a phylogenetic tree was performed, with Treecon software [Plasmodium species. The phylogenetic tree shows that patient’s sequence formed a monophyletic clade with P. cynomolgi independent of the clade formed for P. vivax sequences, adding another evidence that patient was infected with P. cynomolgi , including a gap and a double nucleotide insertion, in the corresponding region of the P. cynomolgi sequence  including two gaps , most of them localized in the 5’-end primer region and just two in the 3’-end  and P. falciparum (30%) [P. knowlesi, morphologically indistinguishable from P. malariae, are frequent [Malaysia is a country endemic for malaria, particularly in the forested, hilly and underdeveloped interior areas of Malaysian Borneo (Sabah and Sarawak states) and Peninsular Malaysia. There is no risk in urban, suburban or coastal areas, including the east coast where the patient lives. Since the Malaria Eradication Programme started in the states of Sabah and Sarawak in 1961  and in Pum (30%) . In someP. cynomolgi, previous reports of infected volunteers with P. cynomolgi showed that fever had different presentations, from afebrile to daily or tertian fever [The symptoms of the patient were non-specific, she experienced episodes of mild to moderate 24-hour cycles of morning fevers with chills and rigor, cough and cold which mimicked a flu-like syndrome. These clinical symptoms were similar to those described in non-natural infection in humans and were usually present only during febrile episodes. These last experimental infections were performed with laboratory strains, which can be less virulent than natural strains . Althougan fever ,25.P. malariae/P. knowlesi was the final diagnosis in the hospital and patient was treated with chloroquine, the fever and symptoms resolved and did not show any complications and no relapse at the time of writing this report. Treatment with primaquine to eliminate hypnozoite forms, characteristic of P. vivax and P. cynomolgi, was not prescribed as patient was diagnosed as P. malariae/P. knowlesi. Infections produced in many volunteers by mosquito bite in the 1950s to 1970s with P. cynomolgi sporozoite showed symptoms of moderate severity which were easily controlled by simple medications or even without treatment [P. cynomolgi in humans are not activated to become hepatic schizonts, which suggests that a specific treatment with primaquine is not necessary to avoid relapses.Despite she lives in a malaria-free area, physicians suspected malaria because the patient experienced the characteristic cyclic fever with chills. A malaria infection by reatment . In thesP. cynomolgi malaria in humans.This report describes the first known case of naturally acquired P. cynomolgi is indistinguishable from P. vivax, and as shown in this report, one of the most used PCR for malaria infection can characterize those infections as P. vivax. Microscopic examination of peripheral blood by thick and thin film is the golden standard for malaria diagnosis and species identification. Despite the high PCR sensitivity and specificity to detect malaria parasites in the blood, it has not been established as a routine diagnostic method in reference central laboratories to correct misdiagnosis in species identification, as well as increasing sensitivity in cases of individuals with low parasitaemia or asymptomatic carriers. As shown in this report and others, misdiagnosis in species identification can be more frequent than expected, especially in relation to non-human malaria, such as P. knowlesi or P. cynomolgi[P. vivax, P. falciparum and P. knowlesi[Morphologically, cynomolgi-6,25, as knowlesi.Plasmodium species could routinely infect humans in Southeast Asia and the correct diagnosis could be missed since it is not possible by microscopy to accurately identify Plasmodium species with similar morphology, such as that observed between P. cynomolgi and P. vivax. The application of new diagnostic methods is necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.Simian The importance of zoonotic malaria transmissible by non-human primates cannot be ignored in view of increasing interactions between humans and the wild animals in the process of urbanization.Oral and written informed consent was obtained from the patient for publication of this case report and any accompanying images after explanation of the report objectives.Plasmodium genus PCR; NM-PCR: Nested multiplex malaria PCR; PCR: Polymerase chain reaction; SSU rRNA: Small subunit ribosomal ribonucleic acid.Bp: Basepairs; DNA: Deoxyribonucleic acid; IMR: Institute for medical research; MAPELAB: Malaria & emerging parasitic diseases laboratory; NG-PCR: Nested The authors declare that they have no competing interests.JMR supervised molecular characterization of parasite, conceived the study, its design and coordination, analysed the data and corrected the manuscript. THT carried out the molecular methods (NM-PCR and NG-PCR), analysed the sequence alignment, the data and drafted the manuscript. SH participated in the design and coordination of the study, and drafted the manuscript. AIJ and NI performed the patient DNA isolation, stained and examined the slides by microscopy and carried out the molecular methods (Snounou-PCR and Singh-PCR assays). ML was the responsible the sequencing and archive blood sample. All authors read and approved the final manuscript.


Plasmodium falciparum-infected red blood cells (iRBCs) have been implicated in antimalarial immunity. To profile the development of this immunity, we have been studying a cohort of Malian children living in an area with intense seasonal malaria transmission.Naturally-acquired antibody responses to antigens on the surface of We collected plasma from a sub-cohort of 176 Malian children aged 3-11 years, before (May) and after (December) the 2009 transmission season. To measure the effect of hemoglobin (Hb) type on antibody responses, we enrolled age-matched HbAA, HbAS and HbAC children. To quantify antibody recognition of iRBCs, we designed a high-throughput flow cytometry assay to rapidly test numerous plasma samples against multiple parasite strains. We evaluated antibody reactivity of each plasma sample to 3 laboratory-adapted parasite lines  and 4 short-term-cultured parasite isolates . 97% of children recognized ≥1 parasite strain and the proportion of IgG responders increased significantly during the transmission season for most parasite strains. Both strain-specific and strain-transcending IgG responses were detected, and varied by age, Hb type and parasite strain. In addition, the breadth of IgG responses to parasite strains increased with age in HbAA, but not in HbAS or HbAC, children.Our assay detects both strain-specific and strain-transcending IgG responses to iRBCs. The magnitude and breadth of these responses varied not only by age, but also by Hb type and parasite strain used. These findings indicate that studies of acquired humoral immunity should account for Hb type and test large numbers of diverse parasite strains. Plasmodium falciparum causes the most severe form of malaria, by some estimates placing more than 3 billion people at risk of disease and killing up to 1 million of them each year  have been variously associated with protection against  malaria -16, and  malaria . A recen malaria . It has  malaria . Specifi malaria -22. Othe malaria ,24. An a malaria -29. Immu malaria ,31.P. falciparum strain-specific agglutination responses, while Gambian adults showed cross-reactive responses  was kindly provided by Dr. Robin Anders . The minimal antibody detection level was 44 ELISA units; all responses below this level were assigned a value of 22 ELISA units.The standardized ELISA was previously described . The absMultiple nonparametric comparisons were made using a Kruskal-Wallis test; if significant differences were found, we used Dunn’s multiple comparison test in follow-up analyses. For each plasma sample, the proportion of iRBCs recognized by IgG (“% recognition”) was calculated. For each parasite strain, we determined a threshold value (mean + 2 standard deviations) for % recognition using data from 4 malaria-naïve American adult plasma samples tested in 5 independent assays . A child with a % recognition value greater than the threshold value was defined as a responder. The proportion of responders in the group of children tested was defined as “% responders.” Fisher’s exact test was used to compare the changes in % responders between pre- and post-transmission season samples. Qualitative data from multiple groups were analyzed using Chi-square likelihood ratio test; if significant differences were found, we calculated odds ratios (ORs) to determine which group was significantly different from the others. Nominal logistic fit was used to analyze data indicating whether or not malaria was experienced, and Proportional hazards fit was used to analyze data indicating the time to first malaria episode. For contingency analysis, Chi-square likelihood ratio test was used to compare 2 parasite strains; p-values were then Holm’s corrected and provided. For each parasite strain, % recognition values for pre- and post-transmission season samples were compared using Wilcoxon matched-pairs signed rank test. Statistical analyses were performed using GraphPad Prism 5  and JMP  statistical software. p-values <0.05 were deemed significant.P. falciparum parasitemia in a thick blood smear at enrollment. No child had detectable parasitemia at the end of the transmission season. The 176 children experienced a mean of 1.23 malaria episodes during the 7-month transmission season. Malaria incidence rates stratified by Hb type and age are shown in vs. 1.47, p=0.0004 for HbAS; 1.57 vs. 1.47, p=0.6769 for HbAC; Kruskal-Wallis test followed by Dunn’s multiple comparison test). Children aged 9-11 years experienced significantly fewer malaria episodes than children aged 6-8 years  or 3-5 years .We analyzed data from 176 Malian children aged 3-11 years who provided a plasma sample before and after the 2009 malaria transmission season. Of the 89 HbAA, 61 HbAS and 26 HbAC children, only 6 (HbAA), 4 (HbAS) and 2 (HbAC) were unmatched for age due to missing samples. Only 3 children  had asymptomatic In a multivariate regression analysis accounting for age and Hb type, we found that the odds of experiencing ≥1 malaria episode decreased significantly with age 2. HbAS,P. falciparum strain at one or both time points, no child’s IgM responded to any of the 7 parasite strains (data not shown).In optimizing the flow cytometry-based assay, we found that antibody-mediated agglutination of iRBCs was minimized in samples with parasitemia <1% and hematocrit <1%. Under these conditions, 1:10 and 1:20 dilutions of plasma produced better signal-to-noise ratios than 1:5 and 1:40 dilutions of plasma (data not shown). To use samples sparingly, all subsequent experiments were performed with 1:20 dilutions of plasma. In assays using sera from 4 malaria-naïve American adults, there was minimal assay-to-assay variation (measured by standard deviation) in the proportion of iRBCs recognized by IgG (“% recognition”) S2. GreaBefore the transmission season, 97.2% (171/176) of the children recognized ≥1 of 7 parasite strains. The frequency of responders ranged from 37% to 77% depending on the parasite strain tested 1. For 3a priori into 3 age groups: 3-5, 6-8 and 9-11 years. Before the transmission season, the % responders to 3 parasite strains  were significantly greater in children 9-11 years old than in those 3-5 years old   2. The %io test) 2. We alio test) 2.vs. 19% for KN1254, p=0.0185; 60% vs. 92% for KN1068, p=0.0008; 55% vs. 15% for CP803, p=0.0002; Chi-squared likelihood ratio test followed by an Odds Ratio test)   3. After=0.0042) 3. No su=0.0042) 3. No otvs. 3.5, p=0.0094, Mann-Whitney test) and after  the transmission season  of combinations tested 3. All sn season 4b, c.Next we investigated whether an IgG response to particular parasite strains before the transmission season correlated with subsequent malaria risk 5. AfterFinally, we measured correlations between the total number of parasite strains recognized by IgG and the IgG titers to 4 recombinant merozoite antigens . Before the transmission season, IgG titers to all 4 antigens were significantly (p<0.05) higher in groups of children recognizing 4, 5 or 6-7 parasite strains than in the group of children recognizing 0-2 parasite strains 5. IgG tAntibodies to iRBC surface antigens are a potentially important component of the immune response against erythrocytic-stage malaria parasites. Previous studies have clearly demonstrated that passive transfer of antibodies can alleviate morbidity and reduce parasite densities in patients with malaria . HoweverOur data suggest that our assay identifies both strain-specific and strain-transcending IgG responses to iRBCs. For example, while only 38% of parasite strain combinations tested before the transmission season showed significant pairwise correlations between IgG responses, this number increased to 62% after the transmission season. This finding, and the observation that IgG responses to parasite strains generally increased over the transmission season, suggests that Malian children actively develop strain-transcending IgG responses to iRBC surface antigens. The “strain-transcending” phenotype can be explained by either 1) the child produced antibody which can recognize multiple parasites, or 2) the child produced multiple strain-specific IgGs, or a mixture of both. Further study is required to answer this issue. This seasonal effect on IgG recognition of iRBCs is consistent with results from Giha et al., who reported an increase in serum agglutination of iRBCs over a transmission season in eastern Sudan . Other pOur assay also detected age effects on IgG recognition of iRBCs. Before the transmission season, IgG recognition of iRBCs  increased with age. However, this age effect was not observed after the transmission season, suggesting that younger children achieved IgG responses that approximated those of their older counterparts. The age effect observed before the transmission season may therefore be the result of waning IgG responses during the preceding dry season. The lack of a significant seasonal effect in older children is presumably due to plateauing of IgG responses to adult levels. Previous studies in Gambian, Sudanese, Nigerian and Tanzanian adults showed that antibodies can recognize and agglutinate diverse parasite strains from Ghana and Tanzania ,30,69. OP. falciparum antigens have only recently been investigated. Using a recombinant protein microarray, Tan et al. found that HbAS and HbAC were not associated with enhanced P. falciparum-specific IgG responses in Malian children [The effects of Hb type on IgG recognition of children . In the children . While wAs in other studies ,72-74, wvar genes switch during in vitro culture, makes it difficult to use these parasite isolates as reagents with stable phenotypes in future studies. Second, we have not identified the parasite antigens recognized by the IgG of children in our study. Recently, however, Chan et al. made a very compelling argument in favor of PfEMP1 being the major target of anti-VSA antibodies [Our study has several limitations. First, the Malian and Cambodian parasite isolates we used are not clonal (data not shown). This finding, and the fact that tibodies . Third, tibodies . Moreovetibodies . Despitetibodies . Since tFigure S1Gating strategy to assess IgG and IgM recognition of parasite-infected red blood cells (iRBCs).Representative gating strategy for iRBCs recognized by IgG or IgM. a, The population of RBCs and iRBCs was gated. b, Gate R1 contains iRBCs stained with the nucleic acid dye Syto 61. The 3 dot plots to the right represent R1-gated iRBC populations reacted with no plasma (c), plasma from malaria-naïve American adults (d), and plasma from malaria-exposed Malian adults (e). IgG and IgM reactivities are shown in the FL1 and FL2 channels, respectively. Corresponding IgG reactivities are shown in the adjacent histograms .(TIF)Click here for additional data file.Figure S2Assay-to-assay variation in % recognition values in both American and Malian adult plasma samples.The proportion of iRBCs recognized by IgG (“% recognition”) was measured by flow cytometry. Sera from 4 malaria-naïve American adults and plasma from 4 malaria-exposed Malian adults were tested against iRBCs infected with Cambodian P. falciparum isolates CP803 and CP806. Each dot represents a % recognition value measured on a separate day and the bar represents the mean.(TIF)Click here for additional data file.Figure S3Differences in % recognition values between paired plasma samples obtained before and after the 2009 transmission season.Paired plasma samples from Malian children were obtained before and after the 2009 transmission season and tested against 7 parasite strains. The proportion of iRBCs recognized by IgG (“% recognition”) was measured by flow cytometry. Tukey plots show the median difference in % recognition  for all 176 children. The dashed line represents 0 on the y-axis. **p<0.01, ***p<0.001 via Wilcoxon match -pairs signed rank test.(TIF)Click here for additional data file.


Plasmodium falciparum parasite density levels.Regulatory T cells (Tregs) are a subset of T cells that play an important role in modulating T effector responses during infectious challenges. The aim of this study was to evaluate possible associations between regulatory gene polymorphisms and the risk of uncomplicated malaria and the control of FOXP3 , IL10RA , IL10RB , STAT6  and TNFRSF18  were genotyped in a cohort of Congolese children. Studied subjects were followed up (passively) during one year. The children who experienced one or several clinical episodes were genotyped as “uncomplicated malaria” group (n=179) and those children who did not experience any episode were genotyped as “asymptomatic children” group (n=138).Twelve regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of rs3024944CC genotype of STAT6 was significantly higher in the group of asymptomatic children compared to that of uncomplicated malaria (P=0.003). Similarly, the minor allele rs3024944C was more prevalent in the group of asymptomatic children (P=0.019). Two novel SNPs were observed including -163T/G (ss491228441) in IL10RA gene and -163C/T (ss491228440) in TNFRSF18 gene. The genotype ss491228441TT and the minor allele ss491228441G of the IL10RA were more frequent in the group of asymptomatic children . The genotype rs11091253CT of the FOXP3 was associated with high parasite density levels. In addition, a new promoter IL10RA variant (ss491228441) contributes to shield against mild malaria.The prevalence of STAT6 promoter polymorphism rs3024944 was associated with uncomplicated malaria, whereas the FOXP3 promoter variant rs11091253 was associated with significant P. falciparum parasitaemia levels. These genetic data may contribute to the understanding of molecular mechanisms that regulate immune response to P. falciparum infections.The study indicated that the  Plasmodium and Plasmodium falciparum being the most virulent species. Plasmodium falciparum-infected individuals show different clinical phenotypes, which range from asymptomatic infections to severe forms of malaria [FOXP3), interleukin 10 receptors alpha and beta (IL10RA and IL10RB), the signal transducer and activator of transcription 6 (STAT6), and the tumour necrosis factor receptor superfamily member 18 (TNFRSF18) [Malaria is one of the major causes of morbidity and mortality in tropical and sub-tropical areas. Approximately 216 million clinical cases of malaria occurred worldwide in 2010, resulting in almost 655,000 deaths [ malaria . The gen malaria -8. A few malaria . ExpressNFRSF18) .FOXP3 belongs to the family of transcription factors that play a role in various cellular processes and is described as an important regulator of natural Tregs development and function [FOXP3 gene are known to be associated with immune dysregulation polyendocrinophathy and enterophathy X-linked syndrome (IPEX) caused by lack of Tregs [IL10RA and IL10RB are two receptor chains forming the human interleukin 10-receptor (IL10R) which belongs to the class II cytokine receptors [IL10RA precursor is to mediate high affinity and ligand binding and subsequent signal transduction, whereas the IL10RB precursor is believed to contribute only in signalling process [IL10RA and IL10RB precursors of the IL10R annul the IL10 mediated immunomodulatory signalling and are strongly associated with inflammatory bowel disorders [IL10RA and IL10RB genes have been shown to be associated with protection against severe malaria in Gabonese children [STAT6 gene, which belongs to the STAT family of transcription factors, has been reported to mediate various cytokine-induced responses. Recent reports have shown that STAT6 gene polymorphisms play crucial roles in controlling helminth infection. More precisely, a promoter variant rs324013 was found to be associated with the control of Schistosoma haematobium in a Dogon population (where S. haematobium is endemic) in Mali [Ascaris lumbricoides[TNFRSF18, also known as glucocorticoid-induced TNF receptor, is a member of TNF superfamily, which plays a key role in the co-stimulatory process for T cell activation for both conventional and CD25+CD4+ T cells. In addition, TNFRSF18 expressed on Tregs was demonstrated to play a crucial role in the maintenance of peripheral tolerance [FOXP3, IL10RA, IL10RB, STAT6 and TNFRSF18 may lead to various immunopathological reactions in auto-immune disorders and/or parasitic infections.function -12. In hof Tregs . IL10RA eceptors . The maj process . Reportsisorders . Recentlchildren . The STA in Mali . Similarbricoides. TNFRSF1olerance . TherefoFOXP3, IL10RA, IL10RB, STAT6 and TNFRSF18 genes were identified in the Gabonese population in Central Africa. These identified SNPs were further validated for their allelic gene expression using transient transfection assays [P. falciparum malaria was investigated. For this purpose, this study utilized a case-control cohort of Congolese children, who were followed up (passively) during one year for surveillance of clinical malaria cases, comprising a group of children who experienced one or several uncomplicated malaria episodes and that of children who had no clinical episode during the follow up. As a second objective, the impact of these SNPs carriage on the P.falciparum parasite density levels was assessed.In a previous investigation, regulatory SNPs in the promoter region of n assays ,19-21. AP. falciparum being the predominant species and Anopheles gambiae the main mosquito vector [et al, unpublished), is a community-based case-control study. The study population consisted of children aged one to nine years permanently living in the study area. All the study individuals belong to Bantu ethnicity and had equivalent risk for infection. Children were recruited between April and June 2010 and passively followed up during one year. At the time of enrolment 4 ml of whole blood were collected from all children and a thick and thin blood smears were performed. The inclusion criteria were: absence of clinical malaria in the previous two weeks and one week after enrolment , negative thick blood smear or positive blood smear with less than 5,000 parasites per microlitre of blood (p/μl), and not being homozygous for sickle cell trait. During the follow up, at any time, when a child was suspected to have fever or any malaria-related symptoms, parents or guardians were invited to present the child at the health centre for clinical examination. In case of malaria (positive thick and thin blood smears and axillary temperature ≥37.5°C), blood sample was collected, parasite density determined and children were treated with artesunate-amodiaquine or artemether-lumefantrine. No data were available about the present or past history of helminth infection.The study was carried out in three districts  of Makélékélé health division in the southern area of Brazzaville. These districts are semi-urban with about 6,000 inhabitants and located along the Congo River. Malaria is transmitted throughout the year with o vector . This stThe ethical approval was given by the Institutional Ethics Committee for Research on Health Sciences of the Republic of Congo. Written informed consent was obtained from parents or guardians of children.Plasmodium species. Asexual parasites were counted against 200 leucocytes and expressed as the number of asexual parasites per μl, assuming the leucocytes count of 8,000/μl of blood.Thick and thin blood films were stained with 10% Giemsa for 15 min and read by two independent, competent microscopists to determine the parasite density and Genomic DNA was extracted from 200 μl of peripheral whole blood samples obtained from patients and asymptomatic children using the commercially available QIAamp DNA Blood Mini Kit  following the manufacturer’s instructions and stored at -20°C until use.-794C/G (ss270137548) and -738C/T (rs11091253), for the FOXP3 gene; -185C/T (rs56356146), and -116T/C (rs7925112) for the IL10RA gene; -978T/G (rs8178433), -754A/G (rs8178435), and -740C/T (rs999788) for the IL10RB gene; -3728C/G (rs3024941), -3430G/A (rs3024943), and -3387G/C (rs3024944) for the STAT6 gene; and -265C/T (ss2080581728), and -199C/T (rs3753344) for the TNFRSF18 gene. Polymerase chain reaction (PCR) amplifications were carried out in 20 μl reaction volumes with 5 ng of genomic DNA, 1X PCR buffer , 0.2 mM of dNTPs, 0.5 mM of each primer and 1 U of Taq DNA polymerase . Thermal cycling parameters are mentioned in Table The promoter region of each of the five targeted genes in all the samples was genotyped for the promoter SNP variants that revealed altered gene expression as reported in earlier investigations. Those SNPs are: C/G ss27037548 andC/G ss27037548 andet al, unpublished). Those data included number of malaria attacks or episodes, parasite density and axillary temperature values.In this study, children without any reported malaria episode during the follow-up period were considered as asymptomatic and were classified in a group as asymptomatic children (as they could possibly be parasite carriers). Similarly, those who experienced at least one clinical malaria attack were classified in another group as uncomplicated malaria. Clinical and parasitological data for each child who had uncomplicated malaria were obtained from the longitudinal cohort study for malaria surveillance  to determine the differences in allele frequencies and genotype distributions between uncomplicated malaria and asymptomatic children adjusted for age and gender. The level of significance was set at P<0.05. In addition, genetic models such as allelic, dominant and recessive were examined to evaluate the contributions of the genotype and allele frequencies using STATA. Kruskal-Wallis tests were employed to analyse the relationship between genotype distributions and the number of episodes as well as the risk of parasite density levels in the group of children with uncomplicated malaria. Genotype or haplotype frequencies were determined by simple gene counting and by using the expectation-maximum (EM) algorithm. The significance of deviation from Hardy-Weinberg equilibrium was tested using the random-permutation procedure as implemented in the Arlequin v. 3.5.1.2 software . Linkageet al, personal communication), samples from 317 children were available for the present study. All the 317 children were successfully followed up during one year, and among them, 179 had at least one clinical malaria episode  whereas 138 did not experience any clinical malaria (forming the group of asymptomatic children). A total of 297 episodes were recorded without any case of recrudescence. No case of severe malaria was recorded and P. falciparum was the only Plasmodium species detected in children with uncomplicated malaria. Characteristics of recruited children are summarized in Table Of the 323 children enrolled in the cohort study for malaria surveillance (Ndounga IL10RA at the position -163T/G (ss491228441) and at the position -163C/T (ss491228440) in the promoter region of TNFRSF18 gene, respectively. The identified novel SNPS were submitted to the SNPper database and appropriate SNP #ss IDs were received. The new SNP IDs for -163T/G of IL10RA and -163C/T variants are rs199832262 and rs150621809 respectively. Association analysis revealed that genotype rs3024944CC of STAT6 gene was observed more in children who did not have clinical malaria episode during the follow up (asymptomatic group) compared to uncomplicated malaria group . The minor allele rs3024944C of STAT6 gene was associated with decreased risk of clinical uncomplicated malaria . Interestingly, the heterozygous genotype ss491228441TG of the IL10RA was also found to be more frequent in asymptomatic children and the homozygous ss491228441TT genotypes were observed to be a risk factor . Also it was revealed that the minor allele ss491228441G of the IL10RA gene, despite it low overall frequency in Congolese children, was significantly more frequent in asymptomatic children compared to uncomplicated malaria group . The other regulatory polymorphisms in the FOXP3, IL10RB and TNFRSF18 loci do not reveal any significant associations either at allele, genotype or at haplotype levels.All the analysed SNPs did not deviate from Hardy-Weinberg equilibrium in each group. Genotype distributions and allele frequencies of these SNP variants are summarized in Table STAT6 (rs3024941/rs30240943/rs3024944) and IL10RA (rs56356146/rs7925112/ss491228441) were observed as a susceptibility factor between both studied groups and are presented in Table STAT6 locus, of which the major haplotype CGG was marginally significant with more prevalence among children who presented at least one clinical episode  . Similarly, five haplotypes were observed for the IL10RA, and the minor haplotype CTG was found to be associated with protection against the disease . Different haplotypes of the other genes were not found to be significantly distributed between both groups of children. The linkage disequilibrium pattern of studied loci in promoter regions of STAT6 and IL10RA are presented in Figure The reconstructed haplotypes of FOXP3 rs11091253 variant and P. falciparum parasitaemia. More precisely, patients carrying the rs11091253CT genotype harboured higher parasite densities compared to those carrying the rs11091253CC or rs11091253TT genotype   showed an association between the P. falciparum infection. Therefore, investigation of human polymorphisms in genes associated with Treg activity may underlie both susceptibility to infection and level of Treg expression.Malaria has exerted selective pressure on the human genome that has led to changes, such as haemoglobin S which confers some resistance against severe malaria . UnderstFOXP3, IL10RA, IL10RB, STAT6 and TNFRSF18 genes in P. falciparum infection outcome among Congolese children. Earlier studies have identified some regulatory SNPs on the promoter loci of these five genes from 40 unrelated Gabonese individuals who had earlier infection episodes with various parasites, including P. falciparum[P. falciparum infection levels were evaluated.The present study evaluated the contribution of promoter SNPs of alciparum,19-21. Talciparum,19-21. TSTAT6 rs3024944C/C genotype was significantly more prevalent in asymptomatic children. This suggests the contribution of this genotype to protection of Congolese children against mild clinical malaria. The SNP rs3024944 is located in a transcription factor-binding site TCF-1 which is a member of family of genes with homology to high mobility group I (HMG) proteins [rs3024944C have low parasite densities compared to those having the major allele rs3024944G, that speculates that the rs3024944C/C variant directs activation of T cells in response to P. falciparum infection, thus contributing to protection against the disease through parasite elimination. In haplotype level, the major haplotype CGG was found to be correlated with the marginal risk of uncomplicated malaria. This emphasizes the role of the SNP rs3024944 on susceptibly/resistance to uncomplicated malaria among Congolese children. This is the first study that shows the correlation between a promoter SNP STAT6 rs3024944 and protection against uncomplicated malaria. However, in previous investigations, the promoter SNP rs324013 was found to be associated with the control of S. haematobium infection levels in a Dogon population in Mali [A. lumbricoides infection levels in Chinese children [The results obtained in this study demonstrated that the proteins ,26. And  in Mali . Similarchildren .IL10RA and TNFRSF18 respectively. In both cases the frequency of the minor allele was low. Meanwhile the minor allele ss491228441G of IL10RA appeared to contribute to protection against uncomplicated malaria. Interestingly, the haplotype CTG involving this minor allele ss491228441G was significantly more present in asymptomatic children. This provides an additional evidence of the involvement of this allele in protection against uncomplicated clinical malaria. However, larger association studies in different malaria endemic areas including children and adults as well as functional studies of this ss491228441 SNP variant would be of great interest to verify the current observations.Two novel promoter SNPs were identified including, ss491228441 and ss491228440 of FOXP3 rs11091253 SNP variant and P. falciparum parasite density levels. In particular, the rs11091253CT genotype was found to be associated with high infection levels, whereas the homozygous for the minor allele, the rs11091253TT genotype, was associated with lower infection levels. This suggests that FOXP3 contributes to the modulation of the immune response against P. falciparum infection. The SNP rs11091253 is located in a putative binding site for the transcription factors nuclear factor kappa B (NF-κB) and c-Rel, which is also a member of NF-κB family. These transcription factors have been demonstrated to be critical for regulation and transcription of FOXP3[rs11091253CT genotype and that of the promoter carrying the rs11091253TT genotype may explain the associations observed in this study. One possible explanation may be that the binding of NF-κB and/or c-Rel with the promoter carrying the rs11091253C/T genotype results in upregulation of FOXP3 which in turn leads to increased number of fully functional Tregs. Consequently, parasite multiplication is facilitated by suppression of either P. falciparum-responsive effector cells or pro-inflammatory cytokine response. This is in line with what was reported by previous study showing that upregulation of FOXP3, and CD4+CD25+ regulatory cells correlates with more rapid parasite growth in human malaria infection [The data also showed the association between the carriage of the  of FOXP3,28. Thernfection .FOXP3, IL10RA, IL10RB, STAT6 and TNFRSF18 genes have been investigated for possible association with uncomplicated malaria and high P. falciparum parasite density among Congolese children. These findings indicate that the STAT6 promoter polymorphism rs3024944 is highly associated with protection against uncomplicated malaria, whereas the FOXP3 promoter variant rs11091253 is more likely to be involved in the control of P. falciparum parasite density levels. In addition, a new SNP detected in the promoter region of IL10RA (ss491228441) contributes to protection against clinical malaria. These findings deserve to be further investigated in a larger population of children in the Republic of Congo but also in other malaria endemic areas.To summarize, selected regulatory SNPs in the promoter region of The authors declare that they have no competing interest.MN designed and performed the field study. VTP designed and supervised the experiments. FKK performed the experiments. FKK, HVT, and VTP analysed data. VTP, FN, and AAA contributed materials/analysis tools. FKK, FN, and VTP wrote the paper. All authors read and approved the final manuscript.