Salmonella (iNTS) infections are commonly associated with Plasmodium falciparum infections, but the immunologic basis for this linkage is poorly understood. We hypothesized that P. falciparum infection compromises the humoral and cellular immunity of the host to NTS, which increases the susceptibility of the host to iNTS infection. We prospectively recruited children aged between 6 and 60 months at a Community Health Centre in Blantyre, Malawi, and allocated them to the following groups; febrile with uncomplicated malaria, febrile malaria negative, and nonfebrile malaria negative. Levels of Salmonella enterica serovar Typhimurium-specific serum bactericidal activity (SBA) and whole-blood bactericidal activity (WBBA), complement C3 deposition, and neutrophil respiratory burst activity (NRBA) were measured. Levels of SBA with respect to S. Typhimurium were reduced in febrile P. falciparum-infected children  compared to nonfebrile malaria-negative children . In relation to SBA, C3 deposition on S. Typhimurium was significantly reduced in febrile P. falciparum-infected children  compared to nonfebrile malaria-negative children . WBBA with respect to S. Typhimurium was significantly reduced in febrile P. falciparum-infected children  compared to nonfebrile malaria-negative children . In relation to WBBA, S. Typhimurium-specific NRBA was reduced in febrile P. falciparum-infected children  compared to nonfebrile malaria-negative children . P. falciparum infection impairs humoral and cellular immunity to S. Typhimurium in children during malaria episodes, which may explain the increased risk of iNTS observed in children from settings of malaria endemicity. The mechanisms underlying humoral immunity impairment are incompletely understood and should be explored further.Invasive nontyphoidal  Salmonella (iNTS) infections, principally by Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis, are estimated to cause over 2.1 million illnesses and 416,000 deaths per year  antibodies to NTS targeting lipopolysaccharide (LPS) are thought to confer some protection against NTS bacteremia in African children , 25, 26.S. Typhimurium correlates with the decline in iNTS infections in childhood in individuals not infected with P. falciparum  compared to nonfebrile malaria-negative children   and at day 14 in convalescence  compared to febrile malaria-negative children   was similar to that seen with febrile malaria-negative children  and nonfebrile malaria-negative children  (S. Typhimurium (ability to kill S. Typhimurium by at least a −1.0 log10 change in S. Typhimurium CFU counts per milliliter) in the acute malaria phase compared to 16/23 (69.5%) at day 30 in convalescence  attained robust SBA with respect to S. Typhimurium at day 30 in convalescence.We have previously shown that acquisition of serum bactericidal activity (SBA) with respect to lciparum , 25. The= 0.052) . SBA wit −1.24]) . SBA wit = 0.39) . Furtherlescence . We alsoS. Typhimurium correlates with age in healthy children   (We have previously shown that acquisition of SBA with respect to children . We founctively)  and B. Ihimurium ) and SBA= 0.39])  to E.S. Typhimurium is mainly mediated by anti-S. Typhimurium IgG antibodies targeting LPS  whereas SBA in febrile nonmalarial children correlated with anti-S. Typhimurium LPS IgG antibody titers   whereas the correlation of SBA with anti-S. Typhimurium LPS IgG antibody titers was superior at day 14 and day 30 in convalescence; however, this was statistically significant only at day 14    and B. Ictively)  to E. Thn = 10) from children (>24 months old) to examine levels of complement C3 and C5b-9 deposition during malaria  than in febrile malaria-negative children  and nonfebrile malaria-negative children   than at day 30 of convalescence  and that the level was similar at day 14 in convalescence    and E. C= 0.113)  and D.S. Typhimurium was significantly lower in febrile P. falciparum-infected children  than in febrile malaria-negative children  but was not significantly different from that seen in nonfebrile malaria-negative children . There were no significant differences in C5b-9 deposition on S. Typhimurium in febrile P. falciparum-infected children  from the levels seen at day 14 in convalescence  and at day 30 in convalescence  (C5b-9 deposition on = 0.084)  and F. TS. Typhimurium is a facultative intracellular organism that requires the action of both cellular immunity and humoral immunity to be effectively controlled. We therefore examined if levels of whole-blood killing of S. Typhimurium were also reduced during P. falciparum infection. We found that whole-blood bactericidal activity (WBBA) with respect to S. Typhimurium was reduced during malaria at the acute stage , day 14 in convalescence , and day 30 in convalescence  and in febrile malaria-negative children  compared to nonfebrile malaria-negative children  ( −0.16]) .S. Typhimurium  and also in febrile malaria-negative children  compared to nonfebrile malaria-negative children   and day 30  in convalescence compared to the acute malaria phase   as it is a key mechanism for intracellular pathogen killing. We found that NRBA was significantly reduced in children during acute malaria  . We obse.7, 20])  but thatP. falciparum infection compromises phagocyte-dependent immunity to NTS in children  and day 30 (n = 41) during convalescence. The uncomplicated-malaria group was comprised of children with acute-phase P. falciparum infection who presented to the hospital for medical care; they were febrile (>37.8°C) at the time of recruitment, had positive malaria rapid diagnostic test and positive malaria blood film results, a Blantyre coma score of 5  assays were performed as previously described . Brieflym D23580 , adjusteS. Typhimurium adjusted to 1.0 × 107 CFU/ml was first opsonized with 1:10 serum from each participating child for 20 min at room temperature (RT) before challenging washed blood cells in a washed-blood-cell and serum-opsonized assay (WBCSOA). All conditions were challenged with S. Typhimurium adjusted to a final concentration of 1.0 × 107 CFU/ml. Colony counts were performed as described for the SBA experiment above.Three conditions were prepared as previously described . For conS. Typhimurium LPS antigen . Plates were washed with PBS containing 0.05% Tween 20 and blocked with 200 μl/well blocking buffer  for 1 h at 37°C. Test serum prepared at 1:20 in dilution buffer (PBS–0.05% Tween 20–1% BSA) was serially diluted 3-fold and incubated at 37°C for 1 h. After washing, 100 μl of 1:2,000 secondary goat anti-human IgG-AP antibodies (Southern Biotech) was added and incubated for 1 h at 37°C. Finally, after washing, 100 μl of SigmaFAST p-nitrophenyl phosphate substrate was added to each plate and the plate was read after 30 min using a BioTek ELx800 reader  at 405 nm.These experiments were performed as previously described . BrieflyS. Typhimurium was gently mixed at 2 × 108 CFU/ml with 45 μl of undiluted serum or PBS (control) at RT for 20 min. Samples were washed twice with 1 ml PBS by spinning for 5 min at 3,300 × g. A 2-μl volume of anti-C3c fluorescein isothiocyanate (FITC)-conjugated antibody (Abcam) was added to 50 μl of pellet to measure C3 deposition. A 1-μl volume of anti-C5b-9 neo-epitope antibody (Abcam) and 2 μl of rabbit anti-mouse FITC-conjugated antibody (Abcam) were added to 50 μl of pellet to measure MAC levels. Samples were washed twice with 1 ml of PBS after 20 min of incubation at RT and fixed with 200 μl of 1% formaldehyde–PBS. Samples were acquired on a CyAN ADP flow cytometer (Beckman Coulter) and analyzed using Flow Jo version 7.6.5.These experiments were performed as previously described , 28. A 5S. Typhimurium at 1.0 × 108 CFU/ml or with a wash solution containing instalmed-salts (control). Samples were then incubated for 10 min at 37°C to allow phagocytosis followed by 10 min of incubation at 37°C after addition of dihydrorhodamine 123 to promote oxidation. The reaction was stopped by application of 1:10 lysing solution for 20 min at RT. Samples were acquired on CyAN ADP flow cytometer (Beckman Coulter) and analyzed using Flow Jo version 7.6.5.A Phagoburst test kit (Glycotope Biotechnology) was modified to measure neutrophil respiratory burst levels as previously described . Whole bS. Typhimurium, percentage of neutrophil respiratory burst-positive cells, and complement deposition were examined for normality of distribution using the D'Agostino and Pearson omnibus normality test. Nonparametric data were compared using the Mann-Whitney U test or the Wilcoxon signed-rank test for paired t tests. Median and interquartile range (IQR) were reported, and a P value of less than 0.05 was considered statistically significant. Spearman's correlation coefficient r was used to determine relationships between bactericidal activity and age and anti-S. Typhimurium LPS IgG antibody titers during malaria.Statistical analyses were performed using GraphPad Prism version 5 . Log10 change in bactericidal activity with respect to


Plasmodium falciparum infection and can cause death especially in children under 4 years of age. RNASE3 (ECP) has been identified as an inhibitor of Plasmodium parasites growth in vitro, and genetic analysis in hospitalized Ghanaian subjects has revealed the RNASE3 +371G/C (rs2073342) polymorphism as a susceptibility factor for cerebral malaria. The +371 C allele results in an Arg/Thr mutation that abolishes the cytotoxic activity of the ECP protein. The present study aims to investigate RNASE3 gene polymorphisms and their putative link to severe malaria in a malaria cohort from Senegal.Severe forms of malaria (SM) are an outcome of p = 0.003, OR 0.43, CI 95% 0.20–0.92). The polymorphisms: +371G/C (rs2073342), +499G/C (rs2233860) and +577A/T (rs8019343) defined a haplotype risk (G-G-T) for malaria severity  .Patients enrolled from hospitals were classified as having either uncomplicated (UM) or severe malaria (SM). The analysis of the RNASE3 gene polymorphisms was performed in 241 subjects: 178 falciparum infected  and 63 non-infected subjects as population control group (CTR). Six frequent SNPs (MAF > 3%) were identified, and one SNP was associated with malaria severity by performing a logistic regression analysis SM vs.UM: RNASE3 +499G/C (rs2233860) under age, sex as covariates and HbS/HbC polymorphisms adjustment  in malaria severity. Malaria is a disease that threatens more than one billion people worldwide and causes hundreds of thousands of deaths each year. A substantial reduction in morbidity and mortality occurred in endemic areas between 2000 and 2015 . The mecMost genetic studies have analysed immune factors implicated in inflammation, sequestration of parasite or vascular occlusion. For most factors studied, an association with SM was not clearly identified or conflicting results were reported . RibonucPlasmodium falciparum growth in vitro [Patients with SM display hypereosinophilia and high levels of RNASE 3 . Moreovein vitro . These oin vitro . More spIn this work, RNASE3 gene polymorphisms was analysed in malaria patients from Senegal (West Africa). RNASE3 gene and its flanking regions were sequenced in samples from a cohort of urban individuals including healthy controls, uncomplicated malaria (UM) and SM subjects. Genetic variations, including single nucleotide polymorphisms (SNPs), were analysed among individuals, with respect to disease status in order to detect and confirm associations with malaria severity.Plasmodium-positive Quantitative Buffy Coat (QBC), a test which is more sensitive than Giemsa-stained thick films [P. falciparum infection. Severe anaemia and cerebral form are the two most “severe form” currently explored, but other complications such as hypoglycaemia, thrombocytopaenia, renal insufficiency, hepatic or even pulmonary oedema may appear alone or in combination. Exclusion criteria included a travel, out of Senegal, in the 3 months prior to hospital admission; and display symptoms that might interfere with the study, such as recent pregnancy or childbirth or prior use of an anti-malarial treatment. The healthy control subjects corresponded to the exposed and uninfected subjects group in the same areas and belonging to Wolof ethnic group. A signed informed consent form was obtained from adult participants and parents or guardians of children involved in the study prior to blood sampling.The study population consisted of black Senegalese-born individuals whose parents and grandparents were born in Senegal and belonging to Wolof ethnic group. Malaria patients were enrolled from participating hospitals and corresponded to subjects with ck films . The malck films . In brieA total of 63 CTR, 82 UM and 96 SM subjects were included in the study Table . This st®Green Master Mix  in a total volume of 25 µl containing 25 ng of genomic DNA (5 ng/µl) and 2.5 µl of each primer (10 μM). The PCR conditions were initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 62 °C for 45 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The amplicons were purified using BioGel P100 gels (Bio-Rad). Sequencing reactions (2 µl of PCR product) were performed using the dye terminator v3.1 method in an ABI PRISMs 3730 DNA Analyzer . Sequencing conditions were: 96 °C for 5 min, 25 cycles of 96 °C for 10 s; 60 °C for 4 min and 15 °C forever, and PCR products were purified with Sephadex G50 superfine columns . Alignment of acquired sequences and SNP discovery were performed using NC_000014.9 as a reference. Analysis was performed with Genalys version 2.0b software [DNA was extracted from whole blood using a Qiagen kit (QIAmp Kit Cat. n°51306) according to the manufacturer’s recommendations. The RNASE3 gene and its flanking sequences were amplified using the primers listed in Table Thesias program [Statistical analysis was performed to evaluate the association between malaria status and RNASE3 polymorphisms. For quality control of association analysis, we excluded the SNPs under the Hardy–Weinberg Equilibrium or with low frequency (MAF < 3%). Differences in allele frequencies among the three groups  were examined using logistic regression analysis. Linkage disequilibrium (LD) was computed for each pair of polymorphisms within the RNASE3 gene using Haploview software . Haploty program . NominalIn the present study, the polymorphism of the RNASE3 gene and its flanking sequences were analysed in a population of 241 Senegalese, including 96 SM, 82 UM and 63 CTR individuals. Six SNPs were identified including: 3 SNPs upstream the unique coding exon 2: RNASE3 –550G/A (rs2284954), RNASE3 –399T/C (rs147413155) and RNASE3 –38C/A (rs2233859); one SNP located within exon 2: RNASE3 +371G/C (rs2073342); and two located in the 3′UTR: RNASE3 +499G/C (rs2233860) and RNASE3 +577A/T (rs8019343)    and p = 0.05  in additive mode, respectively. The +499G/C polymorphism yielded a significant association with SM. For SM vs.UM, the +499G/C exhibit positives signals with the p values p = 0.003  and p = 0.001  in additive and dominant modes respectively. Considering both UM and CTR as unique control groups, the statistical comparison remain significant for +499G/C SNP with a p value p = 0.001 and p = 0.004  in additive and dominant modes, respectively. The haplotypes were estimated using Thesias program. The triplotype estimation from 371G/C-499G/C-577A/T polymorphisms yielded a G-G-T positive signal with p values p = 0.03 when comparing SM vs. Controls (CTR +UM)   using the Mann–Whitney test yielded significant association with parasitaemia levels with 007 Fig. . The higIn this study, an analysis of the RNASE3 (ECP) gene were performed, in order to identify polymorphisms associated with severe malaria in the Senegalese population. Six SNPs, with a MAF greater than 3%, were founded, all of which had been previously described and referred to dbSNP number . Then the interesting findings in this study relate to the two polymorphisms SNPs RNASE3 +371G/C and RNASE3 +499G/C founded to be in strong LD , which induces an Arg/Thr shift. In the other hand, a positive signal was observed with the +499G/C polymorphism located in 3′UTR by comparing SM vs. UM or vs. CTR groups. Additionally, genotypic distribution of RNASE3 +499G/C was then detailed and the +499GC and +499CC genotypes showed significant associations with severity Table . FinallyIn a previous study from Adu et al.  in GhanaThe biological significance of +371G/C polymorphism association, in the particular context of malaria pathogenesis were explained by the potential new glycosylation site of Arg/Thr shift, affecting the cytotoxicity of ECP, and the adhesion of eosinophils to intracellular molecules such as ICAM-1 and to post-capillary vein obstruction during cerebral stages , 17–20.Schistosomiasis, hepatic fibrosis and asthma [Furthermore, a genetic association of the +371G/C polymorphism was assessed in relation to other diseases, such as d asthma , 22, in d asthma , 23.The present study reveals, for the first time, an association of +499G/C transversion with severe malaria. The detailed genotypic distribution of +499G/C showed that +499C allele is a susceptibility factor to malaria severity. Surprisingly, the +499G/C polymorphism was not associated to cerebral malaria in Ghanaian population study , althougTaking into account these results on SNP +499G/C, correlations were conducted to detect association of genotypes with parameters such as parasitaemia, and biological factors . Significant correlations were identified between +499CC genotype with a weak parasitaemia, suggesting a protective effect of 499C allele.G-G-T haplotype for malaria severity.Finally, SNPs in the RNASE 3 gene are commonly described , and mos


There are no data on the burden of malaria in pregnancy (MiP) in Laos, where malaria still remains prevalent in the south.Two cross-sectional surveys were conducted in 2014 to assess the prevalence of MiP in Vapi District, Salavan Province, southern Laos: the first consisted of screening 204 pregnant women during pregnancies [mean (95 % CI) gestational age: 23 (22–25) weeks] living in 30 randomly selected villages in Vapi District; the second was conducted among 331 pregnant women, who delivered during the study period in Vapi and Toumlane District Hospitals and in Salavan Provincial Hospital. Peripheral and placental malaria was detected using rapid diagnostic tests (RDT), thick blood smears (TBS) and real-time quantitative polymerase chain reactions (RT-qPCR). Factors associated with low birth weight (LBW) and maternal anaemia were assessed.Plasmodium vivax infections and 1 was mixed Plasmodium vivax/Plasmodium falciparum infection, among which 9 were sub-microscopic (as not detected by TBS). History of malaria during current pregnancy tended to be associated with a higher risk of MiP . At delivery, two Plasmodium falciparum sub-microscopic infections  were detected  in Vapi District. In both surveys, all infected women stated they had slept under a bed net the night before the survey, and 86 % went to the forest for food-finding 1 week before the survey in median. The majority of infections (94 %) were asymptomatic and half of them were associated with anaemia. Overall, 24 % of women had LBW newborns. Factors associated with a higher risk of LBW were tobacco use  and pre-term delivery . Factors associated with a higher risk of maternal anaemia were no iron supplementation during pregnancy, Lao Theung ethnicity and place of living.In the villages, 12/204 women  were infected with malaria as determined by RT-qPCR: 11 were The prevalence of MiP in this population was noticeable. Most infections were asymptomatic and sub-microscopic vivax malaria, which raises the question of reliability of recommended national strategies for the screening and prevention of MiP in Laos.The online version of this article (doi:10.1186/s12936-016-1492-2) contains supplementary material, which is available to authorized users. In 2007, an estimated 125 million women became pregnant in areas of malaria transmission. Of them 62 %, corresponding to 77 million women, were living in the Asia–Pacific region where malaria transmission is low or unstable . A singlIn Laos, malaria is endemic throughout most of the country, but intensity of transmission is variable, with more intense transmission in remote and forested areas particularly in the south . The disIn the context of persistent malaria transmission in southern Laos, the prevalence of MiP, including asymptomatic and sub-microscopic infection, and its related morbidity in this population were assessed. Epidemiological methods that minimize selection bias as well as highly sensitive methods of detection for malaria were used.The study was conducted in Salavan Province, which is one of the five southern provinces where the reported number of clinical malaria cases has been the highest since 2011 . In this area, malaria is perennial with one peak during the rainy season, which occurs between May and October.Two cross-sectional surveys were carried out simultaneously. One was conducted at facility level to investigate MiP at delivery, at Salavan Provincial Hospital , which is a primary-secondary hospital, and in Vapi  and Toumlane  District Hospitals, which are located in more rural areas  from July to October 2014. Both surveys were part of a larger programme , which aimed to assess the prevalence of asymptomatic and sub-microscopic malarial infections in the general population, including pregnant women, adults and children. This programme was led by the CMPE in Laos, in collaboration with the Salavan Provincial Department of Health, the Research Institute for Development (IRD) and the Lao-Oxford-Mahosot Hospital Wellcome Trust Research Unit (LOMWRU).®)] were determined.At facility level, all women who delivered in one of the three participating maternity clinics between August and October 2014 were included after providing a written informed consent. Women were excluded from the study if they had abortion, complicated/at risk pregnancy requiring a pre-labour emergency or elective caesarean section. The following information was recorded on admission by specifically trained midwives: maternal characteristics , obstetrical history , clinical data related to the current pregnancy , and working in the forest during the current pregnancy. At delivery, gestational age, blood pressure, temperature, and delivery characteristics were recorded. Newborn anthropometric data , gender, Apgar score and congenital abnormalities were recorded. Newborn weight was measured within 1 h after birth using an electronic digital scale with an accuracy of 100 g (SECA Ltd). In addition, maternal peripheral, placental and umbilical cord blood samples were drawn for malaria diagnosis using rapid diagnostic tests (RDT), thick and thin blood smears (TBS) and real-time quantitative polymerase chain reactions (RT-qPCR). Haemoglobin (Hb) level, and proteinuria and glycosuria [using a urine dipstick test  and activities in the forest were collected. Women were screened for malaria using RDT, TBS and PCR whether or not they were symptomatic. Hb level and proteinuria and glycosuria were determined.In both surveys, data quality was monitored by study coordinators on a weekly basis. Women with positive RDTs for malaria as well as anaemic women were treated according to the national guidelines.Plasmodium rRNA, as described by Diallo et al. [P. falciparum line (MR4). PCR were performed in the Microbiology Laboratory of Mahosot Hospital, Vientiane, Laos. A quality control for PCR was performed in Hôpital Bichat, France. DNA extracts of 30 % randomly selected PCR-positive samples and 13 % randomly selected PCR-negative samples were analysed in blind with the FTD (Fast-Track Diagnostics) Malaria PCR, a multiplex PCR for detection of Plasmodium spp. DNA. In case of positive result, the PCR FTD Malaria differentiation was performed to identify the species of Plasmodium. Agreement between both methods for PCR was high (Cohen’s kappa coefficient: 0.92). Agreement was complete for the 48 samples from pregnant women which were analysed.RDT  was performed on all peripheral blood samples. Blood collected on filter papers was dried and conserved at room temperature until DNA extraction using Chelex [o et al. , was use® (community-level survey) or coulter counter (HUMACOUNT 60TS) (facility-level survey).TBSs were stained with Giemsa. All placental blood smears as well as a sub-sample of peripheral blood smears  were read, and parasitaemia was quantified by the Lambaréné method . TBS reaMaternal characteristics were described using proportions  for categorical variables and means (95 % CI) and medians (IQR) for continuous variables. The proportion and its exact Poisson 95 % CI of women infected with malaria using RDT and PCR were calculated.Maternal characteristics associated with malaria during pregnancy (community-level study) were assessed using a modified Poisson regression model . For eacData entry and analysis were performed using Epidata (version 3.1) and Stata (version 13), respectively.P. vivax/P. falciparum infection  of six women (five) per village) were included Table . Women wIn univariate analysis factors significantly associated with MiP were history of malaria during the current pregnancy and a low education level. In multivariate analysis only history of malaria remained associated with MiP, although this association was only marginally significant . The prevalence of maternal anaemia was 51.7 % (106/205) with overall mean (95 % CI) Hb of 10.8 (10.6–11.0) g/dL  in Salavan, 44 (13.3 %) in Vapi and 39 (11.8 %) in Toumlane Hospitals , and the mean (95 % CI) Hb level was 11.4 (11.3–11.6) g/dL   were significantly associated with a reduction in birth weight in univariate analysis , of Lao Theung ethnicity, women who did not use a bed net the night before admission, those who made no ANC visits during pregnancy (compared to women who made ≥four ANC visits) and those who did not have iron supplementation during current pregnancy (compared to women who stated that they had iron supplementation the day before admission) were significantly more likely to be anaemic at delivery. In contrast, primigravidae and women living in Vapi District were less likely to be anaemic Table . In multWhen considering haemoglobin level as a continuous variable, there was a significant reduction in mean Hb level in women living in Toumlane District  and those with a low number of ANC visits (one to three compared to ≥four visits) in multivariate analysis . The low proportion of RTD-positive infections is likely to be due to the low sensitivity of the test for low parasitaemia infections [This survey assessed the burden and epidemiology of MiP in southern Laos, where malaria transmission remains relatively high despite the large-scale distribution of both ITNs and ACT. In Vapi villages, malaria was detected by PCR in almost 6 % (12/204) of pregnant women. The majority of these infections were asymptomatic and sub-microscopic and, therefore, would not have been detected by recommended national screening strategies . All 12 fections . This mafections .P. falciparum using TBS [Only a single prior study had evaluated MiP in Laos, in Vientiane in 1998. This study was conducted in pregnant women with history of fever, who were recruited during ANC visits. Among 68 women, 16 (23.5 %) were diagnosed with sing TBS . The higP. vivax infection, or they may reflect a higher level of exposure to malaria of these women, compared to those who did not report previous infections.In the villages almost 6 % of pregnant women were infected with malaria. This proportion may have been higher if longitudinal follow-up had been used, providing a cumulative proportion of infections during pregnancy. For instance, on the Thai-Burmese border, while the mean malaria prevalence during weekly cross-sectional surveys was 4 % using TBS, the cumulative proportion of women infected with malaria during pregnancy was as high as 22.8 % . A histoP. vivax, which is less likely to sequester in the placenta than P. falciparum. In both surveys, only two out of a total of 14 infections were caused by P. falciparum, and Plasmodium knowlesi was not detected. This is in accordance with the increasing ratio between P. vivax and P. falciparum infections, which is observed in the region [P. vivax compared to P. falciparum infections may also be explained by the inclusion of asymptomatic women, P. falciparum infections being more likely to be symptomatic.At delivery, there was a lower prevalence of sub-microscopic infections, which varied according to antenatal clinic. In Vapi Hospital, the prevalence (4.5 %) was comparable to what had been found in the villages. However, malaria was not detected in pregnant women from Toumlane, although the sample size was very low, nor in women who delivered in Salavan Hospital, which is located in a more urban area than Vapi and where malaria transmission is probably lower. The comparison of these results with those from studies conducted in the Asia–Pacific region is difficult since malaria transmission is highly variable in this region and most of these studies included both symptomatic and asymptomatic pregnant women. On the Thai-Burmese border, where malaria transmission and women’s characteristics may be the most comparable to those in Laos, the prevalence of peripheral and placental malaria at delivery using TBS or PCR ranged from 3.0 to 10.9 % [Plasmodium vivax infection during pregnancy has been associated with poor maternal and child outcomes [P. falciparum. A single episode of vivax or falciparum malaria can have deleterious consequences for the foetus [outcomes , 16, alte foetus . Asymptoe foetus , 18. In e foetus , making e foetus , 20. TheA high proportion of women 24 %) had LBW newborns, mostly due to IUGR. Indeed, newborns were classified as SGA, as a proxy for IUGR, in 23 % of cases with 80 % of LBW babies who were SGA. This is in accordance with the prevalence of LBW and the proportion of LBW due to IUGR in Asia 4 % had L. Since 1The strength of the present analysis is the use of population-based data to assess the prevalence of malaria during pregnancy, limiting selection bias that arises when women are recruited at facility-level. However, the participation in the study of all eligible women in the villages cannot be warranted. Moreover, fewer women than anticipated were recruited both in the villages and at delivery, which may have led to estimating the prevalence of malaria with some inaccuracy. In Vapi and Toumlane District Hospitals all women were included at delivery, which was not the case in Salavan Provincial Hospital where a very few number of women could not be included before an emergency delivery. Finally, the high variability of malaria prevalence in south Laos limits the generalization of our results . The effP. vivax sub-microscopic infections in this population was noticeable. However, these findings cannot be generalized beyond the present study area since malaria transmission is highly heterogeneous within the country [Plasmodium infections that have deleterious consequences during pregnancy and contribute to malaria transmission [P. falciparum and P. vivax in such areas [This survey contributes a first step towards the assessment of MiP in Laos, where the prevalence of  country . Furthersmission . Systemasmission . In addismission  and effich areas . In partch areas .


Kenya is intensifying its national efforts in malaria control to achieve malaria elimination. Detailed characterization of malaria infection among populations living in the areas where the disease is endemic in Kenya is a crucial priority, especially for planning and evaluating future malaria elimination strategy. This study aimed to investigate the distribution and extent of malaria infection on islands in Lake Victoria of Kenya to aid in designing new interventions for malaria elimination. Five cross-sectional surveys were conducted between January 2012 and August 2014 on four islands  in Lake Victoria and a coastal mainland (Ungoye). Malaria prevalence varied significantly among settings: highest in Ungoye, followed by the large island of Mfangano and lowest in the three remaining small islands. Of the 3867 malaria infections detected by PCR, 91.8% were asymptomatic, 50.3% were sub-microscopic, of which 94% were also asymptomatic. We observed geographical differences and age dependency in both proportion of sub-microscopic infections and asymptomatic parasite carriage. Our findings highlighted the local heterogeneity in malaria prevalence on islands and a coastal area in Lake Victoria, and provided support for the inclusion of mass drug administration as a component of the intervention package to eliminate malaria on islands. Plasmodium falciparum by half and the incidence of clinical disease by 40% in endemic Africa between 2000 and 2015P. falciparumRenewed international commitment to control malaria through scale-up of coverage of interventions reduced the prevalence of P. falciparum parasite rate for the population aged 2–10 years old (PfPR2–10) is below 1%. However, despite these control tools, 10.6% (4.3 million) of the population still live in areas with PfPR2–10 ≥ 40%, including areas around Lake Victoria in western Kenyamalaria free Kenya”Kenya implemented a national strategic malaria control plan in 20012 and a surface altitude of 1,135 m above the sea level. In Homa Bay County, Kenya there are two large islands in Lake Victoria namely Rusinga (east) and Mfangano (west) as well as few other small inhabited islands. Given the year-round availability of fresh water, a high density of malaria vectors is found on these islands, as well as along the lakeshore communities79Lake Victoria is the second largest fresh water lake in the world, with a surface area of 69,485 kmThe demographic characteristics of each survey are given in The clinical characteristics of each survey are given in The prevalence of malaria microscopy and PCR in the five surveys is shown in Significant (P < 0.001) intra-island variation in malaria prevalence was also observed among the eight catchment areas on Mfangano Island . PrevaleThe majority of positive cases (89.7% by microscopy and 91.8% by PCR) were not accompanied by fever (i.e. asymptomatic) . The parPlasmodium spp. parasites detectable by microscopy and 3869 participants  by PCR  were double co-infections, contributed mainly by P. falciparum/P. malariae (n = 760). P. falciparum/P. malariae/P. ovale triple co-infections accounted for 22.3%  of mixed-species infections. The species-specific prevalence by microscopy and PCR was as follows: 19.7% vs. 33.4% for P. falciparum, 1.3% vs. 9.6% for P. malariae, and 0.3% vs. 4.0% for P. ovale.Overall, 2076 individuals  had ) by PCR . SpeciesPlasmodium spp. infections by PCR in different geographical settings and age groups is shown in P. falciparum, 1.7–2.8% for P. malariae, and 1.1–1.5% for P. ovale. Furthermore, prevalence rates of all three species were age-dependent (P < 0.001) and lowest in >30 years (1.4–18.7%) . In contrast, P. malariae- and P. ovale-specific prevalence peaked in children 6–10 years of age, at 15.5%  and 7.0%  respectively.The prevalence of 4–18.7%) . P. falcP. falciparum infections detected by microscopy, 10.3%  had observable gametocytes, resulting in a population P. falciparum gametocyte prevalence of 2.02% (95% CI: 1.8–2.3)  and decreasing significantly (P < 0.001) with age  in Ungoye, 10.4% (95% CI: 8.2–12.9) on Mfangano, and 11.2% (95% CI: 7.5–15.9) on the three small islands .Among 2054 1.8–2.3) . The prowith age . Nonethe islands . Among aPlasmodium infection (with any species) for the risk factor analysis  than others (Mrongo and Ugina). In the highland areas, parasite rates were lower in Kangungu than Gulwe. The latter is connected by a small access road to Wakula, where prevalence was high, suggesting that human movement might yet be another contributing factor to the micro-geographical variation in malaria endemicity.Both parasite rates and parasitaemia were strongly age-dependent, although in different ways. While mean malaria parasitaemia was highest among children under five years and decreased with age, prevalence of malaria infections rose throughout childhood and only started decreasing in adolescents and adults. These different patterns may be explained by local mosquito biting behaviour and acquisition of immunity. In addition, the reported use of bed net among school-age children was considerably low (33%) in the lakeside regionConvenience sampling approaches are efficient and cost effective, but are more likely to be flawed by selection bias. In this study, surveys often took place in primary schools, meaning that children of school age (6 to 15 years) were disproportionately represented - the age distribution varied slightly depending on the setting . As thesP. vivax infection was found in any of the surveys by either microscopy or PCR, consistent with the absence in many sub-Saharan populations of the Duffy blood-group-antigen, which is required for red blood cell invasion by P. vivaxP. falciparum, consistent with previous studies conducted in Mbita sub-county1021P. malariae was the second most prevalent species. By both microscopy and PCR, the majority of P. malariae infections were double co-infections with P. falciparum. Triple co-infections of P. falciparum, P. malariae, and P. ovale were more common than P. malariae mono-infection  of malaria infections in the study area were asymptomatic i.e. not accompanied by febrile symptoms. While these asymptomatic infections were most prevalent in children 15 years and under, they were not uncommon in the adult population. As potential gametocyte carriers, this asymptomatic adult population represents an important reservoir for malaria transmission. Many of these asymptomatic infections are present at densities below the limit for microscopic detection and therefore, the use of microscopy as the sole diagnostic method likely leads to an underestimation of the malaria burden. Our study showed that microscopy detected significantly fewer asymptomatic infections when compared to PCR, consistent with several previous findings that molecular-based assays were more sensitive and time-efficient methods for asymptomatic malaria infections34et al.4344The increase in the proportion of sub-microscopic infections by age was contrasted by the decreases in infection prevalence and parasite densities in the population. Parasite density is mediated by host acquired immunityConventional PCR is typically able to detect infections down to approximately one parasite/μl using dried blood spotsIslands present a unique opportunity to interrupt malaria transmission due to the relative geographical isolation and confined population. MDA was previously recognized as an important method of malaria control18In summary, this study highlighted the heterogeneity in malaria prevalence and importance of asymptomatic and sub-microscopic infections on islands and a coastal area in Lake Victoria. We observed significant geographical differences and age-dependency in malaria parasite carriage. The unique findings of malaria epidemiology in our study area may have important implications for the development and implementation of malaria elimination strategy. Taking into account the high and heterogeneous prevalence of asymptomatic and sub-microscopic malaria infections on islands in Lake Victoria, the use MDA to eliminate malaria may be justified. An appropriate package that complements MDA with effective vector control and rapid detection and treatment of new cases has the potential to achieve long-term malaria elimination on islands in Lake Victoria.The study was conducted in accordance with the Declaration of Helsinki and was approved by the Kenyatta National Hospital/University of Nairobi-Ethics and Research Committee in Kenya (No. P7/1/2012) and the Committee on the Ethics of Human Research of Karolinska Institutet in Sweden (Dnr 201271239–31/4). Survey subjects were informed by local interpreters of the purposes and procedures of the study, and a written informed consent was obtained from each adult participant at study registration. In the case of children, written informed consents were obtained from their parents or legal guardians.This study was conducted in Homa Bay County, Kenya and included one large island (Mfangano) and three small islands  in Lake Victoria, and one coastal village (Ungoye) on the mainland . The domAn. gambiae s.s., An. arabiensis and An. funestusAn. gambiae s.s. has experienced a reduction in density of nearly 95% in the Lake Victoria basin over 10 years, and has been replaced by An. arabiensis as the dominant malaria vector species in the mainland part of this region. Nevertheless, An. gambiae s.s is still the predominant malaria vector on islands in Lake VictoriaIn western Kenya a bimodal pattern of rainfall is generally observed, with the long rainy season from March to June and the short rainy season from November to December, but the periods vary each year. Annual rainfall ranges from 700 mm to 1,200 mm. The mean temperature is 25 °C while the maximum temperature is 30 °C, and humidity is relatively high. Malaria prevalence peaks usually lag 1–2 months after the rainy seasons. In the early 1990 s, annual entomological inoculation rates (EIRs) reported between 60 and 300 infectious bites per person per year51Though there are many development initiatives in these areas, poverty is still a major challenge. Almost half of the population in Homa Bay County survives on below the Kenyan poverty line of Kenya Shilling (KSh) 1,562 (USD 15.6) per month2) has a population of approximately 18,600 (HDSS). The island’s terrain is rocky and hilly. The island is easily recognisable from the mainland by the imposing green-clad cliffs of Mount Kwitutu, which rises 562 m above the lake surface from the island’s centre. Mfangano Island is predominantly rural. Nevertheless, it is well connected to Mbita (16 km) in the mainland by a popular ferry service and numerous private, scheduled and unscheduled motorized boat trips. Residents of Mfangano Island rely on the neighbouring Mbita for regional transportation and access to services such as banks. There are six government health facilities  across the island. In contrast to other study sites, on Mfangano there is a sizable Suba speaking population which is becoming rarer due to the intermarriage between the Suba and Luo ethnic tribes. In addition to farming and fishing, tourism is also growing on this island as the connection to and from Mbita is being improved.Lying just east of the border with Uganda, Mfangano ; 66 km2 There were eight catchment points on Mfangano: Ramba, Wakinga and Mrongo along the east coast , Kagungu and Gulwe in the highland , and Wakula, Ugina, and Ringiti Island in the west . These catchment points were selected to represent the different local environments found on the island, from the lakeshore lowland areas to the central highland areas.Each of these three small islands  has a po2) in Nyangwethe area with population of 3,471 (2009 census). The village is connected by an unpaved roadway to the nearby small towns of Sindo (17.2 km) and Mbita Point (31.2 km), each with a sub-district hospital. There is one small public health facility in this area. Ungoye was included for comparative purposes due to its similarity to the islands in environmental characteristics, infrastructure, and access to health services. A recent survey in the Gembe East area revealed a high P. falciparum prevalence of 66.5% by PCRUngoye  is a smaFive cross-sectional surveys were conducted between 2012 and 2014: two after the short rainy season in January-February 2012 and March 2014 and three after the long rainy season in July-August 2012, August 2013, and August 2014. Peak malaria transmission occurs 1–2 months after the rainy season and the mean monthly anopheline vector abundance has been reported to increase by 6- to 8-fold in the rainy season compared to the dry season in the Lake Victoria basinSex and age were recorded for each participant. Axillary body temperature was determined using a digital thermometer . Fever was defined as a temperature exceeding 37.5 °C. Hemoglobin level was measured with the HemoCue Hb 201 analyzer . A measurement below 11 g/dL was classified as anaemic. Children aged 12 years and below were examined for enlarged spleen by AK according to Hackett’s method, regardless of fever or malaria status. Measuring spleen sizes in children has been shown as a useful variable to estimate the burden of malaria transmission in other island settingsP. falciparum gametocytes counts were separately recorded. Parasite density was estimated from parasite counts, assuming that there were 8,000 leukocytes per μl of blood. All slides were independently re-examined by two experienced microscopists blinded to the first microscopy reading results. Discrepancies between the two readings were resolved by a third experienced microscopist.Thin and thick blood smears were prepared on site, stored in slide boxes and transported daily to the main laboratory in Mbita, where thin blood smears were fixed with methanol, and all smears were stained with 3% Giemsa solution for 30 minutes and examined under oil emersion (10 × 100 magnification) by experienced microscopists. Blood smears were defined as negative if no parasites were found after examining 100 high power microscopy fields. For all positive samples, malaria species were identified and asexual parasite forms were counted against 200 leukocytes. Plasmodium infection was detected by a nested PCR protocol that can distinguish the four major human malaria species (except P. knowlesi)For detection by PCR, blood samples (70 μl) withdrawn by 75 mm Micro-hematocrit capillary tube  were spotted on Whatman ET31 Chr filter papers  and dried thoroughly at ambient temperature. The dried blood spots were put in individual zipped plastic bags and stored at −20 °C. DNA was extracted from a quartered blood spot (17.5 μl) using the QIAamp Blood Mini Kit  according to the manufacturer’s instructions. 2) or Fisher’s exact tests. The average enlarged spleen (AES) index for each survey was calculated as the sum of the number of children in each spleen size class multiplied by the class number (0–4), divided by the total number of palpable spleen. Interactions between survey, individual settings, age group and prevalence were examined using conditional logistic regression. Univariate logistic regression was performed to identify risk factors for the outcome of Plasmodium infection as determined by PCR. Sex, age group, geographical area, fever, enlarged spleen and anaemia were considered as individual variables. All variables with a P-value of less than 0.05 from a likelihood ratio test in univariate analyses were entered into a multivariate logistic regression model and stepwise backwards elimination was used to identify the main risk factors for infection.All survey data were double entered into Microsoft Excel spreadsheets and cross-checked for errors. Data were processed and analysed using the STATA/SE 13.1 statistical software package . Differences in participant characteristics and prevalence among age groups and geographical areas were assessed using Chi-squared .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


HIV-exposed, uninfected (HEU) infants suffer high morbidity and mortality in the first year of life compared to HIV-unexposed, uninfected (HUU) infants, but accurate data on the contribution of malaria are limited.The incidence of febrile illnesses and malaria were evaluated in a birth cohort of HEU infants. Infants were prescribed daily trimethoprim–sulfamethoxazole (TS) prophylaxis from 6 weeks of age until exclusion of HIV-infection after cessation of breastfeeding. Infants were followed for all illnesses using passive surveillance and routine blood smears were done monthly. Malaria was diagnosed as a positive blood smear plus fever. Placental malaria was determined by histopathology, placental blood smear and PCR. Risk factors for time to first episode of malaria were assessed using a Cox proportional hazards model. Malaria incidence among HEU infants aged 6–12 months was compared to that in other cohorts of HEU and HUU infants from the same region.Among 361 HEU infants enrolled, 248 completed 12 months of follow-up resulting in 1562 episodes of febrile illness and 253 episodes of malaria after 305 person-years of follow-up. The incidence of febrile illness was 5.12 episodes per person-year (PPY), ranging from 4.13 episodes PPY in the first 4 months of life to 5.71 episodes PPY between 5 and 12 months of age. The overall malaria incidence was 0.83 episodes per person-year (PPY), increasing from 0.03 episodes PPY in the first 2 months of life to 2.00 episodes PPY between 11 and 12 months of age. There were no episodes of complicated malaria. The prevalence of asymptomatic parasitaemia was 1.2 % (19 of 1568 routine smears positive). Infants born to mothers with parasites detected from placental blood smears were at higher risk of malaria . HEU infants in this study had a 2.4- to 3.5-fold lower incidence of malaria compared to HUU infants in other cohort studies from the same area.The burden of malaria in this birth cohort of HEU infants living in a high-transmission setting and taking daily TS prophylaxis was relatively low. Alternative etiologies of fever should be considered in HEU-infants taking daily TS prophylaxis who present with fever.Trial Registration NCT00993031, registered 8 October, 2009 As the number of infants born HIV-exposed but uninfected (HEU) steadily increases throughout Africa , it is iThe objective of this study was to describe the natural history of malaria in a birth cohort of Ugandan HEU infants who were born to mothers who received ART as part of a clinical trial during pregnancy and who themselves received TS prophylaxis starting at ~6 weeks of life, as per current WHO guidelines. The incidence of malaria and all febrile illnesses and the prevalence of asymptomatic parasitaemia at routine visits were measured over the first year of life. Risk factors for malaria in the first year of life were assessed at the time of birth.This was a planned secondary data analysis in a birth cohort of HEU infants born to HIV-infected mothers who were part of a randomized controlled trial of lopinavir/ritonavir versus efavirenz-based ART living in Tororo district, Uganda  . The stuInfants were followed from birth to 1 year of age. At birth, placental malaria status was determined from placental blood and tissue. Infants were seen at monthly routine visits and parents were instructed to bring their infants to a dedicated study clinic, open 7 days a week, for any fever or other illness. All study participants were given a long-lasting, insecticide-treated bed net (LLIN) at birth and prescribed daily TS prophylaxis from 6 weeks of age until they were confirmed to be HIV negative after cessation of breastfeeding. At each monthly visit, adherence to TS prophylaxis was assessed by 3-day recall and parents were asked if infants were sleeping under an LLIN. A thick blood smear for determination of malaria parasitaemia by microscopy was performed at every routine monthly visit.A febrile episode was defined as having a measured tympanic temperature of ≥38.0 °C or a history fever in the past 24 h. Study participants who presented to the study clinic with a febrile episode had a thick blood smear done for the detection of malaria parasites. If the blood smear was positive, they were diagnosed with malaria and a thin blood smear was taken off for identification of parasite species. Infants with uncomplicated malaria were treated with artemether–lumefantrine (AL)  if they were ≥4 months old and weighing ≥5 kg. Infants with uncomplicated malaria who were <4 months old and weighing <5 kg were treated with quinine.Thick and thin blood smears were stained with 2 % Giemsa and examined for malaria parasites by trained microscopists. A blood smear was considered negative when the examination of 100 high power fields did not reveal asexual parasites. All slides were read by a second reader, and a third reader settled any discrepancies. Placental specimens were collected within 30 min of delivery in the hospital . Thick blood smears made from placental blood collected from an incision on the maternal surface of placental tissue, were examined for parasites. Aliquots of approximately 25 µL of placental blood were also placed on filter paper, air dried and stored for DNA extraction and PCR testing for malaria parasites as earlier described . PlacentData were double-entered into Access database  and analysed using Stata version 12 . Follow-up time started at birth and ended at 1 year of age, or the time of premature withdrawal, or when the study was stopped prior to reaching 12 months of age because of limited funding. An incident episode of malaria was defined as having a febrile episode with a positive blood smear not preceded by any treatment for malaria in the prior 14 days. Comparisons of the incidence of malaria between 6 and 12 months of age were made between infants enrolled in this study and other cohorts enrolled in the same study site using the same methodology. Placental malaria status was categorized using a categorical variable as follows: no parasites or pigment detected by any method, only pigment detected by histopathology (no parasites), parasites detected by PCR or histopathology but not placental blood smear, parasites detected by placental blood smear. Time to an infant’s first episode of malaria was estimated using Kaplan–Meier survival analysis. Associations between risk factors assessed at the time of birth and time to first episode of malaria were assessed using a Cox proportional hazards model. In all analyses, a two-sided P value of <0.05 was considered to be statistically significant.This study was approved by the Uganda National Council of Science and Technology, the Makerere University School of Medicine Research Ethics Committee, and the University of California, San Francisco Committee for Human Research. Informed consent was obtained from all mothers at the time of enrolment.Among 386 HEU infants born to 376 HIV-infected women (including ten pairs of twins), 361 infants were included in this analysis . Malaria was diagnosed in 253 (16.2 %) of the febrile episodes, resulting in an overall incidence of malaria of 0.83 malaria episodes PPY. The incidence of malaria steadily increased with age over the first year of life, rising from 0.03 episodes PPY in infants up to 2 months old to 2.00 episodes PPY in infants between 11 and 12 months old  were positive for malaria parasites among 16 different children; six of them developed malaria in the next 30 days, four had malaria during the previous 30 days, three had no further blood smears, and six became smear negative in the absence of anti-malarial therapy.Overall, 108 children (29.9 %) developed at least one episode of malaria with a median time to their first episode of 225 days range 42–357 days). Associations between risk factors assessed at birth and time to a first episode of malaria are presented in Table 57 days. In this birth cohort of HEU infants living in an area of high transmission intensity, the burden of malaria was relatively low in the first year of life compared to HUU infants from the same area. Asymptomatic parasitaemia was rare and all episodes of malaria were uncomplicated, with a gradual increase in incidence from 2 to 12 months of age. Key characteristics of this birth cohort of HEU infants were the provision of LLINs, TS prophylaxis, ART for all mothers during pregnancy, and breastfeeding. These data fill an important gap in understanding the epidemiology of malaria in this vulnerable and growing population of HEU in sub-Saharan Africa, who are receiving the current standard of care, with TS prophylaxis from 6 weeks of life and being born to HIV-infected mothers who received ART. The most accurate data prior to this report came from the breastfeeding, antiretrovirals and Nutrition (BAN) trial, which reported a 48-week incidence of malaria of 0.052 episodes PPY . HoweverAs expected, malaria was very rare in HEU infants up to two months of age in this cohort. Relative protection from malaria in the first weeks of life has been well described among infants living in endemic areas , and is Active placental malaria by blood smear was associated with an increased risk of malaria over the first year of life in this cohort of HEU infants, as has been shown in other studies of HUU infants –27. ThisThe contrasting pattern of the incidence of malaria versus febrile illness over the first year of life has clinical implications for the management of HEU infants living in malaria-endemic regions. In this cohort, the incidence of malaria rose from near zero in the first 2 months of life to two episodes PPY among infants between 11 and 12 months of age, while the incidence of febrile illness increased only slightly during the first 6 months of life and remained steady thereafter. Taken together, these patterns underscore the important use of accurate malaria diagnostics to evaluate all fevers in similar settings. Clinicians evaluating and treating febrile HEU infants, particularly those <2 months old, must not presume fever to represent malaria and should carefully evaluate for non-malarial causes of fever, such as bacterial sepsis and viral infections. Conversely, clinicians must suspect malaria in older febrile HEU infants, even if they are receiving TS. The highest incidence of malaria was seen in infants aged 11–12 months despite receiving daily TS prophylaxis. This could reflect waning protection from maternal antibodies or changes in TS adherence. In either case, the higher rates underscore the need for additional interventions to reduce malaria incidence in similar regions. Infants in this cohort had good clinical outcomes, with no episodes of complicated malaria or malaria-associated deaths among 253 episodes of malaria treated, but that is most likely a result of prompt diagnosis and treatment of malaria in the context of this trial and not available in the most limited resource clinical settings.These results add to existing evidence that supports the importance of daily TS prophylaxis in preventing malaria in HEU children , 11, 12.This study had several limitations. The cohort’s sample size limited the power to evaluate additional risk factors for malaria. Comparisons of malaria incidence among infants who were 6–12 months old from other cohort studies were vulnerable to confounding by temporal differences in malaria transmission which were not adjusted for because long term longitudinal data about temporal changes in malaria transmission in the study area were not available. However, it is notable that the other cohorts were from the same study site and followed with identical malaria diagnostics and study protocols. Finally, there was very little follow-up time when infants were not taking TS and this limited our ability to separately investigate the effect of TS alone on malaria incidence.In this birth cohort of HEU infants living in a malaria-endemic region and taking daily TS prophylaxis, the incidence of malaria was relatively low but increased with increasing age. However, the burden of febrile illnesses was high and remained fairly stable during infancy. Alternative etiologies of febrile illnesses other than malaria should always be considered in HEU infants taking daily TS prophylaxis. While HEU infants living in malaria-endemic regions benefit from the malaria prophylactic efficacy of TS, additional strategies to reduce malaria in the important population are needed.


Plasmodium falciparum but today, evidence points to other Plasmodium species including P. vivax, P. ovale and P. malariae. The purpose of this study was to identify the Plasmodium species in clinical samples from children residing in five epidemiological strata of malaria in Cameroon, so as to advise control policies.Malaria in Cameroon was previously known to be caused solely by Plasmodium species. Statistical analysis performed included the Pearson chi-square test, and statistical significance was set at p < 0.05.One thousand six hundred nine febrile children (≤15 years) were recruited from five epidemiological strata of malaria including the Sudano-sahelian (SS) strata, the High inland plateau (HIP) strata, the South Cameroonian Equatorial forest (SCEF) strata, the High western plateau (HWP) strata and the Coastal (C) strata. Malaria parasites were detected by Giemsa microscopy (GM) while a multiplex polymerase chain reaction (PCR) was used to identify the p = 0.05). However, GM demonstrated a high sensitivity (85.5%) and specificity (100%) and, overall, a perfectly correlated agreement with PCR (97.5%). The prevalence of malaria was significantly higher in children between 60 and 119 months (p < 0.001) and in Limbe  (p < 0.001). Contrariwise, the prevalence of malaria was not associated with gender (p = 0.239). P. falciparum was identified in all (100%) the cases of malaria; P. ovale, P. vivax, P. malariae and P. knowlesi were all absent. No case of mixed infection was identified.The PCR-adjusted prevalence of malaria was 17.6%. The detection rate of PCR was higher than GM (P. falciparum was the only species causing clinical malaria in the target population, which is contrary to studies that have reported P. vivax, P. malariae and P. ovale as causing clinical malaria in Cameroon. Malaria remains a significant public health problem in many countries of the world especially in sub-Saharan Africa (SSA). The WHO estimates that in 2015, there were 214 million cases of malaria and 438,000 deaths attributed to malaria [In Cameroon, malaria is the major cause of morbidity and mortality among the most vulnerable groups including children aged under 5 years 18%), pregnant women (5%), people living with HIV/AIDS (5.5%) and the poor 40%) , 4. In C0% , 4. I%, pregnaPlasmodium. Five species of Plasmodia are known to cause disease in humans, namely, P. falciparum, P. ovale, P. malariae, P. vivax and P. knowlesi. The distribution of the different Plasmodia is not uniform throughout the world; P. vivax is more predominant in Asia [P. falciparum in Africa [P. falciparum is the most virulent species accounting for the majority of deaths [P. vivax malaria is also associated with potentially life-threatening conditions about as often as with a diagnosis of P. falciparum infection [P. falciparum, but an increasing number of studies performed recently report other species of Plasmodia including P. vivax which is the second major species occurring as either single or mixed infection with P. falciparum [P. malariae and P. ovale occur at a lower frequency and in most cases as mixed infection with P. falciparum [P. knowlesi has not been reported to cause malaria in Cameroon. P. knowlesi which was only recently observed to cause human malaria [P. vivax, P. knowlesi also requires the Duffy antigen for infection [P. vivax has been shown to infect some people lacking the Duffy antigen in Cameroon [P. knowlesi may as well evolve to infect Duffy-negative populations.Malaria is caused by parasitic protozoans of the genus  in Asia  and P. fn Africa . Althougf deaths , recent nfection . In Camelciparum , 14. P. lciparum –17. P. k malaria  is the m malaria . Like P.nfection  and is tCameroon , 14. WitPlasmodium species causing malaria in children in Cameroon are limited. With the increasing number of reports of malaria being caused by Plasmodium species other than falciparum in Cameroon, it is important to provide baseline data on the distribution of the different Plasmodium species associated with clinical malaria in children, which will inform control policies. This study was therefore designed to identify the Plasmodium species associated with clinical malaria in children residing in five epidemiological strata of malaria in Cameroon.Studies to identify the Five out of the six epidemiological strata of malaria in Cameroon were randomly selected for this study. Five study sites, each representing the epidemiological strata, were further selected and included: Maroua in the Sudano-sahelian (SS) strata, Ngaoundere in the High inland plateau (HIP) strata, Yaounde in the South Cameroonian Equatorial forest (SCEF) strata, Bamenda in the High western plateau (HWP) strata and Limbe in the Coastal (C) strata Fig. . The chaThis study was a hospital-based cross-sectional study involving children who came to consult in the outpatient department (OPD)/emergency units of regional hospitals in the different study sites. Data was collected between May and November 2015 (to coincide with the rainy season during which transmission is highest), simultaneously in the different study sites.The sample size was estimated using the following formula for sample size calculation as described by Swinscow ;\documenp = prevalence of malaria in Cameroon which is 29% [h is 29% .\documenThus, we recruited 317 participants per study site giving an overall total of 1585 for the 5 sites.Eligible participants were febrile children (≤15 years) who were consecutively recruited as they came to consult in the OPD or emergency unit of the hospitals in the different study sites. Patients not on any anti-malarial treatment for at least 1 week prior to the study commencing were included.About 4 ml of blood was collected from consented participants using aseptic techniques into EDTA test tubes. The uncoagulated blood was used to perform complete blood count (CBC) and prepare thick and thin films for malaria screening by microscopy and for the performance of polymerase chain reaction (PCR).The CBC was performed using the Mindray® Auto haematology analyzer . The white blood cell (WBC) counts were obtained from the CBC results and used in the estimation of malaria parasite density.The prepared blood films were air-dried and stained with 10% Giemsa (1 in 20 dilutions) for 25–30 min . Blood fThe Chelex method  was usedP. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi). We used the Oligonucleotide primer pairs designed by Snounou et al. [P. falciparum, P. vivax, P. malariae and P. ovale in the host’s total DNA. Reaction mix was made in a final volume of 12.5 μL containing 6.25 μL of 2× GoTaq®Hot Start Green master mix , 0.2 μM of each primer , 3.25 μL of molecular grade water  and 2.5 μL of DNA solution served as a template. Positive controls were included for all Plasmodium species. The cycling conditions were as follows: initial heating at 95 °C for 2 min, 43 cycles of heating at 95 °C for 45 s, annealing at 58 °C for 90 s and extension at 72 °C for 60 s, and a final extension at 72 °C for 5 min.Genus- and species-specific sequences have been identified within the small subunit ribosomal DNA genes of all human malaria parasites . The statistical tests performed included the Pearson’s chi-square test for comparison of proportions. Statistical significance was set at One thousand six hundred nine 1609) children met the inclusion criteria and were enrolled; 318, 318, 341, 315 and 317 were enrolled in Bamenda, Limbe, Maroua, Ngaoundere and Yaounde, respectively (Table 609 childp = 0.05). However, GM demonstrated a high sensitivity (85.5%) and specificity (100%) and, overall, a perfectly correlated agreement with PCR (97.5%) in the current study (Table p < 0.001).Among the 1609 participants, 242 and 283 were positive for malaria parasites by Giemsa microscopy (GM) and PCR, respectively. A marginal difference was observed in the detection rate between GM and PCR .Generally, the prevalence of malaria was highest in participants between 60 and 119 months and lowest in participants below 60 months Table . A signiP. falciparum as determined by the multiplex PCR and GM. Neither P. vivax, P. malariae, P. ovale nor P. knowlesi was identified. Furthermore, no mix infection with other Plasmodium species was observed.All (100%) the cases of malaria in this study were caused by Anopheles mosquitoes. The observation of a significant association between the prevalence of malaria and age in the current study is however contrary to some studies that found no association of malaria with age [In the current study, the overall prevalence of malaria was 17.6% as determined by PCR. This prevalence is however lower than the national prevalence of 29% reported in 2012 . This diwith age –34.P. falciparum was the only parasite species identified. This is reminiscent of the WHO report that all (100%) of malaria cases in Cameroon are caused by P. falciparum [P. falciparum was associated with all the cases of clinical malaria in the country [P. vivax ranging from 4% [P. falciparum and P. malariae or P. ovale [P. falciparum was correctly identified as the only Plasmodium species by GM and this confirms the high specificity of GM in the identification of the different Plasmodia. One limitation of using PCR in the diagnosis of malaria is that the parasite DNA can remain in the blood stream long after the infection has been cleared and therefore differentiating an active infection from a recently cleared infection poses a challenge [P. knowlesi was observed. Being one of the first studies to target P. knowlesi in the country serves as a confirmation of its absence.In the current study, lciparum . It is a country –17. The  from 4%  to 14.9% from 4% . The difP. ovale –17. The P. ovale , 41; meaP. ovale , 43. PCRP. ovale , 44, 45.hallenge . As a cohallenge , 48. In In this study, data was collected only during the rainy season during which transmission is usually at its peak. This may have affected the distribution of the different Plasmodia. Studies designed to collect data in both the rainy and the dry season will be paramount to providing a clearer picture of the distribution of the Plasmodia with season. More so, in the current study, participants were recruited only from health care facilities in urban settings and the data generated may therefore not be generalisable to children living in rural areas. Studies will also be needed in rural areas to give a clearer picture.P. falciparum was the only species associated with clinical malaria in the target population, and no mixed infection with other Plasmodium species was identified, which is contrary to earlier studies.A malaria prevalence of 17.6% was observed in the target population. The prevalence was significantly higher in children between 60 and 119 months. On the contrary, gender had no influence on malaria prevalence. A significant association was also observed between the prevalence of malaria and the geographical setting, being highest in Limbe (in the C strata) and lowest in Maroua (in the SS strata).


The mean parasite load for P. vivax and P. falciparum was 14496/µl and 24410/µl, respectively. The abnormal scattergrams of DIFF, WBC/ Baso, IMI channel, and RET-EXT on Sysmex XE-2100 supported 99.2% parasite detection, whereas only 93% of confirmed malaria cases were detected by both rapid tests. About 127 samples were positive by PCR. Since Sysmex XE-2100 automatically detected the presence of malarial parasite with high sensitivity, it can be a good option for presumptive diagnosis in endemic areas. Microscopy remains the gold standard to confirm MP in suspected patients. Rapid diagnostic tests have acceptable sensitivity and specificity.Malaria is the second most prevalent disease in Pakistan resulting in ~30,000 annual deaths. In endemic countries like Pakistan precise and timely diagnosis of malaria is imperative to overcome the associated risks of fatal outcomes. Malarial parasite was screened in 128 malaria suspected patients and 150 healthy controls, by species-specific PCR, microscopy of blood smears, hemoanalyzer Sysmex XE-2100, and rapid test devices . The microscopy detected MP in 126 samples (parasite load/ Plasmodium, namely, P. vivax, P. falciparum, P. malariae, P. knowlesi, and P. ovale. According to an estimate about 40% of the world population lives in high malaria zone [ P. vivax and P. falciparum with P. vivax being more common [ P. falciparum is more severe and may often lead to cerebral malaria and death especially in children. Initially malaria due to P. vivax was generally considered as milder and manageable compared to P. falciparum infection, but recent global reports suggest that P. vivax malaria may cause complications leading to death. The global mortality rate for P. vivax is documented as 0.1–1.6% [ P. falciparum the P. vivax malaria should also be closely monitored to avoid complications and mortality. Thus, timely diagnosis of malaria in endemic areas is vital for early treatment and prevention of fatal outcomes in cases of P. falciparum, P. vivax, or mixed P. falciparum and P. vivax malaria.Malaria is a protozoal disease. It is a parasitic infection of red blood cells. In humans it is generally caused by five different species ofria zone . Pakistae common . Malaria0.1–1.6% . Hence, Due to the limitation of local health service resources, imprecise clinical diagnosis remains the basis of therapeutic care for the majority of febrile patients in remote malaria endemic areas of Pakistan, where laboratory diagnostics is often out of reach. Diagnosis based on clinical features alone has very low specificity and results in overtreatment  and emerµl [The microscopic detection of malarial parasite is generally considered as a gold standard in malaria diagnosis due to low cost and accessibility. Although cheap, specific, and sensitive this procedure requires an expert microscopist and may become unreliable, time-consuming, and laborious at low parasite densities of <1000 parasites/µl . P. falciparum may become complicated and fatal if misdiagnosed or left untreated. In order to detect low level parasitemia and also to detect mono- or coinfection of different parasite species immune-chromatographic/rapid diagnostic testing devices were developed. These devices based on parasite antigens or panspecific aldolases are simple and easy to use. The rapid diagnostic tests (RDTs) for malaria detection are usually based on principle of sandwich ELISA. These are inexpensive and easily available tests and require no prior training. Tests can be performed and results can be interpreted following manufacturer's instructions. Several reports on sensitivity and specificity of various commercial ICT devices are available [ P. falciparum and aldolase of all the Plasmodium species. Reports on poor specificity of these devices for few species like P. ovale led to developing devices based on detection of panspecific parasite lactate dehydrogenase (pLDH) enzyme [Malaria caused byvailable , 13. Dev) enzyme . OptiMal) enzyme .Automated malaria detection by hemoanalyzer is another approach to suspect malaria in febrile patients. Abnormalities in scattergrams of flow-cytometry-based hemoanalyzers like Sysmex XE-2100 and Cell Dyn have been reported as an aid in diagnosing malaria followed by microscopic confirmation .μl [Molecular diagnostic technique like PCR has an edge over the manual microscopy and serodiagnosis by RDTs. It is a reliable technique and can be used for malaria diagnosis. Beside genus specific PCR, species-specific multiplex and nested PCR have been developed for malarial parasite (MP) detection at a threshold of even 1 parasite/μl . The PCRμl .According to the national statistical survey in 2007 malaria results in ~30,000 annual deaths in Pakistan . The disIn this study performance of Sysmex XE-2100, ICT Malaria Combo, and First Response Malaria for early detection of MP was evaluated with microscopy and PCR as gold standard and internal quality control, respectively.This cross sectional study was conducted at National Institute of Blood Diseases and Bone Marrow Transplantation (NIBD), Karachi, Pakistan. The patients and controls were recruited after approval by ethical review committee of NIBD. The study protocol adhered to the tenets of the Declaration of Helsinki.µl) were collected in EDTA tubes from patients admitted at NIBD with clinical suspicion of malarial infection. Following national/WHO guidelines of precise diagnosis prior to treatment for malaria management 128 patients with clinical suspension of malaria were selected for this study. As a control group, sampling from 150 healthy individuals without clinical symptoms of malaria or any other infection or disease was also performed. The control group was confirmed as “malaria negative” by microscopy and RDT at the time of enrollment. The study was conducted over a period of about 9 months from October 2013 to July 2014. Signed informed consent and detailed questionnaire were obtained from the study population.Blood specimens (6000 The complete blood count (CBC) data from Sysmex XE-2100 was recorded following standard machine operating protocol. The data was then analyzed and compared with morphological data to set standard design for automated malaria diagnosis. Sensitivity and specificity of this machine were also evaluated.The microscopic examination of Giemsa/Leishman stained thick and thin blood smear for malaria diagnosis is the gold standard. Thick smear can detect parasite even in low densities since high volume of infected specimen is screened while thin smear helps in species differentiation. For microscopy, both thin and thick smears were prepared immediately in duplicate to avoid any discrepancies in morphological detection of malarial parasites in blood. The smears were stained with 4% Giemsa's/Leishman's stain and observed according to WHO standard guidelines by three independent observers.The immunochromatographic testing was performed using two different RDTs, that is, ICT Malaria Combo and First Response Malaria, on fresh blood samples (not more than three hours old) as per supplier's instructions.Molecular detection of malarial infection based on polymerase chain reaction (PCR) was performed using previously designed primers by Padley et al. 2003. Parasite DNA was extracted from fresh EDTA containing blood using QIAamp DNA Mini Kit . The extracted DNA was amplified and species were identified. The recorded results by agarose gel electrophoresis were then used as quality control to countercheck the data obtained by other diagnostic tests.The data was analyzed by SPSS version 17. The PL was calculated by multiplying number of asexual stages of parasite observed by microscopy with absolute RBCs count per 2500 RBCs. To assess sensitivity and specificity, results of microscopy, automated hemoanalyzer, and RDT were compared with PCR results. The sensitivity was calculated as the proportion of positive test results obtained among samples scored as containing malaria parasites by PCR; the specificity was the proportion of negative test results obtained among samples whose PCR results were negative. Positive and negative predictive values were also calculated as the proportion of true positive or true negative results among all samples scored as positive or negative by PCR, respectively. Youden's J-index was also calculated for comparative performance analysis of different tests. Plasmodium vivax and 19 (14.844%) with P. falciparum while mixed infection of P. vivax and P. falciparum was observed in 18 (14.062%) samples. Parasite load/µl (PL) was also estimated by microscopy to evaluate the degree of severity of malaria. Only parasite load greater than 350 parasites/µl was observed; none of the patients had very low parasitemia  were infected withasitemia . The meaLike other diseases commercially prepared immune-chromatographic rapid diagnostic tests (RDTs) are available to detect malaria. The ICT Malaria Combo and First Response Malaria were compared for their efficacy. Among the two tested devices First Response Malaria seemed to be better with a sensitivity of 91.52%  in basophil region were also observable in approximately all cases of P. vivax, whereas there were no basophils in peripheral film. In case of malaria infection, multiple gray dots in the middle area were observed in the IMI channel despite the absence of immature granulocytes (myelo- and metamyelocyte) and any fluorescent signals above neutrophils in DIFF channel. Furthermore, the presence of gray dots along right side of the box extending vertically down and moving horizontally towards the y-axis in RET-EXT channel was indicative of P. falciparum infection  was missed by PCR. It can thereby be suggested that even PCR may overlook infection with low levels of parasite in blood. This sample was found to be positive by microscopy on third reexamination by expert microscopist.The species-specific PCR, being the internal quality control, was the most sensitive and specific test. It detected 127 malaria positive cases altogether . None ofectively . The PCROn comparison the specificity and sensitivity of microscopy and automated hemoanalyzer were similar . The speµl. It is cheaper when compared with other methods. The only limitation is the risk of human error and thus observer's expertise is required [ P. vivax malaria was missed. This is in line with other studies where cases were either missed or misdiagnosed by microscopy [The present study was designed to evaluate the utility in terms of efficiency of existing routine malaria diagnostic tests compared with gold standard microscopy and PCR as internal quality control. Microscopy being the gold standard was the only test producing quantitative results in the present study. The sensitivity of microscopy by thick smear is 5–10 parasites/required . In the croscopy .Two commercially prepared rapid test devices, that is, ICT Malaria Combo and First Response Malaria, were compared for their efficiency with microscopy as gold standard. Both the test devices were accurate with the accuracy of 93.16% (95% CI: 90.16–96.16) and 93.81% (95% CI: 89.7–97.7), respectively. The First Response Malaria was found to be more efficient device with a sensitivity of 91.52% (95% CI: 87.52–95.52). The sensitivity is comparable with an earlier study by Bharti et al.  on FirstRecent research is directed to detect malaria on the basis of the abnormal scattergrams of flow-cytometry-based automated hemoanalyzers , 24, 25.About 95% malaria cases had abnormal WBC scattergram during the present study. Most common abnormality was found to be graying of eosinophil and neutrophil populations (53.12%). Other common abnormalities were overlapping of eosinophil and neutrophil populations (20.30%) and two eosinophil populations (32.80%).A rightward shift of RBC ghost in WBC/BASO and DIFF scattergrams was also very commonly found in most malaria positive cases. This can be attributed to the presence of extracellular pigment and RBC lysis which are reflected in that area . Pseudoe P. vivax in one sample which was MP negative by microscopy. Additionally, this particular sample was also negative by both RDTs while clear abnormal signals suggesting presence of MP were observed in scattergrams on Sysmex XE-2100. This observation is in line with another study conducted in Pakistan where real-time PCR detected 3 samples missed by microscopy [ P. falciparum/vivax endemic area [µl. The influence of low PL on performance of PCR was also recorded during the present study where MP positive case of P. vivax infection (PL: 386/µl) was found to be false negative . Observer's training is required for identification of abnormal signals in scattergram. It must also be borne in mind that abnormal scattergrams may be observed in other conditions like dengue, basophilic stippling, thalassemia, and chronic myeloid leukemia. Observed scattergram abnormalities can only depict the presence of MP and cannot be used to differentiate between species ofThus, after comparison it can be concluded that the microscopy of thick and thin films remains the gold standard for malaria diagnosis despite chances of human error. The microscopy may be confirmed with PCR since the specificity and sensitivity for PCR are the highest. In remote endemic areas where microscopy due to absence of expert microscopists seems impossible, automated hemoanalyzers can serve as a useful adjunct to timely clinical diagnosis of malaria. The positive signals on hemoanalyzers need further confirmation by microscopy and PCR up to species level at the nearest reference lab to avoid unnecessary treatment, leading to development of drug resistant strains of MP. The comparative high cost of PCR limits its applicability in most diagnostic labs in developing countries, and we hereby recommend it to be added as a mandatory confirmatory test at least at all national reference labs.


Plasmodium falciparum (0.96 – 0.01) was achieved between 2008 and 2012. As part of the elimination campaign, active detection of asymptomatic Plasmodium species by a highly sensitive method was deemed necessary. This study was carried out to determine asymptomatic Plasmodium species carriage by nested PCR in the country, in 2012.Botswana is one of eight SADC countries targeting malaria elimination by 2018. Through spirited upscaling of control activities and passive surveillance, significant reductions in case incidence of Plasmodium species in 14 districts . Venous blood was taken from each participant for a nested PCR detection of Plasmodium species.A cross-sectional study involving 3924 apparently healthy participants were screened for Plasmodium species detected were as follows: Plasmodium falciparum, 0.16 %; Plasmodium vivax, 4.66 %; Plasmodium malariae, (Pm) 0.16 %; Plasmodium ovale, 0 %, mixed infections , 0.055 %; and , 0.027 %, . The high proportion of asymptomatic reservoir of P. vivax was clustered in the East, South Eastern and Central districts of the country. There appeared to be a correlation between the occurrence of P. malariae infection with P. vivax infection, with the former only occurring in districts that had substantial P. vivax circulation. The median age among 2–12 year olds for P. vivax infection was 5 years . The odds of being infected with P. vivax decreased by 7 % for each year increase in age .The parasite rates of asymptomatic Plasmodium species in Botswana, with the exception of P.vivax which was unexpectedly high. This has implication for the elimination campaign so a follow up study is warranted to inform decisions on new strategies that take this evidence into account in the elimination campaign.We have confirmed low parasite rate of asymptomatic  Malaria disease still kills close to half a million people annually with 90 % of the deaths occurring in sub-Saharan Africa . RecentlPlasmodium known to infect humans,  Pf infection is the major known cause in Botswana. It is estimated that 98 % of malaria infections are Pf, while 2 % is attributed to Pv [Plasmodium species are sought out actively for targeted intervention. It is also well established that in malaria elimination countries, as Pf incidence rate goes down Pv incidence rate increases [Plasmodium spp was undertaken in 2012. This was carried out throughout all defined zones of endemicity in the country, using PCR methodology for the case detection. The choice of PCR methodology was informed by the fact that it is well recognized that PCR consistently detects at least twice as many infections as light microscopy and therefore an excellent choice when high sensitivity is desired [In general the trend in transmission in Botswana is seasonal and unstable ranging from high endemicity in the northern part of the country to very low in the southern part of the country Fig. . This laed to Pv . The humed to Pv –9 as thencreases , becausencreases , 12. As  desired , 14. TheStudy sites were selected with the support of Statistics Botswana from five endemic and nine non-endemic districts in Botswana. These were respectively, Okavango, Ngami, Tutume, Boteti and Bobirwa (Endemic areas), and North East, Francistown, Serowe-Palapye, Ghanzi, Kweneng West, Kweneng East, Kgatleng, South East, and Good Hope (epidemic areas) Fig. . Within n = (z2)(r)(1-r)(f)(k)/(p)(ñ)(e2). Using an estimated Pv known prevalence for Africa (r) of 10 %); z statistic of 1.96 for the 95 % Confidence Interval, design effect (f) of 2.4; non response rate (k) of 8 %; the proportion of the population under 12 (p) of 0.34; average household size (ñ)of 3.4; and margin of error (e) of 8 %; a sample size of 897 households was obtained from which the total number of samples were derived based on the population of the districts. Children aged 6–12 years were largely from the primary schools while those aged from 2 to 5 were enrolled from the child welfare clinics and health posts. The subjects from the clinics did not present with any disease except normal growth monitoring follow up visits. All the enrolled children were asymptomatic for malaria. Asymptomatic subjects were individuals with body temperatures <37.5 °C at the time of sample collection and without a history of fever in the preceding 72 h. A subject was enrolled if the individual had no complaints related to malaria, had not left the village for a year at least and parental consent was given for blood to be taken. The sample collection was timed to coincide with the onset and peak of the malaria season in the country.The study included 3624 subjects of ages 2–12 enrolled within a month in May 2012, in the identified catchment area schools, welfare clinics and healthposts, using a multistage sampling strategy. The first stage was a purposeful selection of districts with known malaria transmission, classified as either highly endemic, moderate, low or sporadic, based on recommendations from the National Malaria Control Program (NMCP). In the second stage which was also purposeful, towns within the districts that constituted the focal areas of transmission were selected based on NMCP recommendation, using guidelines on classification of malaria transmission endemicity. This was to ensure that areas of high, moderate, low and sporadic transmissions with distinct characteristics in each community were captured in the sampling process. In the third stage, schools/clinics within each town were purposefully selected using a two stage clustering approach based on population density and adequate cross sectional representation of the communities to avoid any bias. In the final stage, participants were assigned numbers and randomly enrolled following informed parental consent and consent of heads of schools and clinics. The total number of samples obtained per district/town was directly proportional to the estimated population. The formula used in calculating the sample size was: After consent from the guardian or parent of the subjects, a short clinical assessment of tympanic temperature was done and history of malaria and travel requested. Subsequently, basic demographic information on age and sex was documented. A 2.5 to 3 ml venous blood was collected into EDTA vacutainer tubes from which three Dried Blood Spots (DBS) were made unto whatman number 3 filter papers enclosed in zip lock bags with desiccators each. Samples were stored in ice packs (DBS papers were retained at room temperature) and transported to the laboratory within 30 min. Blood was separated by centrifugation into plasma and cell pellets which were frozen at -20 °C and subsequently -80 °C till analyzed.Genomic DNA (gDNA) was isolated from red blood cell pellets  using the QiaAmp DNA Blood mini kit  or Quick-gDNA™ Blood Mini Prep  and quantified with the Fisher Thermo Scientific Nanodrop 2000/2000C USA. All DNA were eluted into 100 μl elution buffer and stored at -80 °C till used for PCR.Plasmodium species were detected by a slight modification  of the double nested PCR procedure targeting conserved species specific regions of the small subunit 18S ribosomal RNA (18S ssRNA) [Plasmodium species DNA, from ATCC, Virginia, USA. The primer sequences were exactly as published previously [All S ssRNA) . The fireviously .2, 50 mM KCl, 250nM of each oligonucleotide primer, 200 μM of each of the four dNTPs and 0.5 units of Dream Taq DNA polymerase. 5 μl of DNA prepared from whole blood was used to initiate the genus specific primary amplification PCR reaction and 2 μl of the product was then used in the secondary amplification including the species specific detection assays. The PCR cycling parameters for the primary amplification were as follows: initial denaturation at 94 °C for 10mins, preceded the cyclin conditions which consisted of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and extension at 72 °C for 1 min for 35 cycles. After the final annealing step followed by extension at 72 °C for four minutes, the reaction was ended. In the second nested PCR, annealing temperature of 62 °C was used. For the species specific amplifications the only changes were initial denaturation at 94 °C for four mins and annealing at 58 °C.All amplification reactions were carried out in a total volume of 25 μl and in the presence of 10 mM Tris-HCl, pH 8.3, 4 mM MgClTo ensure that there was no cross contamination, negative control samples (no DNA template) were randomly included in the run. The consistency of the results was checked by multiple runs of a number of subsets of the samples which were randomly picked.The amplified products were analyzed on 2 % agarose gels by electrophoresis, followed by visualization on a UV transilluminator after ethidium bromide staining.Data were entered into an Excel data sheet and used in SPSS and STATA v11.2  for analysis. Descriptive statistics were provided for relevant demographic covariates. Comparisons of categorical variables were done with chi-square testing or Fisher’s exact test. Factors influencing the likelihood of malaria infection were explored using forward stepwise logistic regression.Plasmodium species. The mean age of all enrolled subjects was 5.48, which was distributed as follows: <2 years, 0.084 %; 2- < 5, 41 %; 5- < 9, 47.27 %; >9 years, 11.65 %; while gender was 51.1 % female. None of the participants had traveled outside the country for the past year.A total of 3624 children across 14 districts were assessed for Plasmodium species infection segregated as follows: Pf, 0.16 %; Pv, 4.66 %; P. malariae (Pm), 0.16 %; P. ovale, 0 %, mixed infections (Pf and Pv), 0.055 %; and (Pv and Pm), 0.027 %. Pv infection accounted for 93.37 % of the Plasmodium species infection in the population. The distribution of Plasmodium species infection by district is shown in Table Pv infection was clustered around East , South East  and Central districts (Kweneng East)  in the Tutume region and 7.10 times higher  in the Kweneng East region than in the rest of the districts that had detectable Pv infection. The median age of those infected with Pv was 5 years  while those not infected with any species of Plasmodium had a median age of 6 years . The age breakdown for those infected with Pv was: <5 years, 72 positives from 1466 specimens (4.91 %); 5- < 9 years, 65 positives from 1688 specimens (3.85 %); 9–12 years, 16 positives from 416 specimens (3.85 %). These differences in positivity between age categories were not statistically significant. However, the odds of being infected with Pv decreased by 7 % for each year increase in age . Districts with higher numbers of Pv infections appeared to have higher numbers of Pm infections as well; Pm infections were only detected in the district with the most Pv transmissions (Kweneng East), the second-most Pv infections (Tutume), and the Francistown district. We did not encounter any problem with contamination of samples.Overall 4.99 % of individuals had Pf reservoir determined in this study is only 0.16 %. However, our identification for the first time of a significant asymptomatic Pv reservoir in the country has brought in a new paradigm for the elimination program that requires immediate attention, in order to sustain the gains made to date and achieve the new target of elimination by 2018. Our findings add to the growing evidence of the presence of Pv in Africa. As has been observed in other countries, the reservoir of Pv was clustered, [An. moucheti and An. vinckei [Pv transmission within specified populations in Botswana and sub-Saharan Africa.The Botswana malaria elimination program has clearly been on track as the asymptomatic ustered,  centerin vinckei , 21. It  vinckei . It appePv in malaria transmission and elimination in sub-Saharan Africa is increasingly drawing attention. Infection of rbcs by Pv is traditionally established to be attributed to positivity for the Duffy antigen, also called Duffy antigen receptor for chemokines (DARC) [Pv is considered an unlikely scenario. However, past and recent reports from several countries in sub-Saharan Africa, including Liberia, Senegal, Equitorial Guinea, Angola, Ethiopia, Cameroon, Kenya and Malagasy, have shown that Duffy negativity alone does not preclude infection with Pv [Pv infections were found [Pv infection, may first be focused on host characteristics within the populations including the environment, parasite genetic variability and available competent vectors. Recently a report on modeling of Pv infection dynamics revealed that up to 90–96 % of Pv infection in endemic areas is due to hypnozoite reactivation [Pm infection and Pv infection in this study  but as to whether they play complimentary roles in infectivity requires further interrogation. Pv appears to show high clonal diversity in endemic regions, so that genotyping approaches will also be necessary in elucidation of the infection dynamics.The role of s (DARC) –25. A si with Pv –31. The  with Pv , so the re found . Furthertivation . A majortivation , 34. We reactive case detection and proactive case detection. In a reactive case detection model, a trigger of a passive case leads to the screening of households including neighbors [Our findings also highlight the fact that active case detection (ACD) in malaria elimination is crucial in the identification of hotspots. ACD is expensive so malaria elimination programs need to think through appropriate models that are cost effective. Two suggested models are eighbors . The limPv asymptomatic infection in Botswana and also affirmed that the malaria control program targeting Pf has indeed been on track. New and concerted strategies to achieve elimination of malaria in 2018 in Botswana are urgently required as are efforts at better understanding of characteristics of susceptible host, environmental factors and Pv biological characteristics.We have provided molecular evidence for the first time of