Plasmodium infections for malaria elimination depends on its size, which is generally considered small in low transmission settings. The precise estimation of this reservoir requires more sensitive parasite detection methods. The prevalence of asymptomatic, sub-microscopic malaria was assessed by a sensitive, high blood volume quantitative real-time polymerase chain reaction method in three countries of the Greater Mekong Sub-region.The importance of the submicroscopic reservoir of Plasmodium falciparum/pan malaria rapid diagnostic tests (RDTs), microscopy and a high volume ultra-sensitive real-time polymerase chain reaction (HVUSqPCR: limit of detection 22 parasites/mL). All villagers older than 6 months were invited to participate.Cross-sectional surveys were conducted in three villages in western Cambodia, four villages along the Thailand–Myanmar border and four villages in southwest Vietnam. Malaria parasitaemia was assessed by P. falciparum, 357 (7 %) with Plasmodium vivax, 56 (1 %) with a mixed infection, and 411 (8 %) had parasite densities that were too low for species identification. A history of fever, male sex, and age of 15 years or older were independently associated with parasitaemia in a multivariate regression model stratified by site.A census before the surveys identified 7355 residents in the study villages. Parasite prevalence was 224/5008 (4 %) by RDT, 229/5111 (5 %) by microscopy, and 988/4975 (20 %) when assessed by HVUSqPCR. Of these 164 (3 %) were infected with Plasmodium reservoir is considerable, even in low transmission settings. Novel strategies are needed to eliminate this previously under recognized reservoir of malaria transmission.Light microscopy and RDTs identified only a quarter of all parasitaemic participants. The asymptomatic The online version of this article (doi:10.1186/s12936-015-0906-x) contains supplementary material, which is available to authorized users. Plasmodium infections in asymptomatic carriers may play a critical role in the elimination of malaria [Eliminating the submicroscopic reservoir of  malaria . In low  malaria . In comp malaria , 4. For A series of cross sectional surveys were conducted along the Thailand–Myanmar border, in Western Cambodia, and Vietnam using a sensitive PCR detection method based on larger blood volumes than conventional PCR methods use . In the Plasmodium vivax and P. falciparum have historically each comprised approximately half the clinical cases, although with recent reductions in overall malaria incidence, P. vivax now predominates [P. falciparum to chloroquine, sulfadoxine-pyrimethamine and mefloquine. More recently, P. falciparum strains with reduced susceptibility to artemisinins have been detected in this region [Surveys were performed in malaria-endemic areas along the Thailand–Myanmar border, in western Cambodia, and south-western Vietnam Fig. . In thesominates . The regs region –14.Fig. In response to large numbers of malaria cases in Myanmar close to the North-western border with Thailand and requests for assistance, health posts with facilities for malaria diagnosis and treatment were installed in several villages. These were the focal point for the conduct of limited cross- sectional surveys in 16 villages to plan optimum control interventions  was collected from all individuals aged ≥5 years, and 500 µL from children aged ≥6 months–5 years. Participants with fever ≥37.5 °C were tested for malaria by rapid diagnostic test (RDT), and were treated if positive according to national guidelines.P. falciparum/Pan one-step RDT was used ; in Thailand–Myanmar border areas and in Vietnam, the SD Bioline Malaria Ag Pf/Pan POCT was used . The RDTs distinguish between P. falciparum infections, non-P. falciparum infections, and no infections. Haemoglobin was measured using Hemocue . Sample processing for the quantitative PCR included separation of plasma, buffy coat, and packed red blood cells, which were frozen and stored at −80 °C. The frozen samples from the Thailand–Myanmar Border and Cambodia were transported monthly on dry ice to the laboratory in Bangkok, Thailand and the samples from the Vietnam sites were shipped to Ho Chi Minh City, Vietnam for DNA extraction and quantitative PCR.The blood samples were stored in a cool box in the field and then transported within 12 h to the local laboratory for further sample processing and RDT assessment. In Cambodia, the Healgen malaria Standard microscopy was performed by microscopists who had at least 5 years experience and/or were confirmed to be Level 2 or better with a WHO 55 slide set. The number of parasites/500 white blood cells was counted on Giemsa-stained thick films.Plasmodium was purified from the thawed packed red blood cells samples. Purified DNA was dehydrated in a centrifugal vacuum concentrator and then suspended in a small volume of PCR grade water resulting in a concentration factor defined by the original blood volumes (100–2000 μL) divided by the resuspension volume (10–50 µL). Two microlitres of resuspended DNA was used as template in the qPCR reaction. The presence of malaria parasites and an estimate of the parasite numbers (genomes) in each sample were assessed by an absolute quantitative real-time PCR (qPCR) method . The 18S rRNA-targeting primers and hydrolysis probes used in the assay have been validated and are highly specific for Plasmodium species [Detailed description, evaluation and validation of the high-volume ultrasensitive real-time polymerase chain reaction (HVUSqPCR) methods have been reported recently in detail . In summ species . The low species .Plasmodium species present using nested PCR protocols specific to P. falciparum (microsatellite marker Pk2), P. vivax (microsatellite marker 3.502) and Plasmodium malariae (18s rRNA) as described previously [Plasmodium spp.).For samples where the HVUSqPCR was positive, an attempt was made to determine the eviously –24. Sampnptrend command in STATA which is based on the Wilcoxon rank-sum test. The detection of parasitaemia was summarized by location, age and gender strata as specified a priori in the analysis plan. Sensitivities and specificities for malaria diagnosis were calculated using HVUSqPCR as the reference standard. Overall and study location specific risk factors for parasite carriage (detected by HVUSqPCR) were assessed by logistic regression with household fitted as a random effect. For all models, a stepwise approach was used and only variables significant at the 0.05 level were retained in the final models. All analyses were performed using Stata, version 13 .For the purposes of analysis, fever was defined as a tympanic temperature >37.5 °C. Anaemia was defined as “None” if the haemoglobin (Hb) was ≥11 g/dL, “Mild” if Hb ≥8–<11 g/dL, and “Moderate” if Hb <8 g/dL. Characteristics of the study population and clinical association with presence of parasitaemia were compared using the Chi squared test. Nonparametric testing for trend was performed using the The studies were approved by the Cambodian National Ethics Committee for Health Research  the Institute of Malariology, Parasitology and Entomology in Ho Chi Minh City (185/HDDD dated 15 May 2013), the Institute of Malariology, Parasitology and Entomology in Qui Nhon (dated 14 Oct 2013) and the Oxford Tropical Research Ethics Committee .A census prior to the surveys identified 7355 residents in 11 study villages, 1766 in three Cambodian villages , 2377 in four villages along the Thailand–Myanmar border  and 3212 in four villages in Vietnam   participants’ blood samples tested positive for the presence of Plasmodium DNA, of which 164/4975 (3 %) were P. falciparum, 357 (7 %) P. vivax, 56 (1 %) were mixed infections and in 411 (8 %) of specimens it was not possible to determine the species because of the low parasite DNA content. The data disaggregated by village are shown in Additional file Plasmodium species other than P. falciparum and P. vivax were detected.Using rapid diagnostic tests (RDT) overall 224/5008 (4 %) participants tested positive  and for P. vivax , the sensitivity of RDTs was 5 % and of microscopy 3 %.Using HVUSqPCR as the reference-standard, the overall sensitivity for RDTs to detect  % Table . The senParasite prevalence detected by HVUSqPCR was lowest in Vietnam , followed by Cambodia  and 34 %; 520/1536) on the Thailand–Myanmar border  with P. vivax infections, 112/3412 (3 %) without parasitaemia and 0/48 with mixed infections . The geometric mean (95 %CI) parasite density in the 47 febrile patients was 3729  parasites/mL, compared to 1151 (905–1464) parasites/mL in afebrile individuals. For both P. falciparum or P. vivax was it impossible to determine a clearly delineated pyrogenic parasite density threshold  participants with 51 905–144 parasitP. falciparum, mild anaemia (Hb 8–<11 g/dL) was present in 30/163 (18 %) participants and moderate anaemia (Hb <8 g/dL) in 7/163 (4 %) (Table P. vivax 39/354 (11 %) had mild anaemia and 5/354 (1 %) had moderate anaemia . Among 3889 participants without evidence of parasitaemia by any detection method 619 (16 %) had mild anaemia and 96 (2 %) had moderate anaemia. Parasitaemic participants without anaemia (n = 819) had a geometric mean (95 % CI) parasite density of 1159 (912–1474) parasites/mL, compared to 608 (295–1253) parasites/ml in those with mild anaemia (n = 137), and 1787  parasites/ml with moderate anaemia. Parasitaemic participants who were febrile on the day of the survey were more likely to have mild anaemia  or moderate anaemia  compared to afebrile participants .In participants carrying %) Table . Among tA multivariate logistic regression model was constructed to identify risk factors for parasite carriage (as detected by HVUSqPCR), which included anaemia, fever at the time of the survey, a history of fever, sex, occupation, and age group. Of these, only a history of fever, male sex, and age equal or older 15 years were independently and significantly associated with parasitaemia Table . CountryPlasmodium parasites. In the GMS and in epidemiologically similar areas in South-East Asia and perhaps beyond the prevalence of malaria infections seems to have been substantially underestimated. The HVUSqPCR Plasmodium  prevalence estimates in 11 villages in the GMS were approximately four times higher than estimates based on microscopy or RDT. The majority of asymptomatic carriers had Plasmodium densities below the lower limits of detection for microscopy, as well as for conventional low volume PCR methods . Parasite DNA detected by HVUSqPCR is likely to represent living parasites, since mRNA coding for Plasmodium species is also detectable in the same patient samples . The findings suggest that the submicroscopic parasite reservoir could be important for transmission between seasons. It will be important to study the longevity and transmissibility of these infections within the human host.This study demonstrates that in areas of the Greater Mekong Subregion (GMS) classified as hypoendemic, a considerable proportion of asymptomatic individuals carry In this study, molecular methods were not used to assess gametocytaemia which would require RNA measurement. This represents a limitation of the study since light microscopy which was used has a much more limited sensitivity and did not detect gametocytaemia . SubmicrThere was substantial heterogeneity between villages in the ratios between RDT, microscopy and HVUSqPCR malaria prevalence rates, which suggests that extrapolation of prevalence from detection rates based on the conventional techniques will be imprecise. The HVUSqPCR technique used in this study has a lower limit of detection of around 100,000 parasites in the entire blood volume of an adult and so will still be unable to detect lower circulating parasitaemias or sequestered non-circulating parasites . Very loParasite densities were lower in the selected villages in Vietnam and Cambodia compared to the Thailand–Myanmar border areas. In the Cambodian study villages all detected parasitaemias were on or below the threshold of parasite densities detectable by microscopy. In assessing these very low parasite densities, false positive PCR results are a concern. A rigorous quality control system was in place throughout the study, and the consistent HVUSqPCR negativity of control samples provide reassurance that the prevalence estimates are not inflated.P. falciparum and in eight specimens P. vivax parasites while the HVUSqPCR was negative. In the PfHRP2-based RDTs for P. falciparum this could be due to persistent antigen after the infection has been eliminated. PfHRP2 is eliminated slowly and thus could accumulate in chronic infections. Alternatively this finding could reflect true false positive RDTs. In 411/988 (42 %) of infections detected by HVUSqPCR, it was not possible to determine the Plasmodium species as there was insufficient Plasmodium DNA to perform the species identifying nested PCRs. Even more sensitive techniques will be needed to determine whether the species composition of these “undetermined infections” is similar to the composition of the identified species.In 38 specimens, RDTs detected The findings of this study have implications for control, elimination, and eradication of malaria, and in particular for the urgent need to contain and eliminate artemisinin resistant falciparum malaria in South-East Asia. Screening and treatment activities will only identify a minority of parasitaemic individuals, and will not eliminate malaria rapidly enough to stop the spread of artemisinin and multidrug resistant malaria . The onl


Plasmodium ovale and Plasmodium malariae as mixed infections with Plasmodium falciparum. In Tshimbulu, Western Kasai, during a humanitarian initiative designed to provide children with free preventive screening and to reduce the local high malaria death rate, accurate species identification was performed,in order to collect unambiguous epidemiological data and to evaluate the reliability of locally applied diagnostics.The literature data on malaria in Western Kasai, DRC, are limited and inadequate. A recent molecular survey there has detected Finger pricks provided fresh blood for microscopic analysis (MA), for rapid diagnostic test (RDT) and for molecular diagnostics (MD). MA and RDT were first performed by the local team and then a re-interpretation of the results (on the same slides and on RDT’s taken pictures) was conducted in Italy, where MD were performed.P. falciparum-positive . MA identified a further four children as positive to P. falciparum and six co-infections with P. ovale. The second RDT evaluation confirmed a similar infection rate (78.2 %) but interpreted as two-band positive a significantly higher share of tests (56.8 %). MA confirmed 80.0 % of the children as malaria positive and, in addition to P. falciparum, identified P. malariae (13.8 %), P. vivax (3.4 %) and P. ovale (2.4 %), and detected Babesia microti in 19 smears. MD confirmed all of the species found (Babesia microti included), classified as mono-infection with P. falciparum a rate of spots comparable to MA revision, and identified all P. ovale as Plasmodium ovale wallikeri. The RDT used locally proved 93.1 % sensitive and 92.1 % specific for P. falciparum.The analysis was conducted on 306 children; RDT found 80.9 % as Plasmodium species, highlighted in this study, identified a sanitary issue which proved to be more alarming than expected, as it was worsened by the unpredictable presence of P. vivax and Babesia microti (never before reported in DRC). Each diagnostic tool showed its point of weakness. Therefore, the most correct approach is by the combined use of different, locally available, diagnostic tools.The malaria prevalence among the children and the presence of four  In the Democratic Republic of Congo (DRC) the Ministry of Health reports malaria as the main cause of morbidity and mortality, mainly affecting pregnant women and children under 5 years of age. This disease accounts for more than 40 % of all outpatient visits and for 40 % of deaths in children <5 years of age, as they are often hospitalized when their anaemia is too severe .Despite the high impact of the disease, the epidemiological data are inadequate, as they mostly derive from local studies that do not allow for national estimates. Only the 2007 demographic data and 2013–2014 health surveys showed that 33.5 % of women and 34.1 % of children aged six to 59 months were positive in molecular testing .Plasmodium knowlesi, which for a long time was falsely diagnosed as P. malariae and later correctly identified in humans only through the use of MD  according to national guidelines based on Malaria Operational Plan Fy 2013 (http://apps.who.int). A further 5 µL of blood were used to perform RDT  and, starting from the fifth day, two drops of blood were drawn and used to impregnate a filter paper for MD.All subjects were examined clinically and axillary temperatures were recorded. Fresh blood was obtained by pricking a child’s finger, and standard malaria thick and thin smears were prepared and stained with 10 % Giemsa solution, following the method described in the WHO Malaria Microscopy Guide , with data on anaemia, fever and course of the infection during therapy.RDT was locally performed according to manufacturer’s instructions (RDT1), and serially numbered. Each of them was read and then photographed within 15–30 min, all in the same place, position and lighting conditions, controlling the match between each image and the related device test. In this way pictures were available for both a second interpretation (RDT2) and for the final comparison of the findings obtained by means of the three methods.Giemsa-stained smears were locally examined (triple blinded) by microscopy at 100× in the hospital laboratory (MA1). A whole thick smear was read before declaring a slide negative. Parasite density was estimated by means of the Plus system, and species identification was confirmed on the corresponding thin film. A further (double) blinded microscopic analysis on 290 undamaged available slides was carried out in Italy to evaluate the level of accordance of the results (MA2).DNA was extracted from a 3 × 3 mm diameter circle of each of the 211 dried blood spots using the commercial kit Dried Blood spot DNA isolation Kit  and following manufacturer’s instructions. In some cases a further DNA extraction was necessary from additional paper circles.Plasmodium species in human blood samples [Plasmodium spp. rRNA gene fragment (ssrRNA 18S) of about 250-bp was amplified. Then a P. falciparum, P. malariae, P. ovale, and Plasmodium vivax species-specific PCR protocol was employed on all genus-specific PCR positive samples, with primers and thermal profiles described in Table 2, 0.2 mM of each dNTP, 1 unit of DNA polymerase  and 5 μL of template. The primer concentrations were 15 pmol of P. falciparum, 17.5 pmol of P. ovale and P. malariae, and 7.5 pmol of P. vivax forward.As previously described for diagnosing  samples , a Plasmt ssrRNA 8S of aboPlasmodium were included in each PCR run as positive control, whereas 5 μL of double-distilled water was employed as negative control. Amplicons were purified using the SureClean kit  following manufacturer’s instructions, and were then directly sequenced with PCR primers in both directions by an external sequencing core service . The resulting chromatograms were analysed and edited. The obtained sequences were compared to those from Plasmodium deposited in GenBank™ and available at the website: http://www.ncbi.nlm.nih.gov by using the BLAST application, and aligned using the software CLUSTAL X  for comparative analysis. In addition, having the MA2 detect the presence of Babesia spp. in several blood samples, the corresponding DNA were further amplified using the primers CRYPTO F (5′-AACCTGGTTGATCCTGCCAGT-3′) and RLB-R2 (5′-CTA AGA ATT TCA CCT CTG ACA GT-3′), which amplify a fragment of approximately 800 bp of the piroplasm 18S ribosomal RNA, using the previously described protocol [To test the specificity of the reaction, 5 μL of DNA extracted from blood samples microscopically positive for protocol . Amplico2 test, and p values <0.05 were considered significant. Cohen’s K index was calculated to evaluate the concordance between the results obtained, for each sample, by each considered diagnostic.Different frequency distributions were evaluated by means of the χIn the last 14 days of November 2014, a total of 306 children were screened for malaria. They were aged from 1 week to <5 years .Prevalence evidenced by each applied method is reported in Table P. falciparum   and 80.9 % (95 % CI 76.5–85.5) of the children were classified as positive to P. falciparum, suspected the co-presence of P. ovale in six smears (2.0 %) , a result which proved to be no statistically different from the one obtained in loco (p = 0.406), although this revision revealed 56.7 and 43.2 % of infected children as two-band and three-band positive, respectively  and identified 222/284 infected children. Therefore, the overall prevalence of P. falciparum, one patient by P. vivax (0.3 %) and 73  with mixed infection, for an overall infection rate of 80.0 % (CI 75.4–84.6), not statistically different from MA1 and RDT2 results . However, on a quality level, MA2 identified P. falciparum as the most prevalent species , followed by P. malariae , P. vivax , and P. ovale .The revision by MA of available thick and thin smears  and, in one case, as a triple infection. As for P. vivax, this species was identified as single (n = 1) and mixed infection . Furthermore, the revision of the slides evidenced the presence of Babesia microti in 19 smears, always associated to P. falciparum and classified, by RDT, as P. falciparum two-band (n = 12) and three-band (n = 6) infection, and ‘invalid test’ (n = 1)  identified by second slide examination (MA2) was significantly different from that evidenced by MA1 , as confirmed by the poor overall concordance (K = 0.08) observed between the results on the available comparable specimen. As expected, MD detected the highest number of infections: 164/198  children proved to be positive  is significantly different (p < 0.0001) only from that detected by MA2 (54.5 %). However, considering the results of MA2 concerning only 164 children positive to MD, the difference in prevalence of mono-infection with P. falciparum is withdrawn: the concordance between the two diagnostics was excellent (K = 0.84).The overall prevalence of single infections with Plasmodium species present in samples classified by RDT1 as two-band and three-band positive. With the exception of 4 % of the samples, which had initially been read as positive, two-band positive results were also caused by mixed infections with P. vivax and Babesia microti, whereas three-band positive by those with P.vivax, P. ovale and P. malariae.Finally, Fig. in loco with all the diagnostic tools, sensitivity and specificity of the tests in loco performed in detecting malaria due to P. falciparum were 93.1 and 92.1 % (RDT1), and 93.2 and 100 % (MA1), respectively.With MD as the gold standard, and considering only the 198 subjects tested P. vivax. Moreover, and for the first time in DRC, human infections with the zoonotic Babesia microti, which may contribute to anaemia, were detected.Epidemiological data on malaria in Western Kasai, DRC, are limited and inadequate, despite this country is suffering the second-highest burden of malaria globally ; the infP. malariae and P. ovale  was confirmed and the additional presence of P. vivax was evidenced. In the studied area, where the lack of the Duffy antigen is widespread, the existence, although at very low prevalence (1.2 %), of this unexpected Plasmodium species should draw attention to the possible clinical and epidemiological consequences. Indeed, it is well known that: i) P. vivax gametocytes are produced earlier than those of P. falciparum [Anopheles mosquito vectors at lower parasite densities than those of P. falciparum, and more efficiently [P. falciparum elimination [P. falciparum, this species is resistant to chloroquine [P. vivax make it necessary to be aware of its geographical distribution in order to plan appropriate control measures, aside from applying a suitable therapy to infected subjects.Comparing the present findings with available data, malaria prevalence 82.8 %) evidenced by MD in children of Tshimbulu pointed out a sanitary problem which turned out to be more alarming 43.7 %) than reported by molecular studies on malaria epidemiology in Western Kasai [.7 % than.8 % evidlciparum , and areiciently ; therefoiciently ; ii) it mination , 19; andPlasmodium species affecting children in Tshimbulu, and Babesia microti is present, even if all of them are in low percentages, which means in loco performed analyses are satisfactory in detecting infection, but not as satisfactory as identifying the species involved.Notwithstanding these relevant findings, the accuracy of diagnoses performed in the limited resource setting of DRC and, then in a research laboratory, was not entirely satisfactory. Indeed, considering that MD was not applied to 106 samples (93 not collected plus 13 not suitable dried spots), and, therefore, in these cases the three methodologies could not be compared, analysis of the results obtained by means of RDT, MA of the blood and MD, suggests the following observations. In respect of overall infection rates, RDT1 and MA1 results were in greater agreement with MD than RDT2 and MA2 findings that, however, were not significantly different from MD , the employed RDT proved to be less sensitive and specific. Moreover, as expected (data sheet informs that three-band positive sample do not means mixed infection), it did not allow identification of mixed infections and, even less so, the species involved in infections, data very important for possible relapses due to the presence of P. vivax and P. ovale and for possible renal damage induced by P. malariae. Considering the additional well-known criticism about the inability to evaluate parasitic load, which is essential to define the severity of the infection, the immediate measures necessary, and the treatment efficiency, RDT cannot be the only employed method.Locally applied RDT demonstrated its usefulness for rapid diagnosis of malaria; however, it is useful to note that at least 1.9 % of infections remained undiagnosed, and Pan weak bands were exposed to doubtful interpretation, probably due to partial corruption during transportation (it is known that test line intensity may decrease with either parasite density and exposure to >70 % humidity and/or >30 °C). Therefore, in respect of the performance assured  may be overlooked or not recognized. The lower (not significant) sensitivity shown by MA2 (80.0 %) when compared to MD (82.8 %), accepting as a true result the presence of 15 microscopically negative smears (as confirmed by negative RDT) in the first group of children unexamined by MD, becomes less relevant, and totally overlaps the results from children examined by both diagnostics (100 % sensitivity). The same applies for mixed infections: several mixed infections detected by MA2 in the first 100 children may have been lost by the unapplied MD, so that the prevalence of mono-infections with P. falciparum stated by MD is only apparently higher than that identified by MA2, as confirmed by the excellent concordance (K = 0.86) about simple and mixed infections observed on the remaining 164 children proved positive to both diagnostics. However, results obtained by this method sometimes proved to be uncorrected. In detail, co-infections of P. falciparum with P. malariae diagnosed during the revision of the slides proved overestimated, may be due to a misidentification of P. falciparum in cases of severe malaria (the data on corresponding children reported a very low Hb concentration). Indeed, due to P. falciparum cyto-adherence, in these cases peripheral blood included trophozoites developed further than usual little rings: they are very alike P. malariae and infection may be identified as co-infection. This pitfall has been overcome during MA1 because the local team, who frequently identify severe malaria, is probably used to recognizing P. falciparum in these conditions. The second (possibly in the absence of corresponding MD identification) misdiagnosis of MA2  occurred in cases of macrocytic anaemia and when slides were badly stained, which make it reasonable that there was an absence of evident Schüffner’s granulations: some single infection with P. falciparum may have been read as possible mixed infection. Finally, Babesia microti has been over identified, probably as it is very similar to P. falciparum when just entered in the erythrocyte. In these circumstances, as well as to distinguish the two major forms of P. ovale, parasite morphology proved unsuitable for a systematic analysis of the relationship between the different species and only PCR-based methods proved to be reliable. However, in this study several underestimated mixed infections diagnosed by MD were observed. It could be strictly linked to the difficulties in extracting DNA from the dried blood spots and, consequently, to the possibility of identifying only the most abundant species. Therefore, MD may present weak spots that affect its performance.Regarding MA, the present findings demonstrate its high performance in detecting all vs 34.1 %) [As for the comparison of the performance shown by the applied malaria tests, the higher (however in this case not significant) sensitivity of MD in respect to MA and RDT, and the ~90 % specificity of the RDT used, reported in previous studies , 20 werePlasmodium species. In addition, the zoonotic Babesia microti, which may contribute to their anaemia, was detected.The findings in Tshimbulu pointed out worrying malaria prevalence in children, mostly without symptoms of the disease, due to four in loco applied diagnostic tools, if a rapid replay of a clinical suspicion is very useful, the importance of both easy interpretation of the test and the identification of mixed infections cannot be underestimated. The RDT used, like most RDT, is exposed to doubtful interpretation and is unable to offer important detail of an infection, therefore, the production of devices that recognize all species, P. knowlesi included, and characterize mixed infections should be promoted.As for the accuracy of Plasmodium species, which is a major issue for all microscopists in any diagnostic setting. This difficulty may be overcome by improving the current practice of microscopy through the use of available on-loan sets of thick and thin malaria smears, self-testing for competency in malaria microscopy [This experience highlighted a satisfactory level of competence in a rural hospital laboratory team in evidencing positive children, but several difficulties in identifying croscopy , and newcroscopy .


The relationship between climatic factors and mosquito abundance is very important to determine parasite activity levels and, therefore, disease risk. Therefore, this study was conducted to investigate the seasonal abundance of anophelines and their association with meteorological variables and disease transmission in two malaria endemic areas of Bangladesh.P. falciparum, P. vivax 210 and P. vivax 247 using ELISA. Pearson correlation and canonical correspondence analyses (CCA) were computed to investigate the associations with species abundance and rainfall, temperature, humidity and malaria cases.Monthly sampling was done from both indoors and outdoors in 12 selected houses using light traps (LTs) and pyrethrum spray (PS) during January, 2011 to January, 2012 in two malaria endemic areas of Bangladesh. Outdoor rainfall, temperature, and relative humidity data of the study areas were collected from the meteorological department of Bangladesh. Mosquitoes were killed with chloroform and identified morphologically under stereoscopic microscopes using taxonomic keys. Samples were tested for CSP of Anopheles were tested for P. falciparum, P. vivax 210 and P. vivax 247 CSP, of which 10 species were found positive. The CSP positive species were An. annularis, An. baimaii, An. barbirostris, An. jeyporiensis, An. karwari, An. minimus s.l., An. philippinensis, An. umbrosus, An. vagus and An. wilmori. Anopheles vagus and An. philippinensis were the dominant species present almost throughout the year with highest peaks in March and smallest peaks in September but An. baimaii and An. willmori were found during monsoon (July -September) only. Lag rainfall and relative humidity were the most significant variables influencing An. baimaii, An. willmori, An. vagus, and An. subpictus density in Kumari area. Abundance of these four species positively related to malaria cases. The effects of temperature were not found as a significant variable on the abundance of anophelines mosquitoes in Bangladesh.A total of 2,443 female anophelines, representing 22 species were captured. Every female Our study demonstrates that the nature of relationship between malaria vector and climatic variables were multifaceted. Detailed studies of vector bionomics, continuous monitoring and malaria transmission dynamics is essential for predicting disease outbreaks and vector control in the region. Anopheles baimaii Sallum and Peyton, 2005 (dirus D), An. minimus s.l. Theobald, 2001, An. philippinensis Ludlow, 1902, and An. sundaicus  are considered as primary and An. aconitus Doenitz, 1902, An. annularis Van der Wulp, 1884, and An. vagus Doenitz, 1902 as epidemic malaria vectors in Bangladesh [An. nigerrimus Giles, 1900, An. subpictus Grassi, 1899, An. barbirostris Van der Wulp, 1884, An. maculates Theobald, 1901, An. jeyporiensis James,1902, An. karwari James, 1902, An. kochi Doenitz,1901, and An. peditaeniatus  as Plasmodium positive.Malaria is one of the most formidable and serious public health problems in Bangladesh . It is eEnvironmental changes have a great bearing on breeding habitats of different mosquito species that control adult population density . MeteoroTwo malaria endemic areas of Bangladesh were selected for the study these are Kumari of Bandarban district  and Sreemangal of Maulvibazar district   and pyrethrum spray (PS) following World Health Organization procedures . CollectPlasmodium falciparum (Pf), P. vivax (Pv) 210 and P. vivax 247 using ELISA as described by Burkot et al. [P. falciparum and two distinct polymorphs of P. vivax: Pv-210 and Pv-247. Four negative controls  and one positive control (supplied by CDC) were included in each microtitre plate. ELISA results (i.e. change in color) were visually judged according to the color intensity , comparable with the positive control. All positive samples were re-tested for confirmation. We considered the sample as CSP-positive, if two of two trials were positive. Recently Durnez et al. [Plasmodium was heat-stable but false positive causal agent was not. Therefore we confirm positive results by a second CSP-ELISA test applying heated the samples at 100°C for 10 minutes.Each mosquito was bisected into head–thorax and abdomen. The anterior part, head, and thorax of the mosquitoes were tested. Samples were tested for CSP of t et al.  and Wirtz et al.  and Bashz et al.  describeEthical clearance was obtained from the head of each village, office of the local government and the ethics committee of Bangladesh Medical Research Council (No. PU/203/11). We took verbal consent from all of the household head for this study because the majority of them are illiterate. The ethics committee approved this consent procedure.Pf and Pv) of the study areas were received from the office of malaria control program and local Hospitals. Local hospitals routinely checked the P. falsiparum cases using rapid diagnostic test (RDT) and P. vivax cases by using thin and thick blood films.Data on the malaria cases  during the monsoon (July-October) is followed by a cool winter period (November-February), then a hot, dry summer (March-June). The winter season is dry and accounts for 2- 4% (average 1.88 mm) of the annual rain. As the winter season progress into the summer hot season, rainfall increase . In the hot summer season, the average maximum temperature is 34°C and the minimum is 21°C. Average maximum temperature in winter is 29°C and the minimum is 11°C. The average monthly relative humidity for the whole year ranges from 63.13 to 87.73%. The relative humidity is over 80% during June to September. March and April are the least humid months.In order to compare counts of female anophelines captured in different periods, we determined the relative density of every mosquito species according to the formula of Kocatas  and SimsAn. vagus  followed by An. philippinensis , An. jeyporiensis , An. karwari  and An. peditaeniatus  respectively. Few number of samples (n < 10) was collected in the following 10 species; An. aitkenii, An. baimaii, An. nigerrimus, An. nivipes, An. pallidus, An. pseudojamesi, An. tessellatus, An. maculatus, An. hyrcanus group, and An. willmori  and PS (n = 178). All 22 species were collected using LTs set indoors (n = 1163), and 20 species were collected with outdoor LTs (n = 1074). In contrast, only eight species were collected using PS (n = 178). Anopheles vagus was captured dominant in both LTs and PS in indoor. Relatively high number of An. karwari (n = 76) and An. peditaeniatus (n = 57) were captured in outdoor  was the dominant species followed by An. jeyporiensis, and An. annularis. Anopheles vagus and An. karwari were captured only in indoors  of female anophelines were captured using light trap set both indoors and outdoors during one year sampling period in Sreemangal. Only six species; atus n = 5 was theGonotrophic stages of mosquito were visually classified as unfed (UF), Blood-fed (BF), semi-gravid (SG), or gravid (G). The highest percentage of specimens was UF , followed by BF , G , and SG .Anopheles were tested for P. falciparum, P. vivax 210 and P. vivax 247 CSP, of which 10 species were found positive. The CSP positive species were An. annularis, An. baimaii, An. barbirostris Van der Wulp, An. jeyporiensis, An. karwari, An. minimus s.l., An. philippinensis, An. umbrosus, An. vagus and An. wilmori. A total of 22 (0.91%) mosquitoes belonging to eight species were found positive for P. falciparum, 10 (0.41%) mosquitoes belonging to five species were positive for Pv-210 and one (0.04%) mosquitoes belonging to one species were positive for Pv-247. Mixed infections were found in 5 females (0.20%) anophelines. In total of P. falciparum and P. vivax infections, the highest infection rate was observed in An. baimaii  followed by An. wilmori , An. barbirostris , An. annularis , An. umbrosus , An. karwari , An. vagus , An. minimus s.l. , An. jeyporiensis , and An. philippinensis  .Every female Anopheles vagus and An. philippinensis were the dominant species present almost throughout the year with major peaks in March and smaller peaks in September but An. baimaii and An. willmori were found during monsoon (July -September) only. Although the population of An. karwari decreased greatly in May and December, it was collected in small numbers throughout the sampling period with peaks in February. Highest and lowest densities of anophelines were found in March and December respectively in Kumari  and An. willmori (p < 0.01) has strong positive correlation with lag rainfall in Kumari. But we did not find any significant relation with rainfall and most other Anopheles species in Kumari as well as in Sreemangal. The effect of temperature were not found as an important variable on the abundance of anophelines mosquitoes in both the study areas except An. karwari . Strong negative correlation between relative humidity and abundance of An. karwari (p < 0.01), An. minimus s.l. (p < 0.01), An. annularis (p < 0.01), and An. jeyporiensis (p < 0.01) were observed in our study. We found significant relation between An. willmori (p < 0.05) and malaria morbidity when Spearman’s correlation analyses was done. Insignificant impact of 1 month lag rainfall and temperature on the malaria morbidity were found in Kumari. Highest number of malaria cases was recorded during July but rainfall was higher during August  in Kumari  and forest fringes [An. willmori breeds in slow running stream margins, rice fields, pits and wells [et al. [Highest peaks of the female anophelines species density were observed during March and smallest peaks during September in Kumari and there were no positive relation with rainfall, and majority of the anopheline species. We did not found any significant correlation with rain fall, and known malaria vector; y Meisch . Investi [et al. did not  [et al.. Usually [et al. for risi fringes  and An. nd wells , seems tnd wells . Olson and wells  and Fock [et al.  reportedWe did not found any significant association with temperature; mosquito density and malaria incidence in our study. Certainly, temperature is directly affecting mosquito breeding, survival, and behavior and also malaria transmission –43. We wet al. [et al. [et al. [P. falciparum and P. vivax and this range of humidity are present throughout the year in Bangladesh. It was also reported that the malaria risk at 80% humidity was double as that of 60% [Though, rainfall is the major key factor to enhance the malaria transmission in several countries –46. Howeet al.  investiget al.  reported [et al.  showed t [et al.  accountet of 60% , 51.Anopheles monthly distribution and malaria prevalence, An. baimaii, An. willmori, An. vagus, and An. subpictus seem to potential vector of malaria in the study area. This hypothesis was supported by their anthropophilic and opportunistic feeding behavior [Plasmodium infection rate [Based on the observation of behavior  and Plasion rate , 9.The study demonstrates that the incidences of malaria all year round in Kumari, Bangladesh, due to the favorable environmental conditions. Therefore, integrated vector management system covering year round should be adopted to reduce of malaria morbidity and mortality in Bangladesh. Additionally, basic and applied research on the ecological, social and economic determinants of the disease is required to promote the regular assessment of a country’s malaria situation. Like other studies, we did not find significant relationships of rainfall and temperature with all anophelines; even so, this may be due to the fact that other studies used diverse methodologies in different regions of the globe, where the vector ecology is pretty different. Multiple environmental factors are responsible for mosquito breeding and malaria infection in the Bangladesh. This study recommends vector ecologists to cautiously consider the complex nature of the relationship between malaria vectors and climate variables. Detailed studies of vector bionomics, continuous monitoring and malaria transmission dynamics is essential for predicting outbreaks of disease and, if necessary, control of pest mosquitoes in Bangladesh.


Plasmodium falciparum and Plasmodium vivax in malaria-endemic area, where multiple episodes are common, is important for intervention programmes.The identification of epidemiological pattern of infection with P. vivax and P. falciparum incidence. Two types of multivariate frailty models were applied , allowing the estimation of adjusted hazard ratios (AHR) of potential risk factors  for species-specific malaria incidence.A longitudinal cohort study based on weekly house-to-house visits was conducted between July 2008 and June 2010 in 2040 children less than 10 years of age, living nearby the Gilgel-Gibe hydroelectric power dam reservoir in order to determine factors associated with increased P. falciparum in 548 children, and 385 due to P. vivax in 316 children. Plasmodiumvivax and P. falciparum malaria incidence rates were 8.2 (95 % CI: 7.3–9.1) and 14.6 (95 % CI: 13.4–15.6) per 1000 children per month, respectively. According to the time-to-recurrent event models, children aged ≥7 years had a lower risk of presenting P. vivax episodes , but a higher risk of P. falciparum episodes, when compared with children under ≤3 years . In addition, P. vivax  and P. falciparum  episodes were respectively 2.7 and 16.9 times more frequent in the dry season than in the long rainy season.Of 2040 children in 96 weeks of follow up, 864 children experienced at least one malaria episode: 685 due to P. vivax episodes mostly observed in younger age groups, and with marked seasonality in the incidence of P. falciparum episodes mainly seen in older children.The analysis of all malaria episodes (first and recurrent episodes) in the malaria cohort suggests different species-specific patterns of malaria disease in children, with mild seasonality in the incidence of The online version of this article (doi:10.1186/s12936-016-1253-2) contains supplementary material, which is available to authorized users. P. falciparum and P. vivax substantially contribute to malaria morbidity in Ethiopia, accounting respectively for 60 and 40 % of the total reported cases [Despite the good progress over the past decade, malaria remains the most important human vector-borne disease worldwide . Accordied cases , 6.P. falciparum, P. vivax can cause relapses due to its ability to produce latent parasite stages (hypnozoites) [P. vivax malaria transmission, given that P. vivax recurrent episodes can be caused either by hypnozoite-triggered relapses, resurgence of erythrocytic parasites (i.e. recrudescence) due to a failure in the treatment, or reinfection of individuals with a new parasite strains following primary infections [Unlike ozoites) , 8. Thisfections .P. falciparum and P. vivax prevalence rates by microscopy in households in close proximity to the Gilgel-Gibe dam [P. falciparum clinical malaria incidence and household distance to the dam [A cross-sectional community-based study conducted in south-western Ethiopia in 2005 reported increased Gibe dam . However the dam .Researchers have often used time-to-first event models to analyse malaria disease data from longitudinal studies –14. ThosP. falciparum and P. vivax) and to generate evidence to strengthen the prevention and control of malaria in the area. The study was implemented as part of several other studies, intended to assess the impact of the Gilgel-Gibe hydroelectric dam in the health and other sectors  following its opening in 2004 [Using a 2-year longitudinal malaria cohort data of children living nearby the Gilgel-Gibe dam in south-west Ethiopia, this study aimed to assess the effect of age, season period, and household distance to the dam on the species-specific clinical malaria incidence  from the dam reservoir shore were randomly selected based on similar eco-topography, access to health facilities, without major impounding water nearby and homogeneous with respect to socio-cultural and economic activities . The maia vector .A longitudinal 2-year malaria cohort study was conducted in children under 10 years old, living in the selected 16 villages around the Gilgel-Gibe hydroelectric power dam. A total of 2040 children aged <10 years were enrolled in July 2008, and then weekly followed up until June 2010. Each child was identified with a unique code, and selected villages and households were geo-referenced using a handheld global positioning system (GPS) device , allowing the estimation of the household distance to the dam.Ethical approval for the study was obtained from Jimma University Research and Ethics Committee. Verbal and written signed informed consent was obtained from the mother or caregiver of each child before enrollment of the child in the study.Plasmodium species.Active case detection (ACD) through weekly household visits allowed the identification and registration of all clinical malaria episodes in the study population during the follow-up period. During the household visits, axillary temperature was taken and the caregiver was asked about fever history. If a child had fever (temperature ≥37.5 °C) or reported a history of fever in the past 24 h, a finger-prick blood sample was taken for immediate diagnosis by microscopy in the same site or at Omo-Nada district Health Center Laboratory. Microscopy diagnosis was conducted by trained laboratory technicians. Thick smears were used to confirm the presence or absence of parasites, whereas the thin smear was used to identify the P.vivax, and artemether-lumefantrine (AL) for P. falciparum twice daily according to the body weight as follows: 5–14 kg, one tablet per dose; 15–24 kg, two tablets per dose; 25–34 kg, three tablets per dose; and adult, four tablets per dose. Treatment adherence was monitored during household visits by asking for the medication packages and the remaining pills.All children with microscopically confirmed malaria were treated according to the national treatment guidelines . TreatmeAbsent children were followed in the next visits and their caregivers were asked about the occurrence of symptomatic episodes and/or the confirmation of malaria by a health facility. In addition, all the health facilities near the study communities were monthly visited to verify their clinical records, checking if any enrolled children had presented a confirmed malaria episode in the past month not being detected during the weekly visits.A. arabiensis during the study period were shown graphically. Mosquitoes were collected in all 16 communities, once a month, from 18:00 to 6:00 h in two selected houses using light trap catches. Further details on the mosquito collection methodology are described elsewhere [The monthly number of episodes by species and data published previously on the density of lsewhere .Malaria episode data of each child were checked carefully to identify a first malaria episode as well as any additional malaria episodes after the first malaria episode (i.e. recurrent episodes). For the time at risk, a child treated for a malaria episode was censured for 21 days in order to prevent double counting of episodes and to allow for any prophylactic effect of the antimalarial treatment .A sub-sample data of four children in Table Conversely, data from children with both one and more malaria episodes were accounted when modelling recurrent episodes. The time at risk for a recurrent episode in each child with recurrent episodes was given by the time between two successive malaria episodes minus 21 days . For instance, the follow-up history of one child is described by the first four rows of Table Malaria incidence rates (by species) were calculated by dividing the number of episodes during the study period by child-months at risk, and expressed as the number of episodes per 1000 child-months at risk. A child-month at risk corresponds to a child that had at least once weekly visit of the total 4 weeks of a specific month. Correspondingly, 95 % confidence intervals (95 % CI) of the incidence rates were calculated assuming that the number of new malaria episodes was Poisson distributed.p value was calculated, using a Wald test. Models were fitted in R version 3.2 using the package ‘frailtypack 2.5.1’ [Frailty models were applied to calculate hazard ratios (HRs) of species-specific incidence using separately time-to first episode data and time-to-recurrent episode data. The following potential risk factors were included in the univariate and multivariate models: gender, age groups , household distance to the dam reservoir (in kilometres), and season . For covariates with more than two categories, a global single k 2.5.1’ .λ (lambda) is the scale parameter, and ρ (rho) the shape parameter.The time-to-first episode data was modelled through a piecewise Weibull frailty model. The piecewise interval was introduced taking into account the hazard rate changes in malaria incidence between the different seasons in the 2 years . This flexible parametric approach has been used in a previous study aiming to model malaria recurrent events, with good results . The twoƟ (theta) while the sub-cluster “individual” random effect variance was estimated by η (eta) from the nested frailty model. For model checking, martingale residual of the frailty model were plotted against covariates at a continuous level, confirming the fit of the data when no systematic or clear pattern is observed [The time-to-recurrent malaria episode data was modelled by nested frailty models allowing to account for the hierarchical clustering of the data by including two nested random effects that act multiplicatively on the hazard function , using aobserved .P. falciparum in 548 children  and 385 episodes of P. vivax in 316 children . Plasmodium falciparum and P. vivax malaria incidence rates were 14.6 (95 % CI: 13.4–15.6) and 8.2 (95 % CI: 7.3–9.1) per 1000 children per month, respectively. Plasmodium falciparum episodes predominated over P. vivax episodes in all selected communities ; episodes by both species peaked during the dry season after an increase of the mosquito density in the previous months , P. falciparum episodes occurred mostly in children older than 7 years (p < 0.001)  slightly outnumbered female children (48.1 %), and the median age at the time of enrolment was 5 years. A total of 1070 clinical malaria episodes were registered during the study period, including 685 episodes of P. vivax episode throughout the study period was slightly lower than the median time between the first and second episodes in 53 children (147 days), and between the second and third episodes in 13 children (172 days). On the other hand, the median time to the first episode in 548 children who had at least one P. falciparum episode (286 days) were higher than the median time between the first and second episodes in 127 children (204 days), and between the second and third episodes in eight children (131 days).Table P. vivax episodes even after controlling for other non-significant potential risk factors such as gender and household proximity to the dam , AHR estimates for the effect of season categories differed slightly between models. P. vivax episodes occurred 1.9 times more frequently in the dry season than in the long rainy season according to the time-to-first event model   that the age of the child at enrolment and season were significantly associated with the incidence of m Tables  and 5. W9) Table , while sP. vivax models, both types of multivariate models for P. falciparum also found that age and season were significantly associated with the incidence of P. falciparum episodes, but with differences in AHR estimates   than the ones obtained from the time-to-first episode model . Gender and distance to the dam were not significantly associated with P. falciparum incidence in both models. Regarding model parameter estimates, ρ values lower than one, indicated that for both species incidence rates decreased with time, but incidence rates for P. vivax (ρ ~ 0.7) during the study period decreased faster than those for P. falciparum (ρ ~ 0.8). The malaria risk variation among villages for P. falciparum (ɵ ~ 0.5) was higher than for P. vivax (ɵ ~ 0.3). Moreover, malaria risk variation among children (η) were higher for P. vivax than for P. falciparum. Additional file 3 shows the fit of the data by plotting martingale residual by the covariate at a continuous level. The lowess estimates did not show a clear pattern, suggesting a good fit.Similarly to s Tables  and 5. Frs Table . ConversP. falciparum and P. vivax malaria episodes occurred in respectively 127 and 53 children along the study period. Plasmodium vivax clinical episodes were more obvious in younger age groups and occurred mostly in the dry season, while P. falciparum episodes predominated in older children and occurred in the dry and moderate season but with a substantial peak in the first months of the dry season. Both P. vivax and P. falciparum incidence rates decreased with time and were not associated with the household proximity to the dam reservoir.This study reports species-specific clinical malaria episode patterns in a 2-year longitudinal cohort study of 2040 children under 10 years old living nearby the Gilgel-Gibe hydropower dam, in south-western Ethiopia. Multiple This is the first study analysing recurrent malaria episodes with two statistical approaches, i.e. time-to-first event models and time-to-recurrent event models with discontinuous risk intervals. Although no major differences in parameter estimates for evaluating risk factors were found between both models using the data at hand, it is not the rule for all health conditions with recurrent episodes and possibly mainly explained by the low occurrence of recurrent malaria episodes in the study. Indeed, a re-analysis of data from a pneumococcal conjugate vaccine trial and the simulation of scenarios found that the difference between effect estimates of the vaccine obtained from time-to-first event models and time-to-recurrent event models was substantial especially when a high incidence of recurrent episodes was noted . MoreoveThe shape model parameter value below one confirmed that malaria incidence rates for both species decreased with time during the study period. A similar decreasing malaria incidence trend has been reported in southwest Ethiopia  and may Although previous studies in Ethiopia and other African countries have reported dams influencing malaria transmission by increasing vector abundance due to breeding sites associated with dam´s floods on the shorelines , 31–33, P. vivax than with P. falciparum. After 5 years of continuous exposure, children can acquire an almost complete clinical immunity to P. vivax, which is characterized by their control of blood-stage parasite densities rather than the acquisition of a significant immunity against infection [P. vivax malaria also decreased significantly faster with age than that the incidence of P. falciparum, while a cohort study in the Brazilian Amazon indicated that P. vivax malaria started to decrease after 5–6 years of residence in the endemic area compared to 8–9 years for P. falciparum [The differences in clinical malaria incidence between species with respect to age may be related to a different species-specific acquisition rate of immunity in the Ethiopian children, which may be related to different exposure levels for both species in the early years of live. Those differences are in line with previous findings from longitudinal studies conducted in co-endemic malaria areas of Papua New Guinea , 14, 35.nfection , 37, andnfection  the incilciparum .Although a weekly identification of symptomatic children and the consequent diagnosis and treatment of malaria infections during the study are likely to have contributed to the decrease of malaria transmission in the area and consequently possibly explain the decrease of the malaria incidence rate in time, it is worth to notice that the decrease may also be associated with the acquisition of immunity in the study children. Further longitudinal studies incorporating the detection and follow-up of both symptomatic and asymptomatic infections in young children are necessary to understand the role of immunity  in the malaria transmission in the co-endemic Ethiopia , 14, 35.P. vivax clinical episodes being less sensitive to seasonal and environmental changes than P. falciparum, an important proportion of the former possibly being hypnozoite-triggered relapses [P. vivax episodes only received CQ, following the national guidelines for areas where the glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is not known and where tests to detect that condition are not available [Many studies have reported high mosquito densities and an increased malaria risk following the rainy season , 40. Aftrelapses –9. This vailable . A previvailable .P. vivax episodes, mostly seen in younger age groups, and with marked seasonality in the incidence of P. falciparum episodes mainly observed in older children. The decision-making and planning of malaria control interventions will have to consider the seasonal patterns of malaria transmission in order to reduce malaria incidence, as well as a species-specific policy.The analysis of all malaria episodes (first and recurrent episodes) found different species-specific patterns of malaria disease in the enrolled children, with mild seasonality in the incidence of


The severity and outcome of malaria is influenced by host immunity in which chemokines such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) play an important role. Previous studies show that variations in the RANTES gene affect RANTES protein production, hence altering host immunity. In this study, the relationship between presence of mutations in RANTES and incidence of malaria in a cohort of children living in a malaria-endemic area of Uganda was determined.This was a longitudinal study comprising of 423 children aged between 6 months and 9 years, who were actively followed up for 1 year. Malaria episodes occurring in the cohort children were detected and the affected children treated with national policy drug regimen. Mutations in the RANTES gene were determined by PCR–RFLP method and their frequencies were calculated. A multivariate negative binomial regression model was used to estimate the impact of RANTES mutations on malaria incidence. In all statistical tests, a P-value of <0.05 was considered as significant.The frequencies of the −403A and In1.1C allele were 53.7 and 19.2 %, respectively. No mutations were found at the −28 locus. After adjustment of incidence rates for age, blood group, insecticide-treated bed net (ITN) use, malaria history and the sickle cell trait, 1n1.1T/C heterozygotes and homozygotes showed a non-significant trend towards higher incidence rates compared to wild-type individuals . Similarly, there was no significant difference in malaria incidence rates between RANTES −403G/A heterozygotes or homozygotes and those without mutations . No relation was seen between RANTES polymorphisms, baseline parasite densities and the time to first re-infection after administration of anti-malaria drugs.This study showed that the −403A mutation occurs in nearly half of the study population and the In1.1C allele occurs in one in every four children. Despite the high frequency of these mutations, there was no clear association with malaria incidence. Other studies evaluating more markers, that could potentially modulate RANTES gene transcription alongside other genetic modifiers of malaria susceptibility, may provide further explanations to these less dramatic findings. Plasmodium falciparum malaria accounts for approximately 198 million clinical cases of malaria worldwide and 584,000 deaths annually [P. falciparum infection [Plasmodium infection. The protective effect of the sickle cell trait (Hb AS) against both uncomplicated and severe malaria disease has been well documented [P. falciparum infection.nfection . The devcumented –8. It isPlasmodium-infected erythrocytes causing occlusion of blood vessels, degradation of cell–cell junctions, blood–brain barrier dysfunction, interfering with formation of blood vessels and formation of blood cells or haematopoiesis [Plasmodium haemozoin by monocytes [Regulatory cytokines and other mediators have also been reported to play a critical role in controlling parasitaemia and subsequent elimination of infection. Interferon-gamma (IFN γ), tumor necrosis factor (TNF), Interleukin 10 (IL-10), IL-17, IL-4, C–C chemokine RANTES , matrix metalloproteinases 8 (MMP8s) and tissue inhibitor of metalloproteinases 1 (TIMP1) have been linked to disease severity in malaria-infected individuals –16. The opoiesis , 17–21. opoiesis , 23. Lowonocytes  or malaronocytes  and haveonocytes .The human RANTES gene is located on chromosome 17q11.2-q12, has a genomic size of 8.8 kb and encodes a protein of 8 kDa . Among tIn several other studies, RANTES polymorphisms have been shown to affect the course of systemic lupus erythematosus , asthma This was a longitudinal study carried out in villages of Iganga district where children aged 6 months to 9 years were enrolled in order to determine malariometric indices during September 2008 and November 2009 towards preparation for the GMZ2 phase II malaria vaccine trial. From September 2008 to October 2008, a team of experienced home visitors approached households to systematically recruit children into the baseline study. Eligible children were enrolled into the study and followed up for a period of 1 year from November 2008 to November 2009. Iganga district is situated in south-eastern Uganda, about 25 km to the north of Lake Victoria and lies at an altitude of about 1138 m above sea level at latitude: 00°36′ North and longitude: 33°30′ East. Malaria transmission is this area is holo-endemic , with transmission peaks seen following the major rains, which normally occur between April to June and September to December . The annP. falciparum parasitaemia plus a tympanic temperature of ≥38.0 °C or a history of fever (within the past 48 h). Thick and thin peripheral blood slides were stained with Giemsa and examined under a microscope. Parasite densities were calculated by counting the number of asexual parasites per 200 white blood cells (WBC) and assuming a WBC count of 8000/µL blood [®) according to the national treatment guidelines and those with severe malaria were admitted for care at Iganga Hospital acute care unit. Time at risk for new infection was considered as the duration of study participation excluding 14 days after each episode of malaria.A baseline survey was carried out and eligible children were enrolled into this cohort. At the time of enrolment, a physical examination was performed by study physicians. Baseline demographic data were collected from guardians through a questionnaire-based interview, including malariometric indices. After the baseline survey, parents or guardians were instructed to bring their children to the malaria clinic based at Iganga Hospital whenever they felt unwell so as to avoid using any drugs not administered or approved by a study physician. A team of field workers visited the enrolled children at home every fortnight. Those children found unwell were referred to the clinic for care or referral in case of severe illness. Malaria was defined as any µL blood . In addiµL blood , 46. ChiGenomic DNA was extracted from blood leukocytes using E.Z.N.A Blood DNA kit as outlined by the manufacturer’s protocol . Approximately 0.1 µg of genomic DNA was used for the genotyping assays.2. After first-round PCR, about 5 µL of each reaction was digested with restriction endonucleases and products were subjected to electrophoresis on 2.5 % agarose gels  and visualized with ethidium bromide. Genotypes were assessed by comparing the sizes of reaction products and controls after digestion.Polymorphisms were detected using polymerase chain reaction (PCR) amplification followed by restriction endonuclease digestion (RFLP) as described elsewhere , 47, 48.2.PCR products from the first round were subjected to a second amplification using DreamTaq DNA polymerase , 0.05 µM of each primer, 200 µM dNTPs and 2.0 mM MgCl2. After second-round PCR, products were digested with restriction endonucleases and subjected to electrophoresis on 2.5 % agarose gels  containing ethidium bromide. As described above, genotypes were assessed by comparing the sizes of reaction products and controls after digestion.Sickle cell genotyping was based on characterization of haemoglobin AS (Hb AS) using nested PCR followed by RFLP as previously described by Parikh et al. . In the Data were cleaned, coded and entered into Microsoft Office Access ™ 2007. Descriptive statistics, Chi square tests and multivariate analysis were carried out using Stata12.0 . Allele and genotype frequencies were calculated in accordance with the Hardy–Weinberg principle . The assThe effect of RANTES polymorphisms on the length of time to first re-infection following administration of anti-malarial drug was estimated using Kaplan–Meier non-parametric survival analysis. The log rank test was used to evaluate differences in the survival times between the different RANTES genotypes. Cox proportion hazard regression model was used to control for other independent predictors of length of time to re infection  and adjusted hazard ratios (aHRs) were calculated. For all statistical tests, two-sided p values of less than 0.05 were assumed to show statistical significance.The clinical study and all study protocols were approved by the School of Medicine Research and Ethics Committee of the College of Health Sciences, Makerere University and by the Uganda National Council for Science and Technology . All participants provided written informed consent. All children received appropriate treatment for other attendant medical conditions.A total of 980 children were screened for baseline studies to determine malariometric indices at Makerere University Iganga-Mayuge Demographic study site, Iganga Hospital during September 2008 and November 2009 Fig.  towards PCR for genotyping of the three RANTES polymorphic sites was performed for all individuals. The success rate was 96 %. Out of the 406 children who were successfully genotyped for the RANTES −403 G/A polymorphism, 224 (55.2 %) had the RANTES GA genotype, 26.1 % carried the AA genotype while the remaining (18.7 %) had the wild type genotype. For the In1.1T/C polymorphisms, only 3.7 % carried homozygous mutations (CC), 30.9 % carried the heterozygous (TC) genotype and a majority (65.4 %) had the wild-type (TT) genotype. The overall prevalence of the respective markers is shown in Table A single across groups test (ANOVA) was initially performed to determine differences in the distribution of malaria incidence across the RANTES genotypes. Crude malaria incidence rates/person/year were 0.91 for In1.1T/C wild-type, 0.94 for heterozygous and 0.73 for homozygous individuals. There was no significant difference in the distribution of malaria incidence across the three genotypes (P = 0.76). Since there are known predictors of malaria incidence that needed to be adjusted for a multivariate negative binomial regression analysis was done in order to evaluate the association between RANTES polymorphisms and malaria incidence. An initial bivariate (unadjusted) analysis identified age, malaria history, ITN use and sickle cell trait . The adjusted incident rate for RANTES −403G/A heterozygotes was 1.09 times the incidence rate for wild-type individuals P = 0.66). Similarly, homozygotes showed a non-significant trend towards higher incidence rates than those carrying the wild type genotype  as shown in Table 6. SimilaThe impact of RANTES polymorphisms upon time to first re-infection after administration of malaria treatment was also evaluated. The average time (weeks) to first re-infection was 13.4 for RANTES In1.1T/C wild-type individuals, 12.7 for heterozygotes and 16.7 for homozygotes. There was no significant difference in the length of time to first re-infection between heterozygote and wild-type genotypes (P = 0.74) or between homozygote and wild-type genotypes (P = 0.34). After adjusting HRs for age, malaria history and ITN use, the hazard for re-infection did not differ significantly between heterozygotes and wild-type genotypes  or between the In1.1C homozygotes and wildtypes   to first re-infection was 10.8 for RANTES −403 G/A wild-type individuals, 13.4 for heterozygotes and 11.4 for homozygotes. The was no difference in the length of time to first re-infection between heterozygotes and wild-type genotypes (P = 0.29) or between homozygotes and wild-type individuals (P = 0.44). Additionally, no significant difference was seen in the hazard for re-infection for wild types vs heterozygotes  or between the wildtypes and −403A homozygotes   in the RANTES gene region were examined. These SNPs have been shown to modulate RANTES gene transcription in vitro  and ofteThe In1.1C allele, which occurs in strong linkage disequilibrium with the −403A allele, has been shown to lead to down-regulation of RANTES and lower levels of the cytokine , 23. LowIn other disease conditions such as HIV where RANTES polymorphisms have been studied, there are still contrasting reports on the influence of these variants on susceptibility and disease progression. The strong down-regulatory activity on RANTES transcription that is afforded by −403 and In1.1 mutations has been shown to lead to increased susceptibility to HIV infection  and incrP. falciparum malaria [In the present study, previous history of malaria prior to recruitment was recorded as well. Most children reported previous experience of malaria, and these showed a higher incidence rate than those who were earlier free from the disease. These findings may suggest a genetic basis for susceptibility to malaria, which of course is most likely multifactorial, and dependent on other markers besides RANTES. A small number of children had many episodes of malaria during the study, up to nine during 1 year. At each incidence, the children were treated and cured from malaria, but they obviously get re-infected rapidly, and obviously do not develop protective immunity. At the same time, many children stayed free from malaria for the whole year, and the majority of those with malaria had only one episode. These children live under very similar conditions, so they should be exposed to infectious mosquito bites at similar rates. In this study, there was no significant relation between the RANTES polymorphisms and malaria incidence. Given that malaria susceptibility involves a complex interplay of other genetic modifiers, such as sickle cell trait, G6PD deficiency, among others , 6, the  malaria , 52 instThere were some limitations to this study. Information on known preventive measures and prior anti-malarial drug use was obtained by questionnaire-based interviews, which could have influenced the outcome of the multivariate analysis since known protective measures unexpectedly scored as risk factors of malaria. Additionally, this study looked at a single gene (RANTES). Additional modifying genetic factors other than the sickle cell trait were not adjusted for.Results of this study show that the −403A mutation occurs in nearly half the study population and In1.1C allele occurs in one in every four children. Despite the high frequency of these mutations, there was no clear association with malaria incidence. Since both polymorphisms lead to down-regulation of RANTES expression and expected lower levels of RANTES, these findings remain hard to explain. There were no mutations at the −28 marker. This marker has previously been shown to lead to increased RANTES expression. Thus, absence of −28 mutation in the Uganda study population is quite surprising. Evaluating other markers that could potentially modulate RANTES gene transcription may provide further explanations to these less dramatic findings. Additionally, since there are several other markers that have been implicated in pathogenesis of malaria, the impact of RANTES polymorphisms alone on malaria incidence may be difficult to evaluate. Therefore, other larger studies looking at the interplay between other genetic modifiers and malaria susceptibility may provide clearer understanding of these findings.


Fever is a common presentation of HIV/AIDS, malaria and acute respiratory tract infections (ARIs) all of which are major causes of childhood morbidity and mortality in sub-Saharan Africa. Correct management requires accurate diagnosis of the cause of fever. This study was carried out to determine prevalence and pattern of in HIV, malaria and ARI in febrile children under 5 years of age.A cross sectional study of all consenting and consecutive children 6- 59 months and their mothers / care-givers seen at the out patient department of Massey Street Children’s, Hospital presenting with fever. Semi structured questionnaire was used to obtain information from the mothers / care-givers and laboratory investigations were done for febrile children using 5mls of venous blood aseptically collected for blood culture, HIV, PCV and Malaria. Plasmids mediated antimicrobial resistance among bacteria isolates was determined. Data obtained from questionnaire, clinical and laboratory diagnoses were analyzed using EPI-INFO version 3.5.1 software.Among 202 patients studied, 186 (92.1%) and 16 (7.9%) were clinically diagnosed as Malaria and ARIs respectively, but laboratory investigations showed 47 (23.3%) positive for malaria, ARIs 31(15.3%), HIV 10 (5.0%) and anaemia 12(5.9%). The results revealed the prevalence of HIV and ARIs co-infection as 10 (5%); HIV and Malaria 10(5%); Malaria and ARIs 27 (13.4%). The entire Malaria positive cases 47 (100%) were due to Plasmodium falciparum. Haemophilus influenzae isolates 5 (100%) and Streptococcus pneumoniae 23 (82.1%) isolates harboured plasmids which conferred multiple antimicrobial resistant on them.Fever in children may be as a result of Infection with HIV/AIDS, Malaria, ARI, or co-infection of any of the two diseases. Home management of fever was very high among mothers / care-givers. The commonest drugs used for treatment of childhood fever by mothers / care-givers were chloroquine and cotrimoxazole. Streptococcus pneumoniae the major organism isolated was highly resistant to cotrimoxazole.