Plasmodium falciparum and Plasmodium vivax co-exist. The present study evaluated the performance of regular microscopy in accurate identification of Plasmodium spp. in febrile patients visiting health facilities in southern Ethiopia.With 75% of the Ethiopian population at risk of malaria, accurate diagnosis is crucial for malaria treatment in endemic areas where P. falciparum, P. vivax or mixed infections using microscopy, were re-evaluated for their infection status by PCR. Finger-prick blood samples were used for parasite genomic DNA extraction. Phylogenetic analyses were performed to reconstruct the distribution of different Plasmodium spp. across the three geographical areas.A cross-sectional study design was employed to recruit study subjects who were microscopically positive for malaria parasites and attending health facilities in southern Ethiopia between August and December 2011. Of the 1,416 febrile patients attending primary health facilities, 314 febrile patients, whose slides were positive for Plasmodium spp. by nested PCR. Among 180 microscopically diagnosed P. falciparum cases, 111 (61.7%) were confirmed by PCR, 44 (24.4%) were confirmed as P. vivax, 18 (10%) had mixed infections with P. falciparum and P. vivax and two (1.1%) were mixed infections with P. falciparum and P. malariae and five (2.8%) were negative for any of the Plasmodium spp. Of 131 microscopically diagnosed P. vivax cases, 110 (84%) were confirmed as P. vivax, 14 (10.7%) were confirmed as P. falciparum, two (1.5%) were P. malariae, three (2.3%) with mixed infections with P. falciparum and P. vivax and two (1.5%) were negative for any of the Plasmodium spp. Plasmodium falciparum and P. vivax mixed infections were observed. Plasmodium malariae was detected as mono and mixed infections in four individuals.Of the 314 patients with a positive thick blood smear, seven patients (2%) were negative for any of the Plasmodium species in the geographical region and has important repercussions in understanding malaria epidemiology and subsequent control.False positivity, under-reporting of mixed infections and a significant number of species mismatch needs attention and should be improved for appropriate diagnosis. The detection of substantial number of false positive results by molecular methodologies may provide the accurate incidence of circulating  Plasmodium, with four different species specifically infecting humans  and parasites of non-human primates, such as Plasmodium knowlesi, infecting humans occasionally. Plasmodium falciparum is the most virulent species contributing to a larger extent to malarial deaths in Africa, including Ethiopia. Plasmodium vivax and P. ovale form resting stages in the liver as hypnozoites and can cause a clinical relapse[Malaria is one of the major causes of mortality and morbidity in tropical and subtropical countries with an estimated 655,000 malaria deaths in 2010, and 91% of these cases were in sub-Saharan Africa[Plasmodium falciparum accounts for 60-70% and P. vivax accounts for 30-40% of malaria cases in Ethiopia, although these proportions might fluctuate on the spatial and temporal scale[Anopheles arabiensis is the main vector widely distributed in Ethiopia, while Anopheles pharoensis, Anopheles funestus and Anopheles nili contribute as secondary vectors[.Ethiopia’s diverse topography and climatic conditions make malaria a seasonal infection covering 75% of the prefecture of the country and malaP. falciparum and P. vivax[Plasmodium species other than P. falciparum and P. vivax. Microscopy remains the golden standard of choice to determine the disease burden. Additionally microscopy can identify febrile patients negative for any Plasmodium infection, thereby directing patients towards an alternative treatment algorithm[Hospitals and health centres in Ethiopia diagnose malaria by microscopy, whereas the health posts employ rapid diagnostic tests (RDT) for diagnosis of  P. vivax. The use P. vivax. Howeverlgorithm,8.Plasmodium species by microscopy[P. malariae and P. ovale infections are frequently overlooked. Dedicated laboratory personnel with good experience remain the key for accurate diagnosis of P. malariae and P. ovale in areas where P. falciparum and P. vivax predominate. Prompt and accurate diagnosis followed by an effective treatment largely depends on the skills of the laboratory personnel, who can spend appropriate time in examining blood film. On the other hand, microscopy techniques fail to detect mixed infections, when one of the Plasmodium species is present at low levels.Microscopy is a valuable technique and has been shown to detect about 75% of malaria infections in high transmission areas and miss up to 88% of infections in low transmission areas. The detcroscopy. In endePlasmodium with higher sensitivity is gaining importance for accurate detection. Methods such as nested PCR are capable of detecting few parasites/μl in blood[Plasmodium DNA by amplifying the 18s ribosomal RNA region of the parasite[Plasmodium are four to eight copies per haploid genome and are scattered on different chromosomes, with two distinct subgroups whose expression is regulated both by type A and type B genes that are expressed in the asexual and sexual stages in the vertebrate host, respectively[Plasmodium and different species reveal genetic heterogeneity in their respective ribosomal regions. This variation, together with the abundance of ribosomes in the parasite, has led to the development of effective diagnostic probes based on SSUrRNA sequences[In recent years, molecular detection of in blood-15. The parasite. The ribectively,17. The Plasmodium spp. Additionally, 18SrRNA genes were subsequently sequenced and phylogenetic relationship was investigated to determine the genetic diversity of the Plasmodium spp.In Ethiopia, previous reports on malaria surveillance were based on microscopic results. The majority of these studies were conducted either with little or no information or with few technical resources-21. In tA health institution-based study targeting febrile patients attending primary health facilities was conducted in southern Ethiopia between August and December, 2011. Malaria transmission in Ethiopia is seasonal with higher transmission peaks in the months between September and December, and between April and May following rainy seasons. The three study sites included in this study were Omo Nada, Bala Wajo and Arba Minch.P. falciparum or P. vivax or with a mixed infection using the existing health care delivery system within the target health centres was approached for consent immediately after receiving their laboratory result. For those who consented to be part of the study, approximately 5–10 μl finger-prick blood sample was collected. No additional clinical assessment was carried out at the time of recruitment.Study participants were recruited from the routine health delivery system. Self-presenting febrile patients attending health centres in Omo Nada (n = 713), Bala Wajo (n = 312) and Arba Minch (n = 391) and whose age was at least six months between August and December 2011 were eligible for the current study. Any patient reported as being infected either with P. falciparum or P. vivax. On collection, the blood spots were air-dried and stored in a separate, clean, sealed, plastic pouch at room temperature for further analysis.In total, 314 finger-prick blood samples were collected for molecular diagnosis from microscopy-positive outpatients in Omo Nada (n = 132), Bala Wajo (n = 99) and Arba Minch (n = 83) from all patients attending the health facilities during the study period. Blood samples were collected in Whatman 3 mm filter papers. These patients were positive in the routine health delivery system using microscopy either for Ethical clearances were obtained from the institutional review boards of Aklilu Lemma Institute of Pathobiology, Jimma University and Armauer Hansen Research Institute. Informed written consent and assent (in the case of children between 12 and 18 years of age) were obtained from each patient or a parent/legal guardian. The study participation was voluntary and had no influence on the treatment provision at the respective health facilities. All patient information were kept confidential.Plasmodium without any quantitative estimation of parasitaemia. In the current study, thin and thick blood films were prepared from finger-prick blood and air-dried, fixed in methanol and subsequently stained for 20–30 minutes in Giemsa. At least 100 thick field films with an oil immersion lens at 100X magnification were examined before a slide was considered negative. All individuals positive for P. falciparum were treated with artemether-lumefantrine (AL), P. vivax-positive patients were treated with chloroquine and mixed infections were treated with AL.Giemsa-stained thick and thin blood films are routinely used in Ethiopian hospitals and health centres for the detection of P. falciparum, P. vivax, P. malariae and P. ovale . The presence or absence of different Plasmodium species was confirmed with representative amplicon size that were species-specific. Samples that failed to amplify were subjected to repeated amplification procedures with different PCR additives.Genomic DNA was isolated using QIAgen DNA Mini Kit blood and tissue  according to the manufacturer’s instructions. The extracted DNA was stored at -20°C until use. Nested PCR assay were carried out as previously described elsewhere[e Table . In briePlasmodium-positive samples were further selected to determine the genetic diversity of the existing subspecies from three collected sites. The selected Plasmodium samples included 11 P. falciparum , 16 P. vivax  and four P. malariae from Omo Nada. PCR products were cleaned up using Exo-SAP-IT  and 1 μl of the purified product were directly used as templates for sequencing, using the BigDye terminator v. 2.0 cycle sequencing kit  on a ABI 3130XL DNA sequencer, according to the manufacturer’s instructions.Thirty-one (n = 31) Plasmodium  obtained from NCBI database were aligned with 31 samples  sequenced for 18srRNA using Codon code Aligner 4.0 software. The phylogenetic tree was reconstructed by Maximum Likelihood method based on Kimura 2-parameter model using the MEGA ver5.2 software[Five 18srRNA-specific DNA sequences for each software with 100Plasmodium DNA by PCR. Of 314 study subjects, 21% were children less than four years of age, 189(60.2%) were male and 125(38.9%) were female. Among 307 PCR-confirmed cases, 125(40.7%) were positive for P. falciparum, 154(50.2%) were positive for P.vivax, two (0.7%) were positive for P. malariae, 24(7.8%) were positive for P. falciparum and P. vivax double infections, two (0.7%) were positive for P. falciparum and P. malariae double infection. Seven study participants remained negative for all species investigated . Of these 154 individuals aged from 5 to 14 years, 81 (52.6%) were infected by P. vivax and 55 (35.7%) infected by P. falciparum. A total of 66 (21%) were children less than four years. The ages of all P. malariae-positive cases that were detected by PCR were below 28 years , cases of P. vivax (131 positive cases) in all the study sites. Three mixed infections of P. falciparum and P. vivax were detected in the samples from Omo Nada. In relative terms the highest numbers of P. falciparum and P. vivax patients were reported from Omo Nada site. Of all 180 microscopically confirmed P. falciparum cases, 111 cases (62%) were mono-infections for P. falciparum, 44 cases (24%) were P. vivax mono-infections, 18 cases (10%) were double infections with P. falciparum and P. vivax, two cases (1%) were double infections with P. falciparum and P. malariae, and five cases (3%) were confirmed negative for any Plasmodium species by the nested PCR. Of all 131 microscopically confirmed P. vivax cases, 110 cases (84%) were P. vivax mono-infections, 14 cases (11%) were mono-infections for P. falciparum, two cases (2%) were detected for P. malariae mono-infection, three cases (2%) were double infections with P. falciparum and P. vivax and two cases (2%) were confirmed negative for any Plasmodium species by the nested PCR. Molecular detection by nested PCR identified that many of the P. vivax cases (44/180) were incorrectly diagnosed as P. falciparum by microscopy. A total of seven negative cases are observed. These seven negative cases were reconfirmed for their status by amplification for Pfmdr1 and Pfcrt alleles. P. ovale was not detected either by microscopy or by nested PCR. In total, 23 mixed infections were detected by nested PCR compared to microscopy-based detection.Comparison of results from microscopy and nested PCR is summarized in Table P. vivax and 15 (4.8%) were P. falciparum gametocyte stage. All the gametocyte stages were identified from 307 study subjects positive for Plasmodium parasites.Of all the 314 study subjects, 32 (10.2%) carried gametocytes. Of which, 17(5.5%) were P. falciparum isolates, 16 P. vivax isolates and four P. malariae isolates) originated from different geographical areas and sequenced for 18srRNA, is summarized in Table Plasmodium isolates is summarized in Table P. vivax isolates from Ethiopia matched in one group whereas all the P. falciparum and P. malariae clustered in another clade. Within the main clade, P. vivax clustered into three, P. falciparum clustered into two and P. malariae placed into one subcluster , thereby directing febrile patients towards prompt treatment that can save lives. The rationale of the study was to assess species mismatch and the specificity of microscopy in differentiating Plasmodium spp. in regions where two virulent species of Plasmodium, namely P. falciparum and P. vivax, co-exist.Early diagnosis and prompt treatment is crucial in controlling malaria infections. Microscopic diagnosis and RDTs are widely used methods for malaria surveillance in Ethiopia. The most fatal species, P. falciparum and P. vivax are the predominant parasite with a prevalence of 55 and 45%, respectively[Plasmodium co-exist. The present study report a prevalence of P. vivax (55%) remained high compared to P. falciparum (45%) during this season. The rationale of the study was to evaluate the limitations of microscopy in differentiating Plasmodium in Ethiopia that may contribute to improve individual’s life by an appropriate administration of anti-malarials. When slide positive cases were compared by molecular detection methodology, a high proportion of P. vivax individuals were misdiagnosed as P. falciparum-positive and were treated with AL, a drug of choice in treating P. falciparum infections. However the treatment of 14 cases (11%) microscopically diagnosed as P. vivax were mono-infections for P. falciparum. These results imply that these individuals were mistreated with chloroquine rather with AL. This may have had a considerable impact on treatment failure as they were misdiagnosed microscopically. Such a scenario may lead to severe malaria-related ailments and to the extent of mortality in these 14 cases. All the laboratories in the study area did not use buffer when preparing Giemsa staining solution. This might be one of the reasons for a high report of species mismatch and low sensitivity of microscopy. pH is an important component of Giemsa stain and pH 7.2 is recommended, as it highlights the Schüffner's dots of P. vivax and Maurer's clefts of P. falciparum, which are essential characteristics for malaria diagnosis by microscopy.In Ethiopia, ectively. In mostPlasmodium, and that helps to rule out false positives and mixed infections[P. falciparum and P. vivax, irrespective of study sites were observed. Of the 21 individuals with double infections, three were reported as P. vivax and 18 were P. falciparum-positive by microscopy. The reason why microscopy failed to detect mixed infections could be due to the presence of higher numbers of parasites of one species relative to the other. Moreover, in laboratories with overwhelming numbers of patients, the workload is not proportional to the laboratory staff involved. Therefore, this leads the laboratory personnel not to utilize adequate time to examine the blood film.Additionally, microscopy methodologies failed to detect multiple infections. As demonstrated by a plethora of other studies that compared microscopy with molecular diagnosis,24-30, nfections,31. A hiP. ovale is one of the prevalent parasites in western Africa[P. ovale were observed in any of the studied three sites. Additionally, P. malariae, a species which is less common in Ethiopia, was detected in four individuals with two as mono-infection and two as double infection with P. falciparum. A total of seven (2%) negative cases were observed. Of these individuals, five were treated for P. falciparum and two for P. vivax. The false positive cases were reconfirmed as negative for other Plasmodium spp. additionally; these false positive samples were subjected to subsequent amplification for both pfmdr1 and pfcrt alleles. Microscopy failed to detect P. malariae mono-infections and mixed infections. The dominance of either P. falciparum or P. vivax can possibly be a reason for the failure to detect P. malariae by microscopy. In addition, the pattern of fever and duration of the erythrocyte 72-hour developmental cycle may contribute to P. malariae low numbers. Although microscopy remains as a gold standard, it has its limitations to differentiate Plasmodium spp., in addition to low parasite counts[P. falciparum or P. vivax infections[Although n Africa, no indie counts. Factorsfections.P. malariae prevalence, including Federal Ministry of Health of Ethiopia. A few of these reported the presence only of P. falciparum and P. vivax[, whereas other reports demonstrated the prevalence of P. malariae in addition to the two dominant species in the country[P. vivax might limit parasitaemia and the pathogenic potential of P. falciparum[P. malariae in the aetiology of human glomerulonephritis is based on circumstantial clinical and epidemiological evidence and on renal biopsies showing granular immune deposits in the glomeruli[Many studies within Ethiopia reported conflicting results on the presence of  P. vivax, whereas country. A studyalciparum. In addilomeruli.8 total body parasites), and the reproducibility of parasite counts and species identification is frequently inconsistent[Microscopic diagnosis of malaria is the most widely used approach for epidemiological studies. The blood smear provides much vital information that helps to correlate the parasite density to clinical malaria. Unfortunately, microscopy detection has both qualitative and quantitative limitations. Blood-smear microscopy reaches its limit of detection when parasitaemia falls below 40 infected red blood cells (IRBC) per μl of blood (10nsistent. Factorsnsistent. False pnsistent.P. vivax and P. falciparum isolates is rather not restricted to one region, but seems to be widespread across different geographical regions within Ethiopia as the isolates from Abra Minch are also found in homology to isolates from Omo Nada. All isolates of P. malariae were represented from the site Omo Nada and were clustered together. Moreover, P. vivax showed a more diverse population than P. falciparum and P. malariae.The phylogenetic relatedness of Plasmodium parasite shall improve diagnosis. Overall, the finding of the current study highlights an important concern in the diagnosis of malaria by microscopy alone in southern Ethiopia and has important repercussions in understanding malaria epidemiology and subsequent control.False positivity, under-reporting of mixed infections and a significant number of species mismatch needs attention and should be improved for accurate diagnosis. Efforts must continue to re-train laboratory staff and provision of appropriate reagents based on the standard staining procedure for the diagnosis of The authors have declared that they have no competing interests.SK designed and performed the field study and experiments with drafting first draft. NB, AA and GM contributed to the study design and study samples and revisions of MS. TPV designed the experiments, contributed to materials and tools, supervised the experiments, data analysis, writing of the MS. All authors read and approved the final manuscript.


Few studies have assessed placental malaria infections from low transmission areas by histopathology to define their impact and underlying mechanisms.Peripheral smears and rapid diagnostic tests (RDTs), placental smears and histological samples, birth weight and gestational age were collected from 2,282 deliveries in three hospitals during a one-year (2006–2007) continuous cross-sectional survey in Madhya Pradesh. Placental histopathology included all 50 cases positive by microscopy or RDT plus 456 randomly selected samples of women negative for malaria by microscopy or RDT. Histological examination included parasites, inflammatory cells, pigment in fibrin, and morphological changes.Plasmodium falciparum infections were associated with stillbirth , lower maternal haemoglobin , lower birth weight  and shorter gestation . Chronic or past infections were not associated with these outcomes. Among the 11 peripheral Plasmodium vivax cases, placental parasites were absent, but they were associated with increased placental polymorphonuclear cells.There were 52 histology-positive cases; 38 (73.1%) active (acute and chronic) and 14 past infections. Intervillous parasitaemia was low (60% had < 1% parasitaemia) and monocytosis mostly mild (63%). Compared with uninfected placentas, acute Malaria associated stillbirth and low birth weight in women with low protective immunity may result, at least in part, from a shortened gestation triggered by acute infection, stressing the importance of early malaria detection. P. falciparum infected erythrocytes to the placental glycosaminoglycan chondroitin sulphate A receptors and associated inflammatory responses. The resulting features of parasite sequestration, increased presence of inflammatory cells, deposition of pigment (haemozoin), and fibrin characterize placental malaria and have been used to classify the chronology of placental infections <11 g/dL) were given haematinic supplementation  in the antenatal clinics. Although at the time of the study weekly chloroquine prophylaxis was recommended, this was not implemented in the participating hospitals.According to a previous survey in Madhya Pradesh in 2007–8, 61% of pregnant women visited ANC at least once and 34% at least three times, whereas 47.1% of women had institutional deliveries. The natper placenta) and processed and stained with haematoxylin and eosin at the National Institute for Malaria Research (NIMR) field laboratory in Jabalpur, India.Surveys were conducted in the delivery units in a hospital in Jabalpur city between September and October 2006 and in hospitals in the towns of Katni  and Maihar  between October 2006 and September 2007 to assess the burden of MiP. Information on demographic parameters, reproductive and malaria morbidity and prevention history during pregnancy, and delivery outcome were obtained. Newborns were weighed to the nearest 10 grams within two hours of delivery on a digital scale . Scales were calibrated weekly with a standard 5 kg weight. Gestational age was assessed within 24 hours of delivery using the Ballard score by staff trained by and under weekly supervision of the study paediatrician. A materHistology slides were examined locally under standard and polarized light microscopy to increase the sensitivity of haemozoin detection. The revP. falciparum and P. vivax; PCR analysis was carried out at the NIMR field station laboratory in Jabalpur of women with a stillbirth, and 20.7% [98/473] of women with a live birth, RR 1.3, 95% CI 0.65-2.6).P. falciparum infections were associated with reductions in birth weight  or mean gestational age  or lower haemoglobin levels  relative to no-infection  was not associated with significant reductions in birth weight , a reduced gestational age  or maternal haemoglobin .Histopathology did not identify any active In the 12 women who were slide smear and peripheral RDT negative but who had parasites detected by histology (sub-patent infection), the mean birth weight, gestational age and maternal haemoglobin levels were lower if both parasites and pigmented monocytes were detected, but numbers were small and the difference was not statistically significant Table .P. falciparum and P. vivax infections and the corresponding impact on adverse birth outcome and maternal anaemia were assessed in an area of low malaria transmission in central India. This was a pregnant population at low risk of malaria and despite the low parasitaemia seen by the low maternal blood geometric mean parasite density and placental parasite percentage, the impact of infection was considerable and independent of the presence of documented of fever.Few studies have described placental malaria associated histopathology outside sub-Saharan Africa. Histological changes associated with P. falciparum malaria than placental or peripheral smear microscopy alone. Approximately one in 40 women (2.4%) that were negative by conventional microscopy  or pLDH-based RDT were found to have active placental P. falciparum infections (acute or chronic) by histology. When this estimate of ‘subpatent’ infections was extrapolated to the overall survey sample of 2282 women, the prevalence of P. falciparum placental malaria increased from 2.2% (50/2282) to 4.5%, doubling the estimate. Placental incision microscopy alone missed about two out of every three infections which could be explained by the largely low parasitaemia. Women with subpatent infections had lower maternal haemoglobin, mean birth weight and gestational age, although the difference did not reach statistical significance, likely because of the small number of women with submicroscopic infections.Based on placental incision microscopy, the overall prevalence of malaria at delivery was 1.0%, and comparable to recent studies in other regions in India,32. As eP. falciparum infections detected by conventional microscopy also had evidence of an active placental histopathology infection. In contrast, no placental parasites could be detected by histology among the 11 women with patent peripheral P. vivax infections and none of the women who were smear negative for peripheral P. vivax had evidence of infection with histopathology. This highlights the importance of using sensitive methods such as placental histology or PCR to detect placental P. falciparum infection as conventional peripheral and placental microscopy smear results alone may markedly underestimate the true burden of P. falciparum malaria in this region.Most  of the women with patent maternal blood P. falciparum infections[P. falciparum were acute rather than chronic or past infections. This might be explained by the low malaria exposure and low host immunity in this population triggering an inflammatory response and early delivery when the placenta becomes infected, although the adverse impact of infection was independent of the occurrence of fever in this study. The low exposure is also reflected by the low parasitaemia within the placenta and the corresponding mild inflammatory response. This contrasts with the dense parasitaemia and the occasionally massive chronic intervillositis and monocyte infiltration reported from highly endemic areas in sub-Saharan Africa[Histopathology is a useful method to grade the chronology of placental fections,4. Compan Africa, but is n Africa and Colon Africa-18. Simin Africa,33.P. falciparum infection was associated with an increase in syncytial knots, focal fibrinoid necrosis and calcification. These changes were also evident in past infections, suggesting they persist after parasites are cleared[Chronic  cleared. Alterna cleared.P. falciparum malaria was associated with maternal anaemia. Acute infection was associated with lower levels of mean haemoglobin (1.4 g/dL), comparable to observations in a study from Malawi[P. falciparum infection on birth weight was considerable, despite the scarcity of parasites within the placenta and the mild degree of placental inflammation. Of all the histological measures, only the presence of parasites and pigmented monocytic infiltration were consistently associated with reduced birth weight and shorter gestation in multivariate analysis. Further analysis showed that the effect on birth weight and gestation was particularly evident among the active infections with pigmented monocyte infiltration, a reduction in birth weight of 356 grams compared to 42 grams in women with presence of parasites alone, especially if this monocyte infiltration was moderate to severe. While mononuclear cells as a source of inflammatory cytokines have been suggested to have a role in poor foetal growth[per se may play a role.Placental m Malawi. Chronicm Malawi,35. Addil growth,36[37-39The observed shortening of the duration of pregnancy with active infections was an important contributor to the reduction in birth weight. It is possible that with the increased risk of acute infections in women with history of fever, the inflammatory response to infections might induce early labour. Thus infections may not last to lead to chronicity. Alternatively clinical symptoms may lead women to seek treatment and prevent chronicity. The average duration of pregnancy was 4 days shorter among women with an active infection and mild monocyte infiltration, and 8 days shorter in women with moderate to severe infiltration. Although this may appear a relatively small difference, the average increase in birth weight between 37 to 40 weeks gestation is estimated at 169 grams per week.P. vivax sequestration in the placenta. Pigment in the fibrin was observed in two peripherally patent P. vivax cases, which is consistent with reports from Thailand and Peru[P. falciparum cannot be excluded. Recent studies on immune aspect of placental malaria in Colombia indicate a possible pathogenic role of P. vivax[Plasmodium vivax cases was the significantly higher presence of polymorphonuclear leukocytes and syncytial knots compared to P. falciparum infected placentas, suggesting a possible placental inflammatory response that might be different from that observed with P. falciparum. In addition, decreased fibrin deposition was seen compared to P. falciparum or uninfected placentas. There was no association, however, between polymorphonuclear cells or increased syncytial knots and birth weight or preterm births, although numbers may have been too small.There was no evidence of and Peru,24, alth P. vivax,18 A notP. vivax mono-infections was too low to draw firm conclusions. Although a single reader examined the sections and she was not blinded to the peripheral, placental microscopy and RDT results, a random sample of positive slides and those corresponding to patent P. vivax cases were reviewed blinded by a senior placental histopathologist in the US and confirmed the accuracy of the results of the first reading. PCR conducted in a subset of samples, provided further confirmation of species and the accuracy of the histological examination. The Ballard score that was used has not been validated in India. It was beyond the scope of this study to assess conditions other than malaria that may affect birth weight, haemoglobin level and prematurity .The study was limited by its cross-sectional design, which did not allow assessing the impact of earlier pregnancy infections via peripheral microscopy. If such infections resolve and are no longer evident as ‘past’ infections but still impact on maternal anaemia and birth outcome, the burden of malaria may have been underestimated. The number of women with P. falciparum infection proved rare in this population. Despite this, a substantial impact of P. falciparum placental infections on birth weight was observed, which in part could be explained by shortening in the average duration of pregnancy. There was no evidence of P. vivax sequestration in the placenta and any histological changes observed were mild, however the number of P. vivax infections were small. These findings with P. falciparum infections highlight the need for early (including subpatent) case-detection and the challenge of malaria control during pregnancy in areas of low malaria transmission to reduce the detrimental impact of malaria during pregnancy in this region.Severe placental inflammation associated with The authors declare that they have no competing interests.RA, DJT, FTK, VU, NS and MD conceived and designed the study. RA led the overall surveys, conducted the histopathology component, data analysis and interpretation under supervision of DJT and FTK. PB and PS conducted the PCR component, RA wrote the first draft of the manuscript with input from DJT and FTK. All authors contributed to the final manuscript. All authors have contributed to, seen, and approved the final, submitted version of the manuscript.


The relationship between malaria and undernutrition is controversial and complex. Synergistic associations between malnutrition and malaria morbidity and mortality have been suggested, as well as undernutrition being protective against infection, while other studies found no association. We sought to evaluate the relationship between the number of malaria episodes and nutritional statuses in a cohort of children below 15 years of age living in a rural community in the Brazilian Amazon.Following a baseline survey of clinical, malaria and nutritional assessment including anthropometry measurements and hemoglobin concentration, 202 children ranging from 1 month to 14 years of age were followed for one year through passive case detection for malaria episodes. After follow-up, all children were assessed again in order to detect changes in nutritional indicators associated with malaria infection. We also examined the risk of presenting malaria episodes during follow-up according to presence of stunting at baseline. Children who suffered malaria episodes during follow-up presented worse anthropometric parameters values during this period. The main change was a reduction of the linear growth velocity, associated with both the number of episodes and how close the last or only malaria episode and the second anthropometric assessment were. Changes were also observed for indices associated with chronic changes, such as weight-for-age and BMI-for-age, which conversely, were more frequently observed in children with the last or only episode occurring between 6 and 12 months preceding the second nutritional assessment survey. Children with inadequate height-for-age at baseline (Z-score < -2) presented lower risk of suffering malaria episodes during follow-up as assessed by both the log-rank test (p =0.057) and the multivariable Cox-proportional hazards regression .P. vivax predominates. Our data all supports that the association presents differential effects for each age group, suggesting distinct pathophysiology pathways. We were also able to demonstrate that undernourishment at baseline was protective to malaria during follow-up. These findings support an intriguing interaction between these conditions in the rural Amazon and the need for a more integrative approach by health systems in endemic areas.Malaria was associated with impaired nutritional status amongst children in an endemic area of the Western Brazilian Amazon where  Plasmodium vivax predominates.Malaria is one of the most serious public health problems in the world, with 3.3 billion people at risk of contracting the disease and almost one million deaths annually, primarily in children younger than five years of age. Undernutrition is also a morbidity of importance to the public worldwide and primarily affects children in tropical regions. In the present study, we sought to analyze the relationship between malaria and undernutrition in children living in a rural Amazonian community where malaria is endemic. Children from 1 month to 14 years of age were followed-up for 12 months through passive case detection . Anthropometric and hemoglobin measurements and active malaria case detection tests (to detect asymptomatic parasitaemia) were conducted at the beginning and end of the follow-up period of 12 months. Children who had at least one episode of malaria during this period presented lower mean anthropometric index scores compared to children who did not have malaria. We concluded that malaria had a negative impact on the nutritional status of children living in an endemic area at the Western Brazilian Amazon, where  Plasmodium vivax. Of these, 263,767 cases were reported in Brazil, mainly in the Amazon region, also with a vast majority of P. vivax (87.8%) [Malaria is one of the most serious public health problems in the world, with 3.3 billion people at risk of contracting the disease and almost one million deaths annually, primarily in children under five years of age . Althoug (87.8%) , 2.It has long been acknowledged that populations residing in malaria-endemic areas generally live under socio-economic conditions leading to poor nutritional status. The groups at highest risk for the adverse effects of malaria, children and pregnant women, are also those most affected by poor nutrition. Although it has been suspected that nutrition may influence susceptibility to the disease or alter its course, there have been comparatively few efforts to comprehensively examine such interactions , 4.Plasmodium and undernutrition [It has been gradually recognized that any infection is associated with a risk of worsening the nutritional status . Whereasutrition , 14, witutrition .P. falciparum is the main species causing malaria. In Latin America, there is scant information regarding the association between infectious diseases and nutrition. A survey performed in Central American countries showed that diarrhea and respiratory diseases worsened the nutritional status of children [The majority of studies addressing the relationship between malaria and nutritional status come from Africa, where children . This nuchildren , 17. In children , 19. Thechildren . A recenchildren , with stchildren .P. vivax and P. falciparum) is endemic. The aim was to evaluate the effect of malaria episodes on the anthropometric nutritional status indicators change and to explore the influence of baseline height-for-age z-scores (HAZ), which more accurately reflects chronic undernutrition with less interference from recent episodes of malaria or other acute conditions) on the risk of subsequent malaria incidence.In the present study, we sought to analyze the occurrence of malaria episodes and undernutrition in a cohort of children living in a rural Amazonian community where malaria . Each participant and his/her parents or legal guardians signed written informed consent forms.The study was approved by the Ethics Committee Board of the 2 and has 31,063 inhabitants. The climate is tropical and humid, with rainfalls ranging from 2,100 to 2,400 mm per annum. The municipality is connected to the capital of the state, Manaus, through a federal road (112 km of distance). Malaria is endemic in this area. The major economic activities are family farming, hunting and fishing. Drinking water comes from rainwater reservoirs or creeks. Garbage collection and sanitation are absent. Two health agents in each community are responsible for health care. The total population of both communities is 790 people, according to the census performed before the beginning of the study, including 300 children ranging from 1 month to 14 years of age.The study was undertaken in two rural communities located in two recently colonized areas devoted to agriculture (Panelão and Castanho Sítio Communities), from May 2008 to May 2011. These settlements are located in the Municipality of Careiro, in the Amazonas State. The municipality has an area of 6,124,300 kmA cohort of 248 children ranging from 1 month to under 15 years of age was recruited for the present study, with 202 completing follow-up. As part of the baseline survey, a questionnaire was completed to collect socio-demographic information, including health history, maternal education, housing, and the number of assets . The housing characteristics were combined using principal component analysis (PCA) as previously described , in ordePlasmodium and did not systematically examined patients for detection of asymptomatic infection.To evaluate whether malaria affects nutritional status, the following outcome variables were used: growth velocity and the weight for height z-scores (WHZ), weight for age z-scores (WAZ), height for age z-scores (HAZ) and body mass index for age (BMI-Z) nutritional index scores, all of them considering the WHO standard cut-offs . MalariaNutritional assessment through anthropometric measurements was performed during the cross-sectional evaluations, at the beginning and at the end of the follow-up. Anthropometric measurements were performed with minimal clothing and no shoes, according to guidelines by the Brazilian Ministry of Health . Weight Body mass index (BMI) was calculated using the program ANTHRO and ANTHRO PLUS . BMI for3 for each child. A sub-sample of blood collected in filter papers was also tested by PCR to allow quality control of the TBS for species-specific diagnosis . A malaria case was considered when the child presented with fever and a positive TBS.The thick blood smear (TBS) was prepared as recommended by the Walker technique  and evalHemoglobin concentration was measured in venous blood using a portable photometer  at the beginning and at the end of the follow-up.Stool samples were taken only at baseline to examine the association between helminth infection and baseline anthropometric indicators. The stool samples were stored in flasks containing 10% formalin as preservative. Flasks were labeled with the patient’s name and date of collection and were kept at room temperature until the end of the month, when all the stool samples were examined. Spontaneous sedimentation  and centChildren were categorized into three age groups for analyses, according to WHO guidelines for use of indicators WAZ, HAZ, WHZ and BMIZ : ages beThe association between stunting (height for age below -2 Z-scores) at baseline and the risk of development of malaria was assessed considering time from enrollment to the first or only malaria episode, using survival analysis techniques with inspection of the Kaplan-Meier survival curve, with performance of log-rank test and computation of adjusted Cox proportional hazard ratios . This waAscaris lumbricoides (26.7%). During the follow-up period, 87 children (43.1%) presented with at least one episode of malaria, and a total of 164 malaria episodes were observed. The remaining 115 children (56.9%) did not develop malaria during the study period. Among the infected subjects, 46 children (52.9%) had one malaria episode, 21 children (24.1%) had two malaria episodes, and 20 children (23%) had three or more malaria episodes. Regarding the Plasmodium species involved, 119 episodes (72.6%) consisted of mono infections with P. vivax, 37 episodes (22.5%) involved P. falciparum alone, and 8 episodes (4.9%) were mixed P. vivax and P. falciparum infections. No episodes of severe malaria occurred amongst the cohort participants.During the 12-month follow-up period, 248 of the 300 children eligible to participate in the cohort study were enrolled; 46 children were lost to follow up and the remaining 202 children were successfully followed . As showThere was an association between final assessment being underweight, low BMI and inadequate growth velocity in children 5–10 years of age who developed malaria during follow-up . Table 3No significant interaction was found between nutritional indicators at baseline and second measurements and malaria for children <5 year of age . For theThe results of the analysis on the risk of malaria episodes related to the baseline nutritional status was assessed only for the or the HAZ indicator as this reflects chronic malnutrition more accurately and are shown on Malaria is one of the main public health problems in several developing countries affecting especially children, a particularly vulnerable population with the highest morbidity and mortality burdens associated with this disease , 34. MalP. vivax is the most prevalent species, the infection may influence the physical development of children. Lee et al. monitored a cohort of children in the Peruvian Amazon and observed that children with P. vivax malaria experienced delayed linear and weight growth [In this study, we show that malaria episodes interferes with the nutritional status of children, with notable reduced linear growth velocity as well as impairment in other indices associated with chronic malnutrition, even after adjustment for other factors. Suffering from one or multiple episodes of malaria had a significant negative effect on the linear growth velocity of children, especially amongst children between 5 and 10 years of age . This important finding suggests that even in areas where malaria transmission is low and t growth . Availabt growth , 37 and t growth –40 are iAlthough we found an association between undernourishment measured by HAZ and the risk of developing malaria, one needs to be careful at implying causality as other unmeasured confounders could be associated, including, for example, improved malaria preventive measures for ill children. We also did not observe a protective effect between baseline anemia and the risk of developing malaria as has been suggested elsewhere . Due to Some studies investigating the pathophysiologic mechanisms suggest that in addition to the anorexia and vomiting caused by acute-phase malaria and the negative nitrogen balance during fever episodes , a lack There were some important limitations in our study. Overall there was a low rate of malaria infection, especially amongst children in the lowest age group, which resulted in low power to examine the association of the infection with nutritional status and may, at least partially, explain the lack of effect observed amongst this particular group. Combined with the possibility of non-differential measurement error of the anthropometric indices, we assume the risk of bias towards a null effect of the association. No assessment of dietary patterns was performed, and although there is evidence of homogeneous habits within the study population, there may be important individual variations that would have a strong impact on the nutritional status of children. The lack of stool examination on the end of follow-up after the decision to treat all patients at inclusion has impaired the capacity to evaluate and control for the important influence that helminths have on the nutritional status of children , 22, espPlasmodium infections. Undernutrition and malaria are important morbidities with relevance to public health; thus, coordinating the actions of malaria control programs and nutrition programs could substantially impact these morbidities among children, meriting further attention by researchers and policy makers.In summary, we found malaria to be associated with the impairment of nutritional status in children, with evidence pointing to specific effects for different age groups, as indicated by anthropometric indexes associated with chronic  and acute (linear growth velocity) nutritional deficits. We were not able to establish a clear association between baseline anthropometric classification and the risk of developing malaria. Bigger cohorts may be necessary especially in areas of low malaria incidence, and future studies should focus on specific nutritional deficiencies that may influence S1 Checklist(DOCX)Click here for additional data file.S1 Dataset(RAR)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children  and 211 Asian adults  presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples.In malaria-endemic areas,  Plasmodium falciparum HRP2 as a measure of parasite biomass [Severe falciparum malaria is a major cause of childhood death in sub-Saharan Africa, where it is a common diagnosis in severely ill children. In areas of moderate and high transmission, malaria parasitemia is also common in apparently healthy children. Because of this, the specificity of a blood slide for malaria is poor, and malaria is consequently overdiagnosed. Postmortem studies indicate that nearly 25% of children dying with a clinical diagnosis of cerebral malaria actually died from other causes , resulti biomass  separateP. falciparum HRP2 is released at the time of schizont rupture, we investigated the hypothesis that plasma concentrations of DNA (reflecting liberated merozoites and degraded parasites) might also reflect the cumulative parasite burden and, thus, the severity of disease in falciparum malaria. Plasma concentrations of bacterial DNA have been shown to correlate with severity in a number of invasive bacterial infections [P. falciparum DNA in plasma or serum [P. falciparum DNA concentrations and malaria severity. We reasoned that DNA released into the plasma by schizont rupture or degradation of sequestered parasites might be a better measure of the previously sequestered biomass and, thus, disease severity than peripheral parasitemia. We assessed the diagnostic performance of plasma P. falciparum DNA concentration, alone and then combined with plasma P. falciparum HRP2 concentration and parasitemia, to distinguish between uncomplicated and strictly defined severe malaria in African children and Asian adults.Because fections –6. Severor serum –9 but haP. falciparum malaria in East Africa. Samples from uncomplicated cases were obtained during longitudinal community studies undertaken between February and August 2008 in Handeni district, northeast Tanzania. These were febrile children with a positive result of a rapid diagnostic test (RDT) for P. falciparum lactate dehydrogenase (pLDH) but no clinical signs or laboratory indicators of severe malaria [The pediatric study involved children with symptomatic  malaria . Samples malaria . Plasma  malaria  and all  malaria  or orallEthics approval was granted by the Oxford Tropical Research and London School of Hygiene and Tropical Medicine ethics committees and relevant local ethical committees. Written informed consent was obtained from all patients or an attending relative.Parasitemia was calculated for children with uncomplicated malaria, using the thick film parasite count per 200 leukocytes and the actual WBC count; if the WBC count was missing, the parasite count was calculated by assuming a WBC count of 8000 leukocytes/μL (count/200 leukocytes × 40) . In the P. falciparum HRP2 concentrations and resulting levels in the pediatric study have been described previously [Baseline plasma specimens were collected in tubes containing ethylenediaminetetraacetic acid and stored at −80°C until testing. The method for measuring plasma eviously , 10.To measure the parasite DNA concentration in each plasma sample, absolute quantitative real-time polymerase chain reaction (qPCR) analysis was performed directly by using plasma (2 μL) as a template without any DNA extraction step, 18S ribosomal RNA–targeting primers, and hydrolysis probes . A CorbeP. falciparum line. Suspensions containing precisely 10 000 ring-stage-infected red blood cells per tube were obtained using fluorescence-activated cell sorting, as described by Malleret et al [r2 > 0.98), with amplification efficiencies of 90%–105%. No nonspecific amplification products were seen on gel electrophoresis and by sequencing qPCR products from a range of field samples . To check cross-contamination of samples during qPCR processing, negative controls (water) were added randomly (8 negative controls per 48 samples). Precision was assessed by 5 independent measurements of 5 samples spanning the range of DNA concentrations encountered , with coefficients of variation ranging from 0.67% to 1.98% (cycle threshold), equivalent to 13.8%–38.2% (parasite genomes/µL).A calibration standard was prepared using a highly synchronized culture of the 3D7 et et al . The staP. falciparum HRP2, and plasma concentrations of P. falciparum DNA were evaluated in Stata v 12.0 , using logistic regression to calculate AUROC values for pairs of explanatory variables.Statistical comparisons between groups were performed using the Mann–Whitney test. Area under the receiver operating characteristic curve (AUROC) analyses for parasitemia, plasma concentrations of Samples were obtained from 435 patients, comprising 224 children in Tanzania and Mozambique  and 211 adults in Bangladesh and India . Baseline characteristics of the 2 groups are shown in Table P. falciparum DNA and P. falciparum HRP2 concentrations and peripheral parasitemia for the respective groups of patients are shown in Figure P. falciparum DNA levels were approximately 40-fold and 10-fold higher in cases of severe malaria, compared with those in cases of uncomplicated malaria, in African children  and Asian adults , respectively.Plasma P. falciparum HRP2 levels were 0.857 and 0.818, respectively. Both methods of assessment were substantially more accurate than parasitemia in differentiating severe from uncomplicated malaria . There was no significant difference in AUROC values between plasma P. falciparum DNA concentrations and P. falciparum HRP2 concentrations . Use of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations led to a further improvement in diagnostic accuracy, compared with use of P. falciparum HRP2 concentrations alone . By comparison, use of parasitemia plus plasma P. falciparum HRP2 concentrations did not significantly improve the AUROC in either children (P = .5) or adults (P = .7).The AUROC values Figure  for seveP. falciparum DNA concentrations in plasma specimens obtained at admission were not significantly associated with mortality within the severe malaria groups . Parasitemia similarly did not predict outcome within the severe group in either location, whereas the plasma P. falciparum HRP2 level predicted outcome in Asian adults  but not in African children .Plasmodium DNA in patients' plasma or serum has been recognized since the introduction of PCR methods for malaria diagnosis and genotyping [P. falciparum DNA concentration are not known but could be tested by centrifugation or filtering to remove different sizes of particulate matter that contain parasite DNA. The kinetics of the production and elimination of plasma parasite DNA have not yet been characterized; studies of human DNA circulation in plasma indicate that rapid hepatic clearance occurs [The presence of species-specific notyping , 8. Thise occurs . The hale occurs .P. falciparum DNA reflected disease severity in falciparum malaria. Plasma P. falciparum DNA levels performed considerably better in both Asian adults and African children than peripheral parasitemia as a measure of disease severity and performed similarly to the recently validated plasma P. falciparum HRP2 as a severity measure [Plasma concentrations of  measure . The welP. falciparum HRP2 and P. falciparum DNA concentrations was significantly more accurate than either measure alone. Additivity in accuracy of diagnostic performance suggests that the 2 markers provide independent measures of severity. This may reflect differing intrinsic mechanisms and kinetics of production and elimination. It might also result from covariates affecting either marker that are independent of parasite biomass, so that the 2 measures complement each other. Potential covariates include P. falciparum HRP2 gene deletion [P. falciparum HRP2 levels in African children found no evidence of this [P. falciparum HRP2 expression [Importantly, the combination of plasma deletion  , differpression , and smapression , 21. DesP. falciparum HRP2 concentrations) had no mortality benefit from artesunate, compared with quinine, which suggests that they may have had another lethal disease  [P. falciparum DNA concentrations between the uncomplicated and severe cases among adults, compared with those observed among children, in this study may be explained by disease severity. The adults with uncomplicated malaria studied in Bangladesh and India were sufficiently ill to warrant admission to a referral hospital , had higher plasma P. falciparum HRP2 and P. falciparum DNA concentrations, and probably had more-severe infections than the African children with uncomplicated malaria recruited in the community-based studies.A clinical definition of severe malaria was used as the gold standard for calculations of diagnostic accuracy (measured as the AUROC). However, in the AQUAMAT study (in which the severe pediatric cases were studied), one third of cases (defined by the ranking of plasma ections) . This suections) . In low-P. falciparum DNA concentrations is currently unlikely to be incorporated into prospective patient management, although rapid advances in PCR technology may change this. The P. falciparum DNA concentration is a useful prognostic measure in research studies and may provide valuable information from previous studies in which plasma or serum samples have been archived. qPCR can be readily adapted to a high-throughput format. Plasma measurements reflecting the sequestered parasite burden are valuable indicators of disease severity in falciparum malaria.Quantitation of plasma


Background. Malaria is a one of the leading causes of morbidity and mortality in tropical countries. Plasmodium vivax (P. vivax) is usually thought to be causing benign malaria with low incidence of complications as compared to Plasmodium falciparum . Methods. This retrospective observational study included malaria patients who were admitted to K.T. Children Hospital and P.D.U. Government Medical College, Rajkot, a tertiary care teaching hospital, Gujarat, western India, during the period January 2012 to December 2012. Inclusion criteria were patients in whom either P. falciparum or P. vivax was positive on rapid malaria antigen test and peripheral blood smear. Patients showing mixed infections were excluded from study. Results. A total of 79 subjects (mean age 5.4 ± 3.6 years) were included in the study. It consisted of 47 P. vivax and 32 P. falciparum cases. The P. vivax cases consisted of 33 (70.2%) males and 11 (19.8%) females while P. falciparum cases consisted of 14 (43.8%) males and 18 (56.2%) females. One patient of each P. vivax and P. falciparum expired. There was no statistical significant difference found between complications such as anemia, thrombocytopenia, liver and renal dysfunction, ARDS, and cerebral malaria between P. vivax and P. falciparum. Conclusion. We conclude that P. vivax monoinfection tends to have as similar course and complications as compared to malaria due to P. falciparum monoinfection. P. falciparum malaria causes more severe disease, mortality and morbidity so intensive measures have been implemented mainly against it. P. vivax malaria has been neglected and mistakenly considered as “Benign” [ P. vivax is able to cause severe disease [ P. vivax at low parasite densities. P. vivax is the most common geographically widespread species of Plasmodium causing malaria in human beings. The objective of this study was to compare the clinical profile and complications of P. vivax with P. falciparum malaria infection in children.According to the UNICEF, at every 30seconds, one child expires due to malaria . It is o“Benign” . But the disease . This maThis retrospective observational study included malaria patients who were admitted to K.T. Children Hospital and P.D.U. Government Medical College, Rajkot, a tertiary care teaching hospital, Gujarat, Western India, during the period from January 2012 to December 2012.Inpatient records from January 2012 to December 2012 were retrieved and scrutinized by using a Performa on the basis of the patient's demographic profile, clinical findings, investigations, treatment, and complications during this 12-month period. The institutional ethical committee approved the study. Inclusion Criteria. All slide positive and rapid diagnostic tests (RDT) that confirmed cases of malaria  admitted and treated in K.T. Children Hospital with age group up to 12 years were included. Exclusion Criteria.These criteria included (1) patients presented with fever  but treated empirically for malaria, (2) mixed infection of PF and PV malaria, and (3) patients presented with clinical features mimicking malaria like dengue fever, sepsis, meningitis were excluded from this study. We also excluded the newborn babies and those patients who died during resuscitation within the first hour in an emergency department before hospital admission formalities were complete. P. falciparum and P. vivax malaria were established by thick and thin film of peripheral blood smear examination under oil immersion with giemsa stain and RDT [ Plasmodium spp. lactate dehydrogenase  and histidine-rich protein 2 . Severe complicated malaria was categorized as per World Health Organization guidelines. Severe complicated malaria in the form of cerebral malaria, severe anemia (Hb < 5 mg/dL), thrombocytopenia (platelet count < 1 lac/cumm), pancytopenia, jaundice (>3 mg/dL), acute renal failure (serum creatinine >3 mg/dL), gastrointestinal tract dysfunction, acute respiratory distress syndrome, and multiorgan dysfunction was included in this study. The level of consciousness was assessed using the modified Glasgow modified Glasgow Coma Scale in patients age <9 months and Glasgow Coma Scale in patients age >9 months. Routine laboratory investigations included a complete blood cell count, peripheral smear examination, blood indices, and platelet count and these were sent immediately after admission of all the patients. Urine examination, liver and renal function tests, coagulation profile, cerebrospinal fluid study, chest radiograph, and blood culture were done whenever it was indicated. The case definition of complicated and severe malaria was taken from the WHO guidelines for treatment of malaria 2010 [The diagnosis and confirmation of species of and RDT . The RDTria 2010 . Anemia Statistical analyses were performed by social package for statistical science (SPSS) version 16. The data of the two groups were compared using the Fisher or chi-square test appropriate for each study parameter. Confidence interval and odds ratio of two groups were also reported. P. vivax and 32 P. falciparum cases. Baseline characteristics of both groups were similar  were included in the study. It consisted of 47 similar . One pat P. vivax and P. falciparum malaria showed no statistical significant difference , jaundice, renal failure, ARDS, and cerebral malaria. These findings of similar complication of P. vivax malaria were also reported by other authors [In this retrospective observational study we report 79 patients with authors , 9. P. falciparum malaria but also encountered in P. vivax malaria. This may be due to multiple factors which include increase in platelet destruction by platelet associated IgG antibody and its consumption [ P. vivax and P. falciparum malarial children. Other authors from India also reported significantly higher proportion of thrombocytopenia in P. vivax. [Thrombocytopenia is a well-known complication ofsumption , 11. We . vivax. , 13. P. vivax while the same were present in 6.2% and 9.3% cases of P. falciparum. Hepatic involvement has been well documented in P. falciparum malaria but also reported in P. vivax malaria. The possible explanation for hepatic involvement is direct injury to liver by parasite leading to malarial hepatitis [Raised liver enzyme and jaundice were present in 10.6% and 6.3% cases ofepatitis –16. P. vivax and P. falciparum cases in our study, respectively. Renal failure was observed commonly in P. falciparum but also has been reported in P. vivax malaria. Renal failure in malaria is caused by parasitized red blood cells leading to mechanical obstruction. Microcirculatory disorders, disseminated intravascular coagulation, fluid loss, and hypoxic or immune-mediated necrosis of renal tubules and glomeruli are the possible mechanisms that may be implicated in vivax infection [Renal failure was encountered in 2.1% and 3.1% ofnfection –20. P. vivax and P. falciparum malaria. One patient of P. vivax died due to ARDS. It is known to occur in P. falciparum malaria due to sequestration but not reported in P. vivax malaria very often. The recent studies have shown that organ specific sequestration, cytokines, and nitric oxide production are mainly responsible for this complication of P. vivax malaria [Acute respiratory distress syndrome (ARDS) has been encountered in 3-4% cases of malaria , 22. P. vivax and 6.2% of P. falciparum cases in our study. Cerebral malaria is a very severe complication and leading to one of the most common causes of mortality of malaria. One of our infected patients of P. falciparum malaria died due to cerebral malaria. Though exact pathogenesis of cerebral malaria in P. vivax remains unknown, few studies suggested that it might be due to sequestration and cytokine mediated cerebral injuries [Cerebral malaria has been observed in 4.2% ofinjuries , 24. P. vivax malaria infection is often underestimated though complications and mortality are almost similar in comparison to P. falciparum malaria. Further large scale studies are required to know the exact pathogenesis of complications of P. vivax malaria. There is an urgent need of public health measures to estimate the burden of P. vivax malaria so that adequate planning and control measures can be taken against this emerging problem.The major limiting factor of our study was its retrospective nature and the small sample size. We believe that P. vivax monoinfection tends to have as severe course and complications as compared to malaria due to P. falciparum monoinfection.The present study concludes that


This study determined the association of TLR4 Asp299Gly and Thr399Ile with uncomplicated and severe malaria among Nigerian children of similar ethnic background in Lagos. The association of these SNPs with high parasite density, malnutrition, hyperpyrexia and anaemia was also investigated.Genomic DNA of the study participants was screened for the genotypes of TLR4 Asp299Gly and Thr399Ile by PCR-RFLP. Anthropometric measurement was performed on the Pf infected children stratified into asymptomatic malaria (control), uncomplicated and severe malaria (case). Parasites were detected by light microscopy and Hardy Weinberg Equilibrium (HWE) of SNP genotypes was also determined.A total of 279 children comprising 182 children  with clinical falciparum malaria and 97 children  with asymptomatic falciparum malaria were enrolled. All the genotypes of both TLR4 SNPs were found in the study population with their minor alleles: 299Gly and 399Ile, found to be 17.6 % and 14.7 % in severe malaria children. Unlike in asymptomatic population, the genotype distribution of TLR4 Asp299Gly SNP was not in HWE in the clinical malaria group but did not condition susceptibility. However, Asp299Gly and Thr399Ile polymorphisms were found to increase the risk of severe malaria 3-fold and 8-fold respectively (P < 0.05). They also increased the risk of severe anaemia, high parasite density and severe malnutrition 3.8 -5.3-fold, 3.3 – 4.4-fold and 4-fold respectively.Based on the above findings, we conclude that TLR4 Asp299Gly and Thr399Ile polymorphisms may modulate susceptibility to severe malaria among Nigerian children of Yoruba ethnic background. Plasmodium falciparum still accounts for 18 % of child deaths [. In 2012, 460,000 deaths out of the estimated 627,000 global malaria deaths were reported for children below 5 years with about 86 % of these deaths occurring in sub-Saharan Africa [Despite a reduction by 54 % in the African region since 2000, malaria due to n Africa . Malarian Africa .TLR4 gene , which spans a genomic region of ~13.3 kb with three exons  [P. falciparum and Trypanosoma cruzi as well as for lipopolysaccharide of gram negative bacteria and other pathogen associated molecular patterns (PAMPS) in gram positive bacteria, fungi and viruses [TLR4: an A to G transition (SNP ID = rs4986790), resulting in Aspartate-Glycine substitution at position 299 (TLR4Asp299Gly) and C to T transition (SNP ID = rs4986791), resulting in threonine-isoleucine substitution at position 399 (TLR4Thr399Ile), to cause altered GPI binding, 50 % reduction in TLR4 expression on the membrane surface of innate immune cells, induce LPS hyporesponsiveness and excessive production of pro-inflammatory cytokines [TLR4 Asp299Gly or Thr399Ile polymorphism with death from septic shock and susceptibility to typhoid fever, tuberculosis, meningitis, chagas disease and respiratory syncytial virus infection in infected infants, children below 5 years and adults [TLR4 polymorphisms in malaria pathogenesis, particularly in other countries with high malaria transmission.Toll-like receptor 4, a major pathogen recognition receptor (PRR) expressed on membrane surface of innate immune cells is genetically encoded by the ih.gov/)  This PRR viruses –12. Meanytokines , 13, 14.d adults , 15. Howd adults –23. Thesd adults , 23. TheTLR4 Asp299Gly and Thr399Ile SNPs are in circulation among the Yoruba tribe [TLR4 Asp299Gly and Thr399Ile polymorphisms in a cohort of P. falciparum infected Nigerian children with susceptibility to clinical and severe malaria.Nigeria is currently among the top there high malaria burden countries in the world with an annual mortality of 300,000 deaths and where falciparum malaria accounts for 30 % of total under-five mortality every year . Despiteba tribe , the rolTLR4 SNPs data from severe malaria cases, children hospitalized at Massey Street Children Hospital, Lagos with laboratory and clinical evidence of severe falciparum malaria between September – October, 2011 were also enrolled into the study. Lagos is located within the Equatorial tropical region where malaria transmission mostly driven by female mosquitoes from the Anopheles gambie complex occurs throughout the year [et al. [et al., unpublished). In this coastal settlement, malaria treatment practices were generally poor among caregivers despite their good knowledge of symptoms of uncomplicated malaria [This was a cross sectional study in which convenience sampling was used to enroll children during two separate malariometric surveys in Lagos between 2009 – 2011, the first survey was conducted in March – August, 2009 in Takway-Bay, Victoria Island, Lagos, while the second survey was conducted during the dry season in January 2011 in Ibeshe Community in Ikorodu. To further obtain the year . In Ibes [et al. , while i malaria . Massey  malaria .Plasmodium falciparum positivity slide results. Asymptomatic malaria was defined as parasitaemia without fever in the previous 48 h or other related malaria symptoms or history of malaria in the preceding two months. Children in the uncomplicated malaria category were those having parasitaemia with fever (axillary temperature > 37.4 °C) other classical symptoms such as headache, chill and sweating plus mild-moderate anaemia (Hb < 11 g/dL - <9 g/dL), while severe malaria category were those with severe anaemia, fever plus one or more of other complications such as prostration, coma, jaundice, and respiratory distress [The study population for this study comprised children (age < 13y) with distress . At the distress  and useddistress . The z-sdistress . The fiedistress . ChildreParasite was detected and speciated by microscopic examination of thick (12 μL) and thin (3 μL) blood smear on grease-free labeled glass slides according to WHO guidelines. The glass slides were prepared in duplicates and read by two independent trained microscopists. A glass slide was considered parasite negative after examining 200 high power fields without seeing one parasite. Parasitaemia was measured by counting the number of parasites against 200–500 leukocytes using the thick blood film and expressed as number of parasites per 1 microlitre of whole blood, assuming 8000 leukocytes per 1 microlitre of blood.A drop of finger pricked blood drawn into a microcuvette was used for the determination of haemoglobin (g/dL) using a Hemocue machine (Hemocue Hb 201). Anaemia was defines as Hb below 11 g/dL and in this study, Hb <7 g/dL was taken as severe malaria.et al. [The salting out protocol of Miller et al.  was usedTLR4 SNPs were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based method using the previously used primers sequences by Nyati et al. [2, 20 pmol of each primer, 1.25 U of Taq DNA polymerase  and 100 ng of each genomic DNA as template. In a thermal cyler , the PCR reaction was then subjected to denaturation at 94 °C for 5 min, followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min and cooling to 4 °C. Template-free water was used as a negative control. After amplification, the purified 4-μL PCR products were subjected to restriction digestion by NcoI restriction endonuclease (Fermentas) for TLR4 Asp299Gly and by HinfI for TLR4 Thr399Ile with 1 mL 10X enzyme buffer (Fermentas) overnight at 37 °C. The PCR products yielded 299 bp and 406 bp respectively, while the digested products yielded same DNA fragments for the wild alleles but decreased to 233 bp and 377 bp fragments for the 299Gly and 399Ile mutant alleles respectively, on 3 % agarose gel after electrophoresis  test using SPSS version 15.0 for windows. Association of TLR4 Asp299Gly and Thr399Ile polymorphisms with clinical and severe malaria was measured by calculating odd ratio (OR) and their 95 % confidence intervals (95 % CI) using dominant inheritance model [i.e. anaemia, severe anaemia, malnutrition, severe malnutrition, high parasite density severe) measured as covariates. Adjustment was performed by including only cases of comparable ages between ASM and clinical malaria for anaemia, malnutrition (WHZ or WAZ or HAZ < −2) and gender or age for severe anaemia, severe malnutrition and high parasite density seen only in clinical malaria cases for the analysis of genotype distribution of the TLR4 SNPs. Hardy-Weinberg Equilibrium (HWE) was conducted by comparing of the observed frequencies of different genotypes of the two TLR4 SNPs with their expected frequencies under HWE for each study group and outcomes with P > 0.05 was considered to be in HWE [Data were double-entered into Microsoft Excel 2008 and Microsoft Access 2008 and validated before analysis using SPSS statistical software for windows version 15.0 . Data were expressed as number and percentages (%), median and range, mean ± standard error of mean (SEM) and 95 % confidence intervals (95 % CI). High parasite density and hyperparasitaemia were defined as parasitaemia > 10,000/uL and > 250,000/uL respectively. Axillary body temperature > 40 °C was indicative of hyperpyrexia, while severe malnutrition was defined as WHZ or WAZ or HAZ < −3 relative to NCHS references. Disparity in mean values between asymptomatic control and clinical malaria case was measured by Student’s ce model . This wae in HWE . GenotypA total of 279 children comprising 182 children  with clinical falciparum malaria and 97 children  with asymptomatic falciparum malaria were enrolled , 5 (2.7 %), 15 (8.2 %) elicited hyperparasitaemia, hyperpyrexia and severe malaria. Between asymptomatic and clinical malarial children there was no statistical significance difference (P < 0.05) in terms of age, sex and being underweight. But variables such as stunting, wasting and axillary body temperature and anaemia occurred more significantly (P <0.05) in symptomatic children (Table TLR4 SNPs were found in the study population (Tables TLR4 SNPs were greater than 10 % (17.6 % and 14.7 % respectively) only in severe malaria children  Table . Further Table . TLR4 gene in the development of clinical and severe malaria among children with Yoruba ethnic background. This selection was based on evidence of existence of the two SNPs of TLR4 among the Yoruba population (http://www.hapmap.org) [P. falciparum malaria, this study has also found all the genotypes of TLR4 SNPs among children of Yoruba ethnicity. However, the difference in the numbers of mutant genotypes of both TLR4 Asp299Gly and Thr399Ile SNPs between asymptomatic and clinical malarial children was observed to be non-significant (P < 0.05), translating to lack of association between carriage of these genetic variants and clinical malaria in the studied children. Our finding is similar to the report of Esposito et al. [TLR4 Asp299Gly SNP and malaria among Burundian children. The MAF of 6 % for 299Gly reported for the uninfected and malarial children by these workers is also similar to 5.4 – 7 % range found in this study. However, unlike in the Burundian children, we found MAF of 17.6 % and 10.4 % for TLR4 Asp299Gly and Thr399Ile among our severe malaria sub-group with genotypes containing these alleles further found to increase the risk of severe malaria 3-fold and 8-fold respectively. Our findings indicate that the two minor alleles 299 Gly and 399Ile of TLR4 SNPs are not protective against severe malaria in the studied children. This is in agreement with the case–control studies conducted in Ghana [et al. [et al. [TLR4 Asp299Gly nor Thr399Ile polymorphism was found to be associated with the risk of severe malaria anaemia and cerebral malaria in children from Uganda and Cameroun [TLR4 Asp299Gly and Thr399Ile polymorphisms reported by these investigators. For instance, in the Cameroun study of 1,862 children, Apinjo et al. [TLR4 polymorphisms in asymptomatic and clinical malaria  children from Bantu, Foulbe and Semi-Bantu ethnic groups. The Ugandan study was conducted among 137 P. falciparum infected hospitalized children and only the frequency of Asp299Gly was above 10 % (i.e. 12.3 %) in the cerebral malaria group that also had a frequency of 1.5 % for the Thr399Ile genotype. This genotype was not found in children with uncomplicated malaria. In Brazil, where temporal changes from low to moderate P. vivax and P. falciparum malaria transmission also occur, contradictory findings, regarding the association of TLR4 Asp299Gly polymorphisms with protection from clinical malaria have been reported [et al. [TLR4Asp299Gly polymorphism among 113 healthy and 535 clinical malaria participants with this polymorphism eliciting protection against clinical malaria, while Soares et al. [TLR4Asp299Gly among their 44 study participants in an earlier study.The present study has examined the role of the two non-synonymous SNPs, rs4986790 (Asp299Gly) and rs4986791 (Thr399Ile), located at the third exon of the map.org) . For theo et al. . The worin Ghana . In the  [et al.  reported [et al. , neitherCameroun . This iso et al.  reportedreported , 20. Wit [et al.  found MATLR4 Asp299Gly and Thr399Ile polymorphisms in malaria endemic countries strongly point to the relevance of other infectious diseases that also provide selection pressure on TLR4. They include tuberculosis, HIV, gram negative bacterial infection, candidiasis, meningitis and respiratory syncytial viral infection [TLR4.Taken together, our findings and previous reports on nfection , 11. SinTherefore, the implications of other TLR4 activated infectious diseases stated above highlights one of the limitations of the present study since the children were enrolled during malariometric surveys and were not clinically and diagnostically examined for candidemia and viral infections that may further influence the phenotypic disposition of TLR4 polymorphism and subsequently impact malaria susceptibility.et al. [TLR4Asp299Gly polymorphism to increase maternal anemia 4.7-fold after adjustment by age. Here, we have found both TLR4 Asp299Gly and Thr399Ile polymorphisms to cause a 3.8 -5.3-fold increased risk of severe anaemia and 3.5 – 4.4-fold increased risk of high parasite density, while Thr399Ile polymorphism alone raised the risk of fever 5-fold. In addition to Mockhenhaupt et al. [In this study and under dorminant model of inheritance, the significant association of the two TLR4 SNPs with clinical malaria syndromes such as fever, severe anaemia and high parasite density was found. This findings indicate that both TLR SNPs elicit functional effects that are related to the pathogenesis of falciparum malaria in the studied children. Similar phenotypic findings were also reported by Mochenhaupt et al.  in Ghanit et al. , The phet et al. . This diTLR4Asp299Gly increased the risk of severe malnutrition 3.7-fold. However, this observation needs to be interpreted with caution because it was not been documented in previous immunogenetic studies of TLR4 and malaria from other countries. Also because of the fact that malnutrition is a common health problem among African children, including Nigeria and many aetiologies have been reported [P. falciparum infection, these rates appear to be lower than the national prevalence rates 5.5 %, 35.7 % and 25.2 % and rates reported for children below 5 years in the northern parts of Nigeria. Another reason for this cautionary interpretation is the fact the present study is cross-sectional in design and is limited in determining the cause and effect relationship between malnutrition and malaria in the studied children. furthermore, the malnutrition investigated in this study exluded micronutrient deficiency such as zinc, iron and vitamin A deficiency, which have been shown to increase the risk of severe anaemia, wasting and dyregulated innate immunity in malarial children [However, of relevance is our finding that the mutant genotypes of reported , 39. In reported , 41. In children .TLR4 Asp299Gly and Thr399Ile polymorphisms among Nigerian children of Yoruba ethnic background may modulate susceptibility to severe malaria and other disease outcomes such as severe anaemia and hyperpyrexia. To further validate the present findings, functional studies of TLR4 polymorphisms, haplotype analysis and investigation of other TLR4 activated infectious diseases in the setting of malaria in this category of Nigerian children are needed.In conclusion, our findings suggest that