Plasmodium offers a free-of-charge reference testing of malaria-positive or doubtful samples to clinical laboratories.The Belgian Reference Laboratory for Plasmodium species-specific PCR) were compared with the final diagnosis from peripheral Belgian laboratories. The Reference Laboratory reports were analysed for all samples submitted between 2013 and 2017. Criteria assessed included the diagnosis of malaria, Plasmodium species identification including mixed infections, and in case of Plasmodium falciparum, the parasite density and the presence of sexual and asexual stages.The final malaria diagnosis from the Reference Laboratory  and Plasmodium ovale and Plasmodium malariae. Submitting laboratories had correctly identified P. falciparum in 95.1% (508/534) samples with P. falciparum single infection. They had correctly diagnosed the species in 62.9% (95/151) single non-falciparum samples and had reported ‘non-falciparum’ in another 26 (17.2%) samples; most errors occurred among P. malariae  and P. ovale . Only one of the 21 mixed Plasmodium species infections had been diagnosed as such by the submitting laboratories; in three of them, P. falciparum had been overlooked. Taken single and mixed infections together, P. falciparum was diagnosed in 98.6% (546/554) samples. Among 471 single P. falciparum samples available for comparison, laboratories had correctly reported parasite densities above 2% in 87.5% (70/80) samples; they had incorrectly reported parasite densities > 2% in an extra 52 (8.9%) samples. Laboratories had correctly reported P. falciparum schizonts and gametocytes in 25.6% (11/43) and 56.7% (17/30) samples, respectively.A total of 947 non-duplicate samples were included. Reference testing confirmed 96.3% (893/927) and 90.0% (18/20) samples submitted as positive and negative, respectively, the two missed diagnoses were samples with P. falciparum, reasonably good diagnosis of non-falciparum infections and acceptable calculation of P. falciparum parasite density.Diagnostic laboratories in a malaria non-endemic setting provided excellent diagnosis of malaria and  Plasmodium species: Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae, and Plasmodium knowlesi. Among them, the most severe with the higher mortality rate is P. falciparum, followed by P. knowlesi [Malaria is a major health problem with 216 million cases and 445,000 deaths worldwide in 2016 [Currently, there is a risk of malaria transmission in 91 countries and 125 million travellers are at risk every year . As intePlasmodium offers a free-of-charge cross-reference testing of malaria-positive and doubtful samples. Clinical laboratories submit samples together with a request form listing their own diagnosis and the Institute of Tropical Medicine ((ITM), Antwerp, Belgium) returns a report on the day of receipt. In the present study, was aimed to assess the accuracy of each final malaria diagnosis made by submitting laboratories by comparing it to the final diagnosis made by the Reference Laboratory.To support diagnostic laboratories in the diagnosis of malaria, the Belgian Reference Laboratory for Plasmodium at the ITM, for the period January 2013 to June 2017. The study was designed to compare the final Plasmodium diagnosis, not to discuss the different diagnostic methods used.The study compares the malaria diagnosis made by Belgian clinical laboratories for the samples they had submitted voluntarily for cross-reference to the Belgian Reference Laboratory for Plasmodium species, parasite density, stages, and results of the malaria rapid diagnostic test (RDT).Samples comprised stained and unstained thick and/or thin blood films, a tube of EDTA-anticoagulated blood and a request form with information about patient identity and country of travel or origin, as well as the diagnosis made by the submitting laboratory, including In case of multiple referrals per patient , only the samples from the first referral were considered, unless the time interval between the successive samples was 2 months or more. Data were considered as ‘insufficient data’ if information from the submitting laboratory for both microscopy and RDT results was lacking.Plasmodium species-specific PCR. Samples were assessed by an expert microscopist according to World Health Organization (WHO) standards for microscopy, with the exception that the Giemsa staining was done with pH 8.0 instead of pH 7.2 [Plasmodium parasites, species identification and parasite density were assessed, as well as the presence of asexual (trophozoites and schizonts) and sexual (gametocytes) stages and pigment in white blood cells (WBC). Two RDTs were carried out: Carestart™  Malaria Pf (pLDH)/Pan (pLDH) and SD Bioline  FK60 Malaria Ag P.f. (HRP-2)/Pan (pLDH). Additionally, SD Bioline FK80 P.f. (HRP-2)/P.v. (pLDH) detecting P. falciparum and P. vivax-specific parasite lactate dehydrogenase was used in case of microscopic identification of P. vivax or P. ovale. Parasite densities were estimated by counting asexual parasites against 200 WBC in thick blood films and using the actual WBC count of the patient for calculation or, when not available, a standard value of 8000 WBC/μl [Reference testing consisted of microscopy, RDTs and f pH 7.2 . PresencPatient data and the final diagnosis made by the Reference Laboratory were extracted from the ITM Reference Laboratory Information System  into an Excel database . Data about the diagnosis recorded by submitting laboratories were manually encoded. Incoherent data were verified against the original submission form. The database used for the analysis was coded and did not contain any patient identity or confidential information.Plasmodium species identification including mixed infections; and, (iii) in case of P. falciparum, the parasite density and the presence of sexual and asexual stages. As most laboratories reported parasite density as % of infected red blood cells (RBC), the reference parasite density expressed per µl was converted to % of infected RBC by dividing by 50,000. The reported parasite density was considered acceptable when the difference with the reference was within 1 log; in addition, the agreement for the parasite density threshold of 2% for severe malaria infection was assessed.Data were analysed with Excel and Graphpad Prism Version 5.01 . Diagnoses made by submitting laboratories were compared to those made by the Reference Laboratory. Criteria assessed included (i) diagnosis of malaria; (ii) P. falciparum 649/893 (72.7%), P. vivax 96/893 (10.8%), P. ovale 77/893 (8.6%), and P. malariae 35/893 (3.9%). The 9 remaining samples were reported as positive without further species reported. The median parasite density for P. falciparum single infections quantified by reference testing was 7110/µl .Out of 1283 submitted samples, there were 1170 non-duplicate first samples, of which 947 (80.9%) had enough data for analysis  represented 69.2% out of 172 laboratories subscribing to the proficiency testing for malaria diagnosis in 2013 , and subP. falciparum and Plasmodium non-falciparum, respectively) and by RDT alone . Of the 20 samples submitted as malaria-negative, 18 (90.0%) were confirmed as negative; the 2 remaining samples were diagnosed by reference testing as P. ovale (parasite density 6583/µl) and P. malariae (the sample received was not appropriate for quantification of parasite density). The number of positive samples that were confirmed as positive increased over time: 94.2% samples were confirmed positive in 2013, 95.8% in 2014, 96.2% in 2015, and 98.0% in 2016 .A total of 927 (97.9%) samples were submitted as malaria-positive, the remaining 20 2.1%) as malaria-negative  malaria positive samples  samples  samples: P. ovale (n = 3) and P. vivax (n = 1). Reference testing further diagnosed 151 samples as single infections by P. vivax, P. ovale, or P. malariae: laboratories had correctly diagnosed the reference species in 95 (62.9%) samples and had reported non-falciparum in another 26 (17.2%) samples; the remaining 30 (19.9%) samples had been incorrectly identified, among which were three misdiagnoses as P. falciparum. Errors were highest among P. malariae  and P. ovale .Among 534 samples diagnosed as n 508 95.% samplesPlasmodium species infections identified at reference testing, submitting laboratories had diagnosed only one as a mixed infection. Most (20) of these samples contained P. falciparum, which had been correctly reported by the submitting laboratories in 17/20 (85%) samples. In three other samples, the presence of P. falciparum had been overlooked. Taken single and mixed infections together, P. falciparum was correctly diagnosed in 98.6% (547/554) samples.Among the 21 samples with mixed P. falciparum samples, submitting laboratories had reported parasite densities, mostly  expressed as % of infected RBC. In 13 cases, only descriptive terms were used  and in one case, the reference laboratory could not reliably assess the parasite density. Among 471 P. falciparum-positive samples available for comparison, 374 (79.4%) of densities recorded by the submitting laboratory were within 1 log of the reference value. Eighty (17.0%) P. falciparum samples had a parasite density > 2%; for 70 of them (87.5%), laboratories had also correctly reported values above 2%. Among the samples incorrectly reported as < 2% (n = 10), parasite densities reported were < 0.5% for four samples. Conversely, laboratories had reported parasite densities > 2% in an extra 52 (8.9%) samples compared to reference testing, with 16 among them counted as < 0.5% by reference testing.For 485/534 (90.8%) single P. falciparum stages ; moreover, all but one (20/21) mixed Plasmodium infections diagnosed at reference testing had been overlooked as mixed infections by submitting laboratories. As shown in this study, mixed Plasmodium infections are rare events but they mostly contain P. falciparum and should be treated accordingly [Malaria diagnosis made by submitting laboratories was confirmed by reference testing in 96.3% of samples and submitting laboratories had missed ordingly .P. falciparum infection and it is used for treatment follow-up. In non-immune travellers, parasite densities exceeding 100,000/µl  point to an increased risk for complications and a need for intravenous treatment [P. falciparum and accuracy was satisfactory. It should be noted, however, that the criterion for acceptability was set broad, in line with the high variation of parasite counting, particularly at low parasite densities [P. falciparum. Almost three-quarters and nearly half of them had not observed P. falciparum schizonts and gametocytes, respectively. The P. falciparum schizont stage is usually restricted to the organ capillaries, and its presence in peripheral blood may alert to an increased risk for complications [P. falciparum gametocytes together with trophozoites indicates a longer standing (> 7–10 days) infection, whereas the unique presence of gametocytes after treatment is a regular finding and not a sign of drug resistance [Quantifying and reporting parasite density is part of standard malaria diagnosis as it reflects the severity of the reatment . Over 90ensities . Most laications . The preP. falciparum species. However, a good performance was noted for the differentiation of non-falciparum species, with most errors observed for P. malariae and P. ovale. As for P. falciparum, errors tended to occur more frequently among mixed versus single infections. The interest of non-falciparum species differentiation is that P. vivax and P. ovale form hypnozoites (dormant stages) in the liver that can cause relapses and need primaquine treatment [P. falciparum) is acceptable pending further species differentiation in a reference laboratory.The identification of non-falciparum species was lower than of reatment . AlthougP. falciparum, the diagnosis of mixed species infections, the estimation of the P. falciparum parasite density, and differentiation between the non-falciparum species. Failures of P. falciparum diagnoses were observed among 11–27.3% [P. falciparum and 2–11.3% of participants reported the presence of malaria parasites on a normal blood film [P. malariae, P. vivax and P. ovale [Plasmodium species  was another consistent finding [Most data for comparison are derived from External Quality Assessments (EQA), reported from the UK 1986–2001) , Canada 11–27.3% . Conversood film . Likewisne study ). Not qu finding , 18 and  finding , 19. Amo86–2001 ,P. falciparum and a poor accuracy for species identification of P. ovale. The Hong Kong study assessed both EQA and cross-checking samples with similar findings as the UK study; in addition, it showed a poorer performance for the cross-checking samples as compared to the EQA samples. A third study assessed routine malaria diagnosis among a cohort of African refugees in Canada in 2000. Main findings were that 5/20 malaria diagnoses were not confirmed by reference testing and that parasite density were reported in only 5/20 samples [Few studies assessed the accuracy of routine malaria diagnosis of clinical laboratories. Two studies had a similar design as the present study, i.e, they cross-checked routine samples submitted to reference laboratories in the UK and Hong Kong , 20. In  samples .P. falciparum and are accurate to rule-in and rule-out the presence of P. falciparum. Recently marketed RDTs designed to detect P. vivax have a 95% sensitivity  to diagnose this species, but RDTs still perform poorly (< 50% sensitivity) for P. ovale and P. malariae [Interestingly, laboratories in this study performed much better than EQAs. This is surprising as EQAs reflect the best rather than day-to-day performance . Severalmalariae , 26. Givmalariae , 24, 25.malariae , and as In the practice of ITM reference testing and as shown in this study, laboratories nearly exclusively submitted malaria-positive samples and only a few doubtful or negative samples. Therefore, the present study did not allow for tracing missed or delayed diagnosis of malaria. Among the other strengths of the study, are the high number and consistent submission of samples over a 4-year period and high representation among clinical laboratories.P. knowlesi-infected samples in the present study. Given its potential fatal complications, and similarly to P. falciparum, species recognition of P. knowlesi and quantification of its parasite density is imperative [P. knowlesi [Of note, there were no perative . Currentknowlesi .P. falciparum. They performed reasonably well in determining P. falciparum parasite density as well as in the diagnosis of non-falciparum species, but fell short in detection of P. falciparum schizonts and gametocytes. The results of this study show a very good performance of malaria diagnosis compared to previous EQA reports from non-endemic settings.This study showed that diagnostic laboratories in malaria non-endemic settings provided excellent diagnosis of malaria and especially the detection of


Adding community-based scheduled malaria screening and treatment to existing intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) strategies did not reduce malaria in pregnancy. Increasing the number of IPTp-SP doses given during pregnancy continues to be a priority. We investigated whether adding community scheduled malaria screening and treatment (CSST) with artemether-lumefantrine by community health workers (CHWs) to standard intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) would improve maternal and infant health.In this 2-arm cluster-randomized, controlled trial, villages in Burkina Faso, The Gambia, and Benin were randomized to receive CSST plus IPTp-SP or IPTp-SP alone. CHWs in the intervention arm performed monthly CSST during pregnancy. At each contact, filter paper and blood slides were collected, and at delivery, a placental biopsy was collected. Primary and secondary endpoints were the prevalence of placental malaria, maternal anemia, maternal peripheral infection, low birth weight, antenatal clinic (ANC) attendance, and IPTp-SP coverage.P = .72). No difference between the study arms was found for peripheral maternal infection, anemia, and adverse pregnancy outcomes. ANC attendance was significantly higher in the intervention arm in Burkina Faso but not in The Gambia and Benin. Increasing number of IPTp-SP doses was associated with a significantly lower risk of placenta malaria, anemia at delivery, and low birth weight.Malaria infection was detected at least once for 3.8% women in The Gambia, 16.9% in Benin, and 31.6% in Burkina Faso. There was no difference between study arms in terms of placenta malaria after adjusting for birth season, parity, and IPTp-SP doses (adjusted odds ratio, 1.06 ; Adding CSST to existing IPTp-SP strategies did not reduce malaria in pregnancy. Increasing the number of IPTp-SP doses given during pregnancy is a priority.NCT01941264; ISRCTN37259296. Malaria causes significant adverse pregnancy outcomes, such as maternal anemia, preterm delivery, low birth weight , and eveThe protective efficacy of IPTp-SP against malaria infection depends on the number of IPTp-SP doses administered, which in turn depends on antenatal clinic (ANC) attendance. In many sub-Saharan African countries, both ANC and IPTp-SP coverage remains low , 6. In aA cluster-randomized controlled trial was designed to establish whether adding community scheduled malaria screening and treatment (CSST) by CHWs to standard IPTp-SP would further reduce placental malaria compared to IPTp-SP alone .A description of the study methods has been published elsewhere . The stuThirty villages (clusters) (village population: 1000–2000) with CHWs in each country were randomly selected from all eligible clusters. Distance from the center of each village to the nearest health facility was calculated and used to group the clusters into 3 distance categories. Randomization was performed using computer-based randomization  and was stratified by the 3 distance categories.Recruitment was done at first ANC . All womCHWs in the intervention arm were trained in malaria case management and malaria in pregnancy, including the benefit of early ANC attendance and IPTp-SP. CHWs were asked to continuously identify all pregnant women and encourage them to attend the ANC as early as possible. Thereafter, at monthly intervals up to the last week of gestation, CHWs performed an RDT at home visits and collected a blood slide, regardless of malaria symptoms. They gave AL to all positive women. Severely ill women were referred to the health center for further care. The CHWs in the control villages did not take part in any of the study training.A blood sample for hemoglobin measurement and on slide and filter paper for later parasitological diagnosis was collected just before delivery, and a placenta biopsy was collected at delivery. Current health status and birth outcomes were collected. All newborns were physically examined and weighed on digital scales immediately after delivery. Gestational age was estimated using the Ballard score .Plasmodium falciparum diagnostic polymerase chain reaction (PCR) ) than in the control arm (14/33 [42.4%]) .Most women in the intervention arm had at least 1 CSST by CHWs, with coverage higher in Burkina Faso (860/900 [96%]) and The Gambia (978/1008 [97%]) compared with Benin (474/542 [87.5%]). Overall, CHWs performed 7236 CSSTs, with an average of 3–4 home visits per woman . The meaeived AL . The numP = .72)  of deliveries in Burkina Faso and The Gambia. There was no difference between intervention and control arms in terms of placenta malaria or for different categories of infection , and thiP = .72) . SimilarP = .72) .P = .7), even after adjusting for several factors  , low birth weight .There was no difference between arms in peripheral maternal infection  . Anemia  P = .3; , mean heThere were 9 maternal deaths (6 in the control arm and 3 in the intervention arm) and 45 perinatal deaths that occurred at time of delivery (21 in control vs 24 in intervention arm). Causes attributed to the SAEs, including maternal and perinatal death, are reported in Antenatal clinic attendance (scheduled visits) was significantly higher in the intervention arm in Burkina Faso but not in The Gambia and Benin . HoweverP < .001), and was more frequent with increasing number of AL treatments , whereas increasing number of AL treatments had the opposite effect (P = .02)  . SimilarAdding CSST by CHWs to the standard IPTp-SP at ANC did not reduce the risk of placental malaria or peripheral malaria infection at delivery. The intervention also aimed at increasing ANC attendance, particularly in early pregnancy, and at identifying and treating infections between scheduled ANC visits. Scheduled ANC attendance did improve in Burkina Faso, suggesting that the intervention had the expected effect. During the trial, the 2013 WHO recommendations of at least 4 ANC visits and of administering IPTp-SP at each of them  had beenIn all countries, the higher number of malaria infections diagnosed in the intervention group and the high proportion of malaria-positive women treated with AL indicate that CHWs implemented the intervention according to the instructions received. In Burkina Faso, RDT sensitivity and specificity performed by CHWs and compared to microscopy was 81.5%  and 92.1% , respectively , furtherThough the intervention did not have a direct effect on the prevalence of past placenta malaria, probably because of infections before the first ANC visit, one would have expected a reduced risk of active or chronic placenta infections. This was not the case, and this may have been due to low adherence to AL treatment. However, CHWs who visited treated women 3 days after CSST, and 96% of women reported having completed the full course; in contrast, a qualitative study carried out in the same study area reported low adherence to antimalarial treatment by pregnant women despite good knowledge about malaria in pregnancy .Increasing doses of IPTp-SP significantly decreased the risk of placental malaria. This effect was mainly seen in Burkina Faso, where women received up to 6 IPTp-SP doses. In The Gambia and Benin, the large majority of women had taken only 2 IPTp-SP doses and in both countries malaria prevalence was lower than in Burkina Faso. In these 2 countries, CSST should have had some effect as the time between IPTp-SP doses was longer than in Burkina Faso, so diagnosing and treating infections during this period should have been beneficial. Malaria screening by the CHWs was done with an RDT whose detection threshold is at most 200 parasites/μL . HoweverPregnant women in the intervention arm had a higher risk of testing malaria positive, not because they had a higher risk of being infected, but rather because they were tested more frequently. Considering that most infections diagnosed in the intervention arm were treated with AL and that the risk of placenta; malaria increased with the number of AL treatments administered, these infections probably represented a small proportion of all infections acquired during pregnancy and it was the undiagnosed infections that had a significant effect on the occurrence of placenta malaria. Receiving multiple AL treatments is probably a marker of a higher malaria risk. There is some controversy on the importance of low-density malaria infections during pregnancy, associated with anemia, lower mean hemoglobin, low birth weight, and premature births in some studies  but not A major strength of this study was the selection of countries based on their varying malaria endemicity: low (The Gambia) vs high (Burkina Faso and Benin), and with varying degrees of SP resistance (high in Benin and moderate in The Gambia and Burkina Faso), which us enables to generalize study findings to West Africa and possibly other sub-Saharan African countries. The study was powered for individual countries for the overall primary outcome; thus, we were able to at least fully investigate the effect of the intervention on placental malaria for the study sites in The Gambia and Burkina Faso. Unfortunately, this was not possible for Benin.https://data.unicef.org/topic/nutrition/low-birthweight/). Being in a trial may have influenced women’s healthcare-seeking behavior, which may explain these lower estimates. There were 45 perinatal deaths and 9 maternal deaths with no difference in mortality between the study arms. Pooling the data together gives a perinatal mortality rate of 10.77 per 1000 live births, and a maternal mortality rate of 215 per 100000 live births. These remain high levels, but comparisons with other sources [The significant number of adverse pregnancy outcomes in both study arms highlights the poor access to timely and adequate care for women in rural and remote areas in sub-Saharan Africa. Both maternal anemia and low birth weight  were below the West African regional estimates, 56%   and 14%  sources  should bIST has been extensively evaluated as an alternative to IPTp-SP, with mixed results . We deciClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary InformationClick here for additional data file.


We assessed the association of mutant allele frequencies of nitric oxide synthase 2 (NOS2) gene at two SNPs (-954 and -1173) with malaria disease severity in children from a malaria endemic area in Southern Ghana.Using children recruited at the hospital, assigned into clinical subgroups of uncomplicated and severe malaria and matching with their “healthy control” counterparts, we designed a case control study. Genomic DNA was extracted and genotyping using Restriction Fragment Polymorphism was done.2; p< 0.05) though this isn’t the case with -1173.A total of 123 malaria cases  and 100 controls were sampled. Their corresponding mean Hbs were 9.6, 9.3 and 11.2g/dl and geometric mean parasite densities of 32097, 193252 and 0 parasites/ml respectively. Variant allele frequencies varied from 0.09 through 0.03 to 0.12 for G-954C and 0.06 through 0.03 to 0.07 for C-1173T in the uncomplicated, severe and healthy control groups respectively. There was a strong linkage disequilibrium between the two alleles (p<0.001). For the -954 position, the odds of developing severe malaria was found to be 2.5 times lower with the carriage of a C allele compared to those without severe malaria (χThe carriage of a mutant allele in the -954 NOS2 gene may have a protective effect on malaria among Southern Ghanaian children. There is accumulating evidence that host genetic factors control and regulate the outcome of malaria disease by regulating immune responses and natural selection over many years may have led to variations in susceptibility in infected individuals. In malaria, much of the variations in disease susceptibility can be attributable to the protective roles of several genetic disorders of the red blood cell such as the sickle cell trait , the thaHost resistance to malaria has been complex, and various associations of genetic polymorphisms with malaria severity have been postulated. Of recent times, the role of cytokines in controlling the intensity and duration of immune responses to malaria infection in different populations has been studied. Polymorphisms in genes encoding such cytokines as the tumor necrosis factor (TNF-α) and the increased risk of cerebral malaria has been documented , 5. GeneNitric oxide (NO) is known to mediate host resistance to infectious organisms  and is pThere has been much speculation about the role of NO in malaria and recent reports identified associations between polymorphisms in the promoter region of the gene encoding NOS2 and malaria disease severity. To date, four polymorphisms in the NOS2 promoter have been associated with variable malarial disease outcomes. These include a CCTTT microsatellite repeat located 2.5 kilobases upstream from the NOS2 transcriptional start site , and thrThough large-scale genomic studies are currently being advocated, the design of these studies does not capture the basic information from Africa and therefore may not be suitable in the African setting. Until these are designed to suit the African settings, a small study of this nature may produce useful information about genes and disease association, since we postulate that NOS2 promoter polymorphism could determine malaria disease severity. In addition, in Ghana, there are three ecological zones with different malaria endemicities and earlier studies looked at SNPs in the savannah belt. This study therefore presents the information about the correlation of NOS2 SNPs in the forest area of Ghana. In this study, using a case-control approach, we hypothesize that in a cohort of Southern Ghanaian children, mutant allele frequencies at two SNPs (-954 and -1173) of the inducible nitric oxide synthase 2 promoter gene correlates positively with malaria disease severity.Approval and ethical clearance for the study was obtained from the Institutional Review Board of the Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana, Legon. (No. IRB 0001276). An information session was presented to the study participants prior to obtaining written consent. Adolescents and children provided assent in addition to the required informed consent of a parent or guardian.The study design was an age and sex-matched case-control study. The study was conducted at the St. Dominic District Hospital at Akwatia in the Eastern Region of Ghana. Study participants were children presenting at the hospital with confirmed malaria after an initial investigation. These children who were designated as cases were matched with their “healthy control” counterparts, who were children in the same age group, recruited from the community. All children who tested positive for sickling  and helminth infections were excluded. Aside cerebral malaria, other causes of encephalopathy including viral encephalitis, uraemia, and hypertensive encephalopathy from glomerulonephritis and head injury from trauma were also excluded. A finger prick blood was taken from both categories of children for the preparation of thick and thin films for microscopy to diagnose malaria and blood was also collected into heparinised capillary tubes  and blotted on chromatographic filter paper  for DNA extraction and subsequent molecular analysis.P. falciparum parasitaemia, with at least one of the following signs; prostration, unrousable coma . For C1173T, fragments were digested with FokI . The Bsal and FokI-digested PCR products were analyzed on 2% agarose gel.DNA was extracted from 5-6mm of filter paper after punching using the Tris-EDTA method. Polymorphisms were detected using nested polymerase chain reaction (nPCR) as described previously . This in2 test was also used to estimate the association between the polymorphisms and the different clinical sub-groups. The SPSS ver. 17 statistical package was used for statistical analysis and p values <0.05 were considered significant.Allelic frequency distribution was tested according to the Hardy-Weinberg equilibrium. The χ2, p<0.05). There was also significant difference  in the mean temperatures for the case and control group. Mean temperatures were highest in the severe malaria cases (38.2) than in the uncomplicated cases and the healthy controls which were 37.8 and 36.7 °C respectively. Plasmodium falciparum was the predominant parasite species in both clinical groups . Four children who had uncomplicated malaria were harbouring Plasmodium malariae while mixed infection of both species was found in five children.In this study, our aim was to use a case-control approach to determine the association of genetic polymorphisms in the NOS2 gene and its relation with malaria disease severity in children in Southern Ghana. This is also the first study in Southern Ghana to document how malaria outcomes are affected by genetic polymorphisms in an endemic population. We found that the G954C polymorphism was present in the Southern Ghanaian population with an allele frequency of 9%. This is consistent with other findings from sub-Saharan African malaria-endemic populations, which varied from 7% to 15% , 18 compWe found evidence for a protective effect of the G954C SNP against severe malaria in this cohort of Ghanaian children. This contrasts earlier findings from Northern Ghana where no evidence was found for the protective effect of the G954C SNP against severe malaria , and in The noted differences in NOS2 promoter polymorphisms and disease severity between our population of Southern Ghanaian (West African) children and the populations of Gabonese  and Tanzanian (East African) children may indicate differences in the genetic backgrounds of each population, differences in the strains of malaria, or differences in the epidemiology of malaria infection in these regions. The functional significance of these conflicting observations is ambiguous, suggesting that the relationship between NOS2 polymorphism and malaria severity is much more complex , 21, ThuP. falciparum infection [Moreover, our findings are pertinent and their interpretation can be attributed to several reasons because the NOS2-954 polymorphism is located in the NOS2 promoter region, which could be an activator of gene expression and nitric oxide production . The hetnfection –26. Genenfection .Among other functions, nitric oxide appears to be protective in endothelial function including reducing blood cell-endothelial cell adhesion in studies both in humans and animal models and ameliorating deleterious cytokine production . CompareS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Placental malaria has been associated with an immune tolerance phenomenon and a higher susceptibility to malaria infection during infancy. HLA-G is involved in fetal maternal immune tolerance by inhibiting maternal immunity. During infections HLA-G can be involved in immune escape of pathogens by creating a tolerogenic environment. Recent studies have shown an association between the risk of malaria and HLA-G at both genetic and protein levels. Moreover, women with placental malaria have a higher probability of giving birth to children exhibiting high sHLA-G, independently of their own level during pregnancy. Our aim was to explore the association between the level of maternal soluble HLA-G and the risk of malaria infection in their newborns. Here, 400 pregnant women and their children were actively followed-up during 24 months. The results show a significant association between the level of sHLA-G at the first antenatal visit and the time to first malaria infection during infancy adjusted to the risk of exposure to vector bites . The level of sHLA-G is a significant predictor of the occurrence of malaria infection during infancy consistent with the hypothesis that mother sHLA-G could be a biomarker of malaria susceptibility in children. This phenomenon is referred as immune tolerance (IT) that may be due to modifications of the newborn immune system development involving at least some cytokines production in cord blood5. These modifications may induce differential immune responses involving IL10 and Interferon-γ during infancy6. A similar phenomenon was described in cases of filariasis infection during pregnancy8. It has been shown that children born to mothers with PM were also at an increased risk for non-malarial fever9. Moreover, a recent result shows that children born to mothers with PM tend to have an increased risk for the first malaria attack but not for subsequent ones10. These observations highlight that it is a very complex phenomenon that could imply immunity to a broader sense, not specific to malaria infection and not only related to PM infection.Children born to mother with placental malaria (PM) seem to have a shorter delay of occurrence of the first malaria infection12 by interacting with immune cells of both innate and adaptive responses14. HLA-G is a non-classical HLA class I antigen, which differs from classical class I molecules in its restricted tissue distribution, diversity of protein isoforms and limited polymorphism16. The soluble isoforms detected in the plasma are shed HLA-G1 (HLA-G1s) and HLA-G511. Apart from pregnancy, HLA-G has been described associated with chronic viral infections, cancer and in vitro fertilization success22. During viruses’ infections, HLA-G can be over-expressed by infected cells to create a tolerogenic environment helping the pathogen to escape immune system23. The same phenomenon has been described in several types of cancers24.Human leucocyte antigen-G (HLA-G) is an immune-modulatory molecule that plays a crucial role in materno-foetal tolerance during pregnancy26 and protein levels28. These last studies have been performed in different populations and geographic areas . It clearly appears that the level of sHLA-G in children during the first year of life is strongly correlated with their own risk of malaria infection during the weeks following the measurement28. The sHLA-G level of a child was also associated with the sHLA-G level of his mother not only at delivery28 but also during the overall pregnancy29. Moreover, mothers with PM have a higher probability of giving birth to a child with a high level of sHLA-G during the first 2 years of life, independently of the mothers’ sHLA-G level29. Altogether, these results are consistent with both the fact that the risk of malaria infection during infancy is associated with the levels of sHLA-G in children and with the potential involvement of HLA-G in the immune tolerance phenomenon described during PM.The association between HLA-G and the risk of malaria has been shown recently by our team at geneticHowever, the direct association between the mothers’ levels of soluble HLA-G and the risk of malaria for their newborns remained unexplored. Here, our aim was to study the association between the mothers’ sHLA-G levels throughout the pregnancy and the risk of malaria during her newborn’s first 24 months of life.P. falciparum at first ANV  and 4.9% at the second. Only two women were infected twice. The prevalence of PM was 10.8%. The mean level of soluble HLA-G at ANV1, ANV2 and at delivery are respectively 10.1 ng/ml (SD = 13.6), 10.6 ng/ml (SD = 14.0) and 17.3 ng/ml (SD = 34.6). Using multivariate linear regression, the level of sHLA-G did not differ significantly according to placental infection  or to peripheral infection at ANV1 , ANV2  and at delivery .Women’s mean age was 25.9 years  and 15.7% were primigravid , 9.0% of them had a LBW, and 14 (3.5%) were preterm.During the study, 284 (71.0%) children developed at least one malaria episode (symptomatic or not): 50% of infected children developed two infections or more. Most of these infections were symptomatic (76.5%). There was no congenital infection.At 12 months, 324 children had been followed-up and after 24 months, 189 infants had moved out of the area or were lost to follow-up. They were considered as censored observations. Finally all 400 infants were included in the survival analysis. The median duration of follow-up was 12 months.All the variables respected the proportional hazards assumption Table .Table 2R−3). Neither placental malaria nor the number of peripheral maternal infections during pregnancy was associated with the risk of first malaria. Infants from the “not Fon” ethnic group had a lower risk of infection . The level of sHLA-G (used as a quantitative variable) at ANV1 was associated with the delay of first malaria infection . At ANV2 and at delivery the association was not significant (p = 0.63 and p = 0.36 respectively). Finally, gender, environmental exposure, ethnic group and all variables concerning maternal HLA-G levels and malaria infections were included.During univariate analysis, high environmental exposure was strongly associated with an increased risk of first infections (p < 10−3) and s-HLAG at ANV1, used as quantitative variable,  remained significantly associated with the risk of first infection. This last result corresponds to the increase of risk of malaria infection when sHLA-G rises by 1 ng/mL. Using sHLA-G as a two-class variable (higher/lower that the median), this association was consistent with a 60% increased risk of presenting a first malaria infection for an infant born to a mother with high level of sHLA-G  (not shown). The Kaplan-Meier curve  were obtained in presence of sHLA-G in the model performed by both logistic regression and random forest (model 2 and model 3) compared to a model without sHLA-G (model 1). The best predictive power has been reached when sHLA-G was considered as a binary variable  by the 2 methods, and the highest AUC was obtained by the random forest method (model 3) . In the present study, substantial variations in malaria vector density have been observed at the level of both village and house, even between houses which are close together. This variability could be explained not only by conventional climatic factors  but also certain environmental factors, i.e. a watercourse nearby, and vegetation index and soil type in the immediate surroundings. The environmental risk of exposure has been taken into account very precisely by means of a predictive model that took into account both individual and environmental factors above-mentioned3. The hypothesis proposed to explain this susceptibility is the existence of an immune tolerance status, although there is no clear and unequivocal explanation of the mechanism involved. The cord blood immune response of children born to mothers with PM is characterized by inducible parasite antigen-specific IL-10-producing regulatory T-cells that can inhibit Th1-type T cell response32. Interestingly, the same immune phenomenon involving an increased level of IL-10 in cord blood has been described during pregnancy geohelminth infections33. Moreover, it has been shown that children born to mothers with PM are also more susceptible to non-malaria fever9 strengthening the hypothesis that immune tolerance could surpass a specific parasite, and that PM could be a biomarker of a more generalized immunosuppressive phenomenon.Several studies have concluded in the existence of a higher susceptibility to malaria for children born to mothers with PM34. A high level of expression of HLA-G, described as an immune checkpoint molecule35, is also reported when cancer, viral or parasitic infections occur37, favoring escape from immune control. Indeed, HLA-G interacts with immunoglobulin-like transcript 2 (ILT2) which is expressed by T and B lymphocytes, natural killer (NK) cells, monocytes/macrophages and dendritic cells, and with ILT4 expressed by monocytes, macrophages, neutrophils and dendritic cells)39. Binding of HLA-G proteins to their inhibitory receptors can affect the function of immune cell populations modulating crucial steps in immunity40 and inducing the differentiation of CD4+ and CD8+ T cells into various subsets of regulatory cells that secrete IL-10 and TGF-β42.Human leukocyte antigen G can inhibit a broad array of immune cells and is strongly involved in fetal maternal tolerance during pregnancy43. In this context, PM may influence both mother and fetus HLA-G production inducing the immunological consequences listed above.The origin of sHLA-G in the mother compartment is not clearly identified. During pregnancy, the source of plasmatic sHLA-G in mothers’ peripheral blood may come from the mother herself and/or from the fetal trophoblast cells. A child, a mother or both genetically committed to produce high levels of HLA-G could be more susceptible to infections. Therefore, the final sHLA-G production may be a combination of fetal–maternal HLA-G genotypes together with the micro-environmental factors that may modulate HLA-G expression43. A high level of sHLA-G represents a tolerogenic environment from the infant’ immune system and increases the risk of infection28. The correlation between the level of mothers’ sHLA-G during pregnancy and in the infant during the first 2 years of life28 could be explained by these genetic factors. Secondly, high levels of sHLA-G observed at ANV1 are induced by the presence of pathogen  escaping the mothers’ immune system and also represents a tolerogenic environment from the infant’s immune system. Infants exposed to infection during pregnancy, combined with a strong immune system regulation on the part of the mother, may develop an attenuated immune response towards pathogens. This state of unresponsiveness or weakness of the infant’s immune system does not seem pathogen-specific but a more generalized phenomenon9.The increased malaria susceptibility of infants born of mothers with a high level of sHLA-G at the first ANV could be explained by several nonexclusive mechanisms. Firstly, genetic variants of the infant involved in the regulation of sHLA-G expression influence the level of sHLA-G in the mother during pregnancy (via trophoblast cells) and in the children during infancyper se. Indeed there is accumulating evidence that, although the pathophysiological mechanisms of early infections are not completely understood yet, their consequences are serious in terms of maternal anemia and low birth weight44. Moreover, it has been shown that even submicroscopic P. falciparum infections can have consequences in terms of low birth weight, prematurity and maternal anemia45. To our knowledge the consequences of malaria infection of the mothers during early pregnancy on the susceptibility of their newborns to infection during infancy have been poorly explored and the results are not convincing46. Our results could be indirectly consistent with the potential existence of such an association and by the involvement of HLA-G in this susceptibility.The absence of associations between the time to the first malaria infection during infancy and the mothers’ levels of sHLA-G at ANV2 and delivery could be explained by the medical care provided to the mother, including the two doses of IPTp against malaria at each antenatal visit. They were also encouraged to attend the clinic at any time, whenever they had any health complaint. These different treatments received during pregnancy may also have modulated HLA-G expression due to the reduction of exposure to infectious diseases. However, independently of the possible effect of IPT or of other treatment received during the follow-up, it is essential to underline the importance of the beginning of pregnancy It has been shown that the level of HLA-G at ANV1 has a significant predictive power of the occurrence of malaria infection during the first 2 years of life. This finding is promising in that it constitutes a strong argument for the idea that maternal HLA-G level could be a biomarker for malaria susceptibility in children. This question must, however, still be developed by complementary studies.Some limitations of the study exist. Due to the constraints imposed by the clinical trial, the follow-up was different before and after the first 12 months. Therefore, there is a possibility that some infections could have been missed during the 1st year because of less extensive monitoring. Nevertheless, this possibility should in particular concern asymptomatic infections since mothers received the instruction to contact health centers if any health problems arose. However under-detection of malaria events is unlikely to be related to the maternal HLA-G level and this should not constitute a major and particularly specific bias in the results. Furthermore, this could explain why the effect of sHLA-G level seems more detectable after 10 months of life. Nevertheless, the same pattern of results was obtained using symptomatic infections alone (p = 0.03).48 and consequently modulate the risk of malaria infection49. We have studied the role played by genetic polymorphism of HLA-G 3′UTR regulatory region. No association was observed between HLA-G 3′UTR variants and the level of sHLA-G expression (data not showed). However we cannot exclude that genetic variants present in HLA-G 3′UTR could modulate HLA-G concentration because the absence of association observed is most likely due to insufficient number of individuals. Moreover, genetic variants in others regulatory regions could also affect HLA-G expression51. Studies combining in vitro functional assays and larger cohorts are needed to better understand the role played by genetic on HLA-G expression.HLA-G genetic polymorphism could influence HLA-G expressionin vitro fertilization22. Our results are consistent with the potential value of sHLA-G from a public health point of view to identify pregnancies that could result in particularly frail newborns. However, we believe it is too early to make some recommendation for the follow-up of women and newborn, and we need to confirm these results in another population including a higher number of pregnant women.The potential advantage of soluble HLA-G as a biomarker has already been proposed in cancer, chronic viral infections and http://clinicaltrials.gov/ct2/show/NCT00811421). The first 400 infants to be delivered were enrolled from January 2010 to June 2011, and followed up for 24 months. HIV-positive, twin pregnancies, stillbirth or fetal abnormalities were excluded. Women were included before the end of 28 gestational weeks (GW) and two doses of Intermittent Preventive Treatment for pregnancy (IPTp) were administered at antenatal visits (ANVs)52. In our analyses ANV1 and ANV2 referred respectively to the ANVs during which the first and the second IPTp doses were given, in compliance with the MiPPAD clinical trial protocol.The present prospective cohort study took part in the framework of the MiPPAD clinical trial  for plasma sHLA-G measurement and malaria diagnosis. After delivery, a placental blood smear was used to assess placental malaria, defined as the presence of asexual Newborns were followed until 24 months. Due to the MiPPAD clinical trial constraints, the follow-up was different before and after 12 months. During the first year of life, at 6, 9 and 12 months children were clinically examined. In case of axillary temperature greater than or equal to 37.5 °C (or a history of fever in the preceding 24 h) a rapid diagnosis test (RDT) and a thick blood smear (TBS) were performed. After 12 months of age, children were visited at home twice a month and the temperature was systematically checked. During the 24-month period, mothers were invited to visit health centers if there was any health problem. During home visits or at the health center, in case of fever or history of fever in the preceding 24 h, a RDT was performed. Monthly between 12 and 24 months, a systematic TBS to detect asymptomatic malaria was also performed. A symptomatic malaria attack was defined as the presence of fever (or a history of fever) and a positive RDT and/or a positive TBS. Malaria attacks were treated with an artemisinin-based combination (arthemeter and lumefantrine), as recommended by the Beninese National Malaria Control Program. An asymptomatic infection was defined as a positive monthly systematic TBS with no fever or history of fever. Overall, at birth (cord blood), 6, 9, 12, 18 and 24 months, blood was collected to perform the same tests evaluated in mothers. All the medications prescribed were free of charge.53. All incubation steps were performed at room temperature, followed by four washes using washing buffer . The plates were incubated for 30 min with the substrate  and absorbance was measured at 450 nm after adding HCL (1 N). Total sHLA-G levels were determined from a five-point standard curve (12.5–200 ng/mL) using dilutions of calibrated HLA-G5 purified from M8-HLA-G5 cell line culture supernatant, and the results were expressed as ng/mL. The detection limit is ~1 ng/mL. A negative and positive control was included in each ELISA plate. The positive control was the supernatant from M8-HLA-G5 cell line culture (with the vector encoding the secreted protein HLA-G5 (M8-HLA-G5)) while the negative control was the supernatant from M8 cell line transfected with the empty vector (M8-pcDNA).). Both M8-HLA-G5 and M8-pcDNA cell lines were previously described54. The precise concentration of HLA-G5 present into the M8-HLA-G5 supernatant was defined using HLA-G5 purified protein55. The methodology to measure sHLA-G1 and -G5 molecules using ELISA has been previously validated the Wet Workshop for measurement of sHLA-G held in Essen, Germany and published56.Soluble HLA-G was quantified using the MEM-G/9 antibody , which recognizes the sHLA-G1, -G5 isoforms and the anti-human β2-microglobulin, as capture and detection antibodies, respectivelyFor survival analysis, time between birth and first malaria infection (symptomatic or not) was defined as primary outcome. After their first malaria infection, children were right-censored. The date of right censoring was the date of first malaria infection or the last available date of follow-up.30.The following variables were used. Gender of the newborn; presence of LBW (birth weight <2500 g); age of the mother; gravidity (primi vs multigravid); ethnic group ; IPTp treatment group; placental malaria infection (presence/absence); number of peripheral malaria infections during pregnancy ; level of sHLA-G at ANV1, at ANV2 and at delivery. Levels of sHLA-G at each measurement were used as quantitative variable. The environmental risk of infection was assessed through quantification of exposure to vector bites. Mosquito catches were performed over two consecutive nights, monthly throughout the course of the study, to assess the density of malaria vectors. Using environmental  and behavioral data , a time- and space-dependent environmental risk of exposure to malaria was assessed for each child by means of a predictive modelp < 0.20). Scaled Schoenfeld residuals were used for testing the proportional-hazards assumption.A Cox regression model was used to assess the effect of PM and of the mothers’ sHLA-G levels on the time of first infection, adjusted on the cofactors mentioned above. First, a univariate Cox analysis was performed to study the association between all covariates and the first malaria infection. Kaplan-Meier curves were obtained to present the probability of occurrence of malaria infection. Secondly, a multivariate analysis was performed to study the association between first malaria infection and PM or mothers’ HLA-G levels, adjusting for the covariates selected in the first step .Complementary, we built a predictive model, designed to evaluate the power of sHLA-G to predict the occurrence of infection during the 2 years follow-up (no infection vs at least one). We performed two machine-learning methods: logistic regression and random forest. For both methods, the same steps were followed. First, the sample was split into a train sample (70% of the data) and a test sample (30% of the data) randomly drawn with stratification according to the malaria infection status to guarantee the same proportion of infection in both samples. At a second step different predictive models were built (see details below) using the train sample and at the last step, the predictive power of each model was evaluated using the test sample through the area under ROC curves (AUC). All the covariates were introduced in the predictive models, which differed only according to the way the sHLA-G variable has been considered: (i) sHLA-G as a binary variable  sHLA-G as a four classes variable (according to the quartiles) and (iii) sHLA-G as a continuous variable. Then, the best sHLA-G variable was selected (i.e. the sHLA-G variable giving the highest predictive power (highest AUC) was selected. At last, three models were compared by testing the difference between the areas under the ROC curves: logistic regression with all covariates except sHLA-G (model 1 considered as the reference model), logistic regression with all covariates including the best sHLA-G variable (model 2) and random forest with all covariates including the best sHLA-G variable (model 3). Finally, the stability of the results has been checked by repeating this last step on 4 other partitions of the data drawn at random . These analyses were performed using R software, (MLR package).Comité Consultatif de Déontologie et d'Éthique (CCDE) of the Institut de Recherche pour le Développement (France) and by the Ethics Committee of the Faculté des Sciences de la Santé de Cotonou in Benin (N° 43/11/2010/CE/FSS/UAC). Written informed consent was signed and a copy was given to each participant with the possibility to withdraw at any time. If the woman could not read, an impartial witness was involved in the process. In addition to the assent of minors, informed consent was obtained from the parents or legal guardians. All the methods were carried out in accordance with the approved guidelines.The study protocol and informed consent were approved by the


Plasmodium falciparum was absent in 100% of examined samples, while aldolase, common to the four types of plasmodial pathogens causing human malaria, was detected in 56% of individuals with no significant difference between the two tested social groups: high officials (HO) and workers (W). It is suggested that the main risk factor was the presence of residences near natural and artificial waterways, which allowed prolonged contact between the vector and human host.The detection of falciparum malaria in ancient Egyptian remains had been performed by many authors using several methodologies including the use of rapid diagnostic tests. Through the immunochromatographic analysis of bony specimens from Giza skeletal collection dated to Old Kingdom, we provide first evidence of non-falciparum malaria in Ancient Egypt. The histidine-rich protein-2 (HRP2) specific to Plasmodium falciparum that is responsible for the highest rate of mortality, while the diseases caused by P. vivax, P. ovale, and P. malariae are considered benign [ Anopheles species responsible for malaria transmission in a given area is greatly influenced by climatic and environmental conditions. A great behavioral variety among different species is reported [Human malaria is an infectious disease that is threatening human health in many parts of the world. Most cases are reported among children under 5 years. Many natural or human-made conditions can increase the chance of malaria transmission. The nutritional deficiencies problems may lead to the complication of the situation, especially among the most susceptible groups: children and pregnant women . The mosd benign . Abundanreported . Social reported .Few studies addressed the issue of malaria diagnosis in archeological human remains. They were carried on ancient Egyptian –13 and Eth Dynasty but also the tombs of high officials (HO) and workers (W) of the Old Kingdom [th Dynasty that comprised an important Nile harbor [The Giza Plateau contains not only the royal pyramids of the 4 Kingdom  and Heite harbor .th-6th Dynasties) using malaria rapid diagnostic tests. Both HO and W were subjected to analysis.The aim of this study is to estimate the prevalence of malaria among Giza community during the Old Kingdom  located west of the Great Pyramid. It was constructed to include the remains of the literate individuals who represented the elites such as princes, princesses, architects, scribes, priests, and temple officials [(1) The Western Cemetery: it is a cemetery of HO  located southeast of the Sphinx. Buried individuals are those who worked not only in royal structures but also in the Giza HO tombs [(2) The Southeast Cemetery: it is the cemetery of W  was added instead of 200 μl to allow doubling the antigen concentration.In each vial containing 50 mg of bony powder, 100 Antigen extraction was performed in the Research and Conservation Centre, Ministry of Antiquities, according to the method described in the study of Fornaciari et al.  with the P.f/Pan Rapid Test Device (Whole Blood) manufactured by ABON Biopharm (Hangzhou) Co. Ltd. This test can detect histidine-rich protein-2 (HRP2) specific to P. falciparum and aldolase common to all species. It was chosen according to the criteria cited in the report of the World Health Organization [Malaria diagnostic test used in this study was ABON™ Plus Malarianization .Using the IBM SPSS statistic software (version 22), statistical analysis was performed in the Centre for Static and Statistical Studies and Consultation, Institute of Statistical Studies and Research, Cairo University. P. falciparum antigen, HRP2. The non-falciparum malarial species were detected in 56% of the tested samples, i.e., detection of aldolase in 47 individuals. Thus, no mixed infection with P. falciparum was reported .The immunochromatographic technique was chosen to diagnose malaria in Giza population based on the positive results obtained from previous studies of Miller et al., Cerutti P. vivax in ancient remains did not meet expected success in spite of presence of historical evidences of malaria endemicity in studied populations, e.g., the researches of Pinello [ P. ovale or P. malariae in ancient bones until now. The findings indicating infection with non-falciparum malaria match the low mortality rate of subadults in the skeletal collection of Giza: the study of Smith [th Dynasty), where falciparum malaria was diagnosed indicated that the largest age group of Amarna bone collection was that of children as it reached 42%, while, according to El-Banna [The results showed that 56% of the Giza population were infected with non-falciparum malaria, while no infection with falciparum malaria was recorded; i.e., only aldolase was detected. To our knowledge, this is the first time that a non-falciparum antigen is detected in archeological human remains, especially the ancient Egyptian bones. In addition, the previous efforts to detect the biomolecules of Pinello  and Kendof Smith  carried El-Banna , the ske P.f/Pan Rapid Test Device is over 99% relative to microscopic technique of malaria diagnosis in clinical samples. The World Health Organization Product Testing Programme that is evaluating malaria tests manufactured by many companies around the world recommends using tests with false-positive rate less than 10%. The false-positive rate of ABON Plus Malaria P.f/Pan Rapid Test Device is 0.4% [Many precautions were considered to ensure the authenticity of results. To decrease the chance of having false-positive and false-negative results bones with sun bleaching, salt encrusting, mold growth, or consolidation materials were excluded from analysis. That is why only 84 individuals were chosen for analysis. In addition, a rapid diagnostic test with a high specificity was chosen. According to the kit enclosed instructions, the specificity of ABON Plus Malaria is 0.4% .an absence of evidence rather than an evidence of absence” as cited by Bianucci et al. [As explained by Sallers and Gomzi , there ai et al.  and Biani et al. . An arguAldolase can persist for days after the clearance of the pathogen from the blood . Thus, i Anopheles vector. As it was demonstrated by Pays [ Anopheles species have been proved to be the vectors of malaria in Modern Egypt: An. pharoensis responsible for the transmission of P. vivax in Nile Delta and Valley and An. sergentii responsible for P. falciparum transmission in Oases [According to the immunochromatographic method used in the current study, it is not possible to determine which species of the three plasmodial pathogens causing non-falciparum malaria was responsible for the disease in Giza population, especially in the absence of textual sources that can be used as indicators for the disease in Giza. However, whatever the species responsible for malaria, the prevalence of the disease must account for the presence of an efficient by Pays  and Kena by Pays , there iin Oases . The pre Anopheles that was found in Giza, it is impossible to expect its behavior, e.g., whether the mosquito was exophagic or endophagic. However, it is expected that individuals experienced prolonged contact with the mosquito breeding sites, which allowed the transmission of the disease.Since it is not possible to determine the species ofAs it was explained by Strouhal , the abuAccording to Love , Giza reSince there is no significant difference between the prevalences among HO and W, it is argued that both were subjected to mosquito bites to the same extent due to their residence near the river, waterways and harbor. It is important to note that, according to Hawass , a groupAccording to Abu-Taleb , the soc e.g., although it was suggested that the excavated bed of the Old Kingdom queen Hetepheres might be designed to bear a bednet [The study of Dahesh et al.  testing a bednet  and the a bednet , there iStudies about fracture treatment and limb amputation that were carried out on the two social groups by Hussien et al.  and ZakiGenerally, the absence of significant difference in malaria prevalence regarding social class in Giza reflects the ability of the disease to affect the whole society by the same degree; i.e., the socioeconomic factors did not affect the non-falciparum malaria prevalence in ancient Giza, while it seems that the presence of residences near natural or artificial waterways represented a risk factor.Using immunochromatographic analysis of archeological bony samples from Giza Necropolis, we provide first evidence of non-falciparum malaria prevalence in an ancient Egyptian population. This finding may enhance knowledge about the history of the disease that still affects many regions around the world.Future work will involve studying the relation between the human-made environmental changes involved with urbanism and agricultural practices in Ancient Egypt and the disease prevalence. This will be performed through the comparison of results from Dynastic Period and Predynastic Period. This may include studying the socioeconomic burden of malaria among agricultural societies and hunter-gatherers.It is recommended to perform aDNA studies on Giza population to detect the species and strains of the non-falciparum pathogen responsible for the disease. The chemical analysis of tetracycline in these archeological bones may extend our knowledge about the relation between dietary behavior and malaria. On a wider level, paleoclimatological researches may add new evidences helping to reconstruct the environmental conditions that permitted the reproduction and the spread of both the vector and the malarial pathogen.


Malaria during pregnancy may threaten the mother's health and cause serious structural damage to the internal architecture of the placenta, which subsequently affects the pregnancy outcome. A better understanding of the impact of malaria parasites on the placenta morphology is crucial for better management of pregnant women and their babies. To assess by stereology the histomorphology of selected placental structures in placenta malaria compared with normal placentae at term. Plasmodium parasites, after which they were classified as study group (Plasmodium positive) or control (Plasmodium negative). Stereological quantification using systematic uniform random sampling technique with test point and intersection counting of photomicrographs were employed to estimate the mean volume densities of syncytial knots, syncytial necrosis, foetal capillaries, and intervillous spaces of the placentae on a total of 1,600 photomicrographs. A total of 10 placentae comprising 5 controls and 5 cases were selected from 50 placentae that were collected at term (38 weeks ± 2 weeks) from the maternal delivery suit of Korle-Bu Teaching Hospital in Accra, Ghana. Blood from the placentae was collected for both rapid diagnostic test and microscopic examinations. Samples collected were examined for Plasmodium, six representing 12% were found to be infected with the parasite by both rapid diagnostic test and microscopy. On stereological assessment, the mean volume density of syncytial knots was significantly higher in the placental malaria group compared with the control placentae at term (P = 0.0080), but foetal capillaries (P = 0.7813), intervillous spaces (P = 0.8078), and syncytial necrosis (P = 0.8249) were not significantly different. Out of the fifty placental samples from the maternal side tested for This preliminary result indicates that placental malaria may cause significant increase in the syncytial knots but not foetal capillaries, intervillous spaces, or syncytial necrosis. This finding signifies early maturation of the placenta and may be crucial in understanding perinatal outcomes. Malaria remains a complex and overwhelming health problem known to contribute significantly to maternal and infant mortality. Pregnant women, in particular, are more susceptible to malaria than the general population . Studies Plasmodium-infected erythrocytes in the intervillous space of the placenta, is one major feature of malaria during pregnancy. Maternal peripheral parasitemia is an indication of malaria during pregnancy. However, it has been observed that while peripheral parasitemia may sometimes remain below microscopic detectable levels, these parasites are detectable in the placenta [Placenta malaria, the accumulation ofplacenta , 6. The placenta , 8.Placental malaria has been associated with different adverse pregnancy outcomes. Some studies reported an association between malaria and the risk of stillbirth , 10. OthStereology is the first choice for obtaining three-dimensional data from two-dimensional profiles . It offeDespite the public health importance of placental malaria, its impact on the placenta histomorphology has not been fully understood. A better understanding is crucial in developing an effective intervention for the control of placental pathology associated with placental malaria. The objective of this study was to assess by stereology the histomorphology of selected placental structures in placental malaria compared with normal placentae at term.2EDTA tubes , UK) for both Plasmodium rapid diagnostic test (RDT) and Giemsa stained smear microscopic examinations, after which it was classified as cases (study group) or controls. The study group was made up of placental samples that were found to be infected with the Plasmodium, while the control group was comprised of Plasmodium negative placenta samples.This was a case control study, involving a purposive sampling of the postpartum placenta of 50 patients. Between 2 and 3 ml of blood from the maternal as well as the foetus side of the placenta was collected and kept in Kμl of a thoroughly mixed whole blood sample (some of which has earlier been used for the RDT) was pipetted and put in the middle of the slide. Using a fine edge glass slide spreader, the blood was spread rapidly at an approximate angle of 45° to get a thin monolayer [A grease free glass slide with frosted end  was used for the thick and thin films. For thin film, 2 onolayer .μl of the blood was pipetted and placed closed to the frosted end of the same slide having the thin film. The 5 μl blood drop was spread circularly to obtain a diameter of between 1 and 2 cm smear. The films were air-dried for three hours at room temperature, after which the thin film was fixed in absolute methanol. Both the thick and thin films were flooded with 3% Giemsa for 30 minutes, followed by washing under running tap water and air-drying. The stained smears were then examined under a light microscope  with oil immersion objective lens with the Plasmodium positive or negative smears leading to classification as case (study group) or control, respectively.For thick film, 5 An immunoblot test kit (Immunetics Inc.) was used for the rapid diagnostic test (RDT). Following the manufacturer's instruction, a drop of the blood collected from the placenta was placed on the sample column of the test kit, after which four drops of the lysing buffer were added, with the results interpreted as RDT positive or RDT negative.A total of ten placentae comprising 5 controls and 5 study group were selected for further examination. After weighing the placenta and recording the volume by liquid displacement, each placenta was then cut into four equal quadrants and by choosing a random starting point, a tissue was sampled full-depth  from each quadrant concentrically from the periphery to the centre.Placenta samples of about 1cm x 1 cm x 1cm cubes were fixed in 10% buffered formalin, pH 7.29, after which the tissues were dehydrated through graded series of ethanol (from 70% through to 95%). The placenta tissues samples were further dehydrated with steps of absolute alcohol, after which they were cleared in two steps of xylene. Tissues samples were infiltrated with molten wax and embedded to form blocks for microtome sectioning. Each placental tissue sample from each quadrant was embedded in one wax block.μm) to expose the whole profile of the tissue using Leica microtome . The tissues were then sectioned at a thickness of 5 μm and four sections were selected systematically and stained with Haematoxylin and Eosin (H&E). The 1st, the 50th, the 100th, and 150th tissue sections were selected from each block . The microscope field was directed to one end of the slide . An eyepiece lens of the microscope was then replaced with a digital microscope eyepiece (Lenovo Q350 USB PC Camera), which was connected to a desktop computer (HP Compaq dx2300 Microtower). The stage was moved two microscope stage units in thea tissue .In total, 1,600 photomicrographs were systematically selected for point counting in the stereological grid . In addi Vv indicates volume density, ƩP is the sum of all test points encountered, (a/p) is the area per point of the stereological grid, t is the thickness of the section, and M is the linear magnification [The volume densities of the syncytial knot, foetal capillaries, villi syncytial necrosis, and intervillous space were determined using point counting with Cavalieri principle . A sterefication . Plasmodium parasites, six (12%) were found to be infected with Plasmodium parasite by both RDT and microscopy, while the rest  were negative for Plasmodium by both diagnostic methods. Therefore, the 12% (Plasmodium positive placenta samples) became the study group, while the 82% (Plasmodium negative placenta samples) became the control group.Out of the total of 50 placenta samples that were tested forWith regard to histomorphological assessment, syncytial necrosis, where the syncytium is shaved off  observed in this study was lower than a previous study conducted in Ghana , which r et al.  who repo[ et al.  in Gabon[ et al.  in Mali.However, similar low placenta malaria prevalence of 12% has been recorded in a study conducted in Tanzania, which involved 984 placental samples . Other sNevertheless, the 12% prevalence of placental malaria suggests that pregnant women are still in the clutches of malaria, despite present control measures like insecticide-treated nets (ITN) and intermittent preventive treatment in pregnancy (IPTp), among others , 29. P. falciparum-infected placentae [The findings of this study were consistent with other studies that also reported increased syncytial knots in thelacentae , 30. Thelacentae . A revielacentae .The strength of our preliminary study is the use of stereology to count placental internal architecture  in 1, 600 photomicrographs of placentae affected by malaria compared with controls. plasmodium-infected placentae being lower than what other studies have reported, as well as the nonsignificant differences observed in some internal architecture of the plasmodium-infected placentae compared to the uninfected ones.A limitation, however, is the inability to tell how long the parasite has been in the placenta. It may be interesting to quantify by stereology these internal structures of the placenta in early exposure to the parasite and that of late infection in future research. Another limitation worth mentioning is the low number of placentae samples used in this preliminary study, since this could possibly account for the percentage ofPlasmodium infections during pregnancy have adverse effect on the internal architecture of the placenta that may also explain perinatal outcomes. Our preliminary results add to the knowledge that placental malaria may cause significant increase in the syncytial knots, which signifies early maturation of the placenta.


It is believed that the current prevalence of malaria in endemic areas reflects selection for the carrier form of sickle cell trait through a survival advantage. Malaria has been incriminated as a great cause of mortality in people with sickle cell disease (SCD). However, people with SCD, a high-risk group, do not benefit from free or subsisized malaria prevention and treatment in Cameroon unlike other vulnerable groups which may be due to insufficient evidence to guide policy makers. This study aimed at describing clinical and socio-demographic characteristics of patients with malaria, determining the prevalence of malaria in hospitalized children and in those with SCD and without, compare frequency of presentation of malaria related complications  between children admitted for malaria with SCD and those without and finally, determing the risk factors for death in children admitted for malaria.p-value ≤0.05.This was a retrospective analysis of admission records of children age 1 to 18 years with a confirmed malaria diagnosis admitted at the Laquintinie Hospital during January 2015 through December 2018. Clinical features, laboratory characteristics and outcome of malarial infections, stratified by SCD status were studied. Patients with HIV infection, malnutrition, renal failure and discharged against medical advice were excluded from the study. Data were analysed using Epi-info 7 software and analysis done. Chi square test, Odds ratios, CI and student’s t test were used to determine association between variables. Statistical significance was set at p-value 0.002). The mean hemoglobin concentration was lower in SCD as compared to non SCD . Overall mortality in SCD was 3.4% and malaria was reponsible for 20.4% of these deaths as compared to the 35.4% in non SCD patients. Convulsion and impaired consciousness were significantly lower in SCD group . Death was significantly higher in SCD patients with malaria as compared to SCD patients admitted for other pathologies .The prevalence of malaria was lower among children with SCD than it was among children without SCD (23.5% vs 44.9%). Similarly, among those with a positive microscopy, the mean parasite density was significantly lower among children with SCD than it was among children without SCD (22,875.6 vs 57,053.6 parasites/ μl with t-value − 3.2, The SCD population has a lower mortality related to malaria compared to the non-SCD population. Meanwhile, within the SCD population, those admitted with malaria are twice more likely to die than those admitted for other pathologies. Jaundice, hepatomegaly and splenomegaly were common in SCD with malaria, however no risk factors for malaria severity or malaria related death was identified. Plasmodium falciparum malaria in sub-Saharan African children [According to WHO, worldwide there was an estimated 219 million malaria cases and 435,000 deaths attributed to malaria in 2017. Approximately 41% of Cameroon population has at least 1 episode a year, with a prevalence of 29% and has an overall mortality of 30–35% [children , 7. SCD children , 9. The children , 11. Camchildren –14. The children . As SCT children . Howeverchildren . DespiteThis was a hospital based retrospective cohort study. The study was conducted at the pediatric unit of the Douala Laquintinie Hospital, which is a second level hospital in which specialized care for SCD patients is offered through the “Centre de Prise en Charge Emmanuel Billong” where approximately 600 patients are being followed. The protocol used for management of severe malaria is artesunate. The pediatric emergency ward and the general pediatric ward were also included. Data from patients admitted between January 2015 to December 2018 were reviewed and analyzed.s discharged against medical advice, who were malnourished, had HIV or renal failure were excluded from the study.Sampling was convenient. We studied malaria in two different populations (SCD and non-SCD population). Patients aged 1 year to 18 years with documented malaria diagnosis were included. PatientThe minimum sample size was calculated in inference from a study carried out in Tanzania by Makani et al., malaria in sickle cell anaemia . A totalRecruitment was done using admission records. The designed data entry form was used to extract the sociodemographic and clinical characteristics as well as laboratory characteristics associated with malaria in both populations and non-malarial diseases only in sickle cell patients. Data was then verified to ensure non-repetitiveness of records and ultimately validated to be used for analysis. In this study, a malaria case was defined as any patient with a positive Rapid Diagnostic Test for malaria, parasites identified on microscopy and/or clinical improvement with just antimalarial treatment. Death from malaria referred to any death in a patient with a confirmed or suspected malaria diagnosis. Severe anaemia was said to be present if subjects had a hemoglobin level < 5 g/dl on full blood count. Hyperparasitemia was defined as parasite density of > 250,000 parasites/ μl and hyperleukocytosis as a white blood cell count of > 15,000/ μl on full blood count.Ethical approval was granted by the Institutional Review Board of the University of Bamenda and administrative approval was obtained from the director of the Laquintinie hospital,p-value ≤0.05. The results were presented in the form of frequency tables and charts.A data spread sheet was created in excel Microsoft 2016 in which collected data was introduced and analysed through Epi-info version 7 software. Through cross tabulation of variables, the data was captured and assessment for frequency tabulation data was analyzed. Chi square test, Odds ratios (OR), Confidence Intervals (CI) and student’s t test were used to determine association between variables. Statistical significance was set at A total of 6563 patients were included in the study, of which 1438 were SCD patients (22%). Of these, 982 SCD cases were admitted, with 526 (53%) being new or first-time admissions and 456 (46%) having readmissions. There was a 10% decline in malaria prevalence between 2015 and 2017 and we noticed an increase of 2% from 2017 to 2018. Fig. In this study, the sex ratio in SCD population was 1.2:1  as compared to 1.3:1 for the non SCD population. The 1-5 years age group had the highest number of cases. Malaria was either the primary or secondary discharge diagnosis in 23% of SCD patients at admission, compared with 49% in the non SCD population.p-value = 0.07).The overall mortality in patients with SCD was 3% as compared to 9% in the non-disease population. Malaria accounted for 35.4% of mortality in the non SCD population compared to 20.4% in the SCD population. Specific malaria mortality was higher in the non-sickle cell disease population (6%) as compared to the sickle cell population (3%) . Splenomegaly and jaundice were found to be greater in the SCD population. . Convulsion and impaired consciousness were significantly lower in patients with SCD . Of the patients who died, the majority were readmitted patients and non-sicklers . More SCD patients had severe anaemia compared to those without . The mean platelet count was significantly higher in the SCD patients as compared to non SCD patient . The mean white blood cell count was lower in patients with SCD relative to patients without SCD (20.3 vs 9.8 × 109/L) though this difference was not statistically significant . Hyperleucocytosis was more prevalence among the SCD group than in non SCD group . The mean parasite density was found to be statistically significantly lower in the SCD group than in the group without SCD . None of the clinical features nor laboratory findings associated with severe malaria was found to be significantly associated with SCD.The mean hemoglobin concentration was significantly lower among patients with SCD at admission as compared to patients without sickle cell disease , splenomegaly , hepatomegaly , were found to be significantly associated with SCD patients with malaria (Table Temperature > 40 °C (9.6% vs 37.7%), prostration , . The mean platelet count was significantly lower in the SCD patients as compared to those without malaria . SCD patients with malaria had a higher risk of dying compared to those admitted for other pathologies  as represented in Table The mean hemoglobin concentration was lower in SCD patients with malaria as compared to those admitted without malaria  but less than 3.2 obtained Nansseu et al., , 20. MeaThe prevalence of SCD was 16.4%. This was considerably higher than other studies carried out by Purohit et al. (8.95%), Komba et al. (1.6%), and Ngolet et al. (10.64%) –25. The Vaso-occlusive crisis was the major cause of admissions, accounting for 31.6% of all hospital admission, and malaria the second with 23.5%. This could be compared to the findings of Mbassi et al., Ngolet et al., and Aluoch et al. who had malaria prevalences of 25.5, 29.8, and 20% respectively , 25, 26.The overall mortality in the sickle cell population was 3.4% and of the patients who died, the highest number of deaths occurred in the 1-5 yr age group. Malaria accounted for 20.4% of these deaths, this finding is similar to the 19.6% deaths from malaria found by Purohit et al. and 16.0% found by Komba et al. , 24. TwoWe found that impaired consciousness and convulsions were lower among the SCD population as compared to the non SCD population. This was similar to findings obtained of Komba et al. and Makani , 24. ThiSplenomegaly, hepatomegaly and icteric sclerae were all higher among the SCD population as compared to those without sickle cell. Studies of Komba et al. and Makani report similar findings , 24. ThiWe found a lower parasite density among SCD patients compared to non SCD population as similarily reported by Makani et al., Aninagyei et al., and Komba et al. , 22, 25.Severe malaria anaemia was higher among patients with SCD as equally demonstrated by Komba et al., Makani et al. and Aninayei et al. (as shown by either a low mean hemoglobin or hemoglobin < 5 g/dl) , 22, 25.p-value 0.04). A high risk of death in SCD patients with malaria as compared to SCD admitted for other pathologies was observed in our study and was equally statistically significant .In our study, we found no clinical nor laboratory characteristics associated with death in SCD population with malaria when compared to non SCD patients. Komba et al.  found noSCD patients in endemic regions are recommended by WHO to receive antimalarial prophylaxis , 34. MalBased on the afore mentioned findings we recommend to maintain a high index of suspicion of malaria in patients with sickle cell anaemia and be prompt to care. Strategies to reduce incidence of and mortality from malaria in this sub population (risk group) should be implemented.