In up to one third of the hospitals in some rural areas of Africa, laboratory services in malaria diagnosis are limited to microscopy by thin film, as no capability to perform thick film exists . A new rapid molecular malaria diagnostic test called Loop-mediated isothermal DNA amplification (LAMP) has been recently validated in clinical trials showing exceptional sensitivity and specificity features. It could be a reliable diagnostic tool to be implemented without special equipment or training.The objective of this proof of concept study was to confirm the feasibility of using LAMP technique for diagnosis of malaria in a rural Ethiopian hospital with limited resources.st to December 31st 2013. A total of 162 patients with a non-focal febrile syndrome were investigated. The diagnostic capability  of rapid malaria tests and microscopy by thin film was evaluated in comparison with LAMP. Eleven (6.79%) out of the 162 patients with fever and suspected malaria, tested positive for LAMP, 3 (1.85%) for rapid malaria tests and none of the eleven cases was detected by thin film microscopy.This study was carried out in Gambo General Hospital, West Arsi Province (Ethiopia), from November 1LAMP can be performed in basic rural laboratories without the need for specialized infrastructure and it may set a reliable tool for malaria control to detect a low level parasitemia. Plasmodium falciparum and Plasmodium vivax [In many areas of Ethiopia, malaria laboratory diagnosis is still based on microscopy. Limitations of blood smear microscopy contributed to failure of the 1950–1960s WHO Global Programme to Eliminate Malaria  with lowum vivax . TherefoGambo Hospital is located at 2.200 m altitude above sea level in the Oromia region, a traditionally malaria-free area. However, many patients affected with malaria coming from the lower slopes of the Rift Valley, at lower altitudes, are assisted and treated in this Hospital. Malaria incidence is lower during the dry season, and higher right after the rainy season when mosquitoes spring up. In this area, a period of low incidence during the previous seasons might have aggravated the events, possibly due to a low level of immunity in the affected population .Studies based in conventional PCR performed in external laboratories are suitable for occasional evaluations, but are uneconomical and require shipping of specimens to reference laboratories. Results are appropriate for evaluating new technologies, but PCR is not an useful tool for internal quality control programs. However, a new rapid and simple molecular diagnostic test called Loop-mediated isothermal DNA amplification (LAMP), does not require special equipment or space distribution in the laboratories, and provide results within 60 minutes –8.A field study was designed in order to compare the standard diagnostic microscopy performed in Gambo Hospital (thin blood film), RDT and LAMP  on site.The study population included every patient referred to the hospital laboratory for malaria diagnosis who was older than two years and attended to the outpatient Department of Gambo Hospital with suspected uncomplicated malaria (axillary temperature > 37.5°C or report of fever in the previous 48 hours) and who did not meet any of the exclusion criteria.Exclusion criteria were: fever ≥ 3 weeks, antimalarial treatment in the previous week, apparent focal infection , infection that could be definitively diagnosed clinically  and patients who had received an immunization in the preceding 48 hours or blood products in the previous 6 months.The study was performed between November 1st and December 31st 2013 for detection of malaria infection in febrile symptomatic individuals during the dry season, in a time with a estimated higher incidence of malaria in this area.Ethics committee approvals were obtained from both the local Research and Publication Committee of the Gambo General Hospital and the Health Unit and Ethical Review Committee of the Ethiopian Catholic Secretary (GH/MSMHF/706). We ensured that the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in the approval by the institution’s human research review committee. We also made sure that oral informed consent was obtained from each patient to participate on a voluntary basis in the study and a questionnaire with complete epidemiological and clinical questions was filled out. Oral informed consent was recorded in each questionnaire and was obtained from the next of kin, caretakers, or guardians on behalf of the minors/children enrolled in this study. Written consent no was obtained because there is a high rate of illiteracy in the population. No economic compensation was granted for participating in the study. All the data were treated confidentially and anonymized.Whole blood was collected from each patient at the time of enrolment by digital pricking; finger prick was performed in the fingerpad of the third or fourth fingers, previously cleaned with 70% ethylic alcohol. This sample was used to carry out routine blood smears, RDTs and LAMP test. Routine blood smears were read by the health facility laboratory personnel as usual; both microscopy and RDT results were provided to the health facility physicians so they could be used for management of the patient according to the usual standard of care. Due to the low sensitivity of thin film, RDT results were given priority over microscopy.P. falciparum and P. vivax are co-endemic  and contained the histidine rich protein-2 (HRP-2) and pan-malarial lactate dehydrogenase (LDH).LAMP results were obtained for the purpose of the study exclusively, and were not used for clinical management. RDTs used were those recommended for an endemic area as Ethiopia, where ® MALARIA Pan/Pf Detection Kit  were followed [The standard operational procedures of the Loopampfollowed . The Loofollowed . BrieflyFresh blood samples collected by finger prick were immediately used for LAMP. Boil and spin method was used in a sample preparation area separated from the amplification area. An aliquot of 60 μl of whole blood of each patient was transferred to the extraction tube and mixed with 60μl of extraction buffer  by vortex for 10 seconds. Extraction tubes containing the samples were placed in the Hot-block (Eppendorf 5350) at 95°C for 5 minutes, later centrifuged at 10,000 g for 3 minutes (mini-centrifuge MCF-2360) and finally 30μl of clear supernatant were transferred to the dilution tube .P. falciparum (Pf) specific primers have been confirmed to be specific for P. falciparum parasites.Two primer sets provided with the kit were designed to detect the mitochondrial DNA of Plasmodium parasites. The Pan specific primers detect a target DNA sequence and are able to detect a wide range of Plasmodium species including the four most common ones that cause human malaria. The As it is required to test both for Pan and Pf, two reaction tubes (one for Pan and other for Pf specific primer) per sample were used in each run (10 samples), plus two reaction tubes for control (one reaction tube for positive control (PC) and a different one for negative control (NC)) (both included in the kit) which allowed a total of 24 reaction tubes.Once DNA was extracted, 30 μl were added in a reaction tube for Pan detection, and 30 extra microliters were added in a different reaction tube for Pf detection. Finally, a NC and a PC were addded in both reaction tubes. All of them were shaken to ensure proper mixing and LAMP reagents dissolving. The tubes were placed immediately into the hot-block at 65°C for 40 minutes (amplification reaction). At the end of amplification reaction, tubes were heated at 80°C for 5 minutes, to terminate the reaction (enzyme inactivation).The bottom of each reaction tube was irradiated with UV lamp (wave length = 240 nm to 260 nm and 350 nm to 370 nm) and observed from the side through dark glasses. For a valid run, green light is emitted by PC and no light is emitted by the NC.A cost and time-to-completion analysis for every method used were done according to Shillcutt S et al . Costs o®)) dose were worked out from the current whole sale price ($2.40)[Costs of an adult ACT (artemether-lumefantrine (Coarteme ($2.40).We set the cost of RDT kits at US$ 0.6–1 and that of microscopy at US$ 0.32–1.27. Microscopy costs are dependent on workload and were based on a range from 1000 to 6800 or more diagnoses per year. To clarify concepts, we assumed that microscopy was used only for malaria diagnosis, but not for other diseases.For the statistical analysis, Software EPIDAT 3.1 was used. Final results of microscopy and RDT were compared to results obtained using LAMP using McNemar’s test; and sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated with 95% confidence interval (CI). Prevalence results were expressed with a 95% CI.In the descriptive study, continuous variables were represented as the mean ± standard deviation (SD), and qualitative variables were expressed as relative frequencies. Statistically relation between lamp-positive and a) quantitative diagnostic parameters were made using Mann-Whitney U test and b) qualitative diagnostic parameters using Chi-squared test. A P-value of <0.05 was considered to indicate statistical significance.Statistically significant differences among the different methods used comparing with LAMP results were found. Eleven out of the 162 patients with fever tested positive for LAMP , 3 for RDTs  (p = 0.0133 McNemar’s test) and none was detected by thin film microscopy  (p = 0.0094 McNemar’s test). Results are shown in The cost-effectiveness of LAMP, RDTs and microscopy relative to presumptive treatment is shown in Malaria incidence has been declining globally in the last decade. However, in countries like Ethiopia, it remains as one of the top health problems and has been reported as one of the three leading causes of morbidity and mortality in the past years . EffortsP. falciparum and P. vivax malaria. LAMP Kit for malaria diagnosis may be used by laboratory technicians without previous training in molecular methods. Knowledge and skills can be acquired during a short training period of less than three days. However, strict adherence to the procedures is necessary to achieve reliable results. Different clinical studies have validated this rapid molecular test in the field with a performance similar to conventional PCR [Recent studies have shown sensitivity values as low as 51% when performing thick and thin film by expert microscopists in comparison with LAMP assays in Uganda . In a pronal PCR . Additioonal PCR  as well onal PCR .P. vivax-P. falciparum and 0% of positive test results for P. falciparum in Pan line, with this density [The results obtained in this study indicate the urgent need of improving the quality of light microscopy in non-certified clinical laboratories and providing RDTs to hospital laboratories with substandard proficiency in malaria microscopy, at least until the laboratory achieves a certification. The poor sensitivity showed by RDTs in comparison with other field studies performed across Africa could indicate that in our small group most positive patients were infected with very low parasitemia (< 200 trophozoites/microliter). Results obtained by the WHO in 2013 showed a 5.7%-86.0% of panel detection score for  density . Moreove density .In accordance with the studies of Patel et al in Thailand and India or Aydin-Schmidt in Zanzibar, this study shows that LAMP can be used in a rural and remote area to detect the presence of very low parasitemia patients not diagnosed by RDTs as well as the convenience of using more powerful diagnostics tools in any strategy aimed to control or eradicate malaria in low transmission areas , 20–21. As previously shown, thick film microscopy and RDTs both become relatively insensitive at parasite densities below 100 parasites/μl. Current surveillance systems which rely on microscopy or RDTs are not sensitive enough to detect asymptomatic and paucisymptomatic carriers who act as reservoirs for malaria transmission and may develop malaria episodes in the future . FurtherPort el al indicated an important limitation, which is the problem of potential unavailability of electricity used for irraditation and heating of thermo-blocks in the field. However, trials using a hand-held battery operated thermoblock were carried out and offered a cheap, simple and fast method of molecular detection of malaria parasites.. MoreoveLAMP may provide reliable results in basic rural laboratories without the need for specialized infrastructure and can represent a powerful tool in malaria control programs.


Malaria in highland areas of Kenya affects children and adults. Local clinicians include symptoms other than fever when screening for malaria because they believe that fever alone does not capture all cases of malaria.Plasmodium falciparum infection on peripheral blood smear in an individual with any screening symptom. Asymptomatic P. falciparum infection was assessed in a cohort at ten time points to determine the extent to which symptomatic episodes with parasitaemia might be attributable to baseline (asymptomatic) parasitaemia in the community.Individuals who presented to dispensaries in a highland Kenya site of low, unstable malaria transmission from 2007–2011 with 1 or more of 11 symptoms were tested by microscopy for malaria. Clinical malaria was defined as asexual P. falciparum parasitaemia was infrequent (0.1%).3,420 individuals were screened for malaria, 634 < 5 years of age and 2,786 ≥ 5 years of age. For the diagnosis of clinical malaria, the symptom of fever had a sensitivity and specificity of 88.9% and15.4% in children <5 years, and 55.8% and 54.4% in children ≥5 years, respectively. Adding the symptom of headache increased sensitivity to 94. 4% in children <5 years and 96.8% in individuals ≥5 years, but decreased specificity to 9.9% and 11.6%, respectively, and increased the number of individuals who would be tested by 6% and 92%, respectively. No combination of symptoms improved upon the presence fever or headache for detection of clinical malaria. In the cohort of asymptomatic individuals, In areas of low, unstable malaria transmission, fever is a sensitive indicator of clinical malaria in children <5 years, but not in older children and adults. Adding headache to fever as screening symptom increases sensitivity of detection in individuals ≥5 years old at the cost of decreased specificity. Screening for symptoms in addition to fever may be required to accurately capture all cases of clinical malaria in individuals ≥5 years old in areas of low malaria transmission. Fever is the universal screening symptom for malaria in research studies -4, but tP. falciparum parasitaemia, which at this point occur almost exclusively in individuals with symptoms that lead them to seek evaluation and treatment at the health dispensaries on site. Individuals with any of eleven symptoms considered consistent with malaria by study clinical officers were tested for malaria by microscopy. In the present study, sensitivity and specificity of individual symptoms and combinations of symptoms for clinical malaria were assessed, with the primary definition of clinical being P. falciparum parasitaemia in any symptomatic individual. Sensitivity and specificity of individual symptoms and combinations of symptoms for alternate definitions of clinical malaria were also assessed, including: (1) P. falciparum parasitaemia with measured fever (axillary temperature ≥37.5°C), (2) P. falciparum parasitaemia ≥ 1,000 parasites/μL with measured fever, or (3) P. falciparum parasitaemia ≥ 4,000 parasites/μL with measured fever.Kipsamoite and Kapsisiywa experienced an atypical situation in 2007, when widespread indoor residual spraying (IRS) with long-lasting insecticides and introduction of artemisinin combination therapy with artemether-lumefantrine (Coartem©) led to a 14-month period with no clinical cases of malaria and possible interruption of malaria transmission . After tThe study was conducted in the Kenyan highland sites of Kipsamoite  and Kapsisiywa , Nandi County, from 2007 to 2011. Elevation ranges from 1,887 m to 2,132 m at these sites. The area has seasonal, unstable malaria transmission and a history of past malaria epidemics and a recent possible interruption of malaria transmission from April 2007 to May 2008, with no clinical cases of malaria identified in that period . PersonsPlasmodium species by microscopic examination of blood smears. The symptoms were determined by local clinical officers and included fever, chills, headache, loss of appetite, vomiting, jaundice, diarrhea, backache, joint pains, nausea, and severe malaise. Microscopy was performed as previously described ) compared to individuals ≥5 years .During the study period, only 3/3,420 smears done were positive for P. falciparum parasitaemia in a person with any of the eleven symptoms, along with the sensitivity and specificity of each symptom, are presented in Table P. falciparum parasitaemia with measured fever (axillary temperature ≥ 37.5°C) are summarized in Additional files P. falciparum parasitaemia in participants seen at these clinics led to high negative predictive values  and low positive predictive values  for all symptoms assessed.The frequency of individual symptoms for clinical malaria, defined as Symptoms with the greatest specificity lacked sensitivity , and those with the greatest sensitivity lacked specificity . Of particular interest, fever in individuals ≥ 5 years was neither sensitive (56%) nor specific (54%).P. falciparum parasitaemia were significantly more likely to have headache, malaise, and chills than those without parasitaemia. Individuals ≥5 years with P. falciparum parasitaemia were also significantly more likely to have fever and vomiting, and significantly less likely to have diarrhoea, than those without parasitaemia. Findings for the outcome of P. falciparum parasitaemia with measured fever (axillary temperature ≥37.5°C) were very similar and are summarized in Additional file Table To assess whether any combination of symptoms could improve on fever for sensitivity and specificity of detection of clinical malaria, a three-step process was undertaken: (1) candidate subsets of symptoms to consider were selected; (2) for each candidate subset of symptoms, a logistic regression model was fitted including those symptoms; (3) the logistic regression results were used to define rules and estimate their sensitivity and specificity. The results of this assessment are summarized in Table P. falciparum infection by only two persons (6%), but increased the number of individuals ≥5 years who would be tested by 1,190 persons (92%).As documented in Tables P. falciparum parasitaemia), having a history of fever or measured fever in the clinic had a sensitivity and specificity of 94% and 11%, respectively, for children <5 years, and 78% and 44%, respectively, for persons ≥5 years.To assess whether more stringent criteria for a definition of clinical malaria would result in different findings, sensitivity and specificity of symptom combinations was assessed for a number of stricter definitions of clinical malaria, as noted in Tables Six hundred and four randomly selected individuals from the site were assessed for presence of asymptomatic parasitaemia in July and November 2007, March, August and October 2008, February, April, August and October 2009, and May 2010. Due to movement of individuals during the study period, different numbers were tested at each time point, but during the three-year period, only five persons out 5,195 tested had asymptomatic parasitaemia (0.1%), with a median parasite density of 240 .P. falciparum parasitaemia would not have been captured as malaria cases had history of fever been the sole screening criterion. A recent study by D’Acremont et al. found that in one area of Tanzania, malaria was a less common cause of fever than other infections, notably respiratory viral infection [Fever is the universal screening tool for clinical malaria in research studies. This is likely appropriate in children <5 years, who constitute the majority of malaria cases in highly endemic areas, because they are more likely to develop fever when they have infection than are older individuals. However in areas such as the highland area of the present study, a substantial proportion of clinical malaria occurs in individuals ≥5 years  and locanfection . The preA history of fever may be absent in individuals ≥5 years with parasitaemia for several reasons. Fever may be less evident in older children and adults than in younger children, whose temperature may be checked or noted by their parent. This is supported by the only modest increase in sensitivity of a history of fever among individuals ≥5 years who had a measured fever at the clinic. In areas of higher transmission, parasitaemia could be incidental rather than causative of symptoms. However in this area, based on the surveys done, one would expect <0.2% of individuals to have asymptomatic parasitaemia, so incidental parasitaemia should account for only a tiny fraction of the parasitaemia in symptomatic individuals seen in the clinic. In addition, history of fever had sensitivity only 65% even for clinical malaria defined as parasitaemia > 4,000 parasites/μL and a measured fever at the clinic, individuals who in this context indisputably have clinical malaria. The Nandi words for each symptom, including fever, were carefully reviewed with our study field assistants, and verified by the clinical officers and the local advisory group, and the symptom of fever was highly sensitive for detection of parasitaemia in young children, so confusion about the term is unlikely. The most likely explanation seems to be that other symptoms precede fever in older individuals. These symptoms bring them to the dispensary, at which time some have measured fever but others remain afebrile. Given the clinical course of malaria, all of these individuals were likely to have a fever eventually, but in a dispensary setting, particularly in an area of low transmission that is working toward elimination, one wants to detect malaria early and accurately. Individuals with malaria who have symptoms other than fever, but are not tested or treated because fever is the sole screening criterion, are likely to go outside the health system, and potentially receive inappropriate treatment.Unfortunately, no symptom combination emerged that would allow us to detect >90% cases of parasitaemia in individuals ≥5 years without screening many more uninfected individuals. A previous study in highland Uganda found that a history of fever or presence of measured fever at the clinic had a sensitivity of 95.7% and a specificity of 25.5% , but theDefinitions that relied on more symptoms were more complex, but did not have better sensitivity than asking about fever or headache, so in the present study site, fever or headache will be used as screening symptoms because they are the best single set of screening symptoms that will capture ≥95% individuals with clinical malaria. An alternative would be to wait for individuals to come in for a repeat visit, since malaria likely will eventually result in fever in all individuals, but this would lead to missed chances to diagnose malaria, to prolonged parasitaemia in symptomatic individuals, and potentially to lost confidence in the health dispensary. The findings of the present study may have applicability to other sites of unstable transmission, and in these areas analysis of symptoms should be done to determine if screening for fever alone can detect >95% of individuals who have clinical malaria.absence of diarrhoea had 96% sensitivity for detection of clinical malaria in individuals ≥5 years. The finding probably reflects a combination of the low incidence of clinical malaria at this site, the frequency of diarrhoea as an illness, the multiple other causes of diarrhoea in this area, and the relatively low frequency of diarrhoea as a symptom of malaria. The study findings contrast with those of Coldren et al., who found a modest association between P. falciparum parasitaemia and a history of diarrhoea in adults from three areas of Kenya with differing malaria transmission intensity [An interesting incidental finding is that the ntensity . The difThe present study documents that in a highland area of unstable transmission, where asymptomatic parasitaemia is rare, the symptom of fever alone is adequate for detection of clinical malaria in individuals <5 years but not in individuals ≥5 years. In areas of high malaria transmission, malaria occurs most often in children and is deadliest in children -21, so aThe authors declare that they have no competing interests.ALM conceptualized the study, conducted literature review, performed data analysis and draftedthe manuscript. PC did data cleaning and initial data analysis. GA and WO reviewed the analysis and manuscript and provided key comments on manuscript revision. JSH conducted data analysis and helped to draft the initial and final manuscript. CCJ provided the study design, and oversaw data analysis, programme implementation, drafting of manuscript, and review of the manuscript. All authors read and approved the final manuscript.Frequency, sensitivity and specificity of particular symptoms for symptomatic Plasmodium falciparum parasitaemia with plus a measured axillary temperature ≥37.5°C in children <5 years of age.Click here for fileFrequency, sensitivity and specificity of particular symptoms for symptomatic Plasmodium falciparum parasitaemia plus a measured axillary temperature ≥37.5°C in individuals ≥5 years of age.Click here for fileOdds ratios of particular symptoms for prediction of symptomatic P. falciparum parasitaemia with a measured axillary temperature ≥37.5°C.Click here for file


Highland areas are hypoendemic zones of malaria and are therefore prone to epidemics, due to lack of protective immunity. So far, Cameroon has not succeeded in implementing a convenient and effective method to detect, prevent and forecast malaria epidemic in these peculiar zones. This monitoring and evaluation study aims to assess the operational feasibility of using the human malaria infectious reservoir (HMIR) and the malaria-attributable fraction of fever episodes (MAFE) as indicators, in designing a malaria epidemic early warning system (MEWS).Longitudinal parasitological surveys were conducted in sentinel health centres installed in three localities, located along an altitudinal transect in Western Cameroon: Santchou (750 m), Dschang  and Djuttitsa . The syndromes of outpatients with malaria-like complaints were recorded and their blood samples examined. The HMIR and the MAFE were estimated and their spatial-temporal variations described.Plasmodium infection in outpatients decreased with increasing altitude; meanwhile the HMIR remained fairly constant, indicating that scarcity of malaria disease in highlands is likely due to absence of vectors and not parasites. In lowland, children carried the heaviest malaria burden in the form of febrile episodes, and asexual parasites decreased with age, after an initial peak in the 0-5 year’s age group; however, they were similar for all age groups in highland. The HMIR did not show any variation with age in the plain; but some discrepancies were observed in the highland with extreme age groups, and migration of populations between lowland and highland was suspected to be the cause. Plasmodium infection was perennial in the lowland and seasonal uphill, with malaria disease occurring here mostly during the short dry season. The MAFE was high and did not change with altitude.The prevalence of asexual It is obvious that a malaria outbreak will cause the sudden rise of HMIR and MAFE in highland, prior to the malaria season; the discrepancy with lowland would then help detecting an incipient malaria epidemic. It is recommended that in designing the MEWS, the National Malaria Control Programme should include these parameters and put special emphasis on: altitude, age groups and seasons. In the lowland regions of sub-Saharan countries, malaria is holoendemic with perennial transmission. On the contrary, highland areas are known to be malaria hypoendemic, due to climate (low temperature and relative humidity), which is not appropriate for anopheline development and their reproductive fitness . NeverthAn epidemic is a sharp increase in malaria incidence among populations in which the disease is scarce; or a sudden high increase in clinical malaria for areas of moderate transmission . For theRecently, modern tools to help decision-makers to predict malaria epidemics were proposed. They rely on the use of: combinations of satellite-derived climate data ,12, weatSince malaria epidemics appear suddenly and terminate within a few months, they require emergency measures that must be implemented as promptly as possible in order to be effective. Moreover, a declaration of an emergency, generally based on the inability of the medical services to cope with demands of patients following malaria outbreaks, must be made. Forecast of epidemics based on indicators preceding elevated infection and disease rates in human populations are required. Such a strategy is likely to be efficient, since there is an elevation of gametocyte prevalence, which can reach 6% in the general population and even more (8 to 10%) in infants, prior to malaria epidemic . In thisMalaria infection tends to be a disease when a defined level of parasitaemia leads to the production of signs and symptoms -28. MalaPlasmodium infection ), among patients who report fever, knowing that these malarial fevers are always associated to other manifestations [Because fever is present in the majority of malaria cases in sub-Saharan Africa, it has been recommended that for all febrile symptoms, a presumptive treatment should be administered . The appstations .Plasmodium infection cases do not present measurable temperature elevation [Although fever is the main characteristic sign of malaria, some levation . These alevation . Asymptolevation . In factlevation ,38. Howelevation . The humPlasmodium extrinsic cycle, the anopheline development and reproductive fitness. Moreover, this absence of immunity may boost the trophozoite-gametocyte transformation ratio (TGR) and extend the longevity of sexual parasites. It is, therefore, likely that early increases in HMIR and MAFE in sentinel health facilities of highland areas will precede the sudden elevation of clinical malaria disease incidence. Thus, monitoring these two parameters using network of health workers in sentinel stations placed in highlands may unveil incipient epidemic and provide good conclusive early warning of malaria outbreaks.The hypothesis of this study is that in normal circumstances, HMIR and MAFE in lowland and highland areas are not different, since the higher number of infective bites found in lowland will result in only low infection in human populations due to naturally acquired immunity; while in highlands, although there is no immunity, there are also low infective bites, because the climate is unfavourable to the The main objective of this study was to evaluate the operational feasibility of using the HMIR and MAFE as indicators to be used by a network of health workers in sentinel stations, while designing a MEWS for an epidemiological surveillance of malaria outbreaks.Anopheles gambiae and Anopheles funestus [Plasmodium falciparum, 2.02% to Plasmodium malariae and 0.68% to Plasmodium ovale [The study took place in the Menoua Division, an outskirt of the Cameroon’s Western highlands, named the Bamileké Plateau. It is a unusual zone as far as topography and climate are concerned, located on the Cameroon Volcanic Line in a savannah landscape within the Guineo-congolese bioclimatic domain as follows: the main dry season (MDS: November to mid-March), the small rainy season (SRS: mid-March to May), the small dry season (SDS: June to July) and the main rainy season (MRS: August to October) . The majfunestus . About 9um ovale . Three sThe village of Santchou  with 37,479 inhabitants , is locaOn top of this sheer cliff, at an altitude of 1,400 m on the Bamiléké plateau, lies the city of Dschang  with 120,207 inhabitants , locatedFinally, Djuttitsa  a village of Nkongni District with 53,367 inhabitants , is locaLongitudinal parasitological surveys were conducted once a week and during 12 months, in three sentinel health centres namely: Santchou District Hospital, Hôpital Saint Vincent de Paul of the catholic mission Secret Heart of Dschang and the Djuttitsa District Hospital, located in the above described localities. Participants were selected among outpatients living in these localities, based on the malaria-like complaints, which have been recorded. The following parameters were also determined: age and axillary temperature (assuming a case definition of fever to be any temperature ≥38°C). Blood samples were collected by finger pricking, then thin and thick blood films were made on the same slide for microscopic examinations.Plasmodium species [The dried slides were brought back to the laboratory where they were fixed by dipping the thin film into the May-Grünwald solution for five seconds and washed with physiological water (0.9% of NaCl). Then both the thick and thin films were stained with 10% Giemsa for 15 minutes . Examina species .A thick blood film was declared negative if no parasite (regardless the stage) was found after examining at least 200 microscope fields . For thePlasmodium-positive and Plasmodium-negative patient groups, as well as the rate of Plasmodium infections, and gametocyte prevalence among population ages and study sites. When in a given locality the difference in the rate of occurrence of a symptom between Plasmodium-positive and Plasmodium-negative patients group was significant, it was assumed that there is a link between the carriage of Plasmodium parasite and the fact of having that symptom at 0.05 level of significance , among seasons and between population ages in each locality were tested using the non-parametric Kruskal Wallis H-test. The difference between the prevalence and means were declared significant when the probability p was < 0.05.The parametric Student t–test was used to compare the mean temperature among Plasmodium infection as recommended by the National Malaria Control Programme.An institutional ethical approval  was obtained from OCEAC, followed by a national ethical clearance  delivered by the National Ethics Committee . The various aspects of the work were conducted in collaboration with the local Health District Authorities. The free and informed consent of patients was requested through individual discussions and group meetings, between the sentinel team and the patients. For those willing to cooperate, a written informed consent was obtained prior to their enrolment in the study. Free malaria treatment with artemether-lumefantrine (Coartem®) was given to all patients referred to the team in case of Plasmodium infected subjects as well as the densities of asexual and sexual parasites at each altitudinal level. The prevalence of overall Plasmodium infections showed a significant reduction along the altitudinal transect . Also, comparison of Plasmodium densities in the three different localities with Kruskal Wallis test showed a decrease .A total of 48 surveys were carried out on the outpatients attending the health centres of the three above described sites, and a total of 677, 2428, and 736 patients were examined in the lowland plain, the highland plateau and uphill respectively. Table χ2 = 55.74, df =2, p =10−8). Likewise comparing the mean density of trophozoites by H test of Kruskal Wallis showed significant reduction with increasing altitude . Concerning the sexual stages, the human malaria infectious reservoir (HMIR) was 2.07% (25.4 gametocytes/μl) in lowland, 0.45% (44.0 gametocytes/μl) in the highland plateau and 1.08% (26.0 gametocytes/μl) at the hill site, showing no significant difference of the HMIR between the three sites .In distinguishing the parasite stages, it was observed that both the prevalence and the density of asexual parasites decreased with increasing altitude from; 36.19% , to 25.04%  and 19.16% , in Santchou, Dschang and Djuttitsa, respectively. Statistical analysis of the asexual parasite prevalence by Chi-square test showed a highly significant reduction of the prevalence with increasing altitude . In Dschang, the prevalence showed no variation in age groups . Likewise in Djuttitsa, this prevalence was almost similar for all the age groups .Figure s higher , then th−3). However, in Dschang as well as in Djuttitsa, the mean parasite densities were fairly similar for all the age groups .The mean of asexual parasite densities also peaked in children less than five years of age in Santchou Figure B and decχ2 = 6.08, p =0.20). However, in Dschang and Djuttitsa, the prevalence of gametocyte carriers was constant only within the age range of 5 to 60 years. On the plateau site, gametocyte index was higher in the extreme age groups (0 – 5 and >60) as compared to 5 – 60 age group . Conversely uphill the gametocyte index was lower in these extreme age groups .The proportion of visits with patent gametocytes in the Mbô plain of Santchou did not show any significant difference with age Figure A, despitPlasmodium gametocytes among population ages in Santchou did not differ significantly. On the plateau and uphill, the pattern of sexual parasite density among age groups was similar to the prevalence. In fact, there was no difference in the gametocyte densities among the age groups of 5 to 60 years. However, on the plateau site, gametocyte density was higher in the extreme age groups (0 – 5 and >60) as compared to 5 – 60 age groups ; conversely uphill the gametocyte density was lower in these extreme age groups .Figure χ2 = 25.22, p =0.51), as well as between seasons . Comparison of parasitaemia with Kruskal Wallis test came to the same conclusion: H =29.40, df =11, p =0.12 between months, and H =16.13, df = 3, p =0.09 between seasons. On the contrary, the prevalence of malaria infections in Djuttitsa was seasonal and occurred from May to August (mainly during the SDS); while the parasite density peaked in September .Figure Plasmodium-infected subjects (PfPR) among the symptomatic populations along the altitudinal transect. This malaria-attributable fraction of fever episodes (MAFE) represented 77.07% in the lowland at Santchou, 75.81% in Dschang and 78.13% in Djuttitsa. There was a significant reduction in the percentage of symptomatic cases among outpatients consulting in health centres with the altitudinal climate variation . However comparing the rate of Plasmodium-infected patients among the febrile individuals (MAFE) showed no significant difference along the altitudinal transect .When considering only the symptomatic patients attending the health centres  used in this study to compare malaria incidence along the transect is a commonly reported index in malaria epidemiology. In fact, it is historically consistent with the classical categories of malaria endemicity  and, therefore, useful for standardization; moreover, it has good biological, epidemiological and statistical properties [Plasmodium prevalence and density among the presumptive malaria cases recorded, with increasing altitude. Considering the classification of malaria zones into endemic categories according to Metsslaar and Van Thiel, Santchou presenting a prevalence of 36.19% is in a mesoendemic malaria zone, Dschang with 25,04% is an intermediate situation, while Djuttitsa with a prevalence of 19.16% is hypoendemic. This PfPR continuous decrement with altitude was already observed in Tanzania [Plasmodium infection in Santchou was also a consequence of the high number of anopheline’s breeding sites. This number was higher in lowland because the hillside gradients provide efficient drainage, resulting in numerous and variable potential breeding sites rather in lowland areas [The operties . In thisTanzania -48. It mTanzania . In factnd areas . MoreoveSurprisingly, malaria prevalence at Djuttitsa was higher than expected from the entomological inoculation rate previously determined . This diSince the prevalence and density of malaria infection decrease with increasing altitude while the gametocyte rate and density do not vary, the scarcity of malaria disease in highlands is likely due to the absence of vectors and not the parasites. In fact, the highland climate is known to reduce the survivorship and reproduction fitness of anopheline mosquitoes ,40. In cAs in most endemic localities of sub-Saharan Africa, several other manifestations were related to malaria, in addition to fever, as the most prominent sign of malaria. However, these signs and symptoms are not specific to malaria as mentioned elsewhere ,38, becaAsexual parasite prevalence and density decrease with age after an initial peak in the 0–5 years age group in the lowland plain of Santchou; meanwhile in Dschang and Djuttitsa where transmission is low, prevalence and density were similar in all age groups. Similar high prevalence in younger age groups have been reported in studies elsewhere in Africa, as in Kenya , in TanzThe gametocyte prevalence and density did not show any variation with age in the Mbô plain of Santchou; however some discrepancy was noted on the plateau and uphill, with extreme age groups (<5 and > 60 years), which represent the non-active age range of the population. It was observed that this age range in Dschang has the tendency of a higher gametocyte index. Conversely, non-active inhabitants of the highland have the trend to lack gametocytes. The conclusion drawn here is that inhabitants of the Bamileké Plateau who are sedentary are exposed to a higher risk of malaria infection, whereas people staying uphill must migrate down to be in contact with malaria parasite. This is in conformity with previous findings indicating that the EIR at Djuttitsa is null . In factPlasmodium infection was perennial throughout the year, but seasonal uphill, with malaria episodes occurring from May to August (i.e. mostly during the SDS), probably due to the abundance of puddles and the alleviation of climate constraints (temperature and relative humidity) on mosquito breeding and survival during the preceding SRS. During the malaria period, which coincides with the peak biting rate of anopheline mosquitoes [In the lowland plain of Santchou and on the Bamiléké plateau, squitoes , there wsquitoes . This issquitoes . StrategMalaria-attributable fraction  is very complex to determine in endemic areas, due to: asymptomatic carriage of malaria parasites and non-malarial fevers, which introduce some bias. In fact, estimates of MAFE should define parasite densities threshold to classify a fever episode as related or not to malaria. In this respect, logistic regression method with model fever risk as continuous function of parasite density was explored, and gave more precise estimates than simple analyses of parasite prevalence . In addiPlasmodium density can cause severe disease at all ages [Herein, a unique and simple method was tested along an altitudinal transect. The malaria-attributable fraction was considered to be the PfPR among the fever episodes, where fever episodes refer to subjects with temperature ≥ 38°C (or reported fever during the preceding days), plus some associated malaria-related symptoms or signs determined in Table all ages . FurtherPlasmodium infection rates . The comment raise is that the higher number of infective bites found in lowland would result in only low infection in human populations due to natural immunity, while in highlands, though there is no immunity, there is also low infective bites due to the climate which is unfavourable to the Plasmodium extrinsic cycle, the anopheline development and their reproductive fitness. It is likely therefore that in the lowland, there are much more pathogens that can cause fever apart from Plasmodium, which is not the case in highland areas. These other pathogens were responsible for 12.11% (50.37 – 38.26) of fever episodes, 14.29% (39.78 – 25.49) and 2.72% (22.96 – 20.24), in Santchou, Dschang and Djuttitsa respectively (Tables After putting the same sampling effort in all of the three sites, a high number of fever episodes were detected in the lowland plain as compared to the highland, but with the same 2.11% 50.7 – 38.26Since the MAFE as well as the HMIR do not vary with altitude, they may be reliable parameters to detect incipient malaria outbreaks in highlands. In fact, it is obvious that an incipient malaria outbreak in the highland would raise their levels in highlands as compared to lowlands. Therefore, in addition to guiding the diagnostic strategies, they can also be used to predict and forecast malaria epidemics.Evidence for the operational feasibility of using the HMIR and the MAFE as indicators in a MEWS was shown. It is recommended that in designing the MEWS in Cameroon, the National Malaria Control Programme should include the HMIR and the MAFE as indicators to be taken into account by a network of health workers in sentinel stations. They should put special emphasis on: altitude of the localities and age groups, and intensify control before and during the SDS, in order to better prevent and forecast an eventual malaria epidemic. Implementation, monitoring and evaluation of this strategy in the sentinel health centres will help confirming the usefulness and cost-effectiveness of these indicators in case a malaria outbreak occur.


Despite significant advancements over the years, there remains an urgent need for low cost diagnostic approaches that allow for rapid, reliable and sensitive detection of malaria parasites in clinical samples. Our previous work has shown that magnetic resonance relaxometry (MRR) is a potentially highly sensitive tool for malaria diagnosis. A key challenge for making MRR based malaria diagnostics suitable for clinical testing is the fact that MRR baseline fluctuation exists between individuals, making it difficult to detect low level parasitemia. To overcome this problem, it is important to establish the MRR baseline of each individual while having the ability to reliably determine any changes that are caused by the infection of malaria parasite. Here we show that an approach that combines the use of microfluidic cell enrichment with a saponin lysis before MRR detection can overcome these challenges and provide the basis for a highly sensitive and reliable diagnostic approach of malaria parasites. Importantly, as little as 0.0005% of ring stage parasites can be detected reliably, making this ideally suited for the detection of malaria parasites in peripheral blood obtained from patients. The approaches used here are envisaged to provide a new malaria diagnosis solution in the near future. Plasmodium are the cause of malaria. During its complex life cycle the parasite infects red blood cells (erythrocytes) and replicates within. The parasite directly ingests hemoglobin from the infected erythrocyte as a source of essential metabolites and energyPlasmodium, only 5 species are known to infect humans; P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum is the most common, and is responsible for more than 90% of malaria deathMalaria is a mosquito-borne infectious disease that affects 3.4 billion people - about half of the world’s populationThe major challenges in current clinical malaria diagnostics are in obtaining a sensitive, robust, fast, and inexpensive measurement from patients’ blood samples. One of the leading causes of the high mortality rate in malaria is the delay in medical diagnosis and treatment7−1 of blood115Giemsa-stained microscopic examination of thick blood smear has remained the gold standard for laboratory diagnosis of malaria for more than a century510R2 of proton present in the bio-samples14151618R2 measurement only takes approximately 1 minute. Recently, we have demonstrated that the presence of paramagnetic malaria pigment or hemozoin crystallites in malaria infected red blood cells (iRBCs) serves as the natural biomarker for malaria detectioniRBCs (hRBCs), which in turn induces a substantial change in the transverse relaxation rate. Nonetheless, MRR detection has a major drawback in that it measures the absolute value of a blood sample instead of detecting a relative change in the R2 value.The miniaturization of magnetic resonance relaxometry (MRR) systems has been attracting considerable interest in recent years due to the promising applications in disease biomarker detection, point-of-care diagnosis, and cancer screening by measuring the transverse relaxation rate, ioniRBCs  is signiiRBCs was made possible with the development of lab on a chip (LOC) or micro total analysis systems (μTAS). Gascoyne et al. exploited the difference in the dielectric properties of iRBCs and hRBCs to perform dielectrophoretic (DEP) separation in a microfluidic chamberiRBCs at a localized position, the application of relatively high electric field causes Joule heating and could dramatically increase the temperature of the suspending bufferet al.et al.iRBCs through magnetic columns (MACS) and magnetic deposition microscopy (MDM), respectively. However, both techniques have a serious limitation in that they were unable to separate ring stage iRBCs, which is important for the human P. falciparum malaria diagnosis. Previously, we have presented a deformability based separation method known as margination, where the stiffer malaria iRBCs are segregated towards the sidewalls of microchanneliRBCs separation.On the other hand, the separation of malaria iRBCs and MRR system for the rapid, label-free detection of malaria parasites. We mitigate the R2 baseline restriction by separating the iRBCs from the hRBCs through margination and compare the relative change in the relaxation rates of the separated blood samples. The diagnostic method that we propose here has similar detection sensitivity to PCR based methods and, at the same time, is one order of magnitude more sensitive than the standard Giemsa microscopic method. We improved the throughput of the microfluidic margination device by employing a multiplex margination design with 7 parallel margination channels, and demonstrated that the device could effectively separate the hRBCs from iRBCs of all infection stages . Due to the margination effect, the stiffer iRBCs are segregated towards the side outlet of the microfluidic channel, while the blood sample collected at the middle outlet is depleted of iRBCs. Thus, the marginated blood sample collected at the middle outlet has a lower R2 value compared to the inlet. The ability to perform relative R2 measurements alleviated the need for establishing a universal R2 baseline for MRR malaria diagnosis.In this work, we develop a malaria diagnosis protocol that combines the use of microfluidics for the separation of iRBC would be of significant benefit for the subsequent diagnostic of malaria, particularly in cases of very low parasitemia levels. In our previous workiRBCs with a single margination channel. To improve this further, we evaluated the margination efficiency of the multiplex margination design  are marginated effectively toward the sidewalls of the microchannel.The efficient enrichment of n design  with 7 piRBCs at various margination flow rates. With the help of a fluorescence activated cell sorting (FACS) flow cytometer, we could characterize the margination efficiency by estimating the parasitemia level of the blood collected from the middle and side outlets accurately. PEF, for single channel flow rate of 1–10 μL min−1 (Re ≈ 0.1–1) for ~1% ring iRBCs. The parasitemia enrichment factor, PEF is defined as the parasitemia ratio between the side and middle outlet. Thus, a higher PEF value would indicate a higher margination sorting efficiency. As the PEF value remains relatively constant throughout the flow rate range, we can infer that increasing the margination flow rate from 1 to 10 μL min−1 does not adversely affect the margination efficiency substantially. Therefore, the flow rate could be increased to improve the throughput of the margination system without compromising the margination performance. With multiplex margination, throughput of the microfluidic separation step is improved to a total flow rate of up to 70 μL min−1, which reduces the margination process time to approximately 15 minutes.Since clinical samples contain only circulating ring stage parasites, we further evaluated the margination efficiency to separate ~1% ring iRBCs at the side outlets are higher than the middle outlets for all three infection stages. In other words, the middle outlet is depleted of iRBCs, while the side outlet is enriched with iRBCs. The comparison of parasitemia enrichment factor for ring, trophozoite and schizont is summarized in iRBCs increases exponentially as the infection stage progresses from ring to trophozoite and schizont . Interestingly, for hRBCs, the side outlet R2 value is approximately 15% higher than the inlet, while the middle R2 is approximately 6% lower than the inlet R2. A similar trend is observed for iRBCs. When the hRBCs are spiked with 0.0005% iRBCs, the R2 value of the side outlet increased further by approximately 7% since the iRBCs are marginated towards the sidewalls.In order to overcome the R2 value is always significantly higher than the inlet, while the middle R2 is lower than the inlet R2 for hRBCs, may indicate that the margination device also separates stiffer and older RBCs from more flexible, younger RBCs. As the average life span of RBCs in vivo is about 120 dayshRBCs circulating in the human blood stream is made up of a distribution of RBCs of various age and deformability. The stiffness of RBC increases substantially during the aging process, and is up to 134% less deformable on day 363+) derivative of Hb, in the senescent RBC4+) state under constant oxidative stress32R2 relaxation rates of the marginated stiff aged RBCs collected at the side outlet.The observed phenomenon, that the side outlet ΔR2 of the separated blood samples even for hRBCs . The box plots show the lower quartile, median, and upper quartile of their respective sample distributions. For all three cases, we observed that, while the side R2 values remains significantly higher than the middle, the middle R2 values has become statistically non-distinguishable from the inlet R2 (two-tailed paired student t-test (n ≈ 10) P-values of 0.739, 0.905, and 0.636, respectively). Due to the variation in R2 values between measurements, it is necessary to take 5–10 measurements per sample to prevent false positives and false negatives, thus the MRR measurement time per sample is approximately 5–10 minutes. Lysing the iRBCs with saponin releases intact P. falciparum parasites from the membrane of erythrocytes and it would therefore be expected that these intact parasites would remain to provide a detectable increase in the R2 values during MRR measurements. If this is indeed correct, we expect that as iRBCs are marginated towards the sidewalls, the middle outlet is depleted of iRBCs. Hence, the middle R2 values would be expected to be significantly lower compared to the inlet while it would be statistically non-distinguishable if the sample contains only hRBCs.Due to the margination of aged RBCs, there exists a substantial relative change in the relaxation rates, or hRBCs . To overR2 values of blood samples collected at inlet, middle and side for an iRBCs sample at parasitemia levels of 5%, 0.5%, and 0.0005%. All these samples contained a mixed infection of 37% ring and 63% schizont. The box plots show the lower quartile, median, and upper quartile of their respective sample distributions. For all three parasitemia levels, the R2 values of the middle is significantly lower than the inlet , while the side R2 is significantly higher than the middle. This confirms that the depletion of iRBC in the middle outlet can be directly measured using the saponin lysis approach even at parasitemia levels as low as 0.0005%, providing us with an important tool for malaria parasite detection. In contrast as shown in iRBCs are marginated towards the side outlet, and although the margination process does not remove all existing iRBCs from the middle channel , trophozoite (T), and schizont (S) stage parasites. Three samples at extremely low parasitemia level of 0.0005% parasitemia with a composition estimated by Giemsa microscopic observation and FACS of (a) ; (b) ; (c)  were tested. R2 values at the inlet, middle, and side for the three samples of iRBCs. In all three cases, the R2 relaxation rates of the sample collected at the middle outlet are significantly lower  compared to the inlet. As seen in P-value is 0.0444) differences between the inlet and middle outlet sample can be detected. This confirms that the malaria diagnostic technique that combines the use of microfluidic separation and MRR system is able to detect early malaria infection at the parasitemia level of 0.0005%, without prior determination of patient-specific healthy baseline.For any diagnostic approach it is crucial that it can detect solely circulating young ring stages as later stage iRBCs and MRR system for a rapid, label-free detection of malaria parasites. The iRBCs are separated from the hRBCs through margination - a naturally occurring hemodynamic phenomenon where more deformable hRBCs experience a lateral migration towards the axial centre of the microchannel, displacing stiffer iRBCs to the sidewalls of the margination channel. Crucially we have demonstrated that this approach is capable of reliably detecting ring stage parasites at parasitemia as low as 0.0005%. In the near future, a Lab-on-a-Chip solution with MRR functionality integrated is envisaged for malaria diagnosis in the clinical setting. Both margination-based microfluidic separation system and MRR detection system have a room for further optimization, which will bring about more sensitive diagnostics, and, potentially, earlier detection of malaria cases. While the cost of the miniaturized MRR system maybe still expensive  for developing nations, the cost per assay (<50 cents) is affordable. Pushing the detection sensitivity of malaria parasite to the lowest level possible would have significant implication in the clinical management of malaria in endemic areas, especially in the light of the possibility of latent, extremely low level parasitemia in non-symptomatic populations.We have successfully developed a malaria diagnosis technique that combines the use of microfluidics for the separation of iRBCs towards the sidewalls  are segregated towards the vessel wall to initiate leukocyte rolling, adhesion and extravasation at the blood vessel wall’s endothelia cells of the injury inflammation siteinset of 25. The mhRBCs are highly deformable, the iRBCs stiffen upon invasion of the large non-deformable intracellular parasite, and changes its morphological, mechanical, and electrical properties43iRBC’s phospholipid bilayer membrane, thus increases the rigidity of the iRBCshRBC, respectively  at 200 mJ cm−2 for 16 s. Subsequently, post exposure bake is carried out at 65 °C for 1 minutes and 95 °C for 2 minutes. The substrate is then developed for 2 minutes. PDMS with a 10:1 mixture ratio for base and curing agent  was casted onto the SU8 master and cured at 80 °C for 8 hours. Inlet and outlets ports were punched with a 0.75 mm PDMS puncher  on the cured PDMS device. Finally, the PDMS device was cleaned in an ultrasonic cleaner with ethanol and DI-water for 15 minutes, respectively. The entire process was repeated to fabricate the PDMS chip that contains the cover layer.The microfluidic margination device was fabricated in polydimethylsiloxane (PDMS) using standard photolithography technique. An approximately 10 μm thick SU8-3005  negative photoresist was spin-coated onto a 4” silicon wafers at 500 rpm for 10 s with an acceleration of 100 rpm sAfter fabricating the PDMS chips for both the margination layer and cover layer, the next step was to assemble the multilayer microfluidic separation device. First, the microfluidics chip that contain the margination layer was bonded onto a pre-cleaned microscope glass slide  with a plasma bonder  for 90 s. Second, the PDMS chip that contains the cover layer was then visually aligned and bonded onto the top surface of the PDMS chip containing the margination layer. The assembled microfluidic device was then kept in an oven at 80 °C for 8 hours to improve the PDMS bonding strength. Lastly, a tubing with a G23 needle end was inserted into the inlet and sealed with epoxy glue  to enhance the sealing.P. falciparum strain 3D7  was cultured at 2.5% haematocrit using human erythrocytes in malaria culture media , 25 mL 1 M HEPES , 2 g NaHCO3 , 5 g AlbuMAX® , 0.05 g of hypoxanthine  in 1 mL of 1 M NaOH, and 25 mg gentamicin  in 1 L milli-Q water)2, 3% O2, and 92% N2. Ring stage parasites were synchronized using 2.5% D-sorbitol, while schizont stage parasites were purified using differential Percoll gradient centrifugation−1 .Healthy non-malarial immune adult volunteer blood donors were recruited, and brought to the National University Hospital of Singapore Blood Bank for phlebotomy services. The protocol was approved by the Institutional Review Board of National University of Singapore (NUS-IRB 10-285). Informed consent was taken from all volunteers, and the methods were carried out in accordance with the approved protocol and guidelines. Whole venous blood was collected in Citrate-Phosphate-Dextrose-Adenine-1 . The white blood cells (leukocyte) were removed via centrifugation and the removal of the buffy coat layer. Malaria parasites, iRBCs reaches ~10%, the infected blood sample is washed with 1 × PBS three times to remove the culture media and resuspended in 1 × PBS at 10% hematocrit. Subsequently, we prepare an infected blood sample at 5% parasitemia by spiking the corresponding amount of iRBCs into hRBCs from a healthy donor. The 5% parasitemia blood sample is then serially diluted with hRBCs to obtain blood samples with lower parasitemia of 0.5%, 0.05%, 0.005%, and 0.0005%. The hematocrit of the blood samples are then adjusted to 45–50% for the microfluidic margination process. The R2 relaxation rates of the blood samples can be measured by two methods: (a) “spin-down”, and (b) “lysis” methods. In the “spin-down” method, the samples are loaded into heparinized microcapillary tubes  via capillary forces. The microcapillary tubes are then sealed with a vinyl-plastic compound  and centrifuged  at 13,000 g for one minutes. A total spun down cell volume of 4 μL is required for each MRR measurement. On the other hand, the procedure for the “lysis” method is similar to the “spindown” method except that the blood cells are lysed before being transferred into the microcapillary tubes for MRR measurements. First, the total volume of blood cells suspended in 1 × PBS required for obtaining a spun down cell volume of 4 μL is determined. Subsequently, the blood sample is centrifuged at 3,000 g for one minute. A portion of the supernatant of the sample is removed such that the total sample volume is 15 μL. The blood sample is then lysed with 5 μL of 1% v/v saponin lysis buffer .When the parasitemia of the −1 to 70 μL min−1 with flexible Tygon© tubing . The flow of the iRBCs and hRBCs are observed under an inverted epi-fluorescence microscope  equipped with a high speed CCD camera . The marginated blood samples are then collected from the middle and side outlets in 1.5 mL microcentrifuge tubes. We evaluated the performance of the margination separation device by running FACS analysis with a BD™ LSR II flow cytometer  on the collected blood samples. The data from the cytometer is then analyzed with FACS Diva  and FlowJo .Prior to the experiment, the microfluidic margination device is primed with 1 × PBS supplemented with 0.5% v/v BSA. The blood sample is filled in a 1 mL glass syringe  and pumped into the margination device with a syringe pump  at total flow rates ranging from 7 μL min1H transverse relaxation rate, R2 of the blood samples from the middle and side outlets are measured with a bench-top nuclear magnetic resonance spectrometer . The microcapillary tubes containing the blood samples are slotted into an impedance matched 1 mm rf-probe tuned at a resonance frequency of 21.57 MHz. The rf-probe is subsequently placed inside a ~0.5 T permanent magnet  and connected to the bench-top spectrometer for proton 1H MRR measurements (R2 relaxation rates are measured using standard Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with 5000 echoes, 200 μs inter-echo time, transmitter power output of 6.25 mW, and 6 μs 90°-pulse length. A total of 24 scans are acquired for signal averaging and the system is maintained at 26 °C via an external temperature controller. The R2 measurement of each blood sample is repeated 5–10 times with independent microcapillary tubes.The proton urements . The R2 How to cite this article: Kong, T.F. et al. Enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection. Sci. Rep. 5, 11425; doi: 10.1038/srep11425 (2015).


P. falciparum malaria infection. Individuals with blood group “A” have been found to be highly susceptible to falciparum malaria whereas blood group “O” is said to confer protection against complicated cases. We analyzed samples from 293 young children less than six years old with malaria in the Korle-Bu Teaching Hospital in Accra, Ghana. It was observed that group O was present in about 16.1% of complicated cases weighed against 40.9% of uncomplicated controls. Individuals with complicated malaria were about twice likely to be of blood groups A and B compared to group O . Blood group O participants with complicated diseases had low parasitaemia compared to the other blood groups (P < 0.0001). This may give blood group O individuals a survival advantage over the other groups in complicated malaria as suggested. Participants with complicated falciparum malaria were generally anaemic and younger than those with uncomplicated disease.The clinical outcome of falciparum malaria in endemic areas is influenced by erythrocyte polymorphisms including the ABO blood groups. Studies have reported association of ABO blood group to resistance, susceptibility, and severity of Plasmodium falciparum malaria is a known cause of morbidity and mortality especially in children of Sub-Saharan Africa [n Africa . The clin Africa , 3 inclun Africa  and othen Africa . All eryn Africa . Individ P. falciparum malaria infection [ P. falciparum malaria cases among blood group “O” individuals have been observed [Numerous associations have been reported between the ABO blood group system and some disease conditions such as skin cancer , schistonfection –15. Indinfection –13. Low observed , 14, 15. P. falciparum malaria infection among the “A,” “B,” “AB,” and “O” blood groups have been attributed to rosetting of parasitized erythrocytes and cytoadherence [ P. falciparum laboratory [These differences in susceptibility and severity ofdherence –19. Rosedherence . Studiesboratory , 22 and boratory . Rosetteboratory , 24, 25. P. falciparum malaria [Some studies have also reported the absence of significant association between ABO blood groups and malaria , 26–30,  malaria . This stThe study was conducted from January to April 2010, at the outpatient department of the Korle-Bu Teaching Hospital in Accra, Ghana. The study was reviewed and cleared by the University of Cape Coast Ethics Review Board. The study was explained to parents/guardians of prospective participants and informed consent was sought from them. A participant was eligible for inclusion into the study if his/her age at the next birthday was 5 years or less and had been diagnosed to have falciparum malaria.Five milliliters (5 mL) of blood sample was collected from each patient into EDTA tubes by trained and licensed medical laboratory technologists. Sterile techniques and disposable, single use materials were used at all times.The hemoglobin level of each participant was determined using a Hematology Analyzer (Abbott Cell-Dyn CD-1800). Giemsa-stained thick and thin blood films were prepared for each sample collected, from which parasite density and species identification were, respectively, determined. Species confirmation of the malaria parasite was done with first response PfHRP-II malaria rapid-diagnostic (RDT) kit. Cerebral malaria  and hyperparasitaemia (with parasite density ≥6%) were considered the primary criteria for defining complicated malaria.ABO blood groups were typed by the agglutination method using commercial antisera .t-test or One-Way ANOVA. In addition, odds ratios and 95% confidence interval were determined. In all the analyses, values were considered to be significant when P ≤ 0.05.Data obtained were analyzed with Minitab Statistical Software version 15. Results were presented as mean ± standard deviation or percentages where appropriate. Mean values were compared using either Student's A total of 293 young children were recruited for the study. Of these, 112 (41.3%) had complicated falciparum malaria, whereas 181 (58.7%) had uncomplicated falciparum cases. The characteristics of the participants are summarized in P < 0.05; Respondents in the complicated group were younger and anaemic and experienced higher parasitaemia compared with their counterparts in the uncomplicated group  of the 112 participants with complicated disease had blood group O, whereas as much as 74 (40.9%) of the 181 participants with uncomplicated disease had blood group O. This observation gives credence to results of previous studies , 35, 36.The low parasitaemia observed in this study, together with reduced rosetting and cytoadherence observed by others , 27–29, Ghanaian children with blood group O may have some protection against complicated falciparum malaria and may possess a survival advantage over their counterparts with other blood groups. However, this protection may be lost at high parasitaemia due to enhanced RBC destruction. Younger children are also more prone to developing complicated falciparum malaria than older ones.


Plasmodium Falciparum And Plasmodium Vivax Attract The Most Focus Because Of Their High Prevalence And Mortality. Knowledge About The Prevalence Of The Cryptic Pathogens Plasmodium Ovale And Plasmodium Malariae Is Limited. Thanks To Recombinant Tools, Their Seroprevalence Was Measured For The First Time, As Well As The Prevalence Of Mixed Infections In A Malaria-Asymptomatic Population In Benin, A Malaria-Endemic Country.Malaria Is A Life-Threatening Pathology In Africa. P. Falciparum, P. Malariae, P. Ovale MSP1, And P. Falciparum AMA1 Were Engineered And Validated On A Biobank With Malaria-Infected Patients (N = 144) Using A Species-Speific ELISA Test (Recelisa). Results Were Compared To An ELISA Using A Native P. Falciparum Antigen (NatELISA).A Panel Of 1,235 Blood Donations Collected Over Ten Months In Benin Was Used For Validation Of The Recombinant Tools. Recombinant P. Falciparum, 94.4%  To rPfMSP1 And rPfAMA1, 56.8%  To rPoMSP1, 67.5% (834/1235) To rPmMSP1 And 45.3% Of The Malaria Seropositive Population Had Antibodies Recognizing The Three Species.Among Microscopically Negative African Blood Donors, 85%  Present Antibodies Directed To Native P. Ovale And P. Malariae Was Found In Asymptomatic Blood Donors. The Proportion Of Mixed Infections Involving Three Species Was Also Unexpected. These Data Suggest That Determining Seroprevalence For These Cryptic Species Is An Appropriate Tool To Estimate Their Incidence, At The Eve Of Upcoming Anti-P. Falciparum Vaccination Campaigns.A High Rate Of Antibodies Against  Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi), which are transmitted by the bite of infected Anopheles mosquitoes. Despite huge efforts to control the disease, resurgence has been observed in many countries due to climate instability, global warming, civil disturbances, drug resistance, and increasing travel between endemic and non-endemic areas[Malaria is one of the most life-threatening diseases in the world. In 2010, 219 million cases were reported globally[P. falciparum and P. vivax are in progress[P. malariae and P. ovale, where prevalence has been insufficiently investigated. To further complicate matters, low levels of parasitaemia and mixed infections can affect diagnosis of P. malariae and P. ovale. Birkenmeyer et al. recently sequenced genes encoding major erythrocyte stage markers of P. malariae and P. ovale, demonstrating the feasibility of Merozoite Surface Protein 1 (MSP1) recombinant production[P. malariae and P. ovale[Plasmodium species and plays a role in red blood cell (RBC) invasions[Plasmodium falciparum AMA1 is a blood-stage antigen that aids in orienting the merozoite during invasion of RBC. Anti-AMA1 antibodies tend to be present in individuals who have acquired natural immunity[Identifying the most affected countries’ direct resources and validating control measures is essential to reducing malaria’s incidence target: 75% by 2015)[progress; not so oduction and use % by 2015nvasions. PlasmodPlasmodium lactate dehydrogenase (pLDH) has been documented as a valuable tool for assessing prevalence in a blood donor population[P. malariae and P. ovale identification. Furthermore, various factors influence the direct detection of parasites, among them parasite clearance due to acquired immunity, drug treatment, season variability and sporadic transmission in low-transmission areas. For this reason, seroprevalence measurement has been explored as an accurate tool for estimating transmission intensity and the potential effects of any measures used to control (and ultimately eliminate) malaria[Plasmodium species appear within days or weeks of erythrocyte invasion, and can persist for months or years reflecting exposure to the parasites[Estimation of malaria prevalence is historically done by optical microscopy but a sensitivity of 50 parasites/μL is insufficient. Furtherpulation. However malaria. Indeed,arasites.P. falciparum antigen and recombinant P. vivax proteins was already developed, exhibiting high specificity and analytical sensitivity  in the detection of Plasmodium antibodies[Immunofluorescence detection of malaria antibodies was until recently the gold standard, but is tibodies. HoweverP. falciparum, P. ovale and P. malariae was reported to establish an ELISA test for the detection of Plasmodium species-specific antibodies. Immunoassay performances were first assessed in a population of Plasmodium-infected French travellers. For the first time, the distribution of the P. malariae and P. ovale in endemic malaria areas in Benin (Western Africa) was evaluated in a blood donor population.In this work, the identification and production of recombinant proteins from P. falciparum (n = 106), P. malariae (n = 12), or P. ovale (n = 26). All results were confirmed by PCR. Every patient was treated and the samples anonymized. This population was used to determine the recombinant ELISA assay’s clinical sensitivity and positive predictive value.Malaria patients returning from endemic countries sera (n = 144), diagnosed and referred at the Strasbourg University Hospital were used for the study. All patients reported fever and were microscopically diagnosed positive for Blood donor samples were collected at the Etablissement Français du Sang d’Alsace (EFS Alsace). Donors were classified as unexposed-to-malaria (192 samples) if their questionnaire responses indicated never having travelled to an endemic area. These samples were used to calculate the test’s specificity and negative predictive value.P. falciparum antigen and recombinant proteins from P. falciparum, P. ovale and P. malariae were used to assess seroprevalence in the Beninese samples. Five donors gave their blood twice in two different seasons (LRS and SRS); they were kept due to the low number of repeat donors, since they were positive for malaria antibodies.Plasma and total blood samples from blood donors without apparent malaria symptoms  were collected over ten months (May 2009 to February 2010) in six Beninese departmental blood centres[P. falciparum MSP1 (Accession Number XP_001352170.1), 356 AA of P. ovale MSP1 (ACZ51239.1), and 350 AA of P. malariae MSP1 (ACZ51237.1) corresponding to the C-terminus region of the protein (42 kD) and a nucleotide construct encoding 448 AA of P. falciparum AMA1 (XP_001348015.1) were commercially synthesized with an Escherichia coli codon bias . Consensus sequences derived from the alignment of several strains for each species and each protein were used for the design of synthetic genes. All genes were inserted in an expression vector containing a Maltose binding protein fusion partner pMAL-c2X® , produced in E. coli expression hosts and purified on amylose resin and DEAEsepharose ®.Nucleotide constructs encoding 373 AA (amino acids) of P. falciparum based on the amplification of the STEVOR gene, a subtelomeric multiple copy gene, was performed according to Filisetti et al.[Plasmodium species were identified using a genus and species-specific nested PCR, in addition to a PCR tailored to a specific P. ovale subspecies [Detection of ti et al.. The othllikeri).P. falciparum antigen produced from in vitro culture (NatELISA)[P. ovale, P. malariae, P. falciparum and rP. falciparum AMA1 (RecELISA) were immobilized on 96-well plates overnight at 4°C in coating buffer and blocked for one hour with PBST  containing 1% BSA  . After washing with PBST, 200 μl of diluent buffer (PBST with BSA 0.1%) were dispensed into each well and 10 μl of serum were incubated for one hour at 37°C. On the same plate, 10 μL of positive controls specific for each species and negative controls in triplicate were added. After three washes with PBST, 100 μl of horseradish peroxidase labelled monoclonal rabbit anti human IgG  were incubated for 30 min at 37°C. After three washes with PBST, 100 μl of TMB plus substrate solution (Tetramethylbenzidine)  were incubated for 15 min at 37°C and the reaction was stopped with 50 μl of 0.5 M sulphuric acid. The absorbance was read within 30 min at 450 nm against 620 nm. Test validation required the positive OD to be >0.500 and the negative OD <0.200. The cut-off value was set at three-fold the negative control wells’ average OD. The antibody (Ab) index of each sample was defined as ratio of its OD value and the cut-off value. The sample was considered positive if the Ab index was >0.7, and negative if the Ab index was ≤0.7.Antibody screening was performed using an in-house ELISA test derived from a commercial assay (DiaMed). Native atELISA) or recomAll recombinant assays were performed on an EVOLIS Microplate System (Bio-Rad) at the Department of Microbiology of the Hôpitaux Universitaires de Strasbourg.P. falciparum native antigen ELISA (NatELISA) and recombinant ELISA (RecELISA) performances were analysed using Chi-squared and t-tests, as detailed in the Results section, below. Significant differences in antibody prevalence depending on the season, observed during follow-up, were assessed using the Chi-squared test. A p-value of less than 0.05 was considered significant. Correlation between antibody titre and ELISA index for the recombinant antigens was calculated using the Pearson correlation test. .Statistical differences between P. falciparum native antigen  was superior to that of the NatELISA  of the population with the NatELISA. Only two mixed infections  were detected by microscopy, confirmed by PCR, and positive with all recombinant proteins  to P. falciparum recombinant rPfMSP1 and rPfAMA1, 56.8%  to the P. ovale recombinant rPoMSP1, and 67.5%  to the P. malariae recombinant rPmMSP1. By summing up all the patients bearing antibodies directed to recombinant proteins, the prevalence was 98.8%   using recombinant proteins were better than those of an ELISA using a native antigen from P. falciparum. In particular, the use of recombinant proteins from the P. ovale and P. malariae species largely enhanced detection rates for those infections. The efficacy of these new tools was optimized using synthetic genes integrating consensus sequences available in GenBank[P. ovale. The surface antigen MSP1 is highly polymorphic due to wide strain variability. This pitfall in the use of recombinant proteins leading to the presentation of limited epitopes to humoral response was previously highlighted[P. ovale strains, now accessible on GenBank, might resolve this problem.The antigenicity of these recombinant proteins was first evaluated in patients infected by  GenBank,5, coverhlighted. A combiP. ovale and 3.1% P. malariae, associated or not to P. falciparum: 1.2% P. falciparum/P. malariae, 2.4% P. falciparum/P. ovale and 0.1% P. falciparum/P. malariae/P. ovale[P. falciparum and 0.15% P. falciparum/P. malariae. Similarly, in Burkina Faso, microscopy detected 72.5% P. falciparum, 13.2% P. malariae and 1.8% P. ovale in a population of 830 children between three and 15 years old, of which 18.8% were febrile[P. ovale and P. malariae. This diagnostic tool has a detection threshold of five to 50 parasites/μL[P. malariae and P. ovale detection sensitivity. Snounou et al., using PCR, were able to detect in a microscopically negative Bissau-Guinean population, 23.3% P. malariae and 6.9% P. ovale[The RecELISA’s strong performance allowed the study of Beninese blood donors. Recent studies using microscopic examination in southern Benin have demonstrated the presence of mixed infection at the following rates: 2% /P. ovale. In this febrile. Microscsites/μL showed t P. ovale infectio P. ovale. Therefo P. ovale. Thus, s P. ovale.P. falciparum antigens, neglecting P. ovale and P. malariae. The use of recombinant proteins of P. malariae and P. ovale allowed revealing high prevalence of antibodies directed to these cryptic parasites. In Benin, an increased production of antibodies directed to P. ovale and P. malariae was observed until the LDS. This may be due to the delayed appearance of these species after the first infection, as has already been observed[P. falciparum (MSP1 and AMA1), seroprevalence increased after the LRS and maintained high levels until the LDS, reflecting the long-lasting presence of IgG directed to rPfAMA1 and rPfMSP1 antigens. Similarly, the seroprevalence against P. falciparum recombinant proteins did not vary between wet and dry seasons in the Gambia[P. falciparum antigen was observed during the SRS. This may be due to a deficit of MSP1 and AMA1 in the native antigen, prepared from a synchronized culture in which an 80% schizont proportion shifted the recognition of the malarial antibodies towards the detection of older stages of P. falciparum. Another hypothesis is that the variation in antibody response is due to differences in antigen concentration and relative proportions of MSP1 in native and recombinant antigenic preparations.However, seroprevalence was exclusively tested using observed. Using re Gambia. InteresPlasmodium in addition to P. falciparum, and that 76% of the investigated healthy population were exposed to more than one species. These results indicate that the prevalence of non-falciparum species is much higher than previously estimated. The levels of antibodies directed to these two cryptic species is low, possibly due to their parasitaemia levels. On the contrary, high levels of antibodies against P. falciparum are observed, corresponding to high parasitaemia levels.This work shows that more than half of the studied population was in contact with other species of P. falciparum infection remains the primary concern, the detection of P. ovale and P. malariae is still important. Although both cause mild infection, P. malariae can cause chronic nephritic syndrome, leading to adverse reactions during treatment and a high rate of mortality[P. falciparum is not fully effective against P. malariae[P. malariae is more prevalent when transmission and infection rates of P. falciparum are lower, probably because of negative interactions with P. falciparum; similar interactions with P. ovale might exist[P. falciparum medication caused the prevalence of P. malariae to rise for four years in Burkina Faso. The authors predicted that in the absence of P. falciparum , P. malariae parasitaemia and gametocyte carriage could increase and maintain severe malaria infection[While ortality. There iortality. Immune  malariae. Furtherht exist. Positivnfection.P. falciparum, other species must be monitored more closely as their presence will remain a serious health concern for exposed populations in endemic countries[Given the advent of vaccine campaigns against ountries.Plasmodium species. Antibody prevalence for all species of Plasmodium will permit detection of residual infections and variations in malaria transmission. Of course, more complex combinations of recombinant Plasmodium proteins should be used in future studies to avoid the disadvantages of antigenic polymorphism and variable individual responsiveness, especially for P. ovale. These kinds of recombinant tools could also be used to enhance blood transmission safety in non-endemic regions[In conclusion, with the elimination of malaria back on the agendas of world agencies, it is necessary to monitor the effects of anti-malarial measures. In this work a standardized, sensitive and specific tool was developed that facilitates measuring and monitoring the transmission intensity of three  regions.The authors declare that they have no competing interests.CDL, PA, AS, and EC designed the study, analysed the data and prepared the report; AS and EC coordinated the study; CDL and PA led the study in each site; LM produced the recombinant proteins; EH, SP, DF, AA, AWP, and JB processed the samples and collected the data in France; NWC, CDA and LA collected the data in Africa. All authors contributed to correcting the report. All authors read and approved the final manuscript.


Methods. A cross-sectional study was conducted at Medani, Sudan. Sociodemographic characteristics were gathered from each parturient woman (163) and malaria was investigated by blood film and placental histology. Protein C, protein S, antithrombin-III, tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibitor-1 levels (PAI-1) were measured using ELISA. Results. One (0.6%), three (1.8), and 19 (11.7%) of the placentae showed active, chronic, and past infection on a histopathological examination, respectively, while 140 (85.9%) of them showed no signs of malaria infection. While the mean [SD] of the protein C, antithrombin-III, and TFPI was significantly lower, there was no significant difference in protein S and PAI-1 levels in women with placental malaria infection (n = 23) compared to those without placental malaria infection (140). In linear regression, placental malaria infection was associated with antithrombin-III. There was no association between placental malaria infections and protein C, protein S, TFPI, and PAI-1 levels. There was no association between hemoglobin, birth weight, and the investigated coagulation and fibrinolysis indicators. Conclusion. This study showed significantly lower levels of protein C, antithrombin-III, and TFPI in women with placental malaria infections.This study aimed to investigate coagulation, fibrinolysis indicators, and malaria during pregnancy.  Malaria during pregnancy is a substantial public health problem in endemic tropical countries, especially sub-Saharan Africa. It has been estimated that approximately 125 million pregnant women live in malaria-endemic areas in sub-Saharan Africa and 32 million of these pregnant women are at risk of malaria –9. Each  P. falciparum malaria especially the severe form can lead to an impairment of the coagulation system which correlates with proinflammatory cytokines  and birth weight , the gestational age  was significantly lower in women with placental malaria infection (n = 23) compared to women who did not have placental malaria infection. Likewise in linear regression, placental malaria infection was not associated with hemoglobin  and birth weight  (While there was no significant difference in the hemoglobin  of the protein C, antithrombin-III, and TFPI was significantly lower, there was no significant difference in protein S and PAI-1 levels in women with placental malaria infection . There was no association between placental malaria infections and protein C, protein S, TFPI, and PAI-1 levels. There was no association between hemoglobin, birth weight and protein C, protein S, antithrombin-III, TFPI, and PAI-1 levels (In linear regression, placental malaria infection was associated with antithrombin-III (−4.091 mg, 1 levels .In linear regression, there was a significant association between protein S, PAI-1, and protein C. Likewise, there was significant association between PAI-1-1, TFPI, and antithrombin-III .The main findings of the current study were as follows: placental malaria affects women regardless of their age and parity. While the levels of protein S and PAI-1 were not different, the levels of protein C, antithrombin-III, and TFPI were significantly lower in women with placental malaria infection than in those without placental malaria infection.The lack of association between age, parity, and placental malaria infection in the current study goes with the previous observations where pregnant women in the different regions of Sudan are susceptible to the peripheral, placental, and submicroscopic malaria regardless of their age or parity , 15, 16.It has recently been shown that both active coagulation and suppressed fibrinolysis are evident at the placental level in association with malaria infection in primigravidae women . TherefoPoovassery and Moore observed that in malaria infected placentae macrophages accumulated and expressed tissue factor, and these macrophages tissue factor expressions in placentae could explain low birth weight in malaria placental infection . In placInterestingly, the “inflammation coagulation cycle” has been observed in preeclampsia which is another health problem during pregnancy and associated with significant maternal morbidity and poor birth outcomes . We haveThere was no association between hemoglobin, birth weight, and the investigated coagulation and fibrinolysis indicators in the current study. Perhaps submicroscopic placental malaria, which was not investigated in the current study rather than histology detected malaria, is the main determinant of low birth weight in this setting . Both miAlthough there was no association between the blood group O and placental malaria infection in the current study, antithrombin-III, TFPI, and PAI-1 levels were significantly lower in women with blood group O. Previous reports showed an increased risk of placental malaria in women with blood type O  that was