Malaria and HIV are two major causes of morbidity and mortality among pregnant women in sub-Saharan Africa. Foetal and neonatal outcomes of this co-infection have been extensively studied. However, little is known about maternal morbidity due to clinical malaria in pregnancy, especially malaria-related fever, in the era of generalized access to antiretroviral therapy and anti-malarial preventive strategies.Plasmodium falciparum. Suspected malaria-related fever was defined as an axillary temperature >37.5°C and the concomitant administration of an anti-malarial treatment in the absence of parasitological investigation.A cohort study was conducted in order to estimate the incidence rate and to determine the factors associated with malaria-related fever, as well as the maternal morbidity attributable to malaria in a high-transmission setting of South Benin among HIV-infected pregnant women. Four-hundred and thirty-two women who participated in a randomized trial testing strategies to prevent malaria in pregnancy were included and followed until delivery, with at least three scheduled visits during pregnancy. Confirmed malaria-related fever was defined as axillary temperature >37.5°C and a concomitant, positive, thick blood smear or rapid diagnostic test for 3 variation = 0.82; CI: 0.71-0.96), antiretroviral treatment started before inclusion  and history of symptomatic malaria in early pregnancy  were associated with the incidence of confirmed or suspected malaria-related fever. More than a half of participants with parasitaemia were symptomatic, with fever being the most common symptom. The crude fraction of febrile episodes attributable to malaria was estimated at 91%.Incidence rate for confirmed malaria-related fever was of 127.9 per 1,000 person-year (PY) : 77.4-211.2). In multivariate analysis, CD4 lymphocytes (Relative Risk (RR) for a 50 cells/mmThis work highlights that malaria is responsible for a substantial morbidity in HIV-infected pregnant women, with cellular immunodepression as a major determinant, and establishes the possible advantage offered by the early initiation of antiretroviral treatment.NCT00970879.PACOME Study has been registered under the number  Plasmodium falciparum has been associated with an increased risk of malaria-related fever among HIV-infected adults living in Uganda, especially in those with severe immunodepression [The consequences of malaria in pregnancy (MiP) for the foetus and the newborn have been extensively studied. MiP increases the risk of low birth weight (LBW), which is the most important determinant of mortality during the first year of life in African infants . In mothpression ,6. The cpression -9, interpression  and resipression  among HIpression ,8,12. Fipression . One exppression . Howeverpression ,16 or anpression  associatP. falciparum infection in HIV-infected pregnant women, especially fever, are scarce. Incidence data on clinical malaria in this population are rare, ancient, and do not reflect the actual standard of care towards antiretroviral therapy access and anti-malarial preventive measures [Despite this body of knowledge, data concerning the maternal morbidity attributable to measures . Moreovemeasures ,12.p with a single dose of SP at each antenatal visit for all pregnant women, irrespectively of their HIV status [P. falciparum infection in this population.To avoid the adverse consequences of MiP, the World Health Organization (WHO) now recommends IPTV status . HoweverV status -22, the V status . This foP. falciparum with two peaks during the rainy seasons, from April to June, and from September to November. The mean entomological inoculation rate was found to be 5.3 infective bites per person and per year in a close rural area [The study took place in the Littoral department of Benin, in Cotonou  and its suburbs, and in Porto Novo , both located on the Atlantic coast of the Guinean Gulf, at the extreme south of the country. Regarding malaria, the south of Benin is characterized by a high perennial transmission of ral area  and the ral area . Among pral area .HIV care centers and antenatal clinics (ANC) from five hospitals were involved in the study: Centre National Hospitalier Universitaire Hubert Koutoukou Maga, Hôpital d’Instruction des Armées de Cotonou, Hôpital de Zone de Suru-Léré, Hôpital Mère Enfant de la Lagune, and Clinique de Porto Novo. The main occupation of the women in this area is informal trading.Midwives were recruited and trained as research assistants to fill questionnaires and to collect blood samples from the study participants. Local midwives, nurses and physicians in charge of the HIV care units and ANC worked in close collaboration with the research assistants.3) or advanced HIV disease (WHO staging ≥2), all women received cotrimoxazole for the prevention of opportunistic infections, associated or not with mefloquine depending on the treatment group. Combined antiretroviral therapy (ART) was prescribed for the prevention of mother-to-child HIV transmission or for maternal health, according to national guidelines. The primary endpoint of the trial was the prevalence of placental malaria. All enrolled women had to be HIV1-infected and between 16 and 28 weeks of gestation, to live in the study area, to have planned to deliver at the hospital, to have signed an informed consent and to have no contra-indication to the study drugs.The study population consisted of 432 women enrolled in the PACOME clinical trial, between December 2009 and December 2011 . The PACAccording to the PACOME trial follow-up, women were evaluated at three antenatal care visits scheduled at least one month apart between 16 and 28, 24 and 32, 28 and 36 weeks, and at delivery. When sick, women were also encouraged to attend the clinic for free-of-charge diagnosis and treatment. Socio-economic data and obstetrical history were recorded at enrolment as well as HIV disease information. At each visit and at delivery, obstetrical, malaria and HIV-related information were also collected. In addition to anti-malarial preventive treatments, intravenous quinine was administered in case of severe malaria. A three-day treatment of artemether-lumefantrine (Coartem® Novartis) was administered in case of non-severe malaria, except for women with asymptomatic or low parasitaemia  who received a dose of mefloquine IPTp the same day. Opportunistic infections of HIV were treated according to national guidelines.P. falciparum and the Plasmodium lactate dehydrogenase (pan pLDH). HIV viral load was planned to be assessed at inclusion and delivery by real-time quantitative PCR using 2000 Abbott thermocyclers  provided by the National AIDS Control Programme, with a 40 copies per milliliter detection threshold. Absolute CD4 count and percentage were planned to be assessed at each visit by Cyflow  or Facscount  cytometer.Laboratory procedures have been extensively described elsewhere . BrieflyFever was defined as an axillary temperature >37.5°C. A case of confirmed malaria-related fever was defined as an axillary temperature >37.5°C and a concomitant, positive thick blood smear, or positive HRP2 band of the RDT in the absence of symptoms or findings indicating other infections. A case of suspected malaria-related fever was defined as an axillary temperature >37.5°C and the concomitant administration of anti-malarial therapy in the absence of parasitological investigation and of symptoms or findings indicating other infections.Data capture, cleaning and validation were performed using Microsoft Access 2003. Data analysis was performed using SAS version 9.2 for Windows  and a two-sided 5% alpha risk.a priori: age, baseline CD4 cell count (as a quantitative variable assessed at inclusion), type of malaria chemoprophylaxis, study centre, parity, season at delivery. A stepwise procedure was used to select the strongest parsimonious models while searching for possible confusion or interaction between covariates. In the final multivariate analysis, a P-value <0.05 was considered significant. A sensitivity analysis was performed for the final model considering confirmed malaria-related fever as the dependent variable, first excluding suspected malaria fever cases from the analysis, then considering them in the control group.First, women’s medical and socio-economic characteristics, the initial clinical presentation and, when known, the diagnoses associated with febrile episodes were described. The diagnoses associated with febrile episodes were compared according to the severity of cellular immunodepression. Incidence rates for confirmed malaria-related fever, suspected malaria-related fever and total malaria-related fever (confirmed or suspected) were estimated and reported symptoms associated with confirmed malaria were described. A sample size of 385 women was necessary to obtain an estimation of the incidence rate of malaria-related fever with 10% relative precision and a confidence level of 95% . After uP. falciparum from positive thick blood smear obtained on the occasion of scheduled visits was estimated. For this purpose, the following equation was used: AFe = (Re – R0)/Re, with AFe the attributable fraction of risk in exposed, Re the risk (of fever) in exposed and R0 the risk (of fever) in unexposed. Exposure was defined as having a positive blood smear for P. falciparum at the considered time point. The risk was defined as the number of either exposed or unexposed women having fever at the considered time point divided by the corresponding exposed or unexposed population (febrile and afebrile) at the same time point.Finally, the crude fraction of febrile episodes attributable to malaria among participants who were infected by The PACOME trial has been ethically approved both in France and in Benin, respectively by the ‘Comité consultatif de déontologie et d’éthique de l’IRD’, and by the ‘Comité national provisoire d’éthique pour la recherche en santé’, of the Beninese Faculté des Sciences de la Santé. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.Between December 2009 and December 2011, 432 participants were randomized in the PACOME study among 533 screened HIV-1 infected pregnant women. Of these, 140 received cotrimoxazole, irrespective of their treatment group, due to severe immunodepression or advanced HIV disease .3, 52% had already initiated ART and HIV RNA viral load was undetectable in 82 (31.9%) women among 257 for whom this assay had been performed. Due to maintenance problems and financial shortages in National AIDS Control Programme laboratory, this test could not be performed for all women. For more details regarding ART regimen received by participating women see Additional file General characteristics of the participants at inclusion are summarized in Table e in 82 3.9% womenP. falciparum among participants.The 432 women who participated in the PACOME study totalized 130.9 person-years (PY) follow-up with a mean duration of 15.8 weeks (SD 5.6). Nine women (2.1%) were lost to follow-up just after their first visit and were not considered in incidence calculation. Recruitment and follow-up in the PACOME study have been detailed elsewhere. Briefly, 393 women (91%) were followed until delivery, with a median of 4 visits (interquartile range: 3 to 5). No death was imputed to the infection with P. falciparum detection . Three febrile episodes were considered as suspected malaria by the caregivers and were treated by anti-malarial treatment. In total, 20 (23%) febrile episodes were referred as confirmed or suspected malaria-related fever. Six (30%) of these episodes, all related to confirmed malaria-related fever, occurred during the period between screening and inclusion of the participants, i.e. before the first intake of the trial’s preventive treatment. Clinical presentations of the other febrile episodes are summarized in Table P. falciparum) and one woman experienced six febrile episodes .Overall, 68 participants (16%) experienced a total of 86 febrile episodes, 17 of whom were associated with P. falciparum were reported during the PACOME trial. The symptoms reported by the women during these infections are summarized in Table P. falciparum and having CD4 < 350/mm3 compared to 26% (5/19) of the women infected with P. falciparum and having CD4 ≥ 350/mm3 .Forty-five non-recurrent episodes of confirmed infection with 3 (31%) than in women with a CD4 cell count ≥350/mm3 . The distribution of febrile episodes and their suspected origin according to CD4 level are summarized in Table On the other hand, the proportion of febrile episodes referred as confirmed or suspected malaria-related fever was higher in women with a CD4 cell count <350/mmP. falciparum parasitaemia were available for 1,130 out of 1,626 scheduled visits. Thirty-one episodes of parasitaemia (non-recurrent positive thick smear) and 42 febrile episodes were recorded during scheduled visits. The distribution of the 1,130 visits according to the presence of parasitaemia or fever is summarized in Table P. falciparum was 91%. In the 32 parasitaemic patients, the association between febrile condition and parasite density was investigated, when data were available. The median parasite density in febrile, parasitaemic patients was 1,000 trophozoïtes/mm3 , n = 10) versus 478 , n = 21) in afebrile, parasitaemic patients .Data regarding Unadjusted incidence rates for febrile episodes, confirmed malaria-related fever and suspected malaria-related fever are displayed in Table 3) or as a quantitative variable (p = 0.03) and having experienced symptomatic malaria (declarative data) at the beginning of the pregnancy (p = 0.001) were associated with confirmed or suspected malaria-related fever.Concerning factors associated with incidence, in univariate analysis, baseline CD4 cell count at inclusion, considered as a dichotomic . Two other cross-sectional studies on malaria-related morbidity during pregnancy were carried out in Eastern Africa in the 1990s. They reported an increased frequency of anti-malarial drugs [p with two doses of SP, in accordance with local guidelines. In both studies, pregnant women had no access to ART. In the present work, almost all women were receiving ART and they all received anti-malarial chemoprophylaxis within the frame of the PACOME trial . A noteworthy difference between the above-mentioned studies and this work is the earliness, the length and the quality of the follow-up. Of note, a similar analysis performed in a real life sample of HIV-infected pregnant women followed-up in the same centers, but not participating in the PACOME trial, provided comparable results for malaria-related fever incidence rates  which occurred before the first intake of the trial’s preventive treatment is a possible explanation. However, a further analysis excluding these cases found similar results. The PACOME trial, whose primary endpoint was the placental infection by eriority . All of Having experienced symptomatic malaria in early pregnancy appeared to be strongly associated with the incidence of malaria-related fever during the follow-up in multivariate analysis. The hypothesis was made that this variable reflects the role of unmeasured factors, for instance, an overall higher exposure to malaria transmission in symptomatic pregnant women. Delivering during the rainy season did not constitute a risk factor for malaria-related fever in this work. The majority of the participants were living in an urban area, less favourable to malaria transmission than a rural setting. Additionally, the time of delivery may not be an adequate indicator of the exposure to malaria associated with seasonal variations throughout the whole pregnancy.In the multivariate analysis of factors associated with confirmed and suspected malaria-related fever episodes, ART initiated before inclusion appeared to be a protective factor, even after adjusting for baseline CD4 count. This could be due to an important gain in CD4 during follow-up for women under ART for a short time before inclusion. The only CD4 cell count included in the analysis was performed at baseline because of frequent missing data for further assessments, so it is not possible to further elucidate this question. This finding represents an additional reason to start ART as early as possible during the pregnancy especially in women newly diagnosed of their HIV infection. Another study carried out previously in the same cohort confirmed the benefit of early ART during pregnancy in order to reach an undetectable HIV viral load at delivery and to minimize the risk of mother-to-child transmission of the virus . The conAn earlier study in Uganda had already shown that ART associated to daily cotrimoxazole was protective against malaria-related fever, compared to daily cotrimoxazole alone, in a population of HIV-infected adults and adjusted for the CD4 cell count . In the P. falciparum on a systematically performed blood smear were febrile at the same time. Interestingly, the frequency of fever among women infected with P. falciparum was almost doubled when the CD4 count was low, although not significantly. The efficacy of malaria preventive measures and a possible lack of power are possible explanations. It could be valuable to systematically record malaria-related maternal morbidity in all longitudinal studies aiming to assess new strategies for the prevention of MiP, especially in vulnerable populations, such as HIV-infected pregnant women. From this point of view, studies including both HIV-infected and uninfected pregnant women are highly suitable to allow direct comparison.MiP in HIV-negative pregnant women living in a high-transmission area is generally considered to be asymptomatic . HoweverIn Benin, malaria-related fever is responsible for a substantial morbidity among HIV-infected pregnant women in spite of generalized access to ART and adequate anti-malarial prevention. More than a quarter of febrile episodes were related to malaria in the study population. This justifies prompt and accurate diagnosis and treatment of malaria within the frame of antenatal care programmes. The severity of immunodepression, which has already been shown to be major risk factor for malaria-related morbidity in HIV-infected adults, is of special concern among HIV-infected pregnant women. As a consequence, women with low CD4 cell count should be prioritized regarding access to ART and anti-malarial chemoprophylaxis.P. falciparum.Indeed, the earliness of ART during the pregnancy could be an efficient measure for the prevention of malaria-related morbidity in this population. From a pathophysiological point of view, the hypothetic restoration of a parity-dependent phenomenon for the acquisition of anti-malarial protective immunity under ART should be further assessed. It could be a strong argument for immediate and definitive ART upon diagnosis of HIV infection for all pregnant women (option B + of the WHO) . It alsoThe authors declare they have no competing interest.AD and LDN carried out the acquisition, the statistical analysis and the interpretation of data and drafted the manuscript. MC participated in the design and the coordination of the study, the acquisition of data, supervised the writing and revised critically the manuscript. JA, KVA, AA, DGK, FA, LDG, and DMZ participated in the acquisition of data. PMG and DMZ participated in the design of the study and revised critically the manuscript. All authors read and approved the final manuscript.Antiretroviral regimen received by PACOME trial participants.Click here for fileFactors associated with confirmed malaria-related fever episodes incidence in the PACOME trial participants: sensitivity analysis including the suspected malaria-related fever episodes (n = 3) in the non malaria fever group.Click here for fileIncidence rates of febrile episodes, confirmed and presumed malaria-related fever episodes for 1000 PY in a real life sample of 174 HIV-infected pregnant-women followed-up in 4 out of 5 PACOME study sites.Click here for file


Microscopic diagnosis of Giemsa stained thick and thin blood films by skilled microscopists has remained the standard laboratory method for the diagnosis of malaria. However, detection and identification of malaria parasites require well trained laboratory personnel.The objective of the study was to evaluate the performance of laboratory technologists and technicians in detecting and identifying malaria parasites in Hawassa town, Southern Ethiopia.P.falciparum, 104/μl; slide 2:P.falciparum, 53404/μl; slide 3 and 4: mixed infection ; slide 5:P.vivax, 23503/μl; slide 6:P.vivax, 400/μl; and slides 7, 8, 9 and 10: negative slides]. Participants were asked to return the responses which were compared with expert microscopist. Agreement in detecting and identifying malaria parasites between participants and expert microscopists was estimated using the Kappa score.A cross-sectional study design was employed among a total of 80 laboratory professionals working in public and private health facilities. A standardized pre-validated slide panel and questionnaires were distributed to laboratory professionals working at eleven health facilities in Hawassa town, Southern Ethiopia. The panels included ten slides for diagnosis, [slide1:The mean age of the participants was 27 (SD = 4.1) years. More than half of the participants (56.9%) were female. Fourteen (19.4%) of the participants correctly reported all the ten distributed slides, whereas 58(80.6%) missed at least one slide. Overall, the sensitivity and specificity of participants in detection of malaria parasites were 82% and 96.5% respectively. The overall agreement between participants and reference readers on detection of malaria parasite was 88% (Kappa = 0.76) while on identification of malaria species was 74.3% (kappa = 0.63). Lower agreement on detection and identification of slides with low parasitic density and mixed infection were observed. Agreement was relatively lower for government health centers . None of the participants reported parasitic load per micro liter method.Agreement of the participants with expert microscopist in the detection of malaria parasites was better than agreement in the identification of different species of malaria. Poor agreement was reported in detection of parasites at a low density and mixed infections. Malaria is the leading public health concern in the Southern Nation Nationalities and People Region (SNNPR) among the regional states of Ethiopia. Epidemiological and ecological data of the region indicate that more than 75% of the area of this region is malarious , 2.Early diagnosis of malaria is a basis for the management of malaria, and key to reducing malaria related mortality and morbidity. Demonstration of the presence of malaria parasites under microscopy prior to treatment with anti-malarial drugs is fundamental to this goal since clinical diagnosis has a poor accuracy and leads to over-diagnosis and increases the risk of anti-malarial drug resistance , 3.Parasites of the Plasmodium species can be detected in stained blood smear by light microscopy. Microscopy is a simple and cost effective for detecting and identifying different stages of Plasmodium species in peripheral blood smear. However, this method is time-consuming and requires expertise especially in patients with low level parasitemia . In manyIn Ethiopia, laboratory diagnosis of malaria used to be performed by malaria microscopists working in malaria control offices. However, after decentralization and integration of independent programs, general laboratory technicians are in charge of malaria diagnosis services at health care facilities , 7. HoweThe current study aimed to evaluate the performance of laboratory professionals in detecting and identifying different stages of Plasmodium species using light microscopy.A cross sectional study design was conducted from November 2013 to January 2014 in Hawassa town. Hawassa is the capital of the Southern Nations Nationalities and Peoples Region (SNNPR), located at 275 km from Addis Ababa, the capital of Ethiopia. The altitude is 1697 m above sea level, the mean annual temperature is 20.9 Celsius, and the annual rainfall is 997.6 mm. According to 2007 census conducted by the Central Statistical Agency of Ethiopia, the total population of Hawassa Town was 258,808 with almost 1:1 male to female ratio. Although malaria occurs throughout the year, the peak malaria transmission occurs during the months of August to December .After validation, ten Giemsa stained thin and thick blood films with questionnaires were delivered to laboratory professionals working at eleven health facilities in Hawassa town. The panels included ten slides for diagnosis, [slide1:For slide examination by professionals, 10 minutes per slide was allocated . It was A structured questionnaire including information on the participating facilities and professionals was distributed. The questionnaire had sub-components like: the socio-demographic characteristics, educational background, service, in service training and routine practice of the professionals, and type and quality of microscope. The questionnaire was first pre-tested on 5% of the participating professionals and was improved as needed before the beginning of the study.Data quality was assured through use of standardized data collection materials, pretesting of the questionnaires, and intensive supervision during data collection by the principal investigator.Data were entered, cleaned and analyzed using SPSS for windows version 16. Level of performance in detection of malaria parasite was compared with independent variables. Association was taken as significant at P < 0.05. Mean, standard deviation, chi-square , sensitivity, specificity, proportion of errors, percent agreement, and kappa score were calculated to assess laboratory professionals’ performance in detecting and identifying different stages of Plasmodium species using light microscopy.The result of the microscopic diagnosis of malaria reported by the participants was evaluated using various parameters. Sensitivity was determined as the ability of participants to diagnose positive blood films; whereas, specificity was calculated for their ability to diagnose negative blood films.P. falciparum parasitemia.[P.vivax and of a mixed infection (reporting single infection in case of mixed parasites) [“Major error” was defined as incorrect diagnosis of malaria, i.e. reporting “negative” in the case of a Plasmodium-positive sample and reporting “positive” in Plasmodium negative samples; and falsely reporting non-falciparum species in the case of sitemia.. “Minor rasites) . The disrasites) .Based on WHO recommendations, participants were classified as: “In training”- when the agreement with the reference reader in detection of malaria parasite was less than 70%; “Competent”- when the agreement was greater than or equal to 70% but less than 80; “Reference”- when the agreement was greater than or equal to 80% but less 90%; and “Expert” -when the agreement was greater than or equal to 90% .Inter-rater agreement is the degree of agreement between two reference readers. It is calculated by computing the sum of true positives and true negatives and then divided by the total.The study was approved by departmental research and ethics review committee (DRERC) of the Addis Ababa University, Department of Medical Laboratory Science. Official letter was written to the participating facilities. Consent forms were prepared in English to be read and signed (if agreed) by the participating health professionals and for blood donors. Interpreters were assigned for non-English speakers among donors. Information obtained about the laboratory professionals from the questionnaires, data capturing formats and the slides were kept totally anonymous. Participants had the right not to participate or to withdraw from the study at any time.A total of 72 out of 80 laboratory professionals responded to the questionnaires with a response rate of 90%. Thirty-two (44.4%) of the participants were from two government hospitals, 912.5%) were from three private hospitals and 31(43.1%) were from six government health centers. The mean age of the participants was 27 (SD = 4.1) years and 41 (56.9%) were female  of the participants used both thick and thin blood films for detection and identification of malaria parasites. On the other hand, a considerable number of participants (23.6%) examined thick films only. More than half (56.9%) of the participants had experience in reporting parasite count using a grading system. There were no problems with the functionality of microscopes and accessibility of reagents in any of the laboratories.Of 72 participants, 14(19.4%) correctly interpreted all ten distributed slides, and 58(80.6%) missed at least one slide. Eighteen (25%) of the participants reported correct results of all positive samples and 67(93.3%) of participants correctly reported all four negative slides  on detection of malaria parasite. Participants also had 80.8% positive agreement for the six positive slides and 77.5% negative agreement for the four negative slides. Comparison across institutions showed lower sensitivity (81.0%) in those working in government health centers with an agreement of 87.0% (Kappa = 0.74) with reference readers Table .Table 3P.vivax and P. falciparum, worst agreement was found for slides with low parasite density . Agreement in detection (88%) was much higher than in identification (74.3%) of malaria species. Comparison across institutions showed that agreement in identification was higher in government hospitals (79%) (Kappa = 0.70). The lowest agreement on identification was found among participants working in government health centers with an agreement of 69% with reference readers (Kappa = 0.56) , 1.4% and 34.3% reported major errors; while, 9.7% and 4.2% reported minor errors respectively. A small proportion  of participants made major errors  on the respective negative slides 7, 8, 9 and 10  for slide 3 and 30(85.7%) for slide 4.Among mixed infection slides, 44.4% and 48.6% of participants reported correct results on slide 3 and 4 respectively. However, 55.6% and 51.4% reported minor errors for the respective slides. Most cases of mixed infection were reported as Based on the WHO grading system, 17(23.6%) of the participants were rated as ‘in training”, 23(31.9%) as “competent”, 14(19.4%) as “reference”, and 18(25%) as “expert” level. Among 56 participants who had two or more years of experience in microscopic diagnosis of malaria, 11(19.6%) were classified as ‘in training’, 18(32.1%) were competent and 13 (23.2%) were “reference” and 14(25%) reached agreement on an “expert” level. Among participants who had less than two years of experience, 6 (37.5%) were classified in the group of “in training”, 5(31.3%) were considered “competent”, 1(6.3%) achieved at “reference” and 4(25%) reached to “expert”. However, there was no statistically significant difference in level of agreement based on experience.Microscopy of Giemsa stained thick and thin blood films is the standard for the diagnosis of malaria. However, interpretation of results requires professional expertise especially when the parasite density is low. In the current study, agreement between reference readers and participants in the detection of malaria parasites was 88% and in the identification of different species of malaria was 74.3%. Fourteen (19.4%) of participants correctly reported all the ten distributed slides, and 58(80.6%) missed at least one slide.Overall, the sensitivity and specificity of laboratory professionals in detecting malaria parasites were 82% and 96.5% respectively. These findings of low sensitivity and relatively high specificity were in agreement with a study conducted in Zambia (88% and 97% respectively) . Low senHowever, our findings of sensitivity and specificity were higher than in other studies conducted in Tanzania, Ethiopia and Haiti, where sensitivity and specificity of detection of malaria parasites by laboratory professionals were  in Tanzania,  in Ethiopia and  in Haiti –21.Our finding of an overall agreement on detection of malaria parasites with reference readers was 0.67 which is defined as ‘substantial’ based on the Kappa index interpretation by Landis and Koch . The oveP. falciparum in the present study was higher than reported from the USA , Hong Kong , Canadia , UK  and Democratic Republic of Congo  [The frequency of errors in the diagnosis of ciparum) , 25–27.Our findings show that false positive results were reported by 6.9% of participants. This finding is higher than the 2.0% false positive result reported in the Canadian study . HoweverP. falciparum in contrast to the study conducted in the Peruvian Amazon where most cases of mixed infections were reported as negative or P. vivax[In the current study, minor errors made on each of two mixed slides were less frequent than reported in studies from the UK where the proportion of minor error was 71% . HoweverP. vivax. This maP. falciparum at a low level of parasitemia (23.2%). This finding is much lower than for slides of P. falciparum with low parasite density reported from the Peruvian Amazon [Participants who had two or more years of experience had poorer agreement for slides with n Amazon . This inn Amazon .The limitation of this study is that we only used proficiency testing slides using unknown panels to evaluate the skill of laboratory professionals under optimal conditions, rather than routine or day to day performance in the diagnosis of malaria. Furthermore, we did not evaluate the performance of the laboratory personnel in regards to the preparation of the smears and the staining of blood films for malaria diagnosis.Participants had low sensitivity and relatively high specificity in the detection of malaria parasites. Agreement of the participants with expert microscopist in the detection of malaria parasites was better than agreement in the identification of different species of malaria. Poor agreement was reported in the detection of parasites at a low density level and mixed infections. Participants from government health centers were found to have lower performances in identification of malaria parasites.


HIV-infected pregnant women are particularly more susceptible to the deleterious effects of malaria infection particularly anaemia. In order to prevent opportunistic infections and malaria, a policy of daily co-trimoxazole prophylaxis without the standard Suphadoxine-Pyrimethamine intermittent preventive treatment (SP-IPT) was introduced to all HIV infected pregnant women in the year 2011. However, there is limited information about the effectiveness of this policy.This was a cross sectional study conducted among HIV-infected pregnant women receiving co-trimoxazole prophylaxis in eight public health facilities in Kinondoni Municipality from February to April 2013. Blood was tested for malaria infection and anaemia (haemoglobin <11 g/dl). Data were collected on the adherence to co-trimoxazole prophylaxis and other risk factors for malaria infection and anaemia. Pearson chi-square test, Fischer’s exact test and multivariate logistic regression were used in the statistical analysis.This study enrolled 420 HIV infected pregnant women. The prevalence of malaria infection was 4.5%, while that of anaemia was 54%. The proportion of subjects with poor adherence to co-trimoxazole was 50.5%. As compared to HIV infected pregnant women with good adherence to co-trimoxazole prophylaxis, the poor adherents were more likely to have a malaria infection  or anaemia . Other risk factors associated with anaemia were advanced WHO clinical stages, current malaria infection and history of episodes of malaria illness during the index pregnancy.The prevalence of malaria was low; however, a significant proportion of subjects had anaemia. Good adherence to co-trimoxazole prophylaxis was associated with reduction of both malaria infection and anaemia among HIV infected pregnant women. Combined malaria and HIV infection during pregnancy increases the susceptibility of the pregnant women to the negative effects of malaria suggesting a synergistic interaction between HIV infection and malaria ,2. HIV-iWHO has recommended a daily dose of co-trimoxazole among HIV-infected pregnant women during the whole pregnancy period in order to prevent opportunistic infections and malaria . ConcurrThis study was carried out in a malaria endemic area characterized by malaria transmission throughout the year . In thisThis cross sectional study reports the prevalence of malaria, anaemia and the associated risk factors among HIV infected pregnant women receiving co-trimoxazole prophylaxis in one district in Tanzania.The design of the study was a cross sectional. It was conducted between February and April 2013 in eight public health facilities in Kinondoni municipality, Dar Es Salaam. Kinondoni has a hot and humid tropical climate with two rainfall seasons: an intense one observed from the month of March to May, and a mild one in November and December. The average temperature ranges from a minimum of 18.1°C to a maximum of 32.1°C. The average annual rainfall is 1,115 mm. A theoretical model based on data for rainfall and temperature characterizes Kinondoni as an area with stable malaria transmission (holoendemic), with transmission occurring during the entire year . MalariaThe targeted subjects were HIV infected pregnant women receiving co-trimoxazole prophylaxis for more than four weeks. Sample size for this study was calculated using the formula for cross-sectional study based on the study done in Uganda . In thatn = Minimum sample size, z = point on standard normal distribution curve corresponding to significance level of 5% , x = previous prevalence of malaria infection among HIV infected pregnant women receiving co-trimoxazole prophylaxis (19%), ϵ = margin of error on x (set at 4%). A total of 420 consented subjects on different trimesters and varied gravidities were enrolled into the study. Pregnant women with sickle cell disease, vaginal bleeding and severe medical conditions were excluded from the study.Kinondoni municipality has a total of 33 public health facilities; among them two are hospitals, one is a health centre and thirty are dispensaries. Cluster sampling was used for selection of health facilities to be included in the study. The health facilities were divided into three clusters, namely hospitals, health centre and dispensaries. The two hospitals and the health centre were included in the study. Five out of the thirty dispensaries were selected using simple random sampling without replacement technique. Each name of the 30 dispensaries was written on a small piece of paper, and then the paper was folded to ‘a ball like’ figure and put in a mug. The mug was thoroughly shaken and five ‘paper balls’ were randomly picked to select the five dispensaries. Two hospitals involved in the study were Mwananyamala and Sinza. A Health Centre involved was Magomeni; and the five dispensaries were Kambangwa, Tandale, Mburahati, Kimara and Mbezi.Due to the limited number of HIV infected pregnant women, all the subjects in the selected clusters who met the inclusion criteria were enrolled in the study.A structured interview schedule was used to obtain information on various characteristics of the subjects and supplemented by information from patients' files. These include age, weight, height, gravidity, marital status, education level, employment status, WHO clinical stage, use of insecticide treated nets (ITN), history of episodes of malaria illness, use of iron supplements, use of deworming drugs, anti-retroviral treatment category (prophylaxis or lifelong), duration of zidovudine use. The subjects were then tested for malaria and anaemia. The blood sample was collected for CD4 assay.Adherence to co-trimoxazole prophylaxis was measured by using an interview schedule adapted from Morisky medication adherence scale Table  13,14].,14.13,14HemoCue Hb 201 + ® machine manufactured by HemoCue AB Ängelholm, Sweden. The tip of the middle finger was cleaned by alcohol swab; then a blood sample was obtained by finger pricking procedure. The Haemocue 201 + ® cuvette was filled with blood, excess blood was cleaned from the cuvette and air bubbles were removed. The cuvette was placed in the device tray and the holder was pushed gently into the photometer. The results were recorded from the digital display.Haemoglobin was measured by SD BIOLINE Malaria Ag P.f/Pan® MRDT manufactured by Standard Diagnostics, Inc, Korea. The blood sample was collected by finger prick method after the tip of the middle finger was cleaned by alcohol swab. The test device was placed on a clean, flat surface. About 5 μl of whole blood were added into the ‘sample well’ of respective test devices using a micropipette supplied with the test device. Four drops of assay diluent were added into the ‘sample diluent well’. All the test results were recorded within 30 minutes.Malaria was tested by using BD FACS Count® Machine (BD Biosciences USA). About 4 ml of whole blood (4 ml) was collected in EDTA collection tubes by standard venipuncture procedure. The collected samples were kept at room temperature (18-25°C) and then transported in a special container. All samples were processed within 30 hours from the time of collection.The CD-4 Count was done at Mwananyamala Hospital by Pregnant women who were diagnosed with malaria or anaemia during the study were managed as per the Tanzania’s Standard Treatment Guidelines through the respective health facilities.Data were double-entered, cleaned and analysed using computer software called IBM SPSS Statistics 20 (IBM Corp.). Dependent variables were malaria infection and anaemia. While the independent variables were the levels of adherence to co–trimoxazole prophylaxis. Other independent variables were socio-demographic factors, WHO clinical stages, CD-4 count bands, gravidity, pregnancy age (trimesters), sleeping under insecticide treated bed nets (ITN), history of episodes of malaria illness, use of iron supplements, the use of de-worming agent and duration of AZT use .Classification of anaemia was based on the recommendation by WHO ; normal Pearson Chi-square Test and Fischer’s Exact Test were used in the univariate analysis between the dependent and independent variables where applicable. Independent variables which showed a statistical significant difference with the outcome variable by univariate analysis were subjected to multivariate logistic regression to determine the predictors of the outcome. The P-value of < 0.05 was considered significant to provide evidence of significant difference or association.Ethical approval for the study was given by the Ethical Committee of the Muhimbili University of Health and Allied sciences (MUHAS). Permission from Kinondoni Municipal Council to conduct the study in the health facilities was granted. Information about the study was delivered to the patients and a written consent was obtained before the study was conducted. Confidentiality was ensured to all individuals who participated in the study.From February to April 2013, a total of 420 HIV infected pregnant women in various trimesters were enrolled into the study. The mean ± SD age of the subjects was 28 ± 5.2 years. Table The study revealed a prevalence of malaria of 4.5% (19/420). Table The prevalence of any anaemia (Hb <11 g/dl) was found to be 54% (227/420); while that of mild-to-moderate anaemia and severe anaemia were 49.3% (207/420) and 5% (21/420) respectively. The overall mean ± SD haemoglobin concentration was 10.3 ± 1.5 g/dl.Table The prevalence of anaemia among subjects with malaria infection was 94.7% (18/19) as compared to 52.1% (209/401) among malaria negative subjects. Malaria infected subjects were 10.4 times more likely to have anaemia as compared to those who had a negative malaria test . Likewise, subjects who had at least one episode of malaria illness during the current pregnancy were 1.8 times more likely to have anaemia as compared with those without a history .The prevalence of anaemia increased as the HIV/AIDS advanced from lower to higher WHO clinical stages (i.e. from stage I to IV). Pregnant women who were on WHO clinical stage III or IV were 2.7 times more likely to have anaemia as compared to those on WHO clinical stage I .This study shows that the prevalence of malaria infection was 4.5% among HIV infected pregnant women receiving co-trimoxazole prophylaxis. A similar low prevalence was obtained in a previous study that was conducted in Malawi by Kapito-Tembo et al. . In thatThe prevalence of malaria infection increased as the level of adherence to co-trimoxazole decreased from good to poor. Pregnant women with poor adherence were almost seven times more likely to have a malaria infection as compared to those with good adherence. We could not find any previous study that investigated the association between the malaria infection and levels of adherence to co-trimoxazole prophylaxis among similar subjects. An explanation for the pattern seen in our study could be the fact that the good adherents had higher exposure to co-trimoxazole and therefore were more protected as compared with the poor adherents.Sleeping under insecticide treated nets (ITN) by the subjects could as well have contributed to the low prevalence of malaria in this study group as it was reported in previous studies -21. ITN In consistent with previous studies ,12,17,23CD-4 Count and WHO clinical staging are parameters which are used to monitor progression of HIV/AIDS ,24. ThisThe present study showed that the prevalence of anaemia among HIV infected pregnant women receiving co-trimoxazole prophylaxis was 54%. Previous studies that were conducted in the Dar Es Salaam city reported higher prevalence compared to the present study. Finkelstein et al.  reportedGood adherence to co-trimoxazole was associated with reduced prevalence of anaemia. A pattern was seen towards the increase in the prevalence of maternal anaemia as the levels of adherence to co-trimoxazole prophylaxis decreased from good to poor. Poor Adherents were almost 1.8 times more likely to have anaemia as compared with good adherents. This could be due to the fact that, well adherent subjects had more exposure to the co-trimoxazole drug and therefore they were more protected from malaria infection and its sequelae particularly anaemia as compared to the poor adherent one. Walker et al. , had preIn consistent with a previous study by Finkelstein et al. , advanceLow CD-4 count and advanced HIV/AIDS stages reflect the chronicity (prolonged existence) of the disease; therefore another cause of anaemia could also be the state of chronic illnesses ,31. The Similar to previous studies ,27,28, dPregnant women who had at least one episode of malaria illness during the current pregnancy were nearly two times more likely to have anaemia as compared with those without a history. In consistent with this study, Nkhoma et al.  reportedIn disagreement with previous knowledge -34; the Similar to a study by Finkelstein et al. , anaemiaOur study had some limitations. We attempted to control the confounders by the multivariate logistic regression; nonetheless, there were potential residual confounding effects from the unmeasured factors like the presence of other infections or pathological conditions. Secondly, because of the cross sectional design of this study, we had only one point of measurement of both the outcomes and exposure; this made it difficult to make a causal inference from the findings. Thirdly, our results may have potentially been affected by information bias because measurement of adherence to co-trimoxazole prophylaxis was done by a self-reported method and no drug levels in the blood were measured. Likewise, the use of iron supplements, de-worming drugs and sleeping under an ITN were self-reported. However, antenatal records were used to verify prescriptions of medications.This study has shown a low prevalence of malaria infection among HIV infected pregnant women using the daily co-trimoxazole prophylaxis; however, a significant proportion of subjects had anaemia. Good adherence to co-trimoxazole prophylaxis was associated with reduction of both malaria infection and anaemia. Routine counselling and provision of health education on the importance of good adherence to medications and the consequences of poor adherence are paramount in addressing the existing problems. Mitigation of advanced HIV/AIDS among pregnant women is also important in combating the problem of anaemia. Approaches like early diagnosis and timely initiation of anti-retroviral treatment could be of benefit. Due to the complexity and multiple etiologies of anaemia; other measures like de-worming, schistosomiasis control and nutritional supplementation should be taken on board.95% CI: 95% confidence interval; AIDS: Acquired immunodeficiency syndrome; ANC: Antenatal clinic; AOR: Adjusted odds ratio; ART: Anti-retroviral treatment; ARV: Anti-retroviral; DNA: Deoxyribonucleic acid; EDTA: Ethylenediaminetetra acetic acid; FACS: Fluorescent activated cell sorting; Hb: Haemoglobin; HIV: Human immunodeficiency virus; HRP-2: Histidine rich protein-2; IPT: Intermittent preventive treatment; MMAS: Morisky medication adherence scale; MRDT: Malaria rapid diagnostic test; PCR: Polymerase chain reaction; pLDH: Plasmodium lactate dehydrogenase; PMTCT: Prevention of mother to child transmission of HIV infection; SD: Standard deviation; SP-IPT: Intermittent preventive treatment by sulphadoxine plus pyrimethamine; SPSS: Statistical package for social sciences; WHO: World Health Organization.The authors declare that they have no any competing interests.VM participated in study design, data collection, data analysis, data interpretation, initiated the manuscript preparation, and participated in the revision process. OM engineered the study design, coordinated data collection, data interpretation and participated in the process of manuscript writing. BN participated in the study design and interpretation of data and manuscript writing. All authors read and approved the final manuscript.VM: Holder of B.Pharm (Hons): Recently completed a Master’s Program in Hospital and Clinical Pharmacy at Muhimbili University of Health and Allied Sciences.OM: Senior Lecturer at the Unit of Pharmacology and Therapeutics, School of Pharmacy, Muhimbili University of Health and Allied Sciences.BN: Lecturer at Department of Parasitology, School of Medicine, Muhimbili University of Health and Allied Sciences.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/15/24/prepub


Plasmodium falciparum affects development of protective immunity and susceptibility to subsequent natural challenges with similar parasite antigens. However, the nature of these effects has not been fully elucidated. The aim of this study was to determine the effect of prenatal exposure to P. falciparum on susceptibility to natural malaria infection, with a focus on median time from birth to first clinical malaria episode and frequency of clinical malaria episodes in the first 2 years of life.Prenatal exposure to P. falciparum in the placenta at time of delivery were defined as exposed, and infants born to mothers without P. falciparum parasites in placenta were defined as unexposed. Placental infection was established by histological techniques. Out of 206 infants recruited, 41 were in utero exposed to P. falciparum and 165 infants were unexposed. All infants were monitored for onset of clinical malaria episodes in the first 2 years of life. The outcome measure was time from birth to first clinical malaria episode, defined by fever (≥37 °C) and microscopically determined parasitaemia. Median time to first clinical malaria episode between exposed and unexposed infants was assessed using Kaplan–Meier survival analysis and comparison was done by log rank. Association of clinical malaria episodes with prenatal exposure to P. falciparum was assessed by multivariate binary logistic regression. Comparative analysis of mean number of clinical malaria episodes between exposed and unexposed infants was done using independent sample t test.A prospective birth cohort study was conducted in Rufiji district in Tanzania, between January 2013 and December 2015. Infants born to mothers with P. falciparum infection on clinical malaria episodes was statistically significant  when compared to other confounding factors. Median time from birth to first clinical malaria episode for exposed and unexposed infants was 32 weeks (95 % CI 30.88–33.12) and 37 weeks (95 % CI 35.25–38.75), respectively, and the difference was statistically significant (p = 0.003). The mean number of clinical malaria episodes in exposed and unexposed infants was 0.51 and 0.30 episodes/infant, respectively, and the difference was statistically significant (p = 0.038).The effect of prenatal exposure to P. falciparum shortens time from birth to first clinical malaria episode and increases frequency of clinical malaria episodes in the first 2 years of life.Prenatal exposure to  Plasmodium falciparum infections, despite strategies put in place for the control of malaria [P. falciparum [P. falciparum during pregnancy may affect newborn susceptibility to subsequent malaria infections, and that infants born to mothers with placental malaria are exposed in utero to P. falciparum antigens [P. falciparum antigen on development of infant protective immunity has been studied and in utero exposed infants have been reported to have limited protective immune response to subsequent natural challenges with the same parasite antigen [Sub-Saharan Africa still faces high rates of infant mortality due to  malaria . In Tanzlciparum . It has antigens –7. The e antigen –10. EpidP. falciparum infection indicate that there remains a need to characterize this phenomenon.A study conducted in Cameroon demonstrated that the prevalence of malaria parasitaemia in infants aged four to 6 months was higher in those born to pm+ mothers compared to the controls , while aP. falciparum antigens and other factors  that could influence susceptibility, with respect to frequency and time from birth to first clinical malaria episode in infants born to pm+ and pm− mothers.This study was designed to systematically and prospectively follow up a birth cohort of infants for a period of 2 years, to delineate the effects of prenatal exposure to Four public health facilities  located in Rufiji District, Coast Region in Tanzania were involved in this study. The area is low-lying, below 500 m above sea level and most of its surface area lies within the fertile flood plain of Rufiji River. Rufiji district typically experiences a long rainy season between February and May and a shorter, less intense one from October to December. Annual rainfall ranges between 800 and 1000 mm in the Rufiji River basin and malaria transmission occurs all year round.P. falciparum in the placenta at the time of delivery confirmed by histopathology was used to categorize the mothers and their infants. Mothers who had P. falciparum in their placenta at time of delivery confirmed by histopathology were defined as placental malaria positive (pm+) and their infants were defined as exposed to P. falciparum, while mothers who had no P. falciparum infection at time of delivery were defined as placental malaria negative (pm−) and their infants were defined as unexposed.This was a prospective birth cohort study which included 206 mother infant pairs with infants aged 0–24 months. The criteria for recruitment of mothers included HIV seronegativity  and confirmation to stay within the study area for a period of 2 years to facilitate follow up of infants to the age of 2 years. In order to maintain confidentiality, all study participants were assigned code numbers. The group assignment of the study participants was based on placental malaria status established by examination of placental tissues using histological techniques. Histological classification was not done in this study but the presence or absence of 3) was collected, by a trained and experienced nurse, from the maternal surface halfway between the placental edge and the insertion of the umbilical cord, within 4 hours of placental expulsion. The placental tissue was fixed in 10 % neutral-buffered formalin. Following fixation, placental tissue was trimmed and placed into histopathological cassettes, processed on an automated tissue processor overnight, embedded in paraffin wax, and then sectioned on a microtome to obtain 5-nm slices. The slices were placed in warm water bath at about 56 °C and transferred onto a microscope slide and left to dry and adhere to the slide. The placental sections were stained using haematoxylin and eosin and were then examined using light microscopic technique. Results were reported as placental malaria positive  after a period of 2 weeks or beyond from the previously treated clinical malaria episode. All Infants who were diagnosed of clinical malaria infection were treated within the routine system at the health facilities. In addition, infants who were diagnosed of non-malaria diseases were attended and provided with standard medical care during the entire study period.Recruited infants were followed-up both at community and facility levels by trained research assistants. Scheduled visits were conducted regularly at 3 months intervals for a period of 2 years from birth while unscheduled visits were conducted whenever recruited infants had signs/symptoms of illness. Clinical examination was conducted by a trained study physician and clinical signs/symptoms were recorded on designated follow up questionnaire forms. Blood smears were collected for determination of clinical malaria infection using light microcopy technique. Clinical malaria was defined as fever (≥37.5 °C) with microscopically determined parasitaemia. Following the initial malaria episode, a new clinical malaria infection was defined as presence of asexual Time to first clinical malaria episode was determined in all recruited infants in order to assess the vulnerability to malaria infection. Specific birth dates for the recruited infants and dates when they experienced clinical malaria episode for the first time were systematically recorded. Time elapsed from birth to diagnosis of clinical malaria episode was defined as the time to first clinical malaria episode. This was recorded for each recruited infant in weeks and the median time for each group was determined. Subsequent malaria episodes experienced by recruited infants were also recorded on the designed forms to determine the frequency of clinical malaria episodes during the first 24 months of life.P. falciparum with socio-demographic characteristics was determined using Chi square test. Comparison of mean number of clinical malaria episodes between exposed and unexposed infants was done using independent sample t test. The median times from birth to first clinical malaria episodes for infants born to mothers with placental malaria (exposed) and infants born to mothers without placental malaria (unexposed) were determined using Kaplan–Meier survival analysis and the comparison was carried out using Log rank test. Binary logistic regression analysis was used to assess the effect of prenatal exposure to P. falciparum on first clinical malaria episode. During the analysis, potential confounders were considered into the multivariate model and these included gravidity, birth weight, age of mother, and infant season of birth. Results were reported as odds ratio where applicable with 95 % confidence intervals. A ‘p’ value of <0.05 was considered to be significant.Double-entry system was employed in data entry process. The data were cleaned and analysed using the Statistical Package for Social Sciences (SPSS), IBM version 20.0. Frequencies and cross-tabulations were used to summarize baseline socio-demographic characteristics between mothers with and without placental malaria  and the association of prenatal exposure to P. falciparum) and 165 infants were born to pm− mothers . Table A total of 206 mother-infant pairs were included in the study. Of these, 41 infants were born to pm+ mothers , while in unexposed infants it was 37 weeks (95 % CI 35.25–38.75) and the difference in the median time was statistically significant  while other factors did not statistically influence the clinical malaria episodes in a multivariate analysis  and behavioral factors are important in malaria transmission and have a direct influence on exposure and susceptibility [Previous studies demonstrated that prenatal exposure to  infants . The cur infants . The fintibility , 23.Although the current study considered some of the factors which could confound the effect of prenatal exposure on susceptibility, it did not incorporate those factors that may have a direct influence on exposure to the parasite.Lack of influence of the other exposure-related factors in this study is supported by the fact that, in the study area there was an ongoing anti-malaria campaign which involved distribution of ITNs at subsidized cost and emphasis was made on use of IPTp-SP for prevention of malaria during pregnancy as part of National Malaria Control Programme. The impact of this campaign in the study area was reflected in the general malaria prevalence which was reported to be 4.8 % . HoweverP. falciparum infection [In the present study, the mean number of clinical malaria episodes in the first 2 years of life was significantly higher in infants exposed to placental malaria than unexposed. These findings are corroborated by earlier studies, which demonstrated that infants born to pm+ mothers were more susceptible to nfection –27.However, a study done in Ghana indicated that the incidence of malaria in infants born to primigravida without placental malaria was significantly lower than that of infants born to primigravida with placental malaria but, there was no significant difference in the incidence of malaria among infants born to multigravida with or without placental malaria . While tP. falciparum were born to primigravida compared to multigravida . The increased vulnerability of primigravida to placental malaria compared to other gravidities is corroborated by a study conducted in Angola [P. falciparum antigen) during early pregnancy were associated with reduced risks for infection and low birth weight [In this study, it was noted that a significantly higher proportion of primigravida were placental malaria-positive compared to multigravida ; hence, the majority of the infants exposed in utero to n Angola , which dn Angola , which rn Angola , 31. It Although a direct influence of gravidity on susceptibility was not demonstrated in this study, one study conducted in the northern part of Tanzania showed that gravidity interacted with prenatal exposure to malaria and played a role in affecting susceptibility of infants to malaria infections . This stP. falciparum significantly influences vulnerability to malaria infection. Infants exposed in utero to parasite antigens succumb early to clinical malaria episodes and experience more frequent clinical malaria episodes than unexposed infants during the first 2 years of life.Prenatal exposure to


Malaria and schistosomiasis often overlap in tropical and subtropical countries and impose tremendous disease burdens; however, the extent to which schistosomiasis modifies the risk of febrile malaria remains unclear.S. haematobium mono-infection, baseline P. falciparum mono-infection, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of 616 children and adults living in Kalifabougou, Mali. Individuals with S. haematobium were treated with praziquantel within 6 weeks of enrollment. Malaria episodes were detected by weekly physical examination and self-referral for 7 months. The primary outcome was time to first or only malaria episode defined as fever (≥37.5°C) and parasitemia . Secondary definitions of malaria using different parasite densities were also explored.We evaluated the effect of baseline S. haematobium transmission, and housing type, baseline P. falciparum mono-infection (n = 254) and co-infection (n = 39) were significantly associated with protection from febrile malaria by Cox regression . Baseline S. haematobium mono-infection (n = 23) did not associate with malaria protection in the adjusted analysis, but this may be due to lack of statistical power. Anemia significantly interacted with co-infection (P = 0.009), and the malaria-protective effect of co-infection was strongest in non-anemic individuals. Co-infection was an independent negative predictor of lower parasite density at the first febrile malaria episode.After adjusting for age, anemia status, sickle cell trait, distance from home to river, residence within a cluster of high S. haematobium and P. falciparum is significantly associated with reduced risk of febrile malaria in long-term asymptomatic carriers of P. falciparum. Future studies are needed to determine whether co-infection induces immunomodulatory mechanisms that protect against febrile malaria or whether genetic, behavioral, or environmental factors not accounted for here explain these findings.Co-infection with  Plasmodium, the agent of malaria, can be important for developing effective malaria vaccines. Past studies have tried to determine if infection with Schistosoma haematobium, which causes urinary schistosomiasis, affects the number of febrile attacks from malaria caused by Plasmodium falciparum in communities where the diseases overlap, but the findings have been inconsistent. Here, we examined 616 healthy people from a village in Mali for symptomless infections with S. haematobium and treated those with infections. We then followed them over a single malaria-transmission season of 7 months during which we diagnosed and treated all febrile malaria attacks. After the season, we examined archived blood collected at enrollment to look for occult P. falciparum infection. The study revealed that people who were infected with both parasites at the beginning of the season were better protected from the malaria attacks than those who were uninfected or infected with either parasite alone. Further studies are needed to confirm these findings and to determine the biological basis for this phenomenon.The parasitic diseases malaria and schistosomiasis are tremendous public health burdens, each affecting over 200 million people worldwide with substantial geographic overlap in sub-Saharan Africa. Understanding how schistosomiasis influences the human immune response to  Plasmodium and the trematode helminth Schistosoma, respectively, impose tremendous public health burdens in tropical and subtropical countries. Whereas malaria afflicts ∼210 million people annually, with ∼0.6 million malaria deaths in 2012 caused primarily by Plasmodium falciparum in sub-Saharan Africa Schistosoma infects ∼240 million people annually, with >90% of cases occurring in Africa Schistosoma haematobium, affects the ureteral or bladder wall and can lead to hematuria-induced anemia, urogenital deformities, bladder cancer, and diminished health-related quality of life Schistosoma, that could impair vaccine efficacy by modulating host immune responses to Plasmodium infection Malaria and schistosomiasis, caused by the protozoan S. haematobium prevalence in Mali was 38.3% but varied widely by region Both malaria and schistosomiasis are endemic to Mali, a landlocked country in West Africa with a population of 14.9 million. Intense, seasonal transmission of malaria occurs over much of the country, with ∼2.1 million malaria cases reported in 2012 S. haematobium and P. falciparum co-infection on the risk of clinical malaria remains unclear. Independent studies have shown that S. haematobium co-infection can either correlate positively P. falciparum parasite density. Although baseline S. haematobium infection decreased the risk of febrile malaria in a prospective cohort study of Malian children P. falciparum carriage at enrollment, which has been associated with a decrease in the subsequent risk of febrile malaria S. haematobiumIn co-endemic settings such as Mali, the impact of S. haematobium mono-infection, asymptomatic P. falciparum carriage  at the end of the six-month dry season, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of Malian children and adults living in an area where both diseases are co-endemic. Individuals diagnosed with urogenital schistosomiasis were treated with praziquantel within 6 weeks of enrollment, prior to the peak of the malaria transmission season. We adjusted for possible confounders of malaria risk, including age, sickle cell trait (HbAS), anemia, and spatial factors as determined by distance from home to river and residence within a cluster of high S. haematobium transmission.To clarify the relationship between urinary schistosomiasis and malaria, we evaluated the effect of baseline ClinicalTrials.gov identifier: NCT01322581). Written, informed consent was obtained from adult participants and from the parents or guardians of participating children.The Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Techniques, and Technology of Bamako, and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health approved this study  and 6% in Kambila in 2006 The study was conducted in the village of Kalifabougou, Mali, which is located 40 km northwest of Bamako, Mali. Kalifabougou is in the savanna ecoclimatic zone where annual rainfall is 800–1,200 mm per year. Among its inhabitants, Bambara is the predominant ethnic group, and ∼90% of residents engage in subsistence farming. Malaria transmission is intense and seasonal, occurring from June through December, with the vast majority of malaria cases caused by The study population has been previously described Plasmodium parasitemia detected by light microscopy were treated according to the National Malaria Control Program guidelines in Mali. The research definition of clinical malaria was parasitemia of ≥2500 parasites/µL, an axillary temperature of ≥37.5°C within 24 hours, and no other cause of fever discernible by physical exam. The primary endpoint was the time to the first or only febrile malaria episode. We also explored secondary definitions of malaria using parasite density thresholds of ≥500, ≥2500, and ≥5000 parasites/µL.After enrollment individuals were followed during the ensuing malaria season for 7 months. Clinical malaria episodes were detected prospectively by self-referral and weekly active clinical surveillance visits which alternated between the study clinic and the participants' homes. All individuals with signs and symptoms of malaria and any level of Thick blood smears were stained with Giemsa and counted against 300 leukocytes. Parasite densities were recorded as the number of asexual parasites/µl of blood based on a mean leukocyte count of 7500 cells/µl. Each smear was read in blinded manner by two certified microscopists of the laboratory team.Schistosoma haematobium eggs were quantified by microscopy after urine filtration with Nytrel filters (Vestergaard Frandsen) from a single urine specimen. Schistosoma mansoni and other geohelminth eggs were detected by microscopy of duplicate fecal thick smears using the Kato-Katz technique Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricodes, and Strongyloides stercoralis) as described previously Urine and stool samples were collected from participants at the time of enrollment, and samples were processed within 24 hours of collection. Plasmodium infection by PCR was done retrospectively at the end of the surveillance period. Detailed methods for PCR detection have been described Plasmodium positive samples were identified as P. falciparum, P. malariae, or both (mixed infections). For each participant, PCR was performed on blood samples in chronological order from enrollment onwards until the first P. falciparum infection was detected.During the scheduled clinic visits, blood was collected by finger prick every two weeks to prepare dried blood spots on filter paper. Detection of asymptomatic http://www.qgis.org/; map provider: glovis.usgs.gov).Geographic coordinates of the study participants' place of residence and the major communal buildings, main roads, and large streams in Kalifabougou were determined using GeoXM global positioning system (GPS) receivers (Trimble). Mapping and determination of distances were performed using ArcView 8.0 software (Esri) and QGIS version 2.0.1 . The Kaplan-Meier survival curve was used to estimate the probability of remaining free of clinical malaria during the surveillance period, and the log-rank test was used to compare the survival curves of different subgroups. The Cox proportional hazards model was applied to evaluate the differences in the risk of febrile malaria between the four subgroups: uninfected (reference group), S. haematobium mono-infection, P. falciparum mono-infection, and co-infection with S. haematobium and P. falciparum. The Cox model includes the following potential confounding variables (age and distance are continuous): age (per year increase), closest distance from home to river , HbAS, mild anemia at baseline, residence within a S. haematobium high-transmission cluster and presence of a metal roof on the participant's home. We also explored a model in which S. haematobium mono-infections at baseline were stratified as light (<10 eggs per 10 ml urine) or heavy (>10 eggs per 10 ml urine) but saw no significant difference in risk between the two groups. Thus, S. haematobium mono-infection was treated as binary covariate for all subsequent regression analyses.Differences in the baseline characteristics between the e groups  and attrS. haematobium infection was included in the model given the differential risk of malaria between S. haematobium infected individuals with and without anemia. The effect of S. haematobium and/or P. falciparum infection on log-transformed parasite density  during first malaria episodes was assessed by multiple linear regression with the following independent variables: HbAS, residence in a S. haematobium high-transmission cluster, and anemia as categorical variables; and log transformations of age and distance from clinic as continuous variables. Missing data were assumed to be missing at random. Statistical significance was defined as a 2-tailed P value of <0.05. Spatial analyses were performed in SaTScan version 9.2 (http://www.satscan.org/). All other analyses were performed in R version 3.0.2 (http://www.R-project.org).Moreover, an interaction term between anemia and P. falciparum and S. haematobium diagnosis, respectively  were PCR positive for P. falciparum at enrollment, and 39 (6.3%) individuals were co-infected with both parasites. Individuals with heavy S. haematobium infections  were no more likely to be co-infected with P. falciparum than those with light infections . Contemporaneous P. falciparum asexual parasite densities by microscopy were similar in both heavy and light S. haematobium infections . Consistent with their recent anti-helminth treatment via MDA, only 31 (5.1%) of individuals had other helminthic infections at enrollment by stool microscopy with a single S. mansoni infection and 30 infections with the non-pathogenic intestinal helminth Hymenolepis nana. For real-time PCR diagnosis of additional helminth infections, only subsets of available samples were analyzed given the overall negative findings  provided blood and urine samples for ectively . Of thesfindings . AdditioS. haematobium infected and uninfected individuals  and were twice as likely to be infected with P. falciparum at enrollment by Fisher's exact test . There was an increase in the attrition rate among individuals who were S. haematobium-infected at the time of enrollment . Gender, spatial measures, sickle cell trait, anemia, and roof type were similarly distributed between those who did and did not complete the study.Of the 616 individuals who provided initial samples for this study, 560 (91%) completed follow up from May 2011 to January 2012. Among the 56 individuals who did not complete the study, 6 individuals (11%) had a clinical malaria episode with one death due to cerebral malaria. Those who remained free of malaria were censored at their last visit. The most common reasons for withdrawing were extended travel outside the study area (50%) and refusal of further blood draws (43%). Three women withdrew due to pregnancy. The attrition rate was highest in adults . Both clusters overlapped substantially; therefore, only the co-infection cluster is shown in P. falciparum infections clustered significantly in two areas centered ∼4.6 km east-northeast  and ∼3.3 km west-southwest  of the clinic  baseline P. falciparum mono-infection, 3) co-infection with both P. falciparum and S. haematobium, and 4) neither infection (uninfected). In the unadjusted analysis  and co-infection  but not with S. haematobium mono-infection .Given that asymptomatic analysis , pairwisS. haematobium high-transmission cluster, and roof type in the Cox proportional hazards model, the protective effect of baseline P. falciparum mono-infection on febrile malaria persisted . Subset analysis of only individuals who were confirmed as negative for other helminth infections by stool PCR . Increased distance from the river was an independent predictor of malaria protection, while age, HbAS, and residence within the S. haematobium high-transmission cluster were associated with a non-significant trend towards reduced malaria risk at the parasite density threshold of ≥2500 asexual parasites/µl .We found a significant interaction between anemia and t anemia . InclusiP. falciparum carriage were strong negative predictors of parasite density, whereas light S. haematobium mono-infection (1–9 eggs/10 ml urine) had no effect on parasite density at the first malaria episode (S. haematobium mono-infection (≥10 eggs/10 ml urine) only suffered from febrile malaria episodes with parasite densities of <500 parasites/µl, and among those episodes, heavy S. haematobium negatively predicted parasite density   .P. falciparum and S. haematobium is one relationship where evaluating confounders may help explain inconsistent findings in the literature. In this prospective cohort study of malaria risk, we accounted for several possible confounders and observed that S. haematobium infection enhances protection from febrile malaria in individuals with asymptomatic P. falciparum carriage.Investigating the relationship between different parasitic infections in co-endemic communities at the population level is challenging due to the possibility of confounding by unknown variables that co-associate with both diseases. The interaction between P. falciparum carriers were more likely to be co-infected with S. haematobium at enrollment, which corroborates previous cross-sectional studies P. falciparum and S. haematobium mono-infections predict protection from malaria, with stronger significance for P. falciparum than S. haematobium (S. haematobium mono-infections). Taken separately, both of these findings are consistent with previous studies done in areas of seasonal malaria transmission In this study, asymptomatic matobium , possiblS. haematobium at baseline . In the adjusted model, asymptomatic P. falciparum carriage and co-infection, but not S. haematobium mono-infection, independently predicted protection from febrile malaria , suggesting that co-infections with gastrointestinal helminths are unlikely to confound our interpretation of the data. Our data demonstrates that heavy S. haematobium infections at baseline predict lower P. falciparum parasite densities at the first malaria episode, suggesting a potential negative interaction between the two parasites. However, we did not observe differences in malaria risk by intensity of S. haematobium infection, perhaps due to the low prevalence of heavy S. haematobium infections in our study (2.1%), a finding consistent with recent praziquantel MDA.Given that we stopped screening individuals for gastrointestinal helminths by stool PCR at only 23% of the cohort due to completely negative findings , it is pS. haematobium infection and malaria risk. A study conducted in Mali reported an association between baseline S. haematobium infection and protection from malaria attacks but did not differentiate between mono-infection and co-infection S. haematobium mono-infection did not influence malaria risk in a malaria vaccine efficacy study conducted in Kenya in which all children were cleared of P. falciparum with anti-malarials immediately prior to surveillance S. haematobium mono-infections were small , suggesting limited power to detect a difference in malaria risk for this group. Indeed, with only 23 cases of S. haematobium mono-infections, we had a 32% probability of detecting a hazard ratio of 0.62 or smaller at a 2-sided significance level of 0.05. While we might have detected more light infections had we examined more than one urine specimen per individual or used a more sensitive molecular diagnostic assay S. haematobium prevalence, are needed to better address whether S. haematobium mono-infection confers protection from febrile malaria per se and also if infection intensity might affect malaria risk.The above findings may help reconcile the disparity between two previous prospective cohort studies of P. falciparum carriage against febrile malaria is suggested by prior studies which demonstrated increased production of the anti-inflammatory cytokine IL-10 in co-infected individuals relative to individuals infected with only P. falciparum by either analysis of circulating plasma cytokines P. falciparum schizont extract P. falciparum-inducible IL-10 responses are upregulated in asymptomatic children with chronic P. falciparum infections S. haematobium infection could further augment anti-inflammatory responses induced by asymptomatic P. falciparum infection, thereby blunting the risk of fever upon subsequent P. falciparum infections.A plausible mechanism of how co-infection enhances the protection conferred by asymptomatic S. haematobium persist for an unknown period of time following clearance of S. haematobium. Although speculative, it is plausible that co-infection induces epigenetic changes that maintain an anti-inflammatory environment—a mechanism described in a mouse model of S. mansoni infection at the level of alternatively activated macrophages S. haematobium infection due to ineffective killing of juvenile worms (a known limitation of praziquantel) or complete drug failure remains a possibility. The latter is less likely given that, to our knowledge, there have been no reports of praziquantel failure in Mali since the initiation of MDA. An immunomodulatory effect of praziquantel per se is another possibility, but this is not supported by our data given the more modest protection seen in individuals who received praziquantel for S. haematobium mono-infection during the same time period as co-infected individuals  S. haematobium cases reside. Here, we used SatScan solely for identifying geographical clusters of disease that can then be used as a proxy for unmeasured confounders in regression models, noting that the spatial statistic employed by SatScan operates ideally when disease information is known for all households rather than a sampling of households. Nevertheless, these findings support a previous study in Kenya which found that intense infections of P. falciparum and S. haematobium clustered together within a subset of individuals even after the authors controlled for behavioral factors related to exposure to both parasites, implicating host susceptibility factors as the reason for this phenomenon S. haematobium transmission cluster as a covariate in our models, we were thus able to adjust for geospatial factors related to S. haematobium infection that we could not directly measure, such as host susceptibility factors, unmapped water sources serving as co-infective reservoirs, and micro-heterogeneity of malaria exposure, the last which has been shown to occur at sites similar to Kalifabougou P. falciparum blood-stage infection between individuals with and without baseline S. haematobium infection , and it is possible that these individuals would be the most susceptible to subsequent malaria episodes. However, since only 4% of screened individuals were excluded due to fever, the potential for bias would be minor. An addition source of bias may be from the higher attrition rates among the S. haematobium-infected individuals (18%), as these individuals may have developed malaria had they remained in the study. This potential bias is mitigated by the fact that 55% of the S. haematobium-infected individuals who withdrew from the study were adults and thus would be less likely to have a malaria episode, and three individuals completed more than 6 months of follow up prior to withdrawal.Potential sources of bias are worth noting. Although we randomly sampled in an age-stratified manner from the entire village-wide census, we enrolled only healthy, afebrile individuals. Thus, we could have excluded individuals with symptomatic infections Click here for additional data file.


Ascaris lumbricoides and Schistosoma haematobium while infection with hookworm or S. mansoni may increase the incidence of malaria and worsen pathology.Helminths and malaria parasites inhabit the same geo graphical areas and coinfection is frequently observed. The immunological interaction between helminth and malaria infections is highly relevant to disease control and elimination, yet is poorly understood and understudied. Chronic helminth infection causes immune hyporesponsiveness and stimulates anti-inflammatory cytokine production, which in turn modulates the host immune response to malaria. In epidemiological studies, many data are conflicting with some reporting protective effects against malaria for P. falciparum malaria infection (thick blood smear and rapid test) and S. haematobium infection (urine microscopy) was performed and treatment was provided to infected children. Children were followed up locally for malaria-like symptoms, actively for two months and passively for a further 2 years. At recruitment, whole blood was collected into Paxgene tubes from a subset of children for transcriptional profiling among uninfected (n = 12), S. haematobium infected (n = 16), malaria infected  or coinfected (n = 13) children. RNA was extracted, amplified and hybridized to Illumina HT12v4 microarrays.We undertook a prospective study of schoolchildren aged 5-16 in an area endemic for schistosomiasis near Lambaréné in Gabon. 425 children were recruited and demographic data was collected. Laboratory analysis for S. haematobium or malaria resulted in relatively insignificant changes to the transcriptome relative to healthy controls at the 10% false discovery level. There was also no difference in gene expression profile between healthy controls and coinfected children. However, between malaria infected and coinfected children there were 240 differentially expressed genes with 180 genes upregulated and 60 downregulated in the coinfected group. Specific genes of relevance to malaria infection, including CD8a, were significantly downregulated in malaria-infected compared to coinfected children. Pathway analysis revealed substantial modulation of gene expression in defence and immune responses and in particular modulation of genes in the Fcγ-receptor-mediated phagocytosis pathway.In the full cohort age, gender and schistosomiasis were not associated with malaria and malaria incidence among these schoolchildren was 0.4 episodes per person per year. Analysis of the microarray data revealed monoinfection with either S. haematobium on P. falciparum malaria in a key population for public health intervention.Confirmatory experiments are underway to confirm these intriguing insights into the immunomodulatory effect of


The large number of deaths caused by malaria each year has increased interest in the development of effective malaria diagnoses. At the early-stage of infection, patients show non-specific symptoms or are asymptomatic, which makes it difficult for clinical diagnosis, especially in non-endemic areas. Alternative diagnostic methods that are timely and effective are required to identify infections, particularly in field settings. This article reviews conventional malaria diagnostic methods together with recently developed techniques for both malaria detection and infected erythrocyte separation. Although many alternative techniques have recently been proposed and studied, dielectrophoretic and magnetophoretic approaches are among the promising new techniques due to their high specificity for malaria parasite-infected red blood cells. The two approaches are discussed in detail, including their principles, types, applications and limitations. In addition, other recently developed techniques, such as cell deformability and morphology, are also overviewed in this article. Plasmodium, and five species are reported for their infections in humans; namely, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi  is the real value of the Clausius–Mossotti factor and Erms is the root mean square of the electric field intensity. The complex Clausius–Mossotti factor is defined by Eq. , which is related to the effective polarizability. The factor depends upon the electrical conductivity and the dielectric permittivity of the particles and the suspension medium, as well as the applied signal frequency. It should be noted that Eq.  and dielectrophoretic crossover approaches . They fop-p at a frequency range from 1 to 5 MHz, resulting in the hRBCs being attracted to the electrode edges, while the iRBCs were pushed to the electrode gaps. As a result, iRBCs were swept away by the hydrodynamic force of the suspension medium resulting in a 200-fold concentration of the iRBCs. The other electrode design employed a spiral array of four parallel electrodes. The electrode was activated by a quadrature-phase sinusoidal signal up to 5 Vp-p in the frequency range from 1 to 15 MHz, while the second electrode created both traditional AC-DEP and travelling wave. After applying the electric signal, hRBCs were trapped at the electrode edges while iRBCs were levitated and carried towards the center of the electrode, resulting in an approximate 1000-fold enrichment of iRBCs.Subsequently, iRBCs were isolated away from hRBCs using two types of electrode configuration  . The sysp-p in the frequency range from 40 to 250 kHz and the successful separation of iRBCs from hRBCs by the DEP-FFF approach was obtained. However, the test of integrated flow-through PCR for malaria diagnosis was not included in that report.In 2004, Gascoyne et al.  proposedRecently, AC-DEP was used not only to discriminate early stage iRBCs from hRBCs but also used to predict cellular mechanical properties . In thisThe AC-DEP method exploits dissimilar dielectric properties between iRBCs and hRBCs for cell discrimination and might be the most appropriate of the different DEP techniques for malaria parasite detection. However, due to the low difference in, and the heterogeneity of, the electrical properties and diameter of iRBCs and hRBCs, the employment of the dielectrophoretic approach is quite limited in malaria parasite research.3O4) or greigie (Fe3S4) [3+ component [−6 mm3 s/kg and 1.80 × 10−6 (relative to water), respectively, at a haemoglobin to haemozoin fraction of 0.5 [With respect to the life cycle of the malaria parasite, after each sporozoite invades a liver cell and undergoes asexual development followed by hepatocyte lysis, the resulting merozoites are swept into the bloodstream to infect RBCs. The parasite digests haemoglobin leaving haem groups that aggregate into an insoluble brown pigment haemozoin. The number and size of haemozoin pigments depends on the developmental stage of the parasite , 81. Acc (Fe3S4) , 84. Addomponent . The magn of 0.5 .μmedium is the magnetic permeability of the suspension medium (N/A2), μparticle is the magnetic permeability of the particles (N/A2), a is the radius of the particles and H is the magnetic field strength (A/m). Considering Eq. (µ0 is the permeability of free space (4π × 10−7 N/A2) and χtarget − χbuffer). From Eq.  and 13 × 13 × 9 mm3 (second chamber). Both chambers were filled with 25-µm diameter stainless wire at 4.6 and 7.6 % density for the first and second chamber, respectively. The channel was placed between a 0.7 T permanent magnet, as shown in Fig. 3 with a microchannel between the dipole magnets [Paul et al. were probably the first research group to apply a magnetophoretic force for separating iRBCs in practice , 87, in  magnets . The cha6 A/m. At this position, where the strongest magnetic field gradient was induced, iRBCs would be forced to move towards the gap between the two magnets perpendicularly to the mainstream of blood flow in the channel  size-based filtration, which is simple and cheap . The proP. falciparum-iRBCs. Moreover, it has been reported that the iRBC deformability could be used to sort P. falciparum-iRBCs at different developmental stages and to enrich the ring-stage by 2500-fold [Plasmodium [In addition to the physical change in RBCs following malaria infection, the reduced deformability of iRBCs is a property that could be employed for malaria detection and separation, at least for 500-fold . Severalasmodium , 96 and asmodium –100.More recent research reported that the high optical absorbance and nanosize of haemozoin could generate a transient vapor nanobubble around haemozoin when exposed to a short NIR picosecond laser pulse. The acoustic signals of these induced nanobubbles provided a transdermal, non-invasive and rapid detection of malaria infection in animals without using any reagents or drawing blood .Several studies have employed a new platform to an existing conventional principle. For example, flow cytometry using standard nucleic acid-based staining methods was employed for a rapid and highly sensitive detection of iRBCs , 103, whGlobally, around 3.2 billion people are estimated to be at risk of being infected and developing malaria, and 1.2 billion people are at a high risk. According to the latest estimation, over 200 million cases of malaria occur around the world annually, and the disease leads to over 500,000 deaths each year. Early detection could save lives and prevent disease outbreaks, as well as allow parasite enrichment for biology studies, which are important topics in malaria research. The conventional microscopic examination of blood smears, antigen-based rapid test and molecular biology-based diagnosis all have some limitations for effective employment in low-resource setting areas. Therefore, researchers around the world are looking for new options. Among various techniques, dielectrophoretic and magnetophoretic principles have recently became attractive possibilities for malaria diagnosis due to the unique changes in the electrical and magnetic properties of iRBCs compared to hRBCs. Moreover, alternative techniques, such as cell morphology and deformability, have been proposed to be suitable bio-markers for the future. Not only is a knowledge of parasite biological properties critical, but the development of an engineering system is also an important factor, and should be studied in parallel. One example is a magnetic-field generating system that could create a high magnetic field gradient over a large area. Another solution might be a combination of two or three detection techniques, which might be able to increase the specificity to iRBCs and so enhance the ability for malaria detection or infected erythrocyte separation.