Transfusion-transmitted malaria (TTM) is a risk of transfusion that has not been well described in malaria endemic regions. The risk of the recipient getting malaria is related to the prevalence of malaria in the blood donors. There is however little information on the prevalence of malaria among donors in Akatsi district of Ghana. Further, the knowledge and practices of healthcare workers to TTM is unknown. The study was undertaken to determine the prevalence of malaria parasite infection among blood donors and to evaluate the knowledge and practices of healthcare workers to TTM in the Akatsi district of Ghana.Plasmodium falciparum, 5 µl of capillary blood was obtained by finger prick from 200 participants . Plasmodium falciparum screening was done using CareStart™ Malaria Antigen kit. To obtain information regarding TTM knowledge and practices, questionnaires were completed by 100 health workers including nurses, doctors and laboratory staff.The study was conducted at Akatsi South District Hospital between May and August 2014. To screen for P. falciparum was the same (10 %) in both donors and controls. All those who were malaria RDT positive were aged 15–25 years. Out of the 100 healthcare workers  surveyed, 45 % of respondents (45/100) had never heard of transfusion-transmitted malaria. Almost all respondents (91 %) had not attended any lecture/seminar/workshop on blood transfusion in the past 12 months. There were 44 respondents (44 %) who wrongly said malaria was being screened for prior to transfusion in their hospital. However, 98.2 % (54/55) of those who had heard about TTM rightly stated that TTM can be prevented.The prevalence of P. falciparum parasitaemia is 10 % in healthy blood donors in the Akatsi district and represents a risk for TTM though the extent of this risk is unclear. Knowledge about TTM in healthcare workers in the district is low. Continuous education and in-service training may be a means to improve TTM knowledge and preventive practices by the health workers in the district.The prevalence of  The global burden of malaria has been considerably reduced over recent years . HoweverSince the first reported case of transfusion-transmitted malaria (TTM) in 1911 , transfuPlasmodium falciparum malaria [Screening healthy donors for malaria in endemic countries poses a diagnostic challenge. This is because currently there is no screening test that is practical, affordable and suitably sensitive for blood banks . Microsc malaria . They haThe knowledge of health workers about TTM has not been previously evaluated. There is very little published data relating to practices and attitudes of health workers to TTM. A study found that practices in a teaching hospital are related to particular departments where doctors worked .The aim of this study was to determine the prevalence of malaria in blood donors by RDT, compared to controls. In addition the knowledge, attitudes and practices of health workers in the district hospital in relation to transfusion malaria was determined.The study was carried out in the Akatsi District Hospital (ADH), a public healthcare facility in the Akatsi South District of the Volta region of Ghana. ADH has staff strength of 140 healthcare workers including two doctors, a medical assistant, a pharmacist and a biomedical scientist. ADH is a 70-bed capacity, with a maternity unit, medical laboratory unit, family planning unit and an outpatient department. It also acts as referral centre for the surrounding primary health facilities in the district. Approximately 450 blood transfusions are carried out each year in ADH. Blood donations are from both voluntary and family replacement donors. The hospital blood bank routinely screens donor blood for hepatitis B, hepatitis C, HIV and syphilis.Blood donors and healthy controls from the community formed the study population to determine prevalence of malaria and health workers in ADH were the study population for the survey conducted. Donors who were aged between 15–55 years, had an haemoglobin (Hb) of more than 12.5 g/dL and were Hepatitis B, Hepatitis C, HIV, and syphilis negative were eligible for enrolment. These donors were consecutively enrolled after obtaining their written informed consent.Healthy controls were also consecutively enrolled from individuals accompanying their relatives to the hospital, people working in the hospital or living in and around the hospital. Participants were excluded from the study if they show any sign of malaria, had fever or had been treated for malaria 4 weeks prior to enrolment. For the health worker survey, the study population was recruited from all health workers of ADH. A list of the names of all 142 health workers in ADH was obtained from the staff office. Workers were then approached individually to obtain their consent to participate in the study. Workers who gave their consent were given the study questionnaire to completely fill out. It was pretested and subsequently modified into a four-part document with a socio-demographics section, a knowledge section and a section on attitudes and practices. Responses to a total of 40 open and closed ended questions were anonymous so as to obtain frank answers without fear of victimization. The study questionnaire used was adapted and modified from a previously reported model .Health workers were given 24 h to complete the questionnaire. Those who did not submit were reminded by phone or followed up in person. In total, 120 questionnaires were systematically distributed but 100 were returned for data entry and analysis.In total, blood samples were taken from 200 subjects, 100 blood donors and 100 controls.P. falciparum. The use of this rapid kit has been previously been reported [Five microlitres of capillary blood sample was obtained from each donor and control participant for malaria parasite screening. Samples were collected at the start of the rainy season, between May and July 2014. This period represents the start of the malaria transmission season. Malaria testing was performed using CareStart™ Malaria Antigen test kit ; a histidine-rich protein (HRP-2) based rapid diagnostic tests (RDT) that detects reported .Data collection was by paper case report sheets. Data were entered electronically and analysed using Statistical Package for Social Sciences (SPSS) for Windows (version 17.0). Prevalence of malaria, determined by malaria RDT positivity in donors and controls was calculated by dividing the number of positive results by the total number of samples.Approval for this study was given by the Committee on Human Research Publication and Ethics of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology in Kumasi, Ghana and the Management of Akatsi District Hospital in the Volta Region of Ghana. Study participants were assured of confidentiality and all data collected remained anonymous.P. falciparum was found to be 10 % in both donors and controls  and were aged between 20–30 years old (77.0 %). The most common job categories were nurses and nursing students followed by laboratory technicians  who correctly knew that TTM is a preventable disease. Almost all respondents 89.1 % correctly mentioned screening of donated blood before donation as a prevention measure Table . Only onFifty percent of respondents (50/100) were directly involved with blood transfusion in Akatsi district hospital. Blood administration 41.0 %) and transfusion monitoring (30.0 %) were the most common activities while donor recruitment (3.0 %) and blood prescription (4.0 %) were the least common transfusion practices that staffs of ADH were involved in (Table 1.0 % andThe majority of the respondents 44.0 %) wrongly stated that the hospital blood bank was screening for malaria parasite in blood donors before blood donation while a quarter of respondents were not sure if screening was being done or not  indicated their willingness to transfuse malaria positive blood when faced with an emergency situation. In contrast, none of the 55 respondents were willing to transfuse hepatitis B positive blood in case of an emergency. The majority of the respondents 92.7 % (51/55) also indicated that they were unwilling to transfuse syphilis positive blood in a case of emergency . This is not surprising and is an indication that the blood donors are a true representation of the healthy adult population in the district. In many African countries the donor population is usually male dominated. However in this study, females were almost half of all donors. In this study, it is unclear why the prevalence of parasitaemia in males was more than double that of females in the control group. Some studies have established that parasitaemia is not significantly associated with the gender of donors. Ukaga et al. [This study showed that the prevalence of s by RDT . Other As by RDT . The pres by RDT . The sens by RDT . This sta et al.  found prThe survey results showed that only 9 % of the respondents had attended any workshop on blood transfusion in the past 12 months. In Ghana, professional bodies require continuous professional development while health institutions encourage service training in all aspect of medicine. In many high-income countries, regular training on blood transfusion is mandatory, reflecting the importance of transfusion for patient safety and treatment. Much more advocacy is needed by the health professions and blood services in Ghana to ensure that transfusion is included in undergraduate curricula and in-service training programmes.Knowledge about TTM was generally low among health workers in Akatsi. Almost half of the health workers had not heard about TTM. Malaria has been neglected a transfusion-transmitted infection, having been substantially overshadowed predominantly by a focus on HIV. Almost all respondents wrongly assumed that screening for malaria was being performed in Ghana. It is likely that the workers assumed that because routine screening takes place for HIV and hepatitis, malaria screening also takes place. It is unclear why a quarter of respondents admitted they were not sure if screening was done in their hospital but did not say so when questioned about screening across Ghana. If this level of misinformation occurs across Ghana then there is a clear need to provide education on TTM and particularly around strategies that can be used to reduce transmission through transfusion and to correct some assumptions that may negatively affect practice. It is interesting to observe that many health workers were unwilling to transfuse hepatitis B or syphilis positive blood but many were willing to transfuse malaria positive blood. Further investigations to determine the rationale for this will be useful.Nearly all respondents (89.1 %) knew the most effective way of preventing TTM. This high level of knowledge could be possibly due to the effective malaria campaigns going on across the country. However, a third of health workers did not think that routine use of anti-malarials in transfusion will ultimately contribute to the spread of drug resistance. Current World Health Organization policies and treatment guidelines recommend that parasitological confirmation of malaria should be done before initiating the treatment . HoweverTTM is a ‘hidden’, neglected and under researched reservoir of ongoing malaria transmission. The recent announcement of a new one billion dollar fund to eliminate malaria means that renewed efforts are needed to understand and reduce the risks of malaria  transmisP. falciparum parasitaemia. This represents a potential risk for transmitting malaria via transfusion. While knowledge about TTM among healthcare workers in the district is low, continues education and in-service training may be a means to improve knowledge and preventive practices by the health workers in the district.Healthy blood donors at the Akatsi district hospital have a 10 % prevalence rate of


Asymptomatic falciparum malaria is associated with poorer cognitive performance in African schoolchildren and intermittent preventive treatment of malaria improves cognitive outcomes. However, the developmental benefits of chemoprevention in early childhood are unknown. Early child development was evaluated as a major outcome in an open-label, randomized, clinical trial of anti-malarial chemoprevention in an area of intense, year-round transmission in Uganda.Infants were randomized to one of four treatment arms: no chemoprevention, daily trimethoprim–sulfamethoxazole, monthly sulfadoxine–pyrimethamine, or monthly dihydroartemisinin–piperaquine (DP), to be given between enrollment (4–6 mos) and 24 months of age. Number of malaria episodes, anaemia (Hb < 10) and neurodevelopment [Mullen Scales of Early Learning (MSEL)] were assessed at 2 years (N = 469) and at 3 years of age (N = 453); at enrollment 70 % were HIV-unexposed uninfected (HUU) and 30 % were HIV-exposed uninfected (HEU).P = 0.02). This relationship was mediated by episodes of anaemia. This regression model was stronger for the HEU than for the HUU cohort. Compared to HUU, HEU was significantly poorer on MSEL receptive language development irrespective of malaria and anaemia (P = 0.01).DP was highly protective against malaria and anaemia, although trial arm was not associated with MSEL outcomes. Across all treatment arms, episodes of malarial illness were negatively predictive of MSEL cognitive performance both at 2 and 3 years of age (Malaria with anaemia and HIV exposure are significant risk factors for poor early childhood neurodevelopment in malaria-endemic areas in rural Africa. Because of this, comprehensive and cost/effective intervention is needed for malaria prevention in very young children in these settings. The adverse effects of both cerebral malaria and severe malaria anaemia in preschool-age children has also been documented , 2. HoweIntermittent prevention treatment (IPT) for malaria in schools and in communities has had cognitive and academic benefits in rural Gambia , westernThe principal study questions are whether: 1) malaria illness and anaemia are related to poorer cognitive development in preschool-age children in an area of high malaria transmission; and, (2) whether perinatally HIV-exposed uninfected (HEU) children more vulnerable to the adverse effects of malarial illness on cognitive development, compared to HIV-unexposed uninfected (HUU) children. Figure  malaria This observational study of neurodevelopmental outcomes was embedded within a malaria chemoprevention randomized controlled trial (RCT) study conducted at the Tororo District Hospital near the Kenyan border in eastern Uganda , 15  during pregnancy and their study children were on nevirapine for 6 weeks and trimethoprim–sulfamethoxazole (TS) prophylaxis for the duration of breastfeeding, which is the standard of care in many African countries, including Uganda . These HEligibility criteria for the HUU children in the original RCT trial included the following : (1) borThe HUU study children in the original RCT trial were enrolled at 4–5 months of age (median = 5.6 months) and randomized to treatment arms at 6 months of age. Both the HEU and HUU children were followed for all their health care needs including monthly routine visits. Chemoprevention was discontinued at 2 years of age and children were followed for one additional year until they reached 3 years of age. The primary outcomes were the incidence of malaria and incidence of moderate-severe anaemia (haemoglobin <80 gm/dL).At enrolment in the original RCT trial, caregivers were given two long-lasting, insecticide-treated bed nets (LLINs). Anthelminthic, iron sulfate and vitamin A were prescribed following Integrated Management of Childhood Illnesses guidelines. During follow-up, children who presented with a documented fever (tympanic temperature >38 °C) or a history of fever in the previous 24 h, had blood obtained by finger prick for a thick blood smear. Episodes of uncomplicated malaria were treated with standard doses of artemether–lumefantrine (AL). Episodes of complicated malaria or treatment failures occurring within 14 days of therapy were treated with quinine. Routine evaluations, including thick blood smears and assessment of adherence to LLINs and study drugs, were done monthly .Figures At 2 years of age, all HEU and HUU children not lost to follow-up in the RCT trials, which had not been hospitalized with a history of severe malaria and with caregivers willing to participate, were co-enrolled into our observational study for neurodevelopmental assessment with the MSEL. Children who had been hospitalized with severe malaria were excluded from the present study because of previous findings documenting the relationship between severe malaria and MSEL performance , 17. As Children’s neurodevelopment was assessed with the Mullen Scales of Early Learning (MSEL) twice; at 2 and again at 3 years of age. This assessment is intended for use in assessing children from birth to 68 months and provides assessment scores for the neurodevelopmental domains of gross motor (up to 33 months), and a composite score for the cognitive scales of visual reception, fine motor, receptive language, and expressive language. This test was designed to be more easily adaptable to resource-limited and cross-cultural settings than the Bayley Scales of Infant Development, considered to be the gold standard of such assessments . At the Research assistants administering the MSEL in the present study were all psychology Bachelor’s degree graduates of Makerere University fluent in the local languages who underwent 1 week of training on the test followed by a month of daily practice testing observation and participation. Following this training and practice period, the head of the assessment team at the study site observed the MSEL administration by the apprentice research assistant and approved the assistant for the assessment of study children contingent on good testing skills and satisfactory completion of an evaluation of MSEL testing skills as observed thereafter on a monthly basis .Socio-economic status (SES) was evaluated in the present study using a questionnaire read to the mother in the local language which asked a series of questions about the physical quality of the dwelling , availability of water and plumbing, availability of electricity and sources of fuel for cooking/heating, material possessions such as common appliances, radio/TV, bicycle, dishes, furniture, and books. The mother was also asked a series of questions about the types of employment for pay for her and the father/husband or other adult household members, as well as domestic animals available to the household. The SES questionnaire asked about food security in the way of sources of meat, fish, poultry, agriculture and garden produce. Finally, the SES questionnaire for the present study included a set of questions about daily caregiving practices in the home for the study child .Measures from the SES questionnaire  along with clinic-based measures of anemia and malaria illness were included along with maternal HIV status in a conceptual model of child neurodevelopment depicted in Fig. Although in the published findings of the RCT study, the principal outcome was malaria illness incidence per person year at risk (PYAR) , 15, couThe principal predictor in the present analyses was incidence of malaria, defined as the number of episodes during the period the chemoprevention was given, and between 2 and 3 years of age after chemoprevention was stopped. The other exposure variable was incidence of anaemia during the study period: the number of incidents of moderate-severe anaemia (haemoglobin <10 gm/dL) during monthly clinic visits during the chemoprevention period (up to 2 years) and during the year following cessation of chemoprevention (between 2 and 3 years of age).Primary analyses for the first research question about the relation of malaria illness and anaemia with cognitive development was addressed using linear mixed effects (LME) models. The numbers of malaria and anaemia episodes before 2 years, and between 2 and 3 years of age were used as time varying covariates. Other time-varying covariates included age, weight for age z-score (WAZ) and number of breastfeeding days in a time period. Fixed (not time-varying) covariates included trial arm, HIV exposure, sex, socio-economic status (SES), and number of observation days prior to randomization. Height-for-age z scores (HAZ) was also added to the regression models Tables , 5 but wThe assumption of linearity of the relationship between the Mullen scores and principal exposure variables  was evaluated using scatterplots. To assess the robustness of findings, sensitivity analyses were performed using several cut-offs in the number of episodes. The results obtained from models with categorical exposure variables were not appreciably different from the results from the models with linear effects.To assess the effect of exposures and the extent to which the effects of the number of malaria episodes were mediated by the anaemia, three LME models were fit.Model 1 related the MSEL scores (one at a time) to time period , number of malaria episodes and other covariates described above. The number of anaemia episodes was not included in this model to assess the main effect of the malaria episodes.Model 2 related the MSEL scores (one at a time) to time period , number of malaria episodes and other covariates. The number of malaria episodes was not included in this model to assess the main effect of the anaemia episodes.Model 3 related the MSEL scores (one at a time) to time period , number of malaria episodes, number of anaemia episodes, and other covariates. This model assessed the effects of malaria and anaemia episodes over and above each other. To establish mediation, the effect of the number of malaria episodes had to be significant in Model 1. If in Model 3 the effect of the number of malaria episodes was no longer significant, then complete mediation was deemed to occur –25. If tP < 0.01). HEU children had fewer bouts of malaria during both the chemoprevention phase (6 months to 2 years)  and post prevention (2–3 years) . The HEU children also had fewer bouts of anaemia before 2 years of age , and fewer exclusively breastfeeding days both before and during the study  , receptive language , and visual reception . The HEU cohort also did more poorly than the HUU children on the MSEL expressive language scale at 2 years of age  (Table The subgroup of children from the RCT cohorts (HUU or HEU) who were not co-enrolled in the neurodevelopmental observational MSEL study did not differ statistically from the present study samples on any of the descriptive measures in Table F = 12.47, P < 0.001 for HUU; F = 9.83, P < 0.001 for HEU). These treatment-arm differences in malaria episodes no longer occurred after treatment was stopped at 2 years of age . After cessation of treatment, there were no longer significant differences among treatment arms on episodes of anaemia. There were no significant differences among treatment arms on the MSEL composite total either following chemoprevention (2 years of age assessment) or a year after chemoprevention (at 3 years of age) (Table Table P = 0.02), but not when controlling for anaemia episodes (P = 0.13), suggesting that anaemia mediates this relationship. The same is true for the MSEL outcomes of fine motor (P < 0.01 to P = 0.03) and receptive language (P = 0.04 to P = 0.15). Similar mediational effects were observed when comparing the relationship of anaemia episodes to MSEL with and without controlling for malaria episodes. This was the case for composite cognitive ability (P = 0.01 to P = 0.10), visual reception (P < 0.01 to P = 0.02) and receptive language (P = 0.03 to P = 0.14) when exploring the malaria and anaemia episode effects . Significant predictors included age (negative association), and time (lower scores at 3 years of age compared to 2 years).Trial arm was P = 0.04 to P = 0.18). A similar mediational effect was observed when evaluating the relationship between anaemia episodes and MSEL fine motor (P = 0.03 to P = 0.14). For Table P = 0.02 to P = 0.10), as well as predictive of receptive language (P = 0.01 to P = 0.05). For this cohort, anaemia episodes only predicted MSEL visual reception as mediated by malaria episodes (P = 0.04 to P = 0.13).Table Within the context of a rigorous RCT study in a high-malaria-exposure area in rural eastern Uganda, strong evidence is presented that repeated episodes of malaria illness, as mediated by anaemia, can diminish cognitive development in very young children. In the present findings, malaria chemoprevention during early childhood is not directly related to child neurodevelopment. However, malaria episodes during and after chemoprevention are significantly predictive of both cognitive and motor development at 2 and 3 years of age. This study is the first to document within the rigorous context of an RCT study that early uncomplicated malaria illness before 2 and 3 years of age can diminish cognitive development in very young African children. Furthermore, this relationship is at least partially mediated by anaemia. However, another form of analysis that might have been used to better understand the mediating effects of anemia is structural equation modeling. Future work could employ this statistical method as well.P < 0.05). These differences may have been due to differing levels of sensitivity of these scales to the developmental effects of HIV-exposure in this setting, depending on which developmental domains blossom most at a given year of age. For example, MSEL language development differences for HEU children compared to HUU children in rural Uganda has been previously observed in the pre-school age years [HIV exposure resulted in lower MSEL scores on composite cognitive performance at 3 years of age, especially receptive and expressive language. MSEL differences were observed at 2 years of age only at the 0.05 significance level on Expressive Language (ge years .It should also be noted that the relationship between episodes of malaria illness and MSEL composite cognitive and receptive language performance for the HEU cohort was more robust than for the HUU cohort in the present study (comparing Tables Despite this more robust relationship, the overall number of episodes of malaria illness and anaemia during and after chemoprevention was lower for the HEU than for the HUU cohorts Table . This isAlthough a significant relationship was observed between episodes of malaria illness and neurodevelopmental outcomes in the present study findings, significant neurodevelopmental differences were not observed among chemoprevention treatment arms, either during or after the treatment phase. This might be partly due to questionable adherence to the treatment arm regimens on the part of the mothers of the children, as witnessed by the results of monitoring of DP blood levels in the RCT trial , 15. CheFinally, socio-economic and educational status can impact on both the reliable use of malaria chemoprevention medications and quality of housing in terms of night-time exposure of study children to mosquitoes . In the The present study findings support other studies with school-age children that conclude that repeated episodes of malaria are associated with poor cognitive scores and low academic performance , 30. EveBecause convenience sampling was used to enroll the original HEU cohort of 200 and the HUU cohort of 400 infants at 4–5 mo of age from the Tororo District Hospital Maternal and Child Health Clinic their birth records were incomplete. Therefore, premature and low-birth weight children could not be excluded and this may have led to developmental delays that could compromise the sensitivity of the MSEL outcomes to the effects of malaria illness frequency and anaemia in the present samples. HEU children in this study area are at significantly greater risk for premature birth and complications, and shorter duration of breast feeding . These rBeyond LLIN distribution programmes, little has been done for very young children in Africa to protect them against malaria. In rural households in Tororo District which have been provided with LLINs, children can still have as high as 310 infectious bites per year with a malaria-positive parasite prevalence rate of 28.7 % .In the longer term, malaria vaccines may have an important role in protecting this important section of the community from malaria. Regardless of the control approach selected, it is important that a comprehensive and aggressive community-wide approach to controlling malaria be incorporated into every public health programme in malaria-endemic regions, as part of the ongoing effort to enhance the health of African school-age children .Early bouts of malaria illness can diminish cognitive development in very young children. These effects of malaria illness on cognition are partly mediated by anaemia, and cognitive development risk from malaria and anaemia seems to be greater in HEU children. This is the case even with children and caregivers who are provided with free and effective medical care and nutritional support in the first years of life. The high level of malaria transmission and the host of other risk factors in such low-resource environments can still overwhelm such medical and nutritional support, even when provided in a conscientious manner in such settings.http://newsroom.msu.edu/site/indexer/3004/content.htm for an overview of this office and http://oip.msu.edu/documents/CDA2001_002.pdf for an example of such an agreement. In addition, we will ask for final approval of all publications from other research groups that include our study data.We will be able to download our de-identified datasets and data definition dictionary from our university data management archive to make them available to other research groups upon request. We will require a collaborative agreement from such groups before making such datasets available, as approved through the Intellectual Property Office at Michigan State University. See


Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) and the 19-kDa C-terminal region of merozoite surface protein-1 (PfMSP-119) are candidate malaria vaccine antigens expressed on merozoites and sporozoites. This investigation was performed to evaluate simultaneously the naturally-acquired antibodies to PfAMA-1 and PfMSP-119 and to compare IgG subclass profiles to both antigens in naturally exposed individuals living in malaria hypoendemic areas in Iran to determine which antigen has better ability to detect sero-positive individuals infected with P. falciparum.19 were expressed in Escherichia coli, and IgG isotype composition of naturally acquired antibodies to the antigens (as single or in combination) was measured by ELISA assay.In this investigation, 101 individuals from the malaria-endemic areas in Iran were examined. PfAMA-1 and PfMSP-119 IgG antibody responses, respectively, and the prevalence of responders did not differ significantly (P > 0.05). Moreover, IgG1 and IgG3 were predominant over IgG2 and IgG4 antibodies and the prevalence of IgG and its subclasses to two tested antigens had no significant correlation with age and exposure (P > 0.05). The present data confirmed that when recombinant PfAMA-1 and recombinant PfMSP-119 antigens were combined in ELISA at equal ratios of 200 ng (100 ng each antigen/well) and 400 ng (200 ng each antigen/well), 86.1% and 87.1% of positives sera were detected among the examined samples, respectively.The result showed that 87.1% and 84.2% of the studied individuals had positive anti-PfAMA-1 and -PfMSP-119. Therefore, these results could support the design of a universal PfAMA-1- and PfMSP-119-based vaccine. Also, both recombinant antigens could be used in combination as reliable serology markers to perform immuno-epidemiological studies in malaria-endemic areas of Iran during elimination strategy. The present information could be of use in control and elimination programmes in Iran and other similar malaria settings.The two tested recombinant antigens are immunogenic molecules, and individuals in low transmission areas in Iran could develop and maintain equal immune responses to PfAMA-1 and PfMSP-1 Plasmodium falciparum is one the most lethal species of malaria parasites that infects humans ) and non-reducing conditions (with SDS and without 2ME) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The concentration of the protein was determined using Bradford’s assay by a spectrophotometer . To confirm the purified recombinant proteins, Western blot assay was carried out by standard protocols using anti-His antibody  and with P. falciparum-infected human sera that reacted with rPfAMA-1 under both reducing and non-reducing conditions. Protein migration at different sizes on SDS-PAGE in the presence and absence of 2ME indicates the presence of a disulfide bound, suggesting that all recombinant proteins had a tertiary shape in their antigens.rPfAMA-1 was expressed in 19 was expressed as described previously [19 protein was expressed in E. coli BL21. Overnight cultures from single colonies of PfMSP-119− specific E. coli were expanded in TB (pH 7.2) containing ampicillin (100 μg/ml) at 37°C with shaking (150 rpm) until an OD of 0.6-0.7 at 600 nm was reached. In addition, the expression of GST-PfMSP-119 was induced with 0.5 mM IPTG . The culture was further grown for 4 h, and the E. coli cells were harvested by centrifugation and kept in −70°C until use. The cell pellet was dissolved in PBS 1× (pH 7.4) and lysed on ice with 10 sonication cycles , each consisting of 20-s pulses at 20-s intervals. The bacterial lysate was centrifuged at 14,000 × g at 4°C for 30 min. The supernatant was incubated with glutathione 4B Sepharose resin  at 4°C for 1 h, and the resin was packed into a column. The column was washed with PBS 1×, pH 7.4 (10-column volume). The bound protein was eluted with a buffer, containing 50 mM Tris–HCl and 10 mM reduced glutathione, pH 8. The fractions containing PfMSP-119 were desalted with Econo-Pac 10 DG columns  according to the manufacturer’s manual and then concentrated with a concentrator . The eluted proteins were analysed under reducing (with 1% SDS and 2% 2ME) and non-reducing conditions (with SDS and without 2ME) by SDS-PAGE (12%), and the concentration of the protein was determined using Bradford's assay by a spectrophotometer .rPfMSP-119, respectively. In priming and boosting, the antigens were then emulsified in complete Freund's adjuvant  and with incomplete Freund's adjuvant  in 1:1 ratio. The mice control groups were immunized with PBS alone, PBS in Freund's adjuvant and rGST alone. The animals were boosted on days 14 and 28 and bled on days 0 (pre-immune), 21, and 35 of first immunization.Inbred BALB⁄c female mice (6–8 weeks old) were obtained from Laboratory Animal Science Department, the Institut Pasteur Iran. Mice groups (n = 7) were immunized subcutaneously at the base of tail with 30 μg and 35 μg of the rPfAMA-1and rPfMSP-119 sera of immunized mice, to recognize the native form of both antigens on merozoite surface and to determine the similarity between epitopes in recombinant forms and corresponding native proteins. For this purpose, multispot slides of parasites were prepared from P. falciparum culture, air-dried and then fixed in cold acetone for 10 min. Polyclonal mouse sera diluted  in PBS were added to the spots and incubated in a wet chamber for 60 min. After washing three times with PBS (pH 7.4), each well was covered with 20 μL of the fluorescein-conjugated anti-mouse polyvalent IgG (1:40) and then left in a wet chamber for 40 min. Again, after washing three times with PBS, coverslips were placed on each slide and examined under a fluorescence microscope  with an oil immersion objective (100×). The serum samples obtained from normal mice were used as negative controls.IFAT assay was performed to test the ability of the anti-PfAMA-1 and anti-PfMSP-119 antigens (as single or in combination) were measured by an ELISA as described previously with some modifications [19 and in combination of rPfAMA-1and rPfMSP-119 (100 ng and/or 200 ng of each antigen/well) or GST alone (as control) in 0.06 M carbonate-bicarbonate buffer (pH 9.6) and then incubated at 4°C overnight. The plates were washed with PBS-Tween (PBS-T) and blocked with bovine serum albumin (BSA)-PBS-0.05% Tween. Serum was added in duplicate at a dilution of 1:200 . After washing with PBS-T, the plates were incubated with horseradish peroxidase-conjugated goat anti-human IgG  at 1:35,000 concentration. Finally, the enzyme reaction was developed with o-phenylediamine dihydrochloride-H2O2  and stopped with 2 N H2SO4. The OD was measured using an ELISA microplate reader  at 490 nm. All samples were re-tested if there was a discrepancy of greater than 20% between the duplicates. Standardization of the plates was achieved using positive-control serum pools on each plate. Background (determined from the wells with either no serum or GST) was subtracted from the mean of each sample, and a cut-off value was determined as the mean plus three standard deviations from the 30 negative control serum samples which were included in each assay.In the present study, IgG antibody responses of individuals during acute infection to rPfAMA-1 and rPfMSP-1P values < 0.05 were considered statistically significant. The sensitivity of each test was measured by dividing the number of positive IgG sera to total numbers of sera obtained from P. falciparum-infected individuals.A database was generated with SPSS 20.0 for windows . As the antibody levels were not normally distributed, non-parametric tests were used. Differences in the proportions of IgG-positive subjects were assessed using the McNemar's test or Chi square comparison of proportions as appropriate. Furthermore, differences between the mean absorbance of antigens alone or in combination were analysed by using Wilcoxon Signed Ranks test or Friedman test as appropriate. The Spearman’s correlation test was also used to assess the association between antibody levels with age as well as exposure. P. falciparum, as a mono-infection, and none of the healthy control individuals had either P. falciparum or P. vivax infection.Based on both microscopy and nested-PCR results, all 101 patients were shown to be infected with 19 produced in mice recognized the native protein present on the surface of P. falciparum merozoite at late schizont (or merozoite) stage with high intensity, as indicated by the grape-like fluorescence pattern  proteins had proper conformation and folding.Anti-rPfAMA-1 and -rPfMSP-1E. coli M15-pQE30, and the purified protein was analysed by SDS-PAGE with a molecular mass of ~55 kDa. The purity of the recombinant proteins was evaluated by Western blot assay. The result showed that expressed proteins migrated at different sizes in the presence and absence of 2ME contained a disulfide bound. Total IgG antibody responses to PfAMA-1 was determined in 101 individuals .Serum samples positive for total anti-rPfAMA-1 IgG were evaluated for IgG subclass responses to rPfAMA-1 antigen. In individuals who were infected with 19 was expressed in E. coli BL21-pGEX-KG, and the purified protein was analysed by SDS-PAGE with a molecular mass of ~37.5 kDa. The purity of the recombinant protein was evaluated by Western blot assay. The result showed that the expressed PfMSP-119 migrated as a single homogeneous band on SDS-PAGE under non-reducing conditions, indicating that it is largely composed of a single conformer. Total IgG antibody responses to rPfMSP-119 was determined in 101 individuals .Serum samples positive for total anti-PfMSP-1r = 0.061, P = 0.544 for IgG; r = 0.042, P = 0.673 for IgG1, and r = 0.014, P = 0.893 for IgG3; Spearman’s correlation test) or age . Likewise, the levels of IgG, IgG1, and IgG3 antibodies to the rPfMSP-119 were not correlated with exposure  or age .The levels of IgG, IgG1, and IgG3 antibodies to rPfAMA-1 were not correlated with exposure  primary infected, individuals with no previous malaria episodes (n = 49) and (ii) individuals with one or more previous malaria episodes (n = 52). The frequency of responders to both antigens did not change significantly when they divided in primary and multiple-infected  confirming that antibody response against PfAMA-1 and PfMSP-119 was established after a single exposure to malaria  1–15 years old (n = 8), 16–30 years old (n = 53), and ≥ 31 years old n = 40). No significant difference was found in the prevalence of positive sera for PfAMA-1 and PfMSP-10. No sigP. falciparum. No response to either antigen was observed in 13.8% of the individuals  and 400 (200 ng each antigen/well), sero-positivity of 86.2% and 87.1% were obtained, respectively . However, when the mean absorbance was compared in paired groups, no significant difference was observed in the mean absorbance of antibodies to combination of rPfAMA-1 and rPfMSP-119 at concentration of either 200 ng or 400 ng .The frequency of individuals with IgG antibodies to combined antigens (100 ng of each antigen/well) was only 86.2% during patent infection with  Figures . The difP. falciparum is the primary objective. Although the prospects of achieving global malaria eradication have been diminished by the limited available intervention tools, vaccines could be considered effectively in these programmes; for instance, they have also vital role in global eradication of smallpox and the elimination of Poliomyelitis from the world. Moreover, for vaccine development, sero-epidemiology studies on various populations with a different genetic background and endemicity in malaria settings would help to understand the host immune responses to malaria parasites as well as the endemicity of the disease. Besides, in malaria elimination programmes, monitoring changes in transmission intensity and identification of residual foci of malaria by using sensitive and reliable tools is very important for measuring the success of the programme. Therefore, the goal of this study was to compare and analyse the naturally acquired antibody responses to two recombinant proteins representing two asexual erythrocytic stage of P. falciparum (PfAMA-1 and PfMSP-119) by human IgG antibodies among naturally exposed individuals living in the malaria hypoendemic setting, Iran. These two proteins not only are the asexual erythrocytic stage vaccine candidates but also are serological markers particularly useful for detection of relative antibody responses in areas of low endemicity [In elimination programmes, reduction in disease and death due to demicity ,58.E. coli, and the results showed that both expressed proteins were folded correctly and suitable for ELISA. The value of using recombinant protein in vaccine development against malaria has been also shown in earlier studies. These surveys demonstrated that the cysteine-rich sequence of PfMSP-119 with two epidermal growth factor (EGF)-like motifs [E. coli in a correctly folded manner [In the present investigation, both antigens were produced in e motifs  could bed manner , and thad manner . In addid manner  located P. vivax (>80%) and P. falciparum (<20%) are prevalent. In the present work, the interaction between the host immune system and parasites showed that 87.1% and 84.2% of the studied individuals had positive anti-PfAMA-1 and -PfMSP-119 IgG antibody responses, respectively, suggesting that both of these expressed antigens are well-recognized asexual-stage parasite antigens. Although the present finding documents that the frequencies of antibodies to both recombinant antigens are almost similar in the areas of unstable transmission, the role of these antibodies in protection to malaria needs further study. However, the absence of such response in about 13% of the individuals could be perhaps explained by unknown human genetic factors [In elimination and eradication strategies, understanding the immunity to malaria parasites is crucial for successful and reliable interventions. The Iranian government aims to eliminate this poverty-related disease from malaria-endemic settings, mostly close to Pakistan and Afghanistan border areas, where both  factors  and/or t factors ,64.19 antigens is important for evaluating protective activity as IgG subclasses differ in their immune effector functions and having such knowledge is important for understanding the immunity to vaccine development. The result of the present study confirmed previous studies [19 IgG1 responses among studied individuals was in contrast to what was reported from Senegalese adults (in Dielmo and Ndiop) that a greater proportion of individuals were anti-MSP-1 IgG3 positive [Analysis of IgG isotype response to the PfAMA-1 and PfMSP-1 studies ,54,65,66positive . This fi19-specific IgG1 was higher among subjects (living in different areas of Brazil) with a long-term exposure to malaria, as compared to the subjects sporadically-exposed [19 IgG1 was predominant, there was also a mixed IgG1/IgG3 response as reported by others [In addition, the present result was in contrast to the previous report that stated the frequency of PfMSP-1-exposed . In thisy others . This hey others ,71. Thery others ,42,65-75P. falciparum requires several years of uninterrupted exposure [19 was not correlated with either age or number of exposure to malaria, which confirms the previous reports [19 are highly immunogenic during natural human infections. The present result was also in agreement with the result of previous studies in low-endemic areas of Senegal [19 [As the interaction between the host immune system and parasites differs based on the degree of the malaria endemicity, it has been suggested that in malaria high-endemic areas, the acquisition of natural immunity to exposure ,76. Howeexposure . In the  Senegal  and West Senegal ,77, wher Senegal  and PfMSegal [19  was obse19, the present study is in favour of using multiple antigens for antibody-based detection in this area and other similar settings during elimination programmes.In the advanced phases of malaria elimination programmes, the technique for assessment of malaria transmission intensity and evaluation of interventions during this effort are highly required. Recently, there has been a recall for elimination of malaria with the scaling-up interventions; therefore, malaria burden and transmission declined across a number of countries -82. In s19-based vaccine in Iran, where malaria is endemic. This study specially demonstrates high level frequencies of antibodies to rPfAMA-1 and rPfMSP-119 among individuals infected with P. falciparum in areas of unstable and low transmission, indicating that these two expressed antigens could be used in combination as serology markers during elimination campaigns in this region.In summary, the present results suggest that the two tested recombinant antigens are immunogenic molecules and useful tools to perform immuno-epidemiological studies in low transmission areas of Iran. These data also provide, for the first time, information on the characteristics of naturally acquired immunity in populations exposed to malaria transmission in Iran. Indeed, it could be beneficial for development and testing of a PfAMA-1 and PfMSP-1


Plasmodiumvivax malaria radical cure. However, in many endemic areas of vivax malaria, patients are treated with PQ without any evaluation of their G6PD status.Glucose-6-phosphate dehydrogenase (G6PD) deficiency causes acute haemolytic anaemia triggered by oxidative drugs such as primaquine (PQ), used for G6PD gen was performed on DNA isolated from G6PD-deficient (G6PDd) subjects through PCR–RFLP and direct DNA sequencing.G6PD deficiency and its genetic heterogeneity were evaluated in northeastern and southeastern areas from Venezuela, Cajigal (Sucre state) and Sifontes (Bolívar state) municipalities, respectively. Blood samples from 664 randomly recruited unrelated individuals were screened for G6PD activity by a quantitative method. Mutation analysis for exons 4–8 of moderately deficient, class III), while DNA analysis showed one or two mutated alleles in 19 of them (79.2 %). The G6PD A-202A/376G variant was the only detected in 17 (70.8 %) individuals, 13 of them hemizygous males and four heterozygous females. Two males carried only the 376A → G mutation. No other mutation was found in the analysed exons.Quantitative biochemical characterization revealed that overall 24 (3.6 %) subjects were G6PDd (average G6PD enzyme activity 4.5 ± 1.2 U/g Hb, G6PD A-202A/376G variant was observed among G6PDd subjects, suggesting a significant flow of G6PD genes from Africa to Americas, almost certainly introduced through African and/or Spanish immigrants during and after the colonization. The data suggest that 1:27 individuals of the studied population could be G6PDd and therefore at risk of haemolysis under precipitating factors. Information about PQ effect on G6PDd individuals carrying mild variant is limited, but since the regimen of 45 mg weekly dose for prevention of malaria relapse does not seem to be causing clinically significant haemolysis in people having the G6PD A-variant, a reasoned weighing of risk–benefit for its use in Venezuela should be done, when implementing public health strategies of control and elimination.The G6PDd prevalence was as low as that one shown by nearby countries. This study contributes to the knowledge of the genetic background of Venezuelan population, especially of those living in malaria-endemic areas. Despite the high degree of genetic mixing described for Venezuelan population, a net predominance of the mild African  G6PD gene, which cause functional variants with many biochemical and clinical phenotypes, affecting about 400 million people worldwide [Plasmodiumvivax malaria and the presumptive anti-relapse therapy in people with extensive exposure to P. vivax.Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an erythro-enzymopathy, recessive and one of the most common X-linked hereditary genetic defects, due to mutations in the orldwide , 2. The orldwide , 4. In gAlthough globally the estimated number of malaria cases decreased from 227 million in 2000 to 198 million in 2013, it remains the most important infectious disease in tropical and sub-tropical areas of the world . ImplemePlasmodium falciparum (16 %), Plasmodium malariae (<1 %), and Plasmodium vivax/falciparum mixed (1.4 %) infections [In the Americas, a substantial reduction >75 %) in the incidence of microscopically confirmed malaria cases was reached in all endemic countries between 2000 and 2013, with the exception of Guyana and Venezuela, for which a significant increase in malaria morbidity was described during the same period [ % in theciparum 1 %, Plasmciparum 1 %, PlasmP. vivax and the only drug with activity against the mature transmission stages of all Plasmodium species [P. vivax, but the haemolytic risk with this long-acting 8-aminoquinoline remains in force [The elimination of reservoirs of infection and reduction of transmission rates are crucial tasks to the success of any malaria eradication programme , 8. PQ t species . However species . Tafenoqin force . Intravain force , 11. PQ in force . Increasin force .Plasmodium infections, in agreement of selection by malaria [G6PD deficiency is still considered the most common of all clinically significant enzyme defects in human biology . G6PD de malaria , althoug malaria . A geost malaria .On the o malaria .G6PD locus shows a considerable degree of genetic heterogeneity and at least 186 distinct alleles involving mutations leading to single amino acid substitutions or deletions, scattered throughout the entire coding and non-coding region, have been identified [G6PD Mediterranean and the G6PD A-202A/376G variants, exist with an increased frequency in certain populations [G6PD A-202A/376G is distributed in relatively homogeneous manner in Africa and the Americas. Although some studies carried out in regions of South and Central America have previously shown heterogeneity of the G6PD variants [G6PD A-202A/376G has been described as the genetic variant most broadly distributed across LA, present in 81.1 % of the deficient individuals surveyed [G6PD Mediterranean563T, a C → T substitution leading to an amino acid change Ser → Phe at position 188, responsible for a severe form of G6PD deficiency, is widely distributed across southern Europe, the Middle East, through Iran, Afghanistan, Pakistan, and much of western India [Over 400 different biochemical variants of G6PD have been described globally , 15, 16.entified , 15, 17.ulations . For exavariants , the G6Psurveyed . This varn India . HoweverTo date, limited studies have been undertaken to ascertain the prevalence of G6PD deficiency in Venezuelan people –27. One G6PD genetic variant identified in apparently healthy individuals from northeastern and southeastern regions of Venezuela to assess the rate and genetic basis of G6PD deficiency in endemic areas of vivax malaria.In this study the prevalence rate of G6PD enzymatic activity was evaluated and the Blood samples from 664 randomly recruited unrelated individuals were collected during the years 2005 and 2006, of which 316 were females and 348 males. The sample represented all of those willing to do the screening and there was thus no bias in terms of gender , age and ethnic origin. All of them were born in Venezuela, had a median age of 24 years (range 1–109 years), represented 1–2 % of the total population in the areas assessed and were living in two of the regions of Venezuela with high malaria incidence: Sifontes municipality , located in the southeast of Bolívar state, near the lowland rain forest and savannas of Guayana, where the most of the population (32.621 inhabitants) is exposed at high risk to malaria due to economic activities, such as agriculture, mining and forest exploitation, and Cajigal municipality, Sucre state (332 subjects), in the northeastern coastal plains, along the Caribbean Sea, largely composed of mangroves, herbaceous and woody swamps, where the population (18.942 inhabitants) lives mainly of fishing, subsistence agriculture, and tourism  through the haemoglobin normalization procedure, following manufacturer’s instructions, and the values used to determine G6PD activity were expressed in U/g Hb (units per gram haemoglobin). The controls used were supplied by the manufacturer in three levels of G6PD activity  at 37 °C, and the cut-off point was 7.14 U/g Hb to define a subject as G6PDd on the basis of adjusted male median G6PD activity, as recommended by Domingo et al. [®, Phoenix Equipment Inc, USA). Samples of subjects with reduced activity were tested for G6PD gene mutations.The blood samples were screened for G6PD enzymatic activity using a quantitative method , according to manufacturer’s recommendations, and used as templates to search for the more common G6PD genetic variants whose mutations are located within the exons 4–8 of G6PD gen. A polymerase chain reaction (PCR) using four pairs of previously described oligonucleotide primers [NlaIII, FokI and MboII endonucleases, in accordance with procedures previously described [® Inc, Korea).Genomic DNA was extracted from G6PDd whole blood samples by using QIAamp DNA Blood Mini kit  when the size sample was less than 5 . Mean, median (both stratified by gender), standard deviation and range of G6PD enzymatic activities were calculated to determine reference values in normal and deficient subjects. Student’s test was applied for comparisons of variable values. Tests were considered significant when p < 0.05.Data were analysed for the comparisons of variables using 2 × 2 tables with χn = 16/337) and among females 2.4 % (n = 8/327), considering 5 % standard error and 95 % confidential intervals  subjects were G6PD-deficient as their G6PD activity level was less than 60 % of the adjusted male median value (Table p < 0.0001). This G6PD deficiency could be classified as moderately deficient enzyme, class III, according to the World Health Organization (WHO) criteria [p = 0.007).All G6PDd subjects detected were asymptomatic. The mean G6PD enzyme activity was of 4.5 ± 1.2 U/g Hb (ranging from 2.6 to 6.2 U/g Hb), significantly lower than that observed in normal subjects  out of 24 G6PDd subjects with deficiency detected biochemically carried the mutated allele 202G → A and 19 (79.2 %) the substitution 376A → G. These results were all confirmed by DNA sequencing. No mutation was detected in five (20.8 %) out of 24 G6PDd subjects, as well as none of the additional mutations studied by DNA sequencing mapping into the exons 4–8. The information is summarized in Table G6PD A-variant of African origin. Thirteen (76.5 %) of them were hemizygous  and four (23.5 %) heterozygous  for each mutation. In addition, the 376A → G allele was detected in hemizygosis in two males  out of 24 subjects were carriers of both 202G → A and 376A → G mutated alleles, that define the P.vivax, allowing implementation of PQ-based malaria radical cure programmes, as well as for the evaluation of new, more effective anti-parasite drugs, potentially able to cause haemolysis, and of appropriate measures of tolerability and risk of severe adverse events, are needed. Moreover, the application of G6PD deficiency tests may dramatically increase the benefits of PQ therapy by permitting the application of relatively higher dose than the current standard regimen, and shortening treatment length.Malaria remains a global health problem. In Venezuela malaria is associated with socio-economic problems and failures in preventive measures and social protection actions. The wide distribution in the world of the G6PD deficiency further hampers malaria control efforts. Epidemiological studies to evaluate the distribution of this enzyme defect, particularly in regions with the highest risk of transmission of There is currently no practical point-of-care field test for the detection of G6PDd subjects, so from a public health perspective uncertainty remains about the best procedures to follow. Studies of cost-effectiveness and budget impact for health services are recommended to support the incorporation of these tests into control programmes aimed at malaria elimination in endemic countries.G6PD mutations involved in the deficiency, G6PD genetic testing could be useful when the benefits of PQ treatment outweigh the risk in patients living in these areas. Moreover, G6PD biochemical tests could fail to detect a fraction of heterozygous subjects, although these have a bigger population of G6PD-normal erythrocytes, and therefore are unlikely to develop a serious haemolytic attack. The determination of the G6PD genetic variant allows more accurate prediction of the risk of haemolysis.Because the haemolytic risk of PQ-based regimens in malaria-endemic regions depends also on the severity of G6PD A-202A/376G genetic variant involved in such enzyme deficiency was identified and confirmed at the molecular level as predominant.In this work the prevalence of G6PD deficiency was evaluated in subjects living in two of the most prevalent malaria regions of Venezuela, and for the first time the African The overall prevalence of G6PD deficiency (3.6 %) observed in the analysed Venezuelan population was low, although it was significantly higher in the sample from Sucre state than that from Bolívar state, which is probably due to higher prevalence of African heredity in the north coastal area of Venezuela, as a result of slave immigration during colonial times . This raThe results obtained here suggest that 1:27 individuals of the studied population could be G6PDd and therefore at risk of haemolysis in the presence of precipitating factors. Low G6PDd subjects frequencies (<10 %) have been described in other LA countries, such as Argentina, Bolivia, Mexico, Perú, and Uruguay, but higher in the Caribbean islands, Guianas, Pacific coastal regions of Colombia and Ecuador, and part of the Brazilian Atlantic coast, which are areas that received the greatest contribution of Africans during the slave trade , 23.G6PD A-202A/376G, widely distributed in Africa where it seems to confer resistance to falciparum malaria [G6PD variant has been reported in Brazil, Mexico, Cuba and Honduras [G6PD gene, which include the most common mutations described in G6PDd subjects of LA countries, it is not possible to exclude those mutations located in other gene regions which can be involved in the deficiency. Even so, it is noteworthy the relatively homogeneous molecular base of the G6PD deficiency found in the analysed sample of Venezuelan population, contrasting with the high heterogeneity reported in several studies that revealed a substantial number of G6PD variants in LA populations, such as the G6PD A-376G/968C, G6PD Santamaría376G/542T, G6PD Seattle844C, G6PD Mediterranean563T, and G6PD Union1360T described in Mexico, Brazil and Cuba [Two mutations were detected in most (71 %) of subjects with biochemically determined deficiency, particularly the 202G → A and the 376A → G, defining together the variant  malaria , 36, and malaria , 37. HigHonduras , 38, butHonduras , 19, 23.and Cuba .G6PD A-202A/376G is the most common variant described among the Spanish population [Although a systematic study from individuals living in other Venezuelan regions would be desirable to better understand the genetic background in the country, the presence of only two G6PD alleles, 202G → A and the 376A → G, in the people studied can be justified. The current Venezuelan population is the result of an intense racial admixture including diverse components, such as Amerindians, Europeans and Africans . HoweverThere has been considerable confusion about the drugs that are capable of producing haemolytic anaemia in patients with G6PD deficiency. The risk of serious harm in any given population exposed to PQ therapy remains a concern. Although a mild methaemoglobinaemia typically occurs with normal PQ dosing, it is probable that other precipitating factors, such as infections or treatment with other drugs than PQ, can cause a slight shortening of the red cell lifespan in G6PDd patients and elicit a haemolytic episode. African A- has frequently been considered a variant associated with mild disease. In subjects carrying a G6PD A- variant, known to be a moderately deficient enzyme, the red cells retain more than 10 % of residual G6PD activity, mostly present in reticulocytes and younger erythrocytes . As a coP.vivax and P.ovale by killing liver stage hypnozoites, is 15 mg daily for 14 days (adult dose). This dose, used in combination with 25 mg of chloroquine, remains the recommended treatment for radical cure of adults with vivax malaria, and is implemented in Venezuela at present. However, P.vivax strains acquired in some parts of the world require a higher dose of PQ to prevent relapse. To achieve reliable eradication of parasites, the CDC has recommended an increase in dose from 15 to 30 mg daily for 14 days for adults [P.vivax not killed by standard therapeutic regimens, has been recommended to treat individuals with a partial G6PD deficiency by a WHO expert committee [vivax malaria, but the implementation of drugs into malaria eradication programmes should go along with a more thorough assessment of the clinical burden of G6PD condition in each region. Because Venezuela does not carry out the test for G6PDd before starting treatment with PQ, its use at higher doses should be applied only after a careful risk/benefit assessment in a known G6PDd individual and under strict medical supervision.On the other hand, the current Food and Drug Administration (FDA)-approved PQ dose, to prevent relapse by r adults ; althougommittee , 47, 48.The prevalence rate of G6PD deficiency was as low as that one shown by nearby countries, and the molecular base was relatively homogeneous. Understanding the G6PD deficiency and its diversity are key issues to analysing evidence of PQ safety in malaria-endemic areas. This is the first survey conducted to identify the genetic variant involved in G6PD deficiency in Venezuelan subjects, and it represents a contribution to the knowledge of the genetic structure of this population.


Plasmodium falciparum, the parasite most often responsible for severe malaria, ranges from asymptomatic parasitaemia to death. The clinical trajectory of malaria is influenced by host genetics and parasite load, but the factors determining why some infections produce uncomplicated malaria and some proceed to severe disease remain incompletely understood.The host response to infection by To identify molecular markers of severe falciparum malaria, human gene expression patterns were compared between children aged 6 months to 5 years with severe and uncomplicated malaria who were enrolled in a case–control study in Bandiagara, Mali. Microarrays were used to obtain expression data on severe cases and uncomplicated controls at the time of acute disease presentation (five uncomplicated and five severe), 1 week after presentation (three uncomplicated and three severe) and treatment initiation, and in the subsequent dry season . This is a pilot study for the first use of microarray technology in Mali.C1q, TLR2, TLR4, TLR8, and CR1 genes. Other genes previously associated with malaria pathogenesis, GZMB, FOS and HSPA6, were also higher among severe cases. TLR2, TLR4, TLR8, CR1, GZMB, FOS, and HSPA6 genes were expressed at lower levels in severe cases at late convalescence.Complement and toll-like receptor (TLR) pathways were differentially expressed, with severe cases showing higher expression of the Overexpression of genes previously associated with uncomplicated malaria was associated with severe disease. Low baseline expression of these genes may represent candidate markers for severe malaria. Despite the small sample size, results of this pilot study offer promising targets for follow-up analyses.The online version of this article (doi:10.1186/s12936-016-1189-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum, the parasite responsible for most severe malaria, is varied and complex, ranging from asymptomatic parasitaemia to death. It has been estimated that out of 400 people who are bitten by P. falciparum infected Anopheles mosquitoes, 200 will be infected, 100 will develop the uncomplicated form of the disease, two will develop severe symptoms, and one will die  and a significance level of p < 0.05 for testing the null hypothesis of whether the two sets are the same. To assess whether any specific biological pathways were over- or under-expressed the probes matching the above criteria were filtered using the Kyoto Encyclopedia of Genes and Genomes [Data were analysed using R statistical software and the BioConductor ‘limma’ package . Preproc Genomes  pathway 2(2) = 1] and a p value of <0.05 and two were housekeeping genes (beta-actin and GAPDH). The selected genes were: CYP1B1, C1QB, CD163, CCR2, CLEC4D, NAIP, TLR2, and IL18R1. Pre-designed primer and probe sets for all genes were obtained from Applied Biosystems [® reagents in accordance with the manufacturer’s recommendations. TaqMan® One-Step RT-PCR Master Mix Reagents Kit (PN 4304437) was used. Briefly, 50 ng of total RNA was used as a template for the one-step RT-PCR assay. The PCR conditions were as follows: 30 min at 48 °C, 15 s at 95 °C, 1 min at 60 °C, 40 cycles. Data were analysed using ABI software to generate delta Ct values, and the delta Ct method was used with GAPDH as Ref. [Real-time PCR was carried out on samples from seven individuals  for whom RNA remained after the microarray analysis . Ten genes were analysed, eight matching the significance threshold of greater than two-fold change [logosystems . RT-PCR  as Ref. . For eacThis study was approved by the ethics committee of the Faculty of Medicine, Pharmacy and Odonto-Stomatology, University of Sciences, Techniques and Technology of Bamako, Mali.Twenty-three uncomplicated malaria controls and 24 severe cases  were enrolled in this study. Five severe cases with cerebral symptoms and five uncomplicated malaria controls were chosen for pilot microarray studies of gene expression on the day of presentation at the clinic (acute illness time point). Three each of controls and cases returned seven days later to submit blood samples for a gene expression analysis a week later . The clinical characteristics of the acute illness and convalescence time point patients in the study are summarized in Table Due to loss to follow-up and development of other disease between the relevant time points, eight different patients were followed to study the late convalescence, low transmission dry season gene expression following either severe cerebral (four patients) or uncomplicated (four patients) malarial disease. These patients were closely followed to assure that no other clinical episodes occurred between their acute illness time point presentation and their late convalescence follow-up. The clinical data for this group are summarized in Table MB21D1, SAMSN1, PLBD1, and MKRN1 genes, had a p value <0.001. These top four genes have not been previously associated with malaria pathogenesis and/or severity. Of the remaining probes, four were in three genes, HSPA6, TLR8 and FOS, previously identified in experiments for malaria severity in human or murine systems [HSPA6 gene , with single association in TLR8 and FOS  , part of the KEGG malaria pathway, had higher expression in the severe cases compared to the uncomplicated malaria group . The other three significant genes associated with malaria in KEGG were the Complement Receptor 1 (2.05-fold higher expression in severe cases) along with glycophorin A and B . Since Staphylococcus aureus infection and leishmaniasis are infectious diseases, and each had a similar odds ratio to malaria, the top three pathways were assessed for any overlap in significant probes. The leishmaniasis pathway had three probes in common with malaria , while Staphylococcus aureus infection had one (CR1). Other significant KEGG pathways of relevance to malaria pathogenesis included complement and coagulation cascades and antigen processing and presentation with odds ratios of 6.4 and 4.5, respectively.A KEGG analysis based on the 215 significant probes showed that the malaria pathway had the second-highest odds ratio Table . A hieraversus severe patients .The top canonical pathways produced by applying Ingenuity Pathway Analysis to significant probes at acute illness were the complement system, toll-like receptor signaling and role of pattern recognition receptors in recognition of bacteria and viruses. Figure versus uncomplicated malaria presentation at acute illness from the section above were evaluated during convalescence. Eleven probes corresponding to eight genes were analysed; these genes were previously found to be experimentally relevant in severe malaria. Figure Longitudinal expression dynamics of the probes relevant to severe TLR probes showed a higher expression in severe malaria cases during acute illness . By convalescence, most of the probes still showed higher expression in severe cases, but by a smaller margin , with the exception of the TLR8 probe which showed a higher expression in the uncomplicated cases (1.36-fold). This trend continued into late convalescence, where all probes showed higher expression in uncomplicated cases as compared to severe . HSPA6 levels were higher in severe cases by 2.56-fold during the acute illness time point, by convalescence uncomplicated cases were 1.17-fold higher and in late convalescence uncomplicated cases were expressed 1.90 times higher. During acute illness, CR1 was expressed at a higher level in severe cases by 2.04, 7 days later during convalescence it was 1.09 times higher is severe cases, and by late convalescence CR1 was expressed at higher levels in the uncomplicated case patients by 1.50-fold. FOS expression was higher in severe cases during acute illness as well as during convalescence, by 2.08 and 1.11, respectively, but by late convalescence, the uncomplicated cases that showed a 1.90-fold higher expression. GZMB had a 2.33-fold higher expression in severe cases during acute illness, 2.04-fold higher expression in severe cases during convalescence, but by the late convalescence time point uncomplicated cases expressed the gene 1.66-fold higher.C1q was the only protein out of the eight candidates that did not follow a similar expression profile. Three probes corresponding to the chains of the C1q protein show a change from having higher expression in the severe patients during acute illness  to showing higher expression in uncomplicated cases during convalescence . By the late convalescence time point, the difference in these probes between uncomplicated and severe cases was seemingly indistinguishable between the groups .The Spearman Rho for the correlation of the GeneChip and RT-PCR expression was 0.87. The Pearson’s correlation between the GeneChip expression data and RT-PCR values was 0.72, but there were not enough observations to assure a normal distribution , where l screen . ExpressThis study suggests that overexpression of genes previously associated with uncomplicated malaria may also be associated with severe disease. Here, models of severe malaria were replicated in humans for the first time, and genes previously thought only to contribute to uncomplicated malaria were implicated in severe disease. In particular, genes in the complement and toll-like receptor pathways were expressed at higher levels in severe cases during acute illness. The longitudinal component of this study demonstrated that 7 days after presentation and treatment, the levels of these complement and toll-like receptor genes were indistinguishable in the uncomplicated and severe malaria patients. However, when expression of these genes was evaluated during the dry season following acute illness, patients with previous severe malaria showed lower levels of expression in these genes. These low baseline values should be evaluated as possible candidate biomarkers for severe malaria.


Plasmodium falciparum histidine-rich protein-2 (PfHRP2) is the most accurate biomarker for severe malaria, but its measurement by ELISA has been considered too unwieldy to incorporate into clinical management.Plasma Plasma samples covering a wide range of PfHRP2 concentrations were applied to rapid diagnostic tests (RDTs). RDTs were read by eye and digital capture, and PfHRP2 concentrations were measured via serial dilution with results compared to ELISA readings.® brand showed the strongest correlation (r2 = 0.963) as well as the lowest inter-observer variability (combined kappa across band intensities for three observers = 0.938). Plasma PfHRP2 measurement via serial dilution showed minimal bias compared to ELISA and acceptable limits of agreement. Three different dilutions of a well characterized set of admission samples from uncomplicated and severe malaria patients studied in a low transmission setting gave an area under the receiver operator characteristic curve of 0.844 in terms of identifying severe malaria.The ParacheckThese studies show that plasma PfHRP2 can be assessed via a single RDT, with application of a plasma dilution of 1:5 or 1:10 providing useful diagnostic information to assist in patient management or clinical trial inclusion. Plasmodium falciparum, diagnosing the cause of severe febrile illness is particularly challenging because of the high background prevalence of malaria parasitaemia. A malaria blood film or rapid diagnostic test (RDT) is often the only diagnostic available, but the presence of parasites is non-specific and malaria is frequently over-diagnosed, resulting in failure to consider and treat other life-threatening conditions [In regions with medium to high intensity transmission of nditions . Approxinditions .P. falciparum histidine-rich protein-2 (PfHRP2), a soluble protein produced by sequestered parasites responsible for severe disease, provides a reliable method to distinguish patients with severe malaria from febrile patients with an alternative cause of severe illness. Plasma PfHRP2 concentrations define malaria severity and prognosis with substantially greater accuracy than parasitaemia in African children [Plasma children –5 and Pachildren . In Africhildren  or malarchildren –9. In Afchildren .Identification of children with low plasma PfHRP2 levels is likely to benefit both individual patients and health systems, indicating the need for additional diagnostic procedures and treatments. Relevant investigations include cerebrospinal fluid examination in comatose children, blood cultures and imaging. Invasive bacterial infection is the most commonly identified alternative cause of severe febrile illness , 12 but Measurement of plasma PfHRP2 has generally been undertaken via ELISA, taking several hours and requiring resources that are beyond the majority of laboratories in malaria endemic regions. Earlier work described the feasibility of using a standard diagnostic RDT to quantify plasma PfHRP2 . Here th≥16-year-old) admitted to hospital with slide- or RDT-confirmed falciparum malaria and enrolled in prospective studies at Chittagong Medical College Hospital, Bangladesh (2011); uncomplicated and severe cases were defined using modified World Health Organization criteria [Characterization of the conditions under which RDTs could be applied was undertaken in two phases with plasma samples from children recruited into the Teule, Tanzania site of the AQUAMAT severe malaria trial comparing quinine and artesunate  Fig. 1)1). The pcriteria . ELISA mcriteria , 18.Fig.™ Malaria Pf kit , Paracheck® Pf Device (V.3) , First Response® Malaria Antigen HRP2 kit  and SD Bioline Malaria Ag Pf kit . A single batch of each RDT was used for all work. All brands passed Phase 2 of Round 1–3 of the WHO testing programme [Four RDT kits solely based on PfHRP2 detection were studied: CoreTM XR+, using the Colorimetric application optimized for faint bands and capturing the maximum intensity of each selected area; on occasions small white spots just prior to the reaction line (areas that remain dry) rendered the mean value for the band an unreliable estimate of band intensity. As well as the PfHRP2 line itself, readings were taken from the background area of the nitrocellulose strip and the positive control band. For non-linear regression, this background intensity was subtracted from the test band result to produce a net reading representing the band strength relative to background.The intensity of bands was assessed by digital scanning and visual assessment. Scanning was undertaken using a BioRad Gel DocAfter application of samples test kits were relabelled using a code and assessed by three independent observers blinded to the identity of each sample. Band intensity was graded as 0 (no visible band), 1 (faint line), 2 (faint band), 3 (clear band weaker than control) and 4 .Given that all tests were undertaken with plasma, it was appropriate to reassess the timeframe over which band strength remained stable visually and by digital scanning. In the first phase of work . Fifty neat samples from Teule, Tanzania, with plasma HRP concentrations ranging from zero to 22,012 ng/ml were applied to each of four kits. The PfHRP2 concentration associated with half-maximal signal in each kit ranged from 36.2 (Paracheck®) to 75.9 ng/ml (First Response®) , indicating very good agreement across the three observers. Identification of strong test bands (category 4) or absent bands (category 0) from other bands appeared to be most robust, while defining bands of intermediate character (categories 1–3) was generally associated with poorer levels of agreement.Results from visual assessment closely agreed with scanning Fig. b. InteroSeven samples with relatively high plasma concentrations (above 7000 ng/ml) were included in order to check for a possible prozone phenomenon, in which band intensity drops due to flooding of the test strip with free antigen. There was no convincing evidence of any reduction in signal in these highly concentrated samples for any of the kits.® kit was then used to test the ability of an RDT to quantitate plasma PfHRP2 in an independent set of 20 clinical samples. Samples underwent half-log serial dilutions in PBS producing 1:3.2, 1:10, 1:32, 1:100 and 1:320 dilutions. The neat sample was also applied if the 1:3.2 dilution failed to produce a strong band. These dilutions were applied in triplicate to Paracheck® kits and digital readings obtained as previously (test band and background). A log dilution-signal curve was plotted and the EC50  calculated  were approximately 1000 ng/ml [The final step was to explore the accuracy of visual judgment using a single RDT to discriminate severe from uncomplicated malaria in 72 patients admitted with malaria in a low transmission setting . Clinical severity assessment was considered as gold standard since incidental malaria in these areas is rare. The visually robust cut-off between a strong band category 4) and lower strength bands (0–3) was used, with a band clearly weaker than positive control indicating a low probability of severe malaria. Choice of possible dilutions was informed by the mid-point of the dynamic range of the Paracheck and loweP. falciparum malaria. A PfHRP2-based RDT to define plasma levels would allow practical assessment in low-resource settings and could be the first field-based test since microscopy to provide information on the contribution of malaria in severe ill children [® RDT appeared to have the tightest correlation with ELISA as well as close inter-observer agreement. The measured agreement also appeared superior to previous studies of dipstick assessment for this purpose [There is still substantial room to refine the diagnosis of severe febrile illness in high transmission settings, and plasma PfHRP2 holds considerable promise in this regard. No other biomarker for cerebral malaria has demonstrated the high positive and negative predictive values seen for plasma PfHRP2 , and it children . The wor purpose .The area under the receiver operator characteristic curve for a single RDT in discriminating severe from uncomplicated malaria was found to be 0.844, based on admission plasma samples from an established cohort of uncomplicated and severe adult malaria patients studied in a low transmission setting  , the prIn summary, this work shows that a single RDT can be used to obtain useful information on plasma HRP2 concentration and hence the underlying cause of severe febrile illness. Application in the field would be enhanced by engineering a simpler testing approach, for example by development of an RDT in which plasma selectively enters the nitrocellulose strip at an appropriate concentration. Incorporation into management algorithms holds promise to refine the diagnosis of severe febrile illness, a common syndrome associated with high mortality.


Plasmodium falciparum. Based on these findings this study investigates the predictive values of blood count alterations during malaria across different sub-populations.Malaria has been shown to change blood counts. Recently, a few studies have investigated the alteration of the peripheral blood monocyte-to-lymphocyte count ratio (MLCR) and the neutrophil-to-lymphocyte count ratio (NLCR) during infection with Cases and controls admitted to the Department of Infectious Diseases and Tropical Medicine from January 2000 through December 2010 were included in this comparative analysis. Blood count values and other variables at admission controlled for age, gender and immune status were statistically investigated.P. falciparum (68%), Plasmodium vivax (21%), Plasmodium ovale (7%) and Plasmodium malariae (4%), and 210 controls. A positive correlation of parasite density with NLCR and neutrophil counts, and a negative correlation of parasite density with thrombocyte, leucocyte and lymphocyte counts were found. An interaction with semi-immunity was observed; ratios were significantly different in semi-immune compared to non-immune patients (P <0.001).The study population comprised 210 malaria patients, infected with The MLCR discriminated best between malaria cases and controls , whereas the NLCR better predicted severe malaria, especially in semi-immune patients (AUC = 0.788).Malaria causes typical but non-specific alterations of the differential blood count. The predictive value of the ratios was fair but limited. However, these changes were less pronounced in patients with semi-immunity. The ratios might constitute easily applicable surrogate biomarkers for immunity. The ratio of neutrophil-to-lymphocyte count (NLCR) was proposed as a parameter of systemic inflammation and stress in severely ill patients in intensive care unit . De Jage setting . The NLC setting .et al. found that the NLCR correlated with parasite density (parasitaemia) in imported cases. NLCR and lymphocytopaenia were investigated as prognostic markers for treatment outcome of severe malaria cases but both were inferior to CRP levels [Thrombocytopaenia, as well as anaemia, are well-known characteristics occurring during and following plasmodial infection ,5. LeucoP levels .et al. (formerly “ML-ratio”) as a parameter for the risk of clinical manifestation of malaria. They concluded that the MLCR could reflect an individual’s protection capacity against clinical manifestation of Plasmodium falciparum malaria [The monocyte-to-lymphocyte count ratio (MLCR) was introduced by Warimwe  malaria .In this study, blood count, NLCR, MLCR, neutrophil-to-monocyte count ratio (NMCR) and other unspecific parameters of malaria cases at admission were investigated and compared to those of healthy controls. Results were controlled for age, gender, immune status, and parasitaemia.A database from the Department of Infectious Diseases and Tropical Medicine (DITM) of the Ludwig-Maximilians-Universität of Munich recording all clinical, epidemiological and laboratory data of patients who presented from January 2000 through December 2010 was used as source for this analysis. The first group (further referred to as “cases”) comprised all laboratory-confirmed malaria patients of both gender and all age groups. Patients with positive bacterial blood cultures, severe underlying diseases and other concomitant diseases were excluded. Individuals matched by gender and age comprised the control group, further referred to as “controls”. A control was defined as a healthy individual without acute or severe underlying chronic diseases at admission who had been admitted for a certificate of fitness before working in tropical regions.Gender, age, origin correlating with immunity against malaria, country of travel, blood count including differentiation, and lactate dehydrogenase (LDH) were included in the main analyses. Liver enzymes , bilirubin and creatinine were used in subanalyses for malaria patients only. Dependent of their birthplace in endemic malaria areas according to the WHO , study pAll cases and controls provided written informed consent that their biological samples and that consequent results may be used for research purposes and publication in a fully anonymized manner. Ethical clearance was approved by the Ethical Committee of the Ludwig-Maximilians-Universität.All laboratory procedures were performed as part of routine diagnostics. Thin and thick blood smears were prepared by specialized laboratory technicians and stained by Romanowski stain (Diff-Quick®) and 10% Giemsa solution for 20 min, respectively. All smears were independently read by two experienced microscopists. Two-hundred fields of thick smears were read, thin smears were used for species discrimination and quantification of parasitaemia expressed in per cent. Blood count was measured with an automated system  and differential blood count was read manually by microscopy. LDH and other liver enzymes were analysed by an external, accredited laboratory.Data were de-identified and transferred from medical report forms into an electronic database by double entry and cleaned by two independent researchers. The homogeneity of both groups was based on age and gender. The first null hypothesis stated that there is no difference of the MLCR in cases compared to controls. Likewise the second null hypothesis was defined with the NLCR as outcome variable. The null hypotheses were assessed by comparing the median ratios in each group using the non-parametric, unpaired, two-sample Mann-Whitney-Wilcoxon test, as no normal distribution was assumed. For further analysis, ratios were categorized and tested in a logistic regression model controlled for gender, age and variables significantly associated with the outcome affirmed by likelihood ratio test. Predictive accuracy was determined by the calculation of the area under the ROC (receiver operating characteristics) curve (AUC). Correlations between continuous variables were analysed by linear regression models if appropriate. All variables were tested for interaction. The statistical software used was Stata, version 11.0 .P. falciparum, 21% with Plasmodium vivax, 7% with Plasmodium ovale, and 4% with Plasmodium malariae. Only two mixed infections with P. falciparum and P. vivax were included in the analysis and counted as P. falciparum; parasitaemia was below 1%.The univariate analysis of baseline data is presented in Additional file P =0.005). Most plasmodial infections were acquired in West Africa (60%), followed by East, Central and Southern Africa (19%), Asia with Pacific region (19%), and Latin America (2%). Additional file Median age of all travellers was 36 years. Matching of very old cases with controls was difficult as controls were recruited from the working population  than SI cases (3%). Ninety per cent of SI travellers returned from East Africa. Patients returning from East Africa were half SI and half NI travellers. In all other regions, SI cases were under-represented. Parasitaemia ranged from 0.1 to 13% in P. falciparum; nine patients had a parasitaemia ≥2%. All other species showed a parasitaemia ≤1%. Parasitaemia in NI cases was similar to densities in SI cases (P = 0.224). Thirty-three P. falciparum and five P. vivax/Plasmodium ovale patients showed signs and symptoms of severe malaria at admission including hyperparasitaemia (≥2%), 3-times elevated liver enzymes and bilirubin ≥ 3.0 mg/dL[The proportion of SI travellers in the control group was negligible (2.4%) but one third of the cases were categorized as SI. 3.0 mg/dL,11. MediP <0.001 each).Median haemoglobin, thrombocyte count, overall leucocyte as well as lymphocyte count were significantly lower in cases Figure . Median Plasmodium malariae, most probably due to the very low sample size . Relative and absolute lymphocyte counts were negatively correlated with parasitaemia . The relation of monocytes with parasitaemia was non-linear. Monocyte count was elevated in uncomplicated malaria but decreased in severe malaria, which was observed in SI and NI cases. In consequence, no evident correlation of parasitaemia with the MLCR or NMCR was found but a strong positive correlation with the NLCR (P = 0.014) could be shown for all cases . Also thrombocytopaenia showed a strong positive correlation with parasitaemia.All correlations if not otherwise mentioned were controlled for age, gender and immune status. A significant positive correlation between parasitaemia and absolute or relative neutrophil as well as band cell count in all cases was shown . The difference was not detectable in acutely ill patients. Median neutrophil and lymphocyte count, and the NLCR were similar in both genders.Median monocyte count was significantly lower in healthy women compared to healthy men. In consequence, healthy women generally had a slightly but significantly lower MLCR and higher NMCR than men . Sensitivity, specificity and the positive predictive value (PPV) for malaria were 59.0, 80.0, and 67.5%, respectively. As SI malaria cases had a significantly lower MLCR, an accordingly lower cut-off was chosen; the lower limit of the 95% CI in malaria patients was 0.15. However, no evident difference between controls and cases was found (P = 0.200).To categorize the MLCRs in NI and SI travellers, the lower limit of the 95% CI in malaria cases in each group was chosen as cut-off Table . MultivaSimilar results could be shown for the NLCR using logistic regression models. A 5.2-times higher risk for malaria was found in NI travellers but no difference for SI travellers Table . HoweverAs described in other studies -7, bloodP. falciparum malaria [The majority of patients with imported  malaria  as well  malaria -16 had lThe association of lymphocyte count and severity of malaria has been controversially reported ,3,17. InLymphocytopaenia is reported to be accompanied by an increase in neutrophil count. This is interpreted as a sign of systemic inflammation and stress, and occurs in many infectious and non-infectious diseases ,15,18.In a recent study, the efficacy of the RTS,S vaccine, measured by malaria episodes during follow-up, was negatively correlated with the MLCR at vaccination . The higet al. found a positive correlation of NLCR and parasitaemia in patients with imported malaria [The differences in neutrophil count and NLCR between malaria patients and controls were distinct Table . Wolfwin malaria . Median  malaria . As normet al.; they propagated a protective effect in malaria by high levels of inducible nitric oxide synthase mRNA that is associated with an increased monocyte count in uncomplicated cases. Patients with severe malaria presented with a low to normal monocyte count, similar to those of controls [The linear correlation of parasitaemia with lymphocytes or neutrophils was not detectable for monocytes Figure . Similarcontrols .The distinct difference of relative white blood cell count and both ratios depending on the immune status against malaria was the most interesting finding in this study. However, data have to be interpreted with caution. Definition of semi-immunity is difficult, especially when accurate data on the exact origin, living conditions, disease episodes in the past, the duration of parasite-free intervals, and antibody titres are missing. This might result in an overestimation of semi-immunity in this group. Semi-immune patients in the NI group seem to be rather unlikely. The similar ratios and differential blood count constellations in all severe cases independent of the theoretical immune status raise the question whether those 11 severe SI patients might have lost immunity. In any way, the striking difference in uncomplicated cases remains.et al. as parameter for the risk of clinical manifestation of malaria in children [The MLCR was measured by Warimwe children . AsymptoP =0.004). The number of children under 15 years was very limited, accurate analysis was impossible but should be performed with a bigger sample size to investigate leucocyte behaviour in children with malaria.The most critical part of the study is the low sample size of healthy SI travellers. All references to this group might not be reliable as shown normal blood values might not be representative. The non-significant lower MLCR in SI controls could be related to the high female proportion (80%) as healthy women in general had a significantly lower MLCR than male controls , P. vivax predominantly in Asia and the Pacific Region (70%), P. ovale mostly in Africa (82%) and P. malariae (75%) mainly in Africa. Almost all P. vivax cases from Africa were imported from Ethiopia.Click here for fileCorrelation of NLCR and parasitaemia. NLCR and parasitaemia were positively correlated. The correlation fitted best with low to moderate parasitaemia. The sample size of cases with high parasitaemia (≥2%) was low.Click here for fileNMCR in different variables. Medians (with 25 and 75% percentiles/IQR) of the NMCR are given. Ranked means were compared by Mann-Wilcoxon-Whitney test and p-values were presented. P-values <0.05 are marked in bold. There is no linearity of monocytes. NMCR behaves contrary to MLCR. There is no difference between severe and uncomplicated by MLCR but between cases and controls. There is only a slight difference between uncomplicated cases and controls by NMCR  but a distinct difference between severe and uncomplicated cases. Discrimination of severe und uncomplicated cases was better by NLCR than NMCR.Click here for file