We conducted a clinical study of pregnant women in Blantyre, Malawi to determine the effect of the timing of malaria infection during pregnancy on maternal, infant and placental outcomes. Women were enrolled in their first or second trimester of their first or second pregnancy and followed every four weeks until delivery. Three doses of sulfadoxine-pyrimethamine were given for intermittent preventive treatment for malaria, and all episodes of parasitemia were treated according to the national guidelines. Placentas were collected at delivery and examined for malaria parasites and pigment by histology. Pregnant women had 0.6 episodes of malaria per person year of follow up. Almost all episodes of malaria were detected at enrollment and malaria infection during the follow up period was rare. Malaria and anemia at the first antenatal visit were independently associated with an increased risk of placental malaria detected at delivery. When all episodes of malaria were treated with effective antimalarial medication, only peripheral malaria infection at the time of delivery was associated with adverse maternal and infant outcomes. One quarter of the analyzed placentas had evidence of malaria infection. Placental histology was 78% sensitive and 89% specific for peripheral malaria infection during pregnancy. This study suggests that in this setting of high antifolate drug resistance, three doses of sulfadoxine-pyrimethamine maintain some efficacy in suppressing microscopically detectable parasitemia, although placental infection remains frequent. Even in this urban setting, a large proportion of women have malaria infection at the time of their first antenatal care visit. Interventions to control malaria early and aggressive case detection are required to limit the detrimental effects of pregnancy-associated malaria.  Pregnancy is a unique period of vulnerability to malaria infection. This increased susceptibility is attributed to the ability of infected erythrocytes to sequester in the developing placenta, causing chronic infection and placental inflammation. In malaria-endemic areas, pregnancy-associated malaria causes maternal anemia and low infant birth weight . BecauseWe hypothesized that the detrimental effects of pregnancy-associated malaria would not be distributed uniformly throughout pregnancy and that the timing of the malaria infection would significantly affect placental pathology as well as clinical outcomes in mothers and infants. We designed a prospective study to examine the effect of timing of malaria infection on placental, maternal and infant outcomes. Within the context of the standard administration of SP IPTp, we enrolled women as early as possible during pregnancy and conducted intensive evaluation of maternal infection status as well as infant and maternal outcomes. We actively screened for malaria infection to determine the rates of microscopically detectable parasitemia during pregnancy and used a combination of histopathology and molecular techniques to detect malaria parasites in the placenta.The study was conducted in Ndirande, a peri-urban township of Blantyre in the southern region of Malawi. Malaria transmission occurs year-round, peaking in the rainy season from December to March. The government health center and antenatal clinic are the only public providers of health care for the catchment area with a population of over 200,000. Participants were recruited from the antenatal clinic. A maternity ward is also located on the health center compound, where deliveries attended to by nurse midwives occur 24 hours a day. Complicated deliveries are referred for obstetric care to Queen Elizabeth Central Hospital, the government referral hospital in southern Malawi, located approximately five kilometers from the health center.All pregnant women residing within the health facility catchment area who were in the first or second pregnancy, at least 15 years old, and less than or equal to 28 weeks gestation were eligible for the study. Women with any major illnesses or who were on chronic antibiotic treatment were excluded from the study. Women known to be HIV-infected were initiated on trimethoprim-sulfamethoxazole prophylaxis according to the national policy, and thus not eligible to receive IPTp with SP; these women were excluded from the study. Informed consent was obtained before conducting any study procedures. On enrollment, study participants were assigned a study identification number. Using a standardized questionnaire, information was collected on demographic and socio-economic characteristics, and past obstetric and medical history. Detailed contact information was also collected and used to trace participants if they did not attend a scheduled visit. Clinical examination included blood pressure, axillary temperature and anthropometric measurements. Assessment of gestational age at enrollment was done using the last menstrual period and measurement of fundal height. A finger prick blood sample was obtained for preparation of blood smears, hemoglobin measurement (HemoCue AB) and storage of a dried blood spot on filter paper. All pregnant women received an insecticide-treated bed net and iron and folic acid supplementation according to standard procedures in the antenatal clinic. The first dose of SP IPTp was given at the enrollment visit or at 20 weeks gestation if enrollment occurred earlier in pregnancy.Study participants were seen at the clinic every four weeks and were encouraged to return every time they were ill. At every routine visit and any visit for an illness suspicious of malaria, a finger prick blood sample was collected to measure hemoglobin levels, to prepare blood smears, and to prepare a filter paper specimen. Women received SP as IPTp up to three times during their pregnancy, separated by at least four weeks. All episodes of malaria parasitemia detected at routine and sick visits were treated with an antimalarial drug in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters), regardless of symptoms. Women with evidence of severe malaria were referred to the hospital for evaluation and treatment. Parasitemia occurring within 28 days of the initial infection was considered to be the same episode.Study participants were encouraged to deliver at the maternity ward at Ndirande Health Centre and those with complications were referred to Queen Elizabeth Central Hospital. After delivery, the placenta was stored and transported to the research clinic where placental blood was collected for parasite DNA detection. Biopsy specimens were preserved in formalin for histopathological processing. Newborns were assessed and gestational age was estimated using a modified Ballard score . InfantsWomen were classified as having anemia if they had a hemoglobin concentration less than 11 g/dL. Anemia was further classified as mild (9.0-10.9 g/dL), moderate (7.0-8.9 g/dL) or severe (<7.0 g/dL). Fever was defined as axillary temperature greater than or equal to 37.5 °C. A birth weight of less than 2500 grams was classified as low birth weight. Gestational age at enrollment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined by combining information from the last menstrual period and the Ballard exam. If the gestational age by last menstrual period and the Ballard exam were less than two weeks apart, menstrual dating was considered the best obstetric estimate of gestational age. If there was a discrepancy of more than two weeks, fundal height was included in the determination of the best estimate and was used for evaluating birth outcomes. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. A gestational age less than 37 weeks at delivery was classified as premature delivery. Birth weight for gestational age, based on the WHO growth curves less than -2 Z-score was considered small for gestational age [Peripheral blood slides were Field stained and examined microscopically using a 100x oil immersion objective to detect and quantify parasitemia. A diagnosis of microscopically-detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. All slides were read by two microscopists and in cases of disagreement between the readings, the conflict was adjudicated by a third expert reader.http://medschool.umaryland.edu/malaria/protocols.asp). Only specimens with parasites detected by histology and confirmed by PCR were considered positive for parasites. Biopsies with parasites or pigment were classified as placental malaria infection.Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with hematoxylin and eosin. Slides were examined for presence of malaria parasites and pigment. If parasites were seen on the slide, a placental blood specimen was used to extract and test for the presence of malaria DNA based on quantitative PCR, as described on our website . Demographic characteristics were described using frequencies and percentages for categorical data and means and 95% confidence intervals (CI) for continuous variables. All data collected from participants between enrollment and delivery or termination was included in the analysis. Episodes of malaria during the follow up period were recorded for participants who had at least one visit beyond the enrollment period. Time under observation began at enrollment and continued until delivery or the last visit prior to study termination, and individuals were considered continuously at risk while under surveillance. Proportions were compared using Fisher’s exact test and odd ratios were calculated using logistic regression. For the analysis of the association between the timing of malaria infection and outcomes, logistic regression was used to compare individuals who experienced malaria infection to individuals who did not experience malaria infection in each trimester. Infections in the second and third trimester were included in the model. Multivariate models were developed when more than one risk factor was associated with the dependent variable of interest. Independent variables were only included in the final model if they had an association with the dependent variable in the univariate analysis with a p-value equal to or less than 0.1.A total of 450 women were enrolled into the study from June 2009 through June 2010. The median age of the participants was 20.1 years (95% CI 19.8-20.4). The majority of women were in their first pregnancy, had received some formal education and had adequate shelter and access to clean water. Approximately half of the women had slept under a bed net on the night before enrollment. The baseline characteristics of the pregnant women at enrollment are listed in Including all 450 participants at enrollment and the 407 women who had at least one follow up visit, 93 episodes of malaria were detected among 79 individuals during enrollment and the antenatal follow up period. The incidence density of malaria infections was 0.6 malaria infections detected per person year of follow up. Seventy percent of women who ever had malaria were initially found to be infected at enrollment (55/79). Twenty-four women who did not have detectable parasitemia at enrollment developed malaria infection during their pregnancy and nine had malaria both at enrollment and later. Most second episodes of malaria occurred within four to eight weeks of the initial diagnosis. Among the 407 participants who had at least one follow up visit, 302 (74.2%) received three doses of SP for IPT and 82 (20.1%) received two doses.Most infections were asymptomatic. Among all cases of peripheral parasitemia detected during the follow up period (excluding three episodes detected at delivery), 13/91 (14%) were accompanied by an objective measure of fever at the time of evaluation and 16/91 (18%) reported a history of fever. Fever detected at the visit and the report of fever in the past 48 hours were independently associated with a four-fold increase in the odds of a positive malaria smear (Odds Ratio (OR) 4.5, 95% CI 2.1-9.9 and OR 4.4, 95% CI 2.2-8.8 respectively).Based on last menstrual period at enrollment, four infections occurred in the first trimester, 63 in the second trimester and 16 in the third trimester. This represented 8.2%, 6.2% and 1.1% of all malaria slides obtained during those trimesters (p<0.001). After excluding the episodes of parasitemia prevalent at enrollment, the incidence of malaria was slightly higher in the second trimester than the third although the difference was not statistically significant (12/591 (2.0%) vs. 16/1398 (1.1%) of smears, p=0.1). The timing of the malaria infection based on the best assessment of gestational age at delivery had a similar distribution, but fewer infections were classified as occurring in the first trimester. Three episodes of peripheral parasitemia were detected at delivery. For two of the participants with malaria at delivery, this was their only episode of malaria and negative malaria smears were obtained within four weeks prior to delivery. For the third participant, this was a positive malaria smear four weeks after a previous episode of parasitemia that was treated with artemether-lumefantrine. The timing of the peripheral malaria infections is shown in  The number of participants with malaria during each trimester based on estimated gestational age at enrollment (blue) and estimated age at delivery (red) is shown, with lighter shading indicating infections detected at enrollment.Histopathology results were available for 306 placentas from singleton deliveries. Four twin deliveries were excluded. Seventy-five (24.5%) had evidence of malaria infection. Of the women who had placental malaria, four (5.3%) had parasites detected, 62 (82.7%) had pigment detected and nine (12.0%) were found to have both parasites and pigment. Among the 60 participants known to have malaria during pregnancy and histopathology results, 47 had positive findings on histology, representing a sensitivity of 78.3% of placental histology for identifying prenatal peripheral parasitemia. There were 248 participants with available placental histopathology results and no positive malaria smears during pregnancy, 220 had no evidence of malaria on placental histology, representing 88.7% specificity. One placenta was collected from a woman who had no malaria smears obtained after enrollment. Participants whose placentas were not analyzed were younger and more likely to be in their first pregnancy than women who did have histopathological results (mean age 19.2 vs. 20.4 years and 34.7% vs. 22.4% primigravid respectively). However, the prevalence of malaria at screening and the incidence of malaria during follow up were the same.No demographic or socio-economic factors were associated with placental malaria, but peripheral malaria and the presence of anemia at enrollment were associated with placental malaria. Infection present at the time of enrollment in antenatal care was more strongly associated with placental infection than malaria occurring during the follow up period. There was a trend towards younger age being associated with placental malaria infection. In multivariate analysis, malaria present at enrollment, malaria during the follow up period and hemoglobin at enrollment were independently associated with placental malaria. Results of univariate and multivariate analysis are shown in The relationship between the timing of malaria infection and specific placental findings is shown in Information on maternal hemoglobin levels at delivery was available for 321 women. The mean hemoglobin level was 12.8 g/dL (95% CI 12.6-13.0). Thirty-five women had anemia at delivery with four (1.3%) women having moderate and 31 (9.7%) having mild anemia. Malaria during pregnancy overall and placental malaria were not associated with differences in maternal hemoglobin. Peripheral malaria at the time of delivery was associated with increased rates of maternal anemia compared to infections during other trimesters .Birth outcomes were available for 330 newborns. Among nine women (2.4%) whose deliveries resulted in stillbirths, eight never had malaria detected during pregnancy or in the placenta whereas one had four episodes of malaria with pigment detected in the placental specimen. Thirteen neonatal deaths occurred. Eleven of the deaths occurred in infants whose mothers did not have any evidence of peripheral or placental malaria during pregnancy One infant was born to a mother who had malaria during the second trimester but whose placenta did not show any infection and another infant was born to a mother who did not have malaria detected during pregnancy but did have malaria pigment found in the placenta.The mean birth weight was 2.8 kilograms (95% CI 2.8-2.9) and the estimated gestational age at delivery was 38.6 weeks (95% CI 38.2-38.9). Fifty (15.1%) infants were born preterm, 58 (17.9%) were low birth weight and 43 (13.3%) were small for gestational age. Birth weight and gestational age were not associated with exposure to malaria during pregnancy overall or in any one trimester except for the finding that mothers with malaria at delivery were at a significantly higher risk of delivering a small for gestational age infant than mothers with malaria at other times during pregnancy . Number of episodes of malaria during pregnancy was also not associated with any of the adverse infant outcomes.In this study the timing of malaria during pregnancy had significant effects on the clinical outcomes of pregnancy. Infection that occurred early in pregnancy, before or just at the onset of the initiation of antenatal care was strongly associated with evidence of placental infection. Lower hemoglobin at first antenatal visit, even in the absence of parasitemia, was a strong predictor of placental malaria. In the context of SP IPTp and also active case detection and treatment, malaria during pregnancy and placental malaria were not associated with adverse maternal or infant outcomes with the exception of the association of peripheral malaria infection found at delivery with small for gestational age infants and maternal anemia.Previous studies have not provided a consistent characterization of the effects of malaria during different periods of gestation on maternal and infant outcomes. A similar study conducted in Malawi, but with two rather than three doses of SP IPT, demonstrated that the number of episodes of malaria during pregnancy was associated with maternal anemia, and malaria during the second trimester was associated with low birth weight . In BeniThe results of this study confirm the moderate usefulness of monitoring placental malaria as a proxy for either peripheral malaria infection during pregnancy or as a surrogate for adverse outcomes associated with pregnancy malaria. The specific histological findings did generally correlate with the expected findings that pigment represents prior infection and parasites represent recent or on-going active infection . HoweverThe prevalence of malaria was highest at the first visit for antenatal care and was infrequently detected during follow up. This points to the continued ability of SP to suppress microscopically detectable blood stage infection. Because there was no untreated control arm for this study, it is not possible to measure the effect of SP IPTp on placental infection. However, the high rate of placental abnormalities associated with malaria infection, particularly pigment deposition, suggests that even three doses of SP IPTp do not provide adequate protection against or cure placental infection. This may be due to poor curative efficacy of SP against malaria in Malawi, like other parts of eastern Africa. Another contributor might be the late timing of the initial SP dose. Women who present with malaria at the initial antenatal visit are at the highest risk of placental infection. The intervention may be administered after parasites have already sequestered in the placenta.Although active peripheral malaria infection at delivery was associated with low birth weight and small for gestational age infants, it is not biologically plausible that a new infection that late in pregnancy would have a significant impact on birth weight. Rather, we suspect that patent parasitemia at delivery may reflect on-going low density parasitemia that was likely occurring throughout pregnancy and led to reduced birth weight. This finding was a result of a small number of cases so further investigation is required to confirm and explore this association.Several features of the study design limit the generalizability of this study. Most women were enrolled late in the second trimester of pregnancy, so we had limited information about the effects of malaria infection during the first half of the pregnancy when placental implantation and transformation of the maternal circulation occurs. Detection and treatment of malaria in the absence of symptoms is not currently standard practice in most malaria endemic countries. However, increasing the attention to the role of asymptomatic infection in adverse outcomes, the pervasiveness of SP resistance and the widespread availability of rapid diagnostic tests may support the addition of this strategy to public health practice. This analysis is limited to the detection of parasites detected through microscopy. Though submicroscopic parasitemia has been shown to be associated with adverse outcomes ,10, we lThe results of this study suggest several key public health implications. First, currently recommended interventions to prevent malaria during pregnancy may be provided too late in pregnancy to achieve the desired impacts. Presentation for antenatal care half way through pregnancy is typical throughout sub-Saharan Africa . HoweverThe association between anemia and placental malaria suggests that screening for anemia at the first antenatal visit may help to identify pregnant women at risk for placental infection. Anemia may be a marker for infections sequestered in the placenta that are not microscopically detectable in the peripheral blood or they may indicate an individual who is frequently infected with malaria. Hemoglobin concentration can be measured from the same finger prick used to diagnose malaria using inexpensive, field-friendly techniques. Further investigations could explore the utility of providing first line antimalarial treatment instead of or in addition to the first dose of SP IPTp.In addition, this study supports the added benefit of screening for malaria infection even in the context of the appropriate administration of SP IPTp. We and others have demonstrated that the ability to detect and treat microscopically detectable malaria during pregnancy prevents the typical detrimental effects of pregnancy-associated malaria ,12. The Malaria infection occurs most frequently early in pregnancy, before women typically seek obstetric care, pointing to the urgent need to expand antimalarial interventions outside of the antenatal clinic. The combination of three doses of SP IPTp, routine screening and treating all detectable malaria infections does not prevent placental infection but appears to mitigate the adverse clinical outcomes typically associated with placental malaria.


Plasmodium vivax is traditionally known to cause benign tertian malaria, although recent reports suggest that P. vivax can also cause severe life-threatening disease analogous to severe infection due to P. falciparum. There are limited published data on the clinical and epidemiological profiles of children suffering from ‘severe malaria’ in an urban setting of India. To assess the clinical and epidemiological profiles of children with severe malaria, a prospective study was carried out during June 2008–December 2008 in the Department of Pediatrics, Guru Teg Bahadur Hospital, a tertiary hospital located in East Delhi, India. Data on children aged ≤12 years, diagnosed with severe malaria, were analyzed for their demographic, clinical and laboratory parameters. All patients were categorized and treated as per the guidelines of the World Health Organization. In total, 1,680 children were screened for malaria at the paediatric outpatient and casualty facilities of the hospital. Thirty-eight children tested positive for malaria on peripheral smear examination (2.26% slide positivity rate). Of these, 27 (71%) were admitted and categorized as severe malaria as per the definition of the WHO while another 11 (29%) received treatment on outpatient basis. Most  cases of severe malaria (n=27) were infected with P. vivax. Among the cases of severe malaria caused by Plasmodium vivax (n=24), 12 (50%) presented with altered sensorium , seven (29.1%) had severe anaemia (haemoglobin <5 g/dL), and 17 (70.8%) had thrombocytopaenia, of which two had spontaneous bleeding (epistaxis). Cases of severe vivax malaria are clinically indistinguishable from severe falciparum malaria. Our study demonstrated that majority (88.8%) of severe malaria cases in children from Delhi and adjoining districts of Uttar Pradesh were due to P. vivax-associated infection. P. vivax should, thus, be regarded as an important causative agent for severe malaria in children. Plasmodium falciparum .The study was conducted among children aged less than or equal to 12 years. Children with a short duration (<3 days) of fever (temperature >38 °C) without any localized symptoms were screened from the paediatric outpatient and casualty.Plasmodium species.The diagnosis of malaria, including identification of species, was established by examination of thick and thin smears of peripheral blood. Slides were prepared, stained with Giemsa, and examined under an oil immersion microscope. At least 100 high-power fields (40X) of the thick smears were examined to rule out malaria. Malarial antigen test (optiMAL) and real-time polymerase chain reaction (PCR) could not be done due to their non-availability at our centre, although these are considered better sensitive methods for the detection of The demographic profile, clinical features, and clinical course of illness were recorded. A baseline haemogram with platelet count, blood sugar, serum creatinine, prothrombin time, partial thromboplastin time, blood lactate, pH estimation, urine examination, and chest skiagram were performed in all the cases. Investigations, such as cerebrospinal fluid examination, urine culture, blood culture, neuroimaging, ultrasonography of abdomen, and liver function tests (serum bilirubin and serum aminotransferases), were conducted where deemed essential.P. vivax. Primaquine was given in the dose of 0.25 mg/kg/day for 14 days in the case of P. vivax and a single dose of 0.75 mg/kg for P. falciparum.Based on the clinical and laboratory parameters, children who were categorized as severe malaria as per the guidelines of the World Health Organization (WHO) were included in the study . ChildreThe study protocol was fully explained to the parents/guardians, and informed written consent was obtained before recruitment.P. vivax, and the remaining three cases were caused by P. falciparum (7.9%). Twenty-seven cases were categorized as severe malaria as per the guidelines of the WHO and were admitted for treatment. The remaining 11 children  received treatment as outpatients.In total,1,680 children were screened for malarial infection in our centre during the study period. Of these, 38 children tested positive for malaria on peripheral smear examination, yielding a slide positivity rate of 2.26%. Of these, 35 (92.1%) were caused by P. vivax was documented in 24 (88.8%) and P. falciparum in three cases (11.1%). The clinical and laboratory profiles of children with severe vivax malaria are presented in the table. The age of the patients with severe vivax malaria (n=24) ranged from eight months to 12 years; 18 (75%) of them were male. Fever was the chief presenting complaint in all the patients (100%), with a median duration of six (range 2-9) days. Other presenting complaints of children with severe vivax malaria were: repeated vomiting , altered sensorium/encephalopathy , seizures , and epistaxis . The clinical signs among these patients included hepatomegaly , splenomegaly , pallor , icterus , and respiratory distress . Thirteen (54%) children had more than one manifestation of severe malaria. Of the 12 children with encephalopathy, eight had multiple convulsions, two had severe anaemia requiring blood transfusion, and one had spontaneous bleeding. Lumbar puncture was done in 10 of the 12 children presenting with altered sensorium, which were all acellular with normal cerebrospinal fluid biochemical parameters.Among the children with severe malaria, Seven (29.1%) patients with severe anaemia (haemoglobin <5 g/dL) required blood transfusion. Thrombocytopenia (platelet <1.5 lakh/dL) was observed in 17 (70.8%) children while acute renal failure (serum creatinine >1.5 mg/dL) was seen in two (8.3%) cases. Of the 24 children with severe vivax malaria, two also had lobar pneumonia requiring intravenous antibiotics (ceftriaxone and cloxacillin). Blood and urine cultures were sterile in all the children.P. falciparum-associated infection died within a few hours of hospitalization.All the patients with severe vivax malaria responded to intravenous quinine. Two patients with acute renal failure were started on intravenous artesunate, followed by oral therapy once the patient started accepting orally, for seven days. All the cases of uncomplicated malaria were treated with oral chloroquine as none of the patients could afford ACT. The outcome of the patients was favourable in all the cases with severe vivax malaria. However, one child diagnosed as cerebral malaria due to P. vivax among children residing in East Delhi and neighbouring districts of Uttar Pradesh during the study period. Clinical features of severe malaria caused by P. vivax were similar to those caused by P. falciparum, which included altered sensorium, severe anaemia, and thrombocytopaenia. These data on severe vivax malaria are in line with those reported from Papau New Guinea and Indonesia (The present study showed a surge of cases of severe malaria caused by ndonesia ,4.P. vivax (et al. showed that vivax malaria can have both sequestration-related and non-sequestration-related complications of severe malaria, including cerebral malaria (P. vivax, although it is well-documented in P. falciparum-associated malaria (P. vivax or by immunological destruction by platelet-associated IgG antibody. We observed thrombocytopaenia in 74.1% of the malarial children, a significantly high proportion, although similar paediatric cases have been reported earlier (Manifestations of severe vivax malaria include severe anaemia, respiratory distress, coma, malnutrition, splenic rupture, thrombocytopaenia, and acute renal failure and shock. Features of severe vivax malaria in our study were cerebral malaria, severe anaemia, and thrombocytopaenia. There has been a recent surge of cases of cerebral malaria caused by P. vivax ,7. Seque malaria . Profoun malaria . It coul earlier .Co-morbidities are common in endemic areas of malaria and could contribute significantly to mortality among cases of severe vivax malaria . Our patP. falciparum was mostly considered the causative agent (Plasmodium infection. Also, the increase in construction activities in and around Delhi has led to an increased breeding of mosquitoes and, hence, a rise in mosquito-borne diseases, including malaria and dengue. Since P. vivax was considered less virulent, surveillance reports in the past had primarily focused on P. falciparum. This could also explain the limited data available on the clinical features of severe malaria due to P. vivax. The emergence of severe malaria due to P. vivax as observed in our study might have important implications for the community with respect to treatment and prevention strategies.There has been an epidemiological change in the pattern of severe malaria in our region where ve agent . A possiP. vivax could have significant implications in planning the malaria-control programmes for the community. The widespread use of ACT for severe malaria as per the guidelines of the WHO could result in the emergence of resistant parasite strains. It may be rational to continue the use of chloroquine for malaria due to P. vivax in chloroquine-sensitive areas. In places where both P. vivax and P. falciparum co-exist, measures need to be equally targeted to P. vivax to decrease morbidity and mortality due to severe malaria.The emergence of severe malaria caused by The present study highlighted the change in the epidemiology of childhood severe malaria in the urban setting of North India. However, annual reporting with multicentric design of prospective studies is required to establish and confirm this trend.


Plasmodium vivax infection by genotyping 1975 children enrolled in three independent epidemiological studies conducted in the Madang area of Papua New Guinea and assessing P. vivax infection and disease in the children.Ivo Mueller and colleagues examined the association of Southeast Asian ovalocytosis with  SLC4A1Δ27) protects against cerebral malaria caused by Plasmodium falciparum; however, it is unknown whether this polymorphism also protects against P. vivax infection and disease.The erythrocyte polymorphism, Southeast Asian ovalocytosis (SAO)  in a cohort of infants aged 3–21 months and a significant 52% reduction in P. vivax (blood-stage) reinfection diagnosed by PCR  and 55% by light microscopy , respectively, in a cohort of children aged 5–14 years. SAO was also associated with a reduction in risk of P. vivax parasitaemia in children 3–21 months  and prevalence of P. vivax infections in children 15–21 months . In a case-control study of children aged 0.5–10 years, no child with SAO was found among 27 cases with severe P. vivax or mixed P. falciparum/P. vivax malaria . SAO was associated with protection against severe P. falciparum malaria  but no effect was seen on either the risk of acquiring blood-stage infections or uncomplicated episodes with P. falciparum. Although Duffy antigen receptor expression and function were not affected on SAO erythrocytes compared to non-SAO children, high level (>90% binding inhibition) P. vivax Duffy binding protein–specific binding inhibitory antibodies were observed significantly more often in sera from SAO than non-SAO children .The association between SAO and P. vivax malaria by a mechanism that is independent of the Duffy antigen. P. vivax malaria may have contributed to shaping the unique host genetic adaptations to malaria in Asian and Oceanic populations.In three independent studies, we observed strong associations between SAO and protection against Please see later in the article for the Editors' Summary. SLC4A1Δ27) in the red blood cell membrane. This defect is carried by up to 35% of people living on the coasts of Papua New Guinea, and as these areas match high malaria endemic areas, it is thought that carrying this defect is associated with improved survival against malaria in these populations—some studies have suggested that SAO is associated with complete protection against cerebral malaria but not other forms of malaria caused by the same type of parasite—P. falciparum.Hereditary blood disorders are widely prevalent in different regions around the world and the type of disorder depends on the population gene pool. For example, sickle cell disease is indigenous to sub-Saharan Africa, and Southeast Asian ovalocytosis (SAO), as the name suggests, to Southeast Asia and the South West Pacific, particularly Malaysia and Papua New Guinea. In SAO, the red blood cells (erythrocytes) are a different shape  from the usual biconcave disc shape due to a genetic defect  a cohort of 1,121 infants aged 3–21 months participating in a clinical drug trial of intermittent preventative treatment of malaria; (ii) a case-control study of 318 children with severe malaria aged 10 years and under; and (iii) a cohort of 206 children aged 5–14 years who took part in a prospective study to evaluate the time of reinfection with all forms of malaria. Given the different nature of these studies, for example, the effect of intermittent treatment for which the researchers had to make statistical adjustments, the researchers analysed the presence of the SAO genetic defect and the incidence of all forms of malaria separately to calculate the association with SAO and malaria in the participants in each study.P. falciparum single infections. Overall, the researchers found that the SAO genetic defect was associated with a 43% reduction in risk of clinical P. vivax episodes in the infants in the first study and a 52%–55% reduction in P. vivax reinfection in children in the third study. Furthermore, from the limited data from the second study, the researchers found that none of the children with P. vivax or mixed P. falciparum/P. vivax malaria had the SAO defect, which may indicate a protective effect.The researchers found that the SAO genetic defect present in 130 infants (11.6%) in the first study and 27 (13.1%) children in the third study. In the case-control study, the researchers found that 28 of the 330 controls (8.5%) had the SAO genetic defect compared to eight of 236 (3.4%) in children with SLC4A1Δ27) may have a protective effect against malaria caused by P. vivax in infants and children of different ages in Papua New Guinea. However, although it seems likely that SAO may alter the ability of the malarial parasite to develop within the red blood cell, this study sheds no further light on the way in which the SAO genetic defect may protect against P. vivax infection and disease and so further studies are needed to investigate possible mechanisms. Importantly, these findings suggest that future studies investigating genetic adaptation of diverse populations around the world, particularly in the Asian Pacific region, should include all forms of human malaria, such as P. vivax, and not exclusively focus on P. falciparum.These findings suggest that the SAO genetic defect Wikipedia has information on malariaWHO provides information on malaria (in English and Spanish)The US Centers for Disease Control and Prevention provide information on malaria and on ovalocytosis MedlinePlus provides links to additional information on  SLC4A1Δ27) has a distribution that is closely correlated with malaria prevalence SLC4A1Δ27 deletion is lethal in the homozygous state SLC4A1Δ27 deletion is thought to be associated with improved survival against malaria in these populations. Indeed, SAO has been associated with complete protection against cerebral but not other forms of severe P. falciparum malaria in previous epidemiological studies in PNG falciparum malaria The populations of the southwest Pacific are highly diverse and exhibit a range of unique red blood cell (RBC) polymorphisms. Within Papua New Guinea (PNG), a variety of RBC variants are found that have geographical patterns paralleling malaria endemicity falciparum parasites are often considered to cause only mild disease, recent data from the island of New Guinea P. vivax infections are associated with severe disease and mortality. In addition, mortality rates of 5% to 15% were regularly observed in patients challenged with P. vivax for therapy of tertiary syphilis P. vivax may be responsible for, or contribute to, natural selection of erythrocyte polymorphisms. This selective pressure is suggested by the emergence of the unique, non-African Duffy-negative allele (ESFY*A) in PNG and the observation that PNG children expressing heterozygotes (FY*A/ESFY*A) are protected from P. vivax blood-stage infection SLC4A1Δ27P. vivax malaria could have contributed to selection of this genetic polymorphism. An early cross-sectional P. vivax and/or P. malariae. While three field studies have investigated associations between SLC4A1Δ27 and prevalence of P. vivax blood-stage infection, no significant relationship with susceptibility to infection was observed Although non-P. falciparum isolates P. knowlesi in vitro, with the suggestion that this is mediated by diminished adherence P. vivax malaria in vivo.In vitro studies have shown that SAO red cells are relatively resistant to invasion by some SLC4A1Δ27) and risk of infection and/or disease with P. vivax, we genotyped 1,975 children participating in three separate studies conducted in the Madang area of PNG: (i) a cohort of infants participating in a clinical trial of intermittent preventative treatment (IPTi) Plasmodium species In order to assess the relationship between SAO  (ICT Combo test), and only RDT positive children treated with arthemeter-lumefantrine . A finger-prick sample was collected from all febrile cases for confirmation of malarial infections by LM and PCR. For clinical management and analysis purposes, malarial illness was defined as axillary temperature >37.5°C, or history of fever in preceding 48 h, plus an infection of any density by LM and/or a positive RDT with speciation subsequently confirmed by PCR. A more detailed description of the study and its primary outcomes is given elsewhere n = 124) and blood  and parents from the PNG North Coast  . Consequently, severe cases with mixed P. falciparum/P. vivax infections by LM or PCR were only included as mixed cases if both species exceeded their respective thresholds. The parasitaemia cut-offs were based on local attributable fraction-based definitions of malaria episodes Between October 2006 and December 2009, a total of 318 children of Madang or Sepik parentage aged 0.5–10 y admitted to the pediatric ward of Modilon Hospital, Madang Town with a diagnosis of severe malaria and 330 age-, location-, and ethnicity- matched healthy controls were enrolled in a case-control study designed to investigate the associations of host genetic polymorphism with protection against severe malaria. Cases were defined as severe malaria if they fulfilled the World Health Organization (WHO) definition of severe malarial illness Plasmodium species infection status by LM and LDR-FMA. All children irrespective of infection status were treated with a 7-d course of artesunate monotherapy (4 mg/kg on day 1 and 2 mg/kg on days 2–7) that successfully cleared all but one P. vivax infection P. vivax reinfections of the blood stream can either be from newly acquired infection via sporozoites or relapsing infections from hypnozoites.The study population of 206 children for this treatment time-to-reinfection study has been described previously Following treatment, children were followed up by active surveillance every 2 wk and passive surveillance at the local health centre for a total of 25 wk. Children were monitored for acquisition of new infections until they completed follow-up, withdrew from the study, or did not provide two consecutive bi-weekly blood samples. At the time of the study, bednet usage in the area was limited, most nets were untreated, and their use was not associated with differences in risk of reinfection At each bi-weekly follow-up visit, a 250-µl finger-prick blood sample was collected from each child for detection of malaria by LM and LDR-FMA. If a child presented with clinical malaria symptoms at a follow-up visit or during the intervening period, a peripheral venous blood sample was taken and treatment given in accordance with 2000 PNG guidelines (amodiaquine [3 d] plus sulfadoxine/pyrimethamine on day 1).These studies were reviewed and approved by institutional review boards of PNG Medical Research Advisory Council, the IMR Institutional Review Board, the Walter & Eliza Hall Institute, and the Veterans Affairs Medical Center .Plasmodium species if the species was detected independently by at least two microscopists and/or subsequent PCR-based analysis confirmed the presence of the species. Densities were converted to the number of parasites per µl of blood assuming 8,000 white blood cells/µl Standard procedures were used for reading the blood smears and estimating parasite densities P. falciparum, P. vivax, P. malariae, and P. ovale) was assessed in all blood samples collected in the two cohorts using a semi-quantitative post-PCR LDR-FMA DNA was extracted using the QIAamp96 DNA Blood kit (Qiagen) from all blood samples. Infection by each of the four human malaria species co-endemic in PNG (SLC4A1Δ27 (Chromosome 17) associated with SAO, glycophorin C exon 3 deletion  associated with Gerbich-negativity −3.7) and 4.2 kb (α−4.2) α-globin (Chromosome 16) deletions associated with α+-thalassaemia Deletion and SNP genotyping of g for 10 min. In order to assess Duffy receptor RBC surface expression for individual blood donors, 1×106 RBC from each sample were incubated with 50 µl (1∶50) of mouse anti-human Fy6 antibody for 15 min at 37°C. Cells were then washed with RPMI-1640, stained with PE-conjugated goat anti-mouse antibody (Molecular Probes), and analyzed using a Becton Dickinson LSR II (Franklin Lakes).Twelve of 21 SAO children participating in the treatment time-to-reinfection study agreed to provide an additional finger-prick blood sample for flow cytometry-based analyses of Duffy antigen on their red cells. For each SAO child, a control sample was collected from a non-SAO (wild-type) child living in the same hamlet. All samples collected were separated into plasma and cell pellets. One sample from an SAO child was subsequently discarded because of excessive cell lysis. Pelleted blood was suspended in 1 ml of 10% DMSO+90% fetal calf serum solution and frozen to −80°C. Prior to antibody staining and protein binding, cells were rapidly thawed for 1 min in 37°C water bath and washed (2×) by first resuspending the red cell pellet in 1.0 ml of RPMI-1640 followed by centrifugation at 600P. vivax parasites to bind to SAO versus non-SAO RBC, we evaluated the level of P. vivax Duffy binding protein (PvDBP) binding using the same set of blood samples as described above 6 washed RBC from each sample were re-suspended in 200 µl of RPMI-1640 and exposed to 0.5 µg of recombinant PvDBP region II  for 15 min at 37°C. Excess protein was removed by centrifugation of erythrocytes at 3,000g for 2 min, resuspended in RPMI-1640 followed by centrifugation at 3,000g for 2 min (2×), and then resuspended in 100 µl of RPMI-1640 to which rabbit anti-PvDBPII antibody  was added and incubated for 30 min at 37°C, washed (2×) as above and resuspended in 100 µl of RPMI-1640 to which 1∶4 to 1∶16 dilution of PE-conjugated goat anti-rabbit antibody . All data were evaluated as normalized mean fluorescence index (nMFI = % of positives cells×MFI) To assess possibility that there are differences in the ability of Measurement of binding inhibitory antibodies (BIAbs) and total antibodies to PvDBPII PvMSP1, PvRBP1, PvRBP2, and PvRBP3 was performed as previously described P. vivax and P. falciparum infections were investigated using generalized estimating equation models (XTLOGIT and XTREG in STATA 10.0) that allowed accurate assessment of both variations in the outcome and the correlations between repeated measurements in individual children (modeled using an exchangeable correlation structure). A semi-robust Huber/White/sandwich estimator of variance was used to assure valid standard errors. Best fitting models were determined by backward elimination using Wald's Chi-square tests for individual variables.The association of malaria incidence rates with SAO genotype in children 3–21 mo was assessed using negative binomial regression. Analyses were adjusted for gender, treatment effect, season (wet versus dry), and village of enrolment (grouped by 12 recruitment zones). The time-at-risk was calculated starting on the date of enrolment until the child either reached the final study time point at 21 mo of age, or was withdrawn from the study Plasmodium spp. infection until they either reached the end point, missed two consecutive bi-weekly follow-ups, were re-treated with antimalarials, or withdrew from the study.In the study of children 5–14 y, log-rank tests were used to assess differences in Kaplan-Meier curves of time-to-first reinfection as detected by LDR-FMA or LM. Cox regression was employed to test for differences in time-to-first reinfection after adjusting for all other factors that were found to be associated with difference in time-to-first reinfection 2 tests. Association of SAO genotype with other common RBC polymorphisms and potential confounders were assessed using χ2 and Student t-tests . The associations between different genetic traits and prevalence of BIAbs against PvDBPII were assessed using Chi-square and Fisher exact tests. Due to non-normality, differences in both expression of the Duffy receptor on RBC and in binding of recombinant PvDBPII on SAO and non-SAO RBCs were compared using non-parametric Mann-Whitney U-tests.Difference in frequency of SAO genotype among severe malaria cases and controls were tested using χAll 95% confidence intervals are model based. Further details on statistical approaches employed are given elsewhere P. vivax and 0.28 P. falciparum episodes/child/year. IPTi with amodiaquine-SP reduced the incidence of P. vivax by 23%  for the first year of follow-up (3–15 mo) and incidence of P. falciparum by 35% , whereas IPTi with artesunate-SP only protected against P. falciparum  but not against P. vivax episodes  p>0.75).A total of 1,121 children  were enrolled in the IPTi trial, with equal numbers in each treatment arm. Of these, 857 were followed up to 21 mo. Over the entire 18-mo follow-up period, children on average experienced 0.74 SLC4A1Δ27 (11.6%), with no significant difference in frequency of SAO among treatment arms (p = 0.30). SLC4A1Δ27 heterozygocity was associated with neither α+thalassaemia nor Gerbich-negative deletion  (p>0.36).SAO genotypes were available for all 1,121 children with 130 heterozygous for  p>0.95) . There wp = 0.0068)  in incidence of P. vivax episodes of any density and a 55% reduction  in incidence of P. vivax episodes with >500 parasites/µl. There were no significant associations with all P. falciparum episodes  .Over the total follow-up period (3–21 mo), SAO was associated with a significant 32% reduction in incidence of any form of malaria  . The pro = 0.89)  or episoP. vivax and P. falciparum were evaluated in 6,269 blood samples collected at scheduled 3-monthly study contacts. Of these, 1,435 (22.9%) and 352 (5.6%) were positive for P. vivax and P. falciparum, respectively, by LDR-FMA and 843 (12.9%) and 197 (3.0%) by LM. Infections with P. malariae and P. ovale were rare even by LDR-FMA . There was no significant difference in the prevalence P. vivax infections in SAO and wild-type children ≤12 mo  and LM . In children of all ages, P. vivax parasite densities (by LM) were significant lower in SLC4A1Δ27 heterozygous children   (p = 0.59).In children 3–21 mo, the prevalence and density of n ≤12 mo . However= 0.011) . By contections) , but parSLC4A1Δ27. SLC4A1Δ27 heterozygous children, were similar in the basic characteristics to wild-type children except that significantly more of them were attending Mugil Elementary School   for the Duffy antigen.Of the 206 children 5–14 y of age enrolled in the treatment-reinfection cohort 106 (51.5%) were female; 116 (56.9%) were over 8 y of age. 27 children (13.1%) were heterozygous for = 0.004) . There wP. falciparum, P. vivax, and P. malariae infections, respectively, over the 26 wk of observation. Similarly, using LDR-FMA diagnosis, the proportions of reinfected children rose to 91.6%, 82%, and 29.3%, respectively Following initial blood-stage malaria therapy, children rapidly acquired LM-detectable blood-stage infections, with 156/192 (81.2%), 102/206 (49.5%), and 17/206 (8.3%) acquiring one or more SLC4A1Δ27 heterozygous and wild-type children had similar times to reinfection with P. falciparumP. vivax and P. malariae reinfections during the follow-up time period  acquired at least one LDR-FMA-positive P. vivax infection compared to 83.8% (149/179) of non-SAO children  over the period of observation. Similarly, P. vivax infections were observed in only 37% (10/27) of SLC4A1Δ27 heterozygotes by microscopy, compared to 54.7% (98/179) of non-SAO children . After correction for all other factors found to be associated with differences in risk of reinfection SLC4A1Δ27 heterozygosity was associated with a significant 52% reduction in time to first LDR-FMA (adjusted hazard ratio (aHR): 0.48, p = 0.003) , as determined by Cox regression analysis. Similarly, SAO was associated with a 71% reduction in time to first LDR-FMA-detectable  P. malariae infections. SAO was however not associated with protection against P. falciparum (While e period . Among S= 0.003)  and 55% lciparum  [8].SLC4A1Δ27 may confer protection against P. vivax may be through reduced expression and altered functionality of the Duffy antigen receptor on RBCs. Accordingly, levels of Duffy expression and ability to bind recombinant PvDBPII were compared on red cells from a subset SAO (n = 11) and non-SAO (n = 12) study children. Erythrocytes with SLC4A1Δ27 showed no significant decrease in expression of the Duffy receptor on the surface . The ability of PvDBPII to bind to the different SAO and non-SAO cells was similar  (A possible mechanism whereby P. vivax in this population SLC4A1Δ27 heterozygous children 6/27 (22.2%) had high levels of PvDBPII BIAbs (>90% strain-transcending binding inhibition p = 0.008) in non-SAO children. Adjusting for the presence of PvDBP-specific BIAbs did not significantly alter the strength of protection of SAO against P. vivax infections detected by either LDR-FMA  or LM .Since we had previously shown that high binding inhibitory antibodies to PvDBPII (>90% inhibitory activity) correlated with protection against P. vivax merozoite surface proteins  were not significantly different in SAO compared with non-SAO children (unpublished data).In contrast to the above findings, total IgG antibodies as measured by ELISA to the main variants of PvDBPII  as well as to other P. falciparum, 34 with P. vivax, ten with P. falciparum plus P. vivax, and one with P. malariae. Of these 264 (83.0%) also fulfilled the pre-defined, more stringent criteria  for the host genetic study: 236 were infected with P. falciparum , 25 with P. vivax (>500/µl), two with P. falciparum plus P. vivax, and one with P. falciparum plus P. malariae. SLC4A1Δ27 genotyping was successfully preformed in cases and healthy community controls.Of 318 cases fulfilling the standard WHO definition of severe malaria, 273 were infected with SLC4A1Δ27 compared with eight of 236 (3.4%) in children with P. falciparum single infections  and 0/27 (0%) for children with P. vivax or mixed P. vivax/P. falciparum infection .28 of 330 (8.5%) health community controls were heterozygous for P. vivax infection (case 1: 429 parasites/µl with prostration and mildly impaired consciousness (Blantyre coma score = 4); case 2: 98 parasites/µl , severe anemia (Hb = 1.7 g/dl), and hyperlactemia (lactate = 6.3 mmol/l) were observed in children with SAO genotype.When all cases fulfilling WHO criteria for severe malaria are considered, two cases of P. falciparum mono-infection, three presented with deep coma  in presence of P. falciparum densities of 4,266, 219, and 309/µl. All three children had clear CSF and negative CSF and blood cultures. These three cases are consistent with the accepted clinical definitions for cerebral malaria caused by P. falciparumAmong the eight SAO children with SLC4A1Δ27 causing SAO was associated with a 43% reduction in risk of clinical P. vivax episodes in a large cohort of infants followed from age 3 to 21 mo and a 52%–55% reduction in P. vivax reinfection diagnosed by PCR-LDR-FMA and LM, respectively, in a cohort of children aged 5–14 y. This is the first in vivo evidence that SAO may afford partial protection against P. vivax malaria in two independent, longitudinal cohort studies. In addition, SAO was associated with a lower P. vivax parasitaemia in children aged 3–21 mo and a reduced prevalence of P. vivax infections in children 15–21 mo. Last but not least, no child SAO genotype was found among 27 cases with severe P. vivax or mixed P. falciparum/P. vivax malaria. However, SAO was not associated with protection against P. falciparum infection and uncomplicated disease. While it was associated with a decreased risk of severe P. falciparum malaria, at least one severe malaria case with SAO genotype was admitted with deep coma (Blantyre Coma score = 2), refuting the earlier assertion that SAO provides complete protection against cerebral P. falciparum malaria This study shows that a deletion in an RBC integral membrane protein SLC4A1Δ27 heterozygous have erythrocytes that are ovalocytic and more rigid than normal P. falciparum cerebral malaria has been considered the most likely cause of selection for SAO P. vivax disease and infections observed in our study opens up the possibility that protection against P. vivax malaria could at least have contributed to the heterozygote advantage of SAO in PNG.SAO is a dominant phenotype with respect to RBC morphology. All individuals who are P. falciparum and P. vivax are associated with severe disease and death in Melanesian populations P. vivax infections were less likely to result in severe symptoms, children admitted with severe P. vivax had the same phenotype as those with severe P. falciparum infections, while those with mixed P. falciparum/P. vivax presented with the most severe illness and the greatest mortality P. vivax-specific mortality benefit. However, the limited number of both SAO children and severe P. vivax infections in our case-control study restricts our ability to assess this association. Larger, appropriately powered studies are therefore required to confirm this finding.It is now well established that both P. vivax infection and disease is unknown. The changes to the RBC membrane and anion transport across the membrane caused by SAO could impact development of malarial parasites in several ways. SAO may alter the ability of the parasite to develop within the erythrocyte. The altered membrane characteristics of SAO erythrocytes may impair the parasite's ability to remodel the RBC surface P. vivax to attach to and/or invade reticulocytes Although SAO reduced parasite densities even in infants aged ≤12 mo, this resulted in a reduced prevalence of infection only in the second year of life. This indicates that SAO cells may not be inherently resistant to P. vivax infection, but that children acquire this protective effect only in concert with increasing acquisition of anti-blood-stage immunity, either by increasing the acquisition and/or by enhancing the protective effect of immune response. A similar interaction between host genetics and age-specific immune status has recently be shown for the sickle cell trait (HbAS), where in cohort of Ugandan children the youngest children were best protected against high density parasitaemia, while only older children were protected against establishment of parasitaemia The mechanism by which SAO may protect against SLC4A1Δ27 allele may induce conformational changes in the normal band 3 protein resulting in the predominance of band 3 hetero-tetramers, higher order hetero-oligomers, and aggregates in SAO RBCs P. vivax via the Duffy antigen that is thought to be part of a 4.1R-based macromolecular complex that contains band 3 as a dimer In contrast to normal RBCs where band 3 is mainly found as dimers, protein expressed from the P. vivax. Thus, while SAO increases the likelihood that high activity Duffy blocking antibodies are acquired, the protection attributed to SAO is distinct from that provided by Duffy blocking antibodies. Further study of SAO-based protection against P. vivax illness, in particular in small children who suffer the highest morbidity burdens of P. vivax morbidity In our studies that further explored the relationship between SAO and the Duffy antigen, fluorescence activated cell sorting (FACS)-based analyses revealed that SAO and non-SAO red cells expressed similar amounts of surface-level Duffy antigen and that PvDBPII bound to SAO and non-SAO cells equally well. Interestingly, we found that while antibody levels to PvDBPII measured by ELISA did not differ, SAO children were 3.3 times more likely than non-SAO children to have high levels of PvDBPII-specific BIAbs. This preferential production of functional BIAbs could occur because critical binding regions of PvDBP might be exposed for a longer period of time during less efficient invasion of SAO cells, making the protein more accessible to the host immune response that generate broadly binding inhibitory antibodies. Adjusting for blocking antibodies did not, however, change the magnitude of the protection provided by SAO against reinfection with P. vivax malaria in shaping the unique RBC polymorphisms found in Asian and Pacific populations. Future studies on host genetic adaptation in the Asia Pacific region should thus not exclusively focus on P. falciparum but need to include other human malaria species.Irrespective of the mechanism by which this mutation evolved, our observations highlight the potential contribution of Table S1Baseline characteristics of SAO and non-SAO children in the infant cohort and association with other common RBC polymorphisms.(DOCX)Click here for additional data file.Table S2Baseline characteristics of SAO and non-SAO children 5–14 y of age in the treatment – reinfection cohort and associations with other common RBC polymorphisms.(DOCX)Click here for additional data file.Text S1P. falciparum cerebral malaria.SAO children with clinical signs and symptoms of (DOCX)Click here for additional data file.


In areas of low malaria transmission, it is currently recommended that a single dose of primaquine  be added to artemisinin combination treatment (ACT) in acute falciparum malaria to block malaria transmission. Review of studies of transmission-blocking activity based on the infectivity of patients or volunteers to anopheline mosquitoes, and of haemolytic toxicity in glucose 6-dehydrogenase (G6PD) deficient subjects, suggests that a lower primaquine dose (0.25 mg base/kg) would be safer and equally effective. This lower dose could be deployed together with ACTs without G6PD testing wherever use of a specific gametocytocide is indicated. Plasmodium vivax or Plasmodium ovale malaria, or as a gametocytocide in Plasmodium falciparum malaria, is the risk of haemolysis in patients who are glucose 6-phosphate dehydrogenase (G6PD) deficient[Primaquine is an important but underused anti-malarial drug. The main limitation to the use of primaquine, either for the radical cure of eficient,2. The peficient. Use of eficient, particueficient. UnfortuP. falciparum. Artemisinin derivatives reduce transmissibility substantially in acute falciparum malaria, largely by killing younger gametocytes , but patients who have high numbers of infectious mature gametocytes at presentation may continue to transmit despite receiving ACTs[P. falciparum gametocytes rapidly[P. falciparum infections may have high blood densities of young stage 5 gametocytes which are not infectious. Furthermore drug effects on transmissibility precede effects on gametocytaemia, probably because of the lag between gametocyte killing and gametocyte clearance, so the transmission-blocking effect may be underestimated[Transmission of malaria requires that a feeding anopheline vector mosquito ingests at least one male and one female gametocyte in a blood meal, and that the subsequent ookinete forms an oocyst in the wall of the mosquito gut which later matures to liberate viable sporozoites which migrate to populate the salivary glands. All the effective anti-malarial drugs, in addition to killing the asexual forms of all human malaria parasites, also kill early developing gametocytes (stages 1 to 3) of ing ACTs-14. The  rapidly. Reducti rapidly. In partstimated. Dose–reP. falciparum to anopheline mosquitoes[The 8-aminoquinoline anti-malarial plasmoquine, the predecessor of primaquine, was discovered in 1924,19. Withsquitoes,22. Evensquitoes-25. Primsquitoes,26. In csquitoes. These oP. falciparum. Primaquine increases the rate of gametocyte clearance but there is a lag phase of >24 hours before clearance accelerates. The effects on transmissibility assessed from mosquito oocyst numbers, and consequent sporozoite numbers and viability, occur much more rapidly than the effects on gametocyte densities  in transmission reduction strategies combined with an ACT.Compared to the volunteer studies, gametocyte densities in malaria infected individuals under natural conditions are generally much lower, with the majority being below the limit of routine microscopy detection,29. InfePrimaquine is safe and generally well tolerated (with food) in G6PD normal subjects, although adult doses over 30 mg are associated with an increased incidence of abdominal discomfort. On the versus females), and (c) the allelic variants of the G6PD gene.The severity of drug-induced acute haemolytic anaemia (AHA) in G6PD deficient persons depends on several factors, the most important of which are: (a) drug dosage, (b) the genotypic combinations at the G6PD locus , there was brisk haemolysis with haemoglobinuria[P. vivax and P. ovale malaria of 0.75 mg base/kg once weekly for eight weeks in patients with the A- type of G6PD deficiency[Primaquine is given once when used as a obinuria,32,35-37obinuria, so if pP. vivax relapse in some 250,000 US troops returning from the Korean war during their trans-Pacific sea voyage. Of these 10% were African-American soldiers[P. falciparum malaria.When primaquine was first introduced in 1951 it was given in a radical curative regimen of 15 mg base per day for two weeks to prevent soldiers. It was i.e. AHA) from this mass drug administration and routine treatment experience was approximately three per million. However, in prospective studies which focussed on safety, the estimated risk of severe adverse effects  was three orders of magnitude higher: 3.9 per thousand[versus continuous administration. The single 0.75 mg base/kg dose has generally been regarded as relatively safe[P falciparum malaria, over a period of two years seven cases of acute haemoglobinuria were observed with severe anaemia in G6PD deficient men and the policy was discontinued[No deaths have been reported following a single dose of primaquine. Primaquthousand. Althougthousand,43. Thesely safe althoughontinued.i.e. relatively little or no haemolysis)[i.e. < 30% G6PD deficient red cells) are unlikely to develop severe AHA.G6PD is X-linked. Males have only one G6PD allele , whereas females have two . Hemizygous males and homozygous females have full expression of G6PD deficiency: the level of G6PD activity in their mature red cells is nearly always ≤15% of normal values. Heterozygous females usually have only partial deficiency, and they have been referred to as having ‘intermediate’ deficiency. Among X-linked genes, G6PD belongs to the majority that are subject to X chromosome inactivation. Therefore ‘intermediate’ deficiency does not mean that each red cell has an intermediate level of G6PD: rather, it is a cumulative average, whereby some red cells have normal levels of G6PD whereas others are G6PD deficient. Moreover, because X-inactivation is a random event which takes place early in embryonic life (Lyonization), the resulting proportions of these two cell types are very variable. The severity of oxidant haemolysis in female heterozygotes will, therefore, vary from that in hemizygous males to that in G6PD normal persons (molysis). UnfortuMany of the >180 G6PD deficiency alleles known are polymorphic, and are common in populations in the tropics  it has been demonstrated that reducing infectivity of treated people in a variety of endemic situations reduces transmission on a community basis;2) further research is done on safety and on adverse haematological effects for both G6PD normal and G6PD deficient subjects;3) more is understood about the proportion of gametocyte carriers who present to receive treatment in a given population and time period4) the cost of the policy balanced against the potential benefit is explored. In any case, patients should be screened for G6PD deficiency and those with variants predisposing to haemolysis should not be given PQ.”These are large and difficult questions which would certainly take many years to answer if the detailed prospective clinical and epidemiological studies requested were undertaken. Whilst the Cochrane review probablyProspective studies should be conducted to confirm the safety of a single dose of primaquine 0.25 mg base/kg as a gametocytocide together with ACT in individuals with G6PD deficiency, and to assess transmission blocking activity dose–response relationships in different geographic areas, particularly in the context of artemisinin resistant falciparum malaria. In the meantime, based on the data reviewed above, there is sufficient evidence on safety and efficacy to support widescale deployment of a single dose of primaquine 0.25 mg base/kg as a gametocytocide without G6PD testing now where it is needed.The authors declare that they have no competing interests.NW conceived of the study, NW and GQ reviewed the transmission blocking studies, LGQ conducted the transmission blocking dose response studies with ACT, LL reviewed the haematological literature. All authors read and approved the final manuscript.


Peptic ulcer disease (PUD) in children remains rare and difficult to diagnose before the onset of complications. We report on a case of a 12-month child with perforated duodenal ulcer, association with malaria. The severity of the febrile presentation and the positive laboratory confirmation of malaria delayed the diagnosis of PUD. Surgical intervention was successful and without significant sequelae. An awareness of the possibility, and a lower threshold for considering PUD in children may help prevent complications. Peptic ulcer disease (PUD) is uncommon and difficult to diagnose in young children and clinical features are often non-specific . We repoA previously healthy 12-month old boy was referred from a clinic to the Kintampo Municipal Hospital . He had presented at the clinic with a 2-day history of fever and vomiting. On admission to the clinic, diagnosis of severe anaemia secondary to Malaria was made. Despite treatment with IV fluid, IM Quinine and a blood transfusion, his condition deteriorated and he began to pass bloody stools. After two days he was referred to hospital for further management.On arrival, the child had a reduced conscious level. He was febrile (39.5°C) and pale with pulse and respiration rates 110 beat/min and 60 breaths/min respectively. Abdominal examination was normal apart from periumbilical tenderness.9. Neither Widal test nor stool cultures were done. Supine abdominal X-ray showed air fluid levels. Erect chest X-ray showed no subphrenic gas. Treatment of severe anaemia secondary to malaria and suspected typhoid was continued. Intranasal oxygen, IV fluids and whole blood were administered along with paracetamol, IM quinine, IV gentamicin and IV metronidazole. After a further 2 days he remained semiconscious and febrile, passing blood clots in his stool. Despite transfusion, haemoglobin remained 4.3g/dL. Abdominal examination remained unremarkable.Blood film was positive for malaria parasites. Haemoglobin was 4.2g/dL and white blood cell count 16.4 × 10An exploratory laparotomy was performed. On entering the peritoneum a large amount of blood-stained fluid was seen. A 1.5cm anterior perforation of the duodenal wall was found, with bleeding from the posterior duodenal wall . The blePostoperatively the child received a further blood transfusion, antibiotics and IV ranitidine. Ten days post-operatively he was discharged. On follow-up he had no sequelae.Helicobacter pylori was performed. The mother denied giving any medication to the child.Due to resource constraint no test for PUD is rare in children with an estimated frequency of 1 in 3000 hospital admissions .PUD in children is classified as primary or secondary depending on the aetiology. Secondary PUD is associated with physiological stress, systemic illness and drugs including non-steroidal anti-inflammatories and steroids. Primary PUD is commonly associated with Helicobacter pylori infection. It can also be idiopathic or associated with conditions causing increased acid secretion , 3.PUD is more commonly secondary in children under ten years of age and primary in the over tens. Secondary PUD occurs in the stomach or duodenum and is more likely to present with complications including perforation and hemorrhage. Primary PUD predominantly occurs in the duodenum and rarely presents with perforation .94% of children with perforated PUD present with acute abdominal signs . Free aiA literature search revealed no reports of perforated duodenal ulcers associated with malaria. In our case, we postulate that physiological stress from severe malaria resulted in secondary PUD progressing to perforation due to characteristics of secondary PUD and diagnostic challenges.PUD remains difficult to diagnose in children prior to complications especially in a resource poor settings. A lower threshold for considering PUD in children may help prevent complications.


Early diagnosis, as well as prompt and effective treatment of uncomplicated malaria, are essential components of the anti-malaria strategy in Madagascar to prevent severe malaria, reduce mortality and limit malaria transmission. The purpose of this study was to assess the performance of the malaria rapid diagnostic tests (RDTs) used by community health workers (CHWs) by comparing RDT results with two reference methods .Eight CHWs in two districts, each with a different level of endemic malaria transmission, were trained to use RDTs in the management of febrile children under five years of age. RDTs were performed by CHWs in all febrile children who consulted for fever. In parallel, retrospective parasitological diagnoses were made by microscopy and PCR. The results of these different diagnostic methods were analysed to evaluate the diagnostic performance of the RDTs administered by the CHWs. The stability of the RDTs stored by CHWs was also evaluated.Plasmodium falciparum in blood at a level of 200 parasites/μl.Among 190 febrile children with suspected malaria who visited CHWs between February 2009 and February 2010, 89.5% were found to be positive for malaria parasites by PCR, 51.6% were positive by microscopy and 55.8% were positive by RDT. The performance accuracy of the RDTs used by CHWs in terms of sensitivity, specificity, positive and negative predictive values was greater than 85%. Concordance between microscopy and RDT, estimated by the Kappa value was 0.83 (95% CI: 0.75-0.91). RDTs stored by CHWs for 24 months were capable of detecting Introduction of easy-to-use diagnostic tools, such as RDTs, at the community level appears to be an effective strategy for improving febrile patient management and for reducing excessive use of anti-malarial drugs. In most countries with endemic malaria, the World Health Organization (WHO) recommends using rapid and reliable diagnostic tests as an essential means of reducing the morbidity and mortality due to malaria, especially in rural areas and for children under five years of age and pregnant women . The diaTo date, microscopic examination remains the gold standard for malaria diagnosis despite its requirements for high-quality microscopes and equipment and for qualified personnel. It is clear that in endemic countries, many rural health care facilities lack the capacity to make accurate microscopic diagnoses ,8. RecenPlasmodium antigens are currently available. Some of these tests are specific for Plasmodium falciparum and detect histidine-rich protein 2 (HRP2). Others combine HRP2 detection with the detection of antigens common to all species, such as lactate dehydrogenase or aldolase (combo RDTs). Combo RDTs can diagnose infections by P. falciparum or by non-P. falciparum malaria parasites .Several immuno-chromatographic tests for detecting specific ®, USA) in health care facilities lacking microscopic diagnostic capability. At the community level, the same recommendations for the treatment of fevers in children under five were put in place several years later. Finally, in 2010, in its effort to improve the management of fever at the community level, the NMCP proposed that RDTs be used by community health workers (CHWs) to diagnose febrile individuals. To do so, the NMCP set up a series of studies to validate this strategy and to answer several key questions regarding the choice of RDT, the cost of this strategy, and the establishment of a RDT quality assurance system. Although the use of RDTs requires basic training for CHWs, RDT performance (in terms of sensitivity and specificity) in the hands of CHWs and the quality of RDTs stored in the field over time have not been evaluated. In this context, the results of a longitudinal study to evaluate the overall performance and quality of RDTs used by CHWs in two regions of Madagascar with endemic malaria: one with stable malaria  and the other with unstable malaria  are reported hereIn 2005, the National Malaria Control Programme (NMCP) of Madagascar revised its national malaria treatment guidelines and replaced chloroquine with artesunate/amodiaquine combination. This change was followed in 2007 by the introduction of RDTs  that evaluated the NMCP's community level recommendations for the treatment of fevers with a fixed combination of artesunate plus amodiaquine in children under five in a region of high malaria transmission  and in a region of low malaria transmission  . RDTs weIn each village, a CHW had to manage about 150 children. CHWs in the study were volunteers who were appointed by the villagers. The CHWs represented a functional relay among the health authorities, NGOs and villagers. The level of training of the CHWs consisted of basic reading and writing capabilities, which were between the third- and fifth-grade educational levels. CHWs in the Moramanga district were supervised by the NGO "ADRA Madagascar" and by staff of the nearest health centre. They had previously received regular training on how to care for fevers at the community level. In exchange for their participation, CHWs received food staples . CHWs in Vohimasy and Manakara, were supervised by the NGO "Inter Aide" under the name of "Maman et Papa conseils." These CHWs had also received training on how to care for fevers at the community level. CHWs in Ampasimanjeva were supervised by the NGO "RTM". In these two communities, agents received help from the villagers to maintain their fields and an annual allocation of rice as compensation for their work. At the beginning of the study, each CHW received a three-day training session on the general and clinical aspects of malaria, on the use of thermometer, the preparation of thick and thin blood smears, and on collecting blood spots on filter paper. Special training was provided for use of the CareStart RDT and the interpretation of the test results.All children in the study were two months of age or more and had experienced a fever with the following criteria: axillary temperature of 37.5°C or fever for at least 24 hours. Children with signs of severe malaria or other infections that were life threatening were excluded from the study and referred to the nearest health centre. At each visit, CHWs collected a finger-prick blood sample to prepare a thin/thick smear and a blood spot. They also performed an RDT according to the instructions received. The samples were then stored at room temperature and protected from light. The results of the RDTs were recorded in a book provided for that purpose. Regardless of RDT outcome, all children were treated with a fixed dose of artesunate/amodiaquine. Every months (each supervisory visit), slides, RDTs and blood spots were transferred to the Malaria Research Unit of Institut Pasteur de Madagascar (IPM).Suitable slides were stained and read by two experienced IPM microscopists. Briefly, after the smear was fixed in methanol, the slide was stained with 10% Giemsa for 10 min, dried, and then examined by microscopy at 100× with an immersion objective. Parasite density  was on the number of asexual parasites per 200 white blood cells (WBC) counted (or per 500 WBC if the parasite count was less than 10 parasites per 200 white blood cells). Parasitaemia was expressed in terms of the number of parasites per μL, considering that the average concentration of WBCs is 8,000 per μL. The smear was declared negative if no parasites were observed after reading 100 microscopic fields. The thin film was used to identify the species of the parasite(s). The slides were read independently by two microscopists. The final parasite density was calculated from the mean estimated parasite densities. For cases in which the difference between the two density estimates was greater than 10%, a third reading was performed by a third microscopist. Quality control was also provided by technicians at the NMCP, who analysed 10% of the slides.DNA was extracted from blood spots using Instagene matrix  according to the manufacturer's instructions. The detection of malaria parasites was assessed by real-time PCR using methods previously described by using RotorGene 3000 thermocycler  . PCR anaP. falciparum and P. vivax  and negative controls.The long-term stability of RDTs kept by CHWs was evaluated according to the protocol developed by WHO/FIND/TDR described in the "Methods manual for laboratory quality control testing of malaria rapid diagnostic tests" . BrieflyAll data were recorded and analysed using EpiInfo software  and MedCalc . A chi-squared test was used to compare the performances of the different diagnostic methods used . A P-value < 0.05 was considered to indicate statistical significance. To evaluate the sensitivity and specificity, the microscopy and RDT results were compared with those of PCR. Sensitivity was calculated as the proportion of positive tests recorded for the samples found positive by the reference method chosen. Specificity was estimated as the proportion of negative tests recorded for samples found negative by the reference method chosen. The positive and negative predictive values  were also calculated as the proportion of results that were "truly positive" or "truly negative" among the samples found to be positive or negative using the reference method chosen. Finally, Kappa values were calculated to estimate the correlation between diagnostic techniques. Kappa values between 0.21 and 0.60 were considered to indicate "moderate agreement"; values between 0.61 and 0.80 indicated a "good fit"; and values greater than 0.80 indicated "excellent agreement".http://www.clinicaltrials.gov and its identification number is NCT00612547.The study was conducted in compliance with the Helsinki Declaration and the laws of Malagasy. The protocol was submitted and approved by the Ethics Committee of National Malagasy (No. 153/SANPF/2007). Signed informed consent was obtained from the parents or legal guardian of the child. If parents or guardians could not write, a fingerprint was made. If parents or caregivers could not read, consent information was read to them in their local dialect. In this case, a witness was required to be present to sign the consent. This protocol was recorded in the registry of clinical trials at Between February 2008 and February 2010, 1,073 children were enrolled in the cohort study as described elsewhere . Of thesP. falciparum. The percentage of children with fever and parasite carriers was approximately two times higher in areas of high transmission  (Tables Of the 529 blood spots collected from February 2008 to February 2010 (97.4% of febrile episodes), 271 (51.2%) were positive only for P. falciparum, which was the only species found. The percentage positive slides did not differ by location. Parasite densities for the positive slides were between 48 and 82,000 parasites per μL. There were no significant differences between the mean parasite densities found in each area  were not suitable for microscopy examination. One hundred ninety seven (37.2%) were positive for .6% were Of the 190 RDTs carried out by CHWs between February 2009 and February 2010, 106 (55.8%) were positive: 94 tests detected both antigens (HRP2 and pLDH) and 12 tests detected only the HRP2 antigen Tables , and 4. Compared to the microscopy analysis, the RDTs produced 174 (91.6%) concordant results and 14 (8.4%) discordant results . The RDT results were similar for the two transmission zones. For the high transmission zone, 91.6% of the RDT results agreed with the microscopy results 8.4% did not . For the low transmission zone, 88.8% of the RDT results were agreed with the microscopy results and 11.2% were discordant . Only six of 11 false-positives RDTs produced only the HRP2 band. Four false-negative RDTs corresponded to samples in which the parasite levels were below 1,000 parasites/μL.RDT performance was evaluated by using either the PCR or microscopy results as reference standards. The analysis was done according to the two transmission zones Tables  and 6. OP. falciparum and negative controls were compliant but the concentration for the 200 parasites/μL. P. vivax control was non-compliant (200 and 2000 parasites/μL).All RDT control samples were found to be compliant before the tests were distributed. After distribution, six-month quality evaluations revealed that all RDT controls were compliant prior to those tested at 18 months and 24 months. For these RDT controls, the P. falciparum and the pLDH of four Plasmodium species: P. falciparum, P. vivax, P. malariae and P. ovale [In Madagascar, the integrated management of febrile syndromes at the community level remains a priority NMCP strategy for providing impoverished populations with diagnosis results and effective anti-malarial treatment within 24 hours after the febrile individual has access to care. In fact, this strategy relies on a system of CHWs and staff working in health centres, which has already proven to be effective for many years. The operational research project, evaluating the use of ACT and the implementation of RDTs, works through a network of CHWs and existing operations, which enabled us to obtain reliable data on the efficacy of "diagnostic" and "therapeutic" agents. Despite encountering difficulties in establishing a consensus for the type of RDT to be used in Madagascar, the NMCP introduced "combo" RDTs, which detect the HRP2 antigen specific for P. ovale ,14,15.P = 0.009).To evaluate the performance of RDTs used by CHWs, their results were compared to those obtained from the two most common methods used to diagnose malaria: PCR, which has the best performance in terms of sensitivity and specificity due to its very low threshold of detection (< 1 parasites/μL); and microscopy, which remains the WHO reference diagnostic despite its higher detection limit (10-50 parasites/μL) . For theP < 0.0001). However, this difference was not evident if microscopy, which detected an infection prevalence of 37.2%, was used as the diagnostic method. This finding demonstrates the importance of using molecular methods to study malaria epidemiology [P. falciparum, the predominant species in Madagascar [From an epidemiological standpoint, malaria burdens in the two study areas were assessed through the use of PCR. On average, 51.2% of the children consulting CHWs were carriers of parasites, and this proportion in area of high transmission was nearly twice as high as that in the low-transmission area  would have been tested again a day or two later by the community worker if clinical signs had persisted. Another encouraging sign for the introduction of RDTs at the community level was the good long-term stability of the RDTs over time, given they were kept in the field conditions by the CHWs. The potential benefits of RDT-negative results  could not be acted upon in the study. However, based on the PCR results, 49% of the children with fever who consulted CHWs were not infected with malaria. There were significantly more of these cases in the low-transmission areas (68.1%) than in the high-transmission area (43.3%). These data show that this testing strategy is solely limited to detect malaria infections and malaria is only a fraction of the fever etiologies seen at the community level. It is clear that further studies should be conducted to determine the agents responsible for non-malarial fever and to develop simple algorithms  to better manage cases of non-malarial fevers.The results, report here, indicate that the management of cases of fever among children under five by CHWs is desirable and feasible in rural areas, such as Madagascar, where many people live a considerable distance from health care facilities. Although the role of CHWs in the care of patients remains a subject of debate, our study suggests RDT use by CHWs improves access to quality care for individuals with uncomplicated malaria. Such a strategy is likely to substantially reduce the prevalence of severe malaria in young children, thereby preventing many deaths, and to reduce the overuse of anti-malarial drugs, which could reduce the risk of emergence and spread of artemisinin-resistant parasites. It remains for the NMCP of Madagascar to accurately assess the costs and benefits of this strategy according to the different epidemiological patterns that exist in Madagascar, especially those of the southern desert and those of the tropical west coast. It will also be necessary to develop new studies to improve the management of non-malarial fevers that produce a negative RDT result.The authors declare that they have no competing interests.DM and AR contributed to the design and coordination of the study, assisted with data entry and interpretation and prepared the manuscript. HR, JAV supervised the field study. RRah and MJ were involved in laboratory work. RRap, JDMR, DMa and PM helped to compose the manuscript and gave constructive. All authors read and approved the final manuscript.