Experimental models show a male bias in murine malaria; however, extant literature on biases in human clinical malaria is inconclusive. Studies in hyperendemic areas document an absence of sexual dimorphism in clinical malaria. Data on sex bias in clinical malaria in hypoendemic areas is ambiguous—some reports note a male bias but do not investigate the role of differential mosquito exposure in that bias. Moreover, these studies do not examine whether the bias is age related. This study investigates whether clinical malaria in hypoendemic regions exhibits a sex bias and whether this bias is age-dependent. We also consider the role of vector exposure in this bias.Retrospective passive clinical malaria datasets (2002–2007) and active surveillance datasets (2000–2009) were captured for the hypoendemic Mumbai region in Western India. To validate findings, passive retrospective data was captured from a primary malaria clinic (2006–2007) in hypoendemic Rourkela (Eastern India). Data was normalized by determining percent slide-positivity rates (SPRs) for males and females, and parasite-positivity distributions were established across age groups. The Mann–Whitney test, Wilcoxon Signed Rank test, and Chi-square analysis were used to determine statistical significances.In both the Mumbai and Rourkela regions, clinical malaria exhibited an adult male bias (p<0.01). A sex bias was not observed in children aged ≤10. Post-puberty, male SPRs were significantly greater than females SPRs (p<0.01). This adult male bias was observed for both vivax and falciparum clinical disease. Analysis of active surveillance data did not reveal an age or sex bias in the frequency of parasite positivity.This study demonstrates an age-dependent sex bias in clinical malaria in hypoendemic regions and enhanced incidence of clinical malaria in males following puberty. Possible roles of sex hormones, vector exposure, co-infections, and other factors in this enhanced susceptibility are discussed. Plasmodium falciparum and P. vivax infections, although some reports note an increased parasite density in pubertal and post-pubertal males Malaria accounts for a major portion of the global disease burden The data on sex bias in hypoendemic areas is equivocal. A few studies observed a skewed male∶female ratio in clinical malaria but did not investigate the phenomenon Anopheles stephensiPlasmodium vivaxP. falciparum, which is spread by An. culicifaciesThe study was conducted primarily in the hypoendemic urban area of Mumbai, situated in Western India. In this region, the principal malarial vector is the indoors- and outdoors-biting The retrospective data was collected and analyzed anonymously, free of any personally identifiable information. Therefore, informed consent was not necessary. Data was collected after obtaining appropriate institutional clearances as required by the Ethics Committee for Research on Human Subjects of Seth GS Medical College and KEM Hospital, Mumbai, and National Institute for Malaria Research.This retrospective study was conducted in the urban Mumbai region of Western India, consisting of Mumbai and Navi Mumbai . The twoP. vivax, and P. falciparum.Diagnosis of clinical malaria was based on presenting symptoms and parasite detection. Parasite identification was done by microscopic examination of Giemsa-stained thick and thin blood smears. Blood was obtained by venous puncture or finger-prick. The data was normalized by determining percent slide-positivity rates (SPRs) for both Records from public and private medical centres for the years 2002–2007 provided the clinical malaria dataset for the Mumbai region. It consisted of 31,616 patients who presented with malaria-like symptoms  at the King Edward Memorial (KEM) Hospital and Kasturba Hospital for Infectious Diseases in Mumbai, and private medical centres in Navi Mumbai. Relapsed/recrudescent patients, repeat blood samples, and pregnant women were excluded from analysis. Most malarial patients (>80%) were treated in the ‘Out Patients Department’ and did not require hospitalization. Both KEM and Kasturba hospitals are government funded and provide access to low-cost medical services. To test for biases caused by under-reporting due to economic considerations, data was also collected from private medical centres in Navi Mumbai that offer higher-cost medical services. Additionally, data was also collected from a government-run free primary malaria referral clinic in Rourkela (2006 and 2007). Majority of the patients reporting to this referral clinic belong to the town and peripheral areas of Rourkela city. Parasite-positive patients from this passive dataset constituted clinically diseased individuals seeking medical intervention for their malarial infection.P. vivax is ∼22 days whereas for P. falciparum it is 35 days. A surveillance cycle of 15 days  is designed to catch most of the secondary cases before the commencement of next cycle http://www.tifr.res.in/~dbs/faculty/sslab/index_files/epidem_data.htm.The malaria surveillance and control program is a house-to-house survey designed to detect suspected cases of malaria that are subsequently confirmed by blood-smear analysis; as per government policy, all positive cases are immediately treated with anti-malarial drugs. The main purpose of this surveillance is to detect changes in trends or distribution in malaria in order to initiate investigative or control measures. Under the malaria surveillance and control program, municipal health workers routinely visit residential areas, slums, construction sites, and municipal schools every fortnight. The incubation interval for Statistical analysis was carried out using GraphPad Instat P. falciparum had too few points for the Wilcoxon Signed Rank test, and as a result, this test could not be applied to that dataset. Passive datasets across years for Mumbai as well as for Rourkela were combined and the two combined datasets analyzed by Chi-square/Fisher's exact test. Mixed infections were rare in both datasets , and these were therefore included in analysis. Statistical significances remained unchanged when the mixed infection data was excluded from analysis.The mean age of menarche for Indian girls has been variously reported to be from age 12.4 to 13.1 years P. vivax was found to be the predominant parasite, accounting for ∼76% (range 69.5–88.7%) of clinical malaria patients P. falciparum accounted for the remaining ∼24% (range 11.3–30.5%). Clinical disease caused by mixed infections was rare; the dataset includes only 18 such instances.P. vivax and P. falciparum ascertained from the active surveillance records were between 0.07–2.5% and 0–2.14% respectively. The age and sex distributions of individuals in the passive and active datasets as well as of those who tested positive for P. vivax and/or P. falciparum are shown in In the active surveillance dataset from Mumbai, the parasite was detected in 257 individuals out of 21,924 persons tested . This paP. falciparum was the predominant parasite, accounting for 69.6% of the infections whereas 30.4% tested positive for P. vivax. There were 17 instances of mixed infections. Active surveillance has been discontinued in Rourkela from July 2007. The SPR based on the data available was ∼2.18% (data not shown).The Rourkela passive data was collected to test our hypothesis regarding an adult male bias in clinical malaria in hypoendemic regions. A total of 27,780 individuals approached the primary malaria clinic in Rourkela for their malaria-like symptoms during 2006 and 2007. Of these, 1,751 (6.30%) tested positive for the parasite . As repoTo assess the existence of a sex bias, we determined the difference between male and female SPRs. SPR is a ratio that represents the percent of malaria positive individuals in a given category and does not suffer from ascertainment bias even if greater number of individuals of a particular sex are included in a given group. Under the hypothesis of neutrality, the difference between male and female SPRs should be distributed around zero. In the Mumbai region, a sex bias was found in the clinical malaria dataset but not in the active surveillance dataset . This waP. falciparum as well. Every age group above age 15 showed a distinct adult male bias .We found that clinical malaria was primarily an adult male disease. Although greater number of males than females reported to medical facilities for their malaria-like symptoms . Field data from the National Institute of Malaria Research suggests the absence of a sex bias in mosquito exposure in Rourkela . Thus our analysis of the active surveillance dataset indicated that the frequency of parasite-positivity in the population was age- and sex-independent.By contrast, sex bias was absent in the active surveillance dataset, whether for lciparum ; p>0.05.lciparum . This ralciparum  or adultlciparum . The p vIn order to determine if the incidence of clinical malaria was related to puberty or reproductive years , we analyzed the clinical malaria data after regrouping it in ≤10, 20–40, and >55 age bins. The results remained essentially unchanged even when we raised the lower limit of the post-menopausal age bin to 60 years of age and distributed the data in ≤10, 20–40, and >60 age bins (data not shown). We used several methods of analyses to arrive at our conclusions. We compared SPRs of male and females in each age group by Mann–Whitney test. SPR data was also transformed to determine the fraction of positives contributed by males and females in a particular class. The use of such transformed data for statistical analysis ensured that there is no ascertainment bias. We also analyzed the datasets using Chi-square analysis which determined if the proportion of positives were significantly different in the groups under consideration. The Mann–Whitney test of the untransformed data gave the most conservative results, and they are reported in p<0.05) lower than that of adult males . However, the most noteworthy feature of clinical malaria for both regions was the dramatic increase in clinical disease in males post-puberty. Our analysis indicates that children ≤10, whether male or female, suffered equally from clinical malaria. In males, the onset of puberty coincided with the beginnings of a significantly increased clinical disease. In contrast to the dramatic increase in clinical disease in post-pubertal males, susceptibility to disease in females remained unchanged post-puberty. Menopause also did not appear to have an effect on susceptibility to clinical disease in women by the most conservative interpretation of the data. Thus, we find that women and children suffered equally from clinical disease and were far less likely to suffer from clinical malaria than adult males.The observed adult male bias was statistically significant, regardless of the method of analysis employed (p<0.05). It also seems to be independent of socio-economic strata. This bias is observed for clinical vivax and falciparum malaria in two distinct hypoendemic regions where the parasite is spread by two different anopheline species. Multiple parasite strains have been shown to co-exist in both Mumbai and Rourkela Plasmodium, clearly establish testosterone-linked susceptibility and estrogen-linked resistance to malaria Puberty is associated with an array of physiological and behavioral changes. Multiple factors, either singly or in combination, could contribute to the observed post-pubertal male bias in this study. We find an increased incidence of clinical malaria with the onset of puberty in males but not females. It is therefore tempting to implicate male sex hormones in this phenomenon. The role of sex hormones in disease susceptibility in human malarial disease remains ambiguous In most epidemiological assessments in hypoendemic areas, the data for the active and passive surveillance is clubbed and reported as the incidence of malaria An. stephensi, the principal vector in the Mumbai region, bites throughout the night, both indoors and outdoors An. culicifacies, the major vector in Rourkela, also bites indoors and outdoors It could be argued that adult males are more likely to be outdoors than adult females, and hence more likely to be bitten by infected mosquitoes. However this seems unlikely. Our analysis of active surveillance data showed that the frequency of parasite-positivity in the population was age- and sex-independent. The biting patterns of malaria vectors in Mumbai and Rourkela are well characterized. Entomological studies have clearly shown that biting activity of Anopheline mosquitoes is sex independent An. gambiensOther changes in male behavior occurring post-puberty such as alcohol and tobacco consumption Our conclusions are likely to be confounded by two major factors:An. stephensi. This suggests that the phenomenon of sex bias in hypoendemic areas may be a general feature for clinical malaria.Ascertainment bias in the clinical malaria dataset: We think that ascertainment bias is unlikely to influence our analysis for the following reasons. First, the adult male bias is observed across both primary and tertiary medical clinics, in low and high-cost medical centers and in two different regions of the country. Second, the bulk of our data for the Mumbai region was captured from KEM and Kasturba hospitals that have special clinical malaria programs. Physicians at these hospitals examine patients and on the basis of clinical assessment, all suspected clinical malaria patients —irrespective of gender—are tested for parasite presence by trained microscopists. Similarly, all patients approaching the primary malaria clinics in Rourkela are tested for the presence of the parasite. It would have been ideal to ascertain the illnesses of the malaria-negative population in our clinical malaria pool and analyze these for a sex bias, however, we captured our data from centers with special clinical malaria programs and primary malaria clinics, neither of which maintain any records on the nature of the patients' non-malaria illnesses. Sur et al. Socio-economic factors: Differences in the health-seeking behavior of males and females is likely to cofound our analysis. Indeed, our clinical malaria dataset showed a distinct male bias in health-seeking behavior, which is in accordance with reports of male-biased health-seeking behavior in the Indian subcontinent vs females). We also do not think economic factors are a likely source of distortion in our analysis as ratios of male∶female SPRs in the clinical dataset were ∼2∶1, irrespective of whether the dataset under consideration is a low-cost, high-cost, primary, or tertiary medical center.Furthermore, we used several different methods of statistical analyses to arrive at this conclusion. Apart from comparing SPRs of males and females, we also stratified the SPR data by age to determine the fraction of positives contributed by males and females in the various age groups and then compared these fractions between males and females within the age group by stringent statistical analysis. This allowed us to avoid ascertainment bias because even though the total number of males approaching medical centers was higher than females, the fraction of positives contributed by males in a given positive pool did not depend on the total number of males tested. The Chi square analysis was also used to determine whether the proportion of positives were statistically significantly different in the given groups. In summary, the use of a proportion avoids ascertainment bias as it does not depend on the total number tested in each class  and Non-monsoon (C) season. B,D, Box plots as in A showing the difference in the percent slide-positivity rates between males and females across age groups who tested positive for P. vivax in the Monsoon (B) and Non-monsoon (D) season in the active surveillance programme. Data were compared with the difference of male/female SPRs expected under the hypothesis of neutrality  and were analyzed with the Mann–Whitney test. Statistically significant values are shown in black. Numbers in red indicate statistically significant p values obtained by Wilcoxon Signed Rank test under the hypothesis that the median of the group did not differ significantly from zero. The test could not be applied to the falciparum data in the active surveillance dataset.(TIF)Click here for additional data file.Figure S2Age distributions of SPRs of males and females in the combined clinical malaria datasets. A, B, Percent slide-positivity rate of P. vivax (A) and P. falciparum (B) attributable clinical disease amongst male  and female  patients across age groups in the combined Mumbai dataset. C, D, Percent slide-positivity rate of P. vivax (C) and P. falciparum (D) attributable clinical disease amongst male  and female  patients across age groups in the combined Rourkela dataset. Statistically significant values obtained by Chi-square analysis between the sexes for a given age group and across age group within the same sex are indicated.(TIF)Click here for additional data file.Figure S3Fraction contributed by males and females to the total positive pool in a particular age group in clinical malaria datasets. A, B, Box plots showing the 25th and 75th percentiles, together with the median, with whiskers showing the minimum and maximum fraction contributed to the total positive pool by males (M) and females (F) across age groups in the Mumbai region for clinical vivax (A) and falciparum (B) malaria. C, D, Box plots showing the 25th and 75th percentiles, together with the median, with whiskers showing the minimum and maximum fraction contributed to the total positive pool by males (M) and females (F) across age groups in the Rourkela region for clinical vivax (C) and falciparum (D) malaria. Statistically significant values obtained by the Mann–Whitney test are indicated.(TIF)Click here for additional data file.


To identify the factors that account for differences in clinical outcomes of malaria as well as its relationship with ethnicity, transmission intensity and parasite density.A prospective study was conducted in nine health facilities in the Centre, Littoral and South West regions of Cameroon, and in three ethnic groups; the Bantu, Semi-Bantu and Foulbe. Children aged one month to 13 years, with diagnosis suggestive of malaria, were recruited and characterized using the WHO definition for severe and uncomplicated malaria. Malaria parasitaemia was determined by light microscopy, haematological analysis using an automated haematology analyser and glucose level by colorimetric technique.P < 0.004) in proportion in the Centre  compared to the Littoral  and South West  regions. Severe anaemia was the most frequent severe disease manifestation, 28.0% (248/885), which was similar in proportion across the three ethnic groups but was more prevalent in females, less than 60 months old, and the Centre region. About 20% (53/267) of the participants presented with respiratory distress, a clinical phenotype independent of age, gender and ethnicity, but highest (P < 0.001) in the Centre  compared to the Littoral  and South West  regions. Uncomplicated malaria constituted 27.7% (255/920) of hospital admissions and was similar in proportion with gender and across the three ethnic groups but more prevalent in older children (≥ 60 months) as well as in the South West region. The density of malaria parasitaemia was generally similar across clinical groups, gender and ethnicity. However, younger children and residents of the Centre region carried significantly higher parasite loads, with the burden heavier in the Semi-Bantu compared to their Bantu (P = 0.009) and Foulbe (P = 0.026) counterparts in the Centre region. The overall study case fatality was 4.8 (47/755), with cerebral malaria being the only significant risk factor associated with death. Severe anaemia, though a common and major clinical presentation, was not significantly associated with risk of death.Of the febrile children screened, 971 of the febrile children screened fulfilled the inclusion criteria for specific malaria clinical phenotypes. Forty-nine (9.2%) children had cerebral malaria, a feature that was similar across age groups, ethnicity and gender but lower (About half of the acutely febrile children presented with severe malaria, the majority being cases of severe malaria anaemia, followed by respiratory distress and cerebral malaria. The latter two were less prevalent in the Centre region compared to the other regions. Cerebral malaria and hyperpyrexia were the only significant risk factors associated with death. Plasmodium falciparum, the deadliest of the malaria parasite species, kills a child every 30 seconds in Africa  respectively  was the most frequent severe manifestation of malaria, 28.0% 248/885) and was similar in proportion across the three ethnic groups but more prevalent in females, less than 60 month of age and the Centre region Table . The mea8/885 andThe total case fatality among children with severe anaemia  was similar to that of children without SMA 5.5%, 28/509) (Table 8/509 (TaAbout 20% (53/267) of participants assessed presented with respiratory distress (RD). The proportion of children with this phenotype was independent of age, gender and ethnicity but highest (P < 0.001) in the Centre  compared to the Littoral  and South West  regions. The geometric mean parasite load of these participants was also similar to that of patients without respiratory distress Table .Uncomplicated malaria (UM) was also a very frequent manifestation constituting 26.3% 242/920) of hospital admissions. This clinical phenotype was similar in proportion with gender and across the three ethnic groups but more prevalent in older children as well as in the South West region (Table 2/920 of P = 0.003), with higher densities in the Semi-Bantu compared to their Bantu (P = 0.009) and Foulbe (P = 0.026) counterparts  parasite loads compared to their male counterparts.The geometric mean density of parasitaemia was generally similar in cerebral malaria , severe malaria anaemia  and uncomplicated malaria  patients as well as with gender and ethnicity. Younger children and residents of the Centre region, however, carried significantly higher parasite loads Table . Furthervs 5.8%), Bantu, Semi-Bantu and Foulbe ethnic groups (5.7% vs 6.0% vs 6.9%) as well as the Centre, Littoral and South West regions (8.7% vs 6.7% vs 4.6%). Their baseline presentations were: age less than 60 months 91.1% (41/45), respiratory distress 46.7% (7/15), severe malaria anaemia 34.9% (15/43), cerebral malaria 29.7% (11/37), hyperpyrexia 27.3% (12/44), hypoglycaemia 21.4% (9/42) and hyperparasitaemia 11.1% (5/45). Bivariate analysis . This was similar in males and females  for future malaria vaccine efficacy trials, the scaling up of the provision of insecticide-treated nets and the adoption of new effective therapeutics. Furthermore, it is not clear how the peripheral parasite density varies with transmission or influences the different types of severe malaria clinical features. This study sets out to determine the factors that account for different clinical outcomes of malaria parasite infection as well as its relationship with ethnicity, transmission intensity and parasite density in young children from three regions with distinct ecological conditions across Cameroon. The results showed that significant hospital admissions and numbers of severe malaria cases exist in the Centre, Littoral and South West regions of Cameroon and that severe malaria in this setting may represent an adequate vaccine trial endpoint ,28.The current study showed that over a period of 48 months, 971 (49.4%) hospital-based malaria cases were recruited, the majority 57.7%, 492/852) aged below 36 months, with a significant decreasing trend towards increasing age  across these ethnic groups in the region. Also, since these individuals live in the same region, location cannot account for the difference observed. The finding seems to suggest that Semi-Bantu are more susceptible compared to the Bantu and Foulbe. Several studies have demonstrated differences in susceptibility to malaria, with the Fulani of West Africa having a lower incidence of malaria than other sympatric groups [Surprisingly, malaria parasitaemia varied with ethnicity in the Centre region Figure . This isc groups  and the c groups . This isc groups . Variousc groups . Such gec groups .vs. 3.7%), age groups (3.3% vs. 3.9%), ethnicity and location. This observation is higher than previous reports in Africa [The overall case fatality in this study was 4.8%; this was similar by gender (3.3% n Africa ,14 but mn Africa ,13. Seven Africa , perhapsEven though mortality impact is the most important public health measure of any vaccine efficacy, it represents a smaller proportion of all disease burdens and will, thus, require large sample size in future trials . Again tA significant seasonal variation of the prevalence of severe malaria was recorded over the study period Figure . The higResults from this study suggest that significant hospital admissions and numbers of severe malaria cases exist in the Centre, Littoral and South West regions of Cameroon. In addition, severe malaria in these areas occurs frequently in the first 24 months of life, the predominant feature being severe anaemia while cerebral malaria and hyperpyrexia were the independent prognostic indicator of death. Although the variation of parasitaemia with ethnicity in the Centre region seems to suggest that Semi-Bantu are more susceptible compared to the Bantu and Foulbe, the differences observed may be pathogen related and not due to a general hyper-reactive immune system as previously reported. Such genetic differences may be less apparent in the Littoral and South West due to extensive ethnic mixing.The authors declare that they have no competing interests; but they had financial support from MalariaGEN and MIMPAC for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work; no other relationships or activities that could appear to have influenced the submitted work.EAA initiated the collaborative project, designed data collection tools, monitored data collection for the surveys, drafted and revised the paper. TOA implemented the surveys, wrote the statistical analysis plan, cleaned and analysed the data, and drafted and revised the paper. RNM implemented the surveys and revised the paper. ANN implemented the surveys and revised the paper. JKA-K implemented the surveys and revised the paper. CNY implemented the surveys and revised the paper. All authors have read and approved the final manuscript.


Malaria in pregnancy is associated with maternal and foetal morbidity and mortality in endemic areas, but information on imported cases to non-endemic areas is scarce.The aim of this study was to describe the clinical and epidemiological characteristics of malaria in pregnancy in two general hospitals in Madrid, Spain.Retrospective descriptive study of laboratory-confirmed malaria in pregnant women at the Fuenlabrada University Hospital and the Príncipe de Asturias University Hospital, in Madrid, over a six- and 11-year period, respectively. Relevant epidemiological, clinical and laboratory data was obtained from medical records.rd, 31% 1st, 16% 2nd. All but one were multigravidae. Three were HIV positive. All were sub-Saharan immigrants: two were recently arrived immigrants and seventeen (89%) had visited friends and relatives. None had taken prophylaxis nor seeked pre-travel advice. Presentation: 16 symptomatic patients , three asymptomatic. Median delay in diagnosis: 7.5 days. Laboratory tests: anaemia (cut off Hb level 11 g/dl) 78.9%  thrombocytopaenia 73.7%, hypoglycaemia 10.5%. All cases were due to Plasmodium falciparum, one case of hyperparasitaemia. Quinine + clindamycin prescribed in 84%. Outcomes: no severe maternal complications or deaths, two abortions, fifteen term pregnancies, no low-birth-weight newborns, two patients were lost to follow-up.There were 19 pregnant women among 346 malaria cases (5.4%). The average age was 27 years. The gestational age (trimester) was: 53% 3Though cases of malaria in pregnancy are uncommon, a most at risk group is clearly defined: young sub-Saharan mothers visiting friends and relatives without pre-travel counselling and recently-arrived immigrants. The most common adverse maternal and foetal effects were anaemia and stillbirth. Given that presentation can be asymptomatic, malaria should always be considered in patients with unexplained anaemia arriving from endemic areas. These findings could help Maternal Health programme planners and implementers to target preventive interventions in the immigrant population and should create awareness among clinicians. Malaria in pregnancy is known to be a primary cause of morbidity and mortality in both mother and foetus in endemic areas. It is in this setting that this form of malaria is being extensively studied and where current knowledge derives from. Information on the incidence and relevance of this particular type of malaria in non-endemic, industrialized areas is scarce -3. ThougPlasmodium falciparum. The region of Madrid registers a proportionally large number of cases: 21-37% of all malaria cases diagnosed in Spain during the period 2006-2009 were reported from Madrid. In 2009, the incidence rate (IR) was 2,0/100,000, three-times higher than the national IR [In Spain, around 350-400 cases of malaria/year have been reported in the past five years to the national communicable diseases database, though it is estimated that the total number of imported cases is over 400, because of under-reporting. The majority are due to Within the region of Madrid, a high proportion of cases are reported from the southern and eastern areas where a large number of migrants originally from endemic areas have settled. The Fuenlabrada University Hospital (FUH) and the Príncipe de Asturias University Hospital (PAUH) are both located in these two particularly migrant populated areas. Nonetheless, as reported by the regional surveillance network, these hospitals diagnosed 53% of malaria cases in Madrid in both 2008 and 2009, despite only 10% of total regional population living in these areas .Although imported malaria to Spain has already been described in several reports from different regions and health care settings and guidelines highlighting the risk and prevention strategies during pregnancy have been published, little is known about the relevance and characteristics of imported malaria during pregnancy ,6.The aim of this study was to describe the clinical and epidemiological characteristics and outcomes of malaria in pregnancy in these two general hospitals in a non-endemic country.st 2000 to June 1st 2011 and January 1st 2005 to June 1st 2011, respectively. Laboratory criteria used for confirmation of diagnosis was the demonstration of asexual forms of malaria parasites in a thick and/or thin blood film. Positive antigen detection  and positive polymerase chain reaction test for Plasmodium nucleic acid was considered additional support to diagnosis. Clinical, epidemiological and laboratory information was collected from medical records.A retrospective descriptive review was conducted on all pregnant women with laboratory confirmed malaria diagnosed in the PAUH and the FUH, during the period January 1Demographic characteristics included age, country of origin, time elapsed since arrival to Spain, reason for travel to endemic area  and recently arrived migrant status. Epidemiological data included: area where malaria was acquired from (last endemic area visited), month, incubation period (number of days elapsed between the last day in endemic area till symptom onset), delay in diagnosis  and written evidence of any pre-travel advice.Clinical data collected included previous medical history (co-morbid conditions), current presentation classified as fever, flu-like, gastrointestinal or respiratory symptoms and other presentations, WHO criteria for severe malaria , any remarkable clinical sign observed at diagnosis and information on chemoprophylaxis (no/inadequately taken prophylaxis or adequate prophylaxis).3), moderate  and severe  thrombocytopenia and any other remarkable laboratory finding at diagnosis. Microbiological data included Plasmodium species, parasitaemia at diagnosis, and results of rapid diagnostic tests and PCR if they had been carried out. Information on the treatment prescribed and clinical and parasitological follow-up at days 3, 7 and 30 after treatment onset was also collected.Laboratory data included anaemia assessment according to WHO standards (anaemia was considered present when haemoglobin (Hb) < 11 g/dl; cut off levels for mild, moderate and severe anaemia were Hb 9-10.9 g/dl, Hb7.1-8.9 g/dl and Hb < 7 g/dl, respectively), platelet count and classification as mild . Continuous variables are reported as mean +/- standard deviation (SD) or median and interquartile range (IR). Categorical variables are reported as frequencies and percentage of each category.There were 19 malaria cases in pregnant women among 346 malaria cases diagnosed in both institutions during the study period (5.4%). Demographical, epidemiological, clinical, microbiological and laboratory characteristics are summarized in Table All but one were multigravidae. All patients were young sub-Saharan African women, mainly from Equatorial Guinea (58%). There were no national travellers. All but two (89%) were VFR. Median time living in Spain of the 15 VFR for whom this information was available was 6 years (IR 10 months-10 years). The two non-VFR patients were recently arrived immigrants to Spain, one month and six months before clinical presentation, respectively.Malaria was acquired in their countries of origin in all cases except for one patient who had visited relatives in another endemic country. There was no written evidence of any pre-travel advice in any case and none had correctly taken chemoprophylaxis. Sixteen patients (83.5%) had acquired malaria travelling during either summer or Christmas holiday periods. No information was available on intermittent preventive treatment.The majority of patients (58%) did not have any other medical condition prior to malaria. There were three HIV positive women: two CDC stage A2 and one A1. Six pregnancies (32%) had previously been classified as high risk. The reasons for this classification were: HIV positivity in three cases, hypertension in one case, gestational diabetes in another case and, finally, one patient was undergoing close follow-up because of a history of fever during her stay in Africa.rd trimester severe anaemia, in one of the HIV positive patients both malaria and microfilariae were observed in the course of a routine 3rd trimester blood test, the other HIV positive patient was found to have malaria after the finding of an unexplained hepatosplenomegaly during a routine obstetric ultrasound. Other than this patient and another patient with splenomegaly, clinical examination was unremarkable in all cases.There were three asymptomatic cases. The predominant clinical syndrome among the symptomatic women was fever (14 patients) with or without associated symptoms. The 2 symptomatic non-febrile women referred asthenia as major complaint. Malaria parasites were detected in the three asymptomatic cases because of different diagnostic work-ups: in one case, the patient was being studied for unexplained 3Date of symptom onset was recorded in 15 of the symptomatic cases. Symptom onset occurred while still in an endemic area in five cases (17 days to 1 day before returning to Spain). Nine patients started referring symptoms once in Spain, ranging from seven to 69 days after the last day in the endemic area . Median delay in diagnosis was 7.5 days (IR 2.7-21.7 days). In 7 cases, diagnosis was made more than one week after symptom onset.None of the patients presented clinical features for severe malaria and only in one case was a laboratory criteria observed, a parasitaemia level over 4%. The most frequent laboratory finding was anaemia, (79%), followed by thrombocytopaenia (74%) and hypoglycaemia .P. falciparum malaria, with one mixed infection. A rapid diagnostic test was used as complementary diagnostic support in 12 cases, and PCR testing was carried out in eight cases, yielding positive results for P. falciparum in all cases. Data on parasitological follow-up was incomplete. Twelve patients had confirmed negative parasitaemia at any time during follow-up.All cases were The majority of cases (84%) were treated with quinine plus clindamycin. Patients with severe anaemia received blood transfusions. There were no maternal complications or deaths. Pregnancy outcomes were favourable in 15 cases: all term pregnancies, no low birth weight newborns. There were two premature abortions. Anaemia was reassessed in 10 patients; in all but one there was an increase in Hb levels after treatment.P. falciparum malaria cases among pregnant women attending the two suburban general hospitals where a large proportion of malaria cases are reported from in the region of Madrid. Results show that, though this presentation is uncommon, a most at risk group is clearly defined: young sub-Saharan mothers visiting friends and relatives without pre-travel counselling and recently arrived immigrants.This study describes the characteristics of 19 Pregnant women are at increased risk for malaria infection. Increased susceptibility has long been recognized and multiple factors have been identified, yet, there are still many gaps in knowledge that need to be addressed. Placental infected erythrocytes differ from those found in peripheral blood because they present surface antigens that act as ligands permitting adhesion to chondroitin-sulphate A receptors in the intervillous space. IgG antibodies against pregnancy specific parasites are gradually developed resulting in a unique form of immunity: parity-specific immunity.Thus, in endemic areas, the clinical impact varies depending on transmission. In highly endemic areas with stable transmission, the frequency and severity of the infection is higher in primigravidae than multigravidae, while, in hypoendemic areas with unstable transmission, women have less opportunity to develop parity-specific immunity and so are equally susceptible across the parity.P. falciparum. In areas of high transmission, severe anaemia is common and is associated with morbidity and mortality. Foetal disease presents as preterm delivery, stillbirth and a higher risk of LBW and consequently increased risk of death in infancy, whether or not maternal symptoms are detected during pregnancy [Malaria is associated with maternal and foetal morbidity and mortality. Percentages of maternal deaths vary depending on studies: up to 23% or up to 17% in hospital-based and community-based studies in Africa, respectively [regnancy . UnfortuIn the United States, basic epidemiological data is available from the Centre for Disease Control surveillance summaries. For instance, in 2005, a total of 21 cases of malaria were reported among pregnant women, representing 4% of cases among women. Among these, 66% were U.S. civilians and only one third reported taking malaria chemoprophylaxis .In Europe, international surveillance networks on imported diseases do not specify the number of pregnant women within female malaria cases in the patient characteristics topic . In SpaiPrevious reports on imported malaria have already described the high density of migrant population within the catchment population of both hospitals and the high proportion of immigrants not taking correct prophylaxis . Given tTable P. vivax in Latin American pregnant women could also possibly be misdiagnosed. Globally, results from the European Network on Tropical Diseases show that the proportion of imported cases caused by P. falciparum in VFR has increased in the last decade. Concomitantly, patient characteristics are different in the other malaria species, where European travellers make up the predominant group. The present and previous studies are consistent with the epidemiological trend in Europe and lead to several questions [There were neither national travellers nor patients from other endemic areas such as Latin America or Asia, though few malaria cases within this other groups have been reported in both hospitals. The large proportion of sub-Saharan immigrants compared to those of different origin and the fact that tropical areas are an uncommon travel destiny among the national low-middle income population attended at our institutions could explain this finding. Less severe presentations due to uestions .First, from the patients' side, are sociocultural and demographic variables influencing on perceived susceptibility and severity of malaria in pregnancy and recognition of malaria and anaemia during pregnancy? Frequently, migrants travelling to their countries of origin mistakenly believe that they retain lifelong immunity and do not seek advice prior to travel nor take appropriate prophylaxis, as was observed in this and previous studies. Also, illness recognition may be hindered and women might confuse malaria symptoms with pregnancy-related symptoms. Socio-cultural aspects on the specific topic of malaria in pregnancy have received little attention in endemic areas . FurtherFrom the health system's side, is there availability and accessibility of health advice in antenatal care clinics and primary care regarding malaria? Health staff attending pregnant immigrant women is crucial in recognizing the potential risk of travel and in considering malaria when a diagnostic work-up is carried out. A large proportion of immigrant patients seek care in general hospitals, situated near their homes, where a tropical diseases department is not always available. Pregnant women are usually attended by obstetricians and general practitioners, who might not be familiarized with this type of malaria. In this study, pregnancies were classified as high risk because of co-morbid conditions and not because of malaria exposure risk. Only in one case exposure was considered, and this was because the patient had had fever during her stay. Awareness of this issue should be raised among physicians, midwives and health policy makers.Regarding clinical presentation, in malaria, nature of effects depends on the patient's background premunition. In pregnancy, parity-specific immunity is crucial and little is known about loss of this specific immunity years after leaving an endemic zone.Among the sixteen symptomatic patients, symptoms were non-specific with a predominance of fever and the median diagnostic delay was 7 days. Previous series have attributed delays to misdiagnoses with common illnesses due to unfamiliarity with the disease . The twoSimilarly, absence of symptoms in three patients might have been due to some degree of pre-existing immunity retained during pregnancy, though time living in Spain ranged widely from four months to six years. Many pregnant women living in high transmission areas are known to remain asymptomatic. It is noteworthy that two asymptomatic patients had anaemia, one of which was severe. In fact, a study in Mozambique found no evidence of association between signs and symptoms suggestive of malaria and the presence of microscopically detected peripheral malaria, but anaemia was the only sign associated with sub-microscopic parasitaemia detected exclusively by PCR, suggesting that low density asymptomatic infections may increase the risk of anaemia .Anaemia (WHO definition: haemoglobin ≤ 11 g/dl) is one of the world's leading causes of disability, affecting nearly half of all pregnant women in the world: 52% in developing countries, 23% in the developed world . A stronP. falciparum malaria in Mozambican pregnant women, risk of maternal anaemia was higher in PCR positive women than in PCR negative (OR 1.92 CI 1.09-3.36) [P. falciparum infections is high even in the absence of parasitaemia [One of the most common causes of anaemia is malaria. In the study on sub-microscopic 09-3.36) . WHO recsitaemia . AnaemiaOther complications observed in the present and in previous reports were hypoglycaemia and splenomegaly. Similarly to the French series, there were no maternal deaths nor other complications apart from those mentioned above, suggesting that probably, this mainly multigravidae group of women retained a certain level of parity-specific immunity years after leaving endemic areas. But adverse foetal outcomes in both series did occur in the form of abortions and preterm delivery of a LBW neonate. One fatal maternal case was described in the U.S. report: this patient was primigravidae, diagnosis was delayed and she progressed towards severe complications (hypoglycaemia and respiratory distress) .Thus, globally, clinical suspicion in a non-endemic area such as Spain may be challenging due to the unfamiliarity with the disease and with the link between anaemia and malaria and the low specificity of clinical complaints and signs. Prompt diagnosis and treatment will depend on illness recognition and the women's health-seeking behaviour based on her perceived susceptibility to malaria.During pregnancy, asymptomatic low parasite densities and parasites sequestered in the placenta are harmful to the mother and foetus, and the sensitivity of microsocopy might be insufficient in these cases. Moreover, parasitaemia can fluctuate and be kept under level of detection by acquired immunity or self-medication, particularly late in pregnancy, and recrudescences can occur later than in non-pregnant patients . This caPatients received treatment following current guidelines and depending on availability of drugs. Following treatment it is necessary to reliably confirm infection and clearance of parasites. In this study, follow up of nearly 40% of patients was not complete to guarantee parasite clearance. But, assessment of the efficacy of treatment is complicated. Except when the treatment occurs near delivery, to know whether current regimens are able to clear placental parasites requires sensitive diagnostic tools. PCR allows the identification of parasitaemia levels below the threshold of microscopy and can be useful in predicting adverse outcomes in pregnancy .Acquired anti-malarial immunity depends not only on the intensity of transmission and the number of previous pregnancies, but also on the presence of conditions that decrease immunity such as HIV infection. HIV infection restricts the development of parity-specific immunity, has an additive effect on LBW and anaemia and has been shown to cause more clinical malaria and higher parasitaemia in perennial transmission areas, and higher rates of severe episodes and mortality in areas with seasonal transmission. Regarding the role of placental malaria in HIV mother to child transmission (MTCT), results are controversial. Both maternal HIV RNA viral load and anaemia are independent risk factors for MTCT . There wThis is a small-sample size, retrospective, hospital-based descriptive study with the limitations that pertain to the nature of the study design. Nevertheless, these findings highlight that falciparum malaria needs to be considered as a differential diagnosis when dealing with pregnant women who might have been exposed in endemic areas, particularly sub-Saharan VFRs, even in the absence of symptoms, and should be especially considered in patients with unexplained anaemia.Uptake of chemoprophylaxis is still low in immigrant travellers. Hence, tailoring health messages to immigrant groups is a priority and access to information on prevention, chemoprophylaxis and health-seeking attitudes should be made available to immigrants in general hospitals.The authors declare that they have no competing interests.BCJ, JMRG and GRM designed the study. All authors complied data and contributed to the bibliographic research. BCJ, JMRG, GRM created the database and interpreted the results. BCJ performed the statistical analysis and drafted the article and JMRG, GRM, PCT and JCG revised it critically. All authors approved the final manuscript.


Leptospira, Rickettsia, O. tsutsugamushi, Dengue- and Influenza virus. At least one pathogen was identified in 73.3% (874/1193) of febrile patient samples. Most frequent pathogens detected were P. vivax (33.4%), P. falciparum (26.5%), pathogenic Leptospira (9.4%), Influenza viruses (8.9%), Dengue viruses (6.3%), O. tsutsugamushi (3.9%), Rickettsia (0.2%), and P. knowlesi (0.1%). In the control group, a potential pathogen was identified in 40.4%, most commonly malaria parasites and Leptospira. Clinic-based diagnosis of malaria RDT-negative cases was poorly predictive for pathogen and appropriate treatment. Additional investigations are needed to understand their impact on clinical disease and epidemiology, and the possible role of therapies such as doxycycline, since many of these pathogens were seen in non-febrile subjects.In the past decade, malaria control has been successfully implemented in Cambodia, leading to a substantial decrease in reported cases. Wide-spread use of malaria rapid diagnostic tests (RDTs) has revealed a large burden of malaria-negative fever cases, for which no clinical management guidelines exist at peripheral level health facilities. As a first step towards developing such guidelines, a 3-year cross-sectional prospective observational study was designed to investigate the causes of acute malaria-negative febrile illness in Cambodia. From January 2008 to December 2010, 1193 febrile patients and 282 non-febrile individuals were recruited from three health centers in eastern and western Cambodia. Malaria RDTs and routine clinical examination were performed on site by health center staff. Venous samples and nasopharyngeal throat swabs were collected and analysed by molecular diagnostic tests. Blood cultures and blood smears were also taken from all febrile individuals. Molecular testing was applied for malaria parasites,  Over the last 13 years there was a steady reduction of around 10% per year for diagnosed cases and 8.5% for case fatality rates P. vivax cases rose among reported cases over this time, from 8% in 2000 to 37% in 2011 Fever is the main clinical symptom of various tropical infectious diseases. In rural areas of developing countries, where diagnostic facilities are limited, etiologies of acute febrile illness remain largely unknown. Malaria has long been considered the most important infectious disease in the Mekong Region in terms of public health impact and it’s elimination has become an international priority with the emergence of artemisinin-resistant parasites along the Thai-Cambodian border In this context, we designed a cross-sectional prospective observational study to investigate the causes of NMFI in three rural areas of Cambodia. Our primary objective was to detect pathogens in samples taken from febrile outpatients in the remote areas where malaria persists, and where malaria RDTs are the only available diagnostic tool for health workers. We also aimed to determine priorities for diagnostic tools applicable in field conditions and guide the improvement of case management algorithms. Additionally, this study aimed to build capacity for operational research and pathology testing within Cambodia and develop a prototype study design useable in similar settings in other tropical countries.The study protocol was reviewed and approved by the Cambodian National Ethics Committee on Health Research. An informed written consent was provided by the parents/guardians of all participants before testing. All samples were anonymized prior to laboratory testing.Three outpatient health centers (HCs) in remote areas in western  and eastern Cambodia  were selected as study sites . These sFrom January 2008 to December 2010, all male and female patients, aged from 7 to 49 years, who had an acute febrile illness , and who were eligible to be tested for malaria by RDT according to the national guidelines  Combo G0131, Access Bio Inc. USA) were invited to participate in the study. Patients with severe illness warranting immediate transfer to a hospital were excluded.In parallel, a group of non-febrile accompanying persons, aged 7–49 years, with a tympanic membrane temperature <38.0°C, no recent history of febrile illness, and providing written informed consent, were recruited as controls.In both groups (cases and controls groups) a physical examination and malaria RDT  COMBO G0131, Access Bio Inc. USA) were performed as per routine practice by the clinical staff. History of illness and results of the physical examination (for case group) were recorded on standardized and simplified forms in Cambodian language, provided by the National Malaria Control Programme . Whole bPlasmodium , Leptospira interrogans 16sRNA sequence (GenBank accession number EU581713), Orientia tsutsugamushi 47 kDa sequence (GenBank accession number L11697) and Rickettsia sp. OmpB and GltA sequences (GenBank accession numbers EF219461 and AB444098). Plasma and throat swab samples were processed for RNA extraction using the QIamp Viral RNA Mini Kit  on 200 µl of plasma and from 140 µl of throat swab-sample, according to the manufacturer’s instructions. In-house multiplex Reverse-Transcriptase (RT) PCR assays were performed on plasma and throat swab RNA extracts, for detection of Dengue , according to the manufacturer’s instructions. Nested-PCRs and nucleotide sequencing were applied to detect DNA sequences of The thresholds of detection for each PCR/RT-PCR were assessed by using dilutions of positive controls . ExternaAvailable laboratory results which may have had a direct bearing on patient management were immediately sent back to the HCs. Immediate decisions on the use of antimalarial or antimicrobial drugs were taken by qualified personnel at the HCs as per usual practice.U test or Kruskal-Wallis method were used for non-parametric comparisons, and Student’s t test or one-way analysis of variance for parametric comparisons. For categorical variables, Chi-squared or Fisher’s exact tests were used to assess significant differences in proportions. Odd ratios (OR) and their 95% confidence intervals (CI) were measured to compare prevalence of pathogens between case and control groups. The Attributable Fraction among case (AFe) was assessed for each pathogen using the following formula: [(OR-1)/OR]×100. Hypothesis testing was made using risk differences, exact 95% confidence intervals, and p-values. A p-value (two-tailed) of less than 0.05 was considered statistically significant.Data were entered and verified using Microsoft Excel software and analyzed using XLSTAT  and MedCalc . The Mann-Whitney From January 2008 to December 2010, 1475 individuals were recruited. Among them, 1193 (80.8%) were febrile patients (case group): 621 in Soun Kouma HC and 650 in Ou Chra HC in Pailin Province and 204 in Snoul HC in Kratie Province, and 282 (19.1%) non-febrile individuals (control group) . As presOf febrile cases, 383/1193 (32.1%) had a positive malaria-RDT on admission  and wereP. vivax  followed by P. falciparum . Both Plasmodium spp. were more frequently detected in the case than in the control group (p<0.01), with AFe values of 72% and 61%, respectively. In 7.4% of all malaria positive samples (56/754), P. falciparum/P. vivax mixed infections were detected without difference in term of frequency between the groups (p = 0.07). Two human cases of the emerging simian malaria parasite P. knowlesi were also detected, one in the case and one in the control group. P. ovale was found only once, in the control group  enrolled individuals were malaria positive by PCR. The observed prevalence was almost 2-fold higher in the case group  compared to the control group . The attributable fraction among cases (AFe) was estimated to 70% . Nucleotol group .All positive malaria RDT individuals  belonged to the case group  and 376/383 (98.2%) of them were confirmed positive by malaria-PCR. In addition, 300 patients from the case group with negative malaria RDT results were also positive by malaria PCR.Leptospira spp. were identified as the second most frequent pathogens. Globally, 167/1475 samples were positive by PCR (11.3%). The ratio between pathogenic and non-pathogenic Leptospira was 140/27. The distribution of the main pathogenic species determined by sequencing was as following: L. interrogans , L. weilii , L. kmetyi , L. kirschneri , L. santarosai , L. wolffii , L. genomospecies1  and L. noguchii   compared to Pailin (29/74). Interestingly, the prevalence of L. interrogans was higher in the control group  than in the case group  , while the prevalence of other pathogenic Leptospira spp. was higher in cases than controls . The AFe were not evaluated.1, 0.7%) . The oveO. tsutsugamushi was detected in 54/1475 samples (3.7%). Its prevalence was not associated with site or with fever. Moreover, 3 samples were positive for other Rickettsia species.Virological tests results were available for 1470 plasma and 1473 throat swab samples. Of the plasma samples, 80/1470 (5.4%) were found positive for Dengue virus type 2 , type 1 , type 4  and type 3 . The overall prevalence was not significantly different between sites, but more frequent in febrile patients  than in non-febrile individuals . The prevalence of type 2 and type 4 was highest in Ou Chra and Soun Komar HC in Pailin province, respectively. The temporal distribution of the dengue cases clearly showed an annual epidemic peaking between May and October .vs. 7/282, 2.5% in control group, p = 0.001). Influenza A cases were more frequent in febrile patients  than in non-febrile individuals . As observed with Dengue, the temporal distribution of Influenza cases clearly showed a seasonal emergence between September and November  and B serotypes  were detected in the throat swabs. The overall prevalence was significantly different between sites: 11.3% in Ou Chra HC, 5.5% in Soun Komar HC and 3.4% in Snoul HC (p<0.01) and between groups , E. coli (n = 1), E. cloacae (n = 5), S. typhi (n = 1) and S. paratyphi (n = 1). In 2 cases (E. cloacae and S. paratyphi) was associated with P. vivax infection. Other positive bacteria growths (98/1128) identifying as Staphylococcus non-aureus, Corynebacteria spp., Pseudomonas fluorescens and Pseudomonas putida were considered as contamination. These results were immediately reported back to the health center and all patients were provided the appropriate antimicrobial treatment.Among 1128 blood cultures collected from febrile patients, only 9/1128 samples (0.8%) containing pathogenic bacteria were considered as evidence of community acquired septicemia (CAS): The distribution and the proportion of pathogens found in samples from febrile patients enrolled in 3 sites are presented in O. tsutsugamushi was present as the only pathogen in 31 samples and in co-infection with other pathogens in 28 cases . Dengue virus was also frequently observed in co-infections , especially with malaria parasites . Similarly, Influenza virus was also frequently detected in co-infections , especially with malaria parasites . Other co-infections included Leptospira (n = 6), O. tsutsugamushi (n = 1), Dengue virus (n = 1), E. cloacae (n = 1) and Rickettsia (n = 1).A substantial proportion of co-infections (more than one pathogen detected by PCR) was observed in this study. vs. 1.4 mg/l, p<0.01). The lowest median CRP concentrations were observed in viral infections (Dengue = 8.2 mg/l and Influenza = 9.6 mg/l) being significantly different to CRP levels in patients with malaria (37.9 mg/l p<0.01) and scrub typhus (26.4 p<0.006). Median CRP concentration was intermediate in febrile patients where no pathogen was detected (6.3 mg/l) or with multiple infections (16.2 mg/l). The sensitivity, specificity, positive predictive (PPV) and negative predictive values (NPV) of CRP>5 mg/ml for bacterial mono-infection versus viral infections were 87.8% (CI95%: 78.2–94.3%), 30.8% (CI95%: 22.3–40.5%), 46.8% (CI95%: 38.3–55.4%) and 78.6% (CI95%: 63.2–89.7%), respectively. The interactive dot diagram analysis indicating the cut-off point with the best separation  was set up at 21.3 mg/L with 52.5% of sensitivity and 84.3% of specificity.Quantitative detection of CRP was performed on 1475 plasma samples. The results are presented in P. falciparum , an important proportion of low-density malaria infections is still missed as demonstrated by PCR in this study. Most of the RDT-negative febrile cases were carrying malaria parasites that were detected by PCR but not by RDT . Although it is not clear if those low-density malaria infections are actually the cause of fever in those patients , these results emphasize the need for more sensitive diagnostic tools for detection and treatment of all infections to target elimination. We observed that most of the RDT-negative cases were clinically diagnosed as UARI and treated apparently indiscriminately with antimicrobial drugs (mainly AMX). Only 14% of them received an effective treatment against the pathogen identified by PCR. These findings underline the need of specific diagnostic tools and clinical training of the health center staff for the effective management of febrile patients and rational use of antimicrobials. Leptospirosis was frequently observed in people with AUFI, regardless the clinical status (case and control groups). This could be due to the recruitment of controls among family members or other accompanying persons, who had been exposed to the same risk-factors as the patients. The high prevalence of leptospirosis detected in this study was consistent with previous reports from similar settings in Thailand, Lao PDR The diagnosis of non-malaria febrile illness in resource-limited rural areas remains challenging A high frequency of dengue fever was observed in our sampled population, mostly adult and rural, while dengue is known to be more common in urban settings and young children P. falciparum and P. vivax) and a second non-malarial pathogen such as Leptospira spp., Rickettsia spp., O. tsutsugamushi or dengue virus. Reports of malaria co-infections with other pathogens such as HIV or helminthes are numerous especially from sub-Saharan Africa. In Southeast Asia, however, reports of co-infections in malaria patients remain rare. Malaria in Southeast Asia is often associated with outdoor occupations including logging, mining and agriculture, activities that also expose people at risk for leptospirosis, rickettsiosis and scrub typhus. In many cases of malaria co-infections, P. vivax was the most frequent species found by PCR (very at low density of infection). These cases could be interpreted as chronic asymptomatic infections with P. vivax plus a simultaneous acute infection with another pathogen. Unfortunately nothing is known about the interactions of these pathogens in co-infections so far, but it can be assumed that multiple infections complicate malaria and may lead to failure to respond to treatment Co-infections were also frequent in this study (∼14%). Most frequently, these included malaria parasites  was run on 10 samples of patients with a history of more than 5 days of fever and 2 had a positive result for JEV and 3 samples were positive for dengue virus. Another viral disease of interest to evaluate in the future is Chikungunya virus infection, of which several outbreaks were reported from Thailand in 2008 and 2009 Although a broad test battery was applied for the detection of pathogens causing fever, it was not possible to detect a causative agent in ∼27% of febrile patients. A variety of other pathogens come into consideration as cause of fever, not all of which were evaluated here. For example, HIV infections, viral hepatitis and tuberculosis were not evaluated due to ethical approval conditions but are likely to contribute to the overall burden of NMFI. Japanese Encephalitis Virus (JEV) was also a possible cause, especially in cases of prolonged fever or additional signs of meningitis. However convalescent serum samples were not available for additional testing. A combined vs. bacterial). However, this correlation is still weak to be used to assign a definite diagnosis and guide treatment decisions. Accessory information such as a full blood cell count and other biomarkers could provide additional useful diagnostic clues that may help to refine decision-making.The clinical evaluation of the subjects recruited for this study has not revealed any predictor symptoms or specific risk factors to differentiate between the different causes of NMFI. The evaluation of CRP-levels, as the only biological marker for infection in this study, did show an informative value for the presence of any infection (versus no pathogen identified), as well as for the cause of the infection  regional disease burden and seasonality of infectious diseases, (ii) clinical condition of patients and severity of disease , and (iii) availability and level of health care facilities and services. Studies should assess disease burden and laboratory gaps across each level of health care. To develop effective clinical guidelines it is imperative to involve physicians, nurses and other health care professionals from various levels of health care as well as health policy makers Figure S1NMFI clinical standardized form.(TIFF)Click here for additional data file.Figure S2Flow chart of cases and controls enrolled in the study sites (January 2008–December 2010).(TIF)Click here for additional data file.Figure S3Temporal distribution of the dengue cases observed in the study sites (January 2008–December 2010).(TIF)Click here for additional data file.Figure S4Temporal distribution of the influenza (A and B) cases observed in the study sites (January 2008–December 2010).(TIF)Click here for additional data file.Table S1Estimated detection thresholds of nucleic acid amplication tests (NAAT) used in the study.(DOCX)Click here for additional data file.


In therapeutic feeding programs (TFP), mid-upper arm circumference (MUAC) shows advantages over weight-for-height Z score (WHZ) and is recommended by the World Health Organization (WHO) as an independent criterion for screening children 6–59 months old. Here we report outcomes and treatment response from a TFP using MUAC ≤118 mm or oedema as sole admission criteria for severe acute malnutrition (SAM).Patient data from September 2007 to March 2009 for children admitted by MUAC ≤118 mm or oedema to a Médecins Sans Frontières (MSF) TFP in Burkina Faso were retrospectively analyzed. Analysis included anthropometric measurements at admission and discharge, program outcomes and treatment response.P<0.001).Of 24,792 patient outcomes analyzed, nearly half  were admitted with MUAC 116–118 mm. Most patients  were 6–24 months old. At admission, 52.7%  of those with MUAC 116–118 mm had a WHZ <−3 SD. At discharge, 89.1%  recovered (15% weight gain or oedema resolution), 7.9%  defaulted, 1.5% (n = 384) failed to respond to treatment, and 1.0% (n = 260) died. Average weight gain was 5.4 g/kg/day, and average MUAC gain was 0.42 mm/day. Patients with MUAC ≤114 mm at admission had higher average daily weight and MUAC gains at discharge compared to those admitted with MUAC 116–118 mm, but those in the latter category required longer lengths of stay to achieve recovery (This analysis suggests that MUAC ≤118 mm as TFP admission criterion is a useful alternative to WHZ. Regarding treatment response, rates of weight and MUAC gain were acceptable. Applying 15% weight gain as discharge criterion resulted in longer lengths of stay for less malnourished children. Since MUAC gain parallels weight gain, it may be feasible to use MUAC as both an admission and discharge criterion. High prevalence of undernutrition in children less than 5 years old results in substantial levels of mortality and overall disease burden in low- and middle-income countries. Severe acute malnutrition (SAM), defined as severe wasting and/or nutritional oedema, is the form of undernutrition associated with the highest mortality risk To implement TFPs in settings where resources and trained health professionals are scarce, simple diagnostic tools are needed. MUAC is a rapid method of assessing nutritional status, without requiring extensive training, supervision, or materials. With simple color-coding and on-the-spot interpretation, MUAC is relatively easy to use and simple to understand for both community health workers and children's caretakers. Errors of measurement associated with MUAC are no more frequent than with either weight or height. Studies have shown that even for minimally trained health workers, intra- and inter-observer reliability of MUAC measurements are at least as good if not superior to other anthropometric indices Although MUAC does increase with age and height In northern Burkina Faso, where food insecurity is a chronic concern, Médecins Sans Frontières (MSF) in collaboration with the Ministry of Health, implemented a TFP in 2007. Some focus group discussions, held by MSF staff with the caretakers of children admitted in TFP, have shown that mothers often do not link the wasting in children to health problems, and are unlikely to spontaneously seek medical help when their children lose weight, which shows the importance of including active case-finding as a component of nutrition programming. Therefore, MSF trained community health workers in the use of the MUAC tape for the purpose of conducting regular active screening and identification of children with SAM within the community. To simplify field operations in a remote, resource-poor setting where malnutrition is highly prevalent, this MSF program used MUAC as both the means for community-based screening and as the case definition of severe wasting that determines admission to therapeutic feeding. All children meeting the admission criteria at community level were referred to the nearest Centre de Santé et de Promotion Sociale [CSPS] for registration, medical examination and initiation of therapeutic feeding. Coherency between the screening tool and the admission criterion minimizes the problem of referral error that often occurs when children are screened by MUAC, but then admitted by WHZ – as these two anthropometric measures do not identify the same group of malnourished children In the Burkina Faso project, MSF piloted the use of a MUAC threshold of ≤118 mm and/or nutritional oedema as the only two admission criteria. The choice of a MUAC admission cut-off at ≤118 mm has been informed by statistical analyses of nutrition surveys conducted by MSF in sub-Saharan Africa that suggest using MUAC >115 mm improves sensitivity for identifying children below WHZ <−3, with minimal decrease in specificity Given the dearth of evidence regarding the use of MUAC as a tool to monitor response to nutritional recovery, the WHO recommends that children admitted by MUAC be discharged according to percent weight gain Here we report on program data from children admitted to TFP by MUAC, focusing on three main issues regarding the current management of SAM: 1) SAM diagnosis and advantages in using a broader MUAC inclusion criterion versus using combined MUAC and WHZ criteria; 2) the effects of the current WHO discharge criteria on treatment duration; and 3) response of MUAC to treatment and potential to use MUAC as a discharge criteria.This study used routine program monitoring data from the MSF nutritional program in Burkina Faso. This program was conducted in collaboration with the Ministry of Health of Burkina Faso via a memorandum of understanding, which is the normal and usual operating procedure for NGOs implementing nutritional programs. No supplementary interventions were given to participants. All data were anonymized when entered into the database and identification numbers were coded. No ethnic or identifying information was included. Individual oral informed consent was obtained from the parent or caregiver at the child's admission to the program.In collaboration with the Ministry of Health, in September 2007, MSF launched a TFP in two health districts of the Northern Region in Burkina Faso: Titao  and Yako . According to the 2006 census, the total populations for Loroum and Passore provinces were 142,853 and 323,222, respectively Nutrition indicators for the Northern Region, assessed in June–July 2008, reflected high levels of both stunting and wasting. Stunting (height-for-age <−2 Z WHO) prevalence among children under 5 was 40.8% and prevalence of wasting (WHZ <−2 WHO) was 11.6%, compared to stunting of 38.7% and WHZ <−2 of 12.4% on a national level As recommended by the national guidelines in Burkina Faso, the nutritional program implemented by MSF was located within the national health system. The program included an inpatient center for complicated cases  terminology) at hospital level, and a series of outpatient units  at CSPS level. As it was not deemed necessary to cover all health structures, some CSPS facilities were selected to deliver A-TFC services according to population density and accessibility. CSPS were identified in such a way that distances remained at an acceptable level and were not considered as a barrier to access for the mothers. The TFP in Yako was composed of 10 A-TFCs (out of the 48 CSPS) and one I-TFC with a capacity of 100 beds located within the district hospital. The TFP in Titao was composed of 12 A-TFCs (out of the 20 CSPS) and one I-TFC with a capacity of 100 beds also at the district hospital.The MSF program trialed a less restrictive MUAC admission cutoff of ≤118 mm, compared to the current WHO-recommended ≤115 mm. Although weight and height were measured for investigative purposes , WHZ tables were not used to determine TFP eligibility. All children aged 6–59 months presenting to one of the 22 CSPS where MSF was present were admitted to the TFP if they had MUAC ≤118 mm and/or oedema. A very small number of special cases  were also admitted upon medical decision.Children were first assessed for bilateral pitting oedema by applying thumb pressure to the dorsum of the foot. All children presenting pitting oedema were eligible for TFP admission regardless of anthropometry, and thus MUAC was not used as an admission criterion if oedema was present. Age was determined either by noting the birth date recorded on the child's vaccination card or by querying the mother. Weight was recorded using a Salter scale to the nearest 100 g, and length was measured in supine position  on a Schorr height board to the nearest 1 mm. MUAC was measured with an MSF-designed tape demarcated in 2 mm increments, using even numbers. MUAC, oedema and weight were measured at each visit, and height was measured at admission and at discharge. Children with severe pathology, anorexia, or oedema extending beyond the dorsum of the foot were considered complicated and admitted directly to the I-TFC. After clinical examination and an appetite test, all non-complicated cases were admitted directly into home treatment with weekly follow-up at A-TFC, and referred to the I-TFC only if they developed complications or failed to respond to treatment.RUTF   was used for treating all outpatients: 2 packets/day for those weighing <8 kg and 3 packets/day for those ≥8 kg. Therapeutic milk formulations F-75 and F-100 were used for initial nutritional therapy in hospitalized children, while medical complications were addressed. For these children, RUTF was introduced once appetite returned and clinical condition had stabilized. All children also received standard treatment in compliance with WHO recommendations: 5-day course of amoxicillin, and one-time doses of vitamin A, folic acid, and albendazole. Any identified medical conditions were treated concurrently, without fee, according to standard MSF protocols for malaria and other common conditions. Screening for HIV and tuberculosis was offered for all children with clinical suspicion and, starting from 2009, all children admitted in the intensive care unit of the I-TFC were routinely offered HIV testing. All necessary treatment and follow-up consultations were offered free of charge.Children receiving nutritional intervention were considered as recovered when all of the following criteria were achieved: weight gain >15% of admission weight; minimum length of stay of 4 weeks; and absence of any associated pathology. If admitted with oedema, children were discharged a minimum of 7 consecutive days after its complete resolution. Children were classified as defaulters if they failed to appear for scheduled weekly appointments for 3 consecutive weeks (in A-TFC) or were absent 3 consecutive days (in I-TFC). Non-response was defined as failure to achieve 15% weight gain after 6 weeks in A-TFC followed by good compliance with treatment during supervised feeding in the I-TFC and exclusion of any associated pathology or chronic disease. Children re-presenting to the A-TFC more than two months after being discharged as recovered from the program were recorded as new admissions; the ones presenting within two months after being discharged as recovered were classified as re-admissions.Routinely collected data of patients admitted and discharged between September 2007 and March 2009 were systematically entered into a database upon discharge and retrospectively analyzed, including date of admission and exit, demographic information , anthropometric measurements at admission and exit, weekly weight and MUAC, symptoms at admission, main morbidities during follow-up, treatment received, and program outcomes .Data were entered daily, using EpiData software, version 3.02 . Data quality was checked by built-in parameters in EpiData that allowed data entry only within specified range, in addition to systematic clean-up at the end of each month when a list of records with aberrant values was generated and compared to the medical file. If a discrepancy occurred, the value on the medical file was entered.Data were analyzed using STATA software, version 10 . Means of weight-for-height Z score (WHZ), weight-for-age Z score (WAZ), height-for-age Z score (HAZ), and MUAC-for-age Z score (MUAZ), using WHO 2006 growth standards, were calculated using WHO Anthro, version 2.0.4 .The total number of records in the database from September 2007 to March 2009 was 26,291. In this analysis, 120 records were excluded because of aberrant or missing data .A total of 26,171 records were analyzed for children admitted to the nutritional program between September 2007 and March 2009. Of all children admitted during this period, 94.7%  were admitted based on MUAC alone, with the remaining 3.8% (991) admitted based on oedema, and 1.5% (388) admitted as special cases. We focus only on children admitted based on MUAC.From November 2007 through April 2008, height at admission was not routinely noted. For this reason, only 18,664 out of the 24,792 admissions by MUAC contributed to determination of WHZ and HAZ upon admission.Among children admitted based on MUAC , close tOf the 18,664 children for whom height data was available, a total of 66.1%  had a WHZ <−3 SD (WHO standard). Of those admitted with MUAC 116–118 mm, mean WHZ was −3.1±0.8 SD and 52.7%  had a WHZ <−3 SD. 76% of the cohort were admissible by either MUAC <115 mm or WHZ <−3 criteria, meaning that almost 1 in 4 children admitted did not meet either currently WHO-accepted anthropometric definition of SAM .Of children admitted to TFP by MUAC, 8% were hospitalized on day of admission in the I-TFC for management of a significant pathology. An additional 10.1% were transferred to I-TFC on a subsequent visit for either illness of poor weight gain, resulting in a total of 81.9% of children who were treated exclusively as out patients .P<0.001)  .Anthropometric measurements for those discharged as recovered showed that all the children admitted by MUAC responded well to treatment and were discharged from the program with improved anthropometric measurements . Among cP<0.001).Average weight gain over the entire treatment was 5.4 g/kg/day across all categories and was inversely related to MUAC upon admission . AverageP<0.001) and a shorter length of stay  than the ones admitted with a WHZ ≥−3  exceeded SPHERE 2011 performance standards Our findings appear also to be the first to document a similar profile in rate of MUAC gain according to initial severity of malnutrition. Whereas previous interventions have reported a mean MUAC gain of 0.3 mm/day (sometimes based on treatment with higher amounts of RUTF) A major challenge in the Burkina Faso program was the use of WHO-recommended 15% weight gain as the discharge criterion. Paradoxically, using >15% weight gain to determine nutritional recovery and program discharge resulted in longer treatment for less malnourished children, as measured by average length of stay in the program, and shorter treatment for more severely malnourished children . The morSince average weekly MUAC gain and average weekly weight gain tracked closely together , MUAC gaThe results reported here must be interpreted in light of several limiting factors. These analyses were performed on program data and cannot therefore be attributed to the entire population of young children in northern Burkina Faso; the findings apply only to those children who were brought by their caregivers to the screening sessions or to the health centers.Although TFP staff were trained and continuously supervised, the quality of measurements is reflective of what one might find in any given large TFP. Because of the MSF MUAC tape design (demarcated in 2 mm increments and using only even numbers), only even numbers were recorded on the patient chart and the maximum eligible admission value was designated as 118 mm. A number of children measuring MUAC = 118 mm would therefore likely have a measurement of 117 mm or 119 mm, if a measuring tape demarcated in 1 mm increments was used. However, given the large cohort size, measurement errors may cancel each other out. For the first 6 months of this program, height was not consistently measured upon admission; during this time the relative proportion of more severely wasted children was higher. Therefore, the reported results in terms of WHZ for the most severely wasted children under-represent this category. Almost all of the children in this cohort are under 2 years of age, and are in a physiologic period of rapid growth, regardless of nutritional status. Rates of MUAC gain may be slower in older children.Large-scale, community-based treatment of severely malnourished children using an adapted MUAC threshold may be able to replace the more complicated anthropometric index of WHZ. Based on simplicity and predictive value of mortality, MUAC-based detection using a fixed cutoff represents an opportunity to easily screen large numbers of children and admit them efficiently to a TFP using the same anthropometric measurement. MUAC-based programs will preferentially identify younger children as malnourished, which is a beneficial bias since children of this group are more vulnerable to illness and at higher risk of death. A more generous inclusion criterion may thus identify malnourished children earlier, when they can be treated easily and effectively, but remain sufficiently specific as evidenced by rapid weight gain. The current WHO recommendation of 15% weight gain as a threshold for discharge is paradoxically disadvantageous for the most severely malnourished children, according to our findings. Beyond use as sole admission criterion, MUAC shows potential as a tool for monitoring response to nutritional rehabilitation as well as a discharge criterion.


Malaria during pregnancy is the main cause of low birth weight (LBW) in the tropics. There are few studies concerning B and T lymphocyte infiltrates in placental malaria infections or their potential association with LBW babies.A case–control study was conducted at the Medani Hospital, Central Sudan. Cases were women who had LBW deliveries  and controls were parturient women with normal birth weight babies. Sociodemographic and medical characteristics were gathered from both groups of women using questionnaires. Cases and controls were investigated for malaria using microscopic blood film analysis, placental histology, and immunohistochemistry for detection of B (CD20) and T lymphocytes (CD3).vs. 24 (24.7%), P = 0.519 of the cases vs. the controls had placental malaria infections on histological examination. Three (3.1%), two (2.1%) and 24 (24.7%) vs. two (2.1%), two (2.1%) and 20 (20.6%) of the placentae showed evidence of acute, chronic and past malarial infections on histopathological examination of the two groups (case–control), respectively, while 68 (70.1%) vs. 73 (75.3%) of them showed no signs of infection; P = 0.420. Women with placental malaria infections had significantly fewer CD20 cell infiltrates  and higher numbers of CD3 cell infiltrates  than those without placental malaria infection. Logistic regression analysis showed that neither placental malaria infections nor CD3 or CD20 were associated with LBW.The two groups (97 in each arm) were well matched in their basic characteristics. There were no malaria-positive blood films in either the cases or the controls. Twenty-nine (30.0%) Significantly higher rates of CD3 T cells and lower rates of CD20 B cells were found in women with placental malaria infections compared with those without such infections. Neither placental malaria infection nor CD3 or CD20 are associated with LBW.http://www.diagnosticpathology.diagnomx.eu/vs/6879723961063755 Malaria is a big public health problem in tropical countries, especially in sub-Saharan African countries where around 125 million pregnant women live in malaria-endemic areas and 32 million of these are at risk of the disease , the latPlasmodium falciparum-infected erythrocytes to the syncytiotrophoblast leads to parasite sequestration in the intervillous space. The parasite adheres specifically to chondroitin sulfate-A expressed on the syncytiotrophoblast  and significantly higher numbers of CD3 cell infiltrates .In comparison to women without placental malaria infections, women with such infections had significantly fewer CD20 cell infiltrates . However, the log of the mean (SD) for CD3 was significantly higher in women with placental infections (n = 50) than in women who had no placental infections (n = 42), .In women where CD20 and CD3 were detected, the log of the mean (SD) of CD20 was not statistically different . Likewise, the mean (SD) of the birth weights was no different in women who had CD20 cell infiltrates  than in those who had no evidence of CD20 cell infiltrates  or those who had CD3 (in both groups = 92) .The mean (SD) for birth weight was 2,375.8 (1548.0) P. falciparum infection nor CD3 or CD20 were associated with LBW or with anemia, Table In this multivariate analysis, neither placental vs. 25.7%) and monocytes and macrophage infiltrates were not associated with birth weight [The main findings of the current study were as follows: Placental malaria infections (the majority of which were past infections) were detected in 27.0% of the women, regardless of their age or parity. Placental malaria infections had fewer and higher numbers of CD20 and CD3, respectively. Neither placental infections nor CD20 and CD3 cell infiltration had an effect on LBW or anemia. In the same hospital , placental malaria infections were detected in placentae regardless of the age or parity of the women and placental malaria infections (detected by histology) were found not to be associated with LBW . We haveh weight . In an ah weight . Yet, ith weight . Likewish weight  observedInterestingly, in the current study, in comparison to women without placental malaria infections, women with such infections had significantly fewer B cell (CD20) infiltrates, and larger numbers of T cell (CD3) infiltrates. This finding supports a published study where Ordi, et al.  observedConsequently, our findings on B cells and T cells, as described in the current study may explain why pregnant women have increased susceptibility to malaria, which is thought to result from pregnancy-related immunomodulation and a Th1/Th2 shift to decreased Th1-type cytokines and increased Th2-type cytokines to prevent rejection of the fetal allograft ,19. TherSignificantly higher rates of CD3 T cells and lower CD20 B cells were detected in malaria-infected placentae than in placentae without such infections. Neither placental malaria infections nor cellular infiltrates were associated with parity or led to reductions in birth weight.The authors’ declare that they have no competing interests.SB and IA designed the study and EME conducted the clinical work. MMS and AAM conducted the laboratory work. IA and SB participated in the statistical analyses. All the authors’ have approved the final paper.