Pulse wave velocity (PWV) is an early marker of arterial stiffness. Low birthweight, infant feeding and childhood nutrition have been associated with cardiovascular disease in adulthood. In this study, we evaluated the association of PWV at 30 years of age with birth condition and childhood nutrition, among participants of the 1982 Pelotas birth cohort.In 1982, the hospital births in Pelotas, southern Brazil, were identified just after delivery. Those liveborn infants whose family lived in the urban area of the city were examined and have been prospectively followed. At 30 years of age, we tried to follow the whole cohort and PWV was assessed in 1576 participants.Relative weight gain from 2 to 4 years was positively associated with PWV. Regarding nutritional status in childhood, PWV was higher among those whose weight-for-age z-score at 4 years was >1 standard deviation above the mean. On the other hand, height gain, birthweight and duration of breastfeeding were not associated with PWV.Relative weight gain after 2 years of age is associated with increased PWV, while birthweight and growth in the first two years of life were not associated. These results suggest that the relative increase of weight later in childhood is associated with higher cardiovascular risk. Cardiovascular disease (CVD) is the leading cause of death worldwide. According to the World Health Organization (WHO), in 2008, 17.3 million deaths were due to CVD and it is estimated that this number will increase to 23.3 million by 2030 [Arterial stiffness increases with age and among young people may accelerate the atherosclerotic process, leading to several pathological conditions, such as decreased vascular compliance, increased systolic blood pressure, hypertrophy and fibrosis of the left ventricle, decreased coronary perfusion and endothelial dysfunction that leads to decreased production of nitric oxide, increasing smooth muscle tonus –8. PulseWith respect to the risk factors of cardiovascular diseases, evidence suggests that early exposures, such as intrauterine growth, birthweight, nutrition status in childhood and infant feeding, may program the development of metabolic cardiovascular risk factors –18. On tIn relation to breastfeeding and timing of introduction of solid foods, Jonge et al. reported that those children who had never been breastfed had higher PWV at 6 years of age, smaller left atrial diameter (LAD) and less left ventricular mass, whereas breastfeeding was not associated with blood pressure . Tauzin This study was aimed at assessing the association between PWV at 30 years of age and infant feeding and nutritional status during pregnancy and early childhood.In 1982, the maternity hospitals located in Pelotas were visited daily and all births identified. The 5914 liveborn infants whose families lived in the urban area were examined and their mothers interviewed. These individuals have been followed several times at different ages. Details of the methods of this cohort are available elsewhere ,27. BetwIn the 2012–13 visit, pulse wave velocity (PWV) was assessed using the Sphygmocor® system , a noninvasive device, which measures the PWV with a tonometric transducer. Participants were asked to refrain from smoking, drinking alcohol, caffeine, eating or physical activity at least 30 minutes before exams. A trained technician took the measurements, after 5 minutes of rest with the participants in the supine position in a quiet environment with controlled temperature (22–24°C). The tonometer was lightly pressed on the participant's skin, after palpation of the right carotid and femoral pulses, with simultaneous recording of the electrocardiogram for synchronization of carotid and femoral pulse wave times.The PWV was calculated from measurements of pulse transit time and distance traveled by the pulse wave. Pulse transit time was estimated using the intersecting tangent foot-to-foot algorithm. The distance traveled by the pulse wave was measured using a flexible tape as the distance from the suprasternal notch to the femoral site of the pulse wave recording and the distance from the carotid site of the pulse wave recording and the suprasternal notch. The PWV was estimated as the distance between the measurement sites divided by the transit time delay between the femoral and carotid pulse wave .In the 2012–13 visit, weight was measured using an electronic scale with a maximum capacity of 150 kg (Tanita®). Height was measured with a portable stadiometer with an accuracy of 0.1 cm.Birthweight was assessed by the hospital staff using pediatric scales that were calibrated weekly by the research team. Gestational age was estimated from the last menstrual period. Birthweight according to gestational age z-score was calculated from the mean and standard deviation of birthweight based on gestational age and sex of the Williams’s reference population . DetaileWeight and length/height measures were transformed into weight-for-age or length/height-for-age z-scores using the WHO growth curves . ConditiThe analyses were performed using Stata 12® statistical package. Sex, race, maternal schooling, income, smoking during pregnancy and birthweight were considered as possible confounders. Analysis of variance (ANOVA) was used to compare means and multiple linear regression to adjust for confounding factors. In the linear regression, we estimated the p-value comparing the differences among the categories of the variable. Furthermore, we also assessed the p-value using the continuous exposure variable.This study was approved by the research ethics committee of the Faculty of Medicine, Federal University of Pelotas, affiliated with the National Council on Ethics in Research of the Ministry of Health. All participants signed an informed consent.In the 2012–13 visit, 3701 individuals were interviewed, and PWV was assessed in 1576 participants . We were not able to measure the PWV in the remaining 2125 subjects due to operational problems. PWV was lower among white individuals (P = 0.01). On the other hand, no association was observed for family income and education. In this cohort that has been prospectively followed since birth, in a southern Brazilian city, we observed that relative weight gain from 2 to 4 years of age was associated with increased PWV, whereas birthweight, relative weight gain in the first two years of life, and linear growth (length/height gain) in childhood were not associated with PWV. Furthermore we did not observe any programming effect of breastfeeding on PWV at 30 years of age.Information on birthweight, gestational age, breastfeeding duration and nutritional status was collected close to the time of its occurrence, reducing the possibility of misclassification. In addition, the absence of any trend in the association between breastfeeding and PWV suggests that it is unlikely that measurement error underestimated an association. By the same token, confounding variables were measured in early childhood, reducing the chance of residual confounding. On the other hand, PWV was not assessed for all subjects who were followed at the age of 30, but PWV examination was independent of socioeconomic status, breastfeeding duration and nutritional status, suggesting that selection bias is unlikely. On the other hand, we were not able to adjust the conditional growth estimates to birth length because information on this variable had not been collected in the perinatal study.With regard to the long-term effects of breastfeeding on the risk of cardiovascular disease, some studies have observed that breastfeeding is inversely associated with cardiovascular risk factors ,30, wherWith respect to childhood growth, it has been reported that growth in early childhood is not associated with an increased risk, whereas weight gain in late childhood is positively associated with cardiovascular risk factors –47. Furt


Malaria, anaemia and under-nutrition are three highly prevalent and frequently co-existing diseases that cause significant morbidity and mortality particularly among children aged less than 5 years. Currently, there is paucity of conclusive studies on the burden of and associations between malaria, anaemia and under-nutrition in Rwanda and comparable sub-Saharan and thus, this study measured the prevalence of malaria parasitaemia, anaemia and under-nutrition among preschool age children in a rural Rwandan setting and evaluated for interactions between and risk determinants for these three conditions.A cross-sectional household (HH) survey involving children aged 6–59 months was conducted. Data on malaria parasitaemia, haemoglobin densities, anthropometry, demographics, socioeconomic status (SES) and malaria prevention knowledge and practices were collected.P = 0.04 for age group 24–35 months, OR = 3.5; P = 0.037 for age group 36–47 months, and OR = 3.03; P = 0.014 for age group 48–60 months, whilst a reduced risk was found among children living in high SES HHs . Risk of anaemia was high among children aged ≥12 months, those with malaria parasitaemia  and children living in HHs of lower SES. Overall, under-nutrition was not associated with malaria parasitaemia. Underweight was higher among males  and children with anaemia .The prevalences of malaria parasitaemia and anaemia were 5.9 and 7.0 %, respectively, whilst the prevalence of stunting was 41.3 %. Malaria parasitaemia risk differed by age groups with odds ratio (OR) = 2.53; In this study group, four in 10 and one in 10 children were found stunted and underweight, respectively, in an area of low malaria transmission. Under-nutrition was not associated with malaria risk. While the high prevalence of stunting requires urgent response, reductions in malaria parasitaemia and anaemia rates may require, in addition to scaled-up use of insecticide-treated bed nets and indoor residual insecticide spraying, improvements in HH SES and better housing to reduce risk of malaria. Malaria, anaemia and under-nutrition are each associated with significant morbidity and mortality, particularly among children in sub-Saharan Africa –3. GlobaAnaemia is characterized by a reduction in haemoglobin concentration causing impairment in meeting the oxygen demands of the body. Anaemia results broadly from either ineffective erythropoiesis or increased loss of erythrocytes or both. The main causes of anaemia include acute or chronic blood loss, nutritional deficiencies  , infectiMalaria causes a substantial proportion of anaemia observed in malaria endemic settings , 16. HowPlasmodiumfalciparum infection has been associated with acute weight loss [Studies elucidating associations between malaria and under-nutrition yield conflicting results ; with soght loss . Additioght loss  and longght loss .Given the extensive temporal and spatial correlation between malaria, anaemia and under-nutrition, any interaction  may lead to synergistic deleterious effects on child health and development. Studies on interactions between malaria, anaemia and under-nutrition particularly among community preschool-aged children are few and inconclusive . Most ofRegarding administration, Rwanda has 30 districts: Each divided into sectors, cells, and villages locally term “umudugudus” (of about 50–100 households). This survey was conducted in 35 villages that are aggregated into five cells that constitute Ruhuha sector, Bugesera District in Eastern Rwanda  and family members to stay in-house. Upon providing consent, the study team (consisting of an interviewer and a laboratory technician) visited the notified HH and administered an interviewer-guided questionnaire to HoHs. In HHs where no member or no HoH or spouse was found present, a return visit was scheduled and attempted within 7 days. All HHs where the study team failed to conduct a survey on the return visit were excluded. Study findings from this larger sector-wide HH survey conducted between June and November 2013 have since been published . For thiA structured questionnaire was administered to the child’s primary caregiver to collect data on (1) demographics ; (2) malaria prevention bed net  and indoor residual spraying (IRS) experience; (3) SES related variables  and HH structural features including type of outside wall, floor and roof materials); and (4) fever management practices. For each HH, location data was captured using a geographic positioning function based on the Samsung Galaxy 2 Android platforms . The questionnaire used was written in English and was field-tested at three sites in order to minimize ambiguity, ensure consistency of comprehension of questions by both interviewers and respondents. Field workers were trained to administer the interviews in the local dialect (Kinyarwanda). Questionnaire data was collected using an electronic format developed using the open source Open Data Kit Collect setup on Android tablets .Measures of under-nutrition indices  were deduced from data on (1) age-in-months as reported by parents, (2) weight measured using UNICEF provided a digital Seca 874 weight scales (seca GmbH & Co. KG.) to the nearest 0.1 kg, and (3) height measured using a mobile measuring Seca 210 (seca GmbH & Co. KG.) mat for children 0 to 99 centimetres and a recumbent length board for children of height > 99 centimetres to the nearest centimetre, respectively.® Hb 301 haemoglobinometer system  according to the product instruction.From all HH members aged ≥6 months, finger-prick blood samples for malaria smear-based diagnosis and haemoglobin measurement were collected. Each smear was stained with 2 % Giemsa, processed and read independently by two study-trained microscopists at Ruhuha Health Centre laboratory. In case of a discrepancy, a tie-breaker third microscopist determined the final result. Expert microscopists at the National Reference Laboratory, Kigali, conducted quality control for all positive slides and a random sample of 5 % of all negative slides. Haemoglobin densities were measured on the spot in the field using a portable automated HemoCuez scores were calculated on the basis of the WHO Global Database on Child Growth and Malnutrition [A blood smear was considered negative when light microscopy examination of 100 high-power fields did not reveal any asexual parasites and considered positive if any asexual parasites were detected on thick blood microscopy. In this study, malaria diagnosis was assessed based on presence of malaria parasites in blood by microscopy only. Data on reported symptoms or clinical signs was not collected. Anaemia was defined as moderate-to-severe haemoglobin levels of <90 g/L as recommended for disease surveillance, especially in areas of high anaemia prevalence . Weight-utrition . Z scorePredictor variables included HoH demographics , cell of residence), malaria prevention practices , roof (iron sheets vs. grass/tents), and outside walls . A HH level SES/wealth index  was generated using 10 indicator variables using principal component analysis .Written informed consent to participate in the study as well to allow study findings to be published in a relevant scientific journal was obtained from the HoH on behalf of all household members including minors. The National Health Research Committee (NHRC) and the Rwanda National Ethics Committee, Kigali, Rwanda (No 384/RNEC/2012) granted ethical and scientific approval for the study protocol.2 test. Associations between predictor variables and primary outcomes were statistically assessed for using both bivariate and multivariate logistic regression analysis. Odds ratios (ORs) and 95 % confidence intervals (CIs) were computed. Any covariate with a p value <0.15 in bivariate analysis were subsequently included in the final multivariable logistic model. Multi-collinearity tests were performed for all potentially correlated variables included in the final multivariate model for each of the five primary outcomes. Any risk estimate with a p-value <0.05 was considered statistically significant after adjustment for HH-level clustering and influence of other variables.Data was collected using handheld android platforms on which open data kit software-hosted electronic questionnaire was loaded. These data was then relayed onto a server. Laboratory and anthropometric data was manually recorded in laboratory registers and later entered into Microsoft Access software. The two datasets were then merged and transferred into STATA 12.1  for analysis. Continuous variables were compared between groups (including stratification by age and sex) using Mann–Whitney U tests, and variable proportions were compared by ΧAs reported in the earlier publication for the larger survey, a total of 4705 HHs were surveyed and of these, data from 12,965  individuals from 3968 (84.3 %) HHs that had complete laboratory and questionnaire data was aggregated in the primary database . From thP.falciparum parasitaemia, moderate-to-severe anaemia and under-nutrition parameters of stunting, wasting and underweight were 5.9 %, 16.4 %, 41.3 %, 8.8 % and 15.8 %, respectively , 18.4–45.7 months); the mean height was 85.8 cm and mean weight was 12.1 kgs. The median number of HH occupants was 5 (IQR 4–6) (Table The proportions of children with co-morbidity were: 23/1882 (1.2 %) with malaria and anaemia; 59/1882 (3.1 %) with any under-nutrition and malaria 82/1882 (4.4 %) with any under-nutrition and anaemia. Only five children were found to have all three conditions concurrently (Table P = 0.034), age groups ; and by cell of residence (10.9 % in Gikundamvura vs. 2.2 % in Ruhuha) (Table P = 0.029) (Table P = 0.059), whose HoH had any education ; and whose house structure walls were made of bricks/cement   higher odds of having anaemia compared to living in Biharwe cell while the risk of anaemia decreased with increasing age group   vs. children in the other four cells with proportions ranging from 5.4 to 6.3 % (Table P ≤ 0.0001) and wasting ; (2) stunting was associated with underweight  but not wasting ; and (3) wasting was associated with underweight  but not stunting . In the final multivariate model, other predictors of (1) stunting were a reported fever history ; living in a house where the HoH belonged to a higher economic group  and living in HHs that has a reported ownership of ≥1 LLIN  (2) wasting were reported IRS applied in the HH , and domestic water source  and (3) underweight were sex , age group, house floor material .Under-nutrition parameters showed varying co-existence patterns Table . In summCollinearity analysis between all variables included in each of final multivariate model for the five primary outcomes showed mean variance inflation factor (VIF) values ranging from 2.17 to 7.23. For each of the five-outcome models, no variable showed a VIF >10: This is the cutoff marker of multicolinearity.At least 4/10 and 1/10 preschool-age children in this rural setting were found stunted and underweight, respectively. Observed malaria parasite rates of <10 % suggest that this area is hypo-endemic for malaria. In this study, the proportion of children aged under 5 years with malaria (6.5 %) was almost two-fold higher than the 3.4 % reported in the same province in 2010 . These dIn this study, risk of malaria increased with increasing age. Findings in this study are consistent with an increasingly observed trend of higher malaria risk among older age groups, as reported previously elsewhere and in this area, following the scale-up of control interventions , 32, 33.vs cement/bricks) showed a significantly higher risk of malaria in this study. Houses whose walls are made of mud have been associated with having more eaves (that support mosquito entry), higher risk of indoor mosquito bites and, by creating cooler and darker conditions in comparison to brick/cement houses, creating a favourable indoor resting environment for mosquitoes [In this study, a protective malaria risk was associated with living in a high SES HHs. However, studies on associations between malaria and SES have hitherto yielded conflicting results, with some indicating no associated effect , 35 whilsquitoes –43.The proportion of children under 5 years found with anaemia in this study (6.8 %) was three-fold higher than the 2.0 % reported for the same province in 2010 . After aAnaemia, but not malaria, was significantly associated with underweight in this study. Evidence for the impact of under-nutrition on development of anaemia in young children living in malaria-endemic areas had been reported previously . PreviouChildren from middle and high SES HHs were found to have a significantly reduced risk of having anaemia than children from low SES HHs. Two plausible reasons for this are the differential nutritional intakes and house structural features that determine risk of indoor malaria transmission between the two SES levels. Presumably, children from low SES HHs are likely to have poorer nutritional intake and also live in houses whose structure are more conducive for indoor malaria transmission, with the increased risk of malaria causing a concurrent deleterious effect on anaemia risk.Among preschool-aged children in the same province in 2010, stunting, wasting and underweight proportions in comparison to findings in this study were 43.9 %, 3.2 % and 11.5 % vs. 41.6 %, 8.8 % and 15.8 %, respectively . Both suOther risk determinants for malaria, anaemia and under-nutrition metrics were also identified. Living in houses where the HoH was not educated and in houses where domestic water was sourced from an open source compared to HHs where domestic water was drawn a closed source were associated with a high risk of malaria infection. Lack of education may likely be associated with low SES status and limited malaria control-associated knowledge and practice factors, which may be related to lower availability and use of malaria control measures like LLINs. Regarding the domestic water sources, open water sources may also serve as potential mosquito breeding sites and hence pose an increased risk. Anaemia risk was interestingly found to be lower by almost 50 % among individuals living in HHs where IRS was carried out. It is plausible that the IRS effect on lower anaemia risk was mediated primariry through reducing malaria risk. However, other unmeasured risk determinants may have contributed to the high risk among individuals from HHs where no IRS was applied of anaemia. Other identified risk determinants for under-nutrition included SES indicator variables  and prior fever experience and malaria control measures (LLIN availability and IRS experience). Given that the study area is predominantly agricultural, HHs where the HoHs reported being a member of an economic group are more likely to have better food security and hence a lower risk of under-nutrition. Individuals who reported having had a fever during the past 6 months are likely to have had malaria: A risk determinant of stunting . The useThis study had several limitations. Being a cross-sectional study design, associations observed may have been confounded by unmeasured factors. Also, causal inferences cannot be drawn from study findings due to the study design employed. The lack of additional haematological assessments including mean cell volume, micronutrients and haemolysis parameters limited the characterization of anaemia types. With regard to under-nutrition, only weight and height measurements were collected. The lack of other under-nutrition related data including skin fold thickness, oedema and body mass index did not allow for a more robust nutritional assessment. In this survey, statistical adjustments for correlation with and between HH members were conducted to ensure study finding were robust. However, given the multi-factorial causal factors associated with anaemia and under-nutrition, data on important covariates including but not limited to helminthic infection, micronutrient levels, co-infections like HIV, genetic haemoglobin disorders and breast feeding need to be collected in future studies to be able to perform a more robust analysis.Study findings pointed to high rates of under-nutrition and anaemia but not malaria parasitaemia in preschool-going children. A strong association between malaria and anaemia but not between malaria and under-nutrition was observed. Although the study design limits the interpretation of cause and effect between these three disease determinants, control of malaria may have a substantial indirect reduction on anaemia burden among preschool-going children in this area. Integrated rather than vertical programmes covering nutritional rehabilitation, malaria control including the scaled up LLIN and IRS coverage, improvements in HH SES and better housing that limits mosquito entry are need to realize optimal child health outputs.


This study aimed to evaluate the prevalence and alteration of hematological parameters in malaria patients with a glucose-6-phosphate dehydrogenase (G6PD) deficiency, in the western region of Thailand, an endemic region for malaria.Data about patients with malaria hospitalized between 2013 and 2015 were collected. Clinical and sociodemographic characteristics such as age and gender, diagnosis on admission, and parasitological results were mined from medical records of the laboratory unit of the Phop Phra Hospital in Tak Province, Thailand. Venous blood samples were collected at the time of admission to hospital to determine G6PD deficiency by fluorescence spot test and detect malaria parasites by thick and thin film examination. Other data such as complete blood count and parasite density were also collected and analyzed.Plasmodium falciparum infections and 217 cases (88.6 %) were diagnosed as P. vivax infections. Seventeen (6.9 %) patients had a G6PD deficiency and 228 (93.1 %) patients did not have a G6PD deficiency. Prevalence of male patients with G6PD deficiency was higher than that of female patients . Among the patients with a G6PD deficiency, two (11.8 %) were infected with P. falciparum, while the remaining were infected with P. vivax. Malaria patients with a G6PD deficiency have higher monocyte counts (0.6 × 103/μL) than those without a G6PD deficiency (0.33 × 103/μL) . Univariate and multivariate analyses also confirmed that malaria patients with a G6PD deficiency have high monocyte counts. The association between G6PD status and monocyte counts was independent of age, gender, nationality, Plasmodium species, and parasite density (P < 0.005).Among the 245 malaria cases, 28 (11.4 %) were diagnosed as P. falciparum infection. In addition, malaria patients with a G6PD deficiency had higher monocyte counts than those without a G6PD deficiency. These findings will help to recognize and diagnose malaria patients with a G6PD deficiency, as well as to identify the risks and protective factors against malaria in endemic areas.This study showed a prevalence of G6PD deficiency in a malaria-endemic area. This study also supported the assertion that patients with G6PD-deficient red blood cells had no protection against the The online version of this article (doi:10.1186/s40249-016-0130-0) contains supplementary material, which is available to authorized users. Please see Additional file G6PD gene result in protein variants with different levels of enzyme activity that are associated with a wide range of biochemical and clinical phenotypes  = 5.0)  = 5.0) .In addition to treatment of malaria with primaquine, drug resistance has remained the greatest challenge to malaria control. Despite a large number of antimalarial drugs available, there is no perfect drug; all have limitations such as poor compliance, side effects, toxicity, and/or resistance. The support from international organizations and governments of some endemic countries should be optimally exploited in various approaches to drug research and development in order to overcome the burden of malaria .This study had several limitations: a small number of patients with a G6PD deficiency; could not obtain data about diseases affecting hematological parameters; and the fluorescent spot test is not a confirmatory test for G6PD. However, the results of this study still provide valuable information relating to G6PD deficiency in patients infected with malaria in endemic regions of Thailand.P. falciparum infection. In addition, malaria patients with a G6PD deficiency had higher monocyte counts than those without a G6PD deficiency, and this was independent of age, gender, nationality, Plasmodium species, and parasite density. These findings will help to recognize and diagnose malaria patients with a G6PD deficiency, as well as to identify the risks and protective factors against malaria in endemic areas.This study showed a prevalence of G6PD deficiency, which becomes apparent from potential hemolytic reactions due to treatment of malaria with primaquine. The study also supported the notion that patients with G6PD deficient-RBCs had no protection against


It seems obvious that the number of pathogens should be important in the pathogenesis of an infectious disease PLOS Pathogens, Nicholas Anstey and colleagues present an analysis of host responses in relation to pathogen load in both Plasmodium vivax and P. falciparum malaria P. falciparum, P. vivax is an important cause of severe malaria, morbidity, and mortality Plasmodium  because iRBCs can adhere to the endothelium, which lines small blood vessels (sequestration), resulting in their under-representation in circulating blood P. falciparum malaria by measuring the plasma concentration of parasite molecules, which are released into the host circulation, and demonstrate that this is a much better predictor of severity than measurement of the circulating parasites alone P. vivax malaria using the plasma concentration of parasite lactate dehydrogenase (LDH) P. falciparum malaria, total P. vivax parasite biomass appears higher in severe than uncomplicated malaria, and also appears to be underestimated by counting parasites in the peripheral blood. Since P. vivax iRBCs exhibit much less endothelial adhesion than P. falciparum iRBCs P. vivax may accumulate outside of the endothelium-lined compartments of the blood, possibly in the slow open circulation of the spleen. This explanation is appealing, but there are some important caveats. First, P. vivax biomass is assumed to be approximately proportional to the plasma concentration of parasite LDH, but this will only be true if this molecule is released evenly throughout the parasite lifecycle or if there is an even distribution of life cycle stages at any point in time . Whilst all subjects had over-representation of younger parasite stages in the blood, there was no difference in this distribution between subjects with severe and uncomplicated P. vivax malaria, meaning that comparison of parasite LDH between groups should be valid. Second, the rate of production and clearance of parasite LDH could vary between subjects with severe and uncomplicated malaria, resulting in differences in plasma concentration that are not solely due to parasite biomass. Thus, parasite LDH probably only gives a semi-quantitative estimate of P. vivax biomass.In this issue of P. falciparum and P. vivax malaria P. vivax malaria, the parasite LDH concentration correlates with the systemic inflammatory response, but not with markers of endothelial activation. In contrast, circulating parasitemia correlates much better with endothelial activation. This is intriguing because it suggests that somehow the circulating parasites are activating the vascular endothelium independent of soluble circulating factors . Does this mean that some sort of non-adhesive interaction between circulating iRBCs and endothelial cells triggers endothelial activation? Further research will be necessary to answer this question. But it may also provide insights into a longstanding debate about whether endothelial sequestration of P. falciparum is the cause, or a consequence, of endothelial activation P. falciparum, these two phenomena are often so closely correlated that they are impossible to separate, but we now know that for P. vivax at least, endothelial activation can occur independent of endothelial parasite sequestration.Despite these limitations, Anstey and colleagues are to be congratulated for applying this methodology to produce a unique assessment of the relationship between parasite biomass and the major determinants of severe malaria pathogenesis: inflammation, sequestration, and vascular endothelial dysfunction Whilst the study by Anstey and colleagues clearly illustrates the importance of assessing pathogen load


The global prevalence of X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is thought to be a result of selection by malaria, but epidemiological studies have yielded confusing results. We investigated the relationships between G6PD deficiency and both malaria and non-malarial illnesses among children in Kenya.Plasmodium falciparum malaria through a case-control study of 2220 case and 3940 control children. Cases were children aged younger than 14 years, who visited the high dependency ward of Kilifi County Hospital with severe malaria between March 1, 1998, and Feb 28, 2010. Controls were children aged between 3–12 months who were born within the same study area between August 2006, and September 2010. We assessed the association between G6PD deficiency and both uncomplicated malaria and other common diseases of childhood in a cohort study of 752 children aged younger than 10 years. Participants of this study were recruited from a representative sample of households within the Ngerenya and Chonyi areas of Kilifi County between Aug 1, 1998, and July 31, 2001. The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria (computed by logistic regression) while for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses (computed by Poisson regression), by G6PD deficiency category.We did this study in Kilifi County, Kenya, where the G6PD c.202T allele is the only significant cause of G6PD deficiency. We tested the associations between G6PD deficiency and severe and complicated 2863 (73%) children in the control group versus 1643 (74%) in the case group had the G6PD normal genotype, 639 (16%) versus 306 (14%) were girls heterozygous for G6PD c.202T, and 438 (11%) versus 271 (12%) children were either homozygous girls or hemizygous boys. Compared with boys and girls without G6PD deficiency, we found significant protection from severe malaria  among G6PD c.202T heterozygous girls but no evidence for protection among G6PD c.202T hemizygous boys and homozygous girls . Median follow-up for the mild disease cohort study was 2·24 years (IQR 2·22–2·85). G6PD c.202T had no effect on other common diseases of childhood in heterozygous girls  or homozygous girls or hemizygous boys , with the sole exception of a marginally significant increase in the incidence of helminth infections among heterozygous girls.Heterozygous girls might be the driving force for the positive selection of G6PD deficiency alleles. Further studies are needed to definitively establish the mechanisms by which G6PD deficiency confers an advantage against malaria in heterozygous individuals. Such studies could lead to the development of new treatments.Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health . Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in the body's defence against oxidant damage that is particularly important in red blood cells.Evidence before this studyWe systematically searched PubMed up to May 22, 2015, for original research articles investigating the relationship between G6PD deficiency and malaria, using the term “((glucose-6-phosphate dehydrogenase deficiency) AND malaria) OR  NOT “review”[publication type]”. Our search returned 564 articles, which we supplemented with secondary citations and our personal collections. Most reports focused on drug treatment of malaria, particularly the effects of primaquine and other antimalarial drugs in people with G6PD deficiency, on diagnostic methods for detecting G6PD deficiency, on G6PD deficiency as a cause of blackwater fever, or were limited to descriptions of the prevalence of G6PD deficiency, and included data from studies from diverse regions with very different genetic backgrounds and malaria epidemiology. We therefore restricted our search to studies done in Africa by the inclusion of the additional search term “AND Africa”, returning 157 articles that were subjected to further appraisal. These studies used a wide range of experimental designs and most had a small sample size (<200); however, 34 reports contained at least some data regarding the association between G6PD deficiency and malaria from clinical or epidemiological studies since 1960, of which eight contained data on asymptomatic parasitaemia, 15 contained data on uncomplicated malaria, and 16 contained data on severe or complicated malaria. These reports reached a range of conclusions about the relationship between G6PD deficiency and malaria, including no significant effect (six reports) or positive or negative associations in various groups of males or females (28 reports). Some studies have considered heterogeneity at the G6PD locus as a potential explanation for the confusing findings from earlier studies, concluding that G6PD deficiency is a balanced polymorphism driven by selection for heterozygous females. The wide range of study designs, diagnostic approaches, and populations under study precluded a formal meta-analysis.Added value of this studyWe investigated the effects of G6PD deficiency on clinical malaria phenotypes ranging from asymptomatic parasitaemia to strictly defined severe malaria, in 7031 children in a malaria-endemic region of Kenya. We considered allelic heterogeneity at the G6PD locus and the possibility of interactions with other common malaria-protective red cell polymorphisms. We found that the effects of G6PD deficiency were limited to severe malaria and malaria-specific mortality, adding new weight to the conclusion that G6PD deficiency is a balanced polymorphism favouring heterozygous girls.Implications of the available evidenceFurther studies are needed to definitively establish the mechanisms by which G6PD deficiency confers an advantage against malaria in heterozygous individuals. Such studies could lead to the development of new treatments. G6PD deficiency has different effects against different subtypes of severe malaria, predisposing to severe malarial anaemia but protecting against cerebral malaria. Moreover, these subtypes vary with malaria transmission intensity and thus between populations and over time, meaning that the net evolutionary pressures for G6PD deficiency might be more complex than previously appreciated.Plasmodium falciparum malaria; second, we did a cohort study to investigate its effect on the risk of uncomplicated clinical malaria and other common diseases of childhood.We examined the effect of G6PD deficiency on the risk of malaria and other childhood diseases in children living within the area served by the Kilifi Health and Demographic Surveillance System in Kenya.P falciparum malaria to the high dependency ward of Kilifi County Hospital between March 1, 1998, and Feb 28, 2010, as cases. Severe malaria was defined by blood-film positivity for P falciparum and the patient having one or more of the following clinical features: prostration (Blantyre Coma Score of 3 or 4), cerebral malaria (Blantyre Coma Score of <3), or respiratory distress .For the case-control part of the study, we included children aged younger than 14 years who were admitted with clinical features of severe or complicated P falciparum parasites of any density; non-malaria fever was defined as an episode of fever in a child whose slide was negative for P falciparum; upper respiratory tract infection was diagnosed in children whose principal symptoms were characterised by rhinitis or pharyngitis and who had no other features of malaria; lower respiratory tract infection was diagnosed in children who fulfilled the WHO clinical criteria for pneumonia if tests and the subsequent clinical course of disease supported this diagnosis; gastroenteritis was defined as diarrhoea (≥three watery stools per day) with or without vomiting (≥three episodes per day); skin infection was diagnosed in children who presented with dermatological conditions, including scabies, boils, and impetigo; helminth infection was diagnosed in children who had a history of passing worms of any species; and severe anaemia was defined as a haemoglobin concentration of less than 50 g/L. The present analysis focuses on 752 children aged younger than 10 years between Aug 1, 1998, and July 31, 2001. Four cross-sectional surveys were done during this period through which the prevalence of P falciparum infection was recorded. Asymptomatic malaria parasitaemia was defined as the presence of a positive P falciparum blood film during these cross-sectional surveys in the absence of fever or symptoms of clinical illness.The design of the cohort study has been described in detail previously.Written informed consent was provided by the parents of all study participants and ethical approval for both studies was granted by the Kenya Medical Research Institute National Ethical Review Committee in Nairobi.Haematological, biochemical, and malaria parasite data were derived as previously described.15The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria whereas for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses, by G6PD deficiency category.For these analyses, we categorised study participants into the three most physiologically meaningful G6PD categories based on G6PD c.202T genotypes: (1) biochemically normal individuals—ie, boys or girls who were either hemizygotes or homozygotes for the wild-type allele at position 202 of the G6PD gene; (2) G6PD c.202T heterozygous girls  with those among controls using logistic regression. We calculated genotype-specific incidence rate ratios (IRRs) for malaria and other diseases in the cohort study using Poisson regression analysis. We did all analyses both with and without adjustment for potential confounders. We tested for interactions between explanatory variables in both studies using the likelihood ratio test. We did all analyses using Stata (version 11.2).We stratified these groups by sex and by genotype at the G6PD 376 locus in secondary analyses. We compared continuous data using Student's None of the funders had a role in the design of the study, the collection, analysis, or interpretation of the data, the writing of the report, or the decision to submit for publication. SU and CMN had full access to the raw data and TNW had full access to all of the data and had the final responsibility to submit for publication.P falciparum malaria and 3940 control participants. 2 p=0·32) and both cases and controls were well balanced with regard to sex . We found no evidence of a significant association between G6PD deficiency category and the incidence of uncomplicated us girls . We detecategory .P falciparum malaria. Conversely, children with G6PD deficiency had a significantly increased risk of severe malaria anaemia. We found no effect of G6PD category on parasite densities or on the incidence of uncomplicated P falciparum malaria or other common diseases of childhood either in the same study or in a cohort study of children living in the same area. The only exception we found was a marginally significant increase in the incidence of helminth infections among heterozygous girls, an observation which may well represent a chance finding given the number of comparisons we made.Through a case-control study done on the coast of Kenya, we found that girls who were heterozygotes for the G6PD c.202T allelic form of G6PD deficiency were significantly protected from severe and complicated The global distribution of G6PD deficiency is thought to be a result of natural selection through providing a survival advantage against malaria.We investigated the effect of G6PD deficiency both on a range of phenotypes of clinical malaria and on the incidence of other common diseases of childhood within a single well-characterised population. Based on data from our case-control study, the risks of severe malaria and within-hospital death among G6PD c.202T heterozygous girls were significantly lower than in G6PD normal children. Conversely, the risks of severe malarial anaemia in G6PD c.202T hemizygous boys and homozygous girls with G6PD deficiency were significantly higher. Data from our cohort study showed no effect of G6PD category on the prevalence or incidence of either uncomplicated malaria or of any other common childhood disease, with the sole exception of a marginally significant increase in the incidence of helminth infections among heterozygous girls. Taken together, our data support the previous conclusion that G6PD deficiency is a balanced polymorphism conferring a selection advantage in heterozygous girls at the expense of a fitness disadvantage in those with G6PD deficiency.18P falciparum infection,P falciparum is the only significant cause of clinical malaria in Kenya; therefore, we were unable to investigate the hypothesis that G6PD deficiency might protect against Plasmodium vivax malaria.Although our conclusions support the results of some previous clinical studies,First, we characterised participants on the basis of G6PD genotype as opposed to G6PD phenotype. The high degree of overlap in enzyme activity between the various haplotypic groupsA second strength of our study is that, by contrast with most previous studies in which participants have been selected on the basis of G6PD genotype, we minimised the possibility of misclassification of G6PD deficiency through earlier studiesUnlike most previous studies, we excluded the possibility of confounding by two other malaria-protective polymorphisms—sickle cell trait and α thalassaemia—both of which are common in our study population. A negative epistatic interaction between sickle cell trait and G6PD deficiency has been reported previously in Mali,Despite having arisen thousands of years ago, there remain no examples of populations in which any of the many G6PD deficiency mutations have been selected to the point of fixation,Our study has several limitations of which perhaps the most important relates to the fact that, to a large extent, our study design was opportunistic and capitalised on the existence of data and samples collected for other purposes. As a result, participants were recruited over a long period during which the transmission of malaria within the study area declined substantially.P falciparum infections does not, on the face of it, support most hypotheses advanced to date. Most have invoked the reduced ability of erythrocytes of people with G6PD deficiency to cope with the oxidant stresses that accompany P falciparum infection, and suggest that this effect might either reduce the ability of parasites to grow and develop or precipitate their early removal once infected.Second, the lack of statistical evidence for an effect of G6PD deficiency on the incidence of uncomplicated malaria or other childhood diseases might simply be a result of a lack of statistical power, either because the study or the effect sizes were too small. Furthermore, being purely epidemiological, our study can provide no definitive insights into the mechanisms by which G6PD deficiency confers malaria protection. Nevertheless, the fact that we found no meaningful difference in parasite densities between G6PD deficiency categories across the full range of Why G6PD deficiency should be associated with an increased risk of severe malaria anaemia is also open to speculation; however, malaria parasites exert oxidant stress on red cellsIn conclusion, more than half a century since the protective effect of G6PD deficiency against malaria was first proposed, the clinical epidemiology of malaria in children with G6PD deficiency has remained confusing. Our study provides strong support for the conclusion that the G6PD c.202T allele has reached its present frequencies in much of sub-Saharan Africa through positive selection for heterozygous girls through a mechanism mediated by malaria. We hope that this growing consensus will lead to a renewed drive to determine the mechanisms involved and to turn this knowledge to new approaches to malaria treatment.


In Zambia, there has been a large scaling up of interventions to control malaria in recent years including the deployment of rapid diagnostic tests (RDTs) to improve malaria surveillance data as well as guide malaria treatment in health facilities. The practical challenge is the impact of RDT results on subsequent management of patients. This study explored the role of RDTs in malaria diagnosis and the health workers’ adherence to test results.An observational prospective study was carried out at health centres in four districts, namely Chibombo, Chingola, Chipata, and Choma. Children under the age of five years with history of fever were recruited and the clinicians’ use of RDT results was observed to establish whether prescriptions were issued prior to the availability of parasitological results or after, and whether RDT results influenced their prescriptions.®. Only 465 (19.4%) of the 2,393 children were prescribed drugs before receiving laboratory results. A total of 76.5% children were prescribed drugs after laboratory results. Children with RDT positive results were 2.66 ) times more likely to be prescribed anti-malarial drugs. Children who presented with fever at admission  were 42% less likely to be prescribed an anti-malarial drug compared to children who had no fever ). It was noted that proportions of children who were RDT- and microscopy-positive significantly declined over the years from 2005 to 2008.Of the 2, 393 recruited children, 2, 264 had both RDT and microscopic results. Two in three (68.6%) children were treated with anti-malarials despite negative RDT results and almost half (46.2%) of these were prescribed CoartemRDTs may contribute to treatment of febrile illness by confirming malaria cases from non-malaria cases in children under the age of five. However, the adherence of the health workers to prescribing anti-malarials to only RDT-positive cases at health facility level will still require to be explored further as their role is crucial in more precise reporting of malaria cases in this era towards malaria elimination as the target. Plasmodium falciparum infection in most endemic countries. ACT is expensive and relies on an insecure supply of raw materials. It is also documented that non-malarial febrile illnesses exert higher childhood mortality across malaria-endemic countries than malaria [The current global policy recommendation is universal access to parasite-based diagnosis of malaria-like febrile illness  due to t malaria . It is fMalaria continues to be a disease of public health importance in Zambia. There has been a large scaling up of interventions to control malaria in recent years . In 2009® compared with SP and CQ, compelled the National Malaria Control Programme (NMCP) to introduce RDTs, firstly as pilot in some districts in 2005 and 2006. Scale up at national level was achieved in 2009, when Zambia made the transition to a strategy of full reporting and treatment using only parasitologically confirmed diagnosis nationwide [Zambia was faced with poor malaria surveillance data caused by inadequate laboratory diagnostic facilities in most parts of the country at the time of introduction of ACT. This, coupled with substantial higher cost of CoartemThe practical challenge is that the impact of RDT results on subsequent management of patients varies. The impact of RDTs on anti-malaria consumption has been demonstrated widely ,7,8 but ® between the clinical paradigm and the RDT with the standard thick blood film microscopy.A study was conducted to explore the role of RDTs in order to determine the adherence  by health workers to test results for the management of febrile illness in children under five years of age in four districts of different epidemiological zones. The study was designed to compare the extent of excessive use of CoartemThe study was conducted from 2005 to 2008 in four districts, namely Chingola, Chibombo, Choma, and Chipata which cover a range of transmission intensity. In each one of these districts, two health facilities were selected. In Chingola, the study was conducted at Chawama clinic and Clinic 1; in Chibombo, at Liteta Hospital and Chisamba clinic. In Choma, the study was conducted at Shampande clinic and Choma General Hospital and in Chipata at Kapata Clinic and Chipata Health Affiliated Clinic (HAC).At the time of the conduct of the study, Chingola, in the Copperbelt province of Zambia with a population of 177,445 (2000 Census), was a district in an area of high malaria transmission with prevalence in the range of 35% in the dry season between May and October and 70% in the wet season, November to April, in children under the age of five years attending outpatient care.Choma with a population of 203,305 (2000 Census) in the Southern Province of Zambia, on the other hand, was in an area of unstable but severe and irregular transmission. The prevalence of malaria in children under five years of age ranged between 33 and 68%. Chipata situated in the Eastern Province of Zambia with a population of 362,132 (2000 Census) was in an area of stable transmission with prevalence rates ranging from 35 to 70%. Chibombo with a population of 242,380 (2000 Census) in the central part of the country was in a medium transmission zone with prevalence ranging from 5 to 45%.The study population was children under the age of five years, weighing more than 10 kg and residing within the catchment area of the recruiting health facility. These children were included in the study based on history (or presence) of fever and being clinically diagnosed as having malaria by the attending clinician at the health facility.The study clinician examined each child who was eligible to enter the study, took history and explained the study protocol to the parent or guardian of the child. Written consent was obtained from all parents or guardians who agreed to have their children participate in the study. Two scenarios were observed and noted by study investigators. The clinician, at his/her own discretion, prescribed an anti-malarial before the patient went to the laboratory for an RDT or at the clinician’s discretion, the patient was sent to the laboratory for a malaria test and anti-malarial prescription was given only after RDT results were made available. The study investigators (blinded to the clinician) took note of the adherence of the attending clinician to await a RDT result before prescribing an anti-malarial or prescribing of the anti-malarial before the availability of the RDT results.All patients were advised to return for review on day 3 and 7. On these days, only blood for microscopy was collected to ensure the patients were fully treated and recovered before being discharged from the sick list.In the laboratory, a finger prick was made on the patient to collect blood for RDT  and microscopy. Only the RDT result was available to the clinician (for those patients whom prescription awaited laboratory results). Blood slides were stained and read later within the health facilities and results entered in the log books.Consistency of operation was required for the study to be conducted in a uniform manner across the country; therefore an element of training study staff was crucial before the study was initiated. This was carried out at a neutral venue, under the coordination of the investigators. The training took seven days and the following comprised the content of the training: to administer the study information and informed consent form; to fill out details on a structured case record form (CRF); how to follow step-by-step the patient flow chart and the aspect of counselling the patient about post-treatment follow-up. The latter included what the parent/guardian should do in the event that the child’s condition post-consultation with study staff worsened; information regarding what treatment the child had been prescribed and counselling for the third and seventh day follow-up visits.The data were entered and initially analysed in EPI INFO (version 3.3.2 of 2005). Consistency and range checks were used to edit the data. Further analysis was conducted using SPSS (version 17.0 of 2008). The Cohen’s Kappa statistic with its 95% confidence interval was used to measure the extent of agreement between RDT and microscopic results. Multivariate logistic regression analysis was conducted to determine independent factors associated with the outcome. Adjusted odds ratios (AORs) and their 95% CIs were reported.®, SP or quinine), prescription given (before or after).The variables used for analysis included the following: fever (axillary temperature of 37.4 degrees Celsius or higher), history of fever, laboratory result by RDT, anti-malarial drugs prescribed  Ethics Review Committee. Technical support throughout the proposal development and planning phase came from MIM/WHO/TDR.1 = 17, Q3 = 36) months. Altogether 2,264 children had both the RDT and microscopic results: 640 were from Chibombo, 584 from Chingola, 398 from Chipata and 642 from Choma.A total of 2,393 children participated in the study of which 52.1% were males. The median age was 25  and noted those who prescribed anti-malarials prior to the availability of laboratory results, it was observed that overall, 465 (19.4%) out of 2,393 children were prescribed anti-malarial drugs before receiving laboratory results by district. About half (53.6%) of the children from Chibombo received anti-malarial prescriptions before receiving laboratory results, while in Chipata 0.9% of the children were prescribed anti-malarials before receiving laboratory results.Table A total of 1,498 (76.5%) of the 1,958 children were given anti-malarial prescriptions after receiving laboratory results  recommends confirmatory diagnosis before treatment in all settings where a diagnostic test is or can be made available. The Ministry of Health in Zambia revised the national guidelines in 2009–2010 to mandate parasitological confirmation of malaria based on microscopy or RDTs in the public and private sectors, with RDTs to be used where microscopy was not available, or where RDT use on an outpatient basis by non-laboratory staff would reduce laboratory workloads .® and lesser of other anti-malarials. Over 77.8% of these patients were not febrile. On counterchecking the parasitological results of the patients who received anti-malarials prior to laboratory results, 84.0% were RDT-negative who should not otherwise have received anti-malarial medication. One assumption that could be made is that this category of health workers (who prescribed for the 19.4% patients) may perhaps not trust the laboratory results yet, or that they remain with the orientation of prescribing an anti-malarial under the Integrated Management of Childhood Illness (IMCI) guidelines . Chibombo district was most affected by prescriber preference to treat with an anti-malarial before the laboratory results (53.6%). It’s encouraging however, to generally note that the number of children in this study who were treated before laboratory results were fewer.This study was carried out before the guidelines were enforced and the assessment of the prescribing practices between availability of laboratory results and anti-malarial prescriptions indicated that only 19.4% of children were prescribed medication prior to receiving laboratory results. This category of clinicians did not wait for the availability of results before prescribing anti-malarials. However, for this smaller number, over 50% of the prescriptions given (i.e. 9.7% of the children) were Coartemper se. Febrile, parasite-negative patients were still prescribed an anti-malarial as much as afebrile patients with RDT-negative results.Of the children whose laboratory results were available before prescriptions were given, those whose results were RDT-positive were 2.66 times more likely to receive an anti-malarial. Nonetheless, for the children who had fever , they were 42% less likely to be prescribed anti-malarial drugs compared to their counterparts who had no fever. This should be interpreted with caution as all children recruited in this study had either a history of fever or were found to be febrile at the time of inclusion in the study. As regards the rationalization of treatment on the basis of fever, this study has confirmed that febrile illness was not the basis for prescription of an anti-malarial Laboratory confirmation of parasite infection is important to the management of malaria and other febrile illness because to properly manage febrile illness, it is necessary for clinicians to accurately know the actual infection status of each patient. As RDTs may be the only available tool for very remote areas in Zambia, the issue of clinicians trusting the results is paramount if confirmation of parasite infection has to remain the basis for prescribing an anti-malarial.et al., that provider non-compliance with test results can lead to reductions in the ability of RDT introduction to reduce ACT consumption and concluded that this may not be a major problem in Zambia [As a follow-up to the observations by Yukich n Zambia , findingThe high levels of malaria clinical diagnoses with anti-malarial prescription in this study were observed across all the districts despite the varied malaria prevalence. In areas of high malaria transmission, where clinical diagnosis may be a marginally more effective predictor of malaria infection, this practice would hardly raise concern. The findings of this study do not allow for the fact that the roll-out of RDTs may contribute to the changing of malaria case definition to mean parasitological malaria and not suspected malaria cases or fever cases.et al. is reassuring in that the incidence of malaria in febrile patients left untreated after seven days post a negative result was significantly lower than the incidence of malaria in the whole cohort [et al., and others [The management of fever in children less than five years in the districts where this study was conducted may be interpreted to be non-compliant to RDT results as there were less actual parasite-positive malaria patients than were treated. The report of Njama-Meya e cohort . This fid others -25).The over-prescription pattern found in Chibombo and the RDT compared to microscopy discrepancies reported in Chipata  mIt is important to underscore that over time in this study, there was a decline in the number of positive malaria cases and anti-malarial prescriptions, confirming the already noted reduction in malaria prevalence countrywide. Therefore, emphasizing the importance for RDT result adherence to allow for interpretation of malaria prevalence over time and treatment of actual malaria cases. This would avoid presumptuous prescriptions and thereby missing febrile cases that would require further investigations and appropriate treatment.RDTs may contribute to treatment of febrile illness in children under the age of five years for malaria and non-malaria cases. However, compliance by the health workers to test results at health facility level will still require further exploration, as their role is crucial in more precise reporting of malaria cases in this era towards the target of malaria elimination. The laboratory staff at health facility level may still require in-service re-orientation in microscopy where these facilities are available for correct interpretation on trends in malaria epidemiology in Zambia.The authors declare that they have no competing interests.All authors read and approved the final manuscript. CM, EMN and CS participated in the conception and design of the study and protocol development. CM, JC and EMN participated in the training. CM, EMN and JC coordinated the data collection, management and participated in the manuscript writing. JC carried out the analyses which were validated by SS who provided overall direction on the analysis. CS conceived the study and provided technical oversight to the design of the study.


Salmonella (NTS) can cause bloodstream infections, referred to as invasive non-typhoidal Salmonella disease (iNTS disease); it can occur in outbreaks and is often preceded by malaria. Data from Central Africa is limited.In sub-Saharan Africa, non-typhoidal Clinical, microbiological and molecular findings of NTS recovered in a blood culture surveillance project (2009–2014) were analyzed.Salmonella Typhimurium and Salmonella Enteritidis together accounted for 88.9 % of pathogens isolated from blood cultures collected during an outreach visit to the affected districts in June 2012. Children infected with Salmonella Enteritidis (33 patient files available) tended to be co-infected with Plasmodium falciparum more often than children infected with Salmonella Typhimurium (40 patients files available) (81.8 % versus 62.5 %). Through the microbiological surveillance project (May 2009–May 2014) 113 unique NTS isolates were collected (28.5 % (113/396) of pathogens); most (95.3 %) were recovered from children < 15 years. Salmonella Typhimurium (n = 54) and Salmonella Enteritidis (n = 56) accounted for 47.8 % and of 49.6 % NTS isolates respectively. Multilocus variable-number tandem-repeat analysis (MLVA) revealed more heterogeneity for Salmonella Typhimurium than for Salmonella Enteritidis. Most  NTS isolates that were available for antibiotic susceptibility testing were multidrug resistant. All isolates were susceptible to ceftriaxone and azithromycin.In March-July 2012 there was an epidemic increase in malaria infections in the Oriental Province of the Democratic Republic of the Congo (DRC). In one referral hospital, overall hospital admissions in June 2012 were 2.6 times higher as compared to the same period in the years before and after (336 versus an average of 128 respectively); numbers of malaria cases and blood transfusions were nearly three- and five-fold higher respectively (317 versus 112 and 250 versus 55). Case fatality rates  peaked at 14.6 %. Salmonella Typhimurium and Salmonella Enteritidis were isolated from blood cultures. Overall, the two serovars showed subtle differences in clinical presentation and genetic diversity.During the peak of an epidemic increase in malaria in the DRC in 2012, a high proportion of multidrug resistant The online version of this article (doi:10.1186/s12879-016-1604-1) contains supplementary material, which is available to authorized users. Salmonella  are associated with self-limiting diarrhea. In sub-Saharan Africa however, they often cause bloodstream infections, which is referred to as invasive non-typhoidal Salmonella disease (iNTS disease). They are among the pathogens most frequently isolated from blood cultures [In industrialized countries, non-typhoidal cultures –4. In facultures . Due to Plasmodium falciparum malaria [Salmonella enterica serovar Typhimurium  and Salmonella enterica serovar Enteritidis .iNTS disease occurs mainly in children and immune-compromised individuals and a number of conditions make individuals more susceptible including malnutrition and  malaria –9. The tStudies about NTS infections in children have been reported mainly from East, West and South Africa , 10–14. In April 2012, local health authorities in the district of Haut-Uélé   to all affected children. Blood culture sampling in the affected districts allowed assessment of a possible concomitant burden of invasive Salmonella infections observed during this period of increased admissions and malaria infections in the Bas-Uélé and Haut-Uélé districts as well as of invasive Salmonella infections recorded in the entire Oriental Province in the period before and after 2012 (2009–2014).The present report aims to describe the clinical, microbiological, and molecular characteristics of the invasive 2 with 8,633,573 inhabitants and comprises four administrative districts: Tshopo , Haut-Uélé, Bas-Uélé and Ituri. Each district is divided into health zones (HZs) with a General Referral Hospital. The four health zones mainly affected by an epidemic increase in malaria in 2012 were located in the districts Haut-Uélé (HZs of Wamba and Isiro) and Bas-Uélé (HZs of Pawa and Poko) , located in the capital of the Oriental Province, has basic laboratory infrastructure performing blood cultures and is participating in the microbiological surveillance network coordinated by the National Institute of Biomedical Research (INRB) in Kinshasa in collaboration with the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. This network monitors the pathogens involved in bloodstream infections and their antibiotic resistance patterns at various sentinel sites in the DRC. CUKIS receives samples from hospitals and health centers in Kisangani and from more remote sites during outbreak investigations. CUKIS is located at 445 km from the HZ Wamba, 519 km from the HZ Pawa, 571 km from the HZ Isiro and 691 km from the HZ Poko.Criteria for blood cultures sampling in patients >28 days of age were: (1) a body temperature of ≥38 °C or ≤35.5 °C; (2) suspicion of severe localized infections; or (3) suspicion of sepsis, typhoid fever, and severe malaria. Additional criteria for collection of blood specimens from women in the neonatal period were premature rupture of membranes, intrapartum fever, low Apgar score, or specific symptoms such as tachypnea, cyanosis, and lethargy. For children (≤14 years old), 1–4 mL of blood was sampled into a pediatric blood culture vial ; for adults, 2 × 10 mL of blood was inoculated into aerobic blood culture vials  and incubated at 35 °C for 7 days. Blood cultures were processed according to standard methods as described elsewhere , 23. AllSalmonella spp. were serotyped using commercial antisera . A selection of the isolates were sent to the National Reference Centre in Belgium for confirmation of serotyping by slide agglutination following the Kauffmann-White scheme [At ITM, isolates biochemically confirmed as e scheme . SelectiSusceptibility testing of the isolates and interpretation of results were done according to the National Committee for Clinical Laboratory Standards (CLSI) guidelines as described elsewhere . AntibioThe methods described apply equally to ongoing surveillance efforts and to the additional site visits done in 2012/2013.gyrA, gyrB, and parC genes as previously described [qnr genes  was determined using PCR [Molecular mechanisms of ciprofloxacin resistance were assessed by amplification and sequencing of the quinolone resistance determining regions (QRDRs) of the escribed . The frasing PCR .Salmonella Enteritidis and Salmonella Typhimurium isolates, MLVA analysis was performed on all isolates as previously described [Salmonella Enteritidis, profiles were attributed based on the number of tandem repeats on five loci in the following order: SENTR7−, SENTR5−, SENTR6−, SENTR4− and SE3-. For Salmonella Typhimurium, profiles were attributed based on the number of tandem repeats on five loci in the following order: STTR9-, STTR5-, STTR6-, STTR10- and STTR3-. Simpson’s index of diversity (D) and Shannon’s indices of diversity (H’) and equitability (E) were calculated to evaluate the discriminatory power and evenness of distribution by using the Biodiversity Calculator [To assess the clonal relatedness among escribed , 29. Forlculator .Salmonella bloodstream infection between 2009 and 2014 were retrieved from patient files. Data recorded included age, gender, weight and geographic origin, clinical presentation, diagnosis upon admission, number of blood transfusions, in-hospital outcome, Hb-value  in June/July 2012 and to the HZ of Pawa in the period October 2012–January 2013. During these and additional visits, clinical data from all children with a documented i method ) and pari method , i.e., Hi method . No dataData on hospital admissions and case-fatality rates were collected from the hospital registers and concerned all patients admitted to the pediatric ward. For the General Referral Hospital of Poko, blood transfusion files for the year 2011 could not be retrieved.Data of rainfall in the Oriental Province (2011–2013) were retrieved from the Global Information and Early Warning System from the Food and Agriculture organization of the United Nations .Demographic, clinical and microbiological data were entered anonymously into an Excel database . Only the first isolate for each patient was considered, isolates recovered from a second blood culture drawn within two weeks after the initial one were considered as duplicates, whereas isolates recovered from a repeat blood culture more than two weeks after the initial one were considered as recurrences (either relapse or repeat infections). Statistical analysis was done with Stata 12 . P values and (adjusted) odds ratio’s (OR) were calculated by univariate and multivariate logistic regression analysis. When one group contained zero observations for a particular variable, the p value was calculated with a two tailed Fisher’s exact test instead.It was decided only to keep the categorical variables anemia and severe anemia rather than the continuous variable of hemoglobin level (g/dL), because of the limitations of the measurement (Sahli method) and because it seemed more relevant clinically. When multiple variables differed statistically between groups at a level of P <0.100 and were also clinically relevant, they were further assessed in a multivariable logistic regression analysis. Diagnoses upon admission were not included in the multivariate analysis. To assess the influence of malaria positivity, initial analysis was done stratified for malaria positivity. Stepwise backward regression was used to eliminate variables that this did not retain their statistical significance. Likelihood ratio testing guided the selection of the final model. A p value of < 0.05 was considered significant.0 701/DPS/PERS/872/2012) who represents the highest health authority as well as from the Chief Medical Officer of the Haut-Uélé district (Reference number: 771/23/ISP/HU.O/2012). The retrieved clinical data were only accessible to the CUKIS collaborator (DF), who is a medical doctor, and were subsequently entered anonymously in an Excel database with restricted access.The study was conducted according to the principles expressed in the Declaration of Helsinki. Ethical approval for the Microbiological Surveillance Study was granted by the Institutional Review Board of the ITM, the Ethics Committee of Antwerp University, and the Ministry of Health of the DRC. In view of the absence of an Ethical committee in Kisangani at the time of the study, written authorization to review the patient files was obtained from the Medical Inspector of the Oriental Province  pathogens, including Salmonella Enteritidis (n = 7) and Salmonella Typhimurium (n = 3) (26.3 % of pathogens). After Salmonella species, the most frequently isolated pathogens were Acinetobacter species and Staphylococcus aureus.In June 2012, at the request of the Ministry of Health, INRB staff did an outreach visit to all four affected HZs. Out of 87 blood cultures sampled, 18 (20.7 %) grew clinically significant organisms (further referred to as pathogens), of which 16 (88.9 %) were NTS, with Salmonella. The annual proportion varied between 25.0 % (2009–2010) and 42.3 % (2012). Salmonella were the most commonly isolated bacteria in both adults and children. Other frequent pathogens were Staphylococcus aureus and Klebsiella pneumoniae. Among the 135 Salmonella isolates, there were five duplicates . Of the 130 first (unique) isolates, there were 17 Salmonella Typhi and 113 NTS isolates.From May 2009 to May 2014, CUKIS processed 3467 blood cultures in total comprising 88.0 % samples (3052/3467) from Kisangani district and 12.0 % (415/3467) from the Haut-Uélé and Bas-Uélé districts, including samples from the outreach visits. In total, 11.4 % (396/3467) blood cultures grew pathogens of which 34.1 % (135/396) were Salmonella Typhimurium and Salmonella Enteritidis were the most frequent serovars accounting for approximately half of NTS isolates each (47.8 and 49.6 % out of 113 isolates respectively) .y) Table . In 2011Salmonella Typhimurium isolates revealed heterogeneity with 23 different profiles across 54 isolates collected between 2009 and 2014 (Simpson’s D = 0.95) . Forty-four of the 54 Salmonella Typhimurium isolates were collected in the Tshopo district , with three major profiles accounting for only 18 isolates  (2-6-11-8-0210 (n = 7), 2-5-10-8-0210 (n = 6), 2-7-10-8-0210 (n = 5)) in this district. Eight Salmonella Typhimurium isolates were collected in the Bas-Uélé district. Of these 8, five belonged to the profile 2-8-12-8-0210, which was not isolated in the Tshopo district.MLVA analysis of the Salmonella Enteritidis isolates revealed less heterogeneity with 13 different profiles (Simpson’s D = 0.71)  and 2-13-4-3-NA (n = 15), accounting for 73.2 % of the tested isolates (Shannon’s H’ = 1.64 and E = 0.64). Thirty-five out of the 56 Salmonella Enteritidis isolates were collected in the health district of Tshopo; 26  of these 35 isolates had the major profile 2-13-3-3-NA. This profile was not isolated in the health districts of Bas-Uélé and Haut-Uélé. Of the 21 Salmonella Enteritidis isolates collected in the Bas-Uélé and Haut-Uélé districts between 2009 and 2014, 17 isolates were collected in the year 2012 of which 10 belonged to the second major profile 2-13-4-3-NA.Multilocus variable-number tandem-repeat analysis (MLVA) of the 56 n = 107) as they were not discussed separately from isolates collected in other provinces of the DRC  were MDR, and with slightly higher resistance rates among Salmonella Typhimurium compared to Salmonella Enteritidis. Two isolates, one Salmonella Typhi and one Salmonella Typhimurium (isolated in 2011 and 2013 respectively), showed decreased ciprofloxacin susceptibility. In the Salmonella Typhi isolate with a MIC-value of 0.19 μg/ml for ciprofloxacin, a gyrA resistance mutation (Asp87-Tyr/Glu133-Gly) was detected; the Salmonella Typhimurium isolate with a MIC of 0.25 μg/ml for ciprofloxacin also showed a gyrA resistance mutation (Asp87-Asn). Both isolates were resistant to nalidixic acid.In contrast to /ml. All isolates were susceptible to ceftriaxone and azithromycin.For all NTS isolates combined, MIC50 and MIC90 values for ciprofloxacin were 0.012 μg/ml and 0.047 μg/ml respectively. The MIC50 value for ciprofloxacin of these isolates was 0.012 μg/ml in the period 2011–2012 and 0.016 μg/ml in the period 2013–2014 while the MIC90 value increased from 0.016 to 0.047 μgClinical charts of children with iNTS diagnosed through ongoing surveillance, as well as during the additional site visits in 2012–2013, were reviewed.Salmonella Typhi (96 months (IQR 48–123); p = 0.000).A total of 91/117 (77.8 %) children with invasive salmonellosis were < 5 years old. As expected, the median age for children with iNTS disease (24 months (IQR 12–36)) was significantly lower as compared to children infected with Salmonella Typhi cases; the diagnosis of typhoid fever was raised in 68.8 % of children with confirmed Salmonella Typhi infection compared to 18.5 % of children with iNTS disease (p = 0.000). This was in line with differences in the clinical presentation: in particular, dyspnea and pallor tended to occur more frequently among children with iNTS disease compared to children with Salmonella Typhi infection (46.7 % vs. 23.1 % and 76 % vs. 38.5 % (p = 0.010) respectively), while vomiting tended to be less frequent (45.3 % vs. 69.2 %). Bivariate analysis also showed significant differences in malaria positivity , anemia , and rate of blood transfusions  between the children with iNTS disease vs. the children with a Salmonella Typhi infection respectively. We included these variables in the multivariate analysis and initially stratified for malaria positivity. In both groups , age (months) was the only variable that remained significant with an OR similar in both groups. We therefore decided to include malaria positivity as a variable in the multivariable analysis. Likelihood ratio testing indicated that the best model included the variables age (adjusted OR 0.95 (95 % CI 0.93–0.97)), malaria positivity (adjusted OR 6.31 (95 % CI 0.99–39.95) and pallor (adjusted OR 5.99 (95 % CI 0.95–37.77). Age remained significant (p = 0.000) indicating that younger age is associated with non-typhoidal Salmonella infection while malaria positivity and pallor were borderline significant (p = 0.050 and p = 0.057 respectively).For 88 out of 124 (71.0 %) children with invasive salmonellosis clinical files were available Tables . There wSalmonella Typhi infection; this did not differ significantly. Seventy per cent (7/10) of deaths due to iNTS disease occurred within the first 24 h after admission.Mortality in children with iNTS disease was 13.3 % (10/75) compared to 7.7 % (1/13) of the children with a Salmonella Typhimurium bloodstream infection (18 months (IQR 12–30)) was lower compared to those with a Salmonella Enteritidis bloodstream infection (24 months (IQR 12–48)) but this difference was not statistically significant (p = 0.128). For all iNTS disease cases combined, malaria represented the most frequent diagnosis upon admission  followed by, in rank of decreasing frequency, typhoid fever, pneumonia and septicemia. Malaria was suspected more often in children with a Salmonella Enteritidis infection compared to children with a Salmonella Typhimurium infection .The median age of children with a Salmonella Typhimurium group .The most common presenting symptom in children with iNTS disease was pallor, observed in 76 % of all children, followed by lethargy, cough, dyspnea, and vomiting. Diarrhea was reported in only 28 % of the children. The spectrum of symptoms did not differ between the different serovars except for seizures which were only observed in the P. falciparum malaria was recorded in nearly 70 % of all children with iNTS disease; this proportion tended to be higher among the Salmonella Enteritidis group compared to the Salmonella Typhimurium group .Laboratory confirmed Salmonella Enteritidis and Salmonella Typhimurium.There was no significant difference in mortality between children infected with Salmonella bloodstream infection reported previous antibiotic exposure.Overall, 35,5 % (39/110) of patients with a Salmonella infections in the Oriental Province during the surveillance period 2009–2014, including data from outreach visits to more remote areas in the province during an epidemic increase of malaria in 2012.The present study describes the clinical, microbiological and molecular characteristics of invasive Salmonella among pathogens from blood cultures varied between 25.0 and 42.3 %, which is consistent with other studies from sub-Saharan Africa [Salmonella Typhimurium and Salmonella Enteritidis that were isolated in equal numbers but with proportions varying over time which is also in line with other studies from sub-Saharan Africa [Salmonella among the pathogens isolated from blood cultures in the health zones affected by the epidemic increase in malaria in June 2012. During subsequent outreach visits between October 2012-January 2013 , this proportion subsequently decreased to only 26.3 %. The latter percentage is more in line with the averages at the provincial level, and suggests that the epidemic increase of malaria was associated with a concomitant increase in Salmonella bloodstream infections. Possibly, the data represented here are an underestimation of the actual number of patients co-infected with malaria and NTS. Other studies have shown that case fatalities are higher for children who are co-infected with NTS compared to those infected with malaria alone and, in the present study, part of them might have died before reaching the hospital [During the surveillance period, the proportion of n Africa –37. Thern Africa , 38, 39.hospital .Salmonella Typhimurium and Salmonella Enteritidis bloodstream infections except for occurrence of seizures but this was probably affected by a small sample size.The clinical presentation of iNTS disease in this study was dominated by pallor, lethargy, gastro-intestinal and respiratory symptoms. A concomitant infection with pneumococci has been forwarded to explain the prevalence of respiratory symptoms during iNTS disease but the dyspnea could also be related to the high prevalence of anemia among children with iNTS disease or to the acidosis due to sepsis in general . There wSalmonella Enteritidis bloodstream infection tended to suffer from concomitant malaria more frequently compared to those infected with Salmonella Typhimurium. We are not aware of other studies that pointed to a particular association of Salmonella Enteritidis with concurrent malaria infection. More extensive and prospective research is needed to evaluate this finding further.Children with a P. falciparum malaria which was also previously reported from Bwamanda in the DRC [P. falciparum malaria predisposes to iNTS disease [Overall, more than two-thirds of the children with iNTS disease tested positive for  the DRC . It is k disease , 41, 42  disease , 43–45.  disease .Besides malaria, other known risk factors for contracting iNTS disease are HIV, sickle cell anemia and malnutrition. It is estimated that 12 % of the hospitalized children in the DRC have sickle cell anemia . For HIVSalmonella Typhi infection, differences with iNTS cases could be explored. Vomiting was more prevalent among children infected with Salmonella Typhi compared to those with iNTS disease. Mtove et al. (2010) had similar observations in a study from rural Tanzania  [Salmonella Typhi had pallor, anemia, need for blood transfusion and were less frequently tested positive for malaria compared to the children with iNTS. In the study of Mtove et al. (2010) children with Salmonella Typhi infections did not show pallor or severe anemia but children with iNTS did in respectively 62.2 and 40.0 % of the cases [Salmonella infection. These results should be interpreted with caution however given the small sample sizes.As there was also clinical data available for 13 children with a e cases) . In bivahe cases . Other she cases , 42. TheUnlike the epidemic increase of iNTS in Kisantu – which was only identified upon retrospect –the Oriental Province health authorities were alerted to the possibility of an increase in iNTS disease in concurrence with the observed increase in malaria cases. As a consequence, empirical antibiotic treatment was installed early on in the epidemic and this may have accounted for the relatively low case-fatality among the microbiologically confirmed iNTS cases in our cohort. This rate is similar to that found during the outbreak in Bwamanda  but loweSalmonella isolates showed decreased ciprofloxacin susceptibility. However, MIC50 and MIC90 values for ciprofloxacin were higher in the period 2013–2014 compared to the period 2011–2012 which is a worrisome trend.Antibiotic susceptibility testing showed high MDR rates among NTS isolates; only two Salmonella Enteritidis isolates showed that two closely related profiles accounted for 73.2 % of all the tested isolates. The two major profiles only differed with one tandem repeat at one single locus suggesting very close relatedness [Salmonella Enteritidis isolates implicated in the epidemic increases in Kisantu and Bwamanda also showed very close relatedness [Salmonella Enteritidis associated with iNTS disease.MLVA profiles of atedness . MLVA pratedness . Whole gSalmonella Typhimurium isolates were more heterogeneous than Salmonella Enteritidis isolates with the two most common MLVA profiles accounting for only 24.1 % of all tested isolates. The major profile isolated from the Haut-Uélé district differed by 1–2 tandem repeats at two loci compared to the major profiles isolated in the Tshopo district. Previous reports on epidemic increases of NTS infections in the DRC did not consider the MLVA profiles for Salmonella Typhimurium. A previous study did assess the population structure of 180 Salmonella Typhimurium isolates collected in the DRC between 2007 and 2011 with CRISPOL typing and found that >96 % belonged to the CT28 group [Salmonella Typhimurium multilocus sequence type (ST) 313 which is associated with bloodstream infections and high rates of antimicrobial drug resistance. Also in other Sub-Saharan countries, the majority of isolates are found to be ST 313 [Salmonella Typhimurium isolates from different parts of the DRC, including isolates from the Oriental Province, Kisantu and Bwamanda.28 group . This gre ST 313 . Future The present study had several limitations, mostly owing to its retrospective nature. First, the outreach visits to the HZs were – due to logistic and administrative constraints – organized at a period considerably after the first alerts of increased admissions and malaria cases. Next, there was no clearly defined population size denominator, precluding calculation of population-based incidence rates. In addition, it could not be ascertained how many children died at home or on their way to the hospital, precluding accurate estimates of case-fatality rates. Furthermore, not all isolates, patients’ files and hospital registers were available for analysis. Other difficulties included accessibility of the affected districts requiring for instance transport of samples by motorbikes on deteriorated roads and subsequent shipment by a small airplane carrier (at a distance of 350 km) to CUKIS, the single diagnostic microbiology laboratory in the Oriental Province.There were also limitations with regard to laboratory data: no distinction could be made between recent (microscopy negative but rapid diagnostic test positive) versus current malaria (microscopy positive), and the quality of malaria diagnosis probably differed per medical center. The limitations of measuring hemoglobin levels with the Sahli method should be kept in mind especially as no quality control for harmonization was performed.Finally, it was not possible to compare the clinical characteristics of children with iNTS disease to the other children admitted, precluding a control group. Also, sampling of the affected health districts was mainly restricted to the year 2012, not allowing for comparisons with years before and after.Sampling during the surveillance period was however constant over time and directed by a set of clinical indications, and clear comparisons with other years could be made. We believe this was also the case for the hospital registers providing compiled data about malaria diagnosis and blood transfusions.Despite the limitations, this study offers valuable data on a neglected disease from an under investigated area collected during a time of great political instability. Previous articles have highlighted the need for studies that combine epidemiological, microbiological and clinical data on iNTS disease . Last, tSalmonella Typhimurium and Salmonella Enteritidis serovars. Whole genome sequencing of NTS isolates from different areas in the DRC, especially those related to local increases in infections, would provide valuable insights. It is obvious that continued blood culture surveillance is imperative to monitor the proportions and distributions of NTS serovars as well as their antibiotic resistance patterns. Last, efforts to control the increase in malaria in this region should be intensified.Future studies should estimate reliable population incidence rates. The development of an (antigen based) rapid diagnostic test would be highly welcome as a tool for diagnosis in low resource settings where microbiological facilities are often absent. More research is also needed on differences between the clinical presentation and outcome of both P. falciparum malaria. A sudden increase in hospital admissions among children < 5 years old, associated with a laboratory-confirmed diagnosis of P. falciparum malaria needs to raise the suspicion of a concomitant increase in NTS infections, particularly if associated with an increase in blood transfusions and case-fatality.The present observations confirm NTS as a leading cause of bloodstream infection in an area holoendemic for salmonella; IQR, interquartile range; ITM, Institute of Tropical Medicine; MDR, multidrug resistance; MLVA, multilocus variable-number tandem-repeat analysis; NTS, non-typhoidal Salmonella; RDT, rapid diagnostic test; TMP-SMX, trimethoprim-sulfamethoxazole(a)OR, (adjusted) Odd’s ratio; CLSI, National Committee for Clinical Laboratory Standards; CSO, clinically significant organisms; CUKIS, University Hospital of Kisangani; DCS, decreased ciprofloxacin susceptibility; DF, Dadi Falay; DRC, Democratic Republic of the Congo; HGR, General Referral Hospital; HZ, health zone; INRB, National Institute of Biomedical Research; iNTS, invasive non-typhoid