Bacterial etiologies of non-malaria febrile illnesses have significantly become important due to high mortality and morbidity, particularly in children. Despite their importance, there are few reports on the epidemiology of these diseases in Tanzania, and the true burden of such illnesses remains unknown. This study aimed to identify the prevalence of leptospirosis, brucellosis, typhoid fever and urinary tract infections and their rate of co-infections with malaria.A cross-sectional study was conducted at Kilosa district hospital in Tanzania for 6 months. Febrile children aged from 2–13 years were recruited from the outpatient department. Patients were screened by serological tests such as IgM and IgG ELISA, and microscopic agglutination test.B. abortus was identified among 26 (7.0%) of patients while B. melitensis was detected in 57 (15.4%) of the enrolled patients. Presumptive typhoid fever due to S. Typhi was identified in thirty eight (10.3%) of the participants and 69 (18.6%) had urinary tract infections. Patients presented with similar symptoms; therefore, the identification of these diseases could not be done based on clinical ground alone. Co-infections between malaria and bacterial febrile illnesses were observed in 146 patients (39.5%). Although antibacterials and/or anti-malarials were prescribed in most patients, some patients did not receive the appropriate treatment.A total of 370 patients were enrolled; of these 85 (23.0%) had malaria parasites, 43 (11.6%) had presumptive acute leptospirosis and 26/200 (13%) had confirmed leptospirosis. Presumptive acute brucellosis due to The study has underscored the importance of febrile bacterial diseases including zoonoses such as leptospirosis and brucellosis in febrile children, and thus such illnesses should be considered by clinicians in the differential diagnoses of febrile diseases. However, access to diagnostic tests for discrimination of febrile illnesses is needed. This would allow febrile patients to receive the correct diagnoses and facilitation of accurate and prompt treatment. B. abortus was identified in 26 (7.0%) and B. melitensis was among 57 (15.4%) of the enrolled patients. Presumptive typhoid fever was observed in 38 patients (10.3%), while 69 (18.6%) had urinary tract infections. Co-infections between bacterial diseases with malaria were observed and thus indicate the necessity of considering multiple infections in patients with malaria. These findings are of relevance to clinicians since they should consider bacterial causes of fever including zoonotic diseases in the routine diagnosis of febrile patients. Also, the findings provide evidence to policy makers and national disease control programs.Understanding the bacterial causes of fever, particularly in the era of reported decline of malaria, is very important. In Tanzania, data on the prevalence of these diseases in several regions is limited. These infections cannot be recognized clinically, and thus the lack of diagnostic tools in most health facilities leads to misdiagnosis and mistreatment of febrile patients. This cross-sectional study was carried out in children at Kilosa district hospital in Tanzania. Among 370 enrolled patients, 85 (23.0%) had malaria parasites, 43 (11.6%) had presumptive acute leptospirosis and 26/200 (13%) had confirmed leptospirosis. Presumptive acute brucellosis due to  Escherichia coli which accounts for 80% of the isolates [Streptococcus pneumonia [S. Typhi) and non-Typhi have been demonstrated as important causes of typhoid fever in Tanzanian children [In recent years, there has been a progressive decline in malaria transmission and morbidity which in line with a decline in proportions of fevers caused by malaria therefore many fever cases are likely to be non-malaria . Febrileisolates . With reneumonia . Salmonechildren . TyphoidLeptospira with several different serovars [Brucella, the most common are B. abortus and B. melitensis. Brucella abortus is mostly associated with cattle while B. melitensis is typically associated with sheep and goats. The infection is transmitted to humans by ingestion of infectious animal products such as milk, dairy products or meat, direct contact with infected animals and their products through skin abrasions or conjunctiva and inhalation of airborne particulates [Furthermore, bacterial zoonotic diseases such leptospirosis and brucellosis have also been reported . Leptospserovars . The disiculates .The diagnosis of bacterial febrile illnesses poses a challenge particularly in resource poor countries where laboratory diagnostic facilities are limited . Most ofThe study was conducted at Kilosa district hospital which is situated in Morogoro region, Tanzania . The disKilosa was selected due to its possession of intensive human activities with livestock and its proximity to wildlife from the Mikumi National Park . Therefoet al. (2006) the sample size (n) was calculated as n = (1.96)2 (0.36)(1–0.36)/(0.05)2 = 354; Total n = 354 + 17.7 = 372 [This study was conducted for 6 months, during rainy season (March—May 2013) and dry season (August—October 2013). The participants were recruited from febrile children who attended outpatient department at Kilosa district hospital. The inclusion criteria were children aged from 2–13 years, axillary temperature ≥37.5°C or rectal temperature ≥ 38.0°C at the time of recruitment. Fever was classified as mild if temperature was 37.5–38.3°C, moderate at 38.4–39.4°C and high at >39.5°C. Children with chronic diseases or those who required immediate attention were excluded. The estimation of the sample size for the assessment of prevalence of febrile illnesses in study area was based on 36%, which is a reported prevalence of malaria and other febrile febrile illnesses such as acute respiratory tract infections and UTIs . The ass.7 = 372 .A trained clinical officer from the hospital collected demographic information, obtained clinical history and performed physical examination for each enrolled participant. The clinical diagnoses and treatment were also performed by the respective clinician and were recorded on a standard assessment form. Blood specimen 5ml of venous blood was collected aseptically from each febrile patient in plain tubes and thereafter serum was separated by centrifugation. In addition urine sample was collected from each patient in sterile tubes and processed immediately. Serum samples were temporarily stored at—20°C at Kilosa district hospital before being transported to Ifakara Health Institute (IHI) where they were kept at–80°C until analyzed.Laboratory investigation was done based on selected bacterial febrile illnesses that might occur within the district. Thus, the selected bacterial febrile illnesses were leptospirosis, brucellosis, urinary tract infection and typhoid fever. In addition, all patients were screened for malaria parasites. Viral causes of fever were also investigated and detailed results have been presented in a separate paper.The presence of malaria parasites was checked by microscopy by a laboratory technician from the respective hospital. Thick and thin blood films were prepared and stained with Giemsa and examined for blood parasites. Each slide was read independently by a second experienced microscopist from IHI and any discrepancies were resolved by a third microscopist. The parasite density was determined by standard methods .Leptospira biflexa containing genus-specific epitopes directed against all Leptospira species. Each set of tests was run with positive and negative controls. The test was valid when the absorbance reading of the above meets the specification of the manufacturer’s and the results were interpreted according to the manufacturer’s recommendation. The sensitivity and specificity of these tests, as determined by the manufacturers, are shown in Leptospira serovars were used; this includes Kenya, Lora, Sokoine and Grippotyhosa  and other serovars were Leptospira reference strains i.e. Hardjo and Hebdomadis. Briefly, live Leptospira cell suspensions were incubated with serially diluted serum specimens in U- bottomed 96-well plates. Negative and positive controls for each serovar were included. The plates with serum–antigen mixture were incubated at 30°C for 2–4 hours before being examined for agglutination under dark field microscopy. To confirm the agglutination titer, serum samples that showed agglutination were further diluted to up to 1: 20,480. The reported titer was the highest dilution of serum that agglutinated at least 50% of the cells for each serovar tested [Leptospira while probable prior leptospirosis exposure was defined as a positive anti-Leptospira IgG ELISA result. Confirmed acute leptospirosis was defined as a positive MAT result.Laboratory diagnosis of leptospirosis was done by the detection of IgM and IgG antibodies in serum by using enzyme-linked immunosorbent assay (ELISA) at IHI, Ifakara, Tanzania. ELISA kits were obtained from Serion Immundiagnostica GmbH (Germany) and the ELISA procedure was carried out as per manufacturer’s instructions. The ELISA kits contain extract from r tested , 29, 30.Brucella IgM-ELISA and NovaLisa™ Brucella IgG-ELISA were used according to the manufacturer’s instructions to detect the presence of Brucella abortus-specific IgM and IgG antibodies in serum samples . The kits contained microtiter strips pre-coated with a lysate antigen of B. abortus. The recommended cutoff values were used to determine negative, positive and borderline results, however, all border line results were considered negative. The sensitivity and specificity of these tests, as determined by the manufacturers, are shown in Brucella suspension for detection of antibodies toward Brucella abortus and Brucella melitensis . The rapid screening test was done by adding one drop of a well-shaken antigen suspension (B. abortus or B. melitensis) on a slide containing 20 μl of undiluted serum. Serum samples which showed a visible agglutination within 1 minute were quantified by tube agglutination test. For the tube method, the serum samples were diluted serially from 1:20 to 1:1280 followed by the addition of a single drop of antigen into the respective tubes. A negative control  was also included. The tubes were incubated at 37°C for 16–20 hours. The positivity cut-off point for tube agglutination reaction was defined as antibody titers ≥ 1:160 [B. abortus and probable prior brucellosis exposure was defined as a positive anti-Brucella IgG ELISA result.NovaLisa™  ≥ 1:160 . PresumpSalmonella Typhi was done by a Widal test which was based on agglutination reaction between S. Typhi somatic lipopolysacharide O antigens and flagellar H antigens [The detection of antibodies toward antigens . This teantigens  and was Urine samples were examined visually and microscopically. Briefly, microscopic examination was performed by centrifugation of urine at 3000 rpm for 3 minutes and thereafter the supernatant was discarded aseptically. The sediment was placed on the slide and a cover slip was applied before being examined under the microscope at 40X objective. An average count of white blood cells (WBC) was taken per number of fields examined. The increased number of leucocytes is mostly observed in urinary tract diseases, and thus presence of ≥5 WBCs per hpf (high-power field) was considered positive .Data were entered into an Access database by an experienced data clerk. The database was designed to ascertain validation rules of each data field. The verification of data entry and data cleaning was done to ensure that clinical data and laboratory findings are matched. The cleaned data were then transferred to STATA using Stat transfer version 9 and statistical analyses were performed using STATA software . The outcome variables were bacterial febrile illnesses i.e. leptospirosis, brucellosis, urinary tract infection, typhoid fever and malaria. Pearson’s Chi-Square test was used to determine the association between categorical variables. An alpha level of 0.05 was used for all tests of statistical significance. In addition, logistic regression model for multivariate analysis was used to assess the relationship between the selected bacterial febrile illnesses and sex, age and season. Interpretation of results was based on the odds ratio and 95% confidence intervals.This study was approved by Institutional Review Board of Ifakara Health Institute (IHI/IRB/No: 01–2013) and Medical Research Coordinating Committee of Tanzania’s National Institute for Medical Research (NIMR/HQ/R.8a/Vol.1X/1472). Children with less than 12 years had written informed consent given from a parent or guardian while children over 12 years provided their own written informed consent which was accompanied by a written consent of a parent or guardian. In addition a verbal assent was also obtained from children aged 7–12 years.A total of 370 febrile children were enrolled, of these 189 (51.1%) were males and 181 (48.8%) were females. Among the enrolled patients, 205 (55.4%) were children aged less than five years and 165 (44.6%) were aged five years and above . One hunPlasmodium falciparum), of these, 45/189 (23.8%) were males and 40/181 (22.1%) were females. Of 85 patients who had malaria, 47/205 (22.9%) were children aged less than 5 years while 38/165 (23.0%) were aged five years and above  were positive for malaria parasites , Kenya 6 (23.1%), Gripotyphosa 6 (23.1), Hebdomadis 6 (23.1%) and Lora 2 (7.7%). However, 2 patients had two Leptospira serogroups; one had Sokoine and Hebdomadis and the other patient had Sokoine and Gripotyphosa. Contrary to presumptive acute leptospirosis, female patients were less likely confirmed with leptospirosis  as compared to males. Probable prior exposure to leptospirosis was observed in 16 (4.3%) of enrolled patients, of these, 10/189 (5.3%) were males and 6/181 (3.3%) were females and all cases occurred during the rainy season  met the definition for presumptive acute leptospirosis . Female y season . The maiy season . The mosy season .B. abortus, including 13/189 (6.9%) males and 13/181 (7.2%) females. Presumptive acute brucellosis was more common among older children (≥ 5 years) than in children aged less than five years . In addition, few cases occurred in dry season as compared to rainy season . The prevalence of B. abortus and B. melitensis by tube agglutination test was 26 (7.0%) and 57 (15.4%), respectively. Similarly, most cases of B. melitensis occurred in children aged ≥ 5 years  even though for B. abortus both age groups were equally affected  met the definition for probable prior brucellosis exposure including 13/189 (6.9%) males and 29/181 (16.0%) females. Among the patients with probable prior brucellosis exposure, 37 had only anti-Brucella IgM antibodies while 5 patients had both IgM and IgG. The prevalence of prior brucellosis, exposure was more in children aged above five years . Similarly, despite the absence of statistical significance, few cases were detected during dry season as compared to rainy season  of all participants met the criteria for presumptive acute brucellosis due to affected . Althougy season . The maineumonia . The comneumonia .Of 370 enrolled participants, 9 children could not provide urine sample. Therefore, among the 361 patients who were screened, 69 (18.6%) had UTIs. Urinary tract infections were three times higher in females than in males . The findings further show that both age groups were equally affected regardless of their age difference. The main diagnoses provided were malaria 11 (15.9%), pneumonia 16 (23.2%) and other diseases 4 (5.8%), . Twelve This study shows that 38 (10.3%) of enrolled patients had presumptive typhoid fever. Presumptive typhoid fever cases were significantly higher in females than in males . In addition, patients aged 5 years and above were represented with a high prevalence, 24 (14.6%) than children aged below 5 years, 14 (6.8%) . It was further noted that the prevalence of presumptive typhoid fever was stable between the rainy and dry season. Thirteen (34.2%) of patients suspected having typhoid fever received anti-malarial drugs while 31 patients (81.6%) were treated with antibiotics. The commonly used antibiotics were co-trimoxazole and amoxicillin .B melitensis  with typhoid fever and 6 (1.6%) with leptospirosis. Also, 11 (3.0%) of patients with typhoid fever had B. melitensis while 9 (2.4%) had presumptive typhoid fever and leptospirosis. Furthermore, twenty (5.4%) of participants could have had more than two diseases. This included malaria, UTIs and B. melitensis 4 (1.1%); typhoid fever, leptospirosis and B. melitensis 4 (1.1%); and Malaria, UTIs and typhoid fever 3 (0.8%), A total of 146 children (39.5%) had a possibility of being infected with more than one disease and of these, 126 (34.1%) could have had two diseases. Among patients diagnosed with malaria, 13 (3.5%) were suspected having acute leptospirosis, 7 (1.9%) had confirmed leptospirosis while 13 (3.5%) could also be infected with typhoid fever, 12 (3.2%) with UTIs, and 9 (2.4%) with litensis . Of the In the present study, we have demonstrated occurrence of non-malarial febrile illnesses due to bacterial infections in children in a malaria-endemic area of Tanzania. This study confirms the importance of some bacterial causes of fever including zoonotic diseases such as leptospirosis and brucellosis. The study also identified the presence multiple bacterial infections as well as co-infections between malaria and bacterial diseases. Furthermore, infected patients were characterized with similar symptoms and thus differential diagnosis could not be made in the absence of diagnostic tests.This study has illustrated that besides malaria, diseases such leptospirosis, brucellosis, typhoid fever and urinary tract infections should be considered in the differential diagnosis of febrile children. It is well documented that in acute leptospirosis or brucellosis, IgM antibodies start appearing during the first week of illness which is usually followed by IgG in the second week , 35, 36.Leptospires in urine. These findings are in line with the results obtained from previous studies which have shown a similar seasonal pattern [Brucella organisms among infected animals which increases environmental contamination with consequences of increasing risk of exposure to infection. In this study, confirmed leptospirosis was detected more often in male patients than in females, which is in agreement with the results of a study conducted in the Netherlands where 90% of reported leptospirosis cases occurred in male patients [Findings from the present study have indicated that leptospirosis and brucellosis occurred more during the rainy season than in the dry season. This could be due to the reason that the rains cause rodents to move to residential areas hence increasing contamination of the environment including surface water through shedding of  pattern , 41. Sim pattern . This ispatients . Kilosa patients , 45.B. melitensis occurred in children aged 5 years and above, similar to findings reported by Tanir et al. (2009) from Turkey and Majali and his colleague from Jordan [et al. (2012) documented highest rates of typhoid fever in both age groups i.e. children aged 5–9 years and 2–4 years old [Our results show that UTI cases were more frequent in females than in males. This could be due to differences in anatomical structure i.e. the shortness of the urethra in women with its close proximity to the anus makes it easier for bacteria to ascend in the urinary tract . Furtherm Jordan , 48. Them Jordan , 50. A pears old . Moreoveears old , 53, 54.Brucella IgG particularly in absence of IgM antibodies [The present findings revealed that some patients had previously been exposed to leptospirosis and brucellosis. It should be noted that several previous reports have indicated the possibility of chronic brucellosis based on detection of anti-tibodies , 56, 57.Our findings indicate the possibility of multiple infections in a considerable number of patients. This included coexistence of malaria and bacterial infections and occurrence of dual or triple bacterial infections. This finding can be compared to the results of a recent study conducted in Tanzania where multiple diagnoses were observed in two thirds of the children . This isDespite being endemic in many developing countries, diseases such as leptospirosis, brucellosis and typhoid fever remain unrecognized, under-diagnosed or under-reported. This was clearly shown by the present study where the main diagnoses were malaria, UTI, pneumonia and upper respiratory tract infections while leptospirosis, brucellosis and typhoid fever were not diagnosed clinically in any patient. Our results show that diarrhea, vomiting, loss of appetite, joint pain, abdominal pain and headache were the common manifestations in malaria patients as well as in patients with bacterial infections. In addition, these symptoms are consistent with several possible diagnoses including viral diseases such as dengue fever and yellow fever , 60. SimAnother finding of this study is the improper management of febrile patients. Patients received antibacterials or anti-malarials but not standard treatment for human brucellosis. In the treatment of human brucellosis, monotherapy and short-term antibiotic regimes are not considered . The mosThe present study has reported the prevalence of some bacterial febrile illnesses in a district hospital which serves as a referral for primary health care facilities in the district and hence provides a good representation of the general population in the district. However, detection of IgM or IgG antibodies in acute serum without considering the convalescent serum could mislead the interpretation of results. A limitation of using a single serum sample in the demonstration of IgM/IgG antibodies is the absence of antibodies in early stages of the infections or the persistence of antibodies from the previous exposure. Another limitation is that we did not conduct culture for typhoid fever and urinary tract infections. However, despite the design of the present study being cross-sectional and some diagnostic tests used were not 100% sensitive or specific, useful information has been gleaned from this study. This information may help the clinicians, policy makers and national disease control programs in planning for better ways for management of such illnesses.Findings from the present study indicate that besides malaria, bacterial diseases such as UTIs and bacterial zoonoses (leptospirosis and brucellosis) are a prominent cause of acute febrile illnesses in Kilosa district, Tanzania. Despite their importance, these diseases are passed unrecognized or are being underreported. Clinical diagnosis is not sufficient to discriminate such illnesses and thus laboratory confirmation is essential. Our findings underline the need to improve strategies for the diagnosis and management of febrile conditions particularly in resource-limited settings and also to refine disease burden estimates of bacterial and other common causes of febrile illnesses in children.S1 Checklist(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Tuberculosis incidence in resource poor countries remains high. We hypothesized that immune modulating co-infections such as helminths, malaria, and HIV increase susceptibility to latent tuberculosis infection (LTBI), thereby contributing to maintaining the tuberculosis epidemic.Adults with sputum-positive tuberculosis (index cases) and their eligible household contacts (HHCs) were recruited to a cohort study between May 2011 and January 2012. HHCs were investigated for helminths, malaria, and HIV at enrolment. HHCs were tested using the QuantiFERON-TB Gold In-Tube (QFN) assay at enrolment and six months later. Overnight whole blood culture supernatants from baseline QFN assays were analyzed for cytokine responses using an 11-plex Luminex assay. Associations between outcomes (LTBI or cytokine responses) and exposures (co-infections and other risk factors) were examined using multivariable logistic and linear regression models.We enrolled 101 index cases and 291 HHCs. Among HHCs, baseline prevalence of helminths was 9% (25/291), malaria 16% (47/291), HIV 6% (16/291), and LTBI 65% (179/277). Adjusting for other risk factors and household clustering, there was no association between LTBI and any co-infection at baseline or at six months: adjusted odds ratio ; p-value) at baseline for any helminth, 1.01 ; hookworm, 2.81 ; malaria, 1.06 ; HIV, 0.74 . HHCs with LTBI had elevated cytokine responses to tuberculosis antigens but co-infections had little effect on cytokine responses. Exploring other risk factors, Th1 cytokines among LTBI-positive HHCs with BCG scars were greatly reduced compared to those without scars: (adjusted geometric mean ratio) IFNγ 0.20 (0.09–0.42), <0.0001; IL-2 0.34 (0.20–0.59), <0.0001; and TNFα 0.36 (0.16–0.79), 0.01.We found no evidence that co-infections increase the risk of LTBI, or influence the cytokine response profile among those with LTBI. Prior BCG exposure may reduce Th1 cytokine responses in LTBI. In 2011, Uganda had an estimated annual TB incidence of 234 per 100 000 population and it ranked 19In the recent past, there has been a particular interest in whether infections endemic to the tropics modulate host immunity, resulting in increased susceptibility to TB and thereby maintaining the TB epidemic, especially in low income countries such as Uganda Mycobacterium tuberculosis (M.tb) is mainly cell mediated and particularly involves T-helper (Th) 1-type cytokines (interferon (IFN) γ, tumour necrosis factor (TNF) α, and interleukin-2 (IL-2)), and to some extent Th17 cytokines Protection against intracellular organisms such as Herpes simplex reactivation During the early erythrocytic stage of malaria infection, there is an increased production of Th1-type cytokines, which not only promotes parasite elimination, but also immunopathology, leading to the symptoms of malaria in-utero, and early in life, causing clinical or sub-clinical disease and thereafter establishing latency Cytomegalovirus infection, which is endemic in developing countries We carried out a longitudinal observational study to determine the association between helminths, malaria, CMV, or HIV and LTBI among household contacts exposed to sputum positive TB with the hypothesis that individuals who are infected with these pathogens, and are at the same time exposed to persons with active TB, would be particularly susceptible to acquiring M.tb infection.This was a cohort study with six months of follow up. Participants were recruited at Kitebi and Kisenyi health centers in Kampala city, Uganda from May 2011 to January 2012. The city is divided into five divisions each with a serving health center. Kitebi health center is located in the peri-urban Rubaga division while Kisenyi health center is found in Kampala central division which is a densely populated slum. Both health centers run out-patient tuberculosis clinics twice a week and offer tuberculosis testing and treatment.Newly diagnosed sputum smear positive patients above 18 years of age attending the clinics were screened for eligibility to participate in the study as index cases. Index cases starting or who had been on TB therapy for one month or less, with household contacts (HHCs), were consecutively included.All available members of the index case's household were screened. Members that had stayed and shared meals with the index case for more than two weeks before the index case was diagnosed with TB irrespective of age and gender were approached to participate in the study.The HHCs were divided into three major groups depending on their LTBI status and all were followed up for six months. Those that were HIV positive or below 5years of age received six months of isoniazid preventive therapy (IPT) according to the WHO guidelines Mansonella perstans parasitology , Hannover, Germany) test (QFN). Cut off for a positive QFN test is >0.35 IU/ml after subtracting the nil (unstimulated) IFNγ production from production in the antigen-stimulated tube, and >0.5 IU/ml after subtracting nil from production in the mitogen-stimulated tube Two hundred and twenty samples of QFN supernatants from the household contacts were analyzed for cytokine responses using an 11-analyte Bio-plex Pro cytokine assay  (Luminex) consisting of Th1 cytokines (detection limits) , IL-2 , TNFα), Th2 cytokines , IL-5 , IL-13 ), regulatory cytokine ), and Th17 cytokines , IL-17f , IL-21 , IL-22 ). These cytokines were measured from the nil (no antigen) and TB antigens ) tubes for each HHC.et al.,A sample size of 145 index cases was chosen. We assumed an average of two HHCs per index case; a prevalence of 10% for malaria, helminth, and HIV infections and 70% for CMV infection; a prevalence of 30% for LTBI in the HHCs without any co-infections; a design effect due to clustering within the households of 1.2; and a loss to follow up of 9%. This sample size would give 80% power to detect as statistically significant increase at the 5% level in LTBI from 30% to 60% among the HHCs with any co-infection using the formula of Fleiss JL The main outcome of this study was infection with latent tuberculosis defined as a positive result to the QFN test at baseline. Similar associations were explored for LTBI at the end of follow up, and for an additional outcome, QFN conversion between baseline and follow up. The four main exposure variables were co-infection with helminths, malaria, HIV and CMV.Other exposure factors assessed were characteristics of the household , of the index case , and of the HHCs . These were selected based on prior social and biological plausibility First unadjusted associations with LTBI were assessed by using cross tabulations and by fitting logistic regression models that took account of the clustering of household contacts using a robust approach. The Wald test was used to assess statistical significance. Then, a multivariable analysis was conducted using a stepwise backward elimination approach to exclude potential confounders, using a liberal p-value of 0.15 to ensure that no important confounder was omitted The Luminex data obtained was adjusted to exclude individual analyte values generated with bead counts of less than 20 as follows: Values above 1 pg/ml but below the lower detection limit (DL) for each cytokine were set to half the lower DL for that cytokine. Values above the upper DL were set to the upper DL. Negative values and values between 0 and 1 pg/ml were set to 1 pg/ml. Since the cytokine responses were skewed, the data were subjected to a logarithmic (to base 10) transformation.Graphs were generated to show cytokine distributions among the groups of interest in the spontaneous (no antigen) and stimulated (TB antigen) data. Despite the log transformation, the data on cytokine responses remained non-normal with a heavy-tailed distribution. We therefore fitted linear regression models to the log-transformed cytokine responses, but used bootstrap resampling methods to estimate the standard errors, in order to investigate associations between the cytokine responses and exposures to different co-infections and other risk factors The study was approved by both the Makerere University Ethical Review Board and Uganda National Council of Science and Technology. All the Index cases gave written informed consent to participate in the study, including permission allowing the team to visit their homes. All adult HHCs gave written informed consent to participate in the study. Parents, next of kin, caretakers or/guardians gave written informed consent on behalf of the minors or children to participate in the study. In addition, children between 10 and 17 years also gave written informed assent to participate in the study.The HHCs who were infected with helminths or malaria were treated with anti-helminthic or anti-malaria drugs, respectively at the health center, as promptly as possible.The study recruited 101 index cases and 291 HHCs who were followed up as detailed in The median age (interquartile range (IQR)) of the index cases was 28 years (23–35 years) and 57% (58/101) were male. Regarding employment, 38% (38/101) were unskilled labourers, 6% (6/101) skilled, 28% (29/101) self-employed and 28% (28/101) lacked employment.The median age (IQR) of the HHCs was 14 years (5–26 years) and 42% (123/291) were male.We assessed 101 households of which 60% (61/101) were one roomed houses. The median number of household members (IQR) was 3 (2–4). Fifty six percent (56/101) of the houses had poor ventilation, 34% (34/101) average ventilation, and 10% (10/101) good ventilation. Over 96% (96/101) used charcoal or firewood as a fuel for cooking. Regarding sanitation, 92% (93/101) had access to a pit latrine. These were being shared by a median (IQR) of 10 (6–15) people. Eighty three percent (84/101) had access to clean water from communal taps. It took an average (SD) of 38 (18) minutes for the household members to get to the study health centers with equal numbers using various transport means which included walking to the health centers (32%), using public motorcycles called boda bodas (32%), and using the public minibus taxis (34%).Among HHCs, the prevalence of helminths was 9% (25/291), malaria 16% (47/291), and HIV 6% (16/291). Of the 16 who were HIV positive, 3 were of known status and were on antiretroviral therapy . Four HHC had both helminths and malaria, 5 HHC had malaria and HIV, and no HHC had all the three infections.Trichuris trichiura (5 cases), Hymenolepis nana (3 cases), Schistosoma mansoni (3 cases), and Ascaris lumbricoides (1 case). There were two HHCs with dual helminth infections; one had both hookworm and Ascaris lumbricoides, and another had Hymenolepis nana and Schistosoma mansoni. None of the HHCs had Mansonella perstans. Over 97% of the HHCs were infected with CMV and therefore associations between this infection and LTBI could not be investigated in the analysis. The prevalence of LTBI (QFN positive) was 65% (179/277), and the rest of the analysis was based on this definition.Fifty nine HHCs provided all the three samples of stool required, 71 provided 2, and 115 provided only one sample. An individual was categorized as helminth infected if any species was detected in at least one of all stool samples available for each HHC. Results were hookworm (13 cases), There were 45 HHCs with missing information on helminth infections, and 21 HHCs with missing information on malaria status. We assessed whether there was any relationship between the missingness and our main outcome (LTBI) by testing for any association between the missing data and LTBI using a Chi-square test. We found no significant difference between the patients with complete and missing data.In the univariable analysis, co-infection with helminths, malaria or HIV was not associated with increased risk of LTBI  but factAfter adjusting for confounders, factors which remained associated with risk of LTBI were closer proximity of HHC to the index case, and HHCs that smoked. HHCs were less likely to have acquired LTBI if their index case was HIV positive .Immunosuppressive co-infections might impair the response in the QFN assay, as well as increasing susceptibility to LTBI and this might mask a true positive association between co-infection and LTBI. Treatment of co-infection with malaria or helminths was possible and we postulated that, if such masking had occurred, treatment might reveal a previously hidden association between co-infection during the period of exposure to the active case and LTBI. Therefore, having treated helminth and malaria co-infections at baseline, we investigated for associations between baseline co-infections and LTBI status at the end of six months of follow up. None of the co-infections was associated with LTBI status at 6 months .Some of the HHC, particularly the older ones, might have been exposed to, and infected with, M.tb before contact with this particular index case, in which case current co-infections, even if chronic, might not be relevant to their LTBI status at baseline. Therefore, the relationship between co-infections at baseline and QFN conversions during the six months of follow up were explored among 19 QFN converters and 52 individuals who remained QFN negative. Small numbers gave this analysis limited power, but there was no suggestion of an association with co-infection (aOR (95% CI) for helminths 0.29 (0.02–3.00), for malaria 0.17 (0.02–1.16) and for HIV 0.24 (0.003–19.2). In this model only male gender aOR 3.44 (95% CI 1.15–10.32) and daily duration of time with the index case greater than six hours 5.42 (1.38–21.3) were associated with QFN conversion.Control responses measured in the mitogen tube were high for all HHCs. Spontaneous cytokine production (measured in the no antigen tube) varied between cytokines, from geometric mean 1.11 pg/ml for IL-4 to 67.35 pg/ml for IFNγ, but did not differ between HHCs with or without LTBI, except for IFNγ . Spontaneous production was subtracted from the amount produced in the TB antigen stimulated tube for further analysis. By definition, HHCs with LTBI had a higher IFNγ response to TB antigens than uninfected contacts in the QFN assay. IFNγ responses to TB antigens measured by Luminex correlated strongly with IFNγ measured in the QFN assay  and so, as expected, IFNγ responses to the TB antigens measured by Luminex were higher in the HHCs with LTBI compared to those without LTBI (p = <0.0001) . SimilarSpontaneous cytokine production was similar among household contacts with and without co-infections except for those with malaria who had raised IL-17f  and IL-21  and those with HIV who had depressed concentrations of IL-10 .Co-infections and other factors could only be expected to influence cytokine responses to TB-antigens among individuals with LTBI, so further analysis was restricted to this subset.In general, co-infections showed little effect on net TB-antigen stimulated cytokine responses, except for increased IL-21 production in HHCs with helminths compared to those without helminths , decreased TNFα levels in malaria versus no malaria , and reduced TNFα production in HIV versus no HIV co-infection .We performed crude and adjusted analyses of the different cytokine responses among the HHCs with LTBI and did not find any association between the helminth co-infections and cytokine responses to TB antigens  but confThe multivariable analyses revealed other factors of interest that were associated with cytokine responses among LTBI infected HHCs. The presence of a BCG scar was associated with markedly low concentrations of all the Th1 cytokines IFNγ, IL-2, and TNFα (aGMR 0.20 (0.09–0.42), 0.34 (0.20–0.59), and 0.36 (0.16–0.79) respectively) but raised IL-10 levels (aGMR 1.23 (1.04–1.45)).Reduced Th2 cytokines were associated with household contacts with higher social-economic status (IL-5: aGMR 0.54(0.31–0.96), IL-13: 0.36(0.18–0.72)). These same cytokines, in the LTBI positive contacts, were also increased with increasing sputum positivity in the index case.In our study, co-infection with helminths, malaria or HIV was not associated with increased risk of LTBI. In keeping with previous studies, the risk of LTBI increased with closer intensity of exposure with the index case, with those HHCs who shared the same bedroom and bed with the index cases, or spent many hours per day with the index case, being at the most risk M. tb-specific antigens although we did not show any specific Th-type bias. Previous studies have found distinct Th1- type responses in LTBI compared to cases with TB disease et al., showed higher concentrations of IFNγ responses in contacts with LTBI compared to those without LTBI The HHCs with LTBI had elevated cytokine responses to When we assessed the effect of co-infections on cytokine responses in the HHCs who had LTBI, we found that malaria and HIV showed reduced cytokine responses across all cytokine families, with statistically significant associations for TNFα, in keeping with their known immune suppressive effects. However, helminth co-infection was not associated with any reduction in cytokine response. The reduced cytokine responses to TB antigens in HIV co-infection, and in particular IFNγ responses, concurs with previous studies We also found that having a BCG scar was associated with low Th1-type but high IL-10 (regulatory) cytokine responses among the HHCs with LTBI. This was an unexpected finding, and could have arisen by chance, but the consistency of the results between the three Th1 cytokines lends weight to the observation. Other studies have also shown a negative association between BCG scar and interferon gamma release assay (IGRAs) positivity, which has been taken to imply protection against LTBI et al., carried out in a different part of Kampala city, found that whereas BCG reduced the risk of tuberculosis infection as was shown by the reduced risk of TST conversions, the household contacts with a BCG scar who acquired LTBI (TST converters) had elevated IFNγ responses to mycobacterial antigen at baseline, compared to TST converters who did not have a BCG scar. However, Whalen and colleagues used M. tuberculosis culture filtrates for stimulation and the tuberculin skin test to diagnose LTBI M. tuberculosis infection. The observed high concentrations of IL-10 found in those with a BCG scar are consistent with findings which show that BCG induced macrophage or dendritic cell dependent production of IL-10 may prevent T-cell induced immunopathology By contrast, a household contact study by Whalen The prevalence of helminths and malaria was low and may have been underestimated, since about half the participants provided only a single sample In addition, the study used the QFN test for the diagnosis of LTBI. This test assumes that the body is able to mount an immune response against the TB antigens resulting in production of IFNγ. The co-infections investigated are associated with immune suppression, in particular HIV and malaria. Infection with HIV has a definite effect on the performance of the tuberculin skin test when used to diagnose LTBI It is plausible that the household contacts with these co-infections were unable to produce adequate amounts of IFNγ making it difficult to see an association with LTBI. This was addressed for helminths and malaria by repeat QFN testing after treatment of the co-infections, and the results did not change. Detailed information on antiretroviral therapy uptake in the HHCs with HIV infection was not available, but there was still no association between HIV and LTBI among the contacts at the end of follow up.We found no evidence that the co-infections in question  increase the risk of latent TB acquisition. We have also shown that BCG could be modulating the Th1 and regulatory immune response profile in LTBI. It is important to further explore the effect of BCG on potential clinical outcomes.


Yemen remains the country with the highest malaria transmission within the Arabian Peninsula and a source of imported cases to neighbouring countries.This study collected samples from individuals resident in a valley in Western Yemen as a baseline to examine infection prevalence for a future trial. As well as rapid diagnostic test (RDT) and microscopy, a filter paper blood spot was collected for molecular and serological analyses.2. Plasmodium falciparum infection prevalence was 12.4, 11.1 and 19.6 % by RDT, microscopy and polymerase chain reaction (PCR), respectively. RDT and microscopy did not detect 45 % of infections present, suggesting many infections were low-density. Infection prevalence and seroprevalence were highly heterogeneous between clusters, with evidence of higher exposure in clusters close to the wadi. The mean multiplicity of infection (MOI) was 2.3 and high heterozygosity and allelic richness were detected.Samples were collected from 2261 individuals from 12 clusters across a study area of approximately 100 kmThis highly diverse parasite population suggests a high degree of transmissibility and coupled with the substantial proportion of low-density infections, may pose challenges for malaria control and elimination efforts.The online version of this article (doi:10.1186/s12936-016-1249-y) contains supplementary material, which is available to authorized users. The Arabian Peninsula, comprising of Bahrain, Kuwait, Oman, Saudi Arabia, Yemen, Qatar and the United Arab Emirates, has made great strides towards malaria elimination over the past 50 years. Of the seven countries in the region, only Saudi Arabia and Yemen still experience ongoing local transmission . Whilst Yemen is one of the poorest and least developed countries in the Arabian Peninsula and the combination of political unrest and widespread resistance to chloroquine, which was the first-line anti-malarial drug until 2005, resulted in a resurgence of malaria during the 1990s, exacerbated by widespread flooding and unusual rain patterns due to the El Niño climate oscillation . SubsequPlasmodium falciparum and Plasmodium vivax antigens in order to estimate transmission intensity and reconstruct historical transmission patterns in the area. Seroconversion rate and antibody dynamics in a population have been shown to correlate with transmission intensity in a number of settings and can be particularly useful in areas of low transmission  and 17 P. malariae infections were found .PCR results were successfully obtained for 2181 samples. Of these, 19.6 % (95 % CI 17.3–21.9) (n = 430) were positive for P. falciparum PCR as the gold standard, RDT had a sensitivity of 45.8 % (41.0–50.6) and a specificity of 95.4 % (94.3–96.3) and P. falciparum microscopy had a sensitivity of 41.6 % (36.9–46.4) and a specificity of 97.1 % (96.3–97.9), although sensitivity for both diagnostic methods varied substantially by cluster, ranging from 0 % in cluster 1 to 78.3 % in cluster 4. In general, sensitivity was higher in clusters of higher prevalence, suggesting more of the infections were high density in these areas , 21.2 % (n = 471) and 31.8 % (n = 729) for MSP-119, AMA-1, or either antigen, respectively. As with the parasitological results, seroprevalence ranged substantially between clusters, with clusters 2 and 5 having the highest prevalence  whilst clusters 1 and 11 were low  , 2.2 % (n = 47) and 3.2 % (n = 75) for MSP-119, AMA-1, or either antigen respectively. None of the four P. vivax infections detected by PCR were antibody positive to either antigen. No children under 1 year of age were seropositive for any P. vivax antigens, with highest seroprevalence detected in 12–15 year olds (5.7 %). As with the other prevalence measures, seroprevalence to P. vivax antigens was highest in the clusters located closest to the wadi , compared to those located further away . Sixty-five percent (n = 49) of P. vivax serological positive samples were also positive for P. falciparum antibodies.Whilst P. falciparum antigen were modelled using a simple reversible catalytic model for clusters within 500 m of the wadi and for those further away. The seroconversion rate in the clusters on the wadi was double that of the ones further away  of the P. falciparum positive samples (N = 430). A total of 84 different msp2 alleles were successfully genotyped, ranging in size from 205 to 609 base-pairs. Of these, 62 % (n = 52) and 38 % (n = 32) belonged to the 3D7 and Fc27 families respectively . All P. falciparum prevalence and seroprevalence measures were strongly correlated with allelic richness  and expected heterozygosity . As previously described, expected heterozygosity was strongly correlated with allelic richness (ρ 0.97 p < 0.001). Interestingly, cluster MOI was not significantly associated with any other measures. Correlations are shown in Additional file At a cluster level, the parasite prevalence measures were strongly associated with Malaria remains a significant public health problem in Yemen. This study demonstrates the wide range of malaria infection prevalence and exposure in communities separated by short distances in a single valley in Western Yemen using a variety of metrics. Parasite prevalence was notably high in some clusters (up to 46 %), although heterogeneity was marked. A large proportion of sub-patent infections were detected, indicating that diagnosis using RDTs or microscopy would leave a significant number of infections untreated. Moreover, the highly diverse nature of the parasite population in this study area is suggestive of a high degree of transmissibility and may pose a barrier for malaria control efforts in the region.An. arabiensis [The marked heterogeneity of infection prevalence in this setting is surprising given the short distance between the clusters sampled i.e. prevalence between cluster 1 (2 %) and 4 (46 %) despite only being 2 km apart Fig. . Heterogabiensis  and one abiensis . In thatAnopheles mosquitoes are still present.The high proportion of sub-patent infections poses challenges for malaria control and elimination efforts. This study observed discordance between microscopy (which is the main diagnostic in Yemen) and RDT, as well as approximately 45 % of infections being missed by these methods. Whilst low parasite density infections are likely to be less infectious to mosquitoes , they stThe high diversity of the parasite population in this region was surprising although is consistent with other studies from Yemen , 29. PreIn this study, there was no association found between MOI and transmission intensity. Whilst many studies have shown that MOI is higher in areas of greater transmission intensity , 20, theSerological data indicates a significant difference in exposure to infection between children born 9 years previously compared with those born more recently, with older children experiencing approximately double the transmission rate. This effect is most apparent in the clusters located within 500 m of the wadi. This difference may be due to an age related behavioural pattern, for example if children over ten spend more time outdoors and/or in high-risk areas, as has been seen observed in South East Asia , 38 and Alternatively, this difference could be indicative of a fall in transmission 9 years previously and in this case enhanced efforts of the malaria control programme, initiated in 2002. However, the fact that samples were only collected from those <16 years means it is difficult to fully reconstruct historical transmission patterns using serological data and it is possible a different pattern would have been apparent if samples from all ages had been examined.P. vivax and P. malariae parasites using PCR, data suggest that P. vivax prevalence is extremely low with no infections detected by microscopy, in line with the national control programme estimates [P. vivax antigens was below 10 % in all clusters, with only a small number of children with high antibody responses to either P. vivax antigen. Several P. malariae infections were identified in the clusters that were tested. This had also been detected in previous molecular studies in Yemen [Plasmodium species has implications for diagnosis, with RDT and microscopy lacking sensitivity for species other than P. falciparum.Despite fewer samples being tested for stimates . Seroprein Yemen . The preAs well as being informative for transmission intensity levels, understanding the genetic diversity of parasites in Yemen may help elimination programmes in the region to accurately classify a case as imported or locally acquired. Such a diverse parasite population may pose challenges for the effectiveness of malaria vaccines, which need to target the most common allelic families present in a region . As has This study used a variety of metrics to identify extreme and relatively focal heterogeneity of malaria infection prevalence over small distances in the foothills of Yemen, including a high proportion of sub-patent infections. The parasite population was highly diverse, even in clusters of low prevalence, with transmission focused close to the wadi. Identifying risk factors for high prevalence, such as proximity to mosquito breeding sites or high proportions of the population travelling to high transmission areas, will enable intervention campaigns to target villages appropriately and may result in a more cost-effective approach to reduce transmission in Yemen.


A detailed understanding of the mechanisms underlying adverse birth outcomes, and the impact of maternal malaria infection on fetal neurodevelopment, may lead to biomarkers to identify at-risk pregnancies and novel therapeutic interventions to prevent these complications.Sixty percent of all pregnancies worldwide occur in malaria endemic regions. Pregnant women are at greater risk of malaria infection than their non-pregnant counterparts and have a higher risk of adverse birth outcomes including low birth weight resulting from intrauterine growth restriction and/or preterm birth. The complement system plays an essential role in placental and fetal development as well as the host innate immune response to malaria infection. Excessive or dysregulated complement activation has been associated with the pathobiology of severe malaria and with poor pregnancy outcomes, dependent and independent of infection. Here we review the role of complement in malaria and pregnancy and discuss its part in mediating altered placental angiogenesis, malaria-induced adverse birth outcomes, and disruptions to the  Plasmodium and includes five species that infect humans: Plasmodium falciparum, P. vivax, P. ovale, P. malaria, and P. knowlesi. Among these, P. falciparum causes the most severe disease and accounts for the majority of malaria-associated deaths , defined by the accumulation of parasitized erythrocytes (PEs) in the placental intervillous space and the infiltration of maternal monocytes/macrophages  variant, VAR2CSA, to the glycosaminoglycan chondroitin sulfate A (CSA) that is expressed on the syncytiotrophoblast lining of the intervillous space , macrophage inflammatory protein (MIP)-1α, and MIP-1β, that recruit other inflammatory mediators and initiate the inflammatory cascade . This loThe complement system is a crucial immune surveillance and innate defense pathway. It is composed of both soluble and membrane bound proteins that cooperate to function in host defense and inflammation. Normally, the complement system is maintained at a basal level of activation but can be further amplified through three major activation pathways: the classical pathway, the mannose-binding lectin (MBL) pathway, and the alternative pathway . The claThe complement system is also an important regulator of several developmental processes, and as such requires tight regulation to prevent excessive activation . RegulatThe complement system plays an essential role in healthy pregnancies  protectiExcessive or dysregulated activation of the complement system can overwhelm these regulatory and protective pathways. Multiple studies have reported an association between complement split products  or mutations in complement regulatory proteins and obstetric complications . An estiFigure 1). For example, IgG has been shown to be deposited on the surface of PEs and C-reactive protein has been shown to be elevated in malaria infection  and pathologic host responses .Optimizing placental vascular development and function and resulting changes in the neumonia . MaternaAngiogenic-modifying compounds are currently being investigated therapeutically for a number of diseases, including anti-angiogenic therapies for metastatic cancer, wound healing, and pathological vasculopathies and retinopathies . Historiin vivo data using the mouse model of cerebral malaria have shown potential utility . However, the high cost of anti-complement antibodies would limit its use in resource-constrained settings and therefore more affordable strategies need to be explored. For example, L-arginine is an essential amino acid in pregnancy and immediate precursor of nitric oxide (NO), an endogenous regulator of placental vascular development . Intervention strategies targeting the L-arginine-NO pathway are being discussed as potential therapeutics for preeclampsia and appear promising due to simple routes of administration, low cost, and a very low toxicity profile in pregnancy . Research has highlighted the pivotal role that the prenatal environment plays in postnatal neurodevelopment as well as later life cognition and behavior . While the specific role that the complement system plays in fetal neurodevelopment continues to be elucidated, this research has important implications for malaria as many exposed pregnancies do not result in a birth phenotype but may still be at risk of adverse neurodevelopmental outcomes.Research in our laboratory using a mouse model of PM, has observed impaired learning and memory, and increased depressive-like behavior in offspring born to malaria-infected dams. This correlated with reduced regional levels of major biogenic amines  in the frontal cortex, temporoparietal cortex, and the striatum. The behavioral phenotype and reduction in circulating levels of neurotransmitters was rescued by genetic and pharmacological blockade of signaling via the C5a-C5aR pathway . ImportaThe majority of all pregnancies worldwide are at risk of malaria infection . These pThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P. falciparum clinical episodes that overlapped the gametocyte hotspots was also identified. Gametocyte positivity was found to be increased in individuals who had been treated with chloroquine, as opposed to other drug treatment regimens, for a clinical P. falciparum episode up to 30 days previously. It seems likely the hotspots were generated by a vicious circle of ineffective treatment of clinical cases and concomitant gametocyte production in a sub-population characterized by an increased prevalence of all the identified risk factors. While rapid access to treatment with an effective anti-malarial can reduce the duration of gametocyte carriage and onward parasite transmission, localised hotspots represent a challenge to malaria control and eventual eradication.Malaria transmission intensity is highly heterogeneous even at a very small scale. Implementing targeted intervention in malaria transmission hotspots offers the potential to reduce the burden of disease both locally and in adjacent areas. Transmission of malaria parasites from man to mosquito requires the production of gametocyte stage parasites. Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (<15 years old) and associated with a clinical episode, blood groups B and O increased risk compared to groups A and AB. A hotspot of clinical  Plasmodium falciparum, the etiological agent of malignant tertian malaria, over the last decade have resulted in a 40–50% decrease in malaria mortality rates. In 2012, there were an estimated 207 million cases of malaria and 627 000 malaria deaths [Global efforts to control and eliminate a deaths , 4 is ca0 [It has long been recognised that there exists very fine scale spatial heterogeneity in the epidemiology of malaria, even at the scale of the within-village community –8. Major0 –13 and t0 .P. falciparum, sexual stage differentiation (gametocytogenesis) from asexual parasites occurs in the blood of the human host. Although all parasite infections produce gametocytes at some point during the infection, their frequency and density varies. In cases of clinical malaria, gametocyte carriage has been associated with a worsening blood environment for the parasite  [Transmission of malaria parasites from man to mosquito depends on the production of gametocyte sexual parasite stages in the human host that are subsequently taken up by a mosquito during a bloodmeal . For P. locytes) –18. Howelocytes) , 20, whelocytes) , 21–23.P. falciparum are more infectious to mosquitoes [We have previously shown that there is a significant human genetic contribution to gametocyte carriage in asymptomatic infections . The sicsquitoes . These fsquitoes . Here weBetween 1990 and 2008, a longitudinal study involving the inhabitants of the village of Dielmo, Senegal, was carried out to identify all episodes of fever. The study design included daily medical surveillance with systematic blood testing of individuals with fever and examination of 200 oil-immersion fields on a thick blood film for malaria parasites (about 0.5 μL of blood). In addition, monthly systematic blood slides were taken from participating individuals to assess the prevalence rate of asymptomatic infections. The village is situated in a Sudan-savannah region of central Senegal, on the marshy bank of a small permanent stream, where anopheline mosquitoes breed all year round , 29. Malad hoc committees of the Ministry of Health, the Pasteur Institute  and the Institut de Recherche pour le Développement .The malaria research program conducted in Dielmo village in Senegal has been ongoing since 1990 as described elsewhere , 29. TheEach individual was given a unique identification code and details of family ties, occupation, and precise place of residence were recorded on detailed maps of each household with the location of each bedroom. All households were visited daily, absenteeism recorded, and the presence of fever or other symptoms assessed. We systematically recorded body temperature at home three times a week (every second day) in children younger than 5 years, and in older children and adults in cases of suspected fever or fever-related symptoms. In cases of fever or other symptoms, blood testing was done at the dispensary by finger prick, and we provided detailed medical examination and specific treatment. Parasitologically confirmed clinical malaria episodes were treated according to national guidelines. From 1990 to 2008, four different drug regimens were implemented: Quinine from 1990 to 1994, Chloroquine from 1995 to 2003, Fansidar (sulfadoxine-pyrimethamine) from 2004 to mid-2006 and Artemisinin-based combination therapy  from mid-2006 to 2008.Parasite positivity was established as follows. Thick blood films were prepared and stained by 3% Giemsa stain. Blood films were examined under an oil immersion objective at x1000 magnification by the trained laboratory technicians and 200 thick film fields were examined to count the number of asexual and gametocyte parasite stages. Asexual parasite densities (per μL) were calculated by establishing the ratio of parasites to white blood cells and then multiplying the parasite count by 8,000, the average white blood cell count per μL of blood.Plasmodium spp. , occurrence of P. falciparum clinical episodes and maximum parasite density. Other variables were individual-dependent including sex, sickle cell trait and the alpha-globin deletion as previously characterized [The outcome variable of interest was whether a bloodslide taken at clinical presentation or collected within the framework of the systematic follow up contained gametocytes, regardless of the disease status or time since last drug treatment. Thus, we assess the prevalence of gametocytes irrespective of the cause. The duration of gametocyte carriage for a single infection in endemic settings can last for at least up to 30 days . To incrcterized . ABO bloIn a second analysis, to address the impact of time since previous drug treatment on gametocyte positivity, the data were not aggregated into trimester.2 distribution, were established. All variables having a P value ≤0.25 were tested in a multivariate analysis  using Genstat ver. 15 (VSN Ltd) to account for the non-independence of individuals because of family relationships and for repeated measures from the same individual . Correlaanalysis . Model sanalysis .For confirmation of the hotspots identified in SaTScan (see below), a GLMM http://www.satscan.org/) [P. falciparum positive blood slides. The program compares the occurrence of gametocyte positive slides over the number of slides read inside a randomly generated cluster circle compared to the rest of the population. The unit of analysis was the “room” within which the individual lived. Reliable knowledge of room location was available for 308 individuals in 117 rooms spread throughout the village (This was performed using Kulldorff's scan statistic in SaTScan (version 9.1.1) (an.org/) . A discr village . An infiWe used the 19-year longitudinal malaria dataset of the Dielmo village cohort, Senegal. From 1990 to 2008, there were 15,567 blood slides read for gametocytes, of which 13,099 were taken during a clinical presentation and 2,468 during a systematic follow-up. This amounted to 2,995 person-trimesters for which parasite positive blood slides were examined for the presence of gametocytes from 544 individuals . The variation in the proportion of blood slides that were positive for gametocytes is shown in P. malariae and P. ovale. By contrast, gametocyte carriage increased with the occurrence of a clinical P. falciparum episode during the trimester, the maximum P. falciparum and P. ovale parasite densities during the trimester and ABO blood group ; this was especially so for children ≤5 years of age . The occurrence of a clinical P. falciparum episode during the trimester  and ABO blood group   increased gametocyte carriage, whereas the Fansidar and ACT drug periods decreased carriage   and ACT drug periods (2006–8) and the number of previous infections with od group . MultivaP<0.001) .We identified two hotspots of gametocyte carriage: the first within a radius of 37m from 1996–2003  that encompassed 33 people in 9 rooms and the second within a radius of 82m from 1995–2002  encompassing 60 people in 23 rooms . These hvs. outside of the hotspots by fitting a GLMM as described in the Statistical analyses section. Both hotspots were confirmed: hotspot 1 ; hotspot 2 .To take into account any bias occurring through the repeated sampling of the same individual, we also analysed the increased risk of gametocyte carriage in individuals within P. falciparum positive bloodslides. Of the remaining 215 individuals for whom “room” information was available and who were thus outside of the hotspots, 170 had at least one P. falciparum positive bloodslide during the same time period. The characteristics, with respect to the identified risk factors, of the individuals with P. falciparum positive bloodslides within these hotspots compared to those outside within the time period can be seen in P. falciparum positive bloodslides occurred in individuals within the cluster who were younger (73% vs. 54% of positive bloodslides occurred in children ≤ 10 years old), who had more P. falciparum clinical episodes  and had more B and O blood groups (83% in hotspot vs 68% outside hotspot). Thus, within the hotspot, there was a higher frequency of P. falciparum positive slides occurring in any of the three risk factor groups. Indeed, although the risk associated with these factors was maintained in both areas ; the effect of ABO blood group tended towards but was not significantly different between sites .We combined the two hotspots to generate individuals within the high gametocyte density hotspot and those outside of the hotspot. Combined together, of the 93 individuals, only 81 had th areas , compariP. falciparum episodes, a cluster analysis of the number of P. falciparum clinical episodes was performed in SaTScan. A hotspot was observed with a radius of 82m from 1995–2003  that entirely overlapped the second gametocyte hotspot, encompassing 28 rooms and 73 people .The overlap of gametocyte positivity during the trimester and the occurrence of a clinical episode during the trimester prompted us to analyse their relationship. Thus, we analysed the entire unaggregated dataset, containing 15,567 vs. treatment >30days ago: In hotspot χ21 = 26.2, P<0.001: outside hotspot χ21 = 74.2, P<0.001). This was not the case for the other treatment regimens, when there was no difference between proportional gametocyte positivity within 30 days since treatment and at later time points .Twenty-six percent of parasite positive blood slides had been taken within 30 days of a treated clinical episode. To assess whether the gametocyte hotspot was a result of prior drug treatment, we compared the proportion of bloodslides that were gametocyte positive according to time since last treatment. As can be seen in 23 = 96.2, P<0.001; Drug Period χ23 = 190.4, P<0.001; ABO blood group χ23 = 16.6, P<0.001; clinical case of P. falciparum within the previous 90 days but excluding that within the previous 30 days χ21 = 6.71, P = 0.01).We excluded blood slides taken within 30 days since last treatment and tested for the effect of the risk factors identified in the original multivariate analysis. The same gametocyte risk was found in all the previously identified risk factors Click here for additional data file.S2 TableShown are the Akaike Information Criterion (AIC) of the model following removal of the variable leading to the largest decrease in AIC at each step Click here for additional data file.


Severe and fatal malaria are associated with dysregulated host inflammatory responses to infection. Chitinase 3-like 1 (CHI3L1) is a secreted glycoprotein implicated in regulating immune responses. Expression and function of CHI3L1 in malaria infection were investigated.Plasmodium falciparum malaria. CHI3L1 levels were compared in children with uncomplicated malaria , severe malarial anaemia  and cerebral malaria  using the Kruskall Wallis-test, and evaluated for utility in predicting fatal (n = 23) versus non-fatal (n = 80) outcomes in severe disease using the Mann Whitney U test, receiver operating characteristic curves, and combinatorial analysis. Co-culture of P. falciparum with human peripheral blood mononuclear cells and the Plasmodium berghei ANKA experimental model of cerebral malaria were used to examine the role of CHI3L1 in severe malaria.Plasma levels of CHI3L1 were quantified in a case–control study of Ugandan children presenting with versus UM (p < 0.001). Among severe malaria cases, CHI3L1 levels at presentation predicted subsequent death (area under receiver operating characteristic curve 0.84 [95% CI 0.76-0.92]) and in combination with other host biomarkers, predicted mortality with high sensitivity (100% [85.7-100]) and specificity (81.3% [71.3-88.3]). Plasmodium falciparum stimulated CHI3L1 production by human peripheral blood mononuclear cells in vitro. CHI3L1 was increased in plasma and brain tissue in experimental cerebral malaria, but targeted Chi3l1 deletion did not alter cytokine production or survival in this model.In children presenting with falciparum malaria, CHI3L1 levels were increased in SMA and CM These data suggest that plasma CHI3L1 measured at presentation correlates with malaria severity and predicts outcome in paediatric SMA and CM, but do not support a causal role for CHI3L1 in cerebral malaria pathobiology in the model tested. Plasmodium falciparum infection in African children. The two major severe malaria syndromes are cerebral malaria (CM), which presents as coma and/or seizures, and severe malarial anaemia (SMA). Case fatality rates for CM and SMA are 18% and 10%, respectively, despite optimal anti-malarial treatment ). CHI3L1 was combined with other biomarkers identified in a previous publication : angiopoP. falciparum infection: uncomplicated malaria , CM (n = 44), and SMA (n = 59) [To determine whether CHI3L1 correlates with disease severity in human malaria, plasma levels of CHI3L1 were compared among groups of Ugandan children presenting to hospital with (n = 59) . DemograNext, it was assessed whether CHI3L1 could prognosticate outcome among children with severe malaria, which could facilitate clinical triage and resource allocation. CHI3L1 levels were higher among children with CM and SMA who subsequently went on to die of their infection, compared to survivors in their respective groups . A cut-point of 179.1 ng/mL had high sensitivity (91.3%) but low specificity (67.5%) for predicting mortality Table . To imprP. falciparum-infected erythrocytes were co-cultured with human peripheral blood mononuclear cells (PBMCs). As a positive control, LPS stimulated CHI3L1 transcription  or anti-inflammatory (IL-10) cytokines  but poor specificity (<70%) for predicting mortality in children with severe malaria. Combining CHI3L1 with other host biomarkers improved predictive accuracy, which has important practical implications in resource-poor health care settings with limited availability of intensive care. Severe malaria patients at higher risk of death could be prioritized for referral and more intensive monitoring and supportive care – although further studies would be required to determine whether these measures could improve outcome. Moreover, in clinical trials of adjunctive therapies for severe malaria, a prognostic test could be used to stratify patients into risk categories, since treatment efficacy can vary depending on risk of death . While cProspective studies in multiple populations are required to confirm the findings in this report. The current study had some limitations, including small sample size and non-consecutive sampling, possibly introducing selection bias. Future studies should also evaluate whether common childhood co-infections associated with elevated CHI3L1, such as pneumonia and schistosomiasis ,45, affePlasmodium falciparum stimulated CHI3L1 production by human PBMCs in vitro. Activated monocytes were likely the main source of CHI3L1 in this system; while CHI3L1 is not expressed by freshly isolated monocytes, differentiation into macrophages – as might be triggered by interaction with P. falciparum – induces its transcription [P. berghei infection, such as neutrophils, which are activated during malaria infection [P. berghei infection in mice susceptible to ECM, potentially due to upregulated transcription in astrocytes [Increased plasma CHI3L1 in severe and fatal human malaria prompted examination of CHI3L1 in malaria models. cription . Additionfection ,51 but etrocytes ,52 or introcytes .Plasmodium falciparum components have been shown to induce these cytokines via activation of pattern recognition receptors such as TLRs [The precise pathways mediating CHI3L1 induction by malaria remain undefined. Pro-inflammatory cytokines such as TNF and IL-1β can stimulate CHI3L1 production ,22. Plas as TLRs -55. Nota as TLRs ,56, thou as TLRs , which p as TLRs . This suChi3l1 deletion on P. berghei infection of ECM-susceptible mice. It was initially hypothesized that CHI3L1 would decrease pathological TH1 inflammation in ECM, and that, similar to the Streptococcal pneumonia model, Chi3l1 deletion would worsen outcome. In fact, Chi3l1 deletion failed to alter measures of TH1 inflammation in ECM. This may be due to differential pathway engagement: CHI3L1 exerted its major effects in the pneumonia model by regulating the NRLP3 inflammasome [Chi3l1 deletion. CHI3L1 could theoretically promote pathological inflammatory responses by supporting survival of immune cells [However, in this study, there was no global effect of ammasome , while iammasome ,59. Altene cells  or activne cells . In the ne cells ,61, wherne cells  and cytone cells ,38,63. Mne cells .Finally, it may simply be technically challenging to detect worsened outcome in ECM-susceptible mice. There are scant examples in the literature of single gene alterations leading to earlier death in this model, and these have been associated with more rapid parasite replication . It may Chi3l1 did not alter key disease parameters or death in the P. berghei model of ECM, these findings do not exclude a role for CHI3L1 in human severe malaria because of model limitations and, potentially, the multiplicity of CHI3L1 functions.In Ugandan children with malaria, plasma levels of CHI3L1 were elevated in severe disease and further increased in those who subsequently died of infection. In combination with other host biomarkers, CHI3L1 holds promise as a prognostic biomarker in severe malaria infection; further studies are warranted to validate this finding. Although deletion of AUROCC: Area under receiver operating characteristic curve; CHI3L1: Chitinase 3-like 1; CM: Cerebral malaria; ECM: Experimental cerebral malaria; PBMC: Peripheral blood mononuclear cells; RBC: Red blood cell; ROC: Receiver operating characteristic; SMA: Severe malarial anaemia; TLR: Toll-like receptor; UM: Uncomplicated malaria.LKE, WCL, and KCK are named inventors on a patent owned by the University Health Network covering biomarkers for early determination of critical response to illness and treatment.LKE conceived of the study, carried out experiments, and drafted the manuscript. CP contributed to study design and in vitro and in vivo studies. ZL carried out in vivo studies. AD, CM, and CMC-G collected human plasma samples and critically revised the manuscript. CGL and JAE generated knockout mice and critically revised the manuscript. WCL and KCK conceived of the study and helped draft the manuscript. All authors read and approved the final manuscript.


Malaria still remains the leading cause of childhood morbidity and mortality in Uganda. Interventions like malaria vaccines which reduce the malaria burden are needed in malaria endemic communities. There is need to establish baseline characteristics in vaccine trial study sites. This study determined the following baseline malariometric indices: spleen rates, bed net use, malaria parasitaemia and malaria episodes in an inception cohort of children aged 12 – 60 months in Iganga district, Uganda.In a longitudinal cohort study, 748 children were enrolled with 397 in an active follow up arm and 351 in a passive arm. The children in the two arms were followed for 6 months to determine the incidence of malaria episodes.The overall baseline spleen rate was 8.2% (61/748) among the study participants. Of the households surveyed, about 36% reported using bed nets and almost 30% of the users had insecticide-treated nets. 274 (36.6%) of the study participants had a history of fever in the past 24 hrs at the time of the baseline survey. All participants had a peripheral blood smear for malaria parasites done at enrollment with 76.8% having the asexual form of malaria parasites. The malaria episodes per child per year were 1.5 and 0.79 in the active and passive follow up arms respectively.There is a high prevalence of malaria asexual parasitaemia in children below five years. The bed net usage still remains low among this population. These baseline malariometric indices have important implication for malaria control interventions. Malaria still remains one of the leading global health problems with more than half a million death per year in Africa, occurring in children below 5 years of age. MalariaIn 2009, the African Malaria Network Trust (AMANET) received a grant from the European and Developing Countries Clinical Trials Partnership (EDCTP), to strengthen capacity in selected African Research institutions in conducting malaria vaccine trials. Makerere University, College of Health Sciences, Uganda, was one of the beneficiaries of the sub-grants awarded by AMANET, but baseline malariometric indices for site characterization to aid in recruitment and follow up of the volunteers were not available. The objective of the current study was to describe the baseline malariometric indices including spleen rate, bed net use, in Iganga district, Uganda. Results from this study were used together with other data to proviPlasmodium falciparum  as the predominant malaria species. Malaria transmission occurs throughout the year but peaks are seen following maximal vector breeding after the rains: periods April to June and September to December. The annual entomologic inoculation rate (EIR) is not known, but is reported to be >500 infective bites/person/year in the neighboring district of Tororo[Our study area in Iganga district comprised of six villages located in the neighborhood of Iganga district hospital. Iganga district is predominantly rural with one government hospital, 10 smaller governmental health facilities, three non-government facilities, 122 drug shops and clinics. Iganga hospital is located about 120 kilometers east of Kampala, the capital city of Uganda. The total population of Mayuge/Iganga is about 70,000 with the under five population being 11,000. Like elsewhere in Uganda, malaria is one of the leading health problems. The climate in Iganga is equatorial, with f Tororo.Anopheles gambiae sensu strictu and their analysis for resistance markers is still ongoing.The study site is a typical rural area, primarily residential, characterized by a high population density with low-income single or two-roomed housing units. The local economy is predominantly dependent on petty commercial activities and small-scale farming. The principal mosquito vector is A representative cohort of 748 children aged 1–5 years participated in a six month longitudinal study to determine the malaria episodes, the normal reference hematological and biochemical ranges which have been reported elsewhere and alsoOver a two month period, teams of four study personnel systematically covered the entire area of the six villages on foot to identify and enumerate all households with help of the local council official of that village. A household was defined as any single permanent or semi-permanent structure acting as the primary residence for a person or group of people. The enrolled households were assigned sequential unique numbers, written on a visible label fixed above the doorway, and residents were given a household identification card. After enumeration, study personnel asked the household head or guardian from each household for verbal consent to participate in a brief demographic survey. Using a standardized questionnaire, demographics and malaria indicator information was collected. If no adult resident was available, that household was excluded from the survey.Household level recruitment was for one eligible child from each household. If a household had more than one eligible child then a raffle was conducted to enroll only one child. From January 2010 to February 2010, experienced home visitors approached households to systematically recruit from the previously generated list. Using handheld GPS receivers they recorded households’ coordinates and a brief description of the study was provided in the appropriate language to parents and/or guardians.Interested potential participants were screened sequentially until the desired sample size was achieved. Initial assessment of eligibility was conducted using a selection criteria of; 1) age one to five years; 2) agreement to come to study clinic for any febrile episode or illness; 3) agreement to avoid medications administered outside the study; 4) agreement to remain in study area during the six months follow up; 5) absence of known chronic disease and 6) written informed consent provided by parent or guardian. Children who passed initial screening were assigned study numbers and underwent a history and physical examination including temperature, height weight measurement and abdominal examination. Severely malnourished children, were exChildren in the passive arm were visited every month by trained field workers to document absences from the study site and find out how they had been in the past four weeks. Parents/guardians were encouraged to bring their child to the malaria clinic if the child was unwell.Children in the active follow up arm were visited twice a week by field workers. A standardized questionnaire was administered for collecting information regarding illnesses that had occurred since the last visit, use of health care facilities and medications used. At each visit, the tympanic temperature was recorded using a digital thermometer. When fever (tympanic temperature of ≥38°C) or history of recent fever (within the last 24 hours) was observed or reported for any study child, a rapid diagnostic test  and microscopy of a stained blood smear were used to screen for malaria and to confirm the presence of malaria parasites respectively. Children found with any peripheral asexual malaria parasitaemia in their blood were treated at the study clinic accordingly following the Uganda national treatment guidelines.Thick blood smears were stained with 2% Giemsa for 30 minutes. Parasite density was estimated by counting the number of asexual parasites per 500 white-blood cells and calculating parasites per μL, using the actual white blood cell count of the subject. A smear was judged to be negative if no parasites were seen after examination of 100 high-powered fields. A second microscopist, who was unaware of the results of the first reading, re-read all slides. A third reviewer resolved discordant results. The presence of sexual forms of the parasites was also determined during slide readings.Open Clinica (https://www.openclinica.com) database. Double entry verification from paper questionnaires was performed to assure accuracy. Categorical variables were compared using the chi-square test and continuous variables using a two-sample t-test. A p-value <0.05 (two-tailed) was considered statistically significant. Analysis was done using SPSS version 10.0 .Baseline data were collected in the field on written forms, verified, checked by investigators and entered into the Written informed consent was obtained from the parent/guardian(s) of children for their participation in the cohort study. The study was approved by Makerere University School of Medicine, Research and Ethics Committee 2009/172 and by the Ugandan National Council of Science and Technology (records no. HS 765).A total of 1161 households were screened, of which 748 were enrolled, 374 (32.2%) households had no children aged less than 5 years, while 39 (3.4%) declined to consent for the study.Table Among the study population, the overall bed net usage among the two arms was reported at 36.4% (273/748) of whom 82.8% (226/273) reported that they were using insecticide treated nets. Overall, 64% (475/748) of the study participants reported that they did not own bed nets or had not used one during the preceding night before the baseline survey. However, there was no significant difference in bed net usage or ownership between the active and passive follow up groups (P = 0.777).P. falciparum malaria parasites in their peripheral blood smears at baseline screening during recruitment  had a dual infection with both P. falciparum and P. vivax. While the median asexual parasite density  reported history of fever in the past 24 hours before the baseline survey was conducted. Overall 61% (457/748) of the study subjects had a positive screening RDT test at enrolment. Fifty eight percent (265/457) of those with a positive RDT were from the active arm. However, out of the 274 subjects with history of fever in the preceding 24 hours, only 66.4% (182/274) had a positive screening RDT malaria test at enrolment. And, the thick blood smears showed that 76.8% (575/748) of the study participants had asexual Total malaria cases for the 6 months follow-up period (March – August 2010) were 892 among the study participants Table . The malLongitudinal studies in well-defined cohorts are critical to improving our understanding of the malaria incidence and associated factors before interventions can be introduced. In the current study, a representative cohort of children from a rural setting where malaria is highly endemic was enrolled.The overall prevalence of malaria parasitaemia at baseline in the study population was 76.8%, this suggests that Iganga is a holoendemic area. This corroborates the general observation that the under fives are more vulnerable to the disease in areas of high transmission. HoweverP. falciparum was the predominant species of malaria parasite identified in this study; with majority 99% of the children having a Plasmodium falciparum mono-infection. The high prevalence of malaria and P. falciparum mono infection noted in this study is an indication of a worsening malaria situation around the study area which might be indicative of poor access or adherence to effective control measures such as the use of insecticide treated nets (ITNs). This is similar to earlier findings reported from other study sites within the region[However, the prevalence of those with symptoms of fever was 36.6% at the baseline survey. This further highlights the fact that some children may have asymptomatic malaria. e region. Only 36e region. This stThe overall spleen rate in this study is 8.2%. This supports the fact that children aged less than five years have a more pronounced immunological response (including splenic enlargement) to malaria. HoweverThe episodes of malaria in the active follow up arm was twice that of the passive arm during the period of observation. This corroborates other reports which have used similar methodology for malariometric indices. This miThis study shows that malaria remains a major health problem among children aged less than five residing in Iganga district, eastern Uganda. The bed net usage still remains low among this population. This baseline study provides the important malariometric data required for vaccine intervention studies to determine their efficacy in our setting.