Maternal and infant predictors of proinflammatory milk immune activity in Kilimanjaro, Tanzania.

OBJECTIVES: The immune system of milk (ISOM) creates a mother-infant immune axis that plays an important role in protecting infants against infectious disease (ID). Tradeoffs in the immune system suggest the potential for both protection and harm, so we conceive of two dimensions via which the ISOM impacts infants: promotion of protective activity and control of activity directed at benign targets. High variability in ISOM activity across mother-infant dyads suggests investment the ISOM may have evolved to be sensitive to maternal and/or infant characteristics. We assessed predictors of appropriate and misdirected proinflammatory ISOM activity in an environment of high ID risk, testing predictions drawn from life history theory and other evolutionary perspectives. METHODS: We characterized milk in vitro interleukin-6 (IL-6) responses to Salmonella enterica (a target of protective immune activity; N = 96) and Escherichia coli (a benign target; N = 85) among mother-infant dyads in rural Kilimanjaro, Tanzania. We used ordered logistic regression and mixture models to evaluate maternal and infant characteristics as predictors of IL-6 responses. RESULTS: In all models, IL-6 responses to S. enterica increased with maternal age and decreased with gravidity. In mixture models, IL-6 responses to E. coli declined with maternal age and increased with gravidity. No other considered variables were consistently associated with IL-6 responses. CONCLUSIONS: The ISOM's capacities for appropriate proinflammatory activity and control of misdirected proinflammatory activity increases with maternal age and decreases with gravidity. These findings are consistent with the hypothesis that the mother-infant immune axis has evolved to respond to maternal life history characteristics.

Does the immune system of milk increase activity for infants experiencing infectious disease episodes in Kilimanjaro, Tanzania?

INTRODUCTION: Multiple studies have reported that milk immune content increases for infants experiencing infectious disease (ID) episodes, suggesting that the immune system of milk (ISOM) offers enhanced protection when needed to combat ID. METHODS: To test the hypothesis that ISOM content and/or activity increases during an infant's ID episode, we characterized milk secretory immunoglobulin A (sIgA; a major ISOM constituent) and in vitro interleukin-6 (IL-6) responses to Salmonella enterica and Escherichia coli, as system-level biomarkers of ISOM activity, in a prospective study among 96 mother-infant dyads in Kilimanjaro, Tanzania. RESULTS: After control for covariates, no milk immune variables (sIgA, Coef: 0.03; 95% CI -0.25, 0.32; in vitro IL-6 response to S. enterica, Coef: 0.23; 95% CI: -0.67, 1.13; IL-6 response to E. coli, Coef: -0.11; 95% CI: -0.98, 0.77) were associated with prevalent ID (diagnosed at the initial participation visit). Among infants experiencing an incident ID (diagnosed subsequent to the initial participation), milk immune content and responses were not substantially higher or lower than the initial visit (sIgA, N: 61; p: 0.788; IL-6 response to S. enterica, N: 56; p: 0.896; IL-6 response to E. coli, N: 36; p: 0.683); this was unchanged by exclusion of infants with ID at the time of initial participation. CONCLUSION: These findings are not consistent with the hypothesis that milk delivers enhanced immune protection when infants experience ID. In environments with a high burden of ID, dynamism may be less valuable to maternal reproductive success than stability in the ISOM.

Evaluation of HIV viral load turnaround time in Moshi, Tanzania.

INTRODUCTION: Viral load measurement is an important gold standard for monitoring anti-retroviral treatment among people living with human immunodeficiency virus. The optimal use of the viral load results for guiding antiretroviral therapy depends on timely availability of the results at the clinic. The objective of the current study was to evaluate the turnaround time and utilization of viral load results in the clinical decision process. METHODOLOGY: This was a retrospective cohort study which involved patients receiving cART from 1 August 2018 to 31 January 2017 at three clinics in Tanzania. Data was extracted from patient files at the clinics and relevant records were kept at the viral load determining laboratory. The data were analysed with the Statistical Package for Social Sciences version 20. RESULTS: 445 subjects had a viral load in test results and 88% had a viral load of > 1,000 copies/mL. The median duration on the current regimen was five years. Median time between the clinics receiving the results and communicating them to the patients was 40 days. Shorter turnaround time was observed for patients with virological failure (p = 0.003). A higher prevalence of virological failure was found in patients monitored at the Kilimanjaro Christian Medical Centre (KCMC) compared to the two primary health clinics (p = 0.04). CONCLUSIONS: The median viral load turnaround time was longer than stipulated by the national Tanzanian guidelines. Interventions that may reduce viral load turn-around-time, including point of care viral load testing, are needed to optimise monitoring of anti-retroviral therapy.

Burden, Clinical Characteristics, Risk Factors, and Seasonality of Adenovirus 40/41 Diarrhea in Children in Eight Low-Resource Settings.

Background: The application of molecular diagnostics has identified enteric group adenovirus serotypes 40 and 41 as important causes of diarrhea in children. However, many aspects of the epidemiology of adenovirus 40/41 diarrhea have not been described. Methods: We used data from the 8-site Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project birth cohort study to describe site- and age-specific incidence, risk factors, clinical characteristics, and seasonality. Results: The incidence of adenovirus 40/41 diarrhea was substantially higher by quantitative polymerase chain reaction than enzyme immunoassay and peaked at ∼30 episodes per 100 child-years in children aged 7-15 months, with substantial variation in incidence between sites. A significant burden was also seen in children 0-6 months of age, higher than other viral etiologies with the exception of rotavirus. Children with adenovirus 40/41 diarrhea were more likely to have a fever than children with norovirus, sapovirus, and astrovirus (adjusted odds ratio [aOR], 1.62; 95% CI, 1.16-2.26) but less likely than children with rotavirus (aOR, 0.66; 95% CI, 0.49-0.91). Exclusive breastfeeding was strongly protective against adenovirus 40/41 diarrhea (hazard ratio, 0.64; 95% CI, 0.48-0.85), but no other risk factors were identified. The seasonality of adenovirus 40/41 diarrhea varied substantially between sites and did not have clear associations with seasonal variations in temperature or rainfall. Conclusions: This study supports the situation of adenovirus 40/41 as a pathogen of substantial importance, especially in infants. Fever was a distinguishing characteristic in comparison to other nonrotavirus viral etiologies, and promotion of exclusive breastfeeding may reduce the high observed burden in the first 6 months of life.

Tradeoffs in milk immunity affect infant infectious disease risk.

Background and objectives: The human immune system has evolved to balance protection against infection with control of immune-mediated damage and tolerance of commensal microbes. Such tradeoffs between protection and harm almost certainly extend to the immune system of milk. Methodology: Among breastfeeding mother-infant dyads in Kilimanjaro, Tanzania, we characterized in vitro proinflammatory milk immune responses to Salmonella enterica (an infectious agent) and Escherichia coli (a benign target) as the increase in interleukin-6 after 24 h of incubation with each bacterium. We characterized incident infectious diseases among infants through passive monitoring. We used Cox proportional hazards models to describe associations between milk immune activity and infant infectious disease. Results: Among infants, risk for respiratory infections declined with increasing milk in vitro proinflammatory response to S. enterica (hazard ratio [HR]: 0.68; 95% confidence interval [CI]: 0.54, 0.86; P: 0.001), while risk for gastrointestinal infections increased with increasing milk in vitro proinflammatory response to E. coli (HR: 1.44; 95% CI: 1.05, 1.99; P: 0.022). Milk proinflammatory responses to S. enterica and E. coli were positively correlated (Spearman's rho: 0.60; P: 0.000). Conclusions and implications: These findings demonstrate a tradeoff in milk immune activity: the benefits of appropriate proinflammatory activity come at the hazard of misdirected proinflammatory activity. This tradeoff is likely to affect infant health in complex ways, depending on prevailing infectious disease conditions. How mother-infant dyads optimize proinflammatory milk immune activity should be a central question in future ecological-evolutionary studies of the immune system of milk.

Population genomics of Bacillus anthracis from an anthrax hyperendemic area reveals transmission processes across spatial scales and unexpected within-host diversity.

Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogen's evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide SNPs among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over 1 year throughout the Ngorongoro Conservation Area, Tanzania, a region hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes (i.e. SNP profiles) within the study area. However, phylogeographical structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis, with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence, as well as co-infection.

Missing and decayed teeth, oral hygiene and dental staining in relation to esophageal cancer risk: ESCCAPE case-control study in Kilimanjaro, Tanzania.

In the African esophageal squamous cell carcinoma (ESCC) corridor, recent work from Kenya found increased ESCC risk associated with poor oral health, including an ill-understood association with dental fluorosis. We examined these associations in a Tanzanian study, which included examination of potential biases influencing the latter association. This age and sex frequency-matched case-control study included 310 ESCC cases and 313 hospital visitor/patient controls. Exposures included self-reported oral hygiene and nondental observer assessed decayed+missing+filled tooth count (DMFT index) and the Thylstrup-Fejerskov dental fluorosis index (TFI). Blind to this nondental observer TFI, a dentist independently assessed fluorosis on photographs of 75 participants. Odds ratios (ORs) are adjusted for demographic factors, alcohol and tobacco. ESCC risk was associated with using a chewed stick to brush teeth (OR 2.3 [95% CI: 1.3-4.1]), using charcoal to whiten teeth (OR 2.13 [95% CI: 1.3, 4.1]) and linearly with the DMFT index (OR 3.3 95% CI: [1.8, 6.0] for ≥10 vs 0). Nondental observer-assessed fluorosis was strongly associated with ESCC risk (OR 13.5 [95% CI: 5.7-31.9] for TFI 5+ v 0). However, the professional dentist's assessment indicated that only 43% (10/23) of participants assessed as TFI 5+ actually had fluorosis. In summary, using oral charcoal, brushing with a chewed stick and missing/decayed teeth may be risk factors for ESCC in Tanzania, for which dose-response and mechanistic research is needed. Links of ESCC with "dental fluorosis" suffered from severe exposure misclassification, rendering it impossible to disentangle any effects of fluorosis, extrinsic staining or reverse causality.

Duration of Postdiarrheal Enteric Pathogen Carriage in Young Children in Low-resource Settings.

BACKGROUND: Prolonged enteropathogen shedding after diarrhea complicates the identification of etiology in subsequent episodes and is an important driver of pathogen transmission. A standardized approach has not been applied to estimate the duration of shedding for a wide range of pathogens. METHODS: We used a multisite birth cohort of children 0-24 months of age from whom diarrheal and monthly nondiarrheal stools were previously tested by quantitative polymerase chain reaction for 29 enteropathogens. We modeled the probability of detection of the etiologic pathogen before and after diarrhea using a log-normal accelerated failure time survival model and estimated the median duration of pathogen carriage as well as differences in subclinical pathogen carriage 60 days after diarrhea onset in comparison to a prediarrhea baseline. RESULTS: We analyzed 3247 etiologic episodes of diarrhea for the 9 pathogens with the highest attributable burdens of diarrhea. The median duration of postdiarrheal carriage varied widely by pathogen, from about 1 week for rotavirus (median, 8.1 days [95% confidence interval {CI}, 6.2-9.6]) to >1 month for Cryptosporidium (39.5 days [95% CI, 30.6-49.0]). The largest increases in subclinical pathogen carriage before and after diarrhea were seen for Cryptosporidium (prevalence difference between 30 days prior and 60 days after diarrhea onset, 0.30 [95% CI, .23-.39]) and Shigella (prevalence difference, 0.21 [95% CI, .16-.27]). CONCLUSIONS: Postdiarrheal shedding was widely variable between pathogens, with strikingly prolonged shedding seen for Cryptosporidium and Shigella. Targeted antimicrobial therapy and vaccination for these pathogens may have a relatively large impact on transmission.

Patterns of acquired HIV-1 drug resistance mutations and predictors of virological failure in Moshi, Northern Tanzania.

Emergence of HIV drug resistance poses a serious risk of inactivity to all currently approved antiretroviral drugs. Profiles of HIV drug resistance mutations (HIVDRM) and virological failure (VF) are not extensively studied in Tanzania. This study aimed to determine HIVDRM and predictors of VF in HIV-infected individuals failing first-line HIV drugs in Moshi, Northern Tanzania. A case-control study was conducted at Kilimanjaro Christian Medical Centre, Mawenzi, Pasua and Majengo health facilities with HIV-care and treatment clinics from October, 2017 to August, 2018. Cases and controls were HIV-infected individuals with VF and viral suppression (VS) respectively. HIV-1 reverse transcriptase and protease genes were amplified and sequenced. Stanford University's HIV drug resistance database and REGA subtyping tool 3.0 determined HIVDRM and HIV-1 subtypes respectively. Odds ratios (OR) with 95% confidence interval (95% CI) investigated predictors of VF. P-value < 5% was considered statistically significant. A total of 124 participants were recruited, of whom 63 (50.8%) had VF, 61 (49.2%) had VS and 82 (66.1%) were females. Median [IQR] age and duration on ART were 45 [35-52] years and 72 [48-104] months respectively. Twenty-five out of 26 selected samples from cases were successfully sequenced. Twenty-four samples (96%) had at least one major mutation conferring resistance to HIV drugs, with non-nucleoside analogue reverse transcriptase inhibitor (NNRTI)-resistance associated mutations as the majority (92%). Frequent NNRTI-resistance associated mutations were K103N (n = 11), V106M (n = 5) and G190A (n = 5). Prevalent nucleoside analogue reverse transcriptase inhibitors-resistance associated mutations were M184V (n = 17), K70R (n = 7) and D67N (n = 6). Dual-class resistance was observed in 16 (64%) samples. Thirteen samples (52%) had at least one thymidine analogue-resistance associated mutation (TAM). Three samples (12%) had T69D mutation with at least 1 TAM. Two samples (8%) had at least one mutation associated with protease inhibitor resistance. Age [aOR = 0.94, 95% CI (0.90-0.97), p < 0.001] and occupation [aOR = 0.35, 95% CI (0.12-1.04), p = 0.059] associated with VF. In conclusion, HIV drug resistance is common among people failing antiretroviral therapy. Resistance testing will help to guide switching of HIV drugs.

Practical and effective diagnosis of animal anthrax in endemic low-resource settings.

Anthrax threatens human and animal health, and people's livelihoods in many rural communities in Africa and Asia. In these areas, anthrax surveillance is challenged by a lack of tools for on-site detection. Furthermore, cultural practices and infrastructure may affect sample availability and quality. Practical yet accurate diagnostic solutions are greatly needed to quantify anthrax impacts. We validated microscopic and molecular methods for the detection of Bacillus anthracis in field-collected blood smears and identified alternative samples suitable for anthrax confirmation in the absence of blood smears. We investigated livestock mortalities suspected to be caused by anthrax in northern Tanzania. Field-prepared blood smears (n = 152) were tested by microscopy using four staining techniques as well as polymerase chain reaction (PCR) followed by Bayesian latent class analysis. Median sensitivity (91%, CI 95% [84-96%]) and specificity (99%, CI 95% [96-100%]) of microscopy using azure B were comparable to those of the recommended standard, polychrome methylene blue, PMB (92%, CI 95% [84-97%] and 98%, CI 95% [95-100%], respectively), but azure B is more available and convenient. Other commonly-used stains performed poorly. Blood smears could be obtained for <50% of suspected anthrax cases due to local customs and conditions. However, PCR on DNA extracts from skin, which was almost always available, had high sensitivity and specificity (95%, CI 95% [90-98%] and 95%, CI 95% [87-99%], respectively), even after extended storage at ambient temperature. Azure B microscopy represents an accurate diagnostic test for animal anthrax that can be performed with basic laboratory infrastructure and in the field. When blood smears are unavailable, PCR using skin tissues provides a valuable alternative for confirmation. Our findings lead to a practical diagnostic approach for anthrax in low-resource settings that can support surveillance and control efforts for anthrax-endemic countries globally.

Epidemiology of Shigella infections and diarrhea in the first two years of life using culture-independent diagnostics in 8 low-resource settings.

Culture-independent diagnostics have revealed a larger burden of Shigella among children in low-resource settings than previously recognized. We further characterized the epidemiology of Shigella in the first two years of life in a multisite birth cohort. We tested 41,405 diarrheal and monthly non-diarrheal stools from 1,715 children for Shigella by quantitative PCR. To assess risk factors, clinical factors related to age and culture positivity, and associations with inflammatory biomarkers, we used log-binomial regression with generalized estimating equations. The prevalence of Shigella varied from 4.9%-17.8% in non-diarrheal stools across sites, and the incidence of Shigella-attributable diarrhea was 31.8 cases (95% CI: 29.6, 34.2) per 100 child-years. The sensitivity of culture compared to qPCR was 6.6% and increased to 27.8% in Shigella-attributable dysentery. Shigella diarrhea episodes were more likely to be severe and less likely to be culture positive in younger children. Older age (RR: 1.75, 95% CI: 1.70, 1.81 per 6-month increase in age), unimproved sanitation (RR: 1.15, 95% CI: 1.03, 1.29), low maternal education (<10 years, RR: 1.14, 95% CI: 1.03, 1.26), initiating complementary foods before 3 months (RR: 1.10, 95% CI: 1.01, 1.20), and malnutrition (RR: 0.91, 95% CI: 0.88, 0.95 per unit increase in weight-for-age z-score) were risk factors for Shigella. There was a linear dose-response between Shigella quantity and myeloperoxidase concentrations. The burden of Shigella varied widely across sites, but uniformly increased through the second year of life and was associated with intestinal inflammation. Culture missed most clinically relevant cases of severe diarrhea and dysentery.

Herpes Simplex virus type 2 seroprevalence and risk factors among adolescents and youth with HIV-1 in Northern, Tanzania.

INTRODUCTION: Herpes Simplex virus type 2 (HSV-2) infection is associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) acquisition and transmission. Individuals co-infected with HIV-1 and HSV-2 may have longer lasting, more frequent and severe outbreaks of herpes symptoms. Previous studies have assessed HSV-2 seroprevalence and associated risk factors in adult populations. However, there is limited data on the HSV-2 seroprevalence among adolescents and youth living with HIV-1. The study aimed to determine the HSV-2 seroprevalence and associated risk factors among adolescents and youth living with HIV-1 at referral hospital setting in Northern Tanzania. METHODOLOGY: A cross-sectional survey was conducted between February and July 2017 among HIV-1-infected individuals aged 10-24 years attending the Child -Centred Family Care Clinic at Kilimanjaro Christian Medical Centre. Blood specimens from 180 individuals were collected for ELISA-based detection of HSV-2 antibodies. Associations between risk factors and HSV-2 seroprevalence were analysed by univariate and multivariate logistic regression models. RESULTS: The overall HSV-2 seroprevalence was 18% (32/180). A significant HSV-2 seroprevalence was noted among adolescents and youth, who reported having had sexual intercourse than those who never had sexual intercourse (28.9% vs 13.3%, p = 0.02). Youths aged 20-24 had six folds higher risk of HSV-2 seroprevalence compared to those aged 10-14 years (AOR = 5.97 95% CI 1.31 - 27.19, p = 0.02). CONCLUSIONS: Our study found that HSV-2 seroprevalence increased by age among adolescents and youth living with HIV-1. Age-specific approaches might play an important role in interventions targeting HSV-2 infection.

Protection From Natural Immunity Against Enteric Infections and Etiology-Specific Diarrhea in a Longitudinal Birth Cohort.

BACKGROUND: The degree of protection conferred by natural immunity is unknown for many enteropathogens, but it is important to support the development of enteric vaccines. METHODS: We used the Andersen-Gill extension of the Cox model to estimate the effects of previous infections on the incidence of subsequent subclinical infections and diarrhea in children under 2 using quantitative molecular diagnostics in the MAL-ED cohort. We used cross-pathogen negative control associations to correct bias due to confounding by unmeasured heterogeneity of exposure and susceptibility. RESULTS: Prior rotavirus infection was associated with a 50% lower hazard (calibrated hazard ratio [cHR], 0.50; 95% confidence interval [CI], 0.41-0.62) of subsequent rotavirus diarrhea. Strong protection was evident against Cryptosporidium diarrhea (cHR, 0.32; 95% CI, 0.20-0.51). There was also protection due to prior infections for norovirus GII (cHR against diarrhea, 0.67; 95% CI, 0.49-0.91), astrovirus (cHR, 0.62; 95% CI, 0.48-0.81), and Shigella (cHR, 0.79; 95% CI, 0.65-0.95). Minimal protection was observed for other bacteria, adenovirus 40/41, and sapovirus. CONCLUSIONS: Natural immunity was generally stronger for the enteric viruses than bacteria, potentially due to less antigenic diversity. Vaccines against major causes of diarrhea may be feasible but likely need to be more immunogenic than natural infection.

Leptospirosis in sugarcane plantation and fishing communities in Kagera northwestern Tanzania.

BACKGROUND: Leptospirosis is a bacterial zoonotic disease of worldwide importance, though relatively neglected in many African countries including sub Saharan Africa that is among areas with high burden of this disease. The disease is often mistaken for other febrile illnesses such as dengue, malaria, rickettsioses and enteric fever. Leptospirosis is an occupational disease likely to affect people working in environments prone to infestation with rodents which are the primary reservoir hosts of this disease. Some of the populations at risk include: sugarcane plantation workers, wetland farmers, fishermen and abattoir workers. In this study we investigated the prevalence of antibodies against Leptospira among sugarcane plantation and factory workers, fishing communities as well as among rodents and shrews in domestic and peridomestic environments within the study areas. METHODS: The study was conducted in Kagera region, northwestern Tanzania and it involved sugarcane plantation workers (cutters and weeders), sugar factory workers and the fishing community at Kagera Sugar Company in Missenyi district and Musira island in Lake Victoria, Kagera, respectively. Blood was collected from consenting human adults, and from rodents and shrews (insectivores) captured live using Sherman traps. Serological detection of leptospiral antibodies in blood serum was carried out by the microscopic agglutination test (MAT). RESULTS: A total of 455 participants were recruited from the sugarcane plantation (n = 401) and fishing community (n = 54) while 31 rodents and shrews were captured. The overall prevalence of antibodies against Leptospira in human was 15.8%. Sugarcane cutters had higher seroprevalence than other sugar factory workers. Prevalent antibodies against Leptospira serovars in humans were against serovars Lora (6.8%), Sokoine (5.3%), Pomona (2.4%), Hebdomadis (1.1%) and Kenya (0.2%). Detected leptospiral serovars in reservoir hosts were Sokoine (12.5%) and Grippotyphosa (4.2%). Serovar Sokoine was detected both in humans and small mammals. CONCLUSION: Leptospirosis is a public health threat affecting populations at risk, such as sugarcane plantation workers and fishing communities. Public awareness targeting risk occupational groups is much needed for mitigation of leptospirosis in the study areas and other vulnerable populations in Tanzania and elsewhere.

Toxoplasma gondii seroprevalence among pregnant women attending antenatal clinic in Northern Tanzania.

BACKGROUND: Acute Toxoplasma gondii infection during pregnancy represents a risk for congenital disease, especially among women without previous exposure to infection. There is, however, a paucity of information about the epidemiology of T. gondii infection in pregnant women in Tanzania. This study aimed to determine the seroprevalence of T. gondii infection and associated demographic, clinical, and behavioral risk factors in pregnant women attending ante-natal clinic (ANC) at Kilimanjaro Christian Medical Center (KCMC), a referral medical center in Northern Tanzania. METHODS: A hospital-based cross-sectional study was carried out from 1 February to 30 April 2017. Data on maternal demographic characteristics, obstetric history, knowledge, and practices related to T. gondii infection were collected from 254 pregnant women attending antenatal care at KCMC. A sample of 4 mL of blood was collected from each participant and sera prepared from each sample. Serum samples were tested for the presence of specific T. gondii IgG and IgM antibodies by indirect Enzyme-Linked Immunosorbent Assay (ELISA). DNA was extracted from whole blood for polymerase chain reaction (PCR) testing, targeting the DNA sequence coding for the Internal Transcribed Spacer 1 (ITS1). RESULTS: The overall T. gondii seroprevalence, including both IgM- and IgG-positive individuals, was 44.5%. Of the 254 tested women, 102 and 23 were seropositive for T. gondii-specific IgG and IgM antibodies respectively and 113 individuals had antibodies of either or both classes. All IgM-positive samples were also tested by PCR, and all were negative. The majority (90%) of the women surveyed had never heard about toxoplasmosis. Consumption of raw vegetables [aOR = 0. 344; 95% CI 0.151-0.784; p = 0.011] and having regular contact with soil [aOR = 0.482; 95% CI 0.268-0.8681; p = 0.015] were both associated with T. gondii antibody status. Inverse relationships with probability of T. gondii exposure were observed, such that these practices were associated with reduced probability of antibody detection. CONCLUSION: Based on serology results, we report widespread exposure to T. gondii infection among pregnant women attending ANC in KCMC. The complex interaction of risk factors for T. gondii infection needs to be studied in larger longitudinal studies.

Use of quantitative molecular diagnostic methods to investigate the effect of enteropathogen infections on linear growth in children in low-resource settings: longitudinal analysis of results from the MAL-ED cohort study.

BACKGROUND: Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings. METHODS: We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding. FINDINGS: Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoeal episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0·14, 95% CI -0·27 to -0·01), enteroaggregative Escherichia coli (-0·21, -0·37 to -0·05), Campylobacter (-0·17, -0·32 to -0·01), and Giardia (-0·17, -0·30 to -0·05). Norovirus, Cryptosporidium, typical enteropathogenic E coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (-0·13 LAZ, 95% CI -0·22 to -0·03 for Shigella; -0·14, -0·26 to -0·02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0·12-0·37 LAZ (0·4-1·2 cm) at the MAL-ED sites. INTERPRETATION: Subclinical infection and quantity of pathogens, particularly Shigella, enteroaggregative E coli, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting. FUNDING: Bill & Melinda Gates Foundation.

Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study.

BACKGROUND: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. METHODS: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. FINDINGS: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0]). INTERPRETATION: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. FUNDING: Bill & Melinda Gates Foundation.

Use of TaqMan Array Cards to Screen Outbreak Specimens for Causes of Febrile Illness in Tanzania.

We describe the deployment of a custom-designed molecular diagnostic TaqMan Array Card (TAC) to screen for 31 bacterial, protozoal, and viral etiologies in blood from outbreaks of acute febrile illness in Tanzania during 2015-2017. On outbreaks notified to the Tanzanian Ministry of Health, epidemiologists were dispatched and specimens were collected, transported to a central national laboratory, and tested by TAC within 2 days. This algorithm streamlined investigation, diagnosed a typhoid outbreak, and excluded dozens of other etiologies. This method is usable in-country and may be incorporated into algorithms for diagnosing outbreaks.

One Health Research in Northern Tanzania - Challenges and Progress.

East Africa has one of the world's fastest growing human populations-many of whom are dependent on livestock-as well as some of the world's largest wildlife populations. Humans, livestock, and wildlife often interact closely, intimately linking human, animal, and environmental health. The concept of One Health captures this interconnectedness, including the social structures and beliefs driving interactions between species and their environments. East African policymakers and researchers are recognising and encouraging One Health research, with both groups increasingly playing a leading role in this subject area. One Health research requires interaction between scientists from different disciplines, such as the biological and social sciences and human and veterinary medicine. Different disciplines draw on norms, methodologies, and terminologies that have evolved within their respective institutions and that may be distinct from or in conflict with one another. These differences impact interdisciplinary research, both around theoretical and methodological approaches and during project operationalisation. We present experiential knowledge gained from numerous ongoing projects in northern Tanzania, including those dealing with bacterial zoonoses associated with febrile illness, foodborne disease, and anthrax. We use the examples to illustrate differences between and within social and biological sciences and between industrialised and traditional societies, for example, with regard to consenting procedures or the ethical treatment of animals. We describe challenges encountered in ethical approval processes, consenting procedures, and field and laboratory logistics and offer suggestions for improvement. While considerable investment of time in sensitisation, communication, and collaboration is needed to overcome interdisciplinary challenges inherent in One Health research, this can yield great rewards in paving the way for successful implementation of One Health projects. Furthermore, continued investment in African institutions and scientists will strengthen the role of East Africa as a world leader in One Health research.

HIV-1 pol diversity among female bar and hotel workers in Northern Tanzania.

A national ART program was launched in Tanzania in October 2004. Due to the existence of multiple HIV-1 subtypes and recombinant viruses co-circulating in Tanzania, it is important to monitor rates of drug resistance. The present study determined the prevalence of HIV-1 drug resistance mutations among ART-naive female bar and hotel workers, a high-risk population for HIV-1 infection in Moshi, Tanzania. A partial HIV-1 pol gene was analyzed by single-genome amplification and sequencing in 45 subjects (622 pol sequences total; median number of sequences per subject, 13; IQR 5-20) in samples collected in 2005. The prevalence of HIV-1 subtypes A1, C, and D, and inter-subtype recombinant viruses, was 36%, 29%, 9% and 27%, respectively. Thirteen different recombination patterns included D/A1/D, C/A1, A1/C/A1, A1/U/A1, C/U/A1, C/A1, U/D/U, D/A1/D, A1/C, A1/C, A2/C/A2, CRF10_CD/C/CRF10_CD and CRF35_AD/A1/CRF35_AD. CRF35_AD was identified in Tanzania for the first time. All recombinant viruses in this study were unique, suggesting ongoing recombination processes among circulating HIV-1 variants. The prevalence of multiple infections in this population was 16% (n = 7). Primary HIV-1 drug resistance mutations to RT inhibitors were identified in three (7%) subjects (K65R plus Y181C; N60D; and V106M). In some subjects, polymorphisms were observed at the RT positions 41, 69, 75, 98, 101, 179, 190, and 215. Secondary mutations associated with NNRTIs were observed at the RT positions 90 (7%) and 138 (6%). In the protease gene, three subjects (7%) had M46I/L mutations. All subjects in this study had HIV-1 subtype-specific natural polymorphisms at positions 36, 69, 89 and 93 that are associated with drug resistance in HIV-1 subtype B. These results suggested that HIV-1 drug resistance mutations and natural polymorphisms existed in this population before the initiation of the national ART program. With increasing use of ARV, these results highlight the importance of drug resistance monitoring in Tanzania.