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Campylobacter species infections have been associated with malnutrition and intestinal inflammation among children in low-resource settings. However, it remains unclear whether that association is specific to Campylobacter jejuni/coli. The aim of this study was to assess the association between both all Campylobacter species infections and Campylobacter jejuni/coli infections on growth and enteric inflammation in children aged 1–24 months. We analyzed data from 1715 children followed from birth until 24 months of age in the MAL-ED birth cohort study, including detection of Campylobacter species by enzyme immunoassay and Campylobacter jejuni/coli by quantitative PCR in stool samples. Myeloperoxidase (MPO) concentration in stool, used as a quantitative index of enteric inflammation, was measured. The incidence rate per 100 child-months of infections with Campylobacter jejuni/coli and Campylobacter species during 1–24 month follow up were 17.7 and 29.6 respectively. Female sex of child, shorter duration of exclusive breastfeeding, lower maternal age, mother having less than 3 living children, maternal educational level of <6 years, lack of routine treatment of drinking water, and unimproved sanitation were associated with Campylobacter jejuni/coli infection. The cumulative burden of both Campylobacter jejuni/coli infections and Campylobacter species were associated with poor growth and increased intestinal inflammation.
The MAL-ED study design and methodology have been previously described19. Briefly, children were enrolled November, 2009 to February, 2012 from the community within 17 days of birth at eight locations: Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru. Children were included if the maternal age was 16 years or older, their family intended to remain in the study area for at least 6 months from enrolment, they were from a singleton pregnancy, and they had no other siblings enrolled in the study. Children with a birthweight or enrolment weight of less than 1500 gm and children diagnosed with congenital disease or severe neonatal disease were excluded. The study was approved by the Research Review Committee and the Ethical Review Committee of icddr,b (Bangladesh), the Local Institutional Review Board at the Federal Universisty of Ceará and the national IRB Conselho Nacional de Ética em Pesquisa (Brazil), the Christian Medical College Institutional Review Board and the Emory University Institutional Review Board (India), the Nepal Health Research Council and Walter Reed Institute of Research (Nepal), the Ethics Committee of Asociacion Benefica PRISMA, the Regional Health Directorate of Loreto and the IRB of Johns Hopkins Bloomberg School of Public Health (Peru), the Ethical Review Committee of Aga Khan University (Pakistan), the Institutional Review Boards at the University of Venda (South Africa), the National Institute for Medical Research (Tanzania), and the Institutional Review Board of the University of Virginia (UAS). Written informed consent was obtained from the parents or legal guardian of every child19,22. All methods were performed in accordance with the relevant guidelines and regulations. Household demographics, presence of siblings, maternal characteristics, and other data on the child’s birth and anthropometry were obtained at enrollment19. The socioeconomic status (SES) of families was assessed at 6, 12, 18, and 24 months. SES score, the water/sanitation, assets, maternal education and income (WAMI) index was developed using composite indicators including the variables such as access to improved water and sanitation, eight selected assets, maternal education, and household income23. Improved water and sanitation were defined following World Health Organization guidelines24. Treatment of drinking water was defined as filtering, boiling, or adding bleach1. Anthropometric measurements and vaccination history were collected monthly. Details of illness and child feeding practices were collected during twice-weekly household visits25. Stool samples were collected monthly and were preserved, transported, and processed at all sites using harmonized protocols26. Child anthropometry was measured using standard scales (seca gmbh & co. kg., Hamburg, Germany). Length-for-age Z score (LAZ) was calculated through the use of the 2006 WHO standards for children27. The Z-score scale, calculated as (observed value – average value of the reference population)/standard deviation value of reference population, is linear and therefore a fixed interval of Z-scores has a fixed length difference in cm for all children of the same age. Z-scores are also sex-independent, thus permitting the evaluation of children’s growth status by combining sex and age groups28. Stool samples were collected without fixative by field workers and raw stool aliquots were kept at −80 °C before nucleic acid extraction. All lab testing was done at the site specific laboratories11,14. Stool samples were assayed for Campylobacter species by enzyme immunoassay (ProSpecT, Remel, Lenexa, KS, USA). In addition, myeloperoxidase (MPO) (Alpco, Salem, New Hampshire) was measured using commercially-available Enzyme Linked Immunosorbent Assay (ELISA) kits following the instructions of the manufacturers1,8. Campylobacter jejuni/coli were detected in the stool samples by quantitative PCR targeting the cadF gene using the TaqMan Array Card (TAC) platform, a compartmentalized probe-based real-time PCR assays for detecting enteropathogens in fecal samples, as previously described22,29. The analytic cutoff of each pathogen was a quantification cycle (Cq) of 35; thus, a Cq < 35 was considered positive20,30. All statistical tests were performed in STATA 14 (Stata Corporation, College Station, TX). Campylobacter burden was defined as the number of pathogens detected divided by the number of stools collected and was scaled divided by (10th vs 90th percentile). Descriptive statistics such as proportion, mean and standard deviation for symmetric data, and median with inter‐quartile range (IQR) for asymmetric quantitative variables were used to summarize the data. Chi-square and proportion test was used to see the association between two categorical variables and t-test was used to see the mean difference between two groups for symmetric distribution. Cumulative incidence of Campylobacter jejuni/coli and Campylobacter species was defined as the proportion of subjects who were infected at least once during the study period. Incidence rates and risk factors associated with Campylobacter detection in surveillance stool samples were calculated using negative binomial regression models due to over dispersion. In the final multiple negative binomial regression model, the following variables were considered for inclusion using stepwise forward selection: child sex, duration of exclusive breastfeeding in months, enrollment weight for age z-score, maternal age in years, maternal education greater than or equal to 6 years, mother having less than 3 living children, routine treatment of drinking water, improved sanitation, and household ownership of cattle/poultry. The MPO values were log‐transformed before the analysis. We excluded children from the Pakistan site for growth analysis, owing to bias noted in a subset of length measurements at this site. Seasonality was calculated via the terms sin(2mπ/12) + cos(2mπ/12), where “m” is the calendar month1,31. Associations between Campylobacter infection and inflammation was estimated using generalized estimating equations to fit regression models after adjusting for seasonality, sex, age, water/sanitation, assets, maternal education, and income (WAMI) index; enrollment length-for-age; maternal height; poultry/cattle in house, some alternative pathogens which were significantly associated with log(MPO) such as enteroaggregative E. coli (EAEC), heat-labile enterotoxin-producing E. coli (LT-ETEC), heat-stable enterotoxin-producing E. coli (ST-ETEC), Shigella/enteroinvasive E. coli (Shigella/EIEC), and site for overall estimate and age in month as time variable32. The Gaussian family with identity link was used for the continuous outcome of log(MPO). To access and compare the associations of Campylobacter jejuni/coli and Campylobacter species infection burden on growth at 24 months of age, we used multi-variable linear regression after adjusting for site and the necessary covariates.
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