BackgroundDiarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.ResultsWe use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.ConclusionsOur findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 ؋ 10 4 ipaH copies per 100 ng of stool DNA for set 1 (n ‫؍‬ 877). One hundred fifty-eight (18%) specimens yielded >2.9 ؋ 10 4 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with >2.9 ؋ 10 4 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 ؋ 10 4 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n ‫؍‬ 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n ‫؍‬ 129) for culture to 17.6% (n ‫؍‬ 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 ؋ 10 4 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic pointof-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.Salmonella enterica serovar Typhi (serovar Typhi) is the cause of typhoid fever, an illness that affects over 20,000,000 individuals worldwide each year, killing over 200, 000 (5, 8, 16). The largest burden of typhoid fever is borne by impoverished individuals in resource-poor areas of the world. Serovar Typhi is a human-restricted invasive enteric pathogen which, after ingestion, crosses the intestinal mucosa, is taken up by gutassociated lymphoreticular tissues, and enters the systemic circulation. Both mucosal and systemic host immune responses are stimulated after infection. Serovar Typhi is an intracellular pathogen, and antibody and cell-mediated immune responses occur after infection or immunization with live oral attenuated typhoid vaccines (10,25,34).Diagnostic tests for typhoid fever often lack sensitivity and/or specificity, especially in areas of the world that are endemic for typhoid fever, where clinically distinguishing typhoid fever from other febrile illnesses is difficult (5, 17, 39). Microbiologic culturing of blood is approximately 30 to 70% sensitive, with the highest sensitivity being associated with an absence of prior use of antibiotics and the culturing of larger volumes of blood, features that complicate this mode of diagnosis in young children (5,6,8,36). Microbiologic culturing of bone marrow aspirates is more sensitive than bl...

Shigellosis, caused by Shigella species, is a major public health problem in Bangladesh. To determine the prevalence and distribution of different Shigella species, we analyzed 10,827 Shigella isolates from patients between 2001 and 2011. S. flexneri was the predominant species isolated throughout the period. However, the prevalence of S. flexneri decreased from 65.7% in 2001 to 47% in 2011, whereas the prevalence of S. sonnei increased from 7.2% in 2001 to 25% in 2011. S. boydii and S. dysenteriae accounted for 17.3% and 7.7% of the isolates respectively throughout the period. Of 200 randomly selected S. sonnei isolates for extensive characterization, biotype g strains were predominant (95%) followed by biotype a (5%). Resistance to commonly used antibiotics including trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, mecillinam and ampicillin was 89.5%, 86.5%, 17%, 10.5%, and 9.5%, respectively. All isolates were susceptible to ceftriaxone, cefotaxime, ceftazidime and imipenem. Ninety-eight percent of the strains had integrons belonging to class 1, 2 or both. The class 1 integron contained only dfrA5 gene, whereas among class 2 integron, 16% contained dhfrAI-sat1-aadA1-orfX gene cassettes and 84% harbored dhfrA1-sat2 gene cassettes. Plasmids of ∼5, ∼1.8 and ∼1.4 MDa in size were found in 92% of the strains, whereas only 33% of the strains carried the 120 MDa plasmid. PFGE analysis showed that strains having different integron patterns belonged to different clusters. These results show a changing trend in the prevalence of Shigella species with the emergence of multidrug resistant S. sonnei. Although S. flexneri continues to be the predominant species albeit with reduced prevalence, S. sonnei has emerged as the second most prevalent species replacing the earlier dominance by S. boydii and S. dysenteriae in Bangladesh.

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BackgroundRapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure.MethodologyTPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment.Principal FindingsTPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO2, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78–97% (73–100, 95% CI), depending on definition of true negative.ConclusionThe TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.

BACKGROUND/OBJECTIVES:To examine the bacteriological quality of complementary foods (CF) and to correlate the results with diarrhoeal morbidity and nutritional status of Bangladeshi children aged 6-24 months. SUBJECTS/METHODS: A total of 212 CF samples were tested, of which 72 were collected immediately before the first time of feeding (p1 h of food preparation) and 140 were collected at second/third time of feeding from 140 households located in urban and rural areas of Bangladesh. Anthropometry, food frequency data and demographic information of the children were collected. RESULTS: Of the first time feeding samples, 3% from each of urban and rural areas were found to be contaminated with faecal coliforms (FC) at X100 CFU/g. E. coli was isolated from 11% and 6% of samples, and B. cereus from 8% and 6% of samples from urban and rural areas, respectively. In contrast, 33% of the second/third time feeding samples from urban areas and 19% from rural areas were contaminated with FC at X100 CFU/g (Po0.05). E. coli was isolated from 40% and 39% of samples, and B. cereus from 33% and 26% of samples from urban and rural areas, respectively. Significantly high numbers of wasted rural children had CF with a high aerobic plate count, which was also significantly associated with diarrhoeal morbidity in children. CONCLUSIONS: Around 40% of CF samples were contaminated with E. coli, which was mainly attributable to food preparation practices. Consumption of contaminated CF appeared to be associated with a higher frequency of diarrhoea and malnutrition in children.

Background.Antibiotic exposure and specimen volume are known to affect pathogen detection by culture. Here we assess their effects on bacterial pathogen detection by both culture and polymerase chain reaction (PCR) in children.Methods.PERCH (Pneumonia Etiology Research for Child Health) is a case-control study of pneumonia in children aged 1–59 months investigating pathogens in blood, nasopharyngeal/oropharyngeal (NP/OP) swabs, and induced sputum by culture and PCR. Antibiotic exposure was ascertained by serum bioassay, and for cases, by a record of antibiotic treatment prior to specimen collection. Inoculated blood culture bottles were weighed to estimate volume.Results.Antibiotic exposure ranged by specimen type from 43.5% to 81.7% in 4223 cases and was detected in 2.3% of 4863 controls. Antibiotics were associated with a 45% reduction in blood culture yield and approximately 20% reduction in yield from induced sputum culture. Reduction in yield of Streptococcus pneumoniae from NP culture was approximately 30% in cases and approximately 32% in controls. Several bacteria had significant but marginal reductions (by 5%–7%) in detection by PCR in NP/OP swabs from both cases and controls, with the exception of S. pneumoniae in exposed controls, which was detected 25% less frequently compared to nonexposed controls. Bacterial detection in induced sputum by PCR decreased 7% for exposed compared to nonexposed cases. For every additional 1 mL of blood culture specimen collected, microbial yield increased 0.51% (95% confidence interval, 0.47%–0.54%), from 2% when volume was ≤1 mL to approximately 6% for ≥3 mL.Conclusions.Antibiotic exposure and blood culture volume affect detection of bacterial pathogens in children with pneumonia and should be accounted for in studies of etiology and in clinical management.