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.

The temporal switching of serotypes from serotype Ogawa to Inaba and back to Ogawa was identified in Vibrio cholerae O1, which was responsible for seasonal outbreaks of cholera in Dhaka during the period 2015 to 2018. In order to delineate the factors responsible for this serotype transition, we performed wholegenome sequencing (WGS) of V. cholerae O1 multidrug-resistant strains belonging to both the serotypes that were isolated during this interval where the emergence and subsequent reduction of the Inaba serotype occurred. The whole-genome-based phylogenetic analysis revealed clonal expansion of the Inaba isolates mainly responsible for the peaks of infection during 2016 to 2017 and that they might have evolved from the prevailing Ogawa strains in 2015 which coclustered with them. Furthermore, the wbeT gene in these Inaba serotype isolates was inactivated due to insertion of a transposable element at the same position signifying the clonal expansion. Also, V. cholerae isolates in the Inaba serotype dominant clade mainly contained classical ctxB allele and revealed differences in the genetic composition of Vibrio seventh pandemic island II (VSP-II) and the SXT integrative and conjugative element (SXT-ICE) compared to those of Ogawa serotype strains which remerged in 2018. The variable presence of phage-inducible chromosomal island-like element 1 (PLE1) was also noted in the isolates of the Inaba serotype dominant clade. The detailed genomic characterization of the sequenced isolates has shed light on the forces which could be responsible for the periodic changes in serotypes of V. cholerae and has also highlighted the need to analyze the mobilome in greater detail to obtain insights into the mechanisms behind serotype switching. IMPORTANCE The switching of serotype from Ogawa to Inaba and back to Ogawa has been observed temporally in Vibrio cholerae O1, which is responsible for endemic cholera in Bangladesh. The serospecificity is key for effective intervention and for preventing cholera, a deadly disease that continues to cause significant morbidity and mortality worldwide. In the present study, WGS of V. cholerae allowed us to better understand the factors associated with the serotype switching events observed during 2015 to 2018. Genomic data analysis of strains isolated during this interval highlighted variations in the genes ctxB, tcpA, and rtxA and also identified significant differences in the genetic content of the mobilome, which included key elements such as SXT ICE, VSP-II, and PLE. Our results indicate that selective forces such as antibiotic resistance and phage resistance might contribute to the clonal expansion and predominance of a particular V. cholerae serotype responsible for an outbreak.

Background.We investigated the performance of polymerase chain reaction (PCR) on blood in the diagnosis of pneumococcal pneumonia among children from 7 low- and middle-income countries.Methods.We tested blood by PCR for the pneumococcal autolysin gene in children aged 1–59 months in the Pneumonia Etiology Research for Child Health (PERCH) study. Children had World Health Organization–defined severe or very severe pneumonia or were age-frequency–matched community controls. Additionally, we tested blood from general pediatric admissions in Kilifi, Kenya, a PERCH site. The proportion PCR-positive was compared among cases with microbiologically confirmed pneumococcal pneumonia (MCPP), cases without a confirmed bacterial infection (nonconfirmed), cases confirmed for nonpneumococcal bacteria, and controls.Results.In PERCH, 7.3% (n = 291/3995) of cases and 5.5% (n = 273/4987) of controls were blood pneumococcal PCR-positive (P < .001), compared with 64.3% (n = 36/56) of MCPP cases and 6.3% (n = 243/3832) of nonconfirmed cases (P < .001). Blood pneumococcal PCR positivity was higher in children from the 5 African countries (5.5%–11.5% among cases and 5.3%–10.2% among controls) than from the 2 Asian countries (1.3% and 1.0% among cases and 0.8% and 0.8% among controls). Among Kilifi general pediatric admissions, 3.9% (n = 274/6968) were PCR-positive, including 61.7% (n = 37/60) of those with positive blood cultures for pneumococcus.Discussion.The utility of pneumococcal PCR on blood for diagnosing childhood pneumococcal pneumonia in the 7 low- and middle-income countries studied is limited by poor specificity and by poor sensitivity among MCPP cases.

Background.Detection of pneumococcus by lytA polymerase chain reaction (PCR) in blood had poor diagnostic accuracy for diagnosing pneumococcal pneumonia in children in 9 African and Asian sites. We assessed the value of blood lytA quantification in diagnosing pneumococcal pneumonia.Methods.The Pneumonia Etiology Research for Child Health (PERCH) case-control study tested whole blood by PCR for pneumococcus in children aged 1–59 months hospitalized with signs of pneumonia and in age–frequency matched community controls. The distribution of load among PCR-positive participants was compared between microbiologically confirmed pneumococcal pneumonia (MCPP) cases, cases confirmed for nonpneumococcal pathogens, nonconfirmed cases, and controls. Receiver operating characteristic analyses determined the “optimal threshold” that distinguished MCPP cases from controls.Results.Load was available for 290 of 291 cases with pneumococcal PCR detected in blood and 273 of 273 controls. Load was higher in MCPP cases than controls (median, 4.0 × 103 vs 0.19 × 103 copies/mL), but overlapped substantially (range, 0.16–989.9 × 103 copies/mL and 0.01–551.9 × 103 copies/mL, respectively). The proportion with high load (≥2.2 log10 copies/mL) was 62.5% among MCPP cases, 4.3% among nonconfirmed cases, 9.3% among cases confirmed for a nonpneumococcal pathogen, and 3.1% among controls. Pneumococcal load in blood was not associated with respiratory tract illness in controls (P = .32). High blood pneumococcal load was associated with alveolar consolidation on chest radiograph in nonconfirmed cases, and with high (>6.9 log10 copies/mL) nasopharyngeal/oropharyngeal load and C-reactive protein ≥40 mg/L (both P < .01) in nonconfirmed cases but not controls.Conclusions.Quantitative pneumococcal PCR in blood has limited diagnostic utility for identifying pneumococcal pneumonia in individual children, but may be informative in epidemiological studies.

Salmonellosis, an acute invasive enteric infection, is endemic in Bangladesh. We analyzed 128,312 stool samples of diarrheal patients to identify Salmonella spp. during 2005–2013. A total of 2120 (1.7%) Salmonella spp. were isolated and the prevalence of Salmonella spp. decreased significantly over time (2→1%, P < 0.001). Among the typhoidal Salmonella (TS) serogroups, S. Typhi was predominant (404, [65.1%]) followed by S. Paratyphi B (139, [22.4%]) and S. Paratyphi A (78, [12.6%]). Of the nontyphoidal Salmonella (NTS) isolates, the serogroup C1 (560, [37%]) was predominant followed by B (379, [25%]), C2 (203, [14%]), E (127, [9%]), and D (94, [6%]). Most of the resistance was found towards nalidixic acid (40%), ampicillin (36%), cotrimoxazole (20%), chloramphenicol (13%), ciprofloxacin (4%), and ceftriaxone (4%). Interestingly, 32% of the isolates showed reduced susceptibility to Cip. Multiantibiotic resistance (MAR, ≥3 drugs) was more common among TS than NTS strains (P < 0.001). Among the representative ceftriaxone-resistant isolates, bla TEM gene was detected among 88% (7/8) of the strains, whereas only one strain of S. Typhi was positive for both bla TEM and bla CTX-M genes. The study reflects higher prevalence of MAR Salmonella spp. and is the first to report the bla TEM gene mediated ESBL production among Salmonellae in Bangladesh. Emergence of MAR Salmonella spp. in particular ESBL strains should be considered a public health concern.

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Background:Escherichia coli is a major extended-spectrum β-lactamase (ESBL)–producing organism responsible for the rapid spread of antimicrobial resistance (AMR) that has compromised our ability to treat infections. Baseline data on population structure, virulence, and resistance mechanisms in E. coli lineages from developing countries such as Bangladesh are lacking.Methods: Whole-genome sequencing was performed for 46 ESBL–E. coli isolates cultured from patient samples at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b)-Dhaka. Sequence data were analyzed to glean details of AMR, virulence, and phylogenetic and molecular markers of E. coli lineages.Results: Genome comparison revealed presence of all major high-risk clones including sequence type 131 (ST131) (46%), ST405 (13%), ST648 (7%), ST410 (4.3%), ST38 (2%), ST73 (2%), and ST1193 (2%). The predominant ESBL gene and plasmid combination were blaCTX–M–15 and FII-FIA-FIB detected in diverse E. coli phylogroups and STs. The blaNDM–5 (9%) gene was present in prominent E. coli STs. One (2%) mcr-1–positive ST1011 E. coli, coharboring blaCTXM–55 gene, was detected. The extraintestinal pathogenic E. coli genotype was associated with specific E. coli lineages. The single nucleotide polymorphism (SNP)-based genome phylogeny largely showed correlation with phylogroups, serogroups, and fimH types. Majority of these isolates were susceptible to amikacin (93%), imipenem (93%), and nitrofurantoin (83%).Conclusion: Our study reveals a high diversity of E. coli lineages among ESBL-producing E. coli from Dhaka. This study suggests ongoing circulation of ST131 and all major non-ST131 high-risk clones that are strongly associated with cephalosporin resistance and virulence genes. These findings warrant prospective monitoring of high-risk clones, which would otherwise worsen the AMR crises.

The emergence of multidrug-resistant (MDR) Escherichia coli (E. coli) clonal lineages with high virulence potential is alarming. Lack of sufficient data on molecular epidemiology of such pathogens from countries with high infection burden, such as Bangladesh, hinders management and infection control measures. In this study, we assessed the population structure, virulence potential and antimicrobial susceptibility of clinical E. coli isolates from Dhaka, Bangladesh. A high prevalence of MDR (69%) and extended-spectrum β-lactamase production (ESBL) (51%) was found. Most E. coli isolates were susceptible to amikacin (95%), meropenem (94%) and nitrofurantoin (89%) antibiotics. A high prevalence of ST131 (22%) and ST95 (9%) followed by ST69 (4%) and ST73 (3%) was observed. Phylogroups B2 (46%), B1 (16%), D (10%) and F (9%) were prominent. blaCTX-M-15 (52%) and blaNDM-1 (5%) were the most prevalent ESBL and carbapenem resistance genes, respectively. Moreover, the predominant pathotype identified was extraintestinal pathogenic E. coli (ExPEC) (41%) followed by enteric pathogens (11%). In conclusion, our results suggest the transmission of clonal E. coli groups amidst diverse E. coli population that are associated with high virulence potential and MDR phenotype. This is of high concern and mandates more efforts towards molecular surveillance of antimicrobial resistance (AMR) in clinically significant pathogens.