Genomic analysis of clinical Aeromonas isolates reveals genetic diversity but little evidence of genetic determinants for diarrhoeal disease.

Aeromonas spp. are associated with a number of infectious syndromes in humans including gastroenteritis and dysentery. Our understanding of the genetic diversity, population structure, virulence determinants and antimicrobial resistance of the genus has been limited by a lack of sequenced genomes linked to metadata. We performed a comprehensive analysis of the whole genome sequences of 447 Aeromonas isolates from children in Karachi, Pakistan, with moderate-to-severe diarrhoea (MSD) and from matched controls without diarrhoea that were collected as part of the Global Enteric Multicenter Study (GEMS). Human-associated Aeromonas isolates exhibited high species diversity and extensive antimicrobial and virulence gene content. Aeromonas caviae, A. dhankensis, A. veronii and A. enteropelogenes were all significantly associated with MSD in at least one cohort group. The maf2 and lafT genes that encode components of polar and lateral flagella, respectively, exhibited a weak association with isolates originating from cases of gastroenteritis.

Methods for estimating the direct and indirect effect of 10 valent pneumococcal vaccine on nasopharyngeal carriage in children under 2 years in Matiari, Pakistan.

Pneumonia is the leading cause of morbidity and mortality in children worldwide. The ten valent pneumococcal vaccine (PCV10) was introduced in Pakistan's Expanded Program on Immunization (EPI) in 2012 as a 3 + 0 schedule without catchup immunization. Nasopharyngeal carriage is taken as a surrogate marker to measure the impact of pneumococcal vaccine on populations. Carriage surveys are necessary to monitor the persistence of Vaccine Type (VT) serotypes, the emergence of Non-Vaccine Type (NVT) serotypes, and their role in both transmission and disease. This article describes various troubleshooting measures which we undertook to adopt the protocol to our setting. We also used an innovative approach to describe various epidemiological parameters of vaccine effectiveness against carriage. It is important to publish these methods to allow for valid regional and temporal comparisons of results in different settings. Thus, in this article, we describe the following methods for isolating upper airway pneumococcal carriage:•Methods for the collection, transport, and storage of nasopharyngeal samples.•Methods for identification and serotyping of pneumococci.•Methods for estimation of the direct and indirect effects of pneumococcal vaccines on nasopharyngeal carriage.

Impact of 10-valent Pneumococcal Conjugate Vaccine (PCV10) on nasopharyngeal carriage in children 2 years of age: Data from a four-year time series cross-sectional study from Pakistan.

The dataset described in this paper was collected for a time-series cross-sectional study exploring the impact of 10-valent Pneumococcal Conjugate Vaccine (PCV10) on nasopharyngeal (NP) carriage in children under 2 years of age from a rural population in Sindh, Pakistan. The study was carried out in two union councils of Matiari - Khyber and Shah Alam Shah Jee Wasi (Latitude 25.680298 / Longitude 68.502711). Data was collected on socio-demographics, clinical characteristics and vaccination status using android phone-based application. NP samples were collected using standard World Health Organisation (WHO) techniques, culture and serotyping was done using sequential Multiplex PCR described by Centre for Disease Control, USA. We looked at the carriage rate of vaccine type (VT) and non-vaccine type (NVT) serotypes over time in vaccinated and unvaccinated children. We additionally looked at the predictors for pneumococcal carriage. The uploaded dataset, available on Mendeley data repository (Nisar, Muhammad Imran (2021), "Impact of PCV10 on nasopharyngeal carriage in children in Pakistan", Mendeley Data, V1, doi:10.17632/t79h6g97gr.1), has 3140 observations in CSV format. Additional files uploaded include a data dictionary and the set of questionnaires. The dataset and accompanying files can be used by other interested researchers to replicate our analysis, carry similar analysis under varying set of assumptions or perform additional exploratory or metanalysis.

Serum amoxicillin levels in young infants (0-59 days) with sepsis treated with oral amoxicillin.

BACKGROUND: WHO recommends simplified antibiotics for young infants with sepsis in countries where hospitalisation is not feasible. Amoxicillin provides safe, Gram-positive coverage. This study was done to determine pharmacokinetics, drug disposition and interpopulation variability of oral amoxicillin in this demographic. METHODS: Young infants with signs of sepsis enrolled in an oral amoxicillin/intramuscular gentamicin treatment arm of a sepsis trial in Karachi, Pakistan, were studied. Limited pharmacokinetic (PK) sampling was performed at 0, 2-3 and 6-8 hours following an index dose of oral amoxicillin. Plasma concentrations were determined by high-performance liquid chromatography/mass spectrometry. Values of ≥2 mg/L were considered as the effect threshold, given the regional minimal inhibitory concentration (MIC) of resistant Streptococcus pneumoniae. RESULTS: Amoxicillin concentrations were determined in 129 samples from 60 young infants. Six of 44 infants had positive blood cultures with predominant Gram-positive organisms. Forty-four infants contributing blood at ≥2 of 3 specified timepoints were included in the analysis. Mean amoxicillin levels at 2-3 hours (11.6±9.5 mg/L, n=44) and 6-8 hours (16.4±9.3 mg/L, n=20) following the index dose exceeded the MIC for amoxicillin (2.0 mg/L) against resistant S. pneumoniae strains. Of 20 infants with three serum levels, 7 showed a classic dose-exposure profile and 13 showed increasing concentrations with time, implying delayed absorption or excretion. CONCLUSION: Amoxicillin concentrations in sera of young infants following oral administration at 75-100 mg/kg/day daily divided doses exceeds the susceptibility breakpoint for >50% of a 12-hour dosing interval.Oral amoxicillin may hold potential as a safe replacement of parenteral ampicillin in newborn sepsis regimens, including aminoglycosides, where hospitalisation is not feasible. TRIAL REGISTRATION NUMBER: NCT01027429.

Use of antibiotics in children younger than two years in eight countries: a prospective cohort study.

OBJECTIVE: To describe the frequency and factors associated with antibiotic use in early childhood, and estimate the proportion of diarrhoea and respiratory illnesses episodes treated with antibiotics. METHODS: Between 2009 and 2014, we followed 2134 children from eight sites in Bangladesh, Brazil, India, Nepal, Pakistan, Peru, South Africa and the United Republic of Tanzania, enrolled in the MAL-ED birth cohort study. We documented all antibiotic use from mothers' reports at twice-weekly visits over the children's first two years of life. We estimated the incidence of antibiotic use and the associations of antibiotic use with child and household characteristics. We described treatment patterns for diarrhoea and respiratory illnesses, and identified factors associated with treatment and antibiotic class. FINDINGS: Over 1 346 388 total days of observation, 16 913 courses of antibiotics were recorded (an incidence of 4.9 courses per child per year), with the highest use in South Asia. Antibiotic treatment was given for 375/499 (75.2%) episodes of bloody diarrhoea and for 4274/9661 (44.2%) episodes of diarrhoea without bloody stools. Antibiotics were used in 2384/3943 (60.5%) episodes of fieldworker-confirmed acute lower respiratory tract illness as well as in 6608/16742 (39.5%) episodes of upper respiratory illness. Penicillins were used most frequently for respiratory illness, while antibiotic classes for diarrhoea treatment varied within and between sites. CONCLUSION: Repeated antibiotic exposure was common early in life, and treatment of non-bloody diarrhoea and non-specific respiratory illnesses was not consistent with international recommendations. Rational antibiotic use programmes may have the most impact in South Asia, where antibiotic use was highest.

Systematic review of the global epidemiology, clinical and laboratory profile of enteric fever.

BACKGROUND: Children suffer the highest burden of enteric fever among populations in South Asian countries. The clinical features are non-specific, vary in populations, and are often difficult to distinguish clinically from other febrile illnesses, leading to delayed or inappropriate diagnosis and treatment. We undertook a systematic review to assess the clinical profile and laboratory features of enteric fever across age groups, economic regions, level of care and antibiotic susceptibility patterns. METHODS: We searched PubMed (January 1964-December 2013) for studies describing clinical features in defined cohorts of patients over varying time periods. Studies with all culture-confirmed cases or those with at least 50% culture-confirmed cases were included. 242 reports were screened out of 4398 relevant articles and 180 reports were included for final review. RESULTS: 96% of studies were from an urban location, 96% were hospital-based studies, with 41% of studies were from South Asia. Common clinical features in hospitalized children include high-grade fever, coated tongue, anaemia, nausea/vomiting, diarrhea, constipation, hepatomegaly, splenomegaly neutrophilia, abdominal distension and GI bleeding. In adults' nausea/vomiting, thrombocytopenia and GI perforation predominate. The case-fatality rate in children under 5 years is higher than school aged children and adolescents, and is highest in Sub Saharan Africa and North Africa/Middle East regions. Multi-drug resistant enteric fever has higher rates of complications than drug sensitive enteric fever, but case fatality rates were comparable in both. CONCLUSIONS: Our findings indicate variability in disease presentation in adults compared to children, in different regions and in resistant vs sensitive cases. Majority of studies are from hospitalized cases, and are not disaggregated by age. Despite higher complications in MDR enteric fever, case fatality rate is comparable to sensitive cases, with an overall hospital based CFR of 2%, which is similar to recent global estimates. This review underscores the importance of further epidemiological studies in community settings among children and adults, and the need for further preventable measures to curtail the burden of disease.

Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED).

BACKGROUND: Most studies of the causes of diarrhoea in low-income and middle-income countries have looked at severe disease in people presenting for care, and there are few estimates of pathogen-specific diarrhoea burdens in the community. METHODS: We undertook a birth cohort study with not only intensive community surveillance for diarrhoea but also routine collection of non-diarrhoeal stools from eight sites in South America, Africa, and Asia. We enrolled children within 17 days of birth, and diarrhoeal episodes (defined as maternal report of three or more loose stools in 24 h, or one loose stool with visible blood) were identified through twice-weekly home visits by fieldworkers over a follow-up period of 24 months. Non-diarrhoeal stool specimens were also collected for surveillance for months 1-12, 15, 18, 21, and 24. Stools were analysed for a broad range of enteropathogens using culture, enzyme immunoassay, and PCR. We used the adjusted attributable fraction (AF) to estimate pathogen-specific burdens of diarrhoea. FINDINGS: Between November 26, 2009, and February 25, 2014, we tested 7318 diarrhoeal and 24 310 non-diarrhoeal stools collected from 2145 children aged 0-24 months. Pathogen detection was common in non-diarrhoeal stools but was higher with diarrhoea. Norovirus GII (AF 5·2%, 95% CI 3·0-7·1), rotavirus (4·8%, 4·5-5·0), Campylobacter spp (3·5%, 0·4-6·3), astrovirus (2·7%, 2·2-3·1), and Cryptosporidium spp (2·0%, 1·3-2·6) exhibited the highest attributable burdens of diarrhoea in the first year of life. The major pathogens associated with diarrhoea in the second year of life were Campylobacter spp (7·9%, 3·1-12·1), norovirus GII (5·4%, 2·1-7·8), rotavirus (4·9%, 4·4-5·2), astrovirus (4·2%, 3·5-4·7), and Shigella spp (4·0%, 3·6-4·3). Rotavirus had the highest AF for sites without rotavirus vaccination and the fifth highest AF for sites with the vaccination. There was substantial variation in pathogens according to geography, diarrhoea severity, and season. Bloody diarrhoea was primarily associated with Campylobacter spp and Shigella spp, fever and vomiting with rotavirus, and vomiting with norovirus GII. INTERPRETATION: There was substantial heterogeneity in pathogen-specific burdens of diarrhoea, with important determinants including age, geography, season, rotavirus vaccine usage, and symptoms. These findings suggest that although single-pathogen strategies have an important role in the reduction of the burden of severe diarrhoeal disease, the effect of such interventions on total diarrhoeal incidence at the community level might be limited.

Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study.

BACKGROUND: Diarrhoeal diseases cause illness and death among children younger than 5 years in low-income countries. We designed the Global Enteric Multicenter Study (GEMS) to identify the aetiology and population-based burden of paediatric diarrhoeal disease in sub-Saharan Africa and south Asia. METHODS: The GEMS is a 3-year, prospective, age-stratified, matched case-control study of moderate-to-severe diarrhoea in children aged 0-59 months residing in censused populations at four sites in Africa and three in Asia. We recruited children with moderate-to-severe diarrhoea seeking care at health centres along with one to three randomly selected matched community control children without diarrhoea. From patients with moderate-to-severe diarrhoea and controls, we obtained clinical and epidemiological data, anthropometric measurements, and a faecal sample to identify enteropathogens at enrolment; one follow-up home visit was made about 60 days later to ascertain vital status, clinical outcome, and interval growth. FINDINGS: We enrolled 9439 children with moderate-to-severe diarrhoea and 13,129 control children without diarrhoea. By analysing adjusted population attributable fractions, most attributable cases of moderate-to-severe diarrhoea were due to four pathogens: rotavirus, Cryptosporidium, enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC; with or without co-expression of heat-labile enterotoxin), and Shigella. Other pathogens were important in selected sites (eg, Aeromonas, Vibrio cholerae O1, Campylobacter jejuni). Odds of dying during follow-up were 8·5-fold higher in patients with moderate-to-severe diarrhoea than in controls (odd ratio 8·5, 95% CI 5·8-12·5, p<0·0001); most deaths (167 [87·9%]) occurred during the first 2 years of life. Pathogens associated with increased risk of case death were ST-ETEC (hazard ratio [HR] 1·9; 0·99-3·5) and typical enteropathogenic E coli (HR 2·6; 1·6-4·1) in infants aged 0-11 months, and Cryptosporidium (HR 2·3; 1·3-4·3) in toddlers aged 12-23 months. INTERPRETATION: Interventions targeting five pathogens (rotavirus, Shigella, ST-ETEC, Cryptosporidium, typical enteropathogenic E coli) can substantially reduce the burden of moderate-to-severe diarrhoea. New methods and accelerated implementation of existing interventions (rotavirus vaccine and zinc) are needed to prevent disease and improve outcomes. FUNDING: The Bill & Melinda Gates Foundation.

Pan-resistant Acinetobacter infection in neonates in Karachi, Pakistan.

BACKGROUND: Pan-resistant Acinetobacter infection has emerged as an important nosocomial pathogen in our inpatient neonates over the past few years. METHODOLOGY: We performed a retrospective chart review during a five-year period (July 2003 - June 2008) of all neonates hospitalized in our neonatal intensive care unit (NICU) who developed Acinetobacter infection to identify mortality-associated risk factors in Acinetobacter neonatal infection. RESULTS: During the five-year study period, 122 cultures from 78 neonates grew Acinetobacter. Source sites of positive culture were in the following descending order: blood (n = 57), trachea (n = 55), tissue/wound/body fluids (n = 4), eye (n = 4), urine (n = 1), and cerebrospinal fluid (n = 1). Twenty-four (31%) patients had Acinetobacter isolated from more than one site. At the time of admission the mean age was 2.08 +/- 4 days and mean weight was 1.77 +/- 0.88 kg; 75% were premature. Pan-resistance (87/122; sensitive only to Polymyxin) was present in 71% of Acinetobacter isolates. Crude mortality rate of this cohort was 47%, while 70% of patients died within four days after positive Acinetobacter culture. We identified weight of less than 1 kg on admission (p 0.06, adjusted Odds Ratio (AOR) 1.53), gestational age 28 weeks or less (p 0.011, AOR 2.88), poor perfusion (p 0.007, AOR 2.4), thrombocytopenia (p 0.01; AOR 1.6) and metabolic acidosis (p 0.01; AOR 1.67) as predictors associated with poor outcome. CONCLUSION: Pan-resistant Acinetobacter infection is exceedingly fatal in newborns, particularly in premature and very low-birth weight neonates. Rational antibiotic use and vigilant infection control in NICUs are key to controlling multi-drug resistant Acinetobacter infection and improving clinical outcome.

Fluoroquinolones for treating typhoid and paratyphoid fever (enteric fever).

BACKGROUND: Fluoroquinolones are recommended as first-line therapy for typhoid and paratyphoid fever (enteric fever), but how they compare with other antibiotics and different fluoroquinolones is unclear. OBJECTIVES: To evaluate fluoroquinolone antibiotics for treating enteric fever in children and adults compared with other antibiotics, different fluoroquinolones, and different durations of fluoroquinolone treatment. SEARCH STRATEGY: In November 2007, we searched the Cochrane Infectious Diseases Group Specialized Register, CENTRAL (The Cochrane Library 2007, Issue 4), MEDLINE, EMBASE, LILACS, mRCT, conference proceedings, and reference lists. SELECTION CRITERIA: Randomized controlled trials of fluoroquinolones in people with blood or bone marrow culture-confirmed enteric fever. DATA COLLECTION AND ANALYSIS: Two authors independently assessed the trials' methodological quality and extracted data. We calculated odds ratios (OR) for dichotomous data with 95% confidence intervals (CI). We analysed trials with greater than 60% children separately from trials of mostly adults. MAIN RESULTS: Of 38 included trials, 22 had unclear allocation concealment and 34 did not use blinding. Four trials included exclusively children, seven had both adults and children, and three studied outpatients. ADULTS: Among primary outcomes (clinical failure, microbiological failure, and relapse), compared with chloramphenicol, fluoroquinolones were not statistically significantly different for clinical failure (594 participants) or microbiological failure (378 participants), but they reduced clinical relapse (OR 0.14, 95% CI 0.04 to 0.50; 467 participants, 6 trials). We detected no statistically significant difference versus co-trimoxazole (82 participants, 2 trials) or azithromycin (152 participants, 2 trials). Fluoroquinolones reduced clinical failure compared with ceftriaxone (OR 0.08, 95% CI 0.01 to 0.45; 120 participants, 3 trials), but not microbiological failure or relapse. Versus cefixime, fluoroquinolones reduced clinical failure (OR 0.05, 95% CI 0.01 to 0.24; 238 participants; 2 trials) and relapse (OR 0.18, 95% CI 0.03 to 0.91; 218 participants, 2 trials). CHILDREN: In children with high proportions of nalidixic acid-resistant strains, older fluoroquinolones increased clinical failures compared with azithromycin (OR 2.67, 95% CI 1.16 to 6.11; 125 participants, 1 trial), with no differences using newer fluoroquinolones (285 participants, 1 trial). Fluoroquinolones and cefixime were not statistically significantly different (82 participants, 1 trial). Trials comparing different durations of fluoroquinolone treatment were not statistically significantly different (889 participants, 9 trials). Norfloxacin had more clinical failures than other fluoroquinolones (417 participants, 5 trials). AUTHORS' CONCLUSIONS: Trials were small and methodological quality varied. In adults, fluoroquinolones may be better for reducing clinical relapse rates compared to chloramphenicol. Data are limited for other comparisons, particularly in children.