The Gut of Healthy Infants in the Community as a Reservoir of ESBL and Carbapenemase-Producing Bacteria.

The recent rapid rise of multi-drug resistant Enterobacteriaceae (MDR-E) is threatening the treatment of common infectious diseases. Infections with such strains lead to increased mortality and morbidity. Using a cross-sectional study, we aimed to estimate the prevalence of gut colonization with extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae among healthy infants born in Pakistan, a setting with high incidence of MDR-E infections. Stool samples were collected from 104 healthy infants between the ages of 5 and 7 months. Enterobacteriaceae isolates were screened for resistance against several antimicrobial classes. Presence of ESBL and carbapenemase genes was determined using multiplex PCR. Sequence types were assigned to individual strains by multi-locus sequence typing. Phylogenetic analysis of Escherichia coli was done using the triplex PCR method. Forty-three percent of the infants were positive for ESBL-producing Enterobacteriaceae, the majority of which were E. coli. We identified several different ESBL E. coli sequence types most of which belonged to the phylogenetic group B2 (23%) or D (73%). The widespread colonization of infants in a developing country with ESBL-producing Enterobacteriaceae is concerning. The multiple sequence types and reported non-human sources support that multiple non-epidemic MDR lineages are circulating in Pakistan with healthy infants as a common reservoir.

Barriers to Implementation of Optimal Laboratory Biosafety Practices in Pakistan.

The primary goal of biosafety education is to ensure safe practices among workers in biomedical laboratories. Despite several educational workshops by the Pakistan Biological Safety Association (PBSA), compliance with safe practices among laboratory workers remains low. To determine barriers to implementation of recommended biosafety practices among biomedical laboratory workers in Pakistan, we conducted a questionnaire-based survey of participants attending 2 workshops focusing on biosafety practices in Karachi and Lahore in February 2015. Questionnaires were developed by modifying the BARRIERS scale in which respondents are required to rate barriers on a 1-4 scale. Nineteen of the original 29 barriers were included and subcategorized into 4 groups: awareness, material quality, presentation, and workplace barriers. Workshops were attended by 64 participants. Among barriers that were rated as moderate to great barriers by at least 50% of respondents were: lack of time to read biosafety guidelines (workplace subscale), lack of staff authorization to change/improve practice (workplace subscale), no career or self-improvement advantages to the staff for implementing optimal practices (workplace subscale), and unclear practice implications (presentation subscale). A lack of recognition for employees' rights and benefits in the workplace was found to be a predominant reason for a lack of compliance. Based on perceived barriers, substantial improvement in work environment, worker facilitation, and enabling are needed for achieving improved or optimal biosafety practices in Pakistan.

Laboratory Methods for Determining Etiology of Neonatal Infection at Population-based Sites in South Asia: The ANISA Study.

BACKGROUND: The Aetiology of Neonatal Infection in South Asia (ANISA) study aims to determine the etiology of neonatal infections in 5 population-based sites in Bangladesh, India and Pakistan. METHODS: The main laboratory challenges in ANISA were selection and consistent implementation of laboratory methods at participating sites with varied infrastructure. The other specific challenges included (1) specimen collection and transport to designated study laboratories and timely processing in rural settings; (2) minimal or nonexistent laboratory facilities at the field sites; (3) obtaining sufficient volumes of blood from enrolled infants aged 0-59 days and (4) caregivers' concerns about collection of clinical specimens from young infants. An additional challenge was selecting an appropriate molecular platform from multiple available options, all with limited field validation, for use in determining infection in young infants. CONCLUSIONS: This article describes how the challenges of specimen collection, transport and processing and implementation of laboratory methods have been addressed in the ANISA study. It also describes the measures taken to improve detection of microorganisms causing young infant infections by enhancing the sensitivity of existing laboratory methods for pathogen detection.

Classification of Blood Culture Isolates Into Contaminants and Pathogens on the Basis of Clinical and Laboratory Data.

The multisite community-based study, Aetiology of Neonatal Infection in South Asia (ANISA), uses blood culture as the gold standard for identifying the etiology of neonatal infection. Considering the importance of this age-old diagnostic tool and the risk of contamination, ANISA has employed rigorous measures to prevent contamination at all stages of blood collection, processing and culture. Because contamination may still occur, an independent expert group evaluates the routinely collected clinical and laboratory data to determine whether a blood culture isolate is a contaminant or a true pathogen. This article describes the methodology used by ANISA to determine whether a blood culture isolate is likely to be a true pathogen or a contaminant in neonatal sepsis.

Implementation of the ANISA Study in Karachi, Pakistan: Challenges and Solutions.

BACKGROUND: Aetiology of Neonatal Infection in South Asia (ANISA) is a multicenter study in Bangladesh, India and Pakistan exploring the incidence and etiology of neonatal infections. A periurban site in Karachi was selected for its representativeness of the general population in neonatal health indicators. An established demographic surveillance system and other infrastructure needed for conducting the study already existed at this site. ANISA presents a unique challenge because of the need to capture every birth outcome in the community within a few hours of delivery to reliably estimate the incidence and etiology of early-onset sepsis in a setting where home births and deaths are common. CONTEXTUAL CHALLENGES: Major challenges at the Karachi site are related to early birth reporting and newborn assessment for births outside the catchment areas, parental refusal to participate, diverse ethnicity of the population, collection of biological specimens from healthy controls, political instability and crime, power outages and blood culture contamination. Some of the remedial actions taken include prolonging working hours; developing counseling skills of field workers; hiring staff with different linguistic abilities from within the study community; liaising with health facilities, key community informants, Lady Health Workers and traditional birth attendants; hiring community mobilizers; enhancing community sensitization; developing contingency plans for field work interruptions and procuring backup generators. The specimen contamination rate has decreased through training, supervision and video monitoring of blood collection procedures with individualized counseling of phlebotomists. CONCLUSION: ANISA offers lessons for successful implementation of complex study protocols in areas of high child mortality and challenging social environments.

Challenges in Implementation of the ANISA Protocol at the Matiari Site, Pakistan.

BACKGROUND: The Aetiology of Neonatal Infection in South Asia (ANISA) study is a population-based study with sites in Bangladesh, India and Pakistan. It aims to determine community-acquired incidence, etiology and associated risk factors for neonatal infections. Matiari, a rural site in Pakistan, was chosen for the study due to its high neonatal mortality rate and the presence of an established pregnancy and birth surveillance system. This article summarizes various challenges, remedial measures taken and lessons learned during the implementation of the ANISA study protocol in the unique rural setting of Matiari where the majority of births take place at home and accessibility to health care is limited. CHALLENGES: Achieving and maintaining project targets of early registration of birth and collection of biological specimens in households have been challenging in Matiari. Capturing births of study subjects that occur outside the catchment areas and those taking place during public holidays and acquiring parental consent for specimen collection from healthy controls require extensive community mobilization. Contamination and power outages that affect the laboratory equipment are 2 of the major logistic challenges faced. We keep track of pregnancy outcomes through mobile phones and reimburse the costs for birth notifications to the caller. We created separate dedicated mobile teams that visit newborns outside the catchment area and carry out possible serious bacterial infection assessments. We also formed mobile teams for specimen collection from residences of newborns as there is no facility for specimen collection at this site. Our study personnel work on holidays and weekends to improve the study's performance. We nurture strong community liaison by employing staff from within the community. We train the study physicians on communication and counseling skills required for overcoming refusal for referral and specimen collection. The contamination rate is controlled by repeated training and supervision and extensive monitoring of phlebotomy activities. The majority of phlebotomy procedures are recorded on video in the field to provide feedback to phlebotomists for improving their performance. CONCLUSION: The contextual challenges faced in field implementation of the ANISA protocol in the rural setting of Matiari are unique. These challenges are being successfully addressed through hard work, strict monitoring and improvisation. This experience can be used for improving study performance in similar settings elsewhere.

Diagnostic methods to determine microbiology of postpartum endometritis in South Asia: laboratory methods protocol used in the Postpartum Sepsis Study: a prospective cohort study.

BACKGROUND: The South Asian region has the second highest risk of maternal death in the world. To prevent maternal deaths due to sepsis and to decrease the maternal mortality ratio as per the World Health Organization Millenium Development Goals, a better understanding of the etiology of endometritis and related sepsis is required. We describe microbiological laboratory methods used in the maternal Postpartum Sepsis Study, which was conducted in Bangladesh and Pakistan, two populous countries in South Asia. METHODS/DESIGN: Postpartum maternal fever in the community was evaluated by a physician and blood and urine were collected for routine analysis and culture. If endometritis was suspected, an endometrial brush sample was collected in the hospital for aerobic and anaerobic culture and molecular detection of bacterial etiologic agents (previously identified and/or plausible). DISCUSSION: The results emanating from this study will provide microbiologic evidence of the etiology and susceptibility pattern of agents recovered from patients with postpartum fever in South Asia, data critical for the development of evidence-based algorithms for management of postpartum fever in the region.

Pneumococcal serotypes and serogroups causing invasive disease in Pakistan, 2005-2013.

While pneumococcal conjugate vaccines have been implemented in most countries worldwide, use in Asia has lagged in part because of a lack of data on the amount of disease that is vaccine preventable in the region. We describe pneumococcal serotypes elicited from 111 episodes of invasive pneumococcal disease (IPD) from 2005 to 2013 among children and adults in Pakistan. Seventy-three percent (n = 81) of 111 IPD episodes were cases of meningitis (n = 76 in children 0-15 years and n = 5 among adults). Serotypes were determined by target amplification of DNA extracted from pneumococcal isolates (n = 52) or CSF specimens (n = 59). Serogroup 18 was the most common serogroup causing meningitis in children <5 years, accounting for 21% of cases (n = 13). The 10-valent pneumococcal conjugate vaccine (PCV 10) or PCV10- related serotypes were found in 61% (n = 47) of childhood (age 0-15 years) meningitis episodes. PCV-13 increased this coverage to 63% (one additional serotype 19A; n = 48). Our data indicate that use of PCVs would prevent a large proportion of serious pneumococcal disease.