Infectious aetiologies of neonatal illness in south Asia classified using WHO definitions: a primary analysis of the ANISA study.

BACKGROUND: Globally, neonatal mortality accounts for almost half of all deaths in children younger than 5 years. Aetiological agents of neonatal infection are difficult to identify because the clinical signs are non-specific. Using data from the Aetiology of Neonatal Infections in south Asia (ANISA) cohort, we aimed to describe the spectrum of infectious aetiologies of acute neonatal illness categorised post-hoc using the 2015 WHO case definitions of critical illness, clinical severe infection, and fast breathing only. METHODS: Eligible infants were aged 0-59 days with possible serious bacterial infection and healthy infants enrolled in the ANISA study in Bangladesh, India, and Pakistan. We applied a partial latent class Bayesian model to estimate the prevalence of 27 pathogens detectable on PCR, pathogens detected by blood culture only, and illness not attributed to any infectious aetiology. Infants with at least one clinical specimen available were included in the analysis. We assessed the prevalence of these aetiologies according to WHO's case definitions of critically ill, clinical severe infection, and infants with late onset, isolated fast breathing. For the clinical severe definition, we compared the prevalence of signs by bacterial versus viral aetiology. FINDINGS: There were 934 infants (992 episodes) in the critically ill category, 3769 (4000 episodes) in the clinical severe infection category, and 738 (771 episodes) in the late-onset isolated fast breathing category. We estimated the proportion of illness attributable to bacterial infection was 32·7% in infants in the critically ill group, 15·6% in the clinical severe infection group, and 8·8% among infants with late-onset isolated fast breathing group. An infectious aetiology was not identified in 58-82% of infants in these categories. Among 4000 episodes of clinical severe infection, those with bacterial versus viral attribution had higher proportions of hypothermia, movement only when stimulated, convulsions, and poor feeding. INTERPRETATION: Our modelled results generally support the revised WHO case definitions, although a revision of the most severe case definition could be considered. Clinical criteria do not clearly differentiate between young infants with and without infectious aetiologies. Our results highlight the need for improved point-of-care diagnostics, and further study into neonatal deaths and episodes with no identified aetiology, to ensure antibiotic stewardship and targeted interventions. FUNDING: The Bill and Melinda Gates Foundation.

Opinions of the Urban Women on Pap test: Evidence from Bangladesh.

Each year, many countries from developed world publishes reports on early cancer detection; which is absolutely absent in most developing countries like Bangladesh.Very limited evidence is found on the role and acceptance of Pap test among the women of Bangladesh in determining cervical cancer. More research and updates are needed relating Pap test in early detection of cervical cancer. Thus the purpose of this study is set to assess the opinions of Bangladeshiurban womentowardsthe Pap test. A questionnaire-based survey of 400 Bangladeshi urban women was evaluated by on their socio-demographic characteristics, knowledgeand attitudes towards Pap testing. In general, the findings reveal that respondents havea good understanding of thepurpose of Pap test screening with 3.92 (Mean score). With 3.54 Mean score,the respondents believed that Pap tests are recommended to women who are married and with 3.45 mean score women believed that Pap tests arerecommended only to those who have children. Generally, respondents possess good knowledge of Pap test and its purpose. These findings can be used in identifying prospect cervical cancer screening significance populations and trend for future intrusion.

The association between daily 500 mg calcium supplementation and lower pregnancy-induced hypertension risk in Bangladesh.

BACKGROUND: Evidence suggests that daily supplementation of 1500 to 2000 mg of calcium during pregnancy reduces pregnancy-induced hypertension (PIH). However, the evidence on the efficacy of low-dose calcium supplementation on PIH is limited. This paper assesses the longitudinal correlation between low-dose calcium intake (500 mg daily) and change in blood pressure during pregnancy among a homogeneous population in terms of hypertension and pre-eclampsia. METHODS: The study followed a retrospective cohort study design, and was carried out among 11,387 pregnant women from 10 rural upazilas (sub-districts) of Bangladesh where maternal nutrition initiative (MNI), implemented by Building Resources Across Communities (BRAC), was ongoing. The modified Poisson regression model was used to estimate the association (risk ratio) between consumption of calcium tablets and PIH. RESULTS: The present research found that women who consumed 500 mg/d calcium tablets for more than 6 months during their pregnancy had a 45% lower risk of developing hypertension compared to those who consumed less calcium (RR = 0.55, 95% CI = 0.33-0.93). CONCLUSIONS: Daily supplementation of 500 mg oral calcium during pregnancy for at least 180 tablets is associated with a considerably reduced risk of PIH, but this study is unable to confirm whether this association is causal. The causal relationship needs to be confirmed through a large scale randomized controlled trial.

Epidemiology and risk factors for pneumonia severity and mortality in Bangladeshi children <5 years of age before 10-valent pneumococcal conjugate vaccine introduction.

BACKGROUND: Pneumonia is the leading infectious cause of morbidity and mortality in young children in Bangladesh. We present the epidemiology of pneumonia in Bangladeshi children <5 years before 10-valent pneumococcal conjugate vaccine introduction and investigate factors associated with disease severity and mortality. METHODS: Children aged 2-59 months admitted to three Bangladeshi hospitals with pneumonia (i.e., cough or difficulty breathing and age-specific tachypnea without danger signs) or severe pneumonia (i.e., cough or difficulty breathing and ≥1 danger signs) were included. Demographic, clinical, laboratory, and vaccine history data were collected. We assessed associations between characteristics and pneumonia severity and mortality using multivariable logistic regression. RESULTS: Among 3639 Bangladeshi children with pneumonia, 61% had severe disease, and 2% died. Factors independently associated with severe pneumonia included ages 2-5 months (adjusted odds ratio [aOR] 1.60 [95% CI: 1.26-2.01]) and 6-11 months (aOR 1.31 [1.10-1.56]) relative to 12-59 months, low weight for age (aOR 1.22 [1.04-1.42]), unsafe drinking water source (aOR 2.00 [1.50-2.69]), higher paternal education (aOR 1.34 [1.15-1.57]), higher maternal education (aOR 0.74 [0.64-0.87]), and being fully vaccinated for age with pentavalent vaccination (aOR 0.64 [0.51-0.82]). Increased risk of pneumonia mortality was associated with age <12 months, low weight for age, unsafe drinking water source, lower paternal education, disease severity, and having ≥1 co-morbid condition. CONCLUSIONS: Modifiable factors for severe pneumonia and mortality included low weight for age and access to safe drinking water. Improving vaccination status could decrease disease severity.

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.

Centralized Data Management in a Multicountry, Multisite Population-based Study.

BACKGROUND: A centralized data management system was developed for data collection and processing for the Aetiology of Neonatal Infection in South Asia (ANISA) study. ANISA is a longitudinal cohort study involving neonatal infection surveillance and etiology detection in multiple sites in South Asia. The primary goal of designing such a system was to collect and store data from different sites in a standardized way to pool the data for analysis. METHODS: We designed the data management system centrally and implemented it to enable data entry at individual sites. This system uses validation rules and audit that reduce errors. The study sites employ a dual data entry method to minimize keystroke errors. They upload collected data weekly to a central server via internet to create a pooled central database. Any inconsistent data identified in the central database are flagged and corrected after discussion with the relevant site. The ANISA Data Coordination Centre in Dhaka provides technical support for operations, maintenance and updating the data management system centrally. Password-protected login identifications and audit trails are maintained for the management system to ensure the integrity and safety of stored data. CONCLUSION: Centralized management of the ANISA database helps to use common data capture forms (DCFs), adapted to site-specific contextual requirements. DCFs and data entry interfaces allow on-site data entry. This reduces the workload as DCFs do not need to be shipped to a single location for entry. It also improves data quality as all collected data from ANISA goes through the same quality check and cleaning process.

Development and Implementation of the ANISA Labeling and Tracking System for Biological Specimens.

BACKGROUND: The Aetiology of Neonatal Infection in South Asia study is a major effort to determine the causes of community-acquired neonatal infections. It involves collecting epidemiological, clinical and laboratory data in 5 sites in 3 countries. The field and laboratory research operations are streamlined to maintain integrity and validity while operating in complex and variable environments. We developed a customized system for implementation of labeling and tracking biological specimen in both rural and urban community settings and integrated into all study laboratories. This report outlines the development and implementation of this harmonized system. DESIGN: The system links and tracks specimens with study participants and results generated from laboratory tests. Each biological specimen and its aliquots are tracked through key steps of the protocol, from collection and transport through molecular testing and long-term storage. CONCLUSION: The labeling and tracking system allows for standardization and monitoring of laboratory processes and improves the accuracy of Aetiology of Neonatal Infection in South Asia data. Community-based scientific projects could greatly benefit by adopting this, or a similar, system for specimen tracking and data linkage.

Using Text Messages for Critical Real-time Data Capture in the ANISA Study.

BACKGROUND: The Aetiology of Neonatal Infection in South Asia (ANISA) study takes advantage of text messaging technology to record information required for randomizing the study population into a control subcohort. The text message system is also used for monitoring various study activities. METHODS: When a child-health worker registers a newborn in the study, she sends a text message to a database server containing the study identification number and newborn's age at the time of registration. For each possible serious bacterial infection case, a study physician also sends a text message to the same server with the age of the young infant at the time of illness assessment. Using this information, a computer-based algorithm randomizes the newborn into a control subcohort. Text messages are also sent to alert the study physicians and study supervisors of a possible serious bacterial infection case being referred to health-care facilities. Phlebotomists working at remote specimen collection sites send text messages to the site laboratory personnel before sending the specimens through porters. DISCUSSION: Real-time data entry and monitoring are challenging for any population-based study conducted in remote areas. Our text messaging system provides an opportunity to overcome this barrier where availability of data entry facilities is limited.

Understanding Bacterial Isolates in Blood Culture and Approaches Used to Define Bacteria as Contaminants: A Literature Review.

BACKGROUND: Interpretation of blood culture isolates is challenging due to a lack of standard methodologies for identifying contaminants. This problem becomes more complex when the specimens are from sick young infants, as a wide range of bacteria can cause illness among this group. METHODS: We used 43 key words to find articles published between 1970 and 2011 on blood culture isolates and possible contaminants in the PubMed database. Experts were also consulted to obtain other relevant articles. Selection of articles followed systematic methods considering opinions from more than 1 reviewer. RESULTS: After reviewing the titles of 3869 articles extracted from the database, we found 307 relevant to our objective. Based on the abstracts, 42 articles were selected for the literature review. In addition, we included 7 more articles based on cross-references and expert advice. The most common methods for differentiating blood culture isolates were multiple blood cultures from the same subject, antibiograms and molecular testing. Streptococcus pneumoniae, Hemophilus influenzae, Neisseria meningitidis and group A and B streptococcus were always considered as pathogens, whereas Bacillus sp., Diphtheroids, Propionibacterium and Micrococcus were commonly regarded as contaminants. Coagulase-negative staphylococci were the most frequent isolates and usually reported as contaminants unless the patient had a specific condition, such as long-term hospitalization or use of invasive devices (catheters). CONCLUSIONS: Inaccurate interpretation of blood culture may falsely guide treatment and also has long-term policy implications. The combination of clinical and microbiological knowledge, patient's clinical history and laboratory findings are essential for appropriate interpretation of blood culture.

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.

Rotavirus Surveillance at a WHO-Coordinated Invasive Bacterial Disease Surveillance Site in Bangladesh: A Feasibility Study to Integrate Two Surveillance Systems.

The World Health Organization (WHO) currently coordinates rotavirus diarrhea and invasive bacterial disease (IBD) surveillance at 178 sentinel sites in 60 countries. However, only 78 sites participate in both surveillance systems using a common sentinel site. Here, we explored the feasibility of extending a WHO-IBD surveillance platform to generate data on the burden of rotaviral diarrhea and its epidemiological characteristics to prepare the countries to measure the impact of rotaviral vaccine. A six-month (July to December, 2012) surveillance, managed by IBD team, collected stool samples and clinical data from under-five children with acute watery diarrhea at an IBD sentinel site. Samples were tested for rotavirus antigen by ELISA and genotyped by PCR at the regional reference laboratory (RRL). Specimens were collected from 79% (n=297) of eligible cases (n=375); 100% of which were tested for rotavirus by ELISA and 54% (159/297) of them were positive. At RRL, all the cases were confirmed by PCR and genotyped (99%; 158/159). The typing results revealed the predominance of G12 (40%; 64/159) genotype, followed by G1 (31%; 50/159) and G9 (19%; 31/159). All in all, this exploratory surveillance collected the desired demographic and epidemiological data and achieved almost all the benchmark indicators of WHO, starting from enrollment number to quality assurance through a number of case detection, collection, and testing of specimens and genotyping of strains at RRL. The success of this WHO-IBD site in achieving these benchmark indicators of WHO can be used by WHO as a proof-of-concept for considering integration of rotavirus surveillance with WHO-IBD platforms, specifically in countries with well performing IBD site and no ongoing rotavirus surveillance.

Epidemiology of Invasive Pneumococcal Disease in Bangladeshi Children Before Introduction of Pneumococcal Conjugate Vaccine.

BACKGROUND: Because Bangladesh intended to introduce pneumococcal conjugate vaccine (PCV)-10 in 2015, we examined the baseline burden of invasive pneumococcal disease (IPD) to measure impact of PCV. METHODS: During 2007-2013, we performed blood and cerebrospinal fluid cultures in children <5 years old with suspected IPD identified through active surveillance at 4 hospitals. Isolates were serotyped by quellung and tested for antibiotic susceptibility by disc diffusion and E-test. Serotyping of culture-negative cases, detected by Binax or polymerase chain reaction, was done by sequential multiplex polymerase chain reaction. Trends in IPD case numbers were analyzed by serotype and clinical syndrome. RESULTS: The study identified 752 IPD cases; 78% occurred in children <12 months old. Serotype information was available for 78% (442/568), including 197 of 323 culture-negative cases available for serotyping. We identified 50 serotypes; the most common serotypes were 2 (16%), 1 (10 %), 6B (7%), 14 (7%) and 5 (7%). PCV-10 and PCV-13 serotypes accounted for 46% (range 29%-57% by year) and 50% (range 37%-64% by year) of cases, respectively. Potential serotype coverage for meningitis and nonmeningitis cases was 45% and 49% for PCV-10, and 48% and 57% for PCV-13, respectively. Eighty-two percent of strains were susceptible to all antibiotics except cotrimoxazole. CONCLUSION: The distribution of serotypes causing IPD in Bangladeshi children is diverse, limiting the proportion of IPD cases PCV can prevent. However, PCV introduction is expected to have major benefits as the country has a high burden of IPD-related mortality, morbidity and disability.

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods.

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of $87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.

Streptococcus pneumoniae serotype-2 childhood meningitis in Bangladesh: a newly recognized pneumococcal infection threat.

BACKGROUND: Streptococcus pneumoniae is a leading cause of meningitis in countries where pneumococcal conjugate vaccines (PCV) targeting commonly occurring serotypes are not routinely used. However, effectiveness of PCV would be jeopardized by emergence of invasive pneumococcal diseases (IPD) caused by serotypes which are not included in PCV. Systematic hospital based surveillance in Bangladesh was established and progressively improved to determine the pathogens causing childhood sepsis and meningitis. This also provided the foundation for determining the spectrum of serotypes causing IPD. This article reports an unprecedented upsurge of serotype 2, an uncommon pneumococcal serotype, without any known intervention. METHODS AND FINDINGS: Cases with suspected IPD had blood or cerebrospinal fluid (CSF) collected from the beginning of 2001 till 2009. Pneumococcal serotypes were determined by capsular swelling of isolates or PCR of culture-negative CSF specimens. Multicenter national surveillance, expanded from 2004, identified 45,437 patients with suspected bacteremia who were blood cultured and 10,618 suspected meningitis cases who had a lumber puncture. Pneumococcus accounted for 230 culture positive cases of meningitis in children <5 years. Serotype-2 was the leading cause of pneumococcal meningitis, accounting for 20.4% (45/221; 95% CI 15%-26%) of cases. Ninety eight percent (45/46) of these serotype-2 strains were isolated from meningitis cases, yielding the highest serotype-specific odds ratio for meningitis (29.6; 95% CI 3.4-256.3). The serotype-2 strains had three closely related pulsed field gel electrophoresis types. CONCLUSIONS: S. pneumoniae serotype-2 was found to possess an unusually high potential for causing meningitis and was the leading serotype-specific cause of childhood meningitis in Bangladesh over the past decade. Persisting disease occurrence or progressive spread would represent a major potential infection threat since serotype-2 is not included in PCVs currently licensed or under development.

Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.

BACKGROUND: PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. METHODOLOGY: Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. CONCLUSIONS: This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Pulsed hydrogen/deuterium exchange MS/MS for studying the relationship between noncovalent protein complexes in solution and in the gas phase after electrospray ionization.

Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.