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

Saha, Samir K. PhD; Islam, Mohammad Shahidul MSc; Qureshi, Shahida M. MSc; Hossain, Belal MSc; Islam, Maksuda BA; Zaidi, Anita K. SM; Modak, Joyanta K. MSc; Al-Emran, Hasan M. MSc; Diaz, Maureen H. PhD; McGee, Lesley PhD; Winchell, Jonas M. PhDfor the ANISA Methods Group

The Pediatric Infectious Disease Journal: May 2016 - Volume 35 - Issue 5 - p S16–S22
doi: 10.1097/INF.0000000000001101
ANISA Supplement

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.

From the *Child Health Research Foundation, Dhaka, Bangladesh; The Aga Khan University, Karachi, Pakistan; and Respiratory Disease Branch, Centers for Disease Control and Prevention, Atlanta, Georgia.

Accepted for publication January 10, 2016.

S.K.S. and A.K.Z. were primarily responsible for the study design. S.K.S., J.M.W., M.H.D., M.S.I. and L.M. designed and developed the TAC panels. B.H., M.I., S.M.Q., J.K.M., M.H.A., M.D. and L.M. participated in different aspects of formative research and implementation of the protocol. S.K.S. drafted the manuscript. All authors reviewed and edited subsequent drafts.

The ANISA study is funded by the Bill & Melinda Gates Foundation (grant no. OPPGH5307). The authors have no conflicts of interest to disclose.

Address for correspondence: Samir K. Saha, PhD, Department of Microbiology, Dhaka Shishu (Children’s) Hospital, Sher-E-Bangla Nagar, Dhaka 1207, Bangladesh. E-mail:

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially.

Although one third of global neonatal deaths occur in Bangladesh, India and Pakistan,1 little is known about the etiology of community-acquired neonatal infections in South Asia. Most previous studies in this region have focused on hospitalized patients and thus included nosocomial infections. The limited available data on community-acquired infections are mainly from outpatient-based clinical studies with limited etiologic characterization.2,3 Finally, the few available population-based studies reporting etiology of neonatal infections have relied solely on bacterial culture for diagnostic evaluation despite significant advances in laboratory diagnostics.4,5

Data on etiology of community-acquired neonatal sepsis in South Asia are scarce,5 which is not surprising considering the numerous challenges in performing an etiology study with newborns in this region. Specific challenges may hinder the investigation of neonatal infections, including (1) lack of laboratory infrastructure and modern diagnostics; (2) reaching infants at home where most births take place; (3) collection of blood from newborns and (4) timely and temperature-controlled specimen transport from remote settings. The Aetiology of Neonatal Infection in South Asia (ANISA) project is a comprehensive population-based study to better understand the etiologies of infection in young infants up to 2 months of age at 5 sites in Bangladesh, India and Pakistan. The approaches and solutions to overcome specific challenges identified in the ANISA study can be extrapolated to other large-scale studies of newborns in similar communities.

In this article, we describe the laboratory methods of ANISA, including the rationale for molecular platform selection and the associated challenges of implementing this testing in difficult field settings. We also discuss measures taken to improve the detection of a wide range of bacteria and viruses through molecular testing of blood and respiratory specimens along with the complementary traditional culture methods employed.

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The ANISA study aims to collect blood and nasopharyngeal-oropharyngeal (NP-OP) swab specimens from all young infants (0–59 days of age) with possible serious bacterial infection (pSBI) in the surveillance area, defined as physician confirmation of any one of the clinical signs outlined by the World Health Organization.6 Cerebrospinal fluid (CSF) is collected from pSBI cases with clinical signs of meningitis.7 Both blood and CSF specimens are tested by bacterial culture and molecular assays, whereas NP-OP specimens are tested by molecular methods only. To understand the significance of detected viruses and bacteria in specimens from pSBI cases, we also enrolled and collected NP-OP and blood specimens from a subset of age- and seasonality-matched healthy controls for molecular testing.8

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Blood and CSF specimens are collected by applying standard clinical procedures.9 In addition, ANISA uses a stringent protocol to reduce the rate of contamination during specimen collection. Adequate volume of blood is an important requirement for successful blood culture, as inadequate volume frequently leads to false-negative results.10 In addition to blood culture, ANISA also aims to collect blood specimens for molecular assays and biobanking, for future testing with new diagnostics. Considering these needs and emphasizing blood culture as the gold standard for detection of infection etiology, ANISA developed a priority algorithm for the allocation of blood for culture, molecular tests and biobanking to ensure that culture bottles are inoculated with the maximum available blood volumes (Table 1). Volume is tracked at each step: the amount of blood drawn is recorded by a phlebotomist; the quantity inoculated in the culture bottle is measured by weighing the preinoculated and postinoculated bottle; and the amount placed in the ethylenediaminetetraacetic acid tube for molecular tests and biorepository are recorded by laboratory personnel.



The primary aim of ANISA is to identify predominant etiological agents causing infection in newborns. Therefore, in addition to blood, we collect respiratory swabs, including both NP and OP swabs, taking into account that some microorganisms are selectively predominant in 1 of the 2 niches.11 We use flocked swabs (Copan Diagnostics, Brescia, Italy), which consist of thousands of short, perpendicular, polyamide bristles for collecting NP-OP specimens.12 This swab can hold a large volume of specimen and spontaneously elutes the whole specimen into liquid medium within a few seconds.13 After collection, both the NP and OP swabs are placed together in the same vial containing universal transport medium (Copan Diagnostics).

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Specimens are collected primarily at community-level clinics. In these settings, caregivers may be apprehensive about the procedures for clinical specimens, and the risk of blood culture contamination remains high. Venous blood is collected before NP-OP swabs to avoid agitating the baby to obtain an optimal volume of blood and limiting the possibility of contamination. CSF specimens are collected only from hospitalized newborns with clinical signs of meningitis.

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Specimen transportation modalities are customized according to the location and transportation facilities available at the specific sites. None of the community-based rural and peri-urban ANISA sites has a comprehensive specimen processing facility in the area of case enrollment. Therefore, sites transport the specimens to the local ANISA site laboratories for processing and testing. Blood culture bottles inoculated at collection sites are transported at ambient temperature and ethylenediaminetetraacetic acid blood and NP-OP specimens at 4°C. For remote sites (Sylhet, Bangladesh, and Matiari, Pakistan), specimens are aliquoted and stored (−20°C) at ANISA field laboratories and transported weekly in liquid nitrogen to other laboratories with molecular testing capacity. For quality control, the temperature of specimens is monitored by placing a thermometer inside the transport containers, and the temperature is recorded in a log book at the times of dispatch and receipt at the laboratory (Fig. 1).



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Although blood culture remains the primary means to determine sepsis etiology, this important diagnostic approach can only support the isolation of certain bacterial species. However, infections may also be caused by viruses and other atypical bacteria that are not able to be recovered using traditional blood culture methods. In addition, sensitivity of blood culture can be compromised because of previous antibiotic therapy, suboptimal blood volume, contamination and/or low levels of bacteremia. To obtain a comprehensive assessment of potential pathogens, we perform real-time polymerase chain reaction (PCR) tests on NP-OP and whole blood specimens for the detection of specific bacteria, viruses and other atypical organisms that may cause infection in newborns in this setting.

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Automated blood culture techniques have improved significantly in recent years to support the growth of fastidious organisms.14 Furthermore, automated blood culture systems reduce workload by limiting subculture; the machine provides a signal for expected growth-positive bottles. Therefore, we use an automated blood culture system and specific bottles for pediatric patients (BACTEC, Becton, Dickinson and Company, Franklin Lakes, NJ or BacT/Alert®3D, Biomerieux, Marcy l'Etoile, France) at all field laboratories.

We also monitor and record the time to positivity (TTP) and delayed vial entry (DVE) and consider these parameters when interpreting blood culture results. To minimize deviation at the sites, ANISA provided a study-specific protocol for standardization and interpretation of blood culture methods across all sites.

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The automated blood culture machine sounds an alarm (beeps) when microbial growth is detected in a blood culture bottle. For beep-positive bottles, the BACTEC system records TTP based on the time the bottle is placed into the instrument to the time of the alarm. This can be used as a proxy for quantitative culture as the value of TTP is inversely proportional to the magnitude of bacteremia, depending on the type of organism.15 During the interim analysis and at the end of the study, duration of TTP along with other parameters will be used in classifying the isolates as true pathogens or contaminants.16

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As per manufacturer’s recommendations, blood culture bottles should be loaded into the BACTEC machine immediately after inoculation. However, real-time loading of bottles is not feasible at most ANISA sites because of the distance between the place of specimen collection and the field laboratories. Studies have shown that DVE up to 12 hours at room temperature has no significant impact on results.17 Considering the possible diversity of pathogens in newborns and environmental temperatures at ANISA sites, we carried out a formative study to mimic the field situation of Sylhet, Bangladesh, where the possibility of DVE is the highest. Blood culture bottles inoculated with 5–10 cfu/mL of Klebsiella pneumoniae or Streptococcus pneumoniae were held at temperatures of 20 and 37°C for 6 and 10 hours, before placing them in the BACTEC machine. Experiments were conducted in duplicate, and average TTP was recorded. Every inoculated bottle resulted in a beep. However, TTP was considerably shorter for specimens with a DVE of 10 hours at 37°C, specifically for K. pneumoniae. On the basis of literature review, manufacturer’s instructions and our formative research findings, we conservatively decided to transport the bottles at room temperature, record the time of blood collection and of loading into the BACTEC machine and subculture the aspirates from blood culture bottles before loading into the BACTEC machine if the delay between collection and loading was more than 8 hours. The subculture step is expected to facilitate earlier isolation of bacteria in bacteremic cases because the organism is expected to multiply significantly during the delay. It should also avoid false beep negativity caused by significant bacterial growth in the bottle before loading it into the machine.

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Immunochromatographic Test

In an automated blood culture system, it is not uncommon to have a machine-positive case (beep positive) without any growth on subculture (beep positive but culture negative [BPCN]). This can be because of erratic functioning of the machine leading to false alarms, or autolysis of bacteria because of delay in subculturing, especially true for S. pneumoniae after its exponential growth phase.18 Because pneumococcus is commonly detected in South Asia in the 0- to 59-day age group,19 and BPCN cases occur more commonly with pneumococcus, we included the pneumococcal BinaxNOW® (Portland, ME) test for all ANISA BPCN specimens to capture cases that are missed by blood culture.

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Culturing Contaminated Specimens on Selective Media

Extensive measures were taken to minimize potential contamination of blood cultures; a maximum of 10% of specimens was selected as the tolerable limit of contamination based on the standard practice guidelines and practical considerations of field laboratory settings. Contamination may preclude the detection of slow-growing true pathogens such as S. pneumoniae and Haemophilus influenzae, which are intrinsically resistant to aminoglycoside and bacitracin, respectively.20,21 We performed culture of all contaminated blood cultures on sheep blood agar with gentamicin and chocolate agar with bacitracin to unmask these pathogens by inhibiting other potentially faster growing contaminating organisms.21

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Molecular Tests

Because blood culture has limited sensitivity, we additionally employ molecular diagnostics to potentially improve detection of specific bacterial and viral etiologies within whole blood. Multipathogen molecular testing is also applied to NP-OP specimens. Although newer molecular methods may improve detection of microorganisms, none of these has proven to be more sensitive than blood culture, particularly in detecting pathogens from neonatal blood specimens.22 However, these methods can use “add-on” tests to increase the overall probability of detecting etiology within blood and NP-OP specimens.

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Selection of Molecular Platform

An appropriate molecular method is important for simultaneous detection of diverse bacteria and viruses (including both RNA and DNA viruses) in multiple specimen types (respiratory and blood) to determine the etiology of pSBI cases. Based on recommendations from the Pneumonia Etiology Research for Child Health study team,23 the following platforms were compared for suitability in the context of ANISA: (1) MassTag PCR, (2) Taqman Array Card (TAC) and (3) Fast-track Diagnostics respiratory pathogen panel (Fast-track Diagnostics, Malta). These methods and platforms were compared based on the performance characteristics (sensitivity/specificity), flexibility in customization for detection of selected pathogens, required specimen volume, user friendliness for laboratories with limited molecular testing experience, risk of contamination and warranty coverage or availability of technical support from manufacturer (Table 2).



We selected the TAC platform (Life Technologies, Foster City, CA) for ANISA.25 This method has the following criteria:

  1. Customized design including multiple parallel singleplex real-time reverse transcriptase PCR assays
    1. Allows testing for select bacteria and viruses predicted to cause infection in newborns rather than a generic panel of potential pathogenic microorganisms.
    2. No loss of sensitivity as often seen during multiplexing.
  2. Minimum specimen volume (50 μL) required for simultaneous testing selected target pathogens on a single card (22 pathogens).
  3. Simple assay setup, including minimal reagent preparation, minimizing the possibility of error during specimen processing.
  4. Closed system with limited possibility of cross-contamination.

In addition, we use separate cards for blood (and CSF) and NP-OP specimens to detect more diverse etiological agents in the same patient (Fig. 2A and B).



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Extraction of Total Nucleic Acid

Optimal extraction of total nucleic acid from blood is challenging as it contains substances that may interfere with nucleic acid extraction or inhibit downstream applications such as PCR. The ANISA laboratory team developed a protocol to improve nucleic acid extraction performance, including implementation of a prelysis treatment step and use of an automated extraction platform (MagNA Pure Compact, Roche Applied Science, Indianapolis, IN) as previously described.25

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Selection of Target Pathogens

Between specific TACs for testing NP-OP and blood specimens, each patient is tested for 28 etiological agents, including 15 bacteria and 13 viruses. NP-OP and blood specimens are tested for 22 and 12 organisms, respectively (Fig. 2A and B). However, the list of definite, probable and potential pathogens for newborn sepsis is more extensive. To generate the most appropriate and comprehensive custom panel of agents for testing respiratory and blood specimens from newborns in South Asia, we performed a literature review to prepare a broad list of the most common pathogens identified in neonatal blood and respiratory (NP-OP) specimens. This list of pathogens was refined by applying the Delphi method. Briefly, a list of potential pathogens for blood and respiratory specimens was shared with 9 external experts to rank the pathogens in hierarchical tiers based on their perceived potential for causing infection in young infants in South Asia. The final list of pathogens for blood and NP-OP cards was selected by the ANISA coordination team and technical advisory group (TAG) members. Additional changes were made to the custom TAC designs, including an increased number of assay replicates on the ANISA TAC for blood specimens (Fig. 2B) and removal of some assays, based on the findings of testing during the study pilot phase (Fig. 2A and B).

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Testing of Specimens from Healthy Control Infants

Detection of certain bacteria and respiratory viruses, particularly in NP-OP specimens, does not alone establish an organism as the cause of a pSBI episode. Some viruses and bacteria may be present as natural colonizers of the respiratory tract. Past studies with respiratory specimens have shown that the prevalence of such organisms can vary across populations and age groups and by season.26 For blood specimens, there is biologic plausibility that molecular tests with higher sensitivity and ability to detect the genome of nonviable organisms may also result in the detection of potential infection-causing organisms’ genome in the blood of clinically healthy young infants.

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The quality of ANISA data, which will ensure correct study conclusions, largely depends on consistent high-quality laboratory performance in multiple field sites in 3 different countries. Identification of inconsistent practices or unexpected laboratory results is a challenge for any large study. The task becomes particularly difficult if the final result has only been noted on a hard copy laboratory book and tests cannot be repeated (Davidson H. Hamer, personal communication, 2009). ANISA data capture forms (DCFs) were created to document the relevant details of every specimen processing step, such as prelysis and nucleic acid extraction specimens and physical, biochemical and serological characterization of blood culture isolates. DCFs are tailored to capture details of specimens identified as contaminated to learn about the contamination and ensure that the recovered organism(s) is properly classified. These DCFs can replace traditional laboratory notebooks; opportunities for transcription errors are limited by built-in checks in the data entry system.27 All laboratory data on DCFs are entered into ANISA databases at the respective sites in real time by the laboratory personnel performing the tests. The data are transferred weekly to the central data server for all sites in Dhaka, Bangladesh, and monitoring reports are generated routinely. These features are intended to minimize the frequency of error during data entry and transfer and to decrease the probability of data loss. Routine monitoring of laboratory data by the ANISA coordination team ensures prompt identification and resolution of errors in specimen collection, transport and testing activities.28

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The ANISA study coordination team is supported by an experienced multidisciplinary TAG that includes clinicians, epidemiologists and laboratory experts in both molecular and microbiologic methods related to bacteria and viruses. The coordination team and TAG utilized a literature review, knowledge of the local demography of participating sites and personal understanding of site-specific needs to design the study procedures described here and throughout this supplement. When necessary and possible, laboratory decisions were also informed by formative research, focused experiments and testing of pilot specimens. The approaches described were developed to address specific challenges faced in ANISA but may be applicable to future population-based etiology studies in resource-limited settings. Laboratory results generated during ANISA will provide additional validation data to support the use of selected methods at the field settings of future studies.

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The authors thank their colleagues at the US Centers for Disease Control and Prevention for their technical assistance in development and implementation of the molecular test systems for ANISA. They also acknowledge the input of experts from around the world for helping them choose the etiological agents to be included in the molecular test platforms.

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ANISA; etiology; neonatal; sepsis; blood culture; molecular diagnostics

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