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

Hossain, Belal MSc; Weber, Martin W. PhD; Hamer, Davidson H. MD; Hibberd, Patricia L. MD, PhD; Ahmed, A. S. M. Nawshad Uddin FCPS; Marzan, Mahfuza MSc; Islam, Maksuda BA; Connor, Nicholas E. MSc; Islam, Mohammad Shahidul MSc; Zaidi, Anita K. SM; Baqui, Abdullah H. DrPH; Bhutta, Zulfiqar A. PhD; Qureshi, Shahida M. MSc; Rafiqullah, Iftekhar MSc; McGee, Lesley PhD; Saha, Samir K. PhD

The Pediatric Infectious Disease Journal: May 2016 - Volume 35 - Issue 5 - p S52–S54
doi: 10.1097/INF.0000000000001107
ANISA Supplement

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.

From the *Child Health Research Foundation, Dhaka, Bangladesh; Child and Adolescent Health Department, World Health Organization, Geneva, Switzerland; Department of Global Health and Center for Global Health and Development, Boston University School of Public Health, Boston, Massachusetts; §Division of Global Health, Massachusetts General Hospital, Boston, Massachusetts; Centre for Child and Adolescent Health, International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh; Department of Paediatrics and Child Health, The Aga Khan University, Karachi, Pakistan; **Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland; and ††Streptococcus Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia.

Accepted for publication January 10, 2016.

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: Davidson H. Hamer, MD, Center for Global Health and Development, Crosstown 3rd Floor, 801 Massachusetts Avenue, Boston, MA 02118. 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.

Even with substantial improvements in global child health, about 6.5 million children younger than 5 years died in 2013. Two thirds (2.8 million) of these deaths occurred in the neonatal period (<28 days of age); 51% (1.4 million) of neonatal deaths are presumed to be because of infections.1,2 However, most estimates of infection-related deaths contain limited direct evidence on infections and their etiology.

As the most recent studies on etiology of neonatal sepsis are from a small population in Bangladesh, the results are not sufficient to inform targeted etiology-specific interventions to reduce deaths from neonatal infection.3,4 To fill this gap, we are conducting the Aetiology of Neonatal Infection in South Asia (ANISA) study in 5 population-based sites in Bangladesh, India and Pakistan. In this study, we collect blood and nasopharyngeal-oropharyngeal swabs from newborns aged 0–59 days who show 1 or more signs of possible serious bacterial infection.5 We test these specimens using both conventional and molecular methods to detect bacterial and viral pathogens.

Among the diagnostic methods for detecting bacterial etiology of neonatal infection, blood culture is still considered the gold standard. In recent years, blood culture methods have improved with the availability of enriched media that have passed quality control measures, with the ability to support the growth of fastidious organisms and neutralize the action of antibiotics present in the specimens. However, all these advances may be jeopardized because of contamination of blood culture leading to false-positive results. The main source of this contamination is skin flora, which often become the source of contamination in blood culture bottles during phlebotomy and specimen processing.6

Skin flora-mediated contamination is a major risk for the ANISA study because the skin surface of a South Asian baby is colonized with a high density of bacteria. Furthermore, these colonizing organisms are diverse, and many are known for being common etiologies of neonatal sepsis.7,8 Considering the aforementioned challenges, ANISA has taken stringent antisepsis measures and introduced monitoring schemes to avoid contamination during phlebotomy. However, despite all efforts, completely eliminating the access of skin flora and/or environmental bacteria into blood specimens during phlebotomy and inoculation of blood culture bottles is not possible. Contamination can also occur at the laboratory, while processing the specimens and culture plates. Taking these issues into account, the ANISA team designed laboratory data capture forms for detailed recording of all laboratory activities. These sources of information facilitate the evaluation of blood culture isolates and thus assist in classifying them as true pathogens, by teasing out contaminants based on the demographic, clinical and microbiologic data.

In this article, we describe the stepwise plan of the ANISA study for dealing with blood culture isolates and for determining their clinical significance for the respective infants.

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The ANISA study group convened a workshop on February 13–14, 2012, in Atlanta to plan the best way to perform valid blood cultures, ranging from phlebotomy to identification of potential pathogens. The workshop participants reached a consensus to designate isolates as (1) contaminants, (2) true pathogens or (3) probable pathogens. This classification was informed by an extensive literature review and experts’ analyses of neonatal infection etiology. Among these 3 groups, the contaminants and the true pathogens (Table 1) were classified using a predetermined approach based on the literature review9 and the opinions of the Technical Advisory Group members. The participants also decided that microbiologic characteristics of the common contaminants would be recorded in a standardized laboratory book and entered into a database for future review.



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This group of isolates is a challenge for the ANISA study for 3 reasons:

  1. Most of the probable pathogens are omnipresent in the South Asian environment.
  2. They frequently colonize the skin of newborns.6
  3. Colonizing organisms may also be causative agents of bloodstream infection in newborns.

The ANISA study introduced different tools for collection of additional data (Table 2) to enrich microbiologic and clinical information and facilitate the classification of probable pathogens as true pathogens or contaminants.



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In the ANISA protocol, growth of multiple organisms in blood culture is considered as a strong sign of contamination. This classification was made based on the study design that involves identifying infants with possible serious bacterial infection by active surveillance in the community and referring the cases to a study physician at the primary stage of disease. Infants having underlying diseases, congenital anomalies, a compromised immune system or recent hospitalization (to eliminate potential nosocomial acquisition) are excluded.

All cases of multiple isolates are thoroughly analyzed and decisions on their classification are made as per the protocol:

  1. Mixed growth with all contaminating organisms are discarded as contaminants.
  2. Any nonskin colonizing pathogen (eg, Streptococcus pneumoniae, Haemophilus influenzae) among mixed growths is considered as a true pathogen.;
  3. Skin colonizing true pathogens (Klebsiella pneumoniae, Escherichia coli, etc) and any mixture of probable pathogens are placed for expert review, as we do for pure isolates of probable pathogens (see above).

As mentioned in the ANISA microbiology methods article,10 specimens with multiple pathogens are also cultured on selective media (gentamicin blood agar for S. pneumoniae and bacitracin chocolate agar for H. influenzae) to determine whether the fastidious slow-growing true pathogens are masked by the fast-growing ones.

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On the basis of information mentioned in Table 2, a 4-member expert team (consisting of 2 infectious disease specialists, a pediatrician and a microbiologist) reviews all cases of probable and definite pathogens and mixed growths from blood culture. The team’s decisions are finalized by consensus. In case of disagreement or insufficient data to reach a conclusion, the expert team requests additional information from the database and the site teams on the respective cases and then reevaluates them. This stepwise review by the experts is based on the multiple criteria including (1) clinical condition of the particular infant at the time of enrollment; (2) identification of the pathogen and its role in causing sepsis; (3) antibiogram with minimal inhibitory concentration level or zone diameter; (4) antibiotic(s) given and their relationship to the susceptibility pattern; (5) clinical condition of the baby in the days after enrollment as a case and (6) finally, in case of treatment failure, the result of the second blood culture.

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The comprehensive evaluation of blood culture isolates will facilitate the identification of true cases of bloodstream infection. This process should generate high-quality data on blood culture-based etiology of neonatal infections and contribute to the analysis of disease burden data on neonatal sepsis and mortality in South Asia.

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1. Baqui AH, El-Arifeen S, Darmstadt GL, et al.Projahnmo Study Group. Effect of community-based newborn-care intervention package implemented through two service-delivery strategies in Sylhet district, Bangladesh: a cluster-randomised controlled trial. Lancet. 2008;371:1936–1944
2. Baqui AH, Darmstadt GL, Williams EK, et al. Rates, timing and causes of neonatal deaths in rural India: implications for neonatal health programmes. Bull World Health Organ. 2006;84:706–713
3. Farzin A, Saha SK, Baqui AH, et al.Bangladesh Projahnmo-2 (Mirzapur) Study Group. Population-based incidence and etiology of community-acquired neonatal viral infections in Bangladesh: a community-based and hospital-based surveillance study. Pediatr Infect Dis J. 2015;34:706–711
4. Darmstadt GL, Saha SK, Choi Y, et al.Bangladesh Projahnmo-2 (Mirzapur) Study Group. Population-based incidence and etiology of community-acquired neonatal bacteremia in Mirzapur, Bangladesh: an observational study. J Infect Dis. 2009;200:906–915
5. Islam MS, Baqui AH, Zaidi AK, et al. Infection surveillance protocol for a multicountry population-based study in South Asia to determine the incidence, etiology, and risk factors for infections among young infants 0 to 59 days old. Pediatr Infect Dis J. 2016;35(Suppl 1):S9–S15
6. Hall KK, Lyman JA. Updated review of blood culture contamination. Clin Microbiol Rev. 2006;19:788–802
7. Mullany LC, Saha SK, Shah R, et al. Impact of 4.0% chlorhexidine cord cleansing on the bacteriologic profile of the newborn umbilical stump in rural Sylhet District, Bangladesh: a community-based, cluster-randomized trial. Pediatr Infect Dis J. 2012;31:444–450
8. Choi Y, Saha SK, Ahmed AS, et al. Routine skin cultures in predicting sepsis pathogens among hospitalized preterm neonates in Bangladesh. Neonatology. 2008;94:123–131
9. Hossain B, Islam MS, Rahman A, et al. Understanding bacterial isolates in blood culture and approaches used to define bacteria as contaminants: a literature review. Pediatr Infect Dis J. 2016;35(Suppl 1):S45–S51
10. Saha SK, Islam MS, Qureshi SM, et al. Laboratory methods for determining etiology of neonatal infection at population-based sites in South Asia: the ANISA study. Pediatr Infect Dis J. 2016;35(Suppl 1):S16–S22

neonate; infection; blood culture; contamination; ANISA

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