Detection of Group B Streptococci in Lim Broth by Use of Group B Streptococcus Peptide Nucleic Acid Fluorescent In Situ Hybridization and Selective and Nonselective Agars

Abstract

The sensitivity, specificity, and positive and negative predictive values for the detection of group B streptococci from Lim enrichment broth with sheep blood agar (SBA), with selective Streptococcus agar (SSA), and by a peptide nucleic acid fluorescent in situ hybridization (PNA FISH) assay were as follows: for culture on SBA, 68.4%, 100%, 100%, and 87.9%, respectively; for culture on SSA, 85.5%, 100%, 100%, and 94.1%, respectively; and for the PNA FISH assay, 97.4%, 98.3%, 96.1%, and 98.9%, respectively.Streptococcus agalactiae, which is a member of the group B streptococci (GBS), is a gram-positive bacterium that can cause invasive disease in newborns (1,2,8). The aim of this study was to compare two types of media and the peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) assay for their abilities to detect GBS from Lim enrichment broth.Two hundred fifty-one genital swab specimens were obtained over a 6-week period from pregnant women as part of their routine diagnostic evaluation (8). All swabs were collected by the clinical staff by the use of routine protocols and sterile double swabs. For this institutional review board-approved study, the following assays were performed in a blinded manner (i.e., the results from each subsequently performed assay were unavailable to the person performing the next assay).Culture on sheep blood agar (SBA) was performed by laboratory staff by the following protocol. The swab specimens (both swabs in the dual swab specimen were used) were directly inoculated onto an SBA plate (plate 1) and then into a vial of Lim selective enrichment broth (which was incubated in 5 to 10% CO 2 at 37°C). Beta-hemolytic colonies were sought the next day, and if any were present, they were subcultured for purity. At this time, the Lim broth was also subcultured onto an SBA plate (plate 2). On the following day (day 3), the cultures plates were again examined and tested to determine if they contained GBS (see below). If GBS were not found on plate 1, then suspect colonies were sought on plate 2; and if any were present, they were subcultured for purity. Any potential GBS from the purity plate that were derived from plate 2 (day 4) were tested to determine if they were GBS.The Lim broth, following the initial phase of incubation described above, was also subcultured onto group A-selective Streptococcus agar (SSA) with 5% sheep blood (no. 221780; BD, Sparks, MD). The package insert indicates that this medium also supports the growth of GBS. This subculture was incubated as described above. On the first day following incubation, beta-hemolytic colonies were sought, and if any were present, they were subcultured for purity onto SBA (and incubated in 5 to 10% CO 2 at 37°C). These were subcultured for purity onto SBA rather than SSA because in our experience the performance of the GBS latex agglutination assay has been less reliable when it is done with colonies from the SSA plates than when it is done with colonies from SBA plates (data not shown). Following another 24 h of incubati...

Usefulness of a Rapid Real-time PCR Assay in Prenatal Screening for Group B Streptococcus Colonization

“…It also requires an experienced technician to identify non-beta-hemolytic GBS. To overcome these problems, more rapid and sensitive techniques have been developed, including DNA probe and nucleic acid amplification tests (NAAT) [13, 14]. Ke et al [15] developed an RT-PCR method based on amplification of a cfb gene fragment that is present in virtually every strain of GBS.…”

Intrapartum GBS screening and antibiotic prophylaxis: a European consensus conference

“…They compare well to subculture on selective differential media [95]. According to CDC's recommendations [2010], as far as the use of available nuclear acid amplification test (NAAT) for GBS after enrichment step increases sensitivity up to 90-100%, they could be used for antenatal screening at 35-37 weeks' gestation [4].…”

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

“…Though not limited to, PNA FISH-based assays have mainly been described for the detection of bacterial pathogens either in FFPE, media, or blood cultures. Many studies have described assays using PNAs for detecting and identifying numerous bacterial and fungal species including of Mycobacterium sp., Staphylococcus sp., E. coli, Pseudomonas sp., Klebsiella sp., Salmonella sp., Listeria sp., Streptococcus sp., Acinetobacter sp., Proteus sp., and others (Cerqueira et al, 2008;Forrest et al, 2006;González et al, 2004;Lefmann et al, 2006;Montague, Cleary, Martinez, & Procop, 2008;Oliveira, Procop, Wilson, Coull, & Stender, 2002;Oliveira et al, 2003;Peleg et al, 2009;Perry-O'Keefe et al, 2001;Reller, Mallonee, Kwiatkowski, & Merz, 2007;Stender et al, 1999). The majority of assays target the rDNA due to its relative abundance, and in the majority of cases, both sensitivity and sensitivity of over 90% were demonstrated.…”