Do NAAT-Based Methods Increase the Diagnostic Sensitivity of Streptococcus agalactiae Carriage Detection in Pregnant Women?

Abstract

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33-63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.

Keywords: GBS; PCR; Streptococcus agalactiae; pregnant women; primers; real-time PCR.

Figure 1

The scheme shows the course of the research and the methods used for GBS–carriage diagnostics in the studied samples. Green and red colors are, respectively, the classical and molecular course of diagnostics in accordance with ASM recommendations [1], taking into account preincubation in liquid TH medium with colistin and nalidixic acid. Gray color is the course of GBS identification using molecular methods, without the preincubation step.

Figure 2

Exemplary results of electrophoresis for GBS detection based on 16S rRNA gene—405 bp (a) and atr gene—780 bp (b). First paths on (a,b)—DNA size marker (100–1000 bp, A & A Biotechnology, Gdańsk, Poland), second paths—positive control (DNA isolated from S. agalactiae ATCC 12386), other paths—the tested samples. Paths number 12 (a) and number 18 (b)—negative controls—nuclease-free water (A & A Biotechnology, Gdańsk, Poland).

Figure 3

The comparison of GBS identification results using the culture method in TH selective medium and NAAT-based methods (based on the cfb, atr and 16S rRNA conserved gene) directly from clinical materials without the preincubation step.

Figure 4

The comparison of GBS identification results by the culture method without preincubation in enrichment broth and by PCR method for atr gene (a) and with (b) preincubation in TH selective broth: blue, percentage/number of positive samples only in PCR; green, percentage/number of positive results both in culture method and PCR (true positive); yellow, percentage of positive results in culture method only.

Figure 5

The comparison of GBS positive identification results by the culture method without preincubation in enrichment broth culture method and real-time PCR method for the cfb gene (a,b) preincubation in TH selective broth:. blue, percentage/number of positive samples only in real-time PCR; green, percentage/number of positive results both in culture method and real-time PCR (true positive), yellow, percentage of positive results in culture method only.

Figure 6

The comparison of GBS identification results using the culture method with preincubation in enrichment broth and NAAT-based methods with preincubation based on the atr gene (conventional PCR) and cfb gene (real-time PCR).