Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR

Abstract

The appropriate clinical applications of pneumococcal polysaccharide vaccines against recent increases in antimicrobial resistant Streptococcus pneumoniae (S. pneumoniae) urgently require accurate analytical methodologies for determining and characterizing the serotypes. The results of current immunological determinations of serotypes with anti-capsular polysaccharide-specific sera are difficult to interpret in terms of quellung changes of the pneumococci. In this study, we applied the multiplex PCR technique for the rapid identification of pneumococci and simultaneous rapid determinations of their serotypes and genotypes that directly correlated with antimicrobial susceptibilities from nasopharyngeal secretions (NPS). Serogroups 6, 19F and 23F were the predominant capsular types of S. pnuemoniae in the NPS samples. Strains of serotypes 19F and 23F frequently had mutations in pbp1a, pbp2x and pbp2b and expressed ermB and mefA; they also were mostly resistant to both penicillin G (PCG) and clarithromycin (CAM). Two NPS samples contained the strain of serotype 19F together with the strain of serotype 23F, although only the strain of serotype 19F was identified by a conventional bacterial culture. Pneumococci were identified in six NPS samples and their serotypes determined by the multiplex PCR, while a conventional bacterial culture failed to identify the pathogens. Our findings suggest that PCR-based serotyping and genotyping can provide an accurate and rapid distribution of pneumococcal serotypes and antimicrobial resistance. The relatively minor populations in the nasopharynx may be determined using molecular techniques.

Effect of Swab Composition and Use of Swabs versus Swab-Containing Skim Milk-Tryptone-Glucose-Glycerol (STGG) on Culture- or PCR-Based Detection of Streptococcus pneumoniae in Simulated and Clinical Respiratory Specimens in STGG Transport Medium

“…For seven of the nine culture-positive clinical specimens, the titer for detection by PCR was at least 2, with median titers that ranged from 2.5 to 4 (varying with the source of material), indicating an excess of pneumococcal DNA in most specimens. However, for certain specimens, such as from patients who received antimicrobial therapy, or for detection of DNA from a second pneumococcal serotype that may be present in small numbers, comparatively small differences in sensitivity may be important (2,16). Saukkoriipi et al (18) successfully amplified and detected pneumococcal DNA from STGG medium containing calcium alginate nasopharyngeal swabs from children, a sample choice with which PCR-based detection exhibited a lower sensitivity in the current study than PCR using the swab itself.…”

“…There was a direct correlation be-tween genome equivalents and the number of colonies on culture; specimens that did not grow S. pneumoniae but were positive by PCR had a low level of genome equivalents (18). Although we acknowledge that some of the positive PCR results for specimens without culturable pneumococci could be false-positive results possibly due to the detection of other alpha-hemolytic streptococci, these results suggest that PCR was more sensitive than culture (2,16,18,19). Whether the swab or STGG medium is the optimal specimen for PCRbased detection and whether swab composition affects the sensitivity of PCR-based detection methods are not known.…”

“…Due to the limitations of culture, nucleic acid amplification techniques such as PCR are being evaluated as diagnostic tests for the detection of pneumococci in nasopharyngeal swabs and other respiratory specimens (2,5,8,14,19,20,22). We sought to maximize the sensitivity of PCR detection by analyzing the effect of the DNA extraction method, namely, the use of a swab or medium containing the swab, and the effect of swab composition on the sensitivity of PCR.…”

“…To detect pneumococcal colonization, an aliquot of STGG medium or the swab is cultured on agar medium. However, it is unclear if culture of the swab or an aliquot of STGG medium containing the swab is optimal for the culture of pneumococci and if swab composition affects the culture of pneumococci.Nucleic acid amplification techniques such as PCR hold promise as sensitive tests for the detection of pneumococci in nasopharyngeal (5, 18, 19) and other (14, 20, 22) respiratory specimens, as well as for the detection of specific pneumococcal serotypes (2,8,16). In a study using calcium alginate nasopharyngeal swabs in STGG medium, S. pneumoniae DNA was detected by real-time quantitative PCR amplification of DNA extracted from STGG medium in 93% of 158 samples that grew S. pneumoniae as well as 41% of S. pneumoniae culture-negative specimens.…”

“…Some of these nonvaccine serotypes may have been present prior to the use of PCV7 but were unrecognized due to limitations in the sensitivity of culture and the difficulty in detecting more than one serotype in a nasopharyngeal specimen. Thus, there is a need for sensitive tests for the detection of colonization with S. pneumoniae, including the detection of simultaneous colonization with more than one serotype (2,6).…”