PCR-based assays for detection of Streptococcus pneumoniae serotypes 3, 14, 19F and 23F in respiratory specimens

Abstract

Current culture-based assays are insensitive for detection of simultaneous respiratory tract colonization by more than one pneumococcal serotype. Separate single-tube, nested PCR-based assays have been developed to detect Streptococcus pneumoniae serotypes 3, 14, 19F and 23F by amplifying unique DNA sequences in the capsular polysaccharide gene cluster of each serotype. Pairs of 27-32-base outer primers and 20-21-base inner primers and a 20-22-base probe were designed to amplify and detect a 200-221-base sequence by dot blotting using the labelled probe. Sensitivity of the assays was 0 . 01-10 fg using chromosomal DNA and < 1 viable cell using DNA extracted from exponential-phase bacteria. Each serotype-specific assay detected chromosomal DNA from all of five to ten clinical isolates of the homologous type and did not detect DNA sequences from any of 190-204 strains from 51-52 different serotypes or 28 non-pneumococcal bacterial strains. Sixteen throat swabs from children that had been cultured for S. pneumoniae were tested in PCR assays following DNA extraction. All of six that grew S. pneumoniae serotype 3, 14, 19F or 23F were positive in the PCR assay for the homologous serotype (and in a PCR assay for sequences in lytA, present in all pneumococci) and were negative in assays for other serotypes. Of eight culturenegative specimens in children not receiving antimicrobials, three were positive for both the lytA assay and an assay for one of the four serotypes, suggesting true positive results; in three others all five PCR assays were negative and, in the remaining two, the lytA assay was positive but each of the four assays for individual serotypes was negative, suggesting either false-positive results or presence of DNA sequences from an S. pneumoniae serotype other than 3, 14, 19F or 23F. These preliminary clinical data suggest that these PCR-based assays are sensitive and specific for detection of individual serotypes of pneumococci and may be used with respiratory tract specimens.

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

“…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.…”

“…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.…”

“…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.…”

Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates

“…Nonetheless, conventional serotyping will have to be performed to differentiate such isolates, although the procedure will be greatly streamlined with the knowledge that after the major multiplex reaction serotype, there are only one to three additional serotypes to screen. The PCR assays described so far have reported similar limitations in developing type specific primers among cross-reacting serotypes (3,13,25). The difference between serotypes 6A and 6B has been correlated to a single nonsynonymous substitution in the putative rhamnosyl transferase gene (wciP) (17), making it impossible to serotype such strains by a PCR-based approach.…”

“…The development of PCR-based serotyping systems has the potential to overcome some of the difficulties associated with serologic testing (3,13). In addition, the development of PCRbased assays for direct detection of select serotypes from clinical specimens could be a valuable aid in surveillance, particularly in situations where culture is insensitive (14,25). Production of capsule is largely controlled by capsular polysaccharide synthesis genes located at the cps locus, typically with the same general genetic organization and flanked by the conserved dexB and aliA genes (21).…”