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.
To detect Streptococcus pneumoniae colonization, the nasopharynx is sampled using a swab placed in skim milk-tryptone-glucose-glycerol (STGG) transport medium, and then the swab specimen or STGG medium is cultured or subjected to PCR. We evaluated the effect of swab composition and compared the sensitivities of detection of culture and PCR using swabs and swab-containing medium. Calcium alginate, Dacron polyester, or rayon-tipped swabs were inoculated with pneumococci or were immersed in nasal wash specimens from children and then placed in STGG medium. Swabs and medium inoculated with pneumococci were cultured. Swabs grew significantly more colonies than medium. The number of colonies cultured from rayon swabs or medium was significantly higher than the number cultured from the calcium alginate swab or medium. The number of colonies from both the Dacron polyester swabs and medium were significantly lower than with either calcium alginate or rayon swabs. When DNA was separately extracted from the calcium alginate swab and medium and subjected to PCR for pneumococcal detection from either S. pneumoniae-inoculated swabs or clinical specimens that grew S. pneumoniae, the sensitivity was at least 10 times higher using the swab. With Dacron polyester or rayon-tipped swabs, there was no consistent difference between the sensitivity of PCR using swabs and that of PCR using medium. Thus, calcium alginate swabs may be superior to STGG medium for the culture and PCR-based detection of S. pneumoniae. For culture, rayon swabs are superior and Dacron polyester swabs are inferior. The sensitivity of the swab and swab-containing medium for culture or PCR detection of S. pneumoniae varies with swab composition.Streptococcus pneumoniae (pneumococcus) is an important pathogen of children and adults (15). Pneumococci frequently and asymptomatically colonize the nasopharynges of infants and young children. Colonizing bacteria are the source of respiratory tract infections such as pneumonia and acute otitis media as well as invasive pneumococcal infections and serve as the reservoir for person-to-person transmission. In 2000, a heptavalent pneumococcal conjugate vaccine (PCV7) was licensed in the United States and recommended for routine administration to all infants (4). Use of this vaccine has resulted in a large decrease in the incidence of invasive pneumococcal infections in young children and a decrease in the prevalence of colonization with vaccine serotypes. The latter fact has resulted in a decrease in invasive disease in adults (21) and in infants too young to have been directly protected by vaccination (13) via herd type immunity. This has been accompanied by an apparent increase in the prevalence of colonization with nonvaccine serotypes (7,10,12). 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 test...
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