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

Bacterial strains, growth, and storage.Clinical isolates of serotype 1 and 19F pneumococci were obtained from Michael Jacobs, Case Western Reserve University. The isolates were subcultured on 5% sheep blood agar plates incubated at 37°C. Colonies were inoculated in 10 ml Todd-Hewitt (TH) broth and incubated at 37°C for ∼2 h to an optical density at 492 nm of 0.4 to 0.5 (exponential phase). A quantitative culture was performed by counting colonies grown after 0.1 and 0.01 ml of a 10⁻⁵ dilution were spread on blood agar plates. Clinical specimens were cultured (i) by plating 0.1 ml of nasal wash specimen on a blood agar plate containing gentamicin, streaking the plate for isolation, and incubating it in 5 to 10% CO₂ (BBL GasPak CO₂ system; Becton Dickinson Microbiology Systems, Sparks, MD) and (ii) by inoculating 1 ml of a modification of Avery's broth (TH broth with 5% rabbit plasma and 0.8% dextrose [1]) with 0.1 ml nasal wash specimen. Inoculated Avery's broth was subcultured onto blood agar plates after incubation at 37°C for 4 to 6 h and overnight. Colonies morphologically consistent with pneumococci were identified by standard methods (17) and serotyped as previously described (16).

Comparison of the sensitivities of methods using a pneumococcus-inoculated swab and STGG medium containing an inoculated swab for the optimal detection of S. pneumoniae by culture.Ten microliters of TH broth containing approximately 2 × 10³ CFU of exponential-phase pneumococci or 10 μl of a 1:10 dilution in TH broth were absorbed onto individual calcium alginate (Fisher Healthcare, Pittsburgh, PA)-, Dacron polyester (COPAN Innovation, Brescia, Italy)-, or rayon (COPAN Innovation, Brescia, Italy)-tipped aluminum swabs that were then placed in tubes containing 1 ml of STGG medium. Control tubes that simulated STGG medium with a 100% release of pneumococci from an inoculated swab were prepared by adding 10 μl of pneumococci directly into STGG medium. After incubation for 30 min at ambient temperature (or at 4°C for 18 h), the tubes were vortexed for 20 to 30 seconds, and then 0.1 ml and 0.01 ml of the inoculated STGG medium were quantitatively cultured by plating and spreading the medium on blood agar plates and incubating the plates overnight. In addition, swabs were cultured by removing the swab from STGG medium, and after excess moisture was removed by pressing and rotating the swab against the inside of the tube above the liquid, the swab was rolled repeatedly across the surface of a blood agar plate, with care taken to spread the inoculum evenly, and the plate was incubated overnight. There was day-to-day variation in the numbers of CFU present in the exponential-phase culture and in the inoculum. In order to compare the results of multiple trials performed on different days, the number of CFU in the inoculum was normalized to 2.1 × 10³ CFU (the median inoculum size) by calculating with a conversion factor (2.1 × 10³ divided by the number of CFU in the inoculum) and by multiplying the results for each culture of pneumococci from the swab or STGG medium by the conversion factor. The values for the median and interquartile numbers of CFU reported in Table 1 reflect the normalized values.

DNA extraction from specimens.Swabs were inoculated with 10 μl of serial 10-fold dilutions of exponential-phase pneumococci, and four methods listed here were tested for the extraction of DNA from 0.2 ml STGG medium or calcium alginate swabs. (i) A High Pure PCR template preparation kit (Roche Applied Science, Mannheim, Germany) was used according to instructions provided by the manufacturer for the isolation of nucleic acids from bacteria or yeasts, with the following modification. The bacterial-lysis step was modified by the addition of 10 μl lysis buffer (0.1% sodium deoxycholate, 0.01% sodium dodecyl sulfate, 0.15 M sodium citrate) to the lysozyme-containing solution. Extracted DNA was suspended in a final volume of 200 μl. (ii) A NucliSens isolation kit (bioMérieux, Durham, NC), which uses an isolation method based on the binding of DNA to silica particles, was used according to the instructions provided by the manufacturer. We also used a silica particle-based DNA extraction and purification method identical to that of the NucliSens isolation kit using reagents prepared in the laboratory as described by Boom et al. (3). With either set of reagents, the final extraction volume was 50 μl. The performance of the NucliSens isolation kit varied between lots, resulting in inconsistent DNA extraction and purification. Therefore, reagents for this silica particle-based DNA extraction and purification method were prepared in the laboratory. For consistent performance of this method, we noted that the sodium thiocyanate solution must be prepared freshly or used within 48 h of preparation. (iii) The STGG medium (after centrifugation at 10,000 × g for 1 min, removal of supernatant, and resuspension in water) or swab in water was boiled in water for 5 min and centrifuged at 10,000 × g for 1 minute, and the supernatant fluid was used. (iv) The specimen was treated with proteinase K (20 μg/ml) and 1% Tween 20, incubated at 55°C for 60 min and then at 95°C for 10 min, and centrifuged at 10,000 × g for 1 min; the supernatant fluid was used.

PCR assays for the amplification of DNA specific for all S. pneumoniae or S. pneumoniae serotype 1 or 19F isolates.PCR for the amplification and detection of serotype 19F DNA was performed as previously described (16) using 10 μl of a sample (extracted DNA) in a 50-μl reaction mixture, except that a nested PCR was not performed and only the inner primers were present in the PCR sample reaction mixture. PCR for serotype 1 was performed as described previously (N. E. Dayan, A. Rizvi, and L. G. Rubin, unpublished data). PCR for the amplification and detection of pneumococci from clinical specimens targeted a region of the autolysin gene, lytA. Left and right primers were GGA AAG ACC CAG AAT TAG GT and GTC TGA GTG GTT GTT TGG TT, respectively. PCR products were detected via agarose gel electrophoresis, and photographs of gels were inspected for the presence of bands with molecular sizes of 178 bp, 208 bp, and 308 bp for serotype 1, serotype 19F, and lytA, respectively.

Comparison of the sensitivities of methods using a pneumococcus-inoculated swab and STGG medium containing an inoculated swab for PCR amplification and detection of S. pneumoniae DNA in simulated clinical specimens.Calcium alginate-, Dacron polyester-, or rayon-tipped aluminum swabs were inoculated with (i) 10 μl of STGG medium containing approximately 2 × 10³ CFU of exponential-phase cultures of a type 1 or 19F isolate or (ii) 10 μl of serial 10-fold dilutions of these isolates and placed in tubes containing 1 ml of STGG medium. After the tubes were incubated at ambient temperature for 30 min and vortexed for 20 to 30 seconds, DNA was separately extracted from the removed swab (after excess moisture was removed by pressing and rotating the swab against the inside of the tube) and from 0.2 ml STGG medium containing the swab by the silica particle-based method. Extracted DNA was subjected to PCR-based amplification and detection as described above.

Comparison of the sensitivities of methods using a swab and STGG medium containing an inoculated swab for the PCR-based detection of S. pneumoniae DNA in clinical specimens.Nasal wash specimens that were submitted to the clinical microbiology laboratory for testing for respiratory syncytial virus and influenza virus antigens in saline were obtained from children suspected of having respiratory tract infections. Only specimens from children who had not received an antimicrobial prior to our obtaining the nasal wash specimen were used in these studies. Specimens were cultured for S. pneumoniae as described above and stored at −70°C. Specimens were thawed, and swabs of each composition were simultaneously immersed in the specimen, removed, and placed in individual tubes containing 1 ml of STGG medium. After being vortexed for 30 seconds, swabs and 0.2 ml of STGG medium were separately processed for DNA extraction and PCR-based amplification and detection. The use of these clinical specimens was approved by the Institutional Review Board of the Long Island Jewish Medical Center.

Comparison of swab and swab-containing STGG medium as specimens for the culture of S. pneumoniae.Culture of calcium alginate swabs (after removal from STGG medium) grew a 3.7-fold-greater number of colonies than 0.1 ml of STGG medium containing the inoculated swab (P < 0.0001) (Table 1). When cultures were repeated after the incubation of STGG medium for 18 h at 4°C, similar results were obtained (data not shown). Compared to the number of colonies grown by the culture of control STGG medium that had been directly inoculated, the culture of STGG medium preincubated with calcium alginate swabs grew only 11% of the number of colonies (Table 1), indicating that a small proportion of viable pneumococci were released from the calcium alginate swab into the STGG medium. Thus, the number of colonies grown on solid medium is significantly higher from the swab than from STGG medium.

Parallel experiments of similar design were performed using Dacron polyester or rayon nasopharyngeal swabs (Table 1). Using Dacron polyester swabs, more colonies were grown from the swab than from the STGG medium, but few colonies were grown from either source and only 1.4% of viable colonies were released from the swab. Results were similar after the culture of swabs and STGG media that had been incubated at 4°C for 18 h (data not shown). Thus, following the inoculation of a Dacron polyester swab, few viable pneumococci were recovered from either the swab or the swab-containing STGG medium. In contrast, using a rayon swab, significantly more colonies were cultured from the swab than from STGG medium, but more colonies were recovered from either the rayon swab or medium than from either the calcium alginate or the Dacron polyester swab. In addition, the median proportion of viable colonies released from the rayon swab was 47%, which was higher than from the calcium alginate or Dacron polyester swab. In limited experiments of similar design using culturing of rayon swabs and STGG media, which were incubated at 4°C for 18 h, similar results were obtained. Unlike the calcium alginate or Dacron polyester swabs, the rayon swabs unraveled in STGG medium and absorbed more liquid; this may have increased the number of colonies grown from rayon swabs on culture but is unlikely to explain the larger number of colonies grown from the STGG medium. Thus, independently of swab composition, more colonies were grown from the swab than from the STGG medium containing the swab. With culturing of either the swab or STGG medium, more colonies were grown using rayon swabs than calcium alginate swabs and more colonies were grown using calcium alginate swabs than Dacron polyester swabs.

DNA extraction from calcium alginate swabs.The four methods for extracting DNA from calcium alginate swabs described in Materials and Methods were compared. When DNA was prepared either by boiling the inoculated swabs in water or by incubating the swab with proteinase K and 1% Tween 20, no amplified DNA was detected after PCR. When DNA was extracted from swabs using a High Pure PCR template preparation kit or silica-based isolation method and subjected to PCR-based amplification and detection, DNA was detected, but detection was at least 100-fold more sensitive using the silica-based method. Therefore, the silica-based method was chosen.

DNA extraction from STGG medium.To determine the optimal method for DNA extraction for PCR-based detection from STGG medium, the inoculation of STGG medium with serial 10-fold dilutions of exponential-phase S. pneumoniae serotype 1 and DNA extraction using the silica-based method and the boiling method, in either water or STGG medium (see Materials and Methods), were compared. In each of two trials, the detection of the PCR product was 100-fold more sensitive using the silica-based method than the method using boiled water (the highest dilution detected was 10⁻⁷ and 10⁻⁶ for the silica-based method and 10⁻⁵ and 10⁻⁴ for the boiling-water method in trials 1 and 2, respectively). No PCR product was detected using DNA prepared by boiling STGG medium (lowest dilution tested, 10⁻²). Therefore, the silica-based method was chosen.

Comparison of swab and swab-containing STGG medium as the source of DNA for the amplification and detection of pneumococcal DNA from simulated clinical specimens.Detection of amplified type 1 or type 19F DNA from the inoculated calcium alginate swab was approximately 100-fold more sensitive than from the STGG medium containing the swab (Table 2). Experiments of identical designs were performed using Dacron polyester and rayon swabs. With Dacron polyester swabs, the sensitivity of detection of pneumococcal DNA from the swab was equal to that of the swab-containing STGG medium (Table 2). With rayon swabs, the sensitivity of DNA amplification and detection was approximately 10-fold higher using DNA from the swab-containing STGG medium than using the swab but the differences were not significant (Table 2). However, the sensitivities of DNA detection methods using swabs of any of the three compositions were similar.

Effect of swab composition on the sensitivity of PCR-based detection of S. pneumoniae DNA from clinical specimens and comparison of the methods using swabs and swab-containing STGG medium.Thirteen clinical specimens were tested, of which four did not grow S. pneumoniae on culture and nine grew S. pneumoniae. Each of the four clinical specimens that did not grow pneumococci was negative for pneumococcal DNA by PCR using primers within lytA. Amplified pneumococcal DNA was detected from all of nine culture-positive specimens, although one was positive using DNA extracted from the original undiluted specimen but not from any of the swabs or STGG media. PCR was performed on serial 10-fold dilutions of extracted DNA from eight culture-positive specimens to compare the sensitivities of the methods using swabs and STGG medium containing the swab as sources of DNA for PCR. Using calcium alginate swabs, the swab was at least 10-fold more sensitive than swab-containing STGG medium for six specimens, equally sensitive and less sensitive for one specimen each (median titers [ranges], 4 [1 to 6] and 3 [1 to 5] for the swab and the medium, respectively). With Dacron polyester swabs, PCRs using the swab and STGG medium exhibited similar sensitivities. The PCR using the swab was more sensitive than the PCR using medium with four specimens, sensitivities were equal with one specimen, and the PCR using medium was more sensitive than the PCR using a swab with three specimens. The median titers (ranges) were 3 (0 to 5) and 2 (1 to 7) for the swab and the medium, respectively. Using rayon swabs, PCR with the swab was at least 10-fold more sensitive than PCR with STGG medium and four specimens and equally sensitive with four specimens. The median titers (ranges) were 3 (1 to 6) and 2.5 (1 to 5) for the swab and the medium, respectively. Thus, with clinical specimens and calcium alginate or Dacron polyester swabs, the sensitivities of PCRs using swabs and swab-containing medium were similar to those observed using simulated clinical specimens. Using rayon swabs with actual clinical specimens, detection was somewhat more sensitive with swabs than with STGG medium, while using simulated clinical specimens, detection from STGG medium was more sensitive than from a swab.