The introduction of effective pneumococcal conjugate vaccines has stimulated interest in understanding aspects of nasopharyngeal carriage among populations receiving the vaccine, since carriage precedes invasive disease. However, using traditional LA/Q for large nasopharyngeal carriage studies is time-consuming, so we sought to use a combination of LA/Q and MP-PCR methods to improve efficiency. Here we have described an algorithm in which we initially isolate pneumococci from NPS stored in STGG medium and then perform MP-PCR supplemented with LA/Q to determine the serotype of the isolated organisms. This strategy resulted in accurate serotyping data for >97% of the isolates compared with standard serotyping methods, simplified the laboratory work by decreasing the number of samples that require serotyping by LA/Q, and provided purified pneumococci for additional tests.
The successful use of genetic approaches to serotype pneumococci began by focusing on a few serotypes of epidemiological importance (4
). As the genetic sequences of more serotypes became available, accurate MP-PCR algorithms to identify an increasing number of serotypes were published (15
). Eventually researchers began manipulating these algorithms to identify prominent serotypes causing IPD in different regions of the world (8
) and using the algorithms to serotype pneumococci from nasopharyngeal samples (1
). The currently published reports describing MP-PCR methods to serotype pneumococci collected from the nasopharynx describe DNA templates prepared directly from STGG medium containing the NPS. These methods allow for identification of pneumococcal serotypes but do not provide purified isolates for antimicrobial susceptibility testing or genetic characterization, which are important parts of a comprehensive program for monitoring carriage of S. pneumoniae
. The algorithm used in this study overcomes this limitation, as the initial step is to isolate and freeze pneumococcal colonies from the STGG medium. These colonies are then available for additional testing, if necessary.
The main disadvantage of using the published genetic approaches for serotyping pneumococci is that the assays currently cannot identify all known capsular types. Many serotypes are resolved to a set of genetically related serotypes (i.e., 6A/6B/6C, 7A/7F, and 15B/15C) rather than an individual serotype. The sequential MP-PCR used in this study can identify 31 different serotypes or sets of serotypes, including NT isolates. However, by initially isolating and freezing the organisms, all known serotypes can be distinguished because samples not identified by MP-PCR or identified as one of a set of serotypes can be removed from storage and grown on BAP and the serotype can be determined using conventional methods. Even though 59% of samples in our study required further LA/Q after completion of the MP-PCR portion of the algorithm, the additional work was simplified with the knowledge of the set of serotypes to which the samples belong.
The high degree of complete concordance (94%) between MP-PCR and LA/Q in our study confirms that MP-PCR can accurately identify serotypes of S. pneumoniae collected from the nasopharynx. Excluding those “not resolved,” MP-PCR correctly identified >97% of samples in our study. Additionally, MP-PCR accurately determined the serotype of 13 samples misclassified by LA/Q and identified a second serotype not found by LA/Q in nine samples.
Of the incorrect MP-PCR results, all but one was the result of misreading the MP-PCR gel; the bands were too close together to be resolved accurately. One of the variables to consider when testing >1,300 samples is how to organize the work to optimize time and resources. With this in mind, we created positive-control mixtures for each reaction, used them as molecular weight markers, and ran the reactions on 4 by 24 (96-well) gels. Organizing the work in this manner had the advantage of optimizing the number of samples tested at any one time; however, it sometimes made it difficult to determine the correct sizes of the unknown bands. Fortunately, the sequential MP-PCR algorithm is flexible and can be easily updated to reflect organizational changes or serotype fluctuations (8
). As part of work done subsequent to this study, we redesigned our MP-PCR algorithm to attempt to make it easier to determine the sizes of the bands in our unknown samples. We increased the number of reactions from six to seven, which decreased the number of serotypes or sets of serotypes in each reaction from five to four. We specifically focused on those serotypes and sets of serotypes listed in (serotypes 3, 10A, 15A/15F, 19A, 21, and 35F/47 and serogroups 9, 11, 18, and 22) which were difficult to identify in this study. For those serotypes we redesigned some of the primer sets so the product sizes were different from those in this study. We also moved most of the serotypes into different reaction mixtures from where they were previously, thus separating the two serotypes that were difficult to discriminate from each other. These changes have led to better band resolution and easier gel interpretation. Data from the updated algorithm have shown a 0.3% (3/975) error caused by misreading the gel. This compares to the 2.7% (31/1,135) error found in this study.
Pai et al. first described three samples that were not amplified by their internal control (cps
locus) primer set (20
). All three of these were NT by traditional serotyping methods. Although NT organisms are not often isolated from cases of IPD, they represent approximately 10% of isolates found in the nasopharynx (6
). In our study they accounted for 12% of all samples, making them more common than any serotype except for serotype 19A. It is important, therefore, to identify these organisms in the first MP-PCR so resources are not spent on the additional testing of samples without a capsule. Based upon the report by Pai et al. (20
), as well as work done in our laboratory previous to this study, we identified all samples not amplified by the primers specific for the cps
locus as suspected NT, and they were removed from subsequent reaction mixtures. All samples containing the gene for the cps
locus were thought to be serotypeable and continued through all six MP-PCR assays or until their serotypes were determined. With one exception, this method correctly identified every sample as either NT or serotypeable. The single exception was a sample that was initially not amplified by the primers specific for the cps
locus but was serotype 21 by LA/Q. Subsequent singleplex PCR analysis confirmed that the sample did contain the cps
locus and the serotype 21-specific gene. It is likely that the DNA template was inadvertently left out of the reaction tube during the initial MP-PCR.
It is important to note that data generated by this method could be slightly different from data generated by LA/Q and will reinforce the serotype frequency distribution used to develop the algorithm. In this study there were seven samples (<1%) where LA/Q identified one capsular type, most likely the more abundant serotype in the sample, and MP-PCR identified another, the serotype or set of serotypes tested earliest in the sequential MP-PCR algorithm. Confirmatory testing showed that both serotypes were present in these samples. In future testing where LA/Q is not completed on every sample, only the serotype that is tested for earliest in the MP-PCR algorithm will be identified. This is acceptable for our large, population-based studies but should be considered by groups doing vaccine studies on an individual level.
This method is also not suitable for detecting multiple colonizing serotypes unless they have different morphologies upon the initial subculture. Samples were not exhaustively searched for multiple serotypes using MP-PCR. If multiple serotypes are present, the more common serotype (the one tested for earliest in the MP-PCR algorithm) will most likely be the one identified.
In March 2010, the U.S. Food and Drug Administration licensed PCV13, and it replaced PCV7 in the Alaska childhood vaccine schedule in April 2010. Continued evaluation of serotype distribution and antimicrobial susceptibility of pneumococci carried in the nasopharynx will be critical for evaluating the impact of this new vaccine. We have described here a combined microbiologic, MP-PCR, and serologic algorithm to serotype S. pneumoniae collected from the nasopharynx and stored in STGG media. This method is accurate and flexible, decreases the number of isolates requiring serotyping by conventional methods, and makes available pneumococcal isolates for antimicrobial susceptibility testing and genetic characterization. We plan to use this algorithm to monitor pneumococcal carriage in Alaska as a part of our program to evaluate the effectiveness of the new PCV13 vaccine, and it could be considered by all laboratories interested in characterizing large numbers of pneumococci either from the nasopharynx or from episodes of IPD.