The analysis of the 229 pneumococcal test strains using the Ibis T5000 biosensor-based PSGS provided a robust demonstration of its capability in identifying and resolving serotypes. The PSGS was designed to segregate 62 out of the 93 currently recognized serotypes into 48 distinct sets of one to three related serotypes. Of 51,498 isolates in our invasive-isolate collection that were assigned Quellung reaction-based serotypes from 1995 to 2011, we found that only 166 (0.3%) were assigned 19 serotypes (2, 10F, 11B, 11C, 16A, 17A, 24A, 24B, 24F, 21, 27, 28A, 28F, 35A, 35C, 36, 39, 41A, and 43) not covered by the T5000 PSGS serotype deduction platform. Of these serotypes, type 21 was the most abundant (57 isolates).
In the present study, 53 serotypes (out of the 76 distinct serotypes actually tested) were, as expected, segregated into 45 distinct sets. For our purposes, it is important to note that within each of the serotype sets 7A/7F, 9A/9V, 11A/11D, 12F/12B/44, 18B/18C, 22F/22A, and 33A/33F shown in , there is only one predominant serotype associated with invasive disease in the United States (unpublished observations). For example, out of our collection of over 51,498 invasive isolates serotyped over the past 16 years through Active Bacterial Core surveillance (3
), serotype 7F is documented from 3,806 isolates while serotype 7A has been found in only 9 isolates. Within this same collection, 1,903 serotype 12F isolates are documented, while only 4 serotype 12B isolates and no serotype 44 isolates have been found. The only primer pair listed in that is incapable of resolving important serotypes from the vantage point of our invasive-disease surveillance is 1-4 (6A/6C-6B/6D), which does not serve to resolve the recently discovered serotype 6C from serotype 6A. Similarly, it cannot resolve serotypes 6B and 6D; however, we have found only two serotype 6D isolates within U.S. invasive-disease surveillance within the past 5 years. There are now PCR assays available that can resolve the 4 serogroup 6 serotypes (8
), which could be added to the PSGS assay.
While the PSGS genotyping component is far more complex than the serotyping component, it has obvious potential. The genotyping component of the PSGS assay is naturally less resolving than conventional sequence-based MLST, since each locus signature captures only a fraction of the information content of the targeted allele. The loss of resolving power is, however, limited, partly because of the compounding effect of the multilocus analysis and partly because the conventional MLST sequence scheme is itself highly redundant. The PSGS was quite efficient in assigning concordant MLST-based genotypes, especially when conventional genotypes were members of the major pneumococcal complexes that are well represented within the global MLST database. It must be noted that we tested the PSGS with extremely rare and unusual strains, often with serotypes that we have never observed outside of our serotype reference strains. There are a number of parameters that dictate the relationship of a PSGS signature to the existing MLST database. These parameters include the relative relatedness of the isolate ST to previously existing STs. This can be reflected by a PSGS signature consisting of a small number of ST components that are distantly related (differing in 2 to 5 loci), as in categories 6 and 7 (). Under these circumstances, we found the actual target sequences within the distantly related STs to be identical or highly conserved (data not shown). All 217 PSGS signatures where the isolate ST was included were found to share highly related target sequences and therefore provided useful genetic identifiers. Only 6 of the total 229 results (shown in , category 9) were found to inaccurately reflect the isolate genotype. The scope of the genetic diversity of the 229 strains used in this study is evidenced by the fact that they were represented by 155 different conventional STs. Forty-one of these genotypes were added to the genotyping database during the past 3 years, and 35 of the study isolate STs were discovered during this work. Nonetheless, although the PSGS genotype output relies upon the completeness of the genotyping database, we feel that its power in providing reasonably specific genotypes for strains in a labor-efficient manner is obviously well suited for large-scale surveillance efforts. This process could be simplified if a database of reference PSGS genotype designations corresponding to specific signatures of varying complexity could be provided as output.
Categories describing the relationship of isolate STs to PSGS components based upon an existing multilocus sequence database
In addition to its utility for pneumococcal isolate typing, we also feel that the PSGS has potential for determining serotypes and genotypes from culture-negative clinical specimens, such as cerebrospinal fluid, that yield ample pneumococcal DNA for conventional PCR assays (1
). It seems particularly suited for detecting mixed serotypes associated with nasopharyngeal carriage of pneumococci using a broth enrichment culture step for specimens prior to DNA extraction (10
). Mixed carriage of multiple pneumococcal serotypes is quite common, and while their detection using conventional PCR assays is very effective, the process requires a great deal of manual manipulation.