Serotyping of Streptococcus pneumoniae isolates from nasopharyngeal samples: use of an algorithm combining microbiologic, serologic, and sequential multiplex PCR techniques.

We evaluated nasopharyngeal carriage of Streptococcus pneumoniae (pneumococci) in nine Alaskan communities and used an algorithm combining microbiologic, serologic, and sequential multiplex PCR (MP-PCR) techniques to serotype the isolates. After microbiological identification as pneumococci, isolates (n = 1,135) were serotyped using latex agglutination and Quellung tests (LA/Q) as well as a series of six sequential MP-PCR assays. Results from the two methods agreed for 94% (1,064/1,135) of samples. Eighty-six percent (61/71) of the discordant results were resolved. Discordant results occurred because (i) the MP-PCR gel was misread (31/61 [51%]), (ii) the LA/Q agglutination was misinterpreted (13/61 [21%]), (iii) two serotypes or sets of serotypes were identified by MP-PCR and only one of the two was identified by LA/Q (9/61 [15%]), (iv) different serotypes or sets of serotypes were identified by LA/Q and MP-PCR and both were correct (7/61 [11%]), and (v) the capsular polysaccharide locus (cps) did not amplify during the initial MP-PCR but was present upon retesting (1/61 [2%]). Overall, isolation of pneumococci followed by MP-PCR quickly and accurately identified pneumococcal serotypes in >97% of samples and made available isolates for additional tests such as antimicrobial susceptibility. Misinterpretation of the MP-PCR gel was identified as the main source of discordance. Increasing the number of MP-PCRs from six to seven and reducing the number of serotypes in each reaction may reduce this error. This method may be of use to laboratories characterizing large numbers of S. pneumoniae samples, especially when antimicrobial susceptibility data are needed.