A new pneumococcal serotype within serogroup 6, 6C, was recently discovered during the development of a monoclonal antibody (MAb)-based typing scheme (7,14), when a subset of conventionally serotyped 6A isolates (CS6As) did not bind to one of the two 6A-specific MAbs used. Subsequent analysis of this CS6A subset revealed a different capsular structure, which was designated 6C (14). The serotype 6C capsular biosynthetic locus (cps) appears to be derived from replacement of the wciN gene at the 6A cps locus with a divergent 6C-specific counterpart that encodes a different glycosyl transferase, resulting in a sugar substitution in the polysaccharide repeating unit (13).The MAb typing system was used to resolve serotype 6C isolates among CS6As recovered during the pre-pneumococcal 7-valent conjugate vaccine (PCV7) year 1999 and post-PCV7 years 2003 to 2006 from areas under continuous surveillance in Active Bacterial Core surveillance (ABCs) in the United States (10). This investigation revealed a marked decrease in the rate of serotype 6A invasive pneumococcal disease (IPD) in the post-PCV7 period that was apparently due to cross-protection mediated by the serotype 6B component included in PCV7.The investigation also revealed a small, yet significant, increase in the rate of serotype 6C IPD. Here, we describe an expedient PCR assay for resolution of serotype 6C and true serotype 6A from CS6As. We extend our recent observations (10)

ᰔIt is important to monitor serotype distribution within the principal reservoir for Streptococcus pneumoniae, which is asymptomatic nasopharyngeal (NP) carriage among children. Monitoring changes of carriage serotype distribution over time can provide insights into the epidemiology of pneumococcal disease.Pneumococci easily autolyze, making carriage studies difficult. NP specimens must be cultured quickly, with pneumococcal cultures immediately frozen at Ϫ70°C in freezing medium (5). We tested silica desiccant packages (SDPs) (Grace Davison, Baltimore, MD) containing 1.5 g of silica powder for storage and transport of NP specimens. SDPs are effective for transporting specimens for delayed culture of several bacterial species (2, 4, 7, 9), but their utility for delayed culture of NP swabs for pneumococcal isolation has not been demonstrated.NP swabs were collected from 302 healthy children ranging from 1 to 15 years of age at a children's homeless shelter in Ranibari, Kathmandu, Nepal. A sterile cotton swab was inserted into the nasopharynx and rolled twice prior to inserting into an SDP and sealing. The length of time between collection of initial and last samples was 8 days. Transportation of NP samples at room or environmental temperature from Kathmandu to the CDC required 72 h. Isolates were stored for up to 13 additional days at room temperature before culture, with a maximal storage duration of 25 days. NP samples were incubated (37°C and 5% CO 2 ) for 4 h in 5 ml of Todd-Hewitt broth containing 3 drops of defibrinated rabbit blood. Cultures were streaked onto Trypticase soy agar II-5% sheep blood agar and incubated for 18 to 24 h.Of 302 NP samples, 184 (61%) contained pneumococci according to alpha-hemolysis, optochin sensitivity, and bile solubility analysis results (Table 1). The level of carriage was high (69 to 83%) within the age groups 1 to 2 years, 3 to 4 years, 5 to 6 years, and 7 to 8 years and incrementally decreased among older ages (Table 1). Carriage among 1-to 10-year-old children (71% [156/221[) (Table 1) was comparable to the carriage previously observed among Nepalese children in the same age group in a different region (84 to 88%) (3). Incrementally decreased carriage was evident among groups of older children (Table 1). We observed no differences in pneumococcal recovery between NP specimens processed after 12 days and those processed after 25 days (data not shown).Of isolates from 184 pneumococcus-positive samples, 160 were serotypeable (43 serotypes) by a Quellung assay using CDC antisera, with one mixed-carriage isolate detected (Table 2). Twenty-five (13.5%) isolates were nontypeable. All isolates were tested by the Etest (AB Biodisk, Sweden) and found to be penicillin susceptible (MIC Յ 068 g/ml). Four of nine conventionally serotyped 6A isolates were subsequently typed as newly discovered serotype 6C (6) by use of 6C-specific PCR (1) but were identified in the present study as representing serotype 6A. Thirty-seven (23%) of 160 isolates were covered by the 7-valent conjugate va...

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