Serotype-Specific Typing Antisera for Pneumococcal Serogroup 6 Serotypes 6A, 6B, and 6C

Abstract

ᰔStreptococcus pneumoniae strains can express one of at least 92 capsular serotypes. To our knowledge, our laboratory is one of two that maintain antisera for resolution of the first 90 discovered pneumococcal serotypes (4; unpublished data). Recently, the evaluation of serogroup 6 isolates using monoclonal antibodies led to the discovery of the 91st serotype, 6C (7), which has become the prevalent invasive serogroup 6 serotype within the United States (2, 9). The pneumococcal 7-valent conjugate vaccine (PCV7) does not protect against it, and it is not included within the newly licensed 13-valent conjugate vaccine. In addition, serotype 6C carriage may also be common within PCV7-vaccinated populations (3). Prior to the discovery of serotype 6C, serotypes 6A and 6B were the 2 known serogroup 6 serotypes. CDC antisera for quellung-based resolution of serotypes 6A and 6B are designated as Danish factors 6b and 6c (DF-6b and DF-6c), respectively. Our original DF-6b antiserum was positive for both 6A and 6C serotypes, preventing their resolution (Table 1).We produced DF-6d antiserum specific to serotype 6C and DF-6b antiserum specific to 6A as follows. New Zealand White female rabbits were inoculated with S. pneumoniae serotypes 6C or 6A formalin-fixed whole cells. The inocula consisted of serotype 6C or 6A cell suspensions (10 8 to 10 9 cells per ml in phosphate-buffered saline), which were administered intravenously via the marginal ear vein (Table 2). Blood was drawn after week 4 of dosing, and serum was evaluated for antibody titer to serotype 6C or 6A and cross-reactivity to serotypes 6A, 6B, and 6C by the quellung reaction. Cross-reactivity was removed from DF-6d by absorbing the serum with formalin-fixed cells of serotypes 6A and 6B. DF-6b antiserum was absorbed with formalin-fixed cells of serotypes 6B and 6C. The resulting DF-6d antiserum was specific for serotype 6C only, and DF-6b was specific to 6A only (Table 1).The specificity of DF-6b, DF-6c, and DF-6d antisera was evaluated with stock strains and 159 clinical isolates, each representing one of the serotypes 6A, 6B, or 6C. For all strains, Neufeld quellung results were compared to PCR testing specific for the serotype 6C wicN 6C locus (2,7,8) with no inconsistencies observed.We have replaced our previous methods with these procedures for producing specific factor sera against serotypes 6A and 6C. We have now learned that Statens Serum Institute (SSI) has also recently produced factor 6d against serotype 6C (5). Our DF-6b, unlike the SSI factor 6b, is now serotype 6A specific and does not cross-react against serotype 6C.Serotype 6C-specific antiserum will be useful for laboratories which utilize quellung testing. Recently a fourth serogroup 6 serotype, serotype 6D, has been discovered in nature (1,6)

Identification of Newly Described Streptococcus pneumoniae Serotype 6D by Use of the Quellung Reaction and PCR

“…Hare et al, who recently reported similar atypical reactions with some serotype 6A isolates, referred to them as "dodgy 6As" and also showed that they were serotype 6C (3). Melnick et al produced 6d antiserum specific for 6C and an absorbed 6b antiserum specific for 6A, which give the same results as ours for serotype 6C (9).…”

“…The use of this factor antiserum for identification of serotype 6C has recently been validated (4,9). We also compared "old" (purchased before 2009) and "new" factor 6b and pool B antisera (purchased in 2009).…”

Epidemiology of Invasive Pneumococcal Disease in Bangladeshi Children Before Introduction of Pneumococcal Conjugate Vaccine

“…11,24 All isolates (2007-2013) and culture-negative cases (2011-2013) of serogroup 6 were screened for 6A, 6B, 6C and 6D by specific antisera and PCR, respectively. [25][26][27] However, serotypes within serogroup 18 and 6, detected by PCR of culture-negative CSF specimens collected during 2007-2010, could not be differentiated. These serogroups were classified to types by extrapolation based on the proportional distribution of serotypes of 18 (A, B, C and F) and 6 (A, B, C and D) among culture-positive isolates from meningitis cases.…”

Serotype and genotype distributions of pneumococcal carriage isolates recovered from Brazilian children attending day-care centres

“…We have recently shown that we are capable of resolving all four known serogroup 6 serotypes, including 6D (Mercado et al, 2011), using our specific factor sera prepared against serotypes 6A, 6B and 6C (Melnick et al, 2010).…”