Comparison of DNA Dot Blot Hybridization and Lancefield Capillary Precipitin Methods for Group B Streptococcal Capsular Typing

Abstract

Group B streptococci (GBS) (Streptococcus agalactiae) are a major cause of sepsis and meningitis in neonates and infants and of invasive disease in pregnant women, nonpregnant, presumably immunocompromised adults, and the elderly. Nine GBS serotypes based on capsular polysaccharide antigens have been described. The serotype distributions among invasive and colonizing isolates differ between pediatric and adult populations and have changed over time. Thus, periodic monitoring of GBS serotype distributions is necessary to ensure the proper formulation and application of an appropriate GBS vaccine for human use and to detect the emergence of novel serotypes. Since the mid-1990s, the proportion of GBS isolates that are nontypeable by standard serologic methods has increased, creating a need for more sensitive typing methods. We describe a typing method that uses DNA dot blot hybridization with probes generated by PCR from the GBS capsular genes for serotypes Ia, Ib, and II to VIII. PCR primers were designed to amplify type-specific GBS capsular gene sequences. Gene probes were constructed from the PCR products and used to classify isolates based on hybridization profiles. A total of 306 previously serotyped invasive and colonizing isolates were used to compare our dot blot capsular typing (DBCT) identification method with Lancefield serotyping (LS). A dot blot capsular type was assigned to 99% (303 of 306) of the isolates, whereas 273 of 306 isolates (89%) were assigned a Lancefield serotype. The overall agreement between the methods was 95% (256 of 270 isolates typeable by both methods). We conclude that the DBCT method is a specific and useful alternative to the commonly used LS method.

Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization

“…Comparison of mPCR/RLB, MS and CS CS has been most commonly used for GBS, but the proportion of non-typable isolates is significant and has increased over time (Borchardt et al, 2004), which could distort the recorded serotype distribution. MS identification is an attractive alternative (Kong et al, 2002;Borchardt et al, 2004).…”

“…In addition, we tested 83 isolates provided by Dr Nicola Jones, Oxford, UK, that had been used previously in a study of multilocus sequence typing (MLST) (Jones et al, 2003) and 260 clinical isolates that have been used in our previous studies (Table 2) (Kong et al, 2002(Kong et al, , 2003. Results of conventional serotyping (CS) were available for all 316 isolates and all had been tested by serotypespecific PCR and/or partial cps sequencing [molecular serotyping (MS)] as previously described (Kong et al, 2002;Borchardt et al, 2004). All isolates were identified as GBS by thefollowing criteria: â-haemolysis on a 5 % horse blood agar, Gram stain showing Gram-positive cocci in pairs or short chains, negative reaction with catalase reagent and Lancefield Abbreviations: CS, conventional serotype/ing; GBS, group B streptococcus; mPCR, multiplex PCR; MS, molecular serotype/ing; RLB, reverse line blot.…”

“…To compare with previously published primer pairs for serotype II-, VII-and VIII-specific PCRs (Borchardt et al, 2004;Cieslewicz et al, 2005) we used cpsIIK, cpsVIIM and cpsVIIIJ, respectively, rather than cpsH as the targets for primers and probes (Kong et al, 2002). We also designed one GBS-specific primer pair and two GBSspecific probes (Ke & Bergeron, 2001).…”

“…Conventional serotyping (CS) requires high-titre serotype-specific antisera (Elliott et al, 2004), results are somewhat subjective and a significant proportion of isolates are nontypable (Borchardt et al, 2004). Molecular serotype (MS) identification methods are attractive because of their high discriminatory power (Edwards et al, 2005) and reproducibility (Borchardt et al, 2004). PCR/sequencing and DNA dot blot hybridizationbased assays have been used to identify GBS serotypes (Kong et al, 2002;Borchardt et al, 2004), but further simplification, without loss of sensitivity and specificity, is needed to make them more practicable for routine use.…”

“…Their distribution varies according to geographic location and study population (Schuchat, 1998). Conventional serotyping (CS) requires high-titre serotype-specific antisera (Elliott et al, 2004), results are somewhat subjective and a significant proportion of isolates are nontypable (Borchardt et al, 2004). Molecular serotype (MS) identification methods are attractive because of their high discriminatory power (Edwards et al, 2005) and reproducibility (Borchardt et al, 2004).…”