Sequetyping Validation of Streptococcus pneumoniae Clinical Isolates

• Introduction: Serotyping of Streptococcus pneumoniae (pneumococcus) is required to monitor epidemiological trends following introductions of pneumococcal conjugate vaccines. However, new techniques are needed to lower the costs and work-load for obtaining pneumococcal serotype information, especially in low-income settings. Here, we validated a modified protocol of sequetyping for S. pneumoniae serotype identification. Methods: Forty-six strains of S. pneumoniae, isolated from blood cultures at the Sahlgrenska University Hospital, Gothenburg, Sweden, were analyzed. Serotypes of the strains were identified by Sanger sequencing of the regulatory gene cpsB of the capsulation locus of S. pneumoniae, using a modification of an earlier protocol (Leung et. al. 2012). To increase the coverage of the 1,000 bp sequence length of the cpsB gene, two additional sequencing primers in the central region of the gene were designed. The sequences were compared to available reference sequences, using the NCBI BLAST function. The results were compared to those obtained by multiplex qPCR, modified from a protocol published by the U.S. Centres of Disease Control and Prevention, and with the results obtained by gel diffusion and/or Quellung reactions, performed by the Public Health Agency of Sweden. Results: In total, 22 different serotypes/serotype groups were acquired by sequetyping of the 46 isolates. Contrary to the 74% of isolates that were serotyped/serotype grouped by qPCR, sequetyping could identify the serotype/serotype group in 98% of the isolates. Of the serotypes correctly identified by sequetyping, 32% were identified to a single serotype, whereas in 68% of the isolates, a single serotype could not be assigned. The serotype groups that could not be differentiated further included 6A/6B/6C/6D and 9A/9V. Conclusion: Sequetyping is a sensitive technique for serotype identification. However, in many cases, the difference between serotypes is detected in only one or two base-pairs, or the sequences are identical, making a definitive serotype identification difficult. To obtain serotype results in these cases, additional PCRs for differentiating particular serotypes/groups are required.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biologisk systematik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biological Systematics (hsv//eng)

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