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J Clin Microbiol. 2012 September; 50(9): 3146–3147.
PMCID: PMC3421822

Potential Nonpneumococcal Confounding of PCR-Based Determination of Serotype in Carriage

Editor: Geoffrey Jagero

LETTER

Monitoring pneumococcal carriage serotype distributions is increasingly used to study pneumococcal biology, disease epidemiology, and vaccine impact. Potentially complicating DNA-based carriage assessment is the well-documented history of genetic recombination between pneumococci and related species that colonize the upper respiratory tract (4).

Recently, when assessing pneumococcal carriage in western Kenya (1) with a multiplex PCR (mPCR) assay for 40 capsular serotypes or serogroups (2), we noted a high frequency of mPCR positivity for cocarried serotypes. This was especially evident in adults who had naso- and oropharyngeal swab specimens combined; from these, 122 of 158 (77%) exhibited 4 or more mPCR serotypes (1). These included 24 of 27 specimens that were negative for the pneumococcus-specific lytA PCR assay (3; unpublished data). The mPCR amplicons sg10F/10C/33C and sg18A/18B/18C/18F occurred among 59 to 80% of adults (1). Sequencing of sg10F/10C/33C amplicons from 11 carriage specimens revealed five 192-bp sg10F/10C/33C sequence types, including three from lytA-negative specimens, which shared 90.1 to 93.8% identity to corresponding pneumococcal reference 10F, 33C, and 35B sequences (Table 1). Analysis of sg18A/18B/18C/18F amplicons from 17 specimens (including three lytA-negative specimens) revealed five sequence types with 89 to 93% identity to the 4 serogroup 18 reference sequences (Table 2). In contrast, sg10F/10C/33C and sg18A/18B/18C/18F mPCR amplicons from carriage specimens that yielded corresponding serotype 18A, 18C, or 10F isolates revealed sequence identity in each instance with the corresponding pneumococcal reference sequence (Tables 1 and and22).

Table 1
Sequence identities (percent) for 192-bp wzx gene sequences from nonpneumococcal species and from carriage specimens that coamplify with targeted PCR assay for pneumococcal sg10F/10Ch
Table 2
Sequence identities (percent) for suspected nonpneumococcal 507-bp wzy gene sequences from carriage specimens that coamplify with targeted PCR assay for pneumococcal sg18A/18B/18C/18F with pneumococcal references 18A, 18C, and 18Fe

Within a reference collection of 54 strains comprised of 16 nonpneumococcal species, we found 5 mPCR-positive results for sg10F/10C/33C within strains of Streptococcus oralis (2 of 3), Streptococcus infantis (1 of 1), Streptococcus gordonii (1 of 5), and Streptococcus salivarius (1 of 3). Strains of Streptococcus mitis (4), Streptococcus pseudopneumoniae (21), Streptococcus parasanguinis (3), Streptococcus sanguinis (3), Streptococcus cristatus (3), Streptococcus vestibularis (2), Streptococcus peroris, Streptococcus australis, Streptococcus intestinalis, Streptococcus oligofermentans, Streptococcus sinensis, and Dolosigranulum pigrum tested negative. Sequence comparisons of the 5 amplicons revealed 87.0 to 95.8% identity to corresponding 10F, 33C, and 35B reference sequences (Table 1). We found that the amplicon from both S. oralis reference strains shared sequence identity with an S. oralis polysaccharide biosynthetic locus (5) and also with the amplicon obtained from lytA-negative specimen 32. We did not find positive mPCR results for sg18A/18B/18C/18F within these reference strains; however, it is unlikely that this collection provided adequate representation of commensal streptococcal species diversity.

In summary, mPCR amplicon sequences for sg10F/10C/33C and sg18A/18B/18C/18F within lytA-negative specimens were divergent compared to the reference pneumococcal serotype sequences (Tables 1 and and2).2). From within the same population, multiple specimens that yielded isolates that expressed the serotypes in question invariably revealed amplicons (from the specimens and isolates) with sequence identity to the corresponding 10F, 18A, and 18C references. We found divergent homologs of a pneumococcal capsular biosynthetic locus within a small nonpneumococcal species collection. We also encountered similar issues in this population with additional vaccine serotypes besides 18C (2, 5, 7F, and 33F) that are vaccine components (unpublished data). Until further clarification, PCR-based serotyping for carriage studies should be employed upon identified pneumococcal isolates only.

This preliminary letter describes our concern. There has been an unforeseen delay in the availability of these carriage specimens to us for isolation and characterization of potentially nonpneumococcal confounding species. While aware that this letter represents an incomplete study, we feel that further delay in conveying this information is unacceptable.

Footnotes

Published ahead of print 3 July 2012

Contributor Information

Maria da Gloria Carvalho, Respiratory Diseases Branch Division of Bacterial Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA.

Muthoni Junghae, International Emerging Infections Program Centers for Disease Control and Prevention Nairobi, Kenya.

Laura Conklin, Respiratory Diseases Branch Division of Bacterial Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA.

Robert F. Breiman, International Emerging Infections Program Centers for Disease Control and Prevention Nairobi, Kenya.

Bernard Beall, Respiratory Diseases Branch Division of Bacterial Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA.

REFERENCES

1. Carvalho MDG, et al. 2012. PCR assessment of carriage among population with high HIV prevalence: frequent colonization, multiple serotypes, and intra-serotype variability within wzy PCR-serotyping target, abstr. 306. 8th Int. Symp. Pneumococci Pneumococcal Dis Iguacu Falls, Brazil, 11 to 15 March 2012.
2. Carvalho MDG, et al. 2010. Revisiting pneumococcal carriage using broth-enrichment and PCR techniques for enhanced detection of carriage and serotypes. J. Clin. Microbiol. 48:1611–1618. [PMC free article] [PubMed]
3. Carvalho MDG, et al. 2007. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J. Clin. Microbiol. 45:2460–2466. [PMC free article] [PubMed]
4. Dowson CG, et al. 1989. Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Proc. Natl. Acad. Sci. U. S. A. 86:8842–8846. [PubMed]
5. Yoshida Y, et al. 2008. Molecular and antigenic characterization of a Streptococcus oralis coaggregation receptor polysaccharide by carbohydrate engineering in Streptococcus gordonii. J. Biol. Chem. 283:12654–12664. [PMC free article] [PubMed]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)