One of the objectives of a recent study by Satola et al. (2) was to use PCR capsule gene typing to evaluate the accuracy of the slide agglutination method for serotyping (SAST) Haemophilus influenzae. Of the 360 invasive H. influenzae isolates tested, 64 (or 17.5%) were found to be inaccurately serotyped by SAST. Since the phosphoglucose isomerase (pgi) genotype has been suggested as a surrogate for the serotype of encapsulated H. influenzae isolates (1), the authors also used pgi genotyping to evaluate their nontypeable (NT) H. influenzae isolates. They found that the pgi alleles detected in their NT H. influenzae strains were primarily those described as being associated with NT strains in the MLST (Multi Locus Sequence Typing) H. influenzae website (http://haemophilus.mlst.net).
In our analysis of invasive H. influenzae isolates (n = 122) from Manitoba, Canada (3), we have also found that strains that belong to different serotypes did not have any common housekeeping gene alleles. Thus, different pgi alleles were found in isolates that belong to different serotypes. And we concluded that no capsule switching was detected among the 122 invasive isolates studied. We also took the opportunity to interrogate the MLST H. influenzae database (accessed 6 June 2007) to examine how specific or accurate the pgi allele can be when used to predict serotype. Among the six H. influenzae serotypes, there were 39 different pgi alleles found. Although, in most cases, the pgi alleles were found to be serotype specific, three alleles were found to be non-serotype specific. Allele 40 was found in a pair of isolates, one H. influenzae type b (Hib) and one Hif; allele 42 was found in another pair of isolates, one Hid and one Hif; and allele 28 was found in numerous Hie strains and in a single Hif isolate (Table 1).
TABLE 1.
pgi alleles of Haemophilus influenzae shared between isolates of different serotypesa
| Allele | Serotype | MLST ST | Strain IDb |
|---|---|---|---|
| 40 | b | ST-286 | ID 450 |
| f | ST-106 | ID 234 | |
| 42 | d | ST-102 | ID 238 |
| f | ST-415 | ID 670 | |
| 28 | e | Numerous STsc | Numerous IDsc |
| f | ST-125 | ID 263 |
Information obtained from the MLST Haemophilus influenzae website (http://haemophilus.mlst.net).
ID, identification number.
ST-17 (ID 44); ST-18 (ID 45); ST-27 (ID 61); ST-28 (ID 62); ST-32 (ID 66); ST-66 (ID 128); ST-67 (ID 129); ST-68 (ID 130); ST-69 (ID 131); ST-121 (ID 259); ST-122 (ID 260); ST-127 (ID 265); ST-129 (ID 267); ST-251 (ID 410); ST-384 (ID 652); ST-386 (ID 653).
Also, of the 169 different sequence types (STs) in the MLST H. influenzae database that were found to be associated with encapsulated strains, most STs were linked to their serotypes, and strains belonging to the different serotypes appeared to have their own sets of STs. Only two related STs that shared five common alleles were detected in two strains of different serotypes: one isolate of serotype b (ST-329; MLST allelic profile: adk = 1, atpG = 1, frdB = 1, fucK = 1, mdh = 31, pgi = 1, and recA = 5) and the other of serotype d (ST-102; MLST allelic profile: adk = 1, atpG = 1, frdB = 1, fucK = 1, mdh = 50, pgi = 42, and recA = 5). Although this may suggest that their relatedness was due to capsule switching, nevertheless, if it occurs, this is not a common event in H. influenzae.
I agree with Satola et al. (2) that accurate serotyping is important in the surveillance of invasive H. influenzae disease in the post-Hib vaccine era. Besides improvement of the laboratory training of personnel and adequate quality control of reagents and methods, other established procedures that avoid the subjectivity of the bacterial slide agglutination test should be explored, such as the indirect whole-cell enzyme-linked immunosorbent assay method that has been developed for the serotyping of Bordetella pertussis (4) and serogrouping of Neisseria meningitidis (5). In addition, MLST of invasive isolates will provide not only indications of the capsular types but also additional information on potential capsule switching among H. influenzae isolates.
Acknowledgments
This study made use of the Haemophilus MLST website (http://haemophilus.mlst.net), developed and maintained by David Aanensen at the Imperial College, London, United Kingdom, and funded by the Wellcome Trust.
Ed. Note: The authors of the published article did not respond.
REFERENCES
- 1.Anyanwu, J. N., C. A. Rodriguez, K. E. Fleming, and E. E. Adderson. 2003. pgi genotyping is a surrogate for serotyping of encapsulated Haemophilus influenzae. J. Clin. Microbiol. 412080-2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Satola, S. W., J. T. Collins, R. Napier, and M. M. Farley. 2007. Capsule gene analysis of invasive Haemophilus influenzae: accuracy of serotyping and prevalence of IS1016 among nontypeable isolates. J. Clin. Microbiol. 453230-3238. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Sill, M. L., D. K. S. Law, J. Zhou, S. Skinner, J. Wylie, and R. S. W. Tsang. 2007. Population genetics and antibiotic susceptibility of invasive Haemophilus influenzae in Manitoba, Canada, from 2000 to 2006. FEMS Immunol. Med. Microbiol. 51270-276. [DOI] [PubMed] [Google Scholar]
- 4.Tsang, R. S. W., M. L. Sill, A. Advani, D. Xing, P. Newland, and H. Hallander. 2005. Use of monoclonal antibodies to serotype Bordetella pertussis isolates: comparison of results obtained by indirect whole-cell enzyme-linked immunosorbent assay and bacterial microagglutination methods. J. Clin. Microbiol. 432449-2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Tsang, R. S. W., and W. D. Zollinger. 2005. Serological specificities of murine hybridoma monoclonal antibodies against Neisseria meningitidis serogroups B, C, Y, and W135 and evaluation of their usefulness as serogrouping reagents by indirect whole cell enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol. 12152-156. [DOI] [PMC free article] [PubMed] [Google Scholar]