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Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays

Binks, Michael J., Temple, Ellinor B., Kirkham, Lea-Ann S., Wiertsema, Selma P., Dunne, Eileen, Richmond, Peter C., Marsh, Robyn L., Leach, Amanda J. and Smith-Vaughan, Heidi C. (2012). Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays. PLoS One,7(3):e34083.

Document type: Journal Article
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IRMA ID cmartelxPUB13
NHMRC Grant No. 1011172
545232
Title Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays
Author Binks, Michael J.
Temple, Ellinor B.
Kirkham, Lea-Ann S.
Wiertsema, Selma P.
Dunne, Eileen
Richmond, Peter C.
Marsh, Robyn L.
Leach, Amanda J.
Smith-Vaughan, Heidi C.
Journal Name PLoS One
Publication Date 2012
Volume Number 7
Issue Number 3
ISSN 1932-6203   (check CDU catalogue  open catalogue search in new window)
Scopus ID 2-s2.0-84859089597
Start Page e34083
Total Pages 8
Place of Publication United States
Publisher Public Library of Science
HERDC Category C1 - Journal Article (DIISR)
Abstract Background
Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination.

Methodology/Principal Findings

Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction.

Conclusions/Significance
Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.
Keywords Gene targeting
Haemophilus
Haemophilus influenzae
Phenotypes
Phylogeography
Polymerase chain reaction
Ribosomal RNA
Species delimitation
DOI http://dx.doi.org/10.1371/journal.pone.0034083   (check subscription with CDU E-Gateway service for CDU Staff and Students  check subscription with CDU E-Gateway in new window)


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