A PCR–High-Resolution Melt Assay for Rapid Differentiation of Nontypeable Haemophilus influenzae and Haemophilus haemolyticus

Pickering, Janessa; Binks, Michael J.; Beissbarth, Jemima; Hare, Kim M.; Kirkham, Lea‐Ann S.; Smith‐Vaughan, Heidi

doi:10.1128/jcm.02191-13

Cited by 16 publications

(23 citation statements)

References 22 publications

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“…Several recent studies have explored the use of PCR screens for the distinction of these species (13,(18)(19)(20)(21)(22)(23). The major challenge in the evaluation of these studies is the lack of a universal delineation of H. influenzae.…”

Section: Discussionmentioning

confidence: 99%

“…However, these methods are labor intensive and too expensive for routine use. A number of studies have, therefore, attempted to identify and evaluate suitable assays for rapid and inexpensive identification of H. influenzae (13,(17)(18)(19)(20)(21)(22)(23). Recently, an evaluation of 9 PCR screening assays was performed on a challenging sample of 60 nasopharyngeal carriage isolates selected to include approximately equal numbers of NTHi, H. haemolyticus, and equivocal strains (23).…”

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Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

2015

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bNonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the "fuzzy species" strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment. Haemophilus influenzae is an important human pathogen involved in respiratory tract infections, such as sinusitis, acute otitis media, pneumonia, and exacerbations in chronic obstructive pulmonary disease (1-5). The majority of these infections are caused by unencapsulated H. influenzae, traditionally designated nontypeable H. influenzae (NTHi). Haemophilus haemolyticus is a close relative of H. influenzae, and the two species colonize the upper respiratory tract of humans. Although H. haemolyticus has, on rare occasion, been isolated from invasive infections (6), several lines of evidence indicate that the pathogenicity of H. haemolyticus is much reduced compared with H. influenzae. While up to 20% of presumptive H. influenzae nasopharyngeal isolates can be identified as H. haemolyticus by molecular characterization (7-9), H. haemolyticus is rarely cultured from middle ear fluid (10, 11), supporting the view that H. haemolyticus, in contrast to NTHi, is a respiratory commensal infrequently associated with otitis media. Reinvestigation of presumptive H. influenzae isolates, cultured from lower respiratory tract samples from cystic fibrosis patients (12) or from unselected clinical samples submitted to the laboratory on suspicion of lower respiratory tract infection (13), detected that Ͻ1% were misidentified strains, further supporting a minor pathogenic role for H. haemolyticus.H. haemolyticus was traditionally identified by its hemolytic action on erythrocytes, but it has become clear that a large proportion of H. haemolyticus strains is nonhemolytic (10,14). Such strains, which are designated as nonhemolytic H. haemolyticus, can only be differentiated from H. influenzae by DNA sequencing or extended phen...

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Section: Discussionmentioning

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Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

2015

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“…Molecular diagnosis through 16S rRNA gene sequencing or PCR with specific genes, such as the Haemophilus protein D gene (hpd) or fuculose kinase gene (fucK), have been shown to be reliable alternatives. The prevalence of either or both of the hpd and fucK genes in H. influenzae can be up to 92% (8)(9)(10)(11). However, molecular testing is expensive and technically demanding.…”

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Rapid Differentiation of Haemophilus influenzae and Haemophilus haemolyticus by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry with ClinProTools Mass Spectrum Analysis

et al. 2017

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Haemophilus influenzae is associated with severe invasive disease, while Haemophilus haemolyticus is considered part of the commensal flora in the human respiratory tract. Although the addition of a custom mass spectrum library into the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system could improve identification of these two species, the establishment of such a custom database is technically complicated and requires a large amount of resources, which most clinical laboratories cannot afford. In this study, we developed a mass spectrum analysis model with 7 mass peak biomarkers for the identification of H. influenzae and H. haemolyticus using the ClinProTools software. We evaluated the diagnostic performance of this model using 408 H. influenzae and H. haemolyticus isolates from clinical respiratory specimens from 363 hospitalized patients and compared the identification results with those obtained with the Bruker IVD MALDI Biotyper. The IVD MALDI Biotyper identified only 86.9% of H. influenzae (311/358) and 98.0% of H. haemolyticus (49/50) clinical isolates to the species level. In comparison, the ClinProTools mass spectrum model could identify 100% of H. influenzae (358/358) and H. haemolyticus (50/50) clinical strains to the species level and significantly improved the species identification rate (McNemar's test, P Ͻ 0.0001). In conclusion, the use of ClinProTools demonstrated an alternative way for users lacking special expertise in mass spectrometry to handle closely related bacterial species when the proprietary spectrum library failed. This approach should be useful for the differentiation of other closely related bacterial species. KEYWORDS MALDI-TOF, Haemophilus haemolyticus, Haemophilus influenzae, ClinProToolsH aemophilus influenzae and Haemophilus haemolyticus are the most prevalent Haemophilus species colonizing the human pharyngeal cavity (1). H. influenzae causes various respiratory tract diseases, such as sinusitis, otitis media, and pneumonia, as well as infective exacerbations of chronic obstructive pulmonary disease and bronchiectasis (2). In contrast, H. haemolyticus colonizes healthy subjects at moderate prevalence and rarely causes infections in the respiratory tract (3-5).Laboratory differentiation of the two species based on phenotypic tests can be difficult. H. haemolyticus and H. influenzae demonstrate similar colony morphologies, and both require hemin (X factor) and NAD (V factor) for growth. Hemolytic activity on

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“…Misidentification of H. haemolyticus as NT H. influenzae has been repeatedly reported by multiple research groups, with up to 40% of isolates being misidentified as NT H. influenzae using classical phenotypic methods in clinical microbiology laboratories (1,(7)(8)(9)(10)(11). The misidentification of H. haemolyticus has a potential impact on accurate assessment of the prevalence of antibioticresistant NT H. influenzae (12).…”

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Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Rishishwar

Sivadas

et al. 2016

J Clin Microbiol

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g Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus. Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus. A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae. Haemophilus haemolyticus is a human commensal that colonizes the respiratory tract. It shares high similarities in morphology, biochemistry, and genetics with nontypeable Haemophilus influenzae (NT H. influenzae) (1-3). NT H. influenzae has emerged as the major cause of invasive H. influenzae disease since the implementation of H. influenzae serotype b (H. influenzae b) vaccine (4). NT H. influenzae infections such as childhood otitis media and respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD) result in an enormous social and economic burden to societies (5). H. haemolyticus was recently reported to cause invasive disease (1). These cases were previously misidentified as NT H. influenzae due to the lack of proper identification methods to discriminate between the two species (6). Reevaluation of NT H. influenzae cases from previous years revealed that about 2% (7/374) of the NT H. influenzae invasive cases reported from Active Bacterial Core surveillance (ABCs) were in fact caused by H. haemolyticus (1).Misidentification of H. haemolyticus as NT H. influenzae has been repeatedly reported by multiple research groups, with up to 40% of isolates being misidentified as NT H. influenzae using classical phenotypic methods in clinical microbiology laboratories (1, 7-11). The misidentification of H. haemolyticus has a potential impact on accurate assessment of the prevalence of antibioticresistant NT H. influenzae (12). Therefore, a rapid and accurate method is needed to distinguish H. haemolyticus from NT H. influenzae and to better understand the epidemiology of H. haemolyticus infections....

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A PCR–High-Resolution Melt Assay for Rapid Differentiation of Nontypeable Haemophilus influenzae and Haemophilus haemolyticus

Cited by 16 publications

References 22 publications

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

Rapid Differentiation of Haemophilus influenzae and Haemophilus haemolyticus by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry with ClinProTools Mass Spectrum Analysis

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

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