Determination of capsulation status in Haemophilus influenzae by multiplex polymerase chain reaction

Nelson, Kevin L.; Smith, Arnold L.

doi:10.1016/j.diagmicrobio.2009.10.005

Cited by 6 publications

(5 citation statements)

References 29 publications

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“…Five were bexA and bexB negative and lacked region II capsulespecific genes, strongly suggesting that these strains are in fact true NTHI strains, i.e., they lack the entire capsule locus. This finding is consistent with other reports of false-positive serotyping of H. influenzae (2,24,31,32), even in reference laboratories.…”

Section: Resultssupporting

confidence: 83%

Use of bexB To Detect the Capsule Locus in Haemophilus influenzae

et al. 2011

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Haemophilus influenzae strains are classified as typeable or nontypeable H. influenzae (NTHI) based upon the presence or absence of capsule. In addition to serotyping, which is subject to false-positive results, typeable strains can be identified through the detection of the capsular export gene bexA and one of six capsule-specific genes, but this method is resource intensive, especially in characterizing large numbers of strains. To address these challenges, we developed a bexB-based method to differentiate true NTHI strains from typeable strains. We validated a PCR-based method to detect bexB in 10 strains whose capsule status was well defined. Among 40 strains that were previously serotype positive in clinical microbiology laboratories, 5 lacked bexA, bexB, and capsule type-specific genes by PCR analysis and thus likely represent false-positive serotyping results. Among 94 additional otitis media, commensal, and serotype b-negative invasive strains, 85 were bexA and bexB negative and 9 contained either a complete or partial capsule locus, i.e., 8 were bexA and bexB positive and 1 was bexA negative but bexB positive. Finally, we adapted the method for use in a high-throughput DNA hybridizationbased microarray method, which showed 98.75 and 97.5% concordance to the PCR methods for bexA and bexB, respectively. In addition, bexB showed 84% or greater nucleotide identity among strains containing the capsule locus. In this study, we demonstrate that bexB is a reliable proxy for the capsule locus and that its detection provides a simple and reliable method for differentiating strains that lack the entire capsule locus from those containing a partial or complete capsule locus.

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Section: Resultssupporting

confidence: 83%

Use of bexB To Detect the Capsule Locus in Haemophilus influenzae

et al. 2011

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“…These 36 isolates were examined using a multiplex PCR for capsule types a-f, and in separate reactions with primers for bexA, the capsule export gene conserved among all encapsulated H. influenzae (Nelson & Smith, 2010). Among those isolates identified as serotype f using the multiplex assay and among those that were also bexA positive, 25 isolates and the ATCC 9833 Hif reference strain were subsequently confirmed to contain a specific type f capsular gene by further PCR amplification (Falla et al, 1994).…”

Section: Confirmation Of Capsular Serotype Fmentioning

confidence: 99%

“…Routine PCR conditions and DNA manipulations were performed as described previously (Erwin et al, 2005). Initial screening of putative type f strains was with a multiplex PCR, using primers and methods reported previously (Nelson & Smith, 2010). Encapsulated control strains were obtained from the American Type Culture Collection comprising capsular types a-f.…”

Section: Introductionmentioning

confidence: 99%

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates

Watson

Nelson

Nguyen

et al. 2013

Journal of Medical Microbiology

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The incidence of invasive infections due to Haemophilus influenzae has decreased significantly in developed countries with high rates of vaccination against H. influenzae serotype b (Hib). This vaccine provides no protection against H. influenzae serotype f (Hif), typically associated with invasive infections in adults with chronic disease and/or immunodeficiency, and rarely in otherwise healthy adults and children. The specific properties of Hif associated with virulence remain largely uncharacterized. A panel of 26 Hif strains consisting of both invasive disease-associated and mucosal surface non-invasive disease-associated isolates was surveyed by DNA fingerprinting, biotyping and PCR detection of hmw1, hmw2, hsf, the hif fimbrial locus and the lipooligosaccharide (LOS) biosynthetic island, and assessment of b-lactamase expression and determination of resistance to the bactericidal activity of normal adult human serum. Repetitive sequence-based PCR fingerprinting differentiated the 26 strains into three clusters, with the majority of isolates (22/26, 84.6 %) clustered into a single indistinguishable group. Most isolates (24/26, 92.3 %) were of biotype I and two isolates produced b-lactamase with detection of a conjugative plasmid, and the isolates displayed a range of resistances to the bactericidal activity of human serum. All 26 isolates carried the adhesin hsf, 21 carried a partial hif fimbrial operon and 4 had the adhesin genes hmw1/2. A LOS biosynthetic island was detected in 20 isolates consisting of the genes lic2BC. It was concluded that Hif has many recognized virulence properties and comprises a relatively homogeneous group independent of the anatomical source from which it was isolated. INTRODUCTIONHaemophilus influenzae, a human-restricted Gram-negative coccobacillus, is a commensal of the upper respiratory mucosa and a pathogen commonly causing airway mucosal disease and occasional invasive disease. Since Margaret Pittman's original description of capsular serotypes among H. influenzae isolates in 1931 (Pittman, 1931), H. influenzae serotype b (Hib) had been the most clinically significant strain causing invasive disease (e.g. meningitis, epiglottitis, septicaemia and osteomyelitis) in previously well infants and children (Aubrey & Tang, 2003). Since the early 1990s, routine administration of the Hib conjugate vaccines (which induce protective levels of anti-capsular antibody) has virtually eliminated Hib disease among infants and young children in developed countries (Agrawal & Murphy, 2011;Ladhani, 2012). However, the vaccine provides no protection from infection due to nonb serotypes, including H. influenzae serotype f (Hif), which remains a rare but significant cause of invasive infection. Given the reduction in Hib carriage among Hib conjugate vaccine recipients, concern exists as to whether invasive disease due to Hif or other non-b encapsulated strains will become more prevalent due to serotype replacement Abbreviations: BLNAR, b-lactamase-negative ampicillin-resistant; CSF, cerebrospinal ...

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“…In this case report, after determining the identity of the isolate as H. influenzae type f by sequencing, we confirmed its capsular status by both latex agglutination and multiplex-PCR-based capsular typing, which has been shown to most accurately classify the capsular type (Nelson & Smith, 2010). It is of note that by 16S rRNA gene sequencing, H. influenzae type f strains are about 2 % distant from the other H. influenzae capsular types; such a distance would be sufficient for some authors to classify H. influenzae type f as a new unique species (Clarridge, 2004).…”

Section: Discussionmentioning

confidence: 57%