Real-Time PCR for Determining Capsular Serotypes of Haemophilus influenzae

Abstract

A two-step real-time PCR assay targeting all six capsulation loci of Haemophilus influenzae (i.e., serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131 H. influenzae clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of H. influenzae a to c (10 1 CFU/PCR) was higher than that for type e (10 3 CFU/PCR) and types d and f (10 4 CFU/PCR), and a broader dynamic range was obtained (5 to 8 log 10 units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of H. influenzae invasive infections.

Microbiological Characterization of Haemophilus influenzae Isolated from Patients with Lower Respiratory Tract Infections in a Tertiary Care Hospital, South India

“…But the capsular genotyping results revealed all 50 strains to be non-typeable. This variation in results could be due to the number of inconsistencies associated with capsular serotyping with slide agglutination test [8,9,14,15]. Hence PCR for capsular serotyping could be more useful [15].…”

“…It is very important to perform capsular typing of H. influenzae, as the capsule is a major virulence factor in the pathogenesis of Keywords: Antibiotic susceptibility testing, Biotyping, Genotyping, Non-typeable strains [8,9]. The amplification of genes involved in capsule expression was found to be rapid, specific, and highly sensitive for H.influenzae detection [8,9].…”

“…The amplification of genes involved in capsule expression was found to be rapid, specific, and highly sensitive for H.influenzae detection [8,9].…”

Evolution of bacterial meningitis diagnosis in Sao Paulo State-Brazil and future challenges

“…Among the types of probes available, the most commonly used are the TaqMan ® linear hydrolysis probes 18,19 . Since last decade many researchers have been publi shing about molecular diagnosis of bacterial meningitis using qPCR in different assay formats, all with excellent performance in detecting DNA from different pathogens [20][21][22][23][24][25] . The traditional microbiological methods, such as the Gram stain or latex agglutination are available for the detection of some agents; however they are not enough sensitive to detect them in small concentrations, a fact usually found in clinical samples from patients undergoing antibiotic therapy 13 .…”

“…The method relies on the detection of specific genes related to the biosynthesis of capsular polysaccharide responsible for different groups and types of Nm and Hi, respectively, and used in previously positive samples for each agent 21,23,24,27 .…”