Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification

Abstract

Summary. Two primer sets were chosen for the detection of Haemophilus inzuenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H . inzuenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H . inyuenzae tested. This primer set also reacted with the closely related species H . haemolyticus and H . aegyptius, and with two of nine H . parainzuenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H . inzuenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR ; 40 were culture-positive for H . inzuenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H . inzuenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H . inzuenzae in CSF.

Evaluation of Methodology for Serotyping Invasive and Nasopharyngeal Isolates of Haemophilus influenzae in the Ongoing Surveillance in Brazil

“…DNA amplification (Perkin-Elmer, Norwalk, Conn.) was carried out as described previously (6), but 55°C was used as the annealing temperature. One primer set derived from the sequence for the gene coding for outer membrane lipoprotein P6, which is present in both capsulated and NC Haemophilus strains, was also used as a control for PCRs (26). PCR products were resolved by electrophoresis (Gibco BRL Life Technologies) on a 1% agarose gel (Sigma Chemical Co., St. Louis, Mo.)…”

The utility of the polymerase chain reaction assay for aetiologic definition of unspecified bacterial meningitis cases

“…The small numbers of H. influenzae meningitis cases during the last few years hindered the evaluation of the bexA primer set. Nevertheless, in previous reports the sensitivity was high and equivalent to those assessed; the primer set was 100% specific (van Ketel et al 1990, Corless et al 2001. Indeed, outside of the MRU, some specimens may have been stored and transported in suboptimal temperature conditions, which might have affected the sensitivity of the PCR assay.…”

“…Bacterial DNA was isolated from the CSF samples with the use of QIAmp DNA Mini Kit (QIAGEN) according to the manufacturer's protocol for DNA purification from Gram-positive bacteria. PCR identification of N. meningitidis, S. pneumoniae and H. influenzae (capsulated) was based on a multiplex assay for the amplification of the crgA, ply and bexA genes, respectively, with specific oligonucleotide primers as previously described (van Ketel et al 1990, Taha 2000, Corless et al 2001. In a further experiment, all the CSF samples that gave negative results in the first round of multiplex PCR were subjected to PCR with a primer set selected by van Ketel et al (1990) from the DNA sequence of a gene (pal) encoding for outer-membrane protein P6 (Deich et al 1988), which can detect any spontaneous capsule-deficient mutants of serotype b (b-) or non-capsulate (NC) strains of H. influenzae.…”

Incidence and spread ofHaemophilus influenzaeon an Antarctic base determined using the polymerase chain reaction

“…PCR has been shown to be useful in the detection of micro-organisms in many other situations where extraction, culture and identification are difficult or time consuming, examples including Mycobacterium tuberculosis [12,13], Chlamydia trachomatis [14], Pneumocystis carinii [15], Mycoplasma pneumoniae [16], Helicobacter pylori [17], Salmonella typhi [18] and Toxoplasma gondii [19]. Although it has previously been used to detect H. inftuenzae in infected cerebrospinal fluid [20] in which it would usually be the only organism present, its use in the detection of this species from a mixed bacterial culture represents a novel approach which may in future prove helpful in both clinical and epidemiological settings. 5 ,ug/ml vancomycin hydrochloride (Sigma) and culturing for 24 h in air+5 % CO2 at 37 'C.…”