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

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.

Figure 1. Sequencing phylogeny of study NTHi isolates.

Radiation trees are presented for (A) 598 bases of the 16S rRNA gene, (B) 545 bases of the recA gene, and (C) the concatenation of these sequences (1143 bases). The trees are rooted by H. parainfluenzae T3T1 as indicated by yellow dots. Blue dots represent isolates that cluster with H. influenzae reference strains, orange dots represent closely related phylogenetic variants, red dots represent likely Hh isolates and green dots represent variants related to H. parainfluenzae. Colours were assigned based on the phylogenetic grouping of the concatenated sequences in radial tree C.

Figure 2. Differentiation of study isolates using omp P6-HRM.

Analysis of publically available sequence identified a SNP in the omp P6 genes of NTHi (G) and Hh (T) corresponding to nucleotide position 402465 of H. influenzae Rd KW20, Accession No. L42023. HRM of the 40 base pair amplicon (402446–402485) surrounding the SNP revealed 2 discrete melt profiles; one that clustered with reference H. influenzae (ATCC 19418) and another that clustered with reference Hh (ATCC 33390).

Figure 3. Neighbour-Joining dendogram and PCR results.

The Neighbour-Joining dendogram of the concatenated 16S rRNA and recA gene sequences is displayed in conjunction with the PCR results and the percent DNA similarity (compared to H. influenzae 86-028NP). The tree is rooted by H. parainfluenzae as indicated by the yellow dots. The blue dots represent the strict NTHi group, the orange dots represent closely related phylogenetic variants, the red dots represent likely Hh isolates and the green dots represent variants related to H. parainfluenzae. Study-defined NTHi was based collectively on the phylogeny, PCR data and DNA similarity as is indicated on the right (✓ = NTHi or ✗ = Hh).