Sequencing phylogeny of study NTHi isolates.

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

PCR screening of respiratory reference isolates.

*<p>Sourced from isolate collection at Menzies School of Health Research, Darwin, NT, Australia.</p

Sensitivity, specificity and ROC curve areas of PCR assays for study-defined NTHi.

<p>Sensitivity, specificity and ROC curve areas of PCR assays for study-defined NTHi.</p

Genbank reference strains.

<p>Sequence information was obtained from 6 reference strains (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a> – December 2010) to facilitate phylogenetic speciation. na = not applicable.</p

Study primers and probes.

<p>All product sizes are based on the NTHi Rd KW20 genome.</p>*<p>product size varies considerably across strains.</p>**<p><i>hpd</i> probes were labelled with Hex at 5′-end, SpC3 at 3′-end, and Black Hole Quencher (BHQ) at the internal “T”. F (forward primer); R (reverse primer); P (probe); HRM (high resolution melt).</p

Neighbour-Joining dendogram and PCR results.

<p>The Neighbour-Joining dendogram of the concatenated 16S rRNA and <i>recA</i> gene sequences is displayed in conjunction with the PCR results and the percent DNA similarity (compared to <i>H. influenzae</i> 86-028NP). The tree is rooted by <i>H. parainfluenzae</i> 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 <i>H. parainfluenzae</i>. Study-defined NTHi was based collectively on the phylogeny, PCR data and DNA similarity as is indicated on the right (✓ = NTHi or ✗ = Hh).</p