Use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections

Abstract

We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.

FIG. 1.

Hybridization results from bacterial cultures and clinical samples. Each hybridization result is presented in duplicate. The positive controls are located in positions D6 and E6. (A) S. pneumoniae hybridization. All three S. pneumoniae-specific oligonucleotides (C2, D2, and E2) gave positive signals. (B) C. diphtheriae hybridization. Positive signals were detected from all C. diphtheriae-specific oligonucleotides (B4, C4, and D4). (C) Hybridization results from a throat swab sample (patient C237). All three S. pyogenes-specific oligonucleotide probes gave a clear signal (A6, B6, and C6). (D) Hybridization results from a middle ear fluid sample (patient C108). Two out of three H. influenzae-specific oligonucleotides (B5 and E4) gave positive signals.

FIG. 2.

Comparison of positive identification results for five pathogens. These results are based on the oligonucleotide array assay detection and conventional bacterial culture. In all, 73 patient samples consisting of 53 middle ear fluid (C100 to C163) and 20 throat swab (C214 to C240) samples were included in this comparison. Each bacterial species is indicated by a specific color.