Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance

Abstract

Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.

Fig. 1.

Mass spectra from DNA amplified from a throat swab (Table 3, sample 5) using each of the 16 surveillance primers (Table 1). (Upper Left) Thumbnail spectra from 16 primers. Each of the PCR wells was calibrated by using an internal standard identical to the bacterial target sequence except for a 2- to 5-nt internal deletion (calibrant peaks are shown in yellow). (Upper Right) Spectrum from a primer pair that targets 23S rDNA. The paired peaks correspond to the sense and antisense strands of the PCR amplicon that are separated under conditions of ionization. The peaks are labeled with base composition of the amplicons and the organism that matches the composition. (Lower Left) Spectrum from a primer pair that targets 16S rDNA. (Lower Right) Spectrum from a primer pair that targets the Bacilli, but not the Proteobacteria.