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.genotyping ͉ group A streptococci ͉ infectious disease ͉ Streptococcus pyogenes ͉ pneumonia

Local changes in rates of groupA Streptococcus disease and antibiotic resistance are associated with geographically widespread strain turnover events

“…The data are consistent with previous reports suggesting that only a small number of the known emm types are generally associated with high virulence and invasive characteristics, with M1, M3 and M5 foremost among them. 3,6,10,14,15,18,23,24 In much the same way that the rise and fall of M75 appears responsible for recent spikes in macrolide resistance, the data suggest that the observed spikes in GAS-associated ARD and invasive disease were associated with the shifting dominance of a particularly virulent strain, M5. The association between this serotype and the widespread rate increases seen among trainees from 2002 through 2007 supports the general hypothesis that virulence is strongly linked to serotype.…”

“…The most common method of direct serological identification is M typing, 16 but this has been widely replaced by inference of M type from emm gene sequence analysis, 17 or more indirectly inferred by various forms of multilocus sequence typing. 18 All of these methods have shown broad concordance to the degree that they overlap in coverage. 19 Analysis of the sample set collected for this study showed very strong correlations between emm type and antibiotic resistance phenotypes, as well as strong tendencies for specific types to be associated with outbreaks.…”

Mitochondrial DNA Mutation Detection by Electrospray Mass Spectrometry

“…Samples 2, 8, 10, and 19 contained variant T8993G, sample 15 contained T8993C, and sample 12 contained G11778A. Samples 3,5,7,9,11,13,14,16,17,18,20, and 21 lacked any of the 7 pathogenic mutations.…”

“…Recently, MS-based nucleic acid protocols have migrated out of MS labs for application in other disciplines, including human and microbial forensics, drug discovery, infectious-disease diagnostics, and biological warfare agent detection (6)(7)(8)(9)(10). Because the masses of the 4 mononucleotides (dAMP, dTMP, dGMP, and dCMP) comprising DNA are known with great accuracy, highly precise measurements of mass can be used to derive a base composition (or a constrained list of base compositions).…”

Targeted proteomics approach to species-level identification of Bacillus thuringiensis spores by AP-MALDI-MS

“…Two promising techniques, real-time PCR and mass spectrometry, target genetic information at different levels: DNA and proteins. The polymerase chain reaction (PCR) has demonstrated unparalleled levels of sensitivity and specificity by targeting DNA for amplification [5][6][7]. Unfortunately, the time required for the PCR reaction hinders rapid detection [5][6][7].…”