Reverse transcription-PCR-electrospray ionization mass spectrometry for rapid detection of biothreat and common respiratory pathogens.

Electrospray ionization mass spectrometry (ESI-MS) analysis of reverse transcription (RT)-PCR amplicons from human respiratory samples allows for broad pathogen identification approximately 8 h after collection. We investigated the performance characteristics of a high-throughput RT-PCR-coupled ESI-MS assay for distinguishing biothreat (BT) agents from common bacterial, fungal, and viral respiratory pathogens in bronchoalveolar lavage (BAL) fluid specimens from subjects with suspected respiratory infections. In a retrospective case series, 202 BAL fluid specimens were collected at the Johns Hopkins Hospital between August 2010 and February 2011 from patients with suspected acute respiratory infections. Samples were processed using standard bacterial, viral, and fungal testing in the clinical microbiology laboratory as part of routine care and then were blindly spiked with either water or nucleic acids from BT organisms (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella spp., Burkholderia spp., and Rickettsia prowazekii) and tested by RT-PCR-ESI-MS. The sensitivities and specificities of RT-PCR-ESI-MS versus standard clinical methods were as follows: for mock BT DNA, 98.5% sensitivity (95% confidence interval [CI], 94.2 to 99.7%) and 100% specificity (95% CI, 93.1 to 100.0%); for bacterial pathogens, 81.8% sensitivity (95% CI, 74.3 to 87.6%) and 73.6% specificity (95% CI, 64.2 to 81.4%); for viral pathogens, 93.3% sensitivity (95% CI, 66.0 to 99.7%) and 97.3% specificity (95% CI, 89.7 to 99.5%); for fungal pathogens, 42.6% sensitivity (95% CI, 29.5 to 56.7%) and 97.8% specificity (95% CI, 91.8 to 99.6%). Our data suggest that RT-PCR-ESI-MS is a useful adjunct to standard culture protocols for rapid detection of both BT and common respiratory pathogens; further study is required for assay validation, especially for fungal detection, and potential implementation.

Performance of three nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by use of self-collected vaginal swabs obtained via an Internet-based screening program.

Use of self-obtained vaginal specimens processed by nucleic acid amplification tests (NAATs) has significantly increased the utilization of nontraditional locations for Chlamydia trachomatis and Neisseria gonorrhoeae screening programs. One important emerging source of such venues includes home-based self-sampling kits available via the Internet. The objective of our study was to evaluate the performance of three commercially available NAATs (Becton-Dickinson ProbeTec SDA, Gen-Probe Aptima Combo2 TMA, and Roche Amplicor PCR) for detection of C. trachomatis and N. gonorrhoeae in vaginal samples obtained via an Internet-based screening program. From July 2004 to August 2005, 500 self-collected vaginal swabs were tested for C. trachomatis and N. gonorrhoeae by using all three NAATs. Another 500 samples were collected between August 2005 and November 2007 and tested by ProbeTec and Combo2; PCR testing was discontinued due to low specificity for N. gonorrhoeae. All tests were conducted according to the manufacturers' procedures; the "gold standard" for an infected C. trachomatis or N. gonorrhoeae patient was defined as > or = 2 positive NAAT results. Of the first 500 swabs submitted, 46 were C. trachomatis infected (9.2%) and 5 were N. gonorrhoeae infected (1.0%), and 3 of these were coinfected (0.6%). All C. trachomatis and N. gonorrhoeae Combo2-positive/ProbeTec-negative samples were confirmed as true positives by an alternative NAAT. For C. trachomatis, ProbeTec, Combo2, and PCR had sensitivities of 82.6%, 100%, and 100%, with specificities of 100%, 100%, and 99.3%, respectively. For N. gonorrhoeae, ProbeTec, Combo2, and PCR had sensitivities of 80%, 100%, and 100%, with specificities of 100%, 100%, and 98.8%, respectively. Of the total 1,000 swabs submitted, 92 were C. trachomatis infected (9.2%) and 15 were N. gonorrhoeae infected (1.5%), and 7 of these were coinfected (0.7%). There were no ProbeTec-positive/Combo2-negative samples. For C. trachomatis, ProbeTec and Combo2 had sensitivities of 81.5% and 100%, with specificities of 100% and 100%, respectively. For N. gonorrhoeae, ProbeTec and Combo2 had sensitivities of 80% and 100%, with specificities of 100% and 100%, respectively. Overall, ProbeTec had 17 C. trachomatis false-negative results (1.7%) and 3 N. gonorrhoeae false-negative results (0.3%), while Combo2 had none. Our results were consistent with the sensitivities and specificities stated by the manufacturers. NAATs perform well for detection of chlamydia and gonorrhea with self-obtained vaginal swabs shipped in a dry state to a laboratory. For 1,000 self-collected vaginal swabs tested by NAATs, the sensitivities for C. trachomatis and N. gonorrhoeae for Combo2 were 100% and 100%, while they were 81.5% and 80%, respectively, for ProbeTec. For 500 PCR samples, the C. trachomatis sensitivity was 100% and the N. gonorrhoeae sensitivity was 100%, with specificities of 99.3% and 98.8%, respectively.

A rabbit model of non-typhoidal Salmonella bacteremia.

Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.