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.

Application of a 16S rRNA PCR-high-resolution melt analysis assay for rapid detection of Salmonella Bacteremia.

Current culture and phenotypic protocols for diagnosing Salmonella infections can be time-consuming. Here, we describe the application of a 16S rRNA PCR coupled to high-resolution melt analysis (HRMA) for species and serotype identification within 6 h of blood sample collection from a patient with Salmonella enterica serotype Enteritidis bacteremia.

Identification of bacterial pathogens in ascitic fluids from patients with suspected spontaneous bacterial peritonitis by use of broad-range PCR (16S PCR) coupled with high-resolution melt analysis.

Spontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBP rely on culture techniques for proper diagnosis, although recent reports suggest that the presence of bacterial DNA in peritoneal fluid in patients with cirrhosis and ascites is an indicator of SBP. A previously published broad-range PCR (16S PCR) coupled with high-resolution melt analysis (HRMA) was compared with standard culture techniques for diagnosis of SBP in 106 peritoneal fluid samples from patients with suspected SBP. The sensitivity and specificity for 16S PCR for detecting eubacterial DNA compared with those of standard culture techniques were 100% (17/17) and 91.5% (85/89), respectively. Overall, HRMA concordance with species identification was 70.6% (12/17), although the 5 samples that were discordant at the species level were SBP resulting from a polymicrobial infection, and species-level identification for polymicrobial infections is outside the capability of HRMA. Both the broad-range 16S PCR and HRMA analysis provide useful diagnostic adjunctive assays for clinicians in detecting and identifying pathogens responsible for SBP.

Comparative analysis of two broad-range PCR assays for pathogen detection in positive-blood-culture bottles: PCR-high-resolution melting analysis versus PCR-mass spectrometry.

Detection of pathogens in bloodstream infections is important for directing antimicrobial treatment, but current culture-based approaches can be problematic. Broad-range PCR assays which target conserved genomic motifs for postamplification amplicon analysis permit detection of sepsis-causing pathogens. Comparison of different broad-range assays is important for informing future implementation strategies. In this study, we compared positive-blood-culture bottles processed by PCR coupled to high-resolution melting curve analysis (PCR/HRMA) and PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to microbiology culture results. Genus-level concordance was 90% (confidence interval [CI], 80 to 96%) for PCR/HRMA and 94% (CI, 85 to 98%) for PCR/ESI-MS. Species-level concordance was 90% (CI, 80 to 96%) for PCR/HRMA and 86% (CI, 75 to 93%) for PCR/ESI-MS. Unlike PCR/HRMA, PCR/ESI-MS was able to resolve polymicrobial samples. Our results demonstrated that the two assays have similar overall concordance rates but may have different roles as potential adjunctive tests with standard blood culture, since each method has different capabilities, advantages, and disadvantages.

Evaluation of the Pan Influenza detection kit utilizing the PLEX-ID and influenza samples from the 2011 respiratory season.

A comparison study was performed between the PLEX-ID and the CDC RT-PCR method for the detection and identification of Influenza A viruses using nasopharyngeal samples (N=75) collected between January and May 2011. Overall agreement was 89.3% (67/75 kappa=0.57 95% CI 0.3-0.89). Positive percent agreement was 92.3% (60/65); negative percent agreement was 70% (7/10). H1N1 pdm09 identified: 42.6% (32/75) and 54.7% (41/75) by PLEX-ID and CDC RT-PCR, respectively. H3N2 identified: 29.3% (22/75) and 32% (24/75) of samples by PLEX-ID and CDC RT-PCR, respectively. Negatives identified: 16% (12/75) and 13.3% (10/75), by PLEX-ID and CDC RT-PCR respectively. For influenza viruses identified as H1N1 pdm09, Influenza A virus A/NEW YORK/15/2009(H1N1 pdm09) was the most prevalent genotype at 50% (16/32), followed by A/CALIFORNIA/05/2009(H1N1 pdm09) at 18.2% (6/32). Updated assay plates containing additional primers designed for H1N1 pdm09 HA and NA genes were utilized for this evaluation. Among H1N1 pdm09 samples, the HA gene was conserved in 96.9% (31/32) of samples. The NA gene was conserved in 96.9% (31/32).

Interventions to mitigate emergency department and hospital crowding during an infectious respiratory disease outbreak: results from an expert panel.

OBJECTIVE: To identify and prioritize potential Emergency Department (ED) and hospital-based interventions which could mitigate the impact of crowding during patient surge from a widespread infectious respiratory disease outbreak and determine potential data sources that may be useful for triggering decisions to implement these high priority interventions. DESIGN: Expert panel utilizing Nominal Group Technique to identify and prioritize interventions, and in addition, determine appropriate "triggers" for implementation of the high priority interventions in the context of four different infectious respiratory disease scenarios that vary by patient volumes (high versus low) and illness severity (high versus low). SETTING: One day in-person conference held November, 2011. PARTICIPANTS: Regional and national experts representing the fields of public health, disease surveillance, clinical medicine, ED operations, and hospital operations. MAIN OUTCOME MEASURE: Prioritized list of potential interventions to reduce ED and hospital crowding, respectively. In addition, we created a prioritized list of potential data sources which could be useful to trigger interventions. RESULTS: High priority interventions to mitigate ED surge included standardizing admission and discharge criteria and instituting infection control measures. To mitigate hospital crowding, panelists prioritized mandatory vaccination and an algorithm for antiviral use. Data sources identified for triggering implementation of these interventions were most commonly ED and hospital utilization metrics. CONCLUSIONS: We developed a prioritized list of potentially useful interventions to mitigate ED and hospital crowding in various outbreak scenarios. The data sources identified to "trigger" the implementation of these high priority interventions consist mainly of sources available at the local, institutional level.

Monitoring seasonal influenza A evolution: rapid 2009 pandemic H1N1 surveillance with an reverse transcription-polymerase chain reaction/electro-spray ionization mass spectrometry assay.

BACKGROUND: The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts. OBJECTIVES: To determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity. STUDY DESIGN: Two sets of pandemic H1N1 samples, 190 collected between April and June of 2009, and 69 collected between October 2009 and January 2010, were processed by the RT-PCR/ESI-MS influenza typing assay, and the spectral results were compared to reference laboratory results and historical sequencing data from the Nucleotide Database of the National Center for Biotechnology Information (NCBI). RESULTS: Strain typing concordance with reference standard testing was 100% in both sample sets, and the assay demonstrated a significant increase in influenza genetic diversity, from 10.5% non-wildtype genotypes in early samples to 69.9% in late samples (P<0.001). An NCBI search demonstrated a similar increase, from 13.4% to 45.2% (P<0.001). CONCLUSIONS: This comparison of early versus late influenza samples analyzed by RT-PCR/ESI-MS demonstrates the influenza typing assay's ability as a universal influenza detection platform to provide high-fidelity pH1N1 strain identification over time, despite increasing genetic diversity in the circulating virus. The genotyping data can also be leveraged for high-throughput influenza surveillance.