scholarly journals Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

2011 ◽  
Vol 77 (18) ◽  
pp. 6570-6578 ◽  
Author(s):  
Sonia E. Létant ◽  
Gloria A. Murphy ◽  
Teneile M. Alfaro ◽  
Julie R. Avila ◽  
Staci R. Kane ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Real Time ◽  
Incubation Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Pcr Analysis ◽  
Content Type ◽  
Pcr Method

ABSTRACTIn the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulentBacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for theB. anthracischromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulentB. anthracisin environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

scholarly journals The ZKIR Assay, a Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Elodie Barbier ◽  
Carla Rodrigues ◽  
Geraldine Depret ◽  
Virginie Passet ◽  
Laurent Gal ◽  
...  
Keyword(s):  
Public Health ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Health Concern ◽  
Complex Matrices ◽  
Content Type ◽  
Novel Method ◽  
Pcr Method

ABSTRACT Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Taxonomically, the K. pneumoniae complex (“Kp”) includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here, we analyzed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR (zur-khe intergenic region) assay, was developed and used to detect Kp in 96 environmental samples. The results were compared to a culture-based method using Simmons citrate agar with 1% inositol medium coupled to matrix-assisted laser desorption ionization–time of flight mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10−1 CFU g−1 after enrichment for 24 h in lysogeny broth supplemented with ampicillin, and it was 1.5 × 103 to 1.5 × 104 CFU g−1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 multilocus sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific, and sensitive novel method to detect the presence of Kp in complex matrices and indicates that Kp isolates from environmental samples differ from clinical isolates. IMPORTANCE The Klebsiella pneumoniae species complex Kp includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic-resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and we show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

scholarly journals Gamma Irradiation Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of Diagnostic Assays

2008 ◽  
Vol 74 (14) ◽  
pp. 4427-4433 ◽  
Author(s):  
Leslie A. Dauphin ◽  
Bruce R. Newton ◽  
Max V. Rasmussen ◽  
Richard F. Meyer ◽  
Michael D. Bowen
Keyword(s):  
Gamma Irradiation ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Pcr Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromosomal Dna ◽  
Unit Reduction

ABSTRACT The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 107 CFU/ml to a dose of 2.5 × 106 rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of B. anthracis chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of B. anthracis. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of B. anthracis spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for B. anthracis could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthracis assays.

scholarly journals Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Kuppuswamy N. Kasturi ◽  
Tomas Drgon
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
False Negative ◽  
Screening Method ◽  
Reference Method ◽  
Rapid Screening ◽  
Content Type ◽  
Pcr Method ◽  
Group D

ABSTRACT The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S. Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method. IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

scholarly journals Comparison and Recommendations for Use of Dientamoeba fragilis Real-Time PCR Assays

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Rory Gough ◽  
John Ellis ◽  
Damien Stark
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Content Type ◽  
Prevalence Studies ◽  
Dientamoeba Fragilis ◽  
Stool Samples ◽  
Pcr Method ◽  
Genetic Signatures

ABSTRACT Dientamoeba fragilis is a gastrointestinal trichomonad parasite whose pathogenicity is yet to be determined. The difficulty involved in microscopically diagnosing D. fragilis in feces led to the development of real-time PCR methodologies for the detection of D. fragilis in stool samples. Prevalence studies in Europe show much higher levels of infection where a laboratory-developed real-time assay is the predominant assay for the detection of Dientamoeba fragilis than in regions that use the EasyScreen assay for detection of gastrointestinal pathogens. The aim of this study was to compare a commercially available Dientamoeba fragilis assay (Genetic Signatures EasyScreen assay) to a widely used laboratory-developed real-time PCR method. Two hundred fifty fecal samples were screened using the laboratory-developed real-time assay on four real-time PCR platforms producing a number of discrepant results. Limit-of-detection studies were undertaken to attempt to resolve sensitivity for each platform tested. The presence or absence of Dientamoeba fragilis DNA in discrepant samples was shown using PCR amplicon next-generation sequencing. Eukaryotic 18S diversity profiling was conducted on discrepant samples to identify the presence or absence of additional protozoan species in samples that may be responsible for cross-reactivity seen in these samples. The results revealed the potential for multiple false-positive results when using the laboratory-developed real-time assay across multiple real-time platforms using manufacturer default settings. This report provides recommendations to resolve these issues where possible and suggestions for future prevalence studies, and it emphasizes the EasyScreen assay as the molecular method of choice as well as the need for standardization of detection assays across all nations screening for D. fragilis.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

scholarly journals Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

2020 ◽  
Author(s):  
zhenhua Guo ◽  
Kunpeng Li ◽  
Songlin Qiao ◽  
Xinxin Chen ◽  
Ruiguang Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Limit Of Detection ◽  
African Swine Fever ◽  
Economic Losses ◽  
Clinical Samples ◽  
Diagnosis Method ◽  
Pcr Method ◽  
And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

scholarly journals The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

2019 ◽  
Author(s):  
Elodie Barbier ◽  
Carla Rodrigues ◽  
Geraldine Depret ◽  
Virginie Passet ◽  
Laurent Gal ◽  
...  
Keyword(s):  
Public Health ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Health Concern ◽  
Complex Matrices ◽  
Animal Pathogens ◽  
Novel Method ◽  
Pcr Method

ABSTRACTKlebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here we analysed 4222 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using SCAI agar medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10-1 CFU g-1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 103 to 1.5 × 104 CFU g-1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates.IMPORTANCEThe Klebsiella pneumoniae species complex (Kp) includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR, which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

scholarly journals Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsui-Kang Hsu ◽  
Jung-Sheng Chen ◽  
Hsin-Chi Tsai ◽  
Chi-Wei Tao ◽  
Yu-Yin Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
In Silico ◽  
Environmental Samples ◽  
Method Development ◽  
Small Volume ◽  
Detection Efficiency ◽  
Genotyping Method ◽  
Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

scholarly journals Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Michael A. Saubolle ◽  
Bette R. Wojack ◽  
Anne M. Wertheimer ◽  
Atehkeng Z. Fuayagem ◽  
Stephen Young ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Clinical Test ◽  
Target Sequence ◽  
Fungal Culture ◽  
Content Type ◽  
Bronchial Wash

ABSTRACT Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's “gold standard,” fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at −70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

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