An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern.

The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System.

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log10 dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System.

Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F. tularensis assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system. F. tularensis DNA in buffer or CFU of F. tularensis was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with F. tularensis Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml F. tularensis in both human and macaque blood. In infected macaques, the assay detected F. tularensis on days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detect F. tularensis in bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.

A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay.

BACKGROUND: Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available. METHODS: We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa. RESULTS: The assay's analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6-0.9) and 84% (95% CI 0.6-0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77-1) and 94% (95% CI 0.7-0.99), respectively. CONCLUSION: This novel approach may permit simple and rapid detection of TB using pediatric stool samples.

Exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB/RIF assay.

The utility of the GeneXpert MTB/RIF (Xpert) assay for detection of Mycobacterium tuberculosis in sputum samples has been extensively studied. However, the performance of the Xpert assay as applied to other readily accessible body fluids such as exhaled breath condensate (EBC), saliva, urine, and blood has not been established. We used the Xpert assay to test EBC, saliva, urine, and blood samples from HIV-negative, smear- and culture-positive pulmonary tuberculosis (TB) patients for the presence of M. tuberculosis. To compare the ability of the assay to perform bacterial load measurements on sputum samples with versus without sample processing, the assay was also performed on paired direct and processed sputum samples from each patient. The Xpert assay detected M. tuberculosis in none of the 26 EBC samples (sensitivity, 0.0%; 95% confidence interval [95% CI], 0.0%, 12.9%), 10 of the 26 saliva samples (sensitivity, 38.5%; 95% CI, 22.4%, 57.5%), 1 of 26 urine samples (sensitivity, 3.8%; 95% CI, 0.7%, 18.9%), and 2 of 24 blood samples (sensitivity, 8.3%; 95% CI, 2.3%, 25.8%). For bacterial load measurements in the different types of sputum samples, the cycle thresholds of the two M. tuberculosis-positive sputum types were well correlated (Spearman correlation of 0.834). This study demonstrates that the Xpert assay should not be routinely used to detect M. tuberculosis in EBC, saliva, urine, or blood samples from HIV-negative patients suspected of having pulmonary tuberculosis. As a test of bacterial load, the assay produced similar results when used to test direct versus processed sputum samples. Sputum remains the optimal sample type for diagnosing pulmonary tuberculosis in HIV-negative patients with the Xpert assay.

Evaluation of Xpert MTB/RIF for detection of tuberculosis from blood samples of HIV-infected adults confirms Mycobacterium tuberculosis bacteremia as an indicator of poor prognosis.

Tuberculosis (TB) remains a leading cause of death among HIV-infected adults, in part because of delayed diagnosis and therefore delayed initiation of treatment. Recently, the Gene-Xpert platform, a rapid, PCR-based diagnostic platform, has been validated for the diagnosis of TB with sputum. We have evaluated the Xpert MTB/RIF assay for the diagnosis of Mycobacterium tuberculosis bacteremia and investigated its impact on clinical outcomes. Consecutive HIV-infected adults with fever and cough presenting to Queen Elizabeth Central Hospital, Blantyre, Malawi, were recruited and followed up for 2 months. At presentation, three sputum samples were examined by smear, culture, and Xpert MTB/RIF assay for the presence of M. tuberculosis and blood was drawn for PCR with Xpert, for mycobacterial culture (Myco/F Lytic), and for aerobic culture. One hundred four patients were recruited, and 44 (43%) were sputum culture positive for M. tuberculosis. Ten were Xpert blood positive, for a sensitivity of 21% and a specificity of 100%. The 2-week mortality rate was significantly higher among patients who were Xpert blood positive than among those who were negative (40% versus 3%; multivariate odds ratio [OR] for death if positive, 44; 95% confidence interval [CI], 3 to 662). This effect persisted on assessment of the mortality rate at 2 months (40% versus 11%; OR, 5.6; 95% CI, 1.3 to 24.6). When screening uncomplicated patients presenting with a productive cough for pulmonary TB, Xpert blood offers no diagnostic advantage over sputum testing. Despite this, Xpert blood positivity is highly predictive of early death and this test rapidly identifies a group of patients in urgent need of initiation of treatment.

Detection of Mycobacterium tuberculosis in blood by use of the Xpert MTB/RIF assay.

We have developed a novel blood lysis-centrifugation approach for highly sensitive Mycobacterium tuberculosis detection in large volumes of blood with the Xpert MTB/RIF assay. One through 20 ml of blood was spiked with 0.25 to 10 CFU/ml of the M. tuberculosis surrogate M. bovis BCG. Multiple replicates of each sample were processed by a new lysis-centrifugation method and tested with the Xpert MTB/RIF assay. The assay was very sensitive with increased blood volumes. In the 20-ml samples, BCG was detected in blood spiked with 10, 5, 1, and 0.25 CFU/ml 100, 100, 83, and 57% of the time, respectively, compared to 100, 66, 18, and 18%, of the time, respectively, in 1-ml blood samples. Assay sensitivity was influenced by the type of anticoagulant used, with acid-citrate-dextrose solution B (ACD-B) providing the best results. A limit of detection of 10 CFU/ml was established with BCG spiked into ACD-B-treated blood, and 92, 36, and 33% of the samples with 5, 1, and 0.5 CFU/ml, respectively, were assay positive. The lysis buffer was stable both at room temperature and at 4°C for 2 months. The assay was tested with blood stored for 8 days without a change in sensitivity as measured by cycle threshold. This new assay format extends the capability of the Xpert MTB/RIF test, enabling up to 20 ml of blood to be tested rapidly for the presence of M. tuberculosis. This approach may be a useful method to detect extrapulmonary tuberculosis and the risk of death in immunocompromised patients.

Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate.

The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.

Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding.

Rifampin resistance in Mycobacterium tuberculosis is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoB gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the M. tuberculosis RRDR by melting temperature (T(m)) analysis. A three-point T(m) code is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted from 589 geographically diverse clinical M. tuberculosis cultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity, 99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using the triple T(m) code. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations when present in at least 40% of the total M. tuberculosis DNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.

Highly sensitive detection of Staphylococcus aureus directly from patient blood.

BACKGROUND: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing-polymerase chain reaction (PCR) platform as a model diagnostic system. METHODOLOGY/PRINCIPAL FINDINGS: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90% positive) as opposed to cell-associated bacteria (in WBCs) (71% samples positive) or free bacterial DNA in plasma (62.5% samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95% CI 0.75-0.96) and 0/59 negative controls with the sodA assay (specificity 100%, 95% CI 0.92-1). CONCLUSIONS: We have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus directly in 1 ml of whole blood, without the need for blood culture.

Containment of bioaerosol infection risk by the Xpert MTB/RIF assay and its applicability to point-of-care settings.

The recently introduced Xpert MTB/RIF assay (Xpert) has point-of-care potential, but its capacity for biohazard containment remained to be studied. We compared the bioaerosols generated by the Xpert assay to acid-fast bacillus (AFB) microscope slide smear preparation. The Xpert assay sample treatment reagent (SR) was also studied for its sterilizing capacity, stability, and effect on assay sensitivity after prolonged treatment. During the preparation of AFB smears, sputum samples spiked with Mycobacterium bovis BCG at 5 × 10(8) CFU/ml produced 16 and 325 CFU/m(3) air measured with an Andersen impactor or BioSampler, respectively. In contrast, neither the sample preparation steps for the Xpert assay nor its automated processing produced any culturable bioaerosols. In testing of SR sterilizing capacity, clinical sputum samples from strongly smear-positive tuberculosis patients treated with SR at a 2:1 ratio eliminated Mycobacterium tuberculosis growth in all but 1/39 or 3/45 samples cultured on solid or liquid medium, respectively. These few unsterilized samples had a mean 13.1-day delay in the time to positive culture. SR treatment at a 3:1 ratio eliminated growth in all samples. SR retained a greater than 6-log-unit killing capacity despite storage at temperatures spanning 4 to 45°C for at least 3 months. The effect of prolonged SR sample treatment was also studied. Spiked sputum samples could be incubated in SR for up to 3 days without affecting Xpert sensitivity for M. tuberculosis detection and up to 8 h without affecting specificity for rifampin resistance detection. These results suggest that benchtop use of the Xpert MTB/RIF assay limits infection risk to the user.

Characterization of Listeria monocytogenes isolates of food and human origins from Brazil using molecular typing procedures and in vitro cell culture assays.

The spreading of diseases through foods is a worldwide concern. Here, molecular and in vitro cell-culture assays were employed to characterize 63 Brazilian Listeria monocytogenes isolates (food, 47; clinical, 16). Serotype 4b was the most predominant (49%) followed by (1/2)b (30%), (1/2)a (10%), (1/2)c (6%), 3c (3%) and 3b (2%). Ribotyping yielded 17 ribopatterns, which were grouped into four phylogenetic clusters. Cluster A comprised of 39/63 isolates primarily of food origin, and clusters B, C and D contained both food and clinical isolates. Isolates were positive for virulence determinants prfA, hlyA and inlA: clinical isolates were more invasive to Caco-2 cells and expressed high levels of inlA transcripts than the food isolates. Highly invasive isolates also provoked more Ped-2E9 cells to die by apoptosis than the weakly-invasive strains. These data demonstrate a strong genetic relatedness among clinical and food isolates and suggest transmission of a subset of L. monocytogenes strains from food to humans.

Label-free detection of multiple bacterial pathogens using light-scattering sensor.

Technologies for rapid detection and classification of bacterial pathogens are crucial for securing the food supply. This report describes a light-scattering sensor capable of real-time detection and identification of colonies of multiple pathogens without the need for a labeling reagent or biochemical processing. Bacterial colonies consisting of the progeny of a single parent cell scatter light at 635 nm to produce unique forward-scatter signatures. Zernike moment invariants and Haralick descriptors aid in feature extraction and construction of the scatter-signature image library. The method is able to distinguish bacterial cultures at the genus and species level for Listeria, Staphylococcus, Salmonella, Vibrio, and Escherichia with an accuracy of 90-99% for samples derived from food or experimentally infected animal. Varied amounts of exopolysaccharide produced by the bacteria causes changes in phase modulation distributions, resulting in strikingly different scatter signatures. With the aid of a robust database the method can potentially detect and identify any bacteria colony essentially instantaneously. Unlike other methods, it does not destroy the sample, but leaves it intact for other confirmatory testing, if needed, for forensic or outbreak investigations.

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line.

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2-3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P=0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44-52 h. A positive correlation (R2=0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes.

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8-89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance.

Analysis of time-resolved scattering from macroscale bacterial colonies.

We investigate the relationship of incubation time and forward-scattering signature for bacterial colonies grown on solid nutrient surfaces. The aim of this research is to understand the colony growth characteristics and the corresponding evolution of the scattering patterns for a variety of pathogenic bacteria relevant to food safety. In particular, we characterized time-varying macroscopic and microscopic morphological properties of the growing colonies and modeled their optical properties in terms of two-dimensional (2-D) amplitude and phase modulation distributions. These distributions, in turn, serve as input to scalar diffraction theory, which is, in turn, used to predict forward-scattering signatures. For the present work, three different species of Listeria were considered: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The baseline experiments involved the growth of cultures on brain heart infusion (BHI) agar and the capture of scatter images every 6 h over a total incubation period of 42 h. The micro- and macroscopic morphologies of the colonies were studied by phase contrast microscopy. Growth curves, represented by colony diameter as a function of time, were compared with the measured time-evolution of the scattering signatures.