Publications by authors named "Soumitesh Chakravorty"

39 Publications

Sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing.

J Med Microbiol 2021 Sep;70(9)

The Public Health Research Institute and Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ 07103, USA.

Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR). The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, =0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, <0.001) or eNAT (67.8 %, =0.0012) and oral swabs in VTM (50 %, <0.0001) or eNAT (58 %, <0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.
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http://dx.doi.org/10.1099/jmm.0.001380DOI Listing
September 2021

A Simple Reverse Transcriptase PCR Melting-Temperature Assay To Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

J Clin Microbiol 2021 Sep 21;59(10):e0084521. Epub 2021 Jul 21.

Public Health Research Institute, Center for Emerging Pathogens, Rutgers New Jersey Medical School, Newark, New Jersey, USA.

The increased transmission of SARS-CoV-2 variants of concern (VOC), which originated in the United Kingdom (B.1.1.7/alpha), South Africa (B1.351/beta), Brazil (P.1/gamma), the United States (B.1.427/429 or epsilon), and India (B.1.617.2/delta), requires a vigorous public health response, including real-time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time-consuming and expensive. Here, we describe a simple, rapid, and high-throughput reverse transcriptase PCR (RT-PCR) melting-temperature () screening assay that identifies the first three major VOCs. RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild-type sequences. After RT-PCR, the VOCs were identified by a characteristic of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (wild type [WT]), B.1.1.7, and B.1.351 variant strains. The assay was then validated using clinical samples. The limit of detection for both the WT and variants was 4 and 10 genomic copies/reaction for the 501- and 484-codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples, as confirmed by Sanger sequencing. We have developed an RT-PCR melt screening test for the major VOCs that can be used to rapidly screen large numbers of patient samples, providing an early warning for the emergence of these variants and a simple way to track their spread.
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http://dx.doi.org/10.1128/JCM.00845-21DOI Listing
September 2021

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

PLoS One 2021 11;16(6):e0252687. Epub 2021 Jun 11.

Department of Medicine, Center for Emerging Pathogens, Rutgers-New Jersey Medical School, Newark, New Jersey, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252687PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195355PMC
June 2021

A Simple RT-PCR Melting temperature Assay to Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

medRxiv 2021 Apr 8. Epub 2021 Apr 8.

Public Health Research Institute; Center for Emerging Pathogens, Rutgers New Jersey Medical School.

Background: The increased transmission of SARS-CoV-2 variants of concern (VOC) which originated in the United Kingdom (B.1.1.7), South Africa (B1.351), Brazil (P.1) and in United States (B.1.427/429) requires a vigorous public health response, including real time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time consuming and expensive. Here, we describe a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature (Tm) screening assay that identifies these three major VOCs.

Methods: RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT), a B.1.17 and a B.1.351 variant strains. The assay was then validated using clinical samples.

Results: The limit of detection (LOD) for both the WT and variants was 4 and 10 genomic copies/reaction for the 501 and 484 codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples as confirmed by Sanger sequencing.

Conclusion: We have developed an RT-PCR melt screening test for the three major VOCs which can be used to rapidly screen large numbers of patient samples providing an early warning for the emergence of these variants and a simple way to track their spread.
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http://dx.doi.org/10.1101/2021.03.05.21252709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987051PMC
April 2021

Evaluation of sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing.

medRxiv 2021 Mar 5. Epub 2021 Mar 5.

Sensitive, accessible, and biosafe sampling methods for COVID-19 reverse-transcriptase polymerase chain reaction (RT-PCR) assays are needed for frequent and widespread testing. We systematically evaluated diagnostic yield across different sample collection and transport workflows, including the incorporation of a viral inactivation buffer. We prospectively collected nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal and oral swabs were placed in both viral transport media (VTM) and eNAT™, a sterilizing transport buffer, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert) test. The sensitivity of each sampling strategy was compared using a composite positive standard. Overall, swab specimens collected in eNAT showed superior sensitivity compared to swabs in VTM (70% vs 57%, P=0.0022). Direct saliva 90.5%, (95% CI: 82%, 95%), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50%, P<0.001) or eNAT (67.8%, P=0.0012) and oral swabs in VTM (50%, P<0.0001) or eNAT (56%, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert test; however, no single sample matrix identified all positive cases.
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http://dx.doi.org/10.1101/2021.03.03.21251172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941657PMC
March 2021

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

medRxiv 2021 Jan 20. Epub 2021 Jan 20.

Public Health Research Institute, 225 Warren Street, Newark, NJ 07103.

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 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 log PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on vero-E6 cell lines 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, preservation, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.
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http://dx.doi.org/10.1101/2021.01.15.21249891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836134PMC
January 2021

Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum.

J Clin Microbiol 2021 02 18;59(3). Epub 2021 Feb 18.

New Jersey Medical School, Rutgers University, Newark, New Jersey, USA

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in and analyzes melting temperatures ( s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific s or patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care.
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http://dx.doi.org/10.1128/JCM.02314-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106700PMC
February 2021

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

PLoS One 2020 8;15(5):e0232343. Epub 2020 May 8.

Department of Medicine, Rutgers New Jersey Medical School, Newark, New Jersey, United States of America.

Background: Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs.

Methods: We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene.

Results: We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases.

Conclusion: This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232343PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7209238PMC
July 2020

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

J Clin Microbiol 2020 Jul 23;58(8). Epub 2020 Jul 23.

Cepheid, Sunnyvale, California, USA.

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 , 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.
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http://dx.doi.org/10.1128/JCM.00926-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383535PMC
July 2020

Automatic Identification of Individual Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

J Clin Microbiol 2019 12 23;58(1). Epub 2019 Dec 23.

Department of Medicine, Rutgers New Jersey Medical School, Newark, New Jersey, USA

Molecular surveillance of rifampin-resistant can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the gene by the use of melting temperature ( ) information from 4 probes which can fall in one of the 9 different assay-specified windows. The large amount of data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the RRDR by utilizing the pattern of the of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific " signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the distances between the test sample and the reference values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.
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http://dx.doi.org/10.1128/JCM.00907-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935902PMC
December 2019

Bacterial Factors That Predict Relapse after Tuberculosis Therapy.

N Engl J Med 2018 Aug;379(9):823-833

From the Department of Medicine, Rutgers-New Jersey Medical School (R. Colangeli, H.J., U.D.C.V., S.C., A.G., D.A.), and the Department of Biostatistics, Rutgers School of Public Health (S.K., R. Connell), Newark; the Center for Infectious Disease Research, Seattle (S.M., D.R.S.); the Centers for Disease Control and Prevention, Atlanta (E.E.S., L.D., W.R.M.K.); Uganda-Case Western Reserve University Research Collaboration, Kampala, Uganda (A.O.); Nucleo de Doencas Infecciosas, Centro de Ciencias da Saude, Universidade Federal do Espirito Santo, Vitoria, Brazil (R.D.); and the Tuberculosis Research Unit, Department of Medicine, Case Western Reserve University School of Medicine and University Hospitals Cleveland Medical Center, Cleveland (W.H.B., J.L.J.).

Background: Approximately 5% of patients with drug-susceptible tuberculosis have a relapse after 6 months of first-line therapy, as do approximately 20% of patients after 4 months of short-course therapy. We postulated that by analyzing pretreatment isolates of Mycobacterium tuberculosis obtained from patients who subsequently had a relapse or were cured, we could determine any correlations between the minimum inhibitory concentration (MIC) of a drug below the standard resistance breakpoint and the relapse risk after treatment.

Methods: Using data from the Tuberculosis Trials Consortium Study 22 (development cohort), we assessed relapse and cure isolates to determine the MIC values of isoniazid and rifampin that were below the standard resistance breakpoint (0.1 μg per milliliter for isoniazid and 1.0 μg per milliliter for rifampin). We combined this analysis with clinical, radiologic, and laboratory data to generate predictive relapse models, which we validated by analyzing data from the DMID 01-009 study (validation cohort).

Results: In the development cohort, the mean (±SD) MIC of isoniazid below the breakpoint was 0.0334±0.0085 μg per milliliter in the relapse group and 0.0286±0.0092 μg per milliliter in the cure group, which represented a higher value in the relapse group by a factor of 1.17 (P=0.02). The corresponding MIC values of rifampin were 0.0695±0.0276 and 0.0453±0.0223 μg per milliliter, respectively, which represented a higher value in the relapse group by a factor of 1.53 (P<0.001). Higher MIC values remained associated with relapse in a multivariable analysis that included other significant between-group differences. In an analysis of receiver-operating-characteristic curves of relapse based on these MIC values, the area under the curve (AUC) was 0.779. In the development cohort, the AUC in a multivariable model that included MIC values was 0.875. In the validation cohort, the MIC values either alone or combined with other patient characteristics were also predictive of relapse, with AUC values of 0.964 and 0.929, respectively. The use of a model score for the MIC values of isoniazid and rifampin to achieve 75.0% sensitivity in cross-validation analysis predicted relapse with a specificity of 76.5% in the development cohort and a sensitivity of 70.0% and a specificity of 100% in the validation cohort.

Conclusions: In pretreatment isolates of M. tuberculosis with decrements of MIC values of isoniazid or rifampin below standard resistance breakpoints, higher MIC values were associated with a greater risk of relapse than lower MIC values. (Funded by the National Institute of Allergy and Infectious Diseases.).
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http://dx.doi.org/10.1056/NEJMoa1715849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317071PMC
August 2018

Molecular Drug-Susceptibility Test for Tuberculosis.

N Engl J Med 2017 12;377(24):2404

Medical University of South Carolina, Charleston, SC

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http://dx.doi.org/10.1056/NEJMc1714218DOI Listing
December 2017

Xpert MTB/RIF Ultra for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study.

Lancet Infect Dis 2018 01 30;18(1):76-84. Epub 2017 Nov 30.

FIND, Geneva, Switzerland.

Background: The Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance.

Methods: In this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection.

Findings: Between Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (-2·7%, -3·9 to -1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance.

Interpretation: For tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity.

Funding: Government of Netherlands, Government of Australia, Bill & Melinda Gates Foundation, Government of the UK, and the National Institute of Allergy and Infectious Diseases.
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http://dx.doi.org/10.1016/S1473-3099(17)30691-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168783PMC
January 2018

Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis.

N Engl J Med 2017 09;377(11):1043-1054

From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda (Y.L.X., L.E.V., R.Y.C., C.E.B.), and Johns Hopkins University School of Medicine, Baltimore (D.T.A., S.E.D.) - both in Maryland; the Center for Emerging and Re-Emerging Pathogens, Rutgers New Jersey Medical School, Newark (S.C., L.E.S., N.D., D.A.); Boston Medical Center and Boston University School of Medicine, Boston (S.L.H., J.J.E.); the International Tuberculosis Research Center, Changwon (T.S., M.L., J.L., S.-N.C.), and the National Medical Center (J.S.J.), Seoul Metropolitan Seobuk Hospital (Y.C.), and the Department of Microbiology, College of Medicine, Yonsei University (S.-N.C.), Seoul - all in South Korea; Henan Provincial Chest Hospital (X.Y., X.M., X.L., X.R., L.L.) and Sino-U.S. Tuberculosis Research Collaboration (H.Z.), Zhengzhou, and Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, School of Basic Medical Science, Fudan University, Shanghai (P.X., Q.G.) - all in China; and the Institute of Infectious Disease and Molecular Medicine and Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa (C.E.B.).

Background: Fluoroquinolones and second-line injectable drugs are the backbone of treatment regimens for multidrug-resistant tuberculosis, and resistance to these drugs defines extensively drug-resistant tuberculosis. We assessed the accuracy of an automated, cartridge-based molecular assay for the detection, directly from sputum specimens, of Mycobacterium tuberculosis with resistance to fluoroquinolones, aminoglycosides, and isoniazid.

Methods: We conducted a prospective diagnostic accuracy study to compare the investigational assay against phenotypic drug-susceptibility testing and DNA sequencing among adults in China and South Korea who had symptoms of tuberculosis. The Xpert MTB/RIF assay and sputum culture were performed. M. tuberculosis isolates underwent phenotypic drug-susceptibility testing and DNA sequencing of the genes katG, gyrA, gyrB, and rrs and of the eis and inhA promoter regions.

Results: Among the 308 participants who were culture-positive for M. tuberculosis, when phenotypic drug-susceptibility testing was used as the reference standard, the sensitivities of the investigational assay for detecting resistance were 83.3% for isoniazid (95% confidence interval [CI], 77.1 to 88.5), 88.4% for ofloxacin (95% CI, 80.2 to 94.1), 87.6% for moxifloxacin at a critical concentration of 0.5 μg per milliliter (95% CI, 79.0 to 93.7), 96.2% for moxifloxacin at a critical concentration of 2.0 μg per milliliter (95% CI, 87.0 to 99.5), 71.4% for kanamycin (95% CI, 56.7 to 83.4), and 70.7% for amikacin (95% CI, 54.5 to 83.9). The specificity of the assay for the detection of phenotypic resistance was 94.3% or greater for all drugs except moxifloxacin at a critical concentration of 2.0 μg per milliliter (specificity, 84.0% [95% CI, 78.9 to 88.3]). When DNA sequencing was used as the reference standard, the sensitivities of the investigational assay for detecting mutations associated with resistance were 98.1% for isoniazid (95% CI, 94.4 to 99.6), 95.8% for fluoroquinolones (95% CI, 89.6 to 98.8), 92.7% for kanamycin (95% CI, 80.1 to 98.5), and 96.8% for amikacin (95% CI, 83.3 to 99.9), and the specificity for all drugs was 99.6% (95% CI, 97.9 to 100) or greater.

Conclusions: This investigational assay accurately detected M. tuberculosis mutations associated with resistance to isoniazid, fluoroquinolones, and aminoglycosides and holds promise as a rapid point-of-care test to guide therapeutic decisions for patients with tuberculosis. (Funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Ministry of Science and Technology of China; ClinicalTrials.gov number, NCT02251327 .).
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http://dx.doi.org/10.1056/NEJMoa1614915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727572PMC
September 2017

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

mBio 2017 08 29;8(4). Epub 2017 Aug 29.

Department of Medicine, Rutgers New Jersey Medical School, Newark, New Jersey, USA

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 mutation detection were tested on DNA or sputum samples spiked with known numbers of H37Rv or BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for 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 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. 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 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.
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http://dx.doi.org/10.1128/mBio.00812-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574709PMC
August 2017

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

J Clin Microbiol 2017 01 28;55(1):291-301. Epub 2016 Dec 28.

Center for Emerging Pathogens, Department of Medicine, Rutgers Biomedical and Health Sciences (RHBS), Rutgers University, Newark, New Jersey, USA

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.
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http://dx.doi.org/10.1128/JCM.01126-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228242PMC
January 2017

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

J Clin Microbiol 2017 01 28;55(1):183-198. Epub 2016 Dec 28.

Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA.

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.
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http://dx.doi.org/10.1128/JCM.01771-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228229PMC
January 2017

A snapshot of the predominant single nucleotide polymorphism cluster groups of Mycobacterium tuberculosis clinical isolates in Delhi, India.

Tuberculosis (Edinb) 2016 09 25;100:72-81. Epub 2016 Jul 25.

Dept. of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, 110007, India.

Several attempts have been made to associate phylogenetic differences among Mycobacterium tuberculosis strains to variations in the clinical outcome of the disease and to drug resistance. We genotyped 139 clinical isolates of M. tuberculosis obtained from patients of pulmonary tuberculosis in North Delhi region. The isolates were analyzed using nine Single nucleotide polymorphism (SNP) markers, spoligotyping and MIRU-VNTRs; and the results were correlated with their drug susceptibility profile. Results of SNP cluster group (SCG) analysis (available for 138 isolates) showed that the most predominant cluster was SCG 3a, observed in 58.7% (81/138) of the isolates with 44.4% (36/81) of these being drug susceptible, while 16% (13/81) were multidrug resistant (MDR). Of the ancestral cluster SCG 1 observed in 19.5% (27/138) of the isolates, 14.8% (4/27) were MDR while 44.4% (12/27) were drug susceptible. SCG 2 formed 5.79% (8/138) of the isolates and 50% (4/8) of these were multidrug resistant (MDR). Spoligotyping subdivided the strains into 45 shared types (n = 125) and 14 orphan strains. The orphan strains were mostly associated with SCG 3a or SCG 1, reflecting the principal SCGs found in the Indian population. SCG 1 and SCG 2 genotypes were concordant with the East African Indian (EAI) and Beijing families respectively. Central Asian (CAS) clade and its sublineages were predominantly associated with SCG 3a. No consistent association was seen between the SCGs and Harlem, T or X clades. The 15 loci MIRU-VNTR typing revealed 123/136 isolates to be unclustered, while 13 isolates were present in 6 clusters of 2-3 isolates each. However, correlating the cluster analysis with patient details did not suggest any evidence of recent transmission. In conclusion, though our study revealed the preponderance of SCG 1 and 3a in the M. tuberculosis population circulating in the region, the diversity of strains highlights the changes occurring within lineages and reemphasizes the importance of cluster investigations in extended studies.
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http://dx.doi.org/10.1016/j.tube.2016.07.007DOI Listing
September 2016

Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance.

PLoS One 2015 4;10(5):e0126257. Epub 2015 May 4.

Department of Medicine, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America.

Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126257PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418795PMC
April 2016

Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

J Clin Microbiol 2015 Jan 22;53(1):43-51. Epub 2014 Oct 22.

Department of Medicine, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA.

Resistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (LJ) absolute concentration, mycobacterial growth indicator tubes (MGIT), and TREK Sensititre MycoTB MIC plate (MycoTB) methods. Target amplicons were also tested for mutations by Sanger sequencing. The SMB assay correctly detected 115/116 mutant and mixed sequences and 487/487 wild-type sequences (sensitivity and specificity of 99.1 and 100%, respectively). Using the LJ method as the reference, sensitivity and specificity for AMK resistance were 92.2% and 100%, respectively, and sensitivity and specificity for KAN resistance were 87.7% and 95.6%, respectively. Mutations in the rrs gene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eis promoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eis promoter mutations with AMK resistance. Among the discordant samples with AMK and/or KAN resistance but wild-type sequence at the target genes, we discovered four new mutations in the whiB7 5' untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB7 5' UTR mutations in KAN resistance.
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http://dx.doi.org/10.1128/JCM.02059-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4290905PMC
January 2015

Importance of cough and M. tuberculosis strain type as risks for increased transmission within households.

PLoS One 2014 2;9(7):e100984. Epub 2014 Jul 2.

Núcleo de Doenças Infecciosas (NDI), Universidade Federal do Espírito Santo (UFES), Vitória, Brazil.

Rationale: The degree to which tuberculosis (TB) is transmitted between persons is variable. Identifying the factors that contribute to transmission could provide new opportunities for TB control. Transmission is influenced by host, bacterial and environmental factors. However, distinguishing their individual effects is problematic because measures of disease severity are tightly correlated, and assessing the virulence of Mycobacterium tuberculosis isolates is complicated by epidemiological and clinical confounders.

Objectives: To overcome these problems, we investigated factors potentially associated with TB transmission within households.

Methods: We evaluated patients with smear-positive (≥2+), pulmonary TB and classified M. tuberculosis strains into single nucleotide polymorphism genetic cluster groups (SCG). We recorded index case, household contact, and environmental characteristics and tested contacts with tuberculin skin test (TST) and interferon-gamma release assay. Households were classified as high (≥70% of contacts with TST≥10 mm) and low (≤40%) transmission. We used logistic regression to determine independent predictors.

Result: From March 2008 to June 2012, we screened 293 TB patients to enroll 124 index cases and their 731 contacts. There were 23 low and 73 high transmission households. Index case factors associated with high transmission were severity of cough as measured by a visual analog cough scale (VACS) and the Leicester Cough Questionnaire (LCQ), and cavitation on chest radiograph. SCG 3b strains tended to be more prevalent in low (27.3%) than in high (12.5%) transmission households (p = 0.11). In adjusted models, only VACS (p<0.001) remained significant. SCG was associated with bilateral disease on chest radiograph (p = 0.002) and marginally associated with LCQ sores (p = 0.058), with group 3b patients having weaker cough.

Conclusions: We found differential transmission among otherwise clinically similar patients with advanced TB disease. We propose that distinct strains may cause differing patterns of cough strength and cavitation in the host leading to diverging infectiousness. Larger studies are needed to verify this hypothesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100984PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079704PMC
February 2015

Tuberculosis Diagnostics in the New Millennium: Role in TB Identification and Control.

Tuberc Res Treat 2012 18;2012:768603. Epub 2012 Dec 18.

Division of Infectious Diseases, Department of Medicine, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, Newark, NJ 07103, USA.

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http://dx.doi.org/10.1155/2012/768603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539344PMC
January 2013

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

J Clin Microbiol 2012 Jul 25;50(7):2194-202. Epub 2012 Apr 25.

Division of Infectious Disease, Department of Medicine, and the Ruy V. Lourenço Center for Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Newark, New Jersey, USA.

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.
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http://dx.doi.org/10.1128/JCM.00143-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405605PMC
July 2012

Highly sensitive detection of Staphylococcus aureus directly from patient blood.

PLoS One 2012 17;7(2):e31126. Epub 2012 Feb 17.

Division for Infectious Diseases, Department of Medicine, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, Newark, New Jersey, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031126PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281916PMC
June 2012

Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay.

J Clin Microbiol 2011 Mar 29;49(3):932-40. Epub 2010 Dec 29.

Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Newark, NJ 07103, USA.

Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (T(m)) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a T(m) shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB T(m) shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.
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http://dx.doi.org/10.1128/JCM.02271-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067712PMC
March 2011

Evaluation of the analytical performance of the Xpert MTB/RIF assay.

J Clin Microbiol 2010 Jul 26;48(7):2495-501. Epub 2010 May 26.

Division of Infectious Disease, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USA.

We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects Mycobacterium tuberculosis and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse M. tuberculosis isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing 13 different rpoB mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79 M. tuberculosis isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding 10(2) to 10(7) CFU of M. tuberculosis into M. tuberculosis-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and 100% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay. M. tuberculosis was not detected until at least 10(8) copies of an MTBDRplus amplicon were spiked into 1 ml of sputum, suggesting that false-positive results would be unlikely to occur.
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http://dx.doi.org/10.1128/JCM.00128-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897495PMC
July 2010

Rapid universal identification of bacterial pathogens from clinical cultures by using a novel sloppy molecular beacon melting temperature signature technique.

J Clin Microbiol 2010 Jan 18;48(1):258-67. Epub 2009 Nov 18.

Division of Infectious Disease, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103, USA.

A real-time PCR assay with the ability to rapidly identify all pathogenic bacteria would have widespread medical utility. Current real-time PCR technologies cannot accomplish this task due to severe limitations in multiplexing ability. To this end, we developed a new assay system which supports very high degrees of multiplexing. We developed a new class of mismatch-tolerant "sloppy" molecular beacons, modified them to provide an extended hybridization range, and developed a multiprobe, multimelting temperature (T(m)) signature approach to bacterial species identification. Sloppy molecular beacons were exceptionally versatile, and they were able to generate specific T(m) values for DNA sequences that differed by as little as one nucleotide to as many as 23 polymorphisms. Combining the T(m) values generated by several probe-target hybrids resulted in T(m) signatures that served as highly accurate sequence identifiers. Using this method, PCR assays with as few as six sloppy molecular beacons targeting bacterial 16S rRNA gene segments could reproducibly classify 119 different sequence types of pathogenic and commensal bacteria, representing 64 genera, into 111 T(m) signature types. Blinded studies using the assay to identify the bacteria present in 270 patient-derived clinical cultures including 106 patient blood cultures showed a 95 to 97% concordance with conventional methods. Importantly, no bacteria were misidentified; rather, the few species that could not be identified were classified as "indeterminate," resulting in an assay specificity of 100%. This approach enables highly multiplexed target detection using a simple PCR format that can transform infectious disease diagnostics and improve patient outcomes.
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http://dx.doi.org/10.1128/JCM.01725-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812257PMC
January 2010

Rifampin resistance, Beijing-W clade-single nucleotide polymorphism cluster group 2 phylogeny, and the Rv2629 191-C allele in Mycobacterium tuberculosis strains.

J Clin Microbiol 2008 Aug 11;46(8):2555-60. Epub 2008 Jun 11.

Division of Infectious Disease, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.

Rifampin resistance is a key prognostic marker for treatment success in tuberculosis patients. Recently, Wang et al. demonstrated that Rv2629 A191C mutations were present in 99.1% of rifampin-resistant and 0% of rifampin-susceptible clinical Mycobacterium tuberculosis isolates and that overexpression of the Rv2629 191C allele in Mycobacterium smegmatis produced an eightfold increase in rifampin resistance. These results suggested that Rv2629 could be a cause of rifampin resistance and a valuable target for rifampin resistance detection assays. We developed a molecular-beacon assay to study the association between Rv2629 191 alleles and rifampin resistance in 246 geographically and phylogenetically diverse clinical M. tuberculosis isolates. The 191C allele was present in 30/98 (30.6%) rifampin-resistant isolates and 25/148 (16.9%) rifampin-susceptible isolates and was more common in isolates from Asia. Phylogenetic analysis demonstrated complete overlap between the 191C allele and single nucleotide polymorphism cluster group 2 (SCG-2), a phylogenetic lineage that corresponds to the Beijing-W clade of M. tuberculosis. All 55 (100%) 191C isolates were SCG-2, while none of the 191 191A isolates were SCG-2 (P < 0.001). No association was found between the 191C allele and rifampin resistance in an analysis that included the SCG type (P = 1.0). Also, in contrast to the findings of Wang et al., we found that overexpression of either Rv2629 191 allele in M. smegmatis did not produce an increase in rifampin resistance. We conclude that the Rv2629 191C allele is not associated with rifampin resistance and that the allele cannot be used as a molecular target to detect rifampin resistance. The allele appears to be an excellent marker for the Beijing-W clade/SCG-2 phylogenetic group.
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http://dx.doi.org/10.1128/JCM.00666-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519504PMC
August 2008

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons.

J Med Microbiol 2007 Oct;56(Pt 10):1356-1362

Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner.
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http://dx.doi.org/10.1099/jmm.0.47265-0DOI Listing
October 2007
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