Detection Of Rifampin Resistance Research Articles

Evaluation of the GeneXpert MTB/RIF Assay for Rapid Diagnosis of Tuberculosis and Detection of Rifampin Resistance in Tuberculous Patients Admitted to Abbassia Chest Hospital

Abstract Background Tuberculosis is considered one of the major infectious diseases especially in the developing countries and a significant diagnostic and therapeutic challenge worldwide. Gene Xpert MTB/RIF assay is a diagnostic test which depends on a real-time polymerase chain reaction for rapid diagnosis of Mycobacterium Tuberculosis and rifampicin resistance in a single test. Aim To evaluate the diagnostic utility of Gene Xpert MTB/RIF assay for rapid diagnosis of pulmonary and extrapulmonary tuberculosis as well as the detection of rifampin resistance in specimens obtained from suspected tuberculous patients in Abbassia Chest Hospital. Patients and Methods Pulmonary and extrapulmonary samples from 77 cases with suspected TB were collected. All retrieved samples were examined with: direct smear microscopy for AFB, culture for Mycobacterium tuberculosis (L.J. media) along with Drug susceptibility testing (D.S.T.) for positive cultures. As well as the gene Xpert MTB/RIF assay and their results were compared to those of both culture and D.S.T. (as gold standard reference methods). Results Gene Xpert MTB/RIF assay showed the highest sensitivity, negative predictive value (NPV) and diagnostic accuracy in detection of pulmonary TB (96.4, 96.4 and 93.1% respectively), with 90%specificity. Xpert MTB/RIF assay showed the lowest sensitivity in diagnosing TB pleural effusion (66.6%) but with the highest specificity and positive predictive value (PPV) (100% for both). As regards diagnosing TB lymphadenitis, gene Xpert MTB/RIF assay showed 75% sensitivity and the lowest specificity (33.3%). Considering rifampin resistance, gene Xpert MTB/RIF assay showed 100% specificity in detection of RIF- Resistance with variable sensitivity according to the type of TB, being the highest in pulmonary TB (90.9% sensitivity). Conclusion Gene Xpert MTB/RIF assay could be used as the first line diagnostic tool for diagnosis of pulmonary TB because of its high diagnostic yield. Yet, it’s not recommended to replace the culture for diagnosing extrapulmonary TB especially in diagnosing tuberculous pleural effusion (pauci-bacillary specimens) which has limited diagnostic utility. Also, the assay showed 100% specificity in diagnosing rifampicin resistance guiding physicians to justifiably starting second line anti-TB treatment.

Comparative evaluation of critical concentrationsfor detecting borderline rifampin resistance in Mycobacterium tuberculosis

Background: Rifampin plays an important role in tuberculosis treatment. In recent years, the introduction of molecular testing techniques has enabled the rapid detection of rifampin resistance, leading to discrepancies with conventional methods. The World Health Organization (WHO) has analyzed mutations in the rpoB gene that induce rifampin resistance and identified certain mutations causing borderline resistance, which are often undetected using conventional tests. Consequently, the WHO has lowered the rifampin resistance criterion concentration from 1.0 to 0.5 μg/mL in 7H10 and MGIT 960. The present study aimed to evaluate the impact of this change in critical concentration on the detection of borderline rifampin resistance in Mycobacterium tuberculosis. Methods: Tuberculosis strains submitted for antituberculosis drug susceptibility testing from May 2021–2022 were analyzed. Three institutions participated; the Seoul Clinical Laboratories used the agar proportion method, whereas the Samsung Medical Center and Seoul National University Bundang Hospital utilized the MGIT 960 system to test both the original and revised concentrations. Mutations were confirmed through rpoB gene sequencing for strains showing discrepancies. Results: A total of 1,596 valid susceptibility tests were conducted during the study period. Rifampin resistance was detected in 35 cases (2.19%) at 1.0 μg/mL and in 38 cases (2.38%) at 0.5 μg/mL, whereas isoniazid resistance was observed in 158 cases (9.90%). Among the three rifampin discrepancy strains, one harbored an H445L mutation, whereas the remaining two exhibited an L452P mutation. These mutations were classified as borderline resistant. Conclusion: Applying the new rifampin critical concentration resulted in a 0.19% increase in resistance rate and an 8.57% increase in detection cases. Additionally, despite testing with large number of rifampin-susceptible strains, no false resistance results were obtained. Therefore, the application of the new critical concentration is considered beneficial for the management of rifampin-resistant tuberculosis.

Evaluation of different diagnostic methods for spinal tuberculosis infection

Background and purposeTuberculosis (TB) is the most fatal infectious disease worldwide. Approximately 24.6% of tuberculosis cases are extrapulmonary and predominantly affect the spine. It is difficult to diagnose spinal TB (STB). We aimed to evaluate the diagnostic performance of the Mycobacteria Growth Indicator Tube (MGIT)-960 culture, T-SPOT.TB, Xpert Mycobacterium tuberculosis complex (MTB)/resistance to rifampin (RIF), and Metagenomic Next-Generation Sequencing (mNGS) to detect STB.MethodsWe assessed 126 patients presumed to have STB using these four methods. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using clinical diagnosis as a reference.ResultsOf the patients, 41 were diagnosed with STB and 85 with non-STB. In the STB group, the sensitivity, specificity, PPV, and NPV of the MGIT-960 culture were 29.3% (12/41), 100% (85/85), 100% (12/12), and 74.6% (85/114), respectively. The sensitivity, specificity, PPV, and NPV of T-SPOT.TB were 92.7% (38/41), 82.4% (70/85), 58.5% (31/53), and 95.9% (70/73), respectively. The sensitivity, specificity, PPV, and NPV of the Xpert MTB/RIF assay were 53.7% (22/41), 100% (85/85), 100% (22/22), and 81.7% (85/104), respectively. The sensitivity, specificity, PPV, and NPV of mNGS were 39.0% (16/41), 98.8% (84/85), 94.1% (16/17), and 77.1% (84/109), respectively. The sensitivity, specificity, PPV, and NPV of mNGS + Xpert MTB/RIF were 73.2% (30/41), 100% (85/85), 96.8% (30/31), and 72.0% (85/118), respectively. The sensitivity, specificity, PPV, and NPV of the mNGS + T-spot assay were 97.6% (40/41), 100% (85/85), 67.9% (38/56), and 75.9% (85/113), respectively. Moreover, the sensitivity, specificity, PPV, and NPV of T-spot + Xpert MTB/RIF were 95.1% (39/41), 100% (85/85), 72.2% (39/54), and 81.0% (85/105), respectively.ConclusionsT-SPOT.TB is the most effective method for diagnosing STB; however, Xpert MTB/RIF is more reliable and can detect RIF resistance. Clinicians can use mNGS to identify pathogens in patients with spinal infections; these pathogens appeared to be more meaningful in guiding the clinical management of patients in the non-STB group. The combination of Xpert MTB/RIF and mNGS can improve the early diagnosis rate and drug resistance detection, reduce the diagnostic cycle, and provide early targeted anti-TB treatment for patients with STB.

Xpert MTB/RIF in the Diagnosis of Mediastinal Tuberculous Lymphadenitis by Endoscopic Ultrasound-Guided Needle Aspiration Techniques: A Systematic Review and Meta-Analysis

Background: Lymphadenopathy is one of the most prevalent clinical manifestations of extrapulmonary tuberculosis. Endosonography is the recommended technique in the diagnostic work-up of mediastinal lymphadenopathies. Xpert MTB/RIF assay is a self-contained cartridge-based fully automated DNA testing platform which can accurately detect both tuberculosis and mycobacterial resistance to rifampicin. A few studies assessed its accuracy for mediastinal lymph node aspirates collected using endosonography. A systematic review of observational studies was performed to provide a pooled estimate of sensitivity and specificity of Xpert MTB/RIF in the diagnosis of mediastinal tuberculous lymphadenitis using endoscopic ultrasound-guided needle aspiration techniques. Methods: A search of the scientific evidence was carried out using PubMed, Embase, and Scopus. Articles describing observational studies on Xpert MTB/RIF in the diagnosis of mediastinal tuberculous lymphadenitis using endoscopic ultrasound-guided needle aspiration techniques were selected. Results: Eight studies met the inclusion criteria. The overall pooled sensitivity was 61% (95% CI = 55–68%; I² = 66.3%; p = 0.004), overall pooled specificity was 89% (95% CI = 85–91%; I² = 90.1%; p < 0.0001). Area under the sROC curve was 0.68. Only one study reported data on rifampin resistance detection and showed a sensitivity of 83.3% and a specificity of 16%. Conclusions: Xpert MTB/RIF shows a good accuracy in the diagnosis of mediastinal mycobacterial lymphadenitis by endosonographic needle aspiration techniques. It should be always recommended for suspected mediastinal tuberculosis.

Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Clinical Isolates from Sputum Samples by High-Resolution Melting Analysis.

The effective management of multidrug-resistant tuberculosis (MDR-TB) and the need for rapid and accurate screening of rifampin (RIF) and isoniazid (INH)-resistant Mycobacterium tuberculosis (Mtb) isolates are the most fundamental and difficult challenges facing the global TB control. The present study aimed to compare the diagnostic accuracy of high-resolution melting-curve analysis (HRMA) in comparison to multiplex allele-specific PCR (MAS-PCR) and xpert MTB/RIF as well as the conventional drug-susceptibility test (DST) and gene sequencing for the detection of INH and RIF resistance in the Mtb isolates. In the present study, a total of 431 Mtb isolates including 11 MDR (%2.55), 7 INH resistance (%1.62), two RIF resistance (%0.46), and 411 sensitive isolates were phenotypically confirmed. HRMA assay identified katG gene mutations and the mabA-inhA promoter region in 15 of 18 INH-resistant samples and rpoB gene mutations were successfully evaluated in 11 out of 13 RIF-resistant samples. The sensitivity and specificity of the HRMA method were 83.3% and 98.8% for INH and 84.6% and 99% for RIF, respectively. The most common mutation in RIF-resistance-determining region (RRDR) occurred at codon 531 (TCG → TTG)(84.6%) and then at codon 513 (CAA → GTA)(7.6%) and 526 (CAC → TAC) (7.6%), which resulted in the amino-acid changes. Also, 88.8% of INH-resistant samples had mutations in the katG gene and the mabA-inhA promoter region, of which the highest mutation occurred at codon 315 (AGC → ACC) of the katG gene. In conclusion, all these results indicated that the sensitivity and specificity of the HRM method were increased when the katG gene and the mabA-inhA promoter region were used as a target.

Identification of mutations in rpoB, pncA, embB, and ubiA genes among drug-resistant Mycobacterium tuberculosis clinical isolates from Iran.

Mycobacterium tuberculosis resistant to effective first-line drugs (FLDs) has challenged national and global tuberculosis control programs. This study aimed to identify mutations in 4 genes related to rifampin, pyrazinamide, and ethambutol resistance among clinical isolates of M. tuberculosis from southwestern Iran. After drug susceptibility testing of 6620 M. tuberculosis clinical isolates by proportional method, a total of 24 FLD-resistant strains were included in the study. Fragments of rpoB, pncA, embB, and ubiA genes were amplified and sequenced to mine the mutations by pairwise alignment with the corresponding M. tuberculosis H37Rv genes. Phenotypic resistance to rifampin, isoniazid, and ethambutol was detected in 67, 54, and 33% (n = 16, 13, and 8) of the isolates, respectively. Of rifampin-resistant isolates, 31% (5/16) were mono-resistant, and 56% (9/16) were multidrug-resistant (MDR). In 100% of rifampin-resistant isolates, mutations were found in the rifampin resistance-determining region (RRDR) of the rpoB, with S450L substitution being the most common, especially in MDRs (77.8%, 7/9). Resistance-conferring mutations in pncA were present in 12.5% (3/24) of FLD-resistant isolates. The embB and ubiA mutations were found in 62.5 and 12.5% (5/8 and 1/8) of ethambutol-resistant isolates, respectively, of which the embB D354A was the most common substitution (37.5%, 3/8). Sixteen distinct mutations were identified, one of which was novel. The sequence analysis of the RRDR segment was the best way to detect rifampin resistance. The rpoB S450L substitution could be a helpful molecular marker to predict MDR. In other genes, no mutation was identified as a reliable marker.

Clinical Validation of the Xpert MTB/RIF Test for Identification of the Mycobacterium tuberculosis Complex in Acid-Fast Bacillus Smear-Positive MGIT Broth Cultures.

The identification of the Mycobacterium tuberculosis complex (MTBC) from smear-positive broth cultures can be achieved using several methods, including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from acid-fast bacilli (AFB) smear-positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampin resistance. The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear-positive MGIT 960 broth cultures (Becton, Dickinson, Sparks, MD). We sought to validate the Xpert MTB/RIF test for use with AFB smear-positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false-positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampin resistance from AFB smear-positive MGIT 960 broth cultures.

Evaluation of the performance of the BD MAX MDR-TB test in the diagnosis of Mycobacterium tuberculosis complex in extrapulmonary and pulmonary samples

ABSTRACT Background The BD MAX MDR-TB is a recently marketed molecular test for detecting Mycobacterium tuberculosis complex (MTC), rifampin, and isoniazid drug resistance. Research Design and Methods This study aimed to evaluate the BD MAX MDR-TB test performance in 933 extrapulmonary and 774 pulmonary samples. Results Test MTC detecting sensitivity was 90.6%, 82.5%, and the specificity was 98.5%, 98.9%, in pulmonary and extrapulmonary samples, respectively. In smear-positive samples, sensitivity, and specificity were 100% for all samples. However, in smear-negative samples, the test’s sensitivity and specificity were 82.3%, 98.5% in pulmonary samples, and 76.7%, 98.9% in extrapulmonary samples. Test sensitivity in detecting isoniazid resistance was 71.4%, specificity 96.8%, and in detecting rifampin resistance was 100%, specificity 93.9%, respectively. Conclusions BD MAX MDR-TB is a reliable, rapid, user-friendly test for detecting MTC in extrapulmonary and pulmonary samples and its resistance toward isoniazid and rifampin. It can be used as an alternative to the Xpert system assays.

Detecting Mycobacterium tuberculosis complex and rifampicin resistance via a new rapid multienzyme isothermal point mutation assay

Simple, rapid, and accurate detection of the Mycobacterium tuberculosis complex (MTBC) and drug resistance is critical for improving patient care and decreasing the spread of tuberculosis. To this end, we have developed a new simple and rapid molecular method, which combines multienzyme isothermal rapid amplification and a lateral flow strip, to detect MTBC and simultaneously detect rifampin (RIF) resistance. Our findings showed that it has sufficient sensitivity and specificity for discriminating 118 MTBC strains from 51 non-tuberculosis mycobacteria strains and 11 of the most common respiratory tract bacteria. Further, compared to drug susceptibility testing, the assay has a sensitivity, specificity, and accuracy of 54.1%, 100.0%, and 75.2%, respectively, for detection of RIF resistance. Some of the advantages of this assay are that no special instrumentation is required, a constant low temperature of 39 °C is sufficient for the reaction, the turnaround time is less than 20 min from the start of the reaction to read out and the result can be seen with the naked eye and does not require specialized training. These characteristics of the new assay make it particularly useful for detecting MTBC and RIF resistance in resource-limited settings.

Diagnostic Performance of GeneChip for the Rapid Detection of Drug-Resistant Tuberculosis in Different Subgroups of Patients.

ObjectiveDrug-resistant tuberculosis (DR-TB) is a growing problem worldwide. The rapid drug susceptibility test (DST) of DR-TB enables the timely administration of a chemotherapy regimen that effectively treats DR-TB. GeneChip has been reported as a novel molecular diagnostic tool for rapid diagnosis but has limited data on the performance of subgroup patients with DR-TB. This study aims to assess the diagnostic value of GeneChip in patients with different sexes, ages, treatment histories, treatment outcomes, and places of residence.MethodsWe recruited newly registered sputum smear-positive pulmonary TB patients from January 2011 to September 2020 in Lianyungang City, Jiangsu Province, China. We applied both GeneChip and DST to measure drug resistance to rifampin (RIF) and isoniazid (INH). The kappa value, sensitivity, specificity, and agreement rate (AR) were calculated. We also applied a Classification and Regression Tree to explore factors related to the performance of GeneChip.ResultsWe observed that sex, age, treatment history, treatment outcomes, and drug resistance type were significantly associated with the performance of GeneChip. For RIF resistance, there was significant accordance in young patients (kappa: 0.79) and cases with the treatment failure outcome (kappa: 0.92). For multidrug resistance (MDR), there was significant accordance in young cases (kappa: 0.77). Compared with previously treated patients, the newly treated patients had a significantly higher AR in detecting RIF resistance (0.97 vs 0.92), INH resistance (0.95 vs 0.89), and MDR (0.98 vs 0.92). The overall sensitivity, specificity, AR and kappa value for the diagnosis of MDR-TB were 0.70 (95% CI: 0.63–0.70), 0.99 (95% CI: 0.98–0.99), 0.98 (95% CI: 0.97–0.98), and 0.72 (95% CI: 0.67–0.78), respectively.ConclusionWe observed a high concordance between GeneChip and DST among TB patients with different characteristics, indicating that GeneChip can be a potential alternative tool for rapid MDR-TB detection.

Performance of Xpert MTB/RIF for the Diagnosis of Extrapulmonary Tuberculosis.

Mycobacterial burden is low in extrapulmonary specimens, making diagnosis and treatment difficult. Xpert MTB/RIF is a real-time PCR assay for the detection of Mycobacterium tuberculosis and rifampin resistance. This study evaluated the performance of the Xpert MTB/RIF assay in extrapulmonary specimens. Acid-Fast Bacilli (AFB) smear, culture, and Xpert MTB/RIF were performed on extrapulmonary specimens. Mycobacterial culture was performed on BACTEC MGIT liquid for 6 weeks and 2% Ogawa medium for 8 weeks. Overall sensitivity and specificity of Xpert MTB/RIF was estimated using culture as a gold standard. Xpert MTB/RIF sensitivity and cycle-threshold (Ct) values according to AFB smear grade were evaluated. The sensitivity, specificity, and concordance of rifampin resistance compared to the phenotypic drug sensitivity test were evaluated. A total of 1,289 specimens were included in the study. The overall sensitivity and specificity of the Xpert MTB/RIF assay were 59.4% (41/69, 95% CI 46.9 - 70.9%) and 99.3% (1,212/1,220, 95% CI 98.7 - 99.7), respectively. Positive predictive value of Xpert MTB/RIF was 83.7% (41/49, 95% CI 69.8 - 92.2) and negative predictive value was 97.7% (1,212/1,240, 95% CI 96.7 - 98.5%). Xpert MTB/RIF assay sensitivity significantly increased with increases in AFB smear grade (p < 0.001). AFB smear grades and Xpert MTB/RIF Ct values were negatively correlated. Rifampin resistance results of Xpert MTB/RIF and culture showed a concordance rate of 97.2%. The Xpert MTB/RIF assay could be used to replace the AFB smear for the diagnosis of extrapulmonary tuberculosis, and has high specificity for the detection of rifampin resistance.

A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay.

Rapid and accurate diagnosis of multidrug-resistant tuberculosis (MDR-TB) is important for timely and appropriate therapy. In this study, a rapid and easy-to-perform molecular test that integrated polymerase chain reaction (PCR) amplification and a specific 96-well microplate hybridization assay, called PCR-ELISA (enzyme-linked immunosorbent assay), were developed for detection of mutations in rpoB, katG, and inhA genes responsible for rifampin (RIF) and isoniazid (INH) resistance and prediction of drug susceptibility in Mycobacterium tuberculosis clinical isolates. We evaluated the utility of this method by using 32 multidrug-resistent (MDR) isolates and 22 susceptible isolates; subsequently, we compared the results with data obtained by conventional drug susceptibility testing and DNA sequencing. The sensitivity and specificity of the PCR-ELISA test were 93.7% and 100% for detecting RIF resistance, and 87.5% and 100% for detecting INH resistance, respectively. These results were comparable to those yielded by commercially available molecular tests such as the GenoType MTBDRplus assay. Based on the aforementioned results, we conclude that the PCR-ELISA microplate hybridization assay is a rapid, inexpensive, convenient, and reliable test that will be useful for rapid diagnosis of MDR-TB, for improved clinical care.

Multidrug Resistant Mycobacterium tuberculosis Among Military and Civilian Personnel seen at a Tertiary Military Hospital, Manila, Philippines (2015-2018).

About one third of the world population is estimated to be infected with Mycobacterium tuberculosis (MTB), and this proportion is expected to be higher in countries with a high tuberculosis (TB) burden. The Philippines is both a high tuberculosis burden and a high multidrug resistant tuberculosis (MDR-TB) burden country. Though TB has been extensively described in the civilian population, there is limited data on TB in the military population. The objectives are: (1) To determine MTB/MDR-TB prevalence among military and civilian patients in the Philippines presenting with clinically suspected TB in a tertiary military hospital and (2) To determine performance of direct sputum smear microscopy (DSSM) using Ziehl-Neelsen (ZN) staining compared to Xpert MTB/RIF real-time reverse transcriptase polymerase chain reaction. Sputum samples were obtained from patients, clinically suspected with TB, and/or with TB associated signs/symptoms. Sputum specimens were tested using DSSM with ZN staining and Xpert MTB/RIF assay (Cepheid, Sunnyvale, California) and patient demographic and clinical data were collected. From March 2015 to December 2018, a total of 795 (173 military personnel [164 active duty and 9 retired]; 618 civilians; and 4 with no data on military/civilian status) patients with TB associated symptoms or clinically suspected with TB were tested. Overall, MTB prevalence was 81/795 (10%). MTB prevalence among active duty and retired military personnel were 27/164 (16%) and 4/9 (44%), respectively while MTB prevalence for civilian patients was 50/618 (8%) (p value=0.0003; OR=2.48 [95% C.I. 1.5-4]). Among active and retired military personnel who tested positive for MTB, rifampin resistance was 4/27 (15%) and 1/4 (25%), respectively, while rifampin resistance for civilian patients was 9/50 (18%) (p value=1; OR=0.88 [95% C.I. 0.26-2.90]). For active duty military personnel, average MTB prevalence (based on Xpert MTB/RIF) covering years 2015-2018 was 21% and ranged from 13% to 35%, while average rifampin resistance among MTB positive active duty military personnel was 15% and ranged from 0% to 25%. Overall sensitivity and specificity of DSSM compared to Xpert MTB/RIF were 70% and 96%, respectively. Positive and negative predictive values of DSSM to accurately categorize MTB in symptomatic cases (with Xpert MTB/RIF as "true positive" reference) were 74% and 95%, respectively. Performance of DSSM varied according to MTB load detected by Xpert MTB/RIF with increasing DSSM sensitivity observed as the MTB load detected by Xpert MTB/RIF increased (p=0.02). This report describes high MTB and MDR-TB prevalence rates among symptomatic military patients with military personnel having higher odds of MTB infection compared to the civilian patients in the study. Since DSSM (ZN) sensitivity greatly varied depending on MTB load, the Xpert MTB/RIF should be used as a first-line diagnostic tool to identify MTB and detect rifampin resistance among presumptive TB cases instead of DSSM (ZN) microscopy.

Gene Xpert MTB/RIF Assay as A New Tool for Tuberculosis Diagnosis and Detection of Rifampin Resistance

Background: Mycobacterium tuberculosis (MTB) is one of the top ten causes of death and the leading cause of a single infectious agent (above HIV/AIDS) Worldwide. Each year millions of people continue to fall sick with TB. Aim: To evaluate the MTB/RIF assay for rapid diagnosis and detection of rifampin resistance of tuberculosis in both positive and negative smears-as well as pulmonary and non-pulmonary clinical specimens. Patients and Methods: A prospective study was conducted on 50 patients (37 pulmonary, 13 extrapulmonary) patients attending the Outpatient Clinic and inpatients of Chest Department, Benha University Hospital, Egypt. Pulmonary samples were analyzed by (Sputum smear exam. by Ziehl Neelsen (ZN) stain, culture on Lowenstein-Jensen(LJ)media, and assessment of rifampin sensitivity). Extrapulmonary samples were analyzed by reference methods (pathological diagnosis) and by GeneXpert. Results: pulmonary patients’ smears were 32 (86.5%) positive and 5 (13.5%) negative. Culture on LJrevealed rifampin sensitivity in 33/37 (89.1%) and rifampin-resistant in 4/37 (10.9%), GeneXpert was positive in 32(86.5%) and negative in 5 (13.5%), rifampin assay was sensitive in 33 (89.1%) and resistant in 4(10.9%). All extrapulmonary patients, (n=13) were positive on GeneXpert and sensitive for rifampin. Conclusion: GeneXpert could be considered as a leading way to early diagnosis, treatment, and prevention of transmission of tuberculosis which could reduce TB- associated morbidity and mortality.

Clinical application of a new-generation Xpert Mycobacterium tuberculosis/rifampin Ultra cartridge

Objective To investigate the clinical value of a new-generation cartridge Xpert MTB/RIF Ultra assay on detection of Mycobacterium tuberculosis (MTB) and rifampin(RIF) resistance. Methods A total of 111 patients from He′nan Provincial Chest Hospital with suspected tuberculosis(TB) and retreated TB were enrolled into this study from March to December 2016, including 33 cases of tuberculosis detection group(CDG) and 78 cases of drug resistant high-risk group (DRG). The sputum samples of patients were collected. The sensitivity and specificity of Xpert MTB/RIF Ultra, sputum smear, solid Lowenstein-Jensen (L-J) culture and mycobacterial growth indicator tube(MGIT) culture for MTB were evaluated. RIF resistance was performed by Xpert MTB/RIF Ultra, traditional phenotypic drug sensitivity test and Xpert MTB/RIF assay. Measurement data were compared using t-test, and categorical data were compared using chi-squared test. Results Using clinical diagnosis result as the standard, in the CDG, the sensitivity of Xpert MTB/RIF Ultra for MTB detection (75.8%) was not significantly different from those of sputum smear (66.7%), L-J culture(63.6%), MGIT culture(75.6%) and Xpert MTB/RIF (66.7%)(χ2=0.67, 1.15, 0.00 and 0.67, respectively, all P>0.05). In the DRG, the sensitivity of Xpert MTB/RIF Ultra (94.9%) was better than those of L-J culture (55.1%) and MGIT culture (80.8%) with statistical significance (χ2=32.8 and 7.25, respectively, both P 0.05). Xpert MTB/RIF Ultra took the shortest time to obtain the final results, which was (1.76±0.18) h and significantly shorter than smear test ([5.04±0.49] h), L-J culture ([31.67±0.56] h), MGIT culture ([22.36±9.68] h), Xpert MTB/RIF ([2.00±0.30] h) (t =16.90, 31.98, 24.38 and 7.05, respectively, all P<0.01). Using culture result as the standard, the sensitivity for MTB detection of Xpert MTB/RIF and Xpert MTB/RIF Ultra were 93.2% and 98.9%, and the specificity of Xpert MTB/RIF and Xpert MTB/RIF Ultra were both 100%. The sensitivity for MTB detection of Xpert MTB/RIF Ultra (52.2%) was significantly better than that of Xpert MTB/RIF (21.7%) in 23 smear-negative pulmonary TB patients with statistical significance (χ2=4.98, P=0.025). Using traditional drug susceptibility test as the standard, the sensitivities for RIF resistance detection of Xpert MTB/RIF and Xpert MTB/RIF Ultra in culture-positive TB patients were 90.9% and 93.2%, respectively, and the specificities were 89.5% and 92.9%, respectively. Conclusions Xpert MTB/RIF Ultra has a higher MTB detection rate than Xpert MTB/RIF in smear-negative pulmonary TB patients. In drug-resistant pulmonary TB patient, MTB/RIF Ultra has high sensitivity, and it takes shorter time to detect MTB and RIF resistance. Thus, Xpert MTB/RIF Ultra has a good application prospect in clinical work. Key words: Mycobacterium tuberculosis; Tuberculosis, pulmonary; Diagnosis; Rifampin; Drug resistance, bacterial; Microbial sensitivity tests; Nucleic acid amplification techniques

Direct Detection of Rifampin-Resistant Mycobacterium tuberculosis in Respiratory Specimens Using Quantamatrix Multiplexed Assay Platform (QMAP) System: A Multicenter Study in Korea.

Rapid and accurate detection of rifampin-resistant Mycobacterium tuberculosis (MTB) is of primary importance for infection control and selection of anti-tuberculosis drugs. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the direct detection of rifampin-resistant MTB in respiratory specimens. A total of 400 respiratory specimens collected from patients with clinically suspected tuberculosis or non-tuberculous mycobacteria (NTM) infections were tested with the culture-based conventional Mycobacterium species identification and QMAP system. Among 400 specimens, 154 samples were evaluated using phenotypic anti-tuberculosis drug susceptibility test (DST) and the QMAP system for the detection of rifampin resistance. Detection agreement rate between the culture-based conventional identification and QMAP system for MTB and NTM according to acid-fast bacillus smear positivity was as follows: 97.0% (131/135) and 93.6% (88/94) in 229 smear-positive samples and 69.4% (25/36) and 73.0% (65/89) in 171 smear-negative samples. Based on culture as the gold standard, the overall sensitivity and specificity of the QMAP system for Mycobacterium identification were 87.3 and 97.8%, respectively. The categorical agreement rate between phenotypic DST and QMAP system for rifampin was as follows: complete agreement, 92.9% (143/154); very major error, 0%; and major error, 0.6% (1/154). The overall sensitivity of the QMAP system for the detection of rifampin resistance was 97.1% (34/35). The QMAP system is a useful screening method for the early diagnosis of tuberculosis and selection of anti-tuberculosis drug, as it may detect rifampin-resistant MTB directly from respiratory specimens.

GeneXpert MTB/RIF for rapid diagnosis and rifampin resistance detection of endobronchial tuberculosis.

Delayed diagnosis and treatment of tuberculosis (TB) contribute to poor outcomes, especially for endobronchial TB (EBTB), which typically leads to tracheobronchial stenosis. Finding rapid and accurate diagnostic tools for EBTB is crucial. GeneXpert Mycobacterium tuberculosis (MTB)/rifampin (RIF) was recommended by the World Health Organization (WHO) as a standard molecular biological diagnostic technique for MTB. The aim of this study was to evaluate the efficacy of GeneXpert MTB/RIF for diagnosing EBTB and for evaluating RIF resistance. Biopsy tissue and bronchial brushings from EBTB patients were prospectively assessed with GeneXpert MTB/RIF. The diagnostic yields of auramine O-stained sputum smears and bronchial brush smears were obtained, and the results were compared with the cultures of sputum and biopsy tissues for MTB. In 61 confirmed cases of EBTB, the sensitivities of sputum smear, bronchial brush smear, sputum culture and tissue culture to diagnose EBTB were 13.1%, 32.8%, 36.1% and 68.9%, respectively. For bronchial brushings and biopsies, our data showed sensitivities of 57.4% and 63.9%, respectively, and a specificity of 100% for GeneXpert MTB/RIF, and these results were superior to those of sputum smears, bronchial brush smears and sputum culture. GeneXpert MTB/RIF for bronchial brushings and biopsies showed complementarity in its diagnostic performance. Resistance to RIF was identified in 17.4% (8/46) of GeneXpert MTB-positive cases. GeneXpert MTB/RIF may enable more rapid EBTB diagnosis and determination of RIF resistance, which are crucial for timely treatment.

Evaluation of a high-intensity fluorescent fluorophage method for diagnosis of drug-resistance in tuberculosis

Objective: To evaluate the high-intensity green fluorescent protein fluorophage Φ(2)GFP10 method for drug susceptibility testing of tuberculosis for isoniazid(INH), rifampin(RIF), and streptomycin(SM). Methods: A total of 128 clinical M. tuberculosis strains were isolated from patients with suspected drug-resistant tuberculosis visiting Beijing Chest Hospital (Beijing, China) from April to June 2014.All of the isolates were tested by the phage assay, while conventional drug susceptibility tests were performing on Lwenstein-Jensen culture medium as reference. Results: The sensitivities of Φ(2)GFP10 assay for INH, RIF, and SM resistance detection were 100.0%, 98.1%(53/54), and 92.6%(50/54), respectively, while their specificities were 84.8%(56/66), 91.9%(68/74), and 91.9%(68/74), respectively. The agreement between the phage assay and the conventional assay for detecting INH, RIF, and SM resistance was 0.92, 0.95 and 0.92, respectively. The Φ(2) GFP10-phage assay could be done within 2 days for RIF and SM, and 3 days for INH. Conclusions: The Φ(2)GFP10-phage method for drug susceptibility test is very sensitive and specific. The method has the potential to be a valuable, rapid and economical screening method for detecting drug-resistant tuberculosis.

Performance of the Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis and rifampin resistance in a low-incidence, high-resource setting.

Performance of the Xpert MTB/RIF assay, designed to simultaneously detect Mycobacterium tuberculosis complex (MTBC) and rifampin (RIF) resistance, has been well documented in low-resource settings with high TB-incidence. However, few studies have assessed its accuracy in low TB incidence settings. We evaluated the performance of Xpert MTB/RIF using clinical sputum specimens routinely collected from suspect pulmonary TB patients over a 4-year time period in San Diego County, California. Xpert MTB/RIF results were compared to acid-fast bacilli (AFB) smear microscopy, mycobacterial culture, and phenotypic drug susceptibility testing (DST). Of 751 sputum specimens, 134 (17.8%) were MTBC culture-positive and 2 (1.5%) were multidrug-resistant (MDR). For the detection of MTBC, Xpert MTB/RIF sensitivity was 89.6% (97.7% and 74.5% in smear-positive and -negative sputa, respectively) and specificity was 97.2%; while AFB smear sensitivity and specificity were 64.9% and 77.8%, respectively. Xpert MTB/RIF detected 35 of 47 smear-negative culture-positive specimens, and excluded 124 of 137 smear-positive culture-negative specimens. Xpert MTB/RIF also correctly excluded 99.2% (121/122) of nontuberculous mycobacteria (NTM) specimens, including all 33 NTM false-positives by smear microscopy. For the detection of RIF resistance, Xpert MTB/RIF sensitivity and specificity were 100% and 98.3%, respectively. Our findings demonstrate that Xpert MTB/RIF is able to accurately detect MTBC and RIF resistance in routinely collected respiratory specimens in a low TB-incidence setting, with comparable performance to that achieved in high-incidence settings; and suggest that under these conditions the assay has particular utility in detecting smear-negative TB cases, excluding smear-positive patients without MTBC disease, and differentiating MTBC from NTM.

Application value of Xpert MTB/RIF in diagnosis of spinal tuberculosis and detection of rifampin resistance

To investigate the application value of Xpert MTB/RIF in diagnosis of spinal tuberculosis and detection of rifampin resistance. The 109 pus specimens were obtained from patients who were primaryly diagnosed as spinal tuberculosis. All of the pus specimens were detected by acid-fast stain, liquid fast culturing by BACTEC MGIT 960 and Xpert MTB/RIF assay to definite the differences in sensitivity and specificity of mycobacterium tuberculosis among detecting methods. Pus specimens obtained by different methods were deteceded by MTB/RIF test to analyze the self-influence on Xpert MTB/RIF test. The result of liquid fast culturing by BACTEC MGIT 960 was used as the gold standard; and the value of Xpert MTB/RIF assay in detecting rifampin resistance was analyzed. The sensitivity of acid-fast stain, liquid fast culturing by BACTEC MGIT 960 and Xpert MTB/RIF assay were 25.92%, 48.15%, 77.78%, respectively. The sensitivity of pus specimens obtained from open surgery, ultrasound positioning puncture and biopsy the sensitivity were 83.78%, 76.47%, 44.68% respectively deteceded by MTB/RIF test. According to the gold standard of the results of liquid fast culturing by BACTEC MGIT 960 assay, the sensitivity and specificity of Xpert MTB/RIF assay in detecting rifampin resistance were 80%(4/5) and 90.70%(39/43), respectively. Xpert MTB/RIF assay has higher value in diagnosis of spinal tuberculosi, and also can detect rifampin resistance. The number of mycobacterium tuberculosis in pus specimens has a great influence in the sensitivity of Xpert MTB/RIF assay.