Detection Of Rifampicin Resistance Research Articles

Epidemiology of Molecular Probes in Xpert MTB/RIF Assay in AJK, Pakistan

Objective: This study aimed to detect rifampicin-resistant tuberculosis cases and assess the frequency of missing probes in different study populations in Azad Jammu and Kashmir (AJK), Pakistan. Methodology: The study was conducted at the State TB Reference Laboratory, District Headquarters Teaching Hospital, Mirpur, AJK. A total of 2,790 specimens collected between March 2016 to August 2019 were analyzed. Pulmonary TB (PTB) accounted for 94% of the cases, while 6% were classified as extra-pulmonary cases. All respiratory and non-respiratory samples underwent fluorescence smear microscopy (AFB) and a real-time PCR test (Xpert MTB/RIF assay) to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance. Results: Among the 2,790 suspected MTB patients, 734 (26%) were confirmed to have MTB using the Xpert MTB/RIF assay, while 564 (20%) tested positive by fluorescence microscopy. Of the MTB-positive patients, 720 (98%) were diagnosed with pulmonary TB, and 14 (2%) had extra-pulmonary TB. Rifampicin resistance (RR) was detected in 66 (9%) cases, with 97% of the resistant cases being pulmonary and 3% extra-pulmonary. The most frequently missing probe was E (Codon 529-533), accounting for 34% of the cases, followed by probe D (Codon 523-529) at 26%. The least frequently missing probe was C (Codon 523-529), observed in 3% of the cases. Probe B (Codon 512-518) was missing in 15.4% of cases, while probe A (Codon 518-523) was missing in 9.4% of cases. Conclusion: The utilization of molecular diagnostic techniques, such as the Xpert MTB/RIF assay, enables rapid identification of MTB and detection of rifampicin resistance. This study provides valuable baseline data on the prevalence of 81 bp mutations in the rpoB gene, highlighting the need for further evaluation of mutation patterns in AJK.

AB1561 COMPARISON OF DIAGNOSTIC PERFORMANCE OF TRUENAT™ MTB PLUS AND XPERT® ULTRA (GX) IN PATIENTS OF OSTEOARTICULAR TUBERCULOSIS (OATB): EXPERIENCE FROM NORTH INDIA

BackgroundDiagnostic delay and drug resistance not only worsen the outcomes of tuberculosis, but are also important impediments to TB elimination efforts. Prompt and accurate diagnosis of osteoarticular tuberculosis (OATB) along with simultaneous detection of drug resistance is crucial to decrease the morbidity and associated sequelae.ObjectivesGiven the need for a near point-of-care test suitable for primary healthcare centers and simultaneous detection of resistance, Truenat MTB Plus (TruPlus), a chip-based real-time polymerase chain reactionassay, was evaluated for the first time for diagnosis of OATB and detection of rifampicin resistance from pus and synovial fluid samples.MethodsTotal of 100 synovial fluid/pus samples (20 microbiologically confirmed OATB[culture-positive], 50 clinically confirmed OATB [culture-negative], and 30 control patients) were subjected to TruPlus assay and Xpert Ultra (GX Ultra)assay; and their performance was compared. Results were evaluated against both culture and composite reference standard.ResultsThe overall sensitivity and specificity of TruPlus in diagnosing OATB was 77.14%(54/70) and 100%, respectively. The sensitivity was 90% (18/20) for microbiologically confirmed cases and 72% (36/50) for clinically confirmed cases. Performance of TruPlus was superior to Xpert ultra (sensitivity = 70%). Overall, sensitivity and specificity of GX ultra was 70% and 100%. However, sensitivity of GX Ultra was 85% in culture confirmed cases and 64% (32/50) in clinically suspected cases of OATB. Both TruPlus and Gx Ultra correctly reported Rifampicin resistance in four cases, when compared with phenotypic DST and rpoB gene sequencing.ConclusionTruPlus, with its greater portability and higher sensitivity than Xpert, could serve as an important tool for diagnosing OATB and rifampicin resistance at outreach endemic areas.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

World Health Organization Guideline on the Management of Tuberculosis in Children: Critical Appraisal, Concerns, and Caution.

In September 2022, the World Health Organization (WHO) published a new guideline for the management of tuberculosis (TB) in children and adolescents. It included eight new recommendations. Xpert MTB/RIF Ultra (Xpert Ultra) has been designated as the preferred initial diagnostic test for pulmonary TB and detection of rifampicin resistance. But its place vis-à-vis the previously recommended GeneXpert has not been clarified. Further, the limited diagnostic accuracy of Xpert Ultra in some biological specimens like nasopharyngeal aspirates, and the inability to report the presence or absence of rifampicin resistance in 'trace' reports has not been addressed. The guideline also recommends a shortened 4-mo treatment regimen for non-severe drug-susceptible TB. This is based on a single trial having several methodological issues that limit its applicability and generalizability. Interestingly, the criteria for designating 'non-severe' TB in the trial is based on smear negativity, whereas the new WHO recommendation is to omit smear microscopy altogether. The guideline also recommends an alternative 6-mo intensive regimen for drug-susceptible TB meningitis, which needs more supportive evidence. The lower age limits for the use of bedaquiline and delamanid have been decreased to less than 6 and 3 y respectively. While this makes it feasible to treat drug resistant TB in children with oral medications, the resource implications need careful consideration. These concerns advocate caution before the WHO guideline recommendations can be universally implemented.

Clinical value of the MeltPro MTB assays in detection of drug-resistant tuberculosis in paraffin-embedded tissues

Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.

Evaluation of Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis and rifampicin resistance

Tuberculosis (TB) is a public health challenge globally, and molecular testing is recommended to expedite diagnosis. Concerns that Xpert MTB/RIF assay (Xpert) may be less sensitive when testing paucibacillary samples led to the development of the Xpert MTB/RIF Ultra assay (Ultra). We evaluated the performance of Ultra against Xpert on clinical samples sent to the national reference laboratory in Singapore. In total, 149 samples collected between January 2019 and November 2020 were analysed. Mycobacterium tuberculosis complex (MTBC) was isolated from 55 cultures. Using culture as the reference standard, Ultra demonstrated higher sensitivity (96.4% vs 85.5%) and marginally lower specificity (88.3% vs 89.4%) compared to Xpert in the full cohort. When considering only paucibacillary specimens such as extrapulmonary and smear-negative samples, similar results were obtained. Reclassifying Ultra trace results (low levels of MTB are detected but no rifampicin resistant result is detected) as negative in the full cohort led to a decrease in sensitivity by 10.9% and a marginal increase in specificity by 1.1%. In instances of low bacillary load, Ultra also identified rifampicin resistance more accurately than Xpert, when corroborated against other methods such as broth microdilution, line probe assay and whole genome sequencing (WGS). One isolate tested rifampicin-resistant using Xpert and Ultra, but was phenotypically susceptible and WGS demonstrated the presence of the silent mutation Thr444Thr. Ultra is more sensitive than Xpert in the detection of MTBC and rifampicin resistance in our local setting. Nevertheless, the results of molecular testing should still be correlated with phenotypic studies.

ДИАГНОСТИЧЕСКАЯ ЗНАЧИМОСТЬ МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКОГО МЕТОДА GENEXPERT MTB/RIF ДЛЯ ДИАГНОСТИКИ ТУБЕРКУЛЕЗА В СРАВНЕНИИ С ТРАДИЦИОННЫМИ МЕТОДАМИ

GeneXpert MTB/Rif assay for rapid diagnosis of tuberculosis and detection of rifampicin resistance can reduce the time to diagnosis and initiation of treatment. Aim. To evaluate the sensitivity and specificity of the GeneXpert MTB/Rif assay compared with traditional methods of detecting Mycobacterium tuberculosis (smear microscopy, culture on liquid and solid media). Material and methods. The results of sputum tests for detection of Mycobacterium tuberculosis (MBT) by bacteriological and molecular genetic methods in 1355 patients diagnosed with tuberculosis during 2016-2020 years were analyzed. Results. The informative value of the GeneXpert MTB/Rif assay was no different to the culture method (for infiltrative TB - 78.4% vs. 80.9%, p=0.138; for disseminated TB, 75.0% versus 79.0%, p=0.384) and higher compared to smear microscopy (for infiltrative TB, 78.4% versus 49.8%, р&lt;0.001; for disseminated TB, 75.0% versus 61.9%, p=009). At high accuracy (85.0%), the sensitivity of the GeneXpert MTB/Rif assay versus the culture on liquid and solid media was 89.1% and specificity – 67.9%. The GeneXpert MTB/Rif method allows for early diagnosis of TB when the clinical and radiological signs are not clear. However, due to low specificity the results of the method should be further confirmed by the classical microbiological methods of diagnostics. Conclusions. The results of this study confirm the high diagnostic value of using GeneXpert MTB/Rif in phthisiology practice, especially for newly diagnosed patients.

Utility of GeneXpert MTB/RIF in the Rapid Diagnosis of Extra Pulmonary Tuberculosis

Introduction: The diagnosis of extrapulmonary tuberculosis (EPTB) is a challenge. World Health Organization (WHO) recommends the use of GeneXpert MTB/RIF assay [Cepheid, United States of America (USA)], using a heminested real-time polymerase chain reaction (PCR) to amplify a Mycobacterium tuberculosis-specific sequence of the rpoB gene for the rapid and simultaneous detection of M. tb complex (MTBC) and resistance to rifampicin from a clinical specimen. The purpose of this study is to evaluate the performance of the GeneXpert MTB/RIF test with conventional mycobacterial culture in EPTB specimens. Materials and methods: This prospective study (February–October 2017, 11 months) includes data on 287 EPTB specimens that were processed by conventional culture on Lowenstein–Jensen (LJ) medium and the rapid molecular-based GeneXpert MTB/RIF assay system. Results: Among the 287 EPTB samples tested, GeneXpert detected the deoxyribonucleic acid of MTBC in 51 samples (17.8%). Standard bacteriological assays, including acid-fast bacilli microscopy and culture, were positive in 26 (9.1%) and 35 (12.1%) specimens respectively. The performance of GeneXpert results was evaluated against culture as a gold standard. The overall sensitivity and specificity of the Xpert assay were calculated to be 94.6 and 94.4%, respectively. The sensitivity of the Xpert assay with tissue specimens was 84.6 and 80.7% specificity, while there was 86.6% sensitivity and 98.1% specificity with the body fluids. Conclusion: GeneXpert had high performance than culture for the EPTB specimen. It can be a useful tool for the early diagnosis of patients with high clinical suspicion of EPTB. The other major advantage of GeneXpert is that it simultaneously detects rifampicin resistance within 2 hours.

Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda.

Proficiency testing (PT) has been hard to set up due to cost limitations and technical capacity. Conventional Xpert MTB/RIF PT programs use liquid and culture spots which require stringent storage and transportation conditions with cross-contamination chances prevalent. These setbacks prompted the use of dried tube specimens (DTS) for Ultra assay PT. For continuity of PT provision, stability of DTS and compatibility with testing protocols when kept for a long period needs to be established. DTS were prepared from known isolates inactivated using a hot air oven at 85°C. 100μl of bacterial suspensions were aliquoted and dried inside a Biosafety cabinet. Panel validation was done to establish the baseline Deoxyribonucleic acid (DNA) concentration in terms of cycle threshold (Ct) value. DTS aliquots were shipped to participants to test and report within six weeks. The remaining DTS were kept at 2-8°C and room temperature for one year with testing at six months. Twenty (20) DTS samples per set remaining at one year were heated at 55°C for two weeks before testing. The means of the different samples were compared to validation data using paired t-tests. Boxplots were designed to visualize the differences in the medians of the DTS. Overall mean Ct value increased by 4.4 from the validation to testing after one year at the different storage conditions. Samples heated at 55°C showed a 6.4 Ct difference from validation data. Testing done at six months on 2-8°C stored items showed no statistical difference. At all the remaining testing times and conditions, P-values were less than 0.008 although the absolute mean Ct when compared showed slight increments and accommodated differences for the detection of MTB and rifampicin resistance. Median values for samples stored at 2-8°C were lower compared to those at room temperature. DTS stored at 2-8°C remain more stable for one year compared to higher temperatures and can be consistently used as PT materials in more than one PT round for biannual PT providers.

Performance of Xpert MTB/RIF and sputum microscopy compared to sputum culture for diagnosis of tuberculosis in seven hospitals in Indonesia.

Tuberculosis (TB) is a major public health concern in Indonesia, where the incidence was 301 cases per 100,000 inhabitants in 2020 and the prevalence of multi-drug resistant (MDR) TB is increasing. Diagnostic testing approaches vary across Indonesia due to resource limitations. Acid-fast bacilli (AFB) smear is widely used, though Xpert MTB/RIF has been the preferred assay for detecting TB and rifampicin resistance since 2012 due to higher sensitivity and ability to rapidly identify rifampicin resistance. However, <1,000 Xpert instruments were available in Indonesia as of 2020 and the Xpert supply chain has suffered interruptions. We compared the performance of Xpert MTB/RIF and AFB smear to facilitate optimization of TB case identification. We analyzed baseline data from a cohort study of adults with pulmonary TB conducted at seven hospitals across Indonesia. We evaluated sensitivity and specificity of AFB smear and Xpert MTB/RIF using Mycobacterium tuberculosis (Mtb) culture as the gold standard, factors associated with assay results, and consistency of Xpert MTB/RIF with drug susceptibility test (DST) in detecting rifampicin resistance. Sensitivity of AFB smear was significantly lower than Xpert MTB/RIF (86.2 vs. 97.4%, p-value <0.001), but specificity was significantly better (86.7 vs. 73.3%, p-value <0.001). Performance varied by hospital. Positivity rate for AFB smear and Mtb culture was higher in subjects with pulmonary cavities and in morning sputum samples. Consistency of Xpert MTB/RIF with DST was lower in those with rifampicin- sensitive TB by DST. Additional evaluation using sputa from primary and secondary Indonesian health centers will increase the generalizability of the assessment of AFB smear and Xpert MTB/RIF performance, and better inform health policy. [https://clinicaltrials.gov/], identifier [NCT027 58236].

Truenat MTB assays for pulmonary tuberculosis and rifampicin resistance in adults

Objectives This is a protocol for a Cochrane Review (diagnostic). The objectives are as follows: To determine the diagnostic accuracy of Truenat assays (MTB, MTB Plus, and MTB RIF Dx) for detecting pulmonary tuberculosis and rifampicin resistance in adults with presumptive pulmonary tuberculosis. Secondary objectives To compare the diagnostic accuracy of Truenat assays (MTB, MTB Plus, and MTB RIF Dx) with that of Xpert MTB/RIF Ultra for the detection of pulmonary tuberculosis and rifampicin resistance in adults with presumptive pulmonary tuberculosis. To summarize the frequency of non‐determinate Truenat results. To determine the diagnostic accuracy of Truenat after repeat testing in people with non‐determinate test results. To investigate potential sources of heterogeneity in the diagnostic accuracy of Truenat for the detection of pulmonary tuberculosis in relation to covariates such as the setting where the index tests were performed, HIV status, previous history of tuberculosis, and sputum smear test status. To investigate heterogeneity in the diagnostic accuracy of Truenat for detecting rifampicin resistance in relation to sputum smear status.

Evaluation of the genexpert Mycobacterium tuberculosis/Rifampicin assay for early detection of extrapulmonary tuberculosis and rifampicin resistance in aligarh region of Northern India

Purpose: Early diagnosis of extrapulmonary tuberculosis (TB) with simultaneous detection of rifampicin (RIF) resistance (RR) directly from the sample is a revolution brought by GeneXpert Mycobacterium TB (MTB/RIF) assay. Line probe assay (LPA) is not recommended for extrapulmonary/nonsputum specimens; GeneXpert MTB/RIF provides a diagnostic option for these. Hence, the following study aimed to evaluate the performance of GeneXpert MTB/RIF assay in nonsputum samples. Materials and Methods: A total number of 642 extrapulmonary and nonsputum respiratory samples were subjected to Ziehl–Neelsen (ZN) staining technique, fluorescent microscopy (FM), and GeneXpert MTB/RIF Assay. All samples were cultured on a solid Lowenstein–Jensen (LJ) medium and culture-positive samples were subjected to GenoType MTBDRplus LPA and drug susceptibility testing by 1% proportion method on a solid LJ medium. Results: GeneXpert MTB/RIF assay detected MTB in 25.9% (166/642) of the samples. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ZN staining technique and FM were 21.52%, 99.35%, 91.89%, and 78.77% and 27.22%, 99.78%, 97.73%, and 80.07%, respectively. Sensitivity, specificity, PPV, and NPV of GeneXpert MTB/RIF in the detection of MTB were 93.75%, 96.53%, 90.36%, and 97.80%, and RIF RR was 92.0%, 97.74%, 88.46%, and 98.48%, respectively. LPA performed on positive culture growth showed 100% sensitivity and specificity in the detection of RIF and isoniazid resistance. Conclusion: GeneXpert MTB/RIF showed high sensitivity and specificity in detecting MTB and simultaneous RIF RR among the nonsputum samples which had a good concordance to LPA. It provides a judicious option for early detection of multidrug-resistant TB in extrapulmonary and nonsputum respiratory samples.

Comparative analysis of cartridge-based nucleic acid amplification test (CBNAAT) with conventional methods in the diagnosis of pediatric tuberculosis at a tertiary care center

BackgroundTuberculosis is an important cause of morbidity and mortality among children. Early diagnosis and treatment in children are challenging, more so in resource-limited, tuberculosis-endemic countries. In 2017, the WHO endorsed the use of CBNAAT for tuberculosis diagnosis. We have undertaken this study to evaluate the diagnostic value of CBNAAT in pediatric tuberculosis in comparison to other methods like microscopic detection of acid-fast bacilli and detection of mycobacteria-by-mycobacteria growth indicator tube (MGIT). Material and methodsThis hospital-based, cross-sectional, observational prospective study was conducted in the department of pediatrics, at a tertiary care center. A detailed history, general physical examination, and relevant physical examination were performed systematically and the findings were noted in the proforma. All necessary basic investigations like CBC, ESR, X-Ray, etc., and advanced investigations like MRI, CT, and FNAC were done as per the requirement of the subjects and the results were mentioned in the study proforma. Sensitivity, specificity, positive and negative predictive value, and diagnostic accuracy were calculated for various methods. A comparison between the two methods was done using the Mc Nemar test. p-value ≤0.05 was taken as statistically significant. All statistical analyses were done using Epi info version 7.2.1.0 statistical software. ResultsAmong 102 children suspected to be suffering from tuberculosis, the maximum number of TB cases were found in the age group of 11–16 years (43.2%), there were 58.2% of females, 58.8% belonged to the rural population, fever (78.4%) was the most common presenting symptom and 35.3% had a history of contact. In the present study, CBNAAT and ZN staining had equal sensitivity (60.8%) and specificity (100%) while the yield for MGIT culture was quite low (sensitivity 37.3%, specificity 100%). ConclusionsCBNAAT as a test was found to be useful, especially for early diagnosis and detection of rifampicin resistance in pediatric tuberculosis against MGIT culture. Since MGIT results become available only after 42 days and have a relatively lower yield so they can be utilized only in a selected clinical situation or in patients with high suspicion of tuberculosis where another test is not able to detect the organisms.

Detection of rifampicin resistance in methicillin-resistant Staphylococcus aureus isolates from the medical centers at Ahmadu Bello University

Objective: The aim of this study was to evaluate the prevalence of rifampicin resistance among methicillin-resistant Staphylococcus (S.) aureus (MRSA) isolates obtained from patients in two health facilities at Ahmadu Bello University, Zaria. Methods: A total of 200 non-duplicate presumptive Staphylococcal isolates were collected from the health facilities. The isolates were confirmed using mannitol salt agar, and biochemical tests were performed to identify S. aureus. The confirmed S. aureus isolates underwent antibiotic susceptibility tests, and the results were interpreted according to the guidelines provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Polymerase chain reaction (PCR) was used to investigate virulence and antibiotic resistance determinant, such as Spa, mecA, and rpoB. Results: This study revealed a S. aureus isolation rate of 22 (11.0%), and 18.2% of these S. aureus isolates were methicillin-resistant, with the highest prevalence of resistance (9.1%) found in wound swab. The results further showed that 75.0% of the MRSA isolates were resistant to rifampicin. The molecular analysis showed that all the isolates carried the virulence gene (Spa), with a prevalence of 10.0% for mecA and 20.0% for rpoB genes, respectively. Conclusions: This study underscores the prevalence of rifampicin resistance among MRSA isolates in medical centers, advocating for the development of alternative strategies to combat the current trends in antibiotic resistance and control of rifampicin-resistant MRSA.

Utility of laboratory diagnostic tests in women suspected of genital tuberculosis attending a tertiary care teaching hospital.

Genital Tuberculosis is a form of extrapulmonary tuberculosis, which if not diagnosed early can lead to complications. The objective of this study was to determine the sensitivity and specificity of Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay in genital tuberculosis (TB) in comparison with culture as a gold standard. The results of the Xpert MTB/RIF assay performed from January 2020 to August 2021 were compared with the results of culture by Mycobacterium Growth Indicator Tube (MGIT) 960. Out of 75 specimens, fluorescent microscopy and liquid culture using MGIT and Xpert assay were positive in 3 (4%), 21 (28%), and 14 (18%), respectively. The sensitivity and specificity of the Xpert MTB/RIF assay were 66.67% and 100%. All smear-positive specimens were positive by culture and Xpert assay. Three specimens were positive by all the tests, i.e., microscopy, culture, and Xpert assay. Fifty-four specimens were negative by microscopy, culture, and Xpert assay. Discordance between the results of culture and Xpert assay was observed in seven specimens which were culture positive and Xpert assay negative. Three (21.42%) out of 21 culture-positive specimens showed monoresistance to rifampicin by Xpert MTB/RIF assay and culture drug susceptibility testing. Xpert MTB/RIF assay showed good sensitivity and specificity compared to liquid culture in genital TB. This test is easy to perform, provides results in 2 h, and can also detect rifampicin resistance, which is a surrogate marker for multidrug-resistant TB. Hence, the Xpert assay can be used under the National TB Elimination Program for early and rapid diagnosis of TB in endometrial specimens to prevent complications like infertility.

Cartridge-based nucleic acid amplification test in drug-resistant Mycobacterium tuberculosis as compared to solid cultures: Is it time to look beyond cartridge-based nucleic acid amplification test?

Background: There is a need to study the performance, validity, and accuracy of cartridge-based nucleic acid amplification test (CB-NAAT) for accessing drug resistance among pulmonary tuberculosis (TB) compared with the solid culture drug susceptibility test (DST).Methods: Patients with symptoms of cough for more than 2 weeks with anyone symptoms such as night sweats, fever, and unintentional weight loss were studied. Cases with previously diagnosed drug-resistant pulmonary TB by sputum CB-NAAT having constitutional symptoms but not on any ATT for a minimum of 2 months were also included in the study. The patient's information, including age, immune surveillance status, clinical features, and chest X-rays, were recorded. Each sputum sample was divided into three aliquots and tested for smear microscopy, liquid culture (LC), and genotypic DST. Results of all three diagnostic modalities were compared with CB-NAAT reports.Results: Of 236 patients with sputum-positive CB-NAAT (n = 236), 49.4% (n = 117) were rifampicin resistant, while 50. 4% (n = 119) were rifampicin sensitive. The genotypic DST assays carried out of all enrolled patients showed that 76.3% (n = 181) patients were resistant to one or more first-line antitubercular drugs (FL ATTs) or second-line (SL) ATTs, while 23.7% (n = 55) patients were sensitive to all ATTs. Among all the study participants, 56.4% (n = 133) of patients had sputum smear-positive by ZN stain, while 88.6% (n = 209) showed growth on LC (BACTEC) media. On concordant analysis of CB-NAAT with DST assays, we found that among 119 CB-NAAT rifampicin-sensitive patients, 66 patients were drug-resistant (DR) TB to any of the FL or SL ATTs. The sensitivity, specificity, positive predictive value, and negative predictive value of CB-NAAT for detecting rifampicin resistance on sputum for pulmonary TB when compared with the gold-standard DST assays were 97.67%, 76.67%, 70.59%, and 98.29%, respectively.Conclusions: This study found that the use of rapid molecular technique (CB-NAAT) in screening DRTB at the community level is suboptimal compared to the gold-standard solid culture method. Although CB-NAAT's sensitivity in detecting DR pulmonary TB is significantly higher, the specificity is lower in that population who have received ATT earlier.

Study on Tuberculosis Treatment and Drug Resistance Testing in a Tertiary Care Center in Central India

National TB Elimination Program (NTEP) in India has planned for free of cost diagnosis and treatment for tuberculosis (TB) patients. This study is carried out to find out the deficiencies in treatment prescribed by practitioners, whether they were as per NTEP guidelines or not, and to assess the use of cartridge-based nucleic acid amplification test (CBNAAT) by practitioners for the detection of rifampicin resistance as per the current guidelines. Aims and objectives: This study was conducted to see the deficiencies in treatment prescribed by practitioners, whether they were as per NTEP guidelines or not, and to assess the use of CBNAAT by practitioners for the detection of rifampicin resistance as per the current guidelines. Materials and methods: The doctor’s prescription of a total of 199 patients who visited the outpatient department (OPD) were assessed, and data was collected as per inclusion and exclusion criteria. Results: Out of the total 199 prescriptions, 83.4% of patients were prescribed antitubercular treatment (ATT) under directly observed treatment short course (DOTS), while in the remaining 16.6% of patients non-DOTS ATT [57% prescribed fixed dosage combination (FDC) and 43% prescribed individual drug] were prescribed by practitioners. CBNAAT was prescribed by only 52.8% of practitioners for TB diagnosis and drug resistance. Conclusion: The method of prescribing non-DOTS ATT prescription drugs was not as per NTEP guidelines, and CBNAAT prescription was also less.

RpoB Gene Mutation Characteristics of Rifampicin-resistant Tuberculosis in Anqing, China

Background: Rifampicin resistant tuberculosis is a serious problem faced by tuberculosis control in China, and rapid detection of rifampicin resistance is urgently needed. Objectives: This study aimed to describe the molecular characteristics and frequency of RNA polymerase β subunit (rpoB) gene mutations in rifampicin-resistant tuberculosis (RR-TB) in the Anqing area. Methods: The rpoB gene fragment was amplified by polymerase chain reaction (PCR), and all isolates were sequenced for mutations in the rpoB gene. The mutations were obtained by comparing the sequencing results with the MUBII database. In addition, logistic regression was used to analyze the relationship between rpoB mutations and rifampicin (RIF) resistance. Results: There were 152 males and 42 females in this study, and the mean age was 56.60 ± 17.91 years. Mutations in the rpoB gene were a risk factor for rifampicin resistance (β = 5.271, P &lt; 0.001 OR = 195.192). Among the 19 RR-TB strains, 16 (84.21%) had mutations in the ropB gene, and three (1.71%) of 175 rifampicin-sensitive strains were mutated. The mutation sites of five strains (31.58%) were at the codon 526 and five strains (31.58%) at the codon 531. However, there were two strains at the codon 513 and two strains at the codon 533 (15.79%), and two strains (10.53%) were double mutations. Conclusions: The mutation characteristics of the rpoB gene in the Anqing area are complex, and rpoB mutation detection can be used as an indicator to screen drug resistance of RIF.

Accuracy of the InnowaveDX MTB/RIF test for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre study

ABSTRACT Early and accurate diagnosis of tuberculosis (TB) is necessary to initiate proper therapy for the benefit of the patients and to prevent disease transmission in the community. In this study, we developed the InnowaveDX MTB/RIF (InnowaveDX) to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance simultaneously. A prospective multicentre study was conducted to evaluate the diagnostic performance of InnowaveDX for the detection MTB in sputum samples as compared with Xpert and culture. The calculated limit of detection (LOD) for InnowaveDX was 9.6 CFU/ml for TB detection and 374.9 CFU/ml for RIF susceptibility. None of the other bacteria tested produced signals that fulfilled the positive TB criteria, demonstrating a species-specificity of InnowaveDX. Then 951 individuals were enrolled at 7 hospitals, of which 607 were definite TB cases with positive culture and/or Xpert results, including 354 smear-positive and 253 smear-negative cases. InnowaveDX sensitivity was 92.7% versus bacteriologically TB standard. Further follow-up revealed that 61 (91.0%) out of 67 false-positive patients with no bacteriological evidence met the criteria of clinically diagnosed TB. Among 125 RIF-resistant TB patients diagnosed by Xpert, 108 cases were correctly identified by InnowaveDX, yielding a sensitivity of 86.4%. Additionally, the proportion of very low bacterial load in the discordant susceptibility group was significantly higher than in the concordant susceptibility group (P = 0.029). To conclude, we have developed a novel molecular diagnostic with promising detection capabilities of TB and RIF susceptibility. In addition, the discordant RIF susceptibility results between InnowaveDX and Xpert are more frequently observed in samples with very low bacterial load.

Progress on diagnosis and treatment of drug-resistant tuberculosis in line with World Health Organization recommendations in six priority countries in the Western Pacific Region.

Diagnosis and treatment of drug-resistant tuberculosis (DR-TB) have radically changed in accordance with recommendations from the World Health Organization (WHO) in the past decade, allowing rapid and simple diagnosis and shorter treatment duration with new and repurposed drugs. A descriptive analysis of the status and progress of DR-TB diagnosis and treatment in six priority countries in the Western Pacific Region was conducted using information from interviews with countries and the WHO TB database. Over the past decade, the use of Xpert MTB/RIF has increased in the six priority countries, in parallel with implementation of national policies and algorithms to use Xpert MTB/RIF as an initial diagnostic test for TB and detection of rifampicin resistance. This has resulted in increases in the number of people diagnosed with multidrug-resistant or rifampicin-resistant TB (MDR/RR-TB). Shorter treatment regimens with new and repurposed drugs have also been adopted for MDR/RR-TB cases, alongside a decentralized model of care, leading to improved treatment outcomes. The Western Pacific Region has achieved considerable progress in the diagnosis and treatment of DR-TB, in line with the evolving WHO recommendations in the past decade. The continued commitment of Member States is needed to address remaining challenges, such as the impact of the coronavirus disease pandemic, suboptimal management and health system issues.

Evaluation of Xpert MTB/RIF Assay, MTB Culture and Line Probe Assay for the Detection of MDR Tuberculosis in AFB Smear Negative Specimens.

The global burden of tuberculosis (TB), particularly with multidrug resistance (MDR), is escalating and has become a major health challenge. It is well known that acid-fast bacilli (AFB) smear-negative TB patients are the major source of spreading TB to healthy individuals when left untreated. Early diagnosis of TB and rapid detection of drug resistance are important for the proper management of drug-resistant TB (DR-TB). Therefore, a laboratory based cross-sectional study was conducted from July to December 2019 at the National Tuberculosis Centre, Thimi, Nepal, with the objective of evaluating the diagnostic performance of Xpert MTB/RIF assay, Mycobacterium tuberculosis (MTB) culture and line probe assay (LPA) for the detection of MDR-TB in AFB smear-negative sputum samples. We evaluated a total of 222 AFB smear-negative sputum specimens, of which 21.6% (n = 48) showed MTB positive with Xpert MTB/RIF assay and, while culturing on Lowenstein-Jensen (LJ) media, 21.2% (n = 47) were MTB culture positive. The sensitivity, specificity, PPV and NPV at 95% confidence interval of Xpert MTB/RIF assay on diagnosing M. tuberculosis from smear-negative specimens were 73% (57-84), 92% (87-96), 71% (59-81) and 93% (89-95), respectively. In addition, the sensitivity of Xpert MTB/RIF assay and LPA in detecting rifampicin resistance was 75% (42-94, 95% CI) and 91.67% (62-99, 95% CI), respectively. The current study also assessed a significant association between the occurrence of pulmonary tuberculosis with different age group, TB history and alcohol consumption. These findings indicate that Xpert MTB/RIF assay and LPA are appropriate methods for early detection and accurate diagnosis of TB and RIF mono-resistant cases.