Detection Of Mycobacterium Tuberculosis Research Articles

Raman-enhanced sensor based on CRISPR-SERS technology for the rapid and hypersensitive detection of Mycobacterium tuberculosis.

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 0.9914. The limit of detection was as low as 4.42pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.

Evaluation of testing face-mask filter samples with LAMP shows high rates of detection in pulmonary TB.

<sec><title>BACKGROUND</title>Detection of Mycobacterium tuberculosis (MTB) in bioaerosols derived from patients with active pulmonary TB is a potential alternative diagnostic method for patients with presumed TB who cannot expectorate sputum.</sec><sec><title>OBJECTIVE</title>To assess the efficacy of a bioaerosol particle collection method to capture MTB and diagnose TB.</sec><sec><title>METHODS</title>A mask-like filter holder (3D mask) with a water-soluble gelatine filter (GF) and one containing a water-insoluble polypropylene filter (PPF) were prepared. Eligible patients wore the 3D mask with GF or PPF within 3 days of starting anti-TB drugs. The GF and PPF filters were collected after 2 and 8 h. DNA was extracted from the filter samples and tested using loop-mediated isothermal amplification (LAMP).</sec><sec><title>RESULTS</title>Filter samples were collected from 57 and 20 patients with and without active pulmonary TB, respectively. The GF and PPF sensitivity was 76.2% and 83.3%, respectively. The specificity of both methods was 100%. Of the 57 patients diagnosed with non-expectorated sputum samples, including suction phlegm, gastric lavage, and bronchial lavage fluid, 55.6% and 50.0% were positive by GF and PPF, respectively.</sec><sec><title>CONCLUSION</title>We present a 3D mask filter sampling method for exhaled bioaerosol particles that can be used in clinical practice to diagnose patients with presumed TB.</sec>.

Quantum Dot-Based Nanosensors for In Vitro Detection of Mycobacterium tuberculosis

Despite the existing effective treatment methods, tuberculosis (TB) is the second most deadly infectious disease, its carriers in the latent and active phases accounting for more than 20% of the world population. An effective method for controlling TB and reducing TB mortality is regular population screening aimed at diagnosing the latent form of TB and taking preventive and curative measures. Numerous methods allow diagnosing TB by directly detecting Mycobacterium tuberculosis (M.tb) biomarkers, including M.tb DNA, proteins, and specific metabolites or antibodies produced by the host immune system in response to M.tb. PCR, ELISA, immunofluorescence and immunochemical analyses, flow cytometry, and other methods allow the detection of M.tb biomarkers or the host immune response to M.tb by recording the optical signal from fluorescent or colorimetric dyes that are components of the diagnostic systems. Current research in biosensors is aimed at increasing the sensitivity of detection, a promising approach being the use of fluorescent quantum dots as brighter and more photostable optical tags. Here, we review current methods for the detection of M.tb biomarkers using quantum dot-based nanosensors and summarize data on the M.tb biomarkers whose detection can be made considerably more sensitive by using these sensors.

Establishment of cross-priming amplification for point-of-care detection of Mycobacterium tuberculosis and non-tuberculosis mycobacteria

ObjectiveTo simplify sputum sample preparation steps and achieve point-of-care testing (POCT) of Mycobacterium tuberculosis (MTB) and non-tuberculosis mycobacteria (NTM) using Cross-Priming Amplification (CPA) technology on portable devices, overcoming the challenges of existing nucleic acid detection technologies that cannot be widely promoted in grassroots settings in China. MethodsEvaluate the liquefying ability of high-concentration guanidine thiocyanate (GTC) for sputum and the effectiveness of MTB inactivation; establish a rapid detection system for MTB and NTM based on CPA technology using EasyNAT integrated detection tubes, with the left amplification zone specific to MTB CPA amplification and the right amplification zone specific to both MTB and NTM CPA amplification. Suspected pulmonary tuberculosis (PTB) patients or patients diagnosed as suspected NTM pulmonary infections specimens collected from the Second Hospital of Longyan, Fujian Province, from September 2022 to September 2023, acid-fast bacilli (AFB) smear, quantitative real-time PCR (RT-PCR) and CPA-POCT were performed. The kappa coefficients was used to evaluate the consistency between the RT-PCR and CPA-POCT. ResultsThe liquefaction effect of 6M GTC on sputum was equivalent to 4 % NaOH, and no MTB growth was observed in the Lowenstein-Jensen medium of sputum samples treated with 6M GTC incorporating the H37Rv strain. The newly established CPA-POCT method showed good agreement with RT-PCR with a positive compliance rate of 86.27 %, a negative compliance rate of 89.36 %, an overall compliance rate of 87.75 %, and a Kappa coefficients of 0.786 (P < 0.05). Conclusion6M GTC can liquefy sputum and render MTB non-viable, eliminating the need for Mycobacterium nucleic acid testing in BSL-2 laboratories; the newly established CPA method can rapidly and accurately distinguish MTB and NTM in the form of POCT, with simple and fast operation, suitable for promotion and application in grassroots medical institutions and remote rural areas.

Evaluation of the STANDARD M10 MDR-TB and MTB/NTM assays for the detection of Mycobacterium tuberculosis , rifampicin and isoniazid resistance, and nontuberculous mycobacteria in a low-incidence setting

ABSTRACT Rapid detection is crucial for tuberculosis (TB) control. GeneXpert (Cepheid) is a widely used PCR system, known for its simplicity, random access, and point-of-care compatibility. SD BIOSENSOR recently introduced a similar system, STANDARD M10, including a Mycobacterium tuberculosis (MTB) and rifampicin (RIF) and isoniazid resistance (herein, MDR-TB) assay and an MTB/nontuberculous mycobacteria (NTM) assay. We evaluated these assays for the potential to replace the established Xpert MTB/RIF Ultra assay in a low-TB incidence setting. We analyzed 160 clinical respiratory samples (45 MTB-positive and 35 NTM-positive) and further 24 drug-resistant MTB, 30 mycobacterial species (2 MTB, 28 NTM), and 37 non-mycobacterial isolates. Compared with culture, clinical sensitivities and specificities for MTB detection were 88.9% (95% confidence interval [CI] = 76.1–95.6%) and 97.4% (CI = 92.3–99.4%) with Xpert Ultra, 88.9% (95% CI = 76.1–95.6%) and 98.3% (CI = 93.5–99.9%) with M10 MDR-TB, and 84.4% (CI = 70.9–94.4%) and 98.3% (CI = 93.5–99.9%) with M10 MTB/NTM, respectively. For NTM detection, M10 MTB/NTM showed sensitivity and specificity of 65.7% (CI = 49.1–79.2%) and 96.8% (CI = 91.8–99.0%). Compared with phenotypic drug susceptibility testing (DST), sensitivity and specificity for detecting RIF resistance were 100% (CI = 77.3–100%) and 95.6% (CI = 84.4–99.6%) with Xpert Ultra, and 100% (CI = 74.9–100%) and 95.5% (CI = 84.0–99.6%) with M10 MDR-TB. M10 MDR-TB showed 92.3% sensitivity (CI = 74.7–99.0%) and 100% specificity (CI = 87.3–100%) for detecting isoniazid resistance. All discrepancies in DST by PCR were concordant with whole-genome sequencing. While M10 MDR-TB demonstrated great potential as an alternative to Xpert Ultra, M10 MTB/NTM had limitations in NTM screening. Additionally, the M10 sputum pretreatment did not inactivate MTB efficiently, which should be considered in process risk assessment. IMPORTANCE The molecular diagnostic STANDARD M10 system is highly analogous to the widely established GeneXpert system, which significantly increases the relevance of this evaluation study in the field of rapid detection of M. tuberculosis . To our knowledge, this is the first clinical evaluation describing the performance of the STANDARD M10 MDR-TB and MTB/NTM assays, including an extensive analytical specificity panel (inclusivity and exclusivity) for the detection of M. tuberculosis , drug resistance, and nontuberculous mycobacteria.

Lite-YOLOv8: a more lightweight algorithm for Tubercle Bacilli detection.

Deep learning is a transformative force in the medical field and it has made significant progress as a pivotal alternative to conventional manual testing methods. Detection of Tubercle Bacilli in sputum samples is faced with the problems of complex backgrounds, tiny and numerous objects, and human observation over a long time not only causes eye fatigue, but also greatly increases the error rate of subjective judgement. To solve these problems, we optimize YOLOv8s model and propose a new detection algorithm, Lite-YOLOv8. Firstly, the Lite-C2f module is used to ensure accuracy by significantly reducing the number of parameters. Secondly, a lightweight down-sampling module is introduced to reduce the common feature information loss. Finally, the NWD loss is utilized to mitigate the impact of small object positional bias on the IoU. On the public Tubercle Bacilli datasets, the mean average precision of 86.3% was achieved, with an improvement of 2.2%, 1.5%, and 2.8% over the baseline model (YOLOv8s) in terms of mAP0.5, precision, and recall, respectively. In addition, the parameters reduced from 11.2 to 5.1 M, and the number of GFLOPs from 28.8 to 13.8. Our model is not only more lightweight, but also more accurate, thus it can be easily deployed on computing-poor medical devices to provide greater convenience to doctors.

Disseminated tuberculosis with osteomyelitis of the right little finger as the first manifestation: a case report

The clinical data of a child with disseminated tuberculosis with osteomyelitis of the right little finger as the first manifestation who was admitted to Tianjin Children's Hospital on April 8, 2023 were retrospectively analyzed. The child, a 14-year-old female, presented with osteomyelitis of the right little finger as the first manifestation. She still had recurrent fever after focal incision and drainage. She was referred to our hospital. The samples from multiple sites were positive for molecular biology detection of Mycobacterium tuberculosis. She was considered as disseminated tuberculosis and was given anti-tuberculosis treatment. The child has recovered well. Pediatric disseminated tuberculosis has variable clinical manifestations and lacks specificity. It is often misdiagnosed and has a high mortality rate. Clinicians should improve their understanding of the disease and ensure early diagnosis and treatment.

Case Report : Brucella infection of total knee arthroplasty - The first case report in an endemic country!

Prosthetic joint loosening due to Brucella spp. is a rare event. In this report, we describe a rare case of prosthetic infection caused by brucella. A 71-year-old woman reported a six-month history of progressing pain in the right prosthetic knee, which had been implanted for arthrosis four years prior to admission, associated with fever. No night sweats were noted. On examination, the right knee appeared slightly swollen and painful on mobilization, and the surgical wound was normal. Blood tests showed increased C-Reactive Protein (CRP) (34mg/L) and erythrocyte sedimentation rate (ESR) (34mm/hr). A CT scan of her right knee was performed, showing loosening of the prosthesis, joint effusion, and osteolysis in contact with the internal tibial component measuring 16mm maximum with bone remodeling of the epiphysis and metaphysis regions. Rose Bengal test was positive for brucellosis, a standard brucella tube agglutination test was positive at a titer of 1/80, blood cultures for brucella were negative and a PCR for detection of Mycobacterium tuberculosis was negative. The patient was diagnosed as having Brucella infection of his total knee arthroplasty. Favorable outcome was observed after medical and surgical treatment. We believe this is the first detailed report of Brucella prosthetic infection in the country despite the endemicity of this type of zoonosis.

Evaluation of Newly Modified Potassium Nitrate Containing Media in Detection and Antibiogram of Mycobacterium tuberculosis

Background: Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and the classic Hansen's strain of leprosy (Mycobacterium leprae) [1]. Tuberculosis is the most common life-threatening opportunistic infection (OI) and AIDS defining illness in developing countries [2]. The routine identification methods of tuberculosis includes, Tuberculin skin test, Chest X-ray, Growth and physical characteristics of the bacteria, automated based system such as BACTEC 460 TB method, Mycobacterium growth indicator tube (MGIT) method, MB/BacT system, as well as molecular based techniques as in Amplification of nucleic acid for detection of Mycobacterium tuberculosis [3-6]. Objectives: To evaluate the validity of newly modified potassium nitrate containing medium (NMK) for rapid detection and antibiogram of M tuberculosis. Material and method: This was a health facility based observational diagnostics retrospective study. 125 sputum specimens were investigated to evaluate the validity of newly modified potassium nitrate containing medium for rapid detection and antibiogram of M tuberculosis. Results: 83/125 (66.4%) showed growth after 5-9 days with a mean of 6.58± 0.977 days NMK - containing medium. Conclusions: The NMK - containing medium showed promising results, it showed a positive growth sings and susceptibility results within 5-9 days of inoculation showing much superior than the golden standard LJ medium.

Research on improved YOLOv8s model for detecting mycobacterium tuberculosis

Accurate identification of Mycobacterium tuberculosis (M. tuberculosis) is a critical step in the diagnosis of tuberculosis. Existing object detection methods struggle with the challenges posed by the varied morphology and size of M. tuberculosis in sputum smear images, which makes precise targeting difficult. To solve these problems, an improved YOLOv8s model is proposed. Specifically, an additional detection head is added to focus on small target information. Second, a multi-scale feature fusion module is introduced to adapt the model to different sizes of M. tuberculosis. In addition, a convolutional layer is added to the Coordinate Attention (CA) module to extract more advanced semantic features. Finally, a self-attention mechanism is added after the CA module to enhance the model's ability to accurately understand and localize the varied morphology of M. tuberculosis. Our model performed well with an average precision of 85.7 % when tested on a publicly available dataset. This clearly demonstrates the effectiveness of our proposed model in M. tuberculosis detection.

Mycobacterium tuberculosis Detection Using CRISPR Technology: An Updated Systematic Review and Meta-analysis.

Rapid and precise detection of Mycobacterium tuberculosis (MTB) is paramount for effective management and control of tuberculosis. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB. A meticulous search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Quality was assessed using the Joanna Briggs Institute checklist. Data synthesis and analyses, including subgroup analyses, were performed with R v 4.3.1, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods. From 389 identified studies, 14 met the inclusion criteria, encompassing 2175 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.93 (95% CI 0.85-0.99) and 0.97 (95% CI 0.94-0.99), respectively. The pooled diagnostic odds ratio was 273.4379 (95% CI 103.3311-723.5794), with an area under the curve of 0.97 for the summary receiver operating characteristic (SROC) curve, denoting excellent diagnostic accuracy. Subgroup analyses illustrated variations in diagnostic metrics based on factors like CRISPR variant utilized, target gene, and pre-amplification methods. For instance, CRISPR-Cas12 exhibited a sensitivity and specificity of 0.93 (95% CI 0.78-0.98) and 0.98 (95% CI 0.93-1), respectively. Moreover, this technology showed a sensitivity of 96% and specificity of 100% in detecting resistant MTB. CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB, with notable variances across different CRISPR variants and methodological approaches. Further studies must be conducted to optimize CRISPR's potential as a diagnostic tool for MTB in a variety of clinical and research settings.

Extended clinical sample incubation in the cepheid Xpert MTB/XDR test sample reagent: Enhancing flexibility and workflows in high-volume laboratories

Cepheid Xpert MTB/RIF ULTRA (ULTRA) and Xpert MTB/XDR are tests for detecting Mycobacterium tuberculosis (MTB) and drug resistance. Both tests involve a sample pre-processing step using the test's sample reagent (SR). The manufacturer recommends a four-hour limit for SR-treated samples prior to testing, posing challenges for high-volume laboratories conducting both tests. Implementing the XDR test as a follow-on to ULTRA positive specimen can be challenging in high-volume laboratories due to the time constraints imposed by the manufacturer's recommendations. To address this issue, this study investigated the impact of extended sample incubation in SR for durations longer than four hours at varying temperature conditions. Pre-characterized MTB isolates with diverse drug susceptibility profiles were incubated up to 36 hours at different temperatures including room temperature (RT), 2-8°C, and -20°C and tested using Xpert MTB/XDR. The study results indicate no adverse effects on sample stability or drug susceptibility detection. This suggests extended incubation could offer flexibility for conducting both tests on a single specimen, benefiting high-throughput laboratories.

Operational feasibility and multi-centric evaluation of ‘TBDetect sputum microscopy kit’ for the direct detection of Mycobacterium tuberculosis in field settings

Background India relies primarily on direct smear microscopy for tuberculosis (TB) diagnosis. However, the low sensitivity of smear microscopy emphasizes the need to improve its performance. We recently described the development of ‘TBDetect’ kit which showed improved performance over direct smear microscopy at National Reference Laboratories (NRLs) in India. Methods The present study was aimed to assess the operational feasibility of ‘TBDetect’ microscopy in field settings. This was evaluated by (i) assessing the performance of ‘TBDetect’ microscopy vs. LED-fluorescence microscopy (LED-FM) on consecutive presumptive pulmonary TB patients (n = 5300) who attended Designated Microscopy Centres (DMCs, n = 13) under 4 NRLs at Bhubaneswar, Bhopal, Chennai, and New Delhi, and (ii) obtaining feedback from Scientists (n = 10) and laboratory technicians (n = 42) using semi-structured questionnaires under the following parameters: feasibility of initiation of ‘TBDetect’ microscopy in DMCs, sample preparation and testing, training, time-to-result, logistics, and troubleshooting. A scoring questionnaire was also used to assess ‘TBDetect’ microscopy vs. LED-FM and statistical significance of the scores was calculated using paired t-test. Results The overall positivity of ‘TBDetect’ microscopy was 10.32% (547/5300) vs. 8.96% (475/5300) of LED-FM at all sites and the increment in positivity was significant (p = 0.019). In addition, ‘TBDetect’ microscopy yielded an increment in smear grade status over LED-FM (p = 0.043). The feedback from the study-in-charge and kit users indicated that ‘TBDetect’ microscopy was easily adapted in point-of-care settings. An analysis of scoring feedback suggested that it was easy to perform and observe in comparison to LED-FM (p < 0.005). Conclusions This study established the feasibility of ‘TBDetect’ microscopy in field settings.

Performance evaluation of the Xpert MTB/XDR test for the detection of drug resistance to Mycobacterium tuberculosis among people diagnosed with tuberculosis in South Africa.

Drug-resistant tuberculosis (TB) poses a significant public health concern in South Africa due to its complexity in diagnosis, treatment, and management. This study assessed the diagnostic performance of the Xpert MTB/XDR test for detecting drug resistance in patients with TB by using archived sputum sediments. This study analyzed 322 samples collected from patients diagnosed with TB between 2016 and 2019 across South Africa, previously characterized by phenotypic and genotypic methods. The Xpert MTB/XDR test was evaluated for its ability to detect resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolones (FLQ), and second-line injectable drugs (SLIDs) compared with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS). Culture, Xpert MTB/RIF Ultra, and Xpert MTB/RIF (G4) tests were performed to determine sensitivity and agreement with this test for TB detection. The sensitivities using a composite reference standard, pDST, and sequencing were >90% for INH, FLQ, amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) resistance, meeting the WHO target product profile criteria for this class. A lower sensitivity of 65.9% (95% CI: 57.1-73.6) for ETH resistance was observed. The Xpert MTB/XDR showed a sensitivity of 98.3% (95% CI: 96.1-99.3) and specificity of 100% (95% CI: 86.7-100) compared with culture, a positive percent agreement (PPA) of 98.8% (95% CI: 93.7-99.8) and negative percent agreement (NPA) of 100.0% (95% CI: 78.5-100.0) compared with G4, and a PPA of 99.5% (95% CI: 97.3-99.9) and NPA of 100.0% (95% CI: 78.5-100.0) compared with Xpert MTB/RIF Ultra for detecting Mycobacterium tuberculosis. The test offers a promising solution for the rapid detection of drug-resistant TB and could significantly enhance control efforts in this setting.

68Ga Radiolabeling of NODASA-Functionalized Phage Display-Derived Peptides for Prospective Assessment as Tuberculosis-Specific PET Radiotracers.

This research presents the development of positron emission tomography (PET) radiotracers for detecting Mycobacterium tuberculosis (MTB) for the diagnosis and monitoring of tuberculosis. Two phage display-derived peptides with proven selective binding to MTB were identified for development into PET radiopharmaceuticals: H8 (linear peptide) and PH1 (cyclic peptide). We sought to functionalize H8/PH1 with NODASA, a bifunctional chelator that allows complexation of PET-compatible radiometals such as gallium-68. Herein, we report on the chelator functionalization, optimized radiosynthesis, and assessment of the radiopharmaceutical properties of [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1. Robust radiolabeling was achieved using the established routine method, indicating consistent production of a radiochemically pure product (RCP ≥ 99.6%). For respective [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1, relatively high levels of decay-corrected radiochemical yield (91.2% ± 2.3%, 86.7% ± 4.0%) and apparent molar activity (Am, 3.9 ± 0.8 and 34.0 ± 5.3 GBq/μmol) were reliably achieved within 42 min, suitable for imaging purposes. Notably, [68Ga]Ga-NODASA-PH1 remained stable in blood plasma for up to 2 h, while [68Ga]Ga-NODASA-H8 degraded within 30 min. For both 68Ga peptides, minimal whole-blood cell binding and plasma protein binding were observed, indicating a favorable pharmaceutical behavior. [68Ga]Ga-NODASA-PH1 is a promising candidate for further invitro/in vivo evaluation as a tuberculosis-specific infection imaging agent.

Analysis of unsuccessful tests and the effect of prolonged clinical sample preprocessing in the GeneXpert MTB/RIF assay

BackgroundThe GeneXpert MTB/RIF (Xpert) assay is a widely used technology for detecting Mycobacterium tuberculosis (MTB) in clinical samples. However, the study on the failure of the Xpert assay during routine implementation and its potential solutions is limited.MethodsWe retrospectively analyzed the records of unsuccessful tests in the Xpert and the GeneXpert MTB/RIF Ultra (Ultra) assays between April 2017 and April 2021 at the Shanghai Public Health Clinical Center. To further investigate the effect of prolonged preprocessing on clinical sputum, an additional 120 sputum samples were collected for Xpert testing after 15 min, 3 h, and 6 h preprocessing. The analysis was performed by SPSS version 19.0 software.ResultsA total of 11,314 test records were analyzed, of which 268 (2.37%) had unsuccessful test results. Among these, 221 (1.95%) were reported as “Error”, 43 (0.38%) as “Invalid”, and 4 (0.04%) as “No result”. The most common clinical specimen for Xpert tests was sputum, accounting for 114 (2.17%) unsuccessful tests. The failure rate of urine specimens was lower than that of sputum (OR = 0.12, 95% CI: 0.02–0.88, χ2 = 6.22, p = 0.021). In contrast, the failure rate of stool specimens was approximately twice as high as that of sputum (OR = 1.93, 95% CI: 1.09–3.40, χ2 = 5.35, p = 0.014). In the prolonged preprocessing experiment, 102 cases (85%) yielded consistent results in Xpert tests. Furthermore, 7 cases (5.83%) detected an increase in MTB load, 8 cases (6.67%) detected a decrease in MTB load, and 3 cases (2.5%) yielded incongruent results in MTB and rifampicin resistance detection.ConclusionsThe primary cause of unsuccessful tests in the Xpert assay was reported as “Error”. Despite varying failure rates depending on the samples, the Xpert assay can be applied to extrapulmonary samples. For paucibacillary specimens, retesting the remaining preprocessed mixture should be carefully considered.

Evaluation of Diagnostic Methods and Rifampicin Resistance in Pulmonary Tuberculosis: A Hospital-Based Study.

Background Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, predominantly affecting the lungs (pulmonary TB) and is a significant public health challenge in India. The study aims to analyze demographic, radiological, and clinical subgroups of pulmonary TB cases, examine the relationship between smear acid-fast bacillus (AFB examination) and cartridge-based nucleic acid amplification test (CBNAAT), evaluate CBNAAT sensitivity for Mycobacterium tuberculosis (MTB) in new and previously treated patients, and determine the proportion of rifampicin resistance. Methods This hospital-based prospective study was conducted among patients diagnosed with pulmonary TB at the Respiratory Medicine Department of a Government Hospital over 16 months (August 2019 to December 2020). The study included 150 diagnosed TB cases (new and previously treated). Data collection encompassed demographic details, clinical symptoms, comorbidities, radiological findings (chest X-ray), and microbiological results (smear AFB examination, CBNAAT). Sputum samples were subjected to Ziehl-Neelsen staining and CBNAAT for MTB detection and rifampicin resistance testing. Statistical analysis was performed using IBM SPSS Statistics version 21.0 (IBM Corp., Armonk, NY, USA). Results Of the 150 patients, 69.3% were male, and 48% were aged 21-40 years. The majority had a BMI of 18.5-24.9 kg/m² (50%) and resided in urban areas (63.3%). Common symptoms included cough (95.3%), fever (80%), and weight loss (74%). Cavitary lesions on chest X-ray were observed in 84% of patients. Smear microscopy detected MTB in 72.7% of cases, while CBNAAT detected MTB in 94% of cases. CBNAAT sensitivity for smear-positive and smear-negative samples was 93.97% and 94.12%, respectively. Rifampicin resistance was found in 3% of new cases and 6% of previously treated cases. The sensitivity of smear microscopy was 77.33%, and the sensitivity of CBNAAT was 94%. Conclusion The study underscores the high burden of pulmonary TB and the utility of CBNAAT in detecting MTB and rifampicin resistance, particularly in smear-negative samples. The findings highlight the necessity of universal drug susceptibility testing (DST) for effective TB management and the importance of addressing drug resistance to improve treatment outcomes.

Reflex Xpert MTB/XDR Testing of Residual Rifampicin-Resistant Specimens: A Clinical Laboratory-Based Diagnostic Accuracy and Feasibility Study in South Africa.

The World Health Organization-approved Xpert MTB/XDR test detects Mycobacterium tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and injectable drugs directly in specimens. This pragmatic, laboratory-based study assessed the diagnostic accuracy and feasibility of a reflex testing approach, where Xpert MTB/XDR was performed on residual specimens previously processed for Xpert MTB/RIF Ultra. Routine respiratory specimens, processed for Xpert MTB/RIF Ultra, were stored in sample reagent buffer at 2°C-8°C. If rifampicin resistant, the residual specimen was assessed for adequate volume (≥2 mL) and tested with Xpert MTB/XDR, with storage time recorded. A second specimen was used for routine and reference standard testing (culture and sequencing). Specimens (99% sputum) from 763 participants submitted to 2 large routine laboratories were included. Xpert MTB/XDR yielded valid resistance detection results in 639 (84%), compared with 507 (66%) for routine testing (difference [95% CI], 18% [13%-22%]). The median turnaround time for results was 23 hours for Xpert MTB/XDR and 15 days for routine testing. While 748 specimens (98%) were ≥2 mL, only 102 (13%) were stored for ≤4 hours. By the reference standard, 284 of 394 (72%) were isoniazid resistant, and 57 of 380 (15%) were fluroquinolone resistant. The sensitivities of Xpert MTB/XDR were 94% (95% CI, 91%-97%) for isoniazid and 91% (81%-97%) for fluoroquinolone resistance detection. The specificities were 98% (94%-100%) and 100% (98%-100%), respectively. Xpert MTB/XDR performed favorably compared with the reference, and the reflex testing approach increased results availability over routine testing, while dramatically decreasing turnaround time from weeks to hours. Laboratory workflow precluded testing within the manufacturer-recommended 4-hour storage time, but longer storage did not appear detrimental.

Insights into mycobacteriome composition in Mycobacterium bovis-infected African buffalo (Syncerus caffer) tissue samples

Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures are resource-intensive, time-consuming, and challenged by heterogeneous populations. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in 60 DNA samples extracted from Mycobacterium bovis infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting Mycobacterium tuberculosis complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. In five samples, we identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the Mycobacterium avium complex (MAC), M. smegmatis, and M. komaniense. The latter Mycobacterium species was described in South Africa from bovine nasal swabs and environmental samples from the Hluhluwe-iMfolozi Park, which was the origin of the buffalo samples in the present study. This finding suggests that exposure to environmental mycobacteria may confound detection of MTBC in wildlife. In conclusion, our approach represents a promising alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables rapid differentiation of heterogeneous mycobacterial populations, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.

Role of GeneXpert in the diagnosis of extrapulmonary tuberculosis.

The World Health Organization endorsed the cartridge-based nucleic acid amplification test Xpert MTB/RIF (GXP) for the diagnosis of tuberculosis (TB). Studies about GXP efficiency in extrapulmonary TB (EPTB) are scarce. Hence, we decided to study the role of GXP in EPTB. This prospective observational study, conducted in the pulmonary medicine department of a tertiary care hospital after ethics committee permission, recruited 200 EPTB patients. The diagnosis of TB was achieved with the help of clinico-radiological correlation with microbiological test positivity. Acid-fast bacilli (AFB) culture was treated as the comparative gold standard. Patients who had no or incomplete data were excluded from the study. Data was analyzed to calculate the sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of TB and the detection of rifampicin resistance. The majority of cases were women (126 patients: 63%). The mean age was 23.71 years. On GXP, 130 (65%) had detected mycobacterium tuberculosis (MTB), and 70 (35%) did not. Adding AFB culture data, 168 (81.5%) showed microbiological evidence of TB, and 32 (18.5%) were negative. On the drug susceptibility test, 131 cases were rifampicin-sensitive, 32 were rifampicin-resistant TB, and in 5 cases, data was unavailable. The most common extrapulmonary site of involvement was the lymph node, with 94 patients (47%). The most common lymph node involved was the cervical lymph node, with 70 patients (74.5%). The sensitivity, specificity, positive predictive value, and negative predictive value of GXP in EPTB collectively were 76.68%, 86.48%, 96%, and 45.7%, respectively. GXP is useful for the rapid detection of EPTB and the identification of rifampicin resistance, especially in a high-prevalence country like India.