Detection Of Mycobacterium Tuberculosis Research Articles

Sequential testing with Xpert MTB/RIF assay for diagnosis of tuberculous meningitis in Maharaj Nakorn Chiang Mai University Hospital

Early diagnosis and appropriate treatment are essential for reducing morbidity and mortality in tuberculous meningitis (TBM). This study aimed to evaluate the diagnostic performance of the Xpert MTB/RIF assay for the diagnosis of TBM in patients with subacute lymphocytic meningitis. This cross-sectional study included 65 cerebrospinal fluid (CSF) specimens from patients at Maharaj Nakorn Chiang Mai University Hospital, Thailand, between January 2015 and March 2016. Mycobacteria growth indicator tube (MGIT) culture was used as the reference standard. Sensitivity, specificity, and agreement between Xpert MTB/RIF and MGIT culture were calculated. Sequential testing using a TBM score, followed by Xpert MTB/RIF was also analyzed. Xpert MTB/RIF demonstrated 83.33% sensitivity (95% CI 57.19–98.22) and 96.23% specificity (95% CI 87.02–99.54). Agreement between Xpert MTB/RIF and MGIT culture was 93.85% (p < 0.001), with a kappa score of 0.80 (95% CI 0.60–0.99). Sequential testing with a TBM score cut-off of 6, followed by Xpert MTB/RIF improved specificity from 96.23 to 97.15%. The Xpert MTB/RIF assay is a rapid and valuable tool for detecting Mycobacterium tuberculosis in centrifuged CSF specimens. A diagnostic algorithm incorporating the TBM score enhances performance, balancing sensitivity and specificity, and could improve patient outcomes in resource-limited settings.

Optimal processing of tongue swab samples for Mycobacterium tuberculosis detection by the Xpert MTB/RIF Ultra assay.

Tongue swabs represent a potential alternative to sputum as a sample type for detecting pulmonary tuberculosis (TB) with molecular diagnostic tests. The methods used to process tongue swabs for testing in the World Health Organization-recommended Xpert MTB/RIF Ultra (Xpert Ultra) assay vary greatly. Here, we aimed to identify the optimal tongue swab processing for Xpert Ultra testing. We compared four methods for treating dry tongue swabs with the Xpert Sample Reagent (SR) mixed with various concentrations of Tris-EDTA-Tween to treatment with SR alone or to a commonly used heat inactivation protocol. In each condition, swabs obtained from volunteers without TB were placed into test buffer spiked with known amounts of Mycobacterium tuberculosis (Mtb) H37Rv-mc26230. Swabs processed with 1:1 diluted SR buffer had the lowest Mtb limit of detection (LOD) at 22.7 CFU/700 µL (95% confidence interval (CI) 14.2-31.2), followed by 2:1 diluted SR buffer at 30.3 CFU/700 µL (95% CI 19.9-40.7), neat SR at 30.9 CFU/700 µL (95% CI 21.5-40.3), and SR prefilled in the Xpert Ultra at 57.1 CFU/700 µL (95% CI 42.4-71.7). Swabs processed using the heat-based protocol had the highest LOD (77.6 CFU/700 µL; 95% CI 51.2-104.0). Similar findings were observed for the LOD of RIF susceptibility. The 2:1 diluted SR buffer condition produced similar LODs when swabs were tested in the presence of sputum matrix or phosphate buffer saline. Further studies are needed to assess the performance of this processing protocol in a clinical setting.IMPORTANCEXpert MTB/RIF Ultra (Xpert Ultra) is approved by the World Health Organization for the diagnosis of tuberculosis (TB). This test is typically performed using sputum specimens obtained from people with presumptive TB. In order to inactivate Mycobacterium tuberculosis (Mtb) and aid liquefaction, sputum must be mixed with Xpert SR prior to transfer into the Xpert Ultra. However, some people under evaluation for TB are unable to produce sputum. Alternative sample types for TB diagnosis would, therefore, be of value. Oral swabs, including tongue swabs, have shown promise, but there are technical challenges associated with sample processing. In this study, several new tongue swab processing conditions were evaluated by utilizing SR, either neat or diluted in buffer. The ability of Xpert Ultra to detect TB was improved under these conditions compared with the previously published heat-processing method; processing steps were simplified; and technical challenges were overcome.

Diagnostic value of interferon-γ release assay in patients with COPD complicated with pulmonary tuberculosis

BackgroundThe common diagnostic methods for tuberculosis have been showing reduced sensitivity among chronic obstructive pulmonary disease patients. This study was conducted to evaluate and analyse the diagnostic value of an interferon-γ release assay in COPD patients complicated with pulmonary tuberculosis.MethodsA nested case-control study was conducted on 123 COPD patients hospitalized at the Fifth Hospital of Shijiazhuang, Hebei Province, from January 2019 to June 2021. Thirty-one patients with active pulmonary tuberculosis complicated with COPD composed the observation group (Group A), 31 patients with nonactive pulmonary tuberculosis complicated with COPD composed the COPD control group (Group B), and 31 patients with active pulmonary tuberculosis not complicated with COPD composed the non-COPD control group (Group C). An interferon-γ release assay, a purified protein derivative of tuberculin (PPD) test, an anti-tuberculosis antibody test, a test of Mycobacterium tuberculosis by sputum smear microscopy and a test of Mycobacterium tuberculosis by PCR method were used to test patients in each group. The positive detection rates generated from the five test methods were compared and analysed.ResultsIn COPD patients complicated with active pulmonary tuberculosis, the differences in the percentage of patients with positive interferon-γ release between the PPD test, anti-tuberculosis antibody test, Mycobacterium tuberculosis by sputum smear microscopy and PCR test results were statistically significant.ConclusionIn patients with COPD complicated with active pulmonary tuberculosis, the percentage of patients who were positive according to the interferon-γ release assay was higher than that according to the sputum smear microscopy, PCR detection of Mycobacterium tuberculosis in sputum specimen, and detection of anti-tuberculosis antibodies. COPD-related complications did not affect the T-SPOT; the greater the T-SPOT value was, the greater the likelihood of active TB. For patients who are T-SPOT positive but clinically considered to have inactive tuberculosis, regular follow-ups should be performed to observe changes in the patient’s condition.

Enhanced detection of Mycobacterium Tuberculosis using nanogold-based silver staining enhancement

Enhanced detection of Mycobacterium Tuberculosis using nanogold-based silver staining enhancement

PCR-Free Self-Calibrated Ratiometric Electrochemical Genosensor Utilizing a Dual-Signal Amplification Approach for Genomic Detection of Mycobacterium tuberculosis

Abstract Tuberculosis (TB) remains a critical global health challenge. While many electrochemical (EC) biosensors have been developed for Mycobacterium detection, most relied on PCR amplification strategies. This study introduced a PCR-free, self-calibrated ratiometric electrochemical (REC) genosensor leveraging dual-signal amplification for the detection of Mycobacterium tuberculosis (MTB). Electroactive silver nanoparticles (AgNPs) were modified onto screen-printed gold electrodes, followed by chemisorption of a methylene blue (MB)-tagged peptide nucleic acid (PNA) probe targeting MTB genomic DNA. MTB presence triggered structural changes in the probe, reducing the MB redox current (IMB) as the detection signal, while the Ag current (IAg) diminished as an internal calibration marker. This ratiometric mechanism minimized system errors, enhancing detection reliability. The genosensor achieved a broad detection range (3.5 fM–35 nM) with a detection limit of 1.59 fM for non-amplified MTB DNA, showing high specificity, reproducibility, and accuracy comparable to standard diagnostic methods. It effectively detected MTB in simulated sputum samples, demonstrating its potential for reliable, rapid, and amplification-free TB diagnostics.

Efficacy of Xpert MTB/RIF assay in detecting Mycobacterium tuberculosis in samples with different results by smear and culture in a coastal city with high incidence of tuberculosis

BackgroundTuberculosis (TB) is a global problem that seriously jeopardizes human health. Among them, the diagnosis and treatment of smear- or culture-negative TB patients is a challenge. The Xpert MTB/RIF (Xpert) assay has been reported to be a novel molecular diagnostic tool for rapidly detecting TB. Still, there is limited data on this assay's performance in subgroups of TB patients. This study aimed to evaluate the diagnostic value of the Xpert method in patients with different smear and culture results and to assess its efficacy for rifampicin resistance (RR) detection.MethodsWe retrospectively collected data from 1,721 patients with a clinical diagnosis of tuberculosis. Smear, Xpert, and traditional solid culture methods were used to detect TB infection and explore the detection rate of Xpert in the grouping of results from different smear and culture methods. Information on RR detected by the Xpert method and proportional method of drug sensitivity test (DST) was also recorded and kappa values, sensitivity, and specificity were calculated.ResultsWe observed that among the three methods, the Xpert method had the highest detection rate of 66.8%, followed by the culture method at 56.0% and the smear method had the lowest at 40.0%. The detection rate of Xpert was 98.3% (642/653) when both smear and culture were positive, 85.1% (296/348) when only one of the two methods, smear and culture, was positive, and 29.4% (212/720) when both smear and culture were negative. The Xpert method and DST showed a high agreement (κ = 0.92) for RR detection. The highest mutation rate was observed for probe E (64.7%), and the least number of probe C mutations occurred (1.5%).ConclusionThe Xpert method has high detection efficiency. It has good diagnostic value in detecting MTB and RR, especially in cases where traditional culture and sputum smear results are negative, and significantly reduces the rate of missed diagnosis.

Clinical value of Metagenomic Next- and Third-Generation Sequencing applied in ultrasound-guided puncture biopsy for diagnosing lymph node tuberculosis.

The aim of this study was to assess the clinical utility of metagenomic next-generation sequencing (mNGS) and metagenomic third-generation sequencing (mTGS) in diagnosing cervical lymph node tuberculosis through analysis of lymph node tissue. This study recruited 101 patients with suspected cervical lymph node tuberculosis and took samples under ultrasound guidance. Parallel culture, X-pert, mNGS and mTGS workflows to each sample were performed. Taking clinical diagnosis as the gold standard, We comparatively compared diagnosis performance of the four methods. Clinically, 76 cases were diagnosed as lymph node tuberculosis and 20 cases were non-lymph node tuberculosis. Compared with clinical diagnosis, the sensitivities of mNGS and mTGS were 89.47% and 82.89%, respectively, significantly higher than those of culture and X-pert which were 43.28% and 68.42%, respectively (P<0.05). The specificity of mNGS and X-pert reached 100%, while those of culture and mTGS were 93.75% and 95%, respectively. And mNGS alone identified 7 species of bacteria, 3 species of viruses, and 5 species of fungi, and identified more mixed infections. Particularly, besides Mycobacterium tuberculosis detection, mNGS may be superior to mTGS for the detection of fungi. Pathogen identification of mNGS and mTGS is less affected by previous anti-tuberculosis drug usage. mNGS and mTGS play a crucial role in the rapid diagnosis and accurate treatment on Cervical lymph node tuberculosis.

Design and Optimization of a Highly Sensitive Surface Plasmon Resonance Biosensor for Accurate Detection of Mycobacterium Tuberculosis

Design and Optimization of a Highly Sensitive Surface Plasmon Resonance Biosensor for Accurate Detection of Mycobacterium Tuberculosis

Amplification-free detection of Mycobacterium tuberculosis using CRISPR-Cas12a and graphene field-effect transistors.

Current molecular tests for tuberculosis (TB), such as whole genome sequencing and Xpert Mycobacterium tuberculosis/rifampicin resistance assay, exhibit limited sensitivity and necessitate the pre-amplification step of target DNA. This limitation greatly increases detection time and poses an increased risk of infection. Here, we present a graphene field-effect transistor (GFET) based on the CRISPR/Cas system for detecting Mycobacterium tuberculosis. The CRISPR/Cas12a system has the ability to specifically recognize and cleave target DNA. By integrating the system onto the FET platform and utilizing its electrical amplification capability, we achieve rapid and sensitive detection without requiring sample pre-amplification, with a limit of detection (LoD) as low as 2.42 × 10-18 M. Cas12a-GFET devices can differentiate 30 positive cases from 56 serum samples within 5 minutes. These findings highlight its immense potential in future biological analysis and clinical diagnosis.

Diagnostic accuracy of self-collected tongue swabs for Mycobacterium tuberculosis complex detection in individuals being assessed for TB in South Africa using the Xpert MTB/RIF Ultra assay.

Tongue swabs (TS) have shown potential for detecting Mycobacterium tuberculosis complex (MTBC) through downstream molecular testing. Analytical performance varies, depending on the processing protocol and the molecular test used. This study aimed to first investigate ease-of-use of TS collection in addition to acceptability by individuals being assessed for TB and second to determine the performance of self-collected TS on the Xpert MTB/RIF Ultra (Ultra) assay (Cepheid, Sunnyvale, CA, USA) for MTBC and Rifampicin (RIF) resistance detection. The ease-of-use of TS collection and acceptability by study participants was assessed through completion of a survey questionnaire. Analytical performance of self-collected TS on the Ultra assay was determined by comparing results to a liquid culture reference result and to Ultra on sputum. Results were additionally stratified by HIV and smear status. Of the 399 survey respondents, all were happy with the TS collection procedure with minimal discomfort reported. Data analysis was performed on 321 specimens. The sensitivity of the Ultra TS assay for MTBC detection was 78.1% (95% CI: 66.0-87.5) while the specificity was 100% (95% CI: 98.6-100). Test performance was better in individuals with a higher bacillary load, with some variability. On HIV-positive individuals, TS performance is ∼30% lower than that on sputum but also slightly better than smear microscopy while the performance of TS among HIV-negative individuals is similar to sputum (93% vs 97%). For RIF resistance profiling, the Ultra TS assay showed 41/41 (100%) concordance with phenotypic drug susceptibility testing. Although self-collected TS have lower sensitivity compared to sputum, their ease of use and high acceptability make them a valuable additional specimen type for molecular TB testing. Self-collection can reduce the burden on healthcare workers and increase access to testing, particularly for individuals who are too busy to visit healthcare facilities or fear stigma.

The Imperative Role of Xpert® Mycobacterium Tuberculosis Complex/Resistance to Rifampin (MTB/RIF) in Rapid Diagnosis of Pulmonary and Extrapulmonary Tuberculosis.

Background The Xpert® mycobacterium tuberculosis complex/resistance to rifampin (MTB/RIF) test is widely recognized for its ability to provide rapid and accurate results for diagnosing tuberculosis (TB) in both pulmonary and extrapulmonary samples. Its utility is especially critical in smear-negative and paucibacillary cases where traditional diagnostic methods often fail. This study aimed to evaluate the role of Xpert® MTB/RIF in diagnosing TB in various pulmonary and extrapulmonary samples and TB in smear-negative pulmonary samples. Methods This retrospective study analyzed records from 2017 to 2021, focusing on presumptive TB patients who underwent Xpert® MTB/RIF testing at a tertiary care hospital. Data from 1174 samples (671 pulmonary and 503 extrapulmonary) were included. The Xpert® MTB/RIF reports were assessed for Mycobacterium tuberculosis (MTB) detection rates across different sample types. Results Of the pulmonary samples, MTB detection was highest insputum samples, 122 (48.41%), followed bybronchial washing samples, 49 (16.33%). In smear-negative pulmonary cases, Xpert® MTB/RIF detected MTB in 72 (16.55%) samples, demonstrating its utility in paucibacillary cases. The test also proved valuable in diagnosing extrapulmonary TB, where symptoms are often atypical. Conclusion Xpert® MTB/RIF is a reliable tool for the rapid and accurate diagnosis of pulmonary and extrapulmonary TB, including smear-negative and paucibacillary cases. Its application can significantly enhance the timely diagnosis and treatment of TB, especially in resource-limited settings.

Effectiveness of Biological and Surgical Sampling for Detection of Mycobacterium Tuberculosis

Introduction. Tuberculosis remains a significant public health issue, ranking second only to COVID-19 in terms of mortality from infectious diseases, with more than 10 million individuals affected annually. Despite a decline in incidence rate, its diagnosis continues to be challenging, particularly in cases with negative results from microbiological and histological examinations.Aim. To compare the effectiveness of surgical diagnostic methods for tuberculosis during the pre-analytical phase of bacteriological verification.Materials and methods. The retrospective study, conducted at the Republican Clinical Tuberculosis Treatment Dispensary of the Republic of Bashkortostan, analyzed data from 1272 patients who were hospitalized in the Tuberculous Pulmonary Surgery Unit from 2021 to 2023 and underwent bacteriological diagnostics, including microscopy and polymerase chain reaction.Results and discussion. The results indicate that out of 1272 surgeries, 65.2% involved resection procedures, with confirmed tuberculosis infection in 83.1% of patients. Bronchoalveolar lavage identified mycobacteria in 153 patients (20.5%), whereas sputum collection revealed them in 106 patients. The new collection method, although less effective, demonstrated a detection rate of 15.7% through molecular genetic analysis.Conclusion. Surgical diagnostic methods for tuberculosis appeared to be effective, detecting mycobacterium tuberculosis in 83.1% of cases, surpassing the standard sputum collection method, which has a detection rate of 10.8% via molecular genetic analysis. Bronchoalveolar lavage proved to be more productive, yielding 20.5% positive results on a molecular genetic basis, 11% microscopically, and 8% through culture. Although the new method is less effective, it is recorded as being more accurate than the standard, with detection rates of 15.7% molecularly, 6.6% microscopically, and 5.2% through culture.

Drug-resistant pulmonary tuberculosis with surgical treatment: clinical forms, histological and macroscopic aspects

OBJECTIVE. To clarify the clinical forms of drug-resistant pulmonary tuberculosis with surgical treatment and their relationship with the morphological activity of specific inflammation and the detection of Mycobacterium tuberculosis (MBT) in the surgical material. MATERIALS AND METHODS. The study had a cohort retrospective character. The two groups of patients were studied – with multidrug-resistant tuberculosis (n=27) and pre-extensively drug-resistant tuberculosis (n=25). All patients after completion of a standardized 6-month or individualized chemotherapy was performed surgery. The results of morphological examination namely clarification of pulmonary tuberculosis clinical forms and the morphological degree of activity of a specific inflammatory process were analyzed. A comparative analysis of MBT detection by the cultural method in the surgical material depending on the forms of pulmonary tuberculosis and its degree of activity was performed. RESULTS AND DISCUSSION. Regardless of the drug-resistant profile, different forms of pulmonary tuberculosis were presented in both study groups. In general, cases of multiple tuberculomas (24; 46.2 %) were quantitative prevailed with predominance in group 1. In second place in frequency was diagnosed fibrocaseous tuberculosis (10; 19.2 %) at that cases of pre-extensively drug-resistant tuberculosis (7; 28.0 %) more than twice prevailed. A diagnosis of residual post-tuberculosis changes in the lungs (7; 13.5 %) was at third place, which also prevailed in group 2 (6; 24.0 %). According of morphological examination, it was found that only a quarter of patients at the time of surgery achieved remission of the disease (12; 23.1 %) with a predominance of group 1 (8; 32.0 %). The progression of specific inflammation persisted (18; 34.6 %) in a third of observations in both groups. During surgery operating material was collected for cultural research, which was conducted in 41 cases (78.8 %). In most cases (30; 57.7 %), MBT was not detected, the largest proportion occurred on lung tuberculoma (17; 32.7 %). In almost quarter of all cases, the MBT was detected in surgical material (12; 23.1 %). The share of MBT detection was much higher in fibrocaseous tuberculosis (4; 40.0 %), compared to cases of solitary tuberculomas (4; 16.7 %). The MBT was also detected in residual post-tuberculosis changes – only in the group of pre-extensively drug-resistant tuberculosis (3; 12.0 %). It is established that the morphological activity of the inflammatory process does not clearly correlate with cases of cultural detection of MBT. CONCLUSIONS. The use of modern chemotherapeutic regimens for the treatment of tuberculosis with multidrug resistance and pre-extensively drug-resistant tuberculosis during, in the main course of therapy does not allow to achieve complete abacylation in such patients, which causes additional surgical treatment. In cases of pre-extensively drug-resistant tuberculosis, the development of more severe forms of lung tuberculosis and the lower preoperative effectiveness of specific anti-tuberculosis therapy was observed.

Clinical features of Tibetan adolescent tuberculosis at high altitudes: a retrospective study

BackgroundAdolescent pulmonary tuberculosis (TB) is considered inadequately recognized and underreported at high altitudes. This study aimed to investigate the clinical features of adolescent pulmonary TB patients at high altitudes in China.MethodA retrospective analysis was performed at Linzhi People’s Hospital. Patients recruited were newly diagnosed with pulmonary TB. Detailed information, including demographics, medical history, presenting symptoms, laboratory results and pulmonary images, was collected. Sputum samples were collected to detect Mycobacterium tuberculosis via the GeneXpert MTB/RIF assay.ResultA total of 63 adolescents and 192 adults were recruited. Compared with those in the adult group, typical TB-related manifestations were significantly less common in the adolescent group (all P < 0.05). The adolescent group had significantly more subclinical TB (23.8% vs. 8.3%, P = 0.001) and a higher previous tuberculosis exposure rate (38.8% vs. 8.3%, P < 0.001) than did the adult group. The erythrocyte sedimentation rate was significantly higher in the adolescent group than in the adult group (P = 0.026). Compared with adult patients, adolescent patients presented a lower rate of pleural thickening (P < 0.01). Compared with active adolescent patients, there were more female than male in the adolescent subclinical TB group (12/15 vs 27/48) and the positive rate of the sputum GeneXpert test was higher in subclinical TB group (6/15 vs 8/48).ConclusionAdolescent patients with pulmonary TB at high altitudes were prone to subclinical TB and have a higher previous TB exposure rate. Sputum GeneXpert test and ESR were important for the diagnosis of subclinical pulmonary TB.

Laboratory validation of a simplified DNA extraction protocol followed by a portable qPCR detection of M. tuberculosis DNA suitable for point of care settings.

Tuberculosis, caused by Mycobacterium tuberculosis, is a treatable and curable disease, and yet remains one of the leading causes of death worldwide. Diagnosis is essential to reducing the number of cases and starting treatment, but costly tests and equipments that require complex infrastructure hamper their widespread use as a tool to contain the disease in vulnerable populations as well countries lacking resources. Therefore, it becomes necessary to develop new technological approaches to molecular methods as well as screening tests that can be rapidly conducted among people presenting to a health facility to differentiate those who should have further diagnostic evaluation for TB from those who should undergo further investigation for non-TB diagnoses. The present study aimed to evaluate two experimental DNA extraction methods from clinical samples (FTA card versus sonication) followed by analysis in a portable qPCR instrument (the Q3-plus). The FTA card-based protocol showed 100% sensitivity and specificity, while the sonication protocol showed 80% sensitivity and 89% specificity when compared to the traditional gold standard culture. The portable protocol, comprised by the FTA card method and the portable instrument Q3-Plus, showed sensitivity and specificity of 92% and 61%, respectively, when compared to culture, and 75% and 81%, respectively, when compared to the standard TB case classification. The ROC curve showed an AUC of 0.78 (p<0.001) for the portable protocol and 0.93 (p<0.001) for the GeneXpert Ultra. The limit of detection (LOD) for Mycobacterium tuberculosis (H37Rv strain) detection in spiked samples obtained using the portable protocol (FTA card and Q3-Plus) was 19.3 CFU/mL. As an added benefit, using the FTA card facilitates sample handling, transport, and storage. It is concluded that the use of the FTA card protocol and the Q3-Plus yields similar sensitivity and specificity as the gold standard diagnostic tests and case classification. We suggest that the platform is suitable to use as a point of care tool, assisting in the screening of tuberculosis in hard-to-reach or resource-limited areas.

Diagnostic accuracy of LiquidArray MTB-XDR VER1.0 for the detection of Mycobacterium tuberculosis complex, fluoroquinolone, amikacin, ethambutol, and linezolid susceptibility.

Drug susceptibility testing (DST) is essential for starting people on effective tuberculosis (TB) regimens. No published data exists for the high-throughput LiquidArray MTB-XDR (LA-XDR) test, which detects Mycobacterium tuberculosis complex (MTBC) and fluoroquinolone, amikacin, ethambutol, and linezolid susceptibility (latter two have no rapid DSTs available). We enrolled people (n=720) with presumptive TB who provided two sputa for a Xpert MTB/RIF Ultra and a culture (MTBC reference standard). Phenotypic DST and Sanger sequencing were the composite reference standards. LA-XDR on the manual FluoroLyse and automated GenoXtract-fleXT (fleXT) DNA extraction methods were compared. For MTBC, LA-XDR had similar sensitivities (85-87%) and specificities (99%) using each extraction method. Drug susceptibility sensitivities varied: 94% (95% CI: 86, 98) for fluoroquinolones, 64% (45, 80) for amikacin, and 88% (79, 93) for ethambutol (specificities 97-100%). 6/7 (86%) resistant linezolid isolates were detected. LA-XDR with fleXT had indeterminate proportions of 21/251 (8%), 2/251 (1%), 63/251 (25%), and 93/251 (37%) for fluoroquinolones, ethambutol, amikacin, and linezolid, respectively (amikacin and linezolid indeterminates higher with Fluorolyse-extracted DNA). In a hypothetical population of 100 smear-negative people with fluoroquinolone-resistant TB undergoing fleXT DNA extraction, 24/100 (24%) would be missed (one extraction error, two invalid results, 15 MTBC-negative, six fluoroquinolone-indeterminate, one false-susceptible). LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high fluoroquinolones and ethambutol resistance sensitivity, moderate amikacin resistance sensitivity, and promise for linezolid resistance, for which more data are needed. Improved LA-XDR MTBC detection would reduce missed resistance.

Study on the early diagnostic value of nanopore sequencing in alveolar lavage fluid smear-negative pulmonary tuberculosis.

The objective of this study was to investigate the early diagnostic value of nanopore sequencing in alveolar lavage smear-negative pulmonary tuberculosis (PTB). A prospective study was conducted on patients hospitalized at Zhuzhou Central Hospital from October 2021 to June 2022 and suspected to have PTB. Alveolar lavage fluid specimens were collected from these patients and simultaneously subjected to centrifugal bacterial collection smear method in a sandwich cup, bifidobacteria solid culture (referred to as culture), Mycobacterium tuberculosis-DNA (TB-DNA), and nanopore sequencing for detection of Mycobacterium tuberculosis. These patients were ultimately diagnosed with smear-negative PTB. The final clinical diagnosis was used as a reference to compare the diagnostic efficacy of Nanopore sequencing, culture, and TB-DNA for PTB. The results showed that the positive rates detected by nanopore sequencing, culture, and TB-DNA in the 103 suspected tuberculosis patients were 73.8%, 13.6%, and 33.0%, respectively. The sensitivity of nanopore sequencing was significantly higher than culture (P < 0.001) and TB-DNA (P < 0.001) for the identification of smear-negative PTB. Similarly, the positive rate of TB-DNA was also significantly higher than that of culture (P = 0.001). In conclusion, nanopore sequencing exhibited the highest sensitivity for the rapid diagnosis of Mycobacterium tuberculosis in alveolar lavage fluid specimens, using clinical diagnosis as a reference standard, and it could improve the PTB clinical diagnosis.

An electrochemical biosensor for the detection of tuberculosis specific DNA with CRISPR-Cas12a and redox-probe modified oligonucleotide

BackgroundThe development of a robust and accurate point-of-care platform for the detection of tuberculosis (TB) biomarkers is important for disease control. In the current study, the detection principle relies on the shredding of PES-modified non-specific ssDNA (Poly T) in the presence of target DNA IS6110, a reliable biomarker for TB diagnosis by the CRISPR-Cas12a mechanism. Cas protein has great potential in the detection of nucleic acids. ResultsHerein, we developed a biosensing platform by utilizing the trans cleavage activity of CRISPR-Cas12a into an electrochemical biosensor. Square wave voltammetry technique is used for the analysis of the fabricated biosensing platform. In the presence of target DNA, the trans cleavage activity is observed by a nonspecific ssDNA substrate, PolyT chain. Various concentration of target DNA is tested on the constructed biosensor, the fabricated biosensor successfully detected TB target DNA by trans cleavage of PES-modified poly T. This novel biosensor was able to detect the target DNA, IS6110 with the limit of detection of 14.5 nM within 60 min by trans-cleavage activity of CRISPR-Cas12a and the results revealed the potential of Cas12a-based biosensors as a diagnostic platform. SignificanceThis is the first study reporting the CRISPR-Cas12a-based electrochemical sensor for TB. The developed CRISPR-Cas12a endonuclease-based electrochemical biosensor provides a potentially powerful platform for the accurate detection of Mycobacterium tuberculosis.

Evaluation of the Cerebrospinal Fluid (CSF)-Truenat Assay: A Novel Chip-Based Test in the Diagnosis and Management of Tubercular Meningitis at a Tertiary Care Hospital.

Tuberculosis (TB) continues to be a major health concern that has a significant impact on morbidity and mortality worldwide. Tubercular meningitis (TBM) may be fatal due to its severe neurological outcomes if not diagnosed and treated promptly. The newer molecular diagnostic techniques have brought significant advancements in the detection of Mycobacterium tuberculosis (MTB). One such test is the cerebrospinal fluid (CSF)-Truenat assay which offers several advantages over traditional methods and provides results within a few hours. This is crucial for timely intervention and can further improve patient outcomes. We have evaluated its diagnostic accuracy, its utility, and its usefulness in treatment decisions. A cross-sectional study was conducted on 150 patients. The CSF samples were analyzed by both cartridge-based nucleic acid amplification test (CBNAAT) and Truenat (Molbio Diagnostics Private Limited, Verna, Goa, India). Brain imaging was also performed. Patients were divided into four groups, i.e., definite, probable, possible, and non-TBM. Truenat-positive cases were considered definite, and their efficacy and clinical utility for diagnosing and managing TBM were compared with CBNAAT. A comparative analysis between CBNAAT and Truenat showed concordance for positive results. But it also identified some discrepancies, particularly in cases where CBNAAT was positive and Truenat was negative. Overall, Truenat demonstrated strong diagnostic performance with a sensitivity of 83.75%, a specificity of 88.57%, and overall accuracy of 86% as compared to CBNAAT. The study highlighted the role of Truenat in improving diagnostic accuracy and guiding cost-effective treatment strategies for TBM as compared to CBNAAT. As molecular tests alone cannot detect bacilli in most cases, a combination of clinical, microbiological, and radiological parameters is also obligatory for diagnosing TBM.

Comparison of Two Staining Methods (Ziehl–Neelsen Staining and Auramine O Staining) in Detecting Mycobacterium tuberculosis in Cytology Samples: A Cross-sectional Study

Comparison of Two Staining Methods (Ziehl–Neelsen Staining and Auramine O Staining) in Detecting Mycobacterium tuberculosis in Cytology Samples: A Cross-sectional Study