Detection Of MTB Research Articles

Computer vision applications for the detection or analysis of tuberculosis using digitised human lung tissue images - a systematic review

ObjectiveTo conduct a systematic review of the computer vision applications that detect, diagnose, or analyse tuberculosis (TB) pathology or bacilli using digitised human lung tissue images either through automatic or semi-automatic methods. We categorised the computer vision platform into four technologies: image processing, object/pattern recognition, computer graphics, and deep learning. In this paper, the focus is on image processing and deep learning (DL) applications for either 2D or 3D digitised human lung tissue images. This review is useful for establishing a common practice in TB analysis using human lung tissue as well as identifying opportunities for further research in this space. The review brings attention to the state-of-art techniques for detecting TB, with emphasis on the challenges and limitations of the current techniques. The ultimate goal is to promote the development of more efficient and accurate algorithms for the detection or analysis of TB, and raise awareness about the importance of early detection.DesignWe searched five databases and Google Scholar for articles published between January 2017 and December 2022 that focus on Mycobacterium tuberculosis detection, or tuberculosis pathology using digitised human lung tissue images. Details regarding design, image processing and computer-aided techniques, deep learning models, and datasets were collected and summarised. Discussions, analysis, and comparisons of state-of-the-art methods are provided to help guide future research. Further, a brief update on the relevant techniques and their performance is provided.ResultsSeveral studies have been conducted to develop automated and AI-assisted methods for diagnosing Mtb and TB pathology from digitised human lung tissue images. Some studies presented a completely automated method of diagnosis, while other studies developed AI-assisted diagnostic methods. Low-level focus areas included the development of a novel μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT scanner for soft tissue image contract, and use of multiresolution computed tomography to analyse the 3D structure of the human lung. High-level focus areas included the investigation the effects of aging on the number and size of small airways in the lungs using CT and whole lung high-resolution μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT, and the 3D microanatomy characterisation of human tuberculosis lung using μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT in conjunction with histology and immunohistochemistry. Additionally, a novel method for acquiring high-resolution 3D images of human lung structure and topology is also presented.ConclusionThe literature indicates that post 1950s, TB was predominantly studied using animal models even though no animal model reflects the full spectrum of human pulmonary TB disease and does not reproducibly transmit Mtb infection to other animals (Hunter, 2011). This explains why there are very few studies that used human lung tissue for detection or analysis of Mtb. Nonetheless, we found 10 studies that used human tissues (predominately lung) of which five studies proposed machine learning (ML) models for the detection of bacilli and the other five used CT on human lung tissue scanned ex-vivo.

Rapid and sensitive detection of Mycobacterium tuberculosis using nested multi-enzyme isothermal rapid amplification in a single reaction.

In this study, we have successfully developed a method called nested multi-enzyme isothermal rapid amplification (nestMIRA) for the detection of Mycobacterium tuberculosis (Mtb). This method involves a two-step thermostatic amplification process in the same tube and can be read using fluorescence and lateral flow dipstick (LFD) assays. It is known to be rapid, specific, and highly sensitive. Our method has shown promising results in the detection of clinical specimens, and we believe that it can be a valuable tool for the rapid detection of Mtb in a clinical setting.

Design of THz Refractive Index-Based Diamond Shaped Biosensor Using Gated Axial Attention Network for MTB Detection

MTB is usually diagnosed using sophisticated and expensive methods that include molecular and microscopic examination, making the process tedious and lengthy. This research proposes an innovative approach to enhance MTB detection through a novel biosensor design that integrates Terahertz refractive index measurements with a Gated NasNet Running City Game Axial Attention Network. The proposed diamond-shaped biosensor harnesses the unique properties of Terahertz waves to measure refractive index variations in biological tissues, specifically targeting MTB infections. A thorough optimization process was carried out to obtain an optimal sensor design, which included optimizing several design aspects such as size and materials. The sensor exhibits exceptional characteristics, including processing time of 0.1 s for 40000 iterations and 0.04 Confinement loss among other models. To optimize the weight parameters and reduce computational complexity, the Gated NasNet Running City Game Axial Attention Network is integrated into this research. The model demonstrates impressive performance of nearly 99.1% sensitivity in detecting MTB. The exceptional results indicate the substantial capability of the proposed biosensor for quick and accurate detection of MTB. The sensor’s exceptional performance metrics, along with its simple design, signify a significant breakthrough in the realm of biosensing technology.

Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method.

Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.

Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat‐Cas13a point‐of‐care RNA testing

AbstractMycobacterium tuberculosis (MTB) is the causal pathogen of tuberculosis (TB). Rapid and accurate detection of live MTB is important for transmission control and patient treatment. Here, we described a clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13a‐based molecular diagnosis approach for rapid and specific detection of live MTB. This detection method, which we termed CRISPR‐Live‐MTB, contained two consecutive reactions including nuclear acid sequence‐based amplification (NASBA) and CRISPR‐Cas13a collateral cleavage reaction. CRISPR‐Live‐MTB could efficiently detect MTB single‐stranded RNA (ssRNA) in 2 hours with high specificity over double‐stranded DNA (dsDNA). Importantly, CRISPR‐Live‐MTB exhibited a limit of detection of 2.4 copies for MTB ssRNA, which was 1000 times lower than that of the clinically used NASBA method. Moreover, lateral flow was integrated into the CRISPR‐Live‐MTB method to enable point‐of‐care testing application with a sensitivity of 95% and a specificity of 100%. Overall, our study demonstrated the feasibility of CRISPR‐Live‐MTB as a rapid, sensitive, and specific approach for live MTB detection.

Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection.

Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1pg/μl. Its general sensitivity and specificity were 76.62and 60.53%, respectively, with kappa index agreement of 0.166.

Comparison of Xpert MTB RIF (G4) With Xpert MTB (Ultra) in Diagnosis of Mycobacterium Tuberculosis in Clinical Samples

Abstract Background: Tuberculosis (TB) continues to be a major cause of morbidity and mortality worldwide. The main strategy to fight the pandemic is the development of rapid, accurate diagnostic tests, which helps to reduce the time for initiation of therapy. In 2010, WHO recommended the use of Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) (G4), which is a cartridge-based semi-automated nucleic acid amplification test (NAAT) as a primary test with an aim to improve the detection of MTB. Later on, to improve the diagnostic efficacy, the new and improved cartridges called Xpert Ultra were introduced in 2017. Materials and Methods: One thousand and sixty-nine clinical samples were utilized for the study for microscopy, culture, and NAATs (Xpert MTB/RIF G4 and Ultra). The samples were randomly allocated. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated while assuming trace results as positive and negative in two separate scenarios. Results: The sensitivity of Xpert MTB/RIF Ultra cartridge was higher (100%) than Xpert MTB/RIF G4 cartridge (95.23%) and the specificity of Ultra cartridge was lower (71.79%) when compared to G4 cartridge (91.63%). However, the specificity of Xpert Ultra cartridge improved by reclassifying the “trace” interpretation as negative (89.1%). The NPV was 100% and 99.09% for Xpert Ultra cartridge and the Xpert G4 cartridge, respectively, and did not change by reclassifying the “trace” interpretation as negative. In contrast, the PPV varied greatly between the two tests, 26.66% for Xpert Ultra cartridge and 66.66% for Xpert G4 cartridge. The PPV of Xpert Ultra cartridge was improved to 48.48% by including “trace” samples in the negative pool. Conclusion: Xpert Ultra and G4 shows good performance as a first line diagnostic test. Caution should be exercised when interpreting ‘trace’ results in a country like India without parallel confirmatory tests like microscopy and culture. Data is limited on this newly incorporated value assigned in the automated report. Conducting further studies on results interpreted as ‘trace’ will help in improving the guidelines and as well as utilisation of such tests.

State-of-the-art detection of Mycobacterium tuberculosis in blood during tuberculosis infection using phage technology

Tuberculosis (TB), an aerosol-transmitted infection caused by Mycobacterium tuberculosis (Mtb), remains the commonest cause of death globally, from an infectious bacterial disease. Nine years on from the launch of the World Health Organization (WHO)'s END-TB strategy, disease incidence rates are stubbornly unchanged [1]. While this represents, in part, a reversal of improving trends caused by the COVID-19 pandemic, it also reflects the fragility and inadequacy of healthcare systems to sustain TB control [2]. Although multifactorial, a key reason for this is the ineffectiveness of existing clinical tools to meet the two key objectives of the END-TB strategy-(i) early diagnosis and treatment of TB disease (to limit onward transmission); and (ii) disease prevention through screening for asymptomatic TB infection (TBI). Meeting both objectives will rely on the development of new biomarkers with high accuracy, but the global nature of the TB problem also requires that new tests are rapid, low cost and can be measured in patients by sampling from universally accessible sites. In this review, we will present the accumulating evidence for circulating Mtb in both TB disease and asymptomatic TBI and discuss the potential utility of novel bacteriophage-based technology for blood-based detection of Mtb.

DdPCR provides a sensitive test compared with GeneXpert MTB/RIF and mNGS for suspected Mycobacterium tuberculosis infection.

The Metagenomics next-generation sequencing (mNGS) and GeneXpert MTB/RIF assay (Xpert) exhibited a sensitivity for tuberculosis (TB) diagnostic performance. Research that directly compared the clinical performance of ddPCR analysis, mNGS, and Xpert in mycobacterium tuberculosis complex (MTB) infection has not been conducted. The study aimed to evaluate the diagnostic performance of ddPCR compared to mNGS and Xpert for the detection of MTB in multiple types of clinical samples. The final clinical diagnosis was used as the reference standard. Out of 236 patients with suspected active TB infection, 217 underwent synchronous testing for tuberculosis using ddPCR, Xpert, and mNGS on direct clinical samples. During follow-up, 100 out of 217 participants were diagnosed with MTB infection. Compared to the clinical final diagnosis, ddPCR produced the highest sensitivity of 99% compared with mNGS (86%) and Xpert (64%) for all active MTB cases. Twenty-two Xpert-negative samples were positive in mNGS tests, which confirmed the clinical diagnosis results from ddPCR and clinical manifestation, radiologic findings. Thirteen mNGS-negative samples were positive in ddPCR assays, which confirmed the clinical final diagnosis.ddPCR provides a higher sensitive compared to Xpert and mNGS for MTB diagnosis, as defined by the high concordance between ddPCR assay and clinical final diagnosis.

Estimating Limit of Detection of M. Tuberculosis DNA as an Analyte Released from a Chemically Coated Solid Matrix and Using a WHO-approved CB-NAAT Platform

The limit of detection of Mtb (Mycobacterium tuberculosis) bacilli was evaluated by detection of released DNA from a chemically treated cellulose matrix. Loopful of the Mtb cells were suspended in phosphate buffer (pH 6.8), DNA was extracted from it and quantified using a commercial Mtb real time PCR quantitation kit. Synthetic sputum, prepared as the matrix of choice was mixed with defined number of Mtb bacilli and spotted onto the cellulose matrix. Mtb DNA from the matrix was released into a DNA-release buffer and detected using a WHO-approved CB-NAAT detection platform. One hundred and fifty, 500 and 1000 but not 10 Mtb bacilli/mL of synthetic sputum could be detected by this protocol. The result underlined the potential of cellulose matrix as a potent, room temperature, Mtb-infected sputum transportation, storage and DNA-release device for sensitive detection of Mtb using Mtb Xpert Ultra CB-NAAT as the WHO-approved Mtb test of choice.

A new method for the detection of Mycobacterium tuberculosis based on the CRISPR/Cas system

ObjectMycobacterium tuberculosis (MTB) is a bacterium that can cause zoonoses by aerosol transmission. Tuberculosis (TB) caused by MTB heavily burdens world public health security. Developing efficient, specific, convenient, and inexpensive MTB assays are essential for preventing and controlling TB.MethodsIn this study, we established a specific detection method for MTB using the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) system, combined with recombinase mediated isothermal nucleic acid amplification (RAA) to improve the sensitivity of the detection system and achieve “two-level” amplification of the detection signal. The sensitivity and specificity of RAA combined with the CRISPR/Cas system were analyzed. Using BACTEC 960 culture as the gold standard for detecting MTB, we established the TB-CRISPR technique by testing 504 samples from patients with suspected TB.ResultsMTB H37Ra could be seen as low as 3.13 CFU/mL by the CRISPR-Cas12a system targeting IS6110. With BACTEC960 culture (120 positives and 384 negatives) as the gold standard, the sensitivity of the TB-CRISPR technique was 0.883 (0.809–0.932), and the specificity was 0.940 (0.910–0.961). According to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) reached 0.944 (0.914–0.975) within 95% CI. The positive likelihood ratio (PLR) was 14.747 (9.870-22.035), and the negative likelihood ratio (NLR) was 0.124 (0.076–0.203). The positive predictive value (PPV) was 0.822 (0.742–0.881), and the negative predictive value (NPV) was 0.963 (0.937–0.979).ConclusionTB-CRISPR plays an essential role in the molecular diagnosis of TB. The whole detection time is less than 1.5 h. It is easy to operate and does not need complex instruments. It is of great significance for the rapid detection of MTB and the clinical diagnosis of TB.

A-248 Evaluation of NextGene™ MTB/NTM Detection Kit for Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Sputum and Culture Specimens

Abstract Background Tuberculosis, an infectious disease, is a major cause of deteriorating health, and, until SARS-CoV-2 (COVID-19) became a pandemic disease, a major cause of death worldwide. The recently developed NextGene™ MTB/NTM Detection Kit is designed for detection of Mycobacterium tuberculosis complex (MTB) using IS6110 genes and MPB64 genes, and for detection of nontuberculous mycobacteria (NTM) using 16S rRNA genes for resolution of false positive and false negative results, and for development of more sensitive and specific real-time PCR products. The purpose of this study is to examine the clinical sensitivity and specificity of the NextGene™ MTB/NTM Detection Kit in order to verify its clinical efficacy. Methods A total of 446 residual clinical specimens were obtained from 274 sputum and 172 culture samples collected from July to August 2021. Diagnostic tests were performed in order to confirm that these residual samples were positive and negative; 344 specimens were positive for MTB or NTM, and 102 specimens were confirmed as negative for both MTB and NTM. Measurement of positive/negative percent agreement values and kappa coefficients was performed for comparison of diagnostic performance between the NextGene™ and Advansure assays. Positive and negative controls were included in each run. Results Of the 446 samples (274 sputum and 172 culture samples), 344 specimens showed a positive result and 102 specimens showed a negative result. Results of the comparison tests were all consistent for detection of MTB and NTM. Both MTB and NTM met the criterion for evaluation of target validity of kappa value 0.8 or higher in sputum and culture samples. Results of the evaluation of clinical sensitivity and specificity were consistent with both clinical diagnoses. Conclusion This study was conducted in order to examine the clinical sensitivity and specificity of the NextGene™ MTB/NTM Detection Kit in order to verify its clinical validity. Residual samples from sputum and culture specimens from patients who were suspected being infected that were confirmed positive or negative for MTB and NTM were used in this study with the goal of establishing the already commercially available licensed product as a control reagent for comparative analysis of the clinical performance of the NextGene™ MTB/NTM Detection Kit. Compared to existing kits that are widely used, consistent results from comparison of the NextGene™ MTB/NTM Detection Kit have been reported with comparable clinical sensitivity and specificity, thus it is regarded as showing the same performance.

Evaluation of Xpert MTB/RIF Ultra for the Diagnosis of Extrapulmonary Tuberculosis: A Retrospective Analysis in Saudi Arabia

The incidence of extrapulmonary tuberculosis (EPTB) in low- and middle-income countries, as well as, high-income countries has increased over the last two decades. The acid-fast bacillus (AFB) smear test is easy to perform and cost-effective with a quick turnaround time but the test has low sensitivity. Culture remains the gold standard for detecting TB; however, it has low sensitivity and slow bacterial growth patterns, as it may take up to 6 to 8 weeks to grow. Therefore, a rapid detection tool is crucial for the early initiation of treatment and ensuring an improved therapeutic outcome. Here, the Xpert Ultra system was developed as a nucleic acid amplification technique to accelerate the detection of MTB in paucibacillary clinical samples and endorsed by the World Health Organization. From March 2020 to August 2021, Xpert Ultra was evaluated for its sensitivity and specificity against EPTB and compared with those of the routinely used Xpert, culture, and AFB tests in 845 clinical samples in Saudi Arabia. The results indicate the overall sensitivity and specificity of Xpert Ultra to be 91% and 95%, respectively, compared with the Xpert (82% and 99%, respectively) and AFB smear (18% and 100%, respectively) tests. The results also indicated that despite the low microbial loads that were categorized as trace, very low, or low on Xpert Ultra, yet, complete detection was achieved with some sample types (i.e., 100% detection). Consequently, Xpert Ultra has great potential to replace conventional diagnostic approaches as a standard detection method for EPTB.

Evaluation of Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis in Southwest China.

The purpose of this study was to determine the diagnostic efficacy of Xpert MTB/RIF assay for rapid diagnosis of Tuberculosis (TB) and detection of rifampicin (RIF) resistance in patients suspected of having EPTB, assessing it against traditional culture and drug susceptibility test (DST) by proportional method, and the ability to predict multidrug resistance TB by Xpert MTB/RIF assay. In this study, the Xpert MTB/RIF assay was applied to 1,614 extrapulmonary specimens. Compared with TB culture and Composite Reference Standard (CRS), the Xpert MTB/RIF assay had a high sensitivity and specificity for detection of EPTB. Depending on the culture method or CRS as the standard, sensitivity of the Xpert MTB/RIF assay for detection of MTB in pleural effusion, cerebrospinal fluid, thoracic drainage fluid and throat swabs specimens were lower than that of other specimens. According to the experimental results, we have reason to believe that Xpert MTB/RIF assay is a rapid and simple technique with high sensitivity and specificity for diagnosing EPTB and detecting drug resistance in variety of specimens. Xpert MTB/RIF assay combined with DST maybe identify more cases of multi-drug resistant tuberculosis (MDR-TB).

Efficacy of TrueNat cartridge extracted DNA for detecting drug resistant tuberculosis by line probe assay

BackgroundCurrently for diagnosing Mycobacterium tuberculosis and its drug resistance, two sputum samples are required. One of them is subjected to TrueNat™ and if positive the other sample is subjected to line probe assay (LPA). This study was done to evaluate whether TrueNat extracted DNA can be directly used for performing LPA in a diagnostic laboratory setting to decrease patient turn-around time. MethodsTotal 45 smear positive sputum samples were subjected to TrueNat™ MTB detection and first and second line (FL and SL) LPA testing in parallel. DNA extracted by Trueprep® Cartridge was also tested by LPA and results were compared. Further, TrueNat extracted DNA from 20 samples was divided into 6 aliquots each, two of which were stored at 4 °C, 37 °C and 55 °C (under humidification) each. One aliquot from stored DNA at each temperature was used for FL & SL LPA on day three and the other on day eight. The blots thus obtained were compared with those of conventional LPA at day 1. ResultsFor FL-LPA, TrueNat extracted DNA gave valid results for all 45 (100%) samples but conventionally extracted DNA could give results for 44 (97.8%) samples. Likewise, for SL-LPA, valid results were obtained for 40 (88.9%) and 35 (77.8%) samples respectively using TrueNat extracted DNA and conventionally extracted DNA respectively. All samples with invalid LPA results had Ct values ≥ 28 by TrueNat PCR. LPA results were obtained for all the 20 samples using stored DNA at all temperatures and duration. ConclusionsTrueNat extracted DNA can be used for performing LPA under field conditions for selected samples.

Newer TB diagnostics: An update

In recent years, nucleic-acid amplification tests (NAATs), which are highly specific and sensitive, have helped to transform the TB diagnostic landscape. According to the WHO 2021 Guidelines on Diagnostics, the NAATs used in TB diagnosis at the point of care (POC) include Xpert MTB/RIF a cartridge-based test manufactured by Cepheid, and Truenat a chip-based test manufactured by Molbio. Other POC tests that are expected to be implemented in near future include Xpert Omni and Xpert MTB/XDR. The use of line probe assay is involved at the level of reference labs for the detection of MTB and its resistance to first-line (Isoniazid and Rifampicin) and second-line (fluoroquinolones and second-line injectables) drugs. When the currently available NAATs detect mutations for drug resistance at a particular region of MTB sequence, the Whole genome sequencing (WGS) platform demonstrates the exceptional potential for reliable and comprehensive resistance prediction for MTB isolates, by multiple gene regions or whole genome sequence analysis allowing for accurate clinical decisions.

Diagnostic accuracy of Xpert MTB/RIF Ultra and culture assays to detect Mycobacterium Tuberculosis using OMNIgene-sputum processed stool among adult TB presumptive patients in Uganda.

Stool is a potential sample for diagnosing Mycobacterium tuberculosis (Mtb) in patients with difficulty in expectorating. However, high mycobacterial culture contamination rates and Xpert MTB/RIF Ultra test error rates on stool samples have limited its use. OMNIgene SPUTUM (OM-S) is a sample transport reagent with characteristics of sputum decontamination while maintaining viable Mtb. We evaluated the impact of OM-S on Mtb diagnostic yield from stool using smear microscopy, Xpert MTB/RIF Ultra, and culture among presumptive TB patients. Paired stool and expectorated sputum samples were collected from consecutive Ugandan adults undergoing diagnostic evaluation for pulmonary TB between June 2018 and June 2019. Stool was divided into 2 portions: one was homogenized in OM-S (OM-S stool) and the other in PBS (PBS stool) as control. Both sputum and processed stool were tested for Mtb using concentrated smear fluorescence microscopy (CFM), Xpert MTB/RIF Ultra (Xpert) and Mycobacteria Growth Indicator Tube (MGIT) culture. Sensitivity, specificity, and predictive values for each test were calculated against sputum MGIT culture as the reference standard. Of the 200 participants, 120 (60%) were male, 73 (37%) were HIV positive (median CD4 120 cells/uL (IQR 43-297)) and 128 (64%) had confirmed pulmonary TB by sputum MGIT culture. Seven (4%) OM-S stool Xpert samples reported errors while 47 (25%) and 103 (61%) were contaminated on OM-S stool MGIT and PBS stool MGIT, respectively. OM-S stool MGIT was able to accurately diagnose 56 of the contaminated PBS stool MGIT samples compared to only 5 of the contaminated OM-S stool MGIT samples diagnosed by PBS stool MGIT. Sensitivity (95% Confidence Interval, CI) 89% (83-94) for OM-S stool Xpert was higher compared to that of OM-S stool MGIT 60% (51-69) and PBS stool MGIT 42% (32-52). Specificity (95%CI) 91% (82-97) was also higher for OM-S stool Xpert compared to OM-S stool MGIT 64% (51-75) and PBS stool MGIT 26% (16-38). Stool processed with OM-S showed potential to improve Mtb diagnostic yield and reduce rates of indeterminate results when tested on Xpert and MGIT culture. The method may thus be of value in Mtb detection among patients with difficulty to expectorate.

Comparison of gastric lavage/sputum and stool specimens in the diagnosis of pediatric pulmonary tuberculosis- A pilot study

Background and ObjectiveGlobal TB report 2021 mentions 11 % prevalence of pediatric TB, whereas 5.65% of the cases were reported from India in 2020. India features in the list of TB high burden countries, HIV-TB high burden and MDR-TB high burden countries. The diagnosis of pulmonary tuberculosis in children is difficult as they tend to swallow the sputum, invasive techniques of gastric aspirates needs to be followed and the disease itself is paucibacillary. The disease progresses rapidly in young children and hence rapid diagnosis is needed. Obtaining appropriate respiratory samples for diagnosis is difficult especially in primary care settings. Stool sample is easy to obtain and since children swallow sputum, it can be used to diagnose pulmonary tuberculosis. With this background, a pilot study was planned to evaluate the accuracy of the Xpert MTB/RIF assay for the detection of MTB in stool specimens obtained from pediatric pulmonary TB patients confirmed either by gastric lavage(GL) or sputum(SP) Xpert MTB/RIF assay. In addition, the results of microscopy of stool specimen were compared with that of gastric lavage/ sputum (GL/SP) specimen by Ziehl-Neelsen (ZN) and fluorescent light-emitting diode (LED) staining. Material and methodsA prospective study was carried out on 50 GL/SP Xpert MTB/RIF assay positive children (0-14 years). Stool specimens from these children were processed for Xpert MTB/RIF assay. The GL/SP and stool specimens were processed for ZN and Auramine O fluorescent microscopy as well. ResultsFluorescent staining detected acid fast bacilli (AFB) in 24 GL/SP and 16 stool specimens as compared to 20 GL/SP and 10 stool specimens by ZN staining. Stool Xpert MTB/ RIF assay was positive in 29 out of 50 children. Rifampicin resistance was detected in 13 of the 50 (26%) GL/SP specimens. Of these 13 children, rifampicin resistance was detected in 7 stool specimens, rifampicin indeterminate resistance was detected in one specimen and in the remaining 5 children, M.tuberculosis was not detected in stool. ConclusionStool is a good non-invasive specimen for the detection of pulmonary TB in children, especially in remote areas, where invasive techniques cannot be performed for sample collection.

Assessment of Smear-Positive Pulmonary Tuberculosis and Associated Factors among Patients Visiting Health Facilities of Gedeo Zone, Southern Ethiopia: A Cross-Sectional Study.

Tuberculosis (TB) was one of the top causes of ill health and the leading cause of deaths worldwide until the coronavirus (COVID-19) pandemic. Hence, this study is aimed at assessing the prevalence of sputum smear-positive TB and associated factors among TB-suspected patients attending in Gedeo Zone health facilities, Southern Ethiopia. A facility-based cross-sectional study was conducted among 220 TB-suspected patients in Gedeo Zone health facilities from July 01 to Sep 30, 2021. Patients were grouped as smear positive if one sputum out of two was positive or two sputum smears became positive. Various descriptive statistics were computed using the SPSS-25, and factors to smear positivity were identified by multivariable logistic regression analysis. Odds ratio at 95% CI and p values < 0.05 were considered as indicators of statistical association. The overall prevalence of smear-positive TB in Gedeo Zone health facilities was 18.2%, which is significantly high, and the MTB detection rate of GeneXpert was 29.5%. Contact with a TB patient, cigarette smoking, and previously treatment for TB were factors significantly associated with smear-positive TB. The prevalence rate of smear-positive PTB in the study area was 18.2% and 29.5% by direct sputum AFB and sputum GeneXpert, respectively. As a result, we recommend intervention on the identified associated risk factors and further studies to ascertain risk factors and their magnitude at the community level.