Mycobacterium Tuberculosis DNA Research Articles

Whispers in the wind: Face mask sampling for Mycobacterium tuberculosis detection in children with pulmonary tuberculosis.

Recently, face mask sampling (FMS) confirmed detection of Mycobacterium tuberculosis DNA from exhaled breath in adults with TB. To date, no study has evaluated the use of FMS to detect pulmonary Tuberculosis (TB) in children. We developed a method for FMS of M. tuberculosis-specific DNA in children and performed a clinical exploration to assess feasibility in children. Face masks were spiked, analysed on GeneXpert-Ultra, qPCR, and tNGS. Children with pulmonary TB were asked to wear three modified FFP2 masks for 30 minutes as part of an exploratory clinical study. Experiments with H37Ra M. tuberculosis strain showed a limit of 95% detection of 3.75 CFU (4.85-3.11; 95%CI) on GeneXpert-Ultra. Ten children with pulmonary TB participated in the clinical study. M. tuberculosis-specific DNA was detected on none of the face masks. Paediatric FMS has a low limit of detection for M. tuberculosis-specific DNA in vitro. However, M. tuberculosis DNA was not detected in any of thirty masks worn by children with pulmonary TB. This suggests that FMS in this form may not be more effective for detecting M. tuberculosis in children with TB than existing methods.

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Detection of <i>M. tuberculosis</i> Using Cartridge-Based Technology in Patients with Negative Sputum Microscopy Results in a Regional Laboratory

The objective: to evaluate results of M. tuberculosis detection and testing drug susceptibility to rifampicin using molecular genetic methods in tuberculosis patients with a negative sputum smear microscopy result.Subjects and Methods. Pulmonary tuberculosis patients registered in Arkhangelsk Region from 2016 to 2020 were enrolled in the study; those patients were notified as new cases and relapses with a negative sputum smear microscopy result before treatment and had results of molecular genetic testing (MGT) (Xpert MTB/RIF MTB/RIF) and BACTEC MGIT 960 available.Results. 479 people (40%) were registered as pulmonary tuberculosis cases with a negative sputum smear microscopy result. MGT was performed in 327 (68%) patients. A positive result of MGT was received in 120 (37%) patients, a positive result of BACTEC MGIT 960 was received in 165 (50%) people. Concordance of results was observed in 70% of cases. In 136 (41%) people, both tests did not detect Mycobacterium tuberculosis, in 95 (29%) both tests showed a positive result. Discrepancies between the results of two tests were observed in 30% of cases. In 71 (22%) cases, culture growth was observed in BACTEC MGIT 960 with negative results of MGT. In 8% of cases, DNA of Mycobacterium tuberculosis was detected, but there was no culture growth on the nutrient medium. 43/120 (36%) cases of resistance to rifampicin were detected. The median time from sputum collection to initiation of MDR-TB treatment based on sputum MGT results made 18 (IQR 10–29) days. Molecular genetic testing makes it possible to promptly detect the majority of TB patients with a negative sputum smear result, and reduce the time for drug susceptibility testing, which allows this testing to be used as the first test when examining for pulmonary TB.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

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.

Detection of Mycobacterium tuberculosis DNA in CD34+ peripheral blood mononuclear cells of adults with tuberculosis infection and disease

ObjectivesTo investigate whether Mycobacterium tuberculosis (Mtb) DNA is detected in peripheral blood mononuclear cells (PBMC) of subjects with tuberculosis (TB) or TB infection (TBI) living in a low-burden country. MethodsWe prospectively enrolled 57 patients with TB, 41 subjects with TBI, and 39 controls in Rome, Italy. PBMC were isolated, cluster of differentiation (CD)34+ and CD34− cells were immunomagnetic separated, DNA was extracted, and digital polymerase chain reaction for IS6110 and rpoB sequences was used to detect Mtb DNA in PBMC subsets and unfractionated PBMC. ResultsWe detected Mtb DNA at a low copy number in CD34+ cells in 4o f 30 (13%) patients with TB, 2 of 24 (8%) subjects with TBI, and 1 of 24 (4%) controls. Mtb DNA was detected in unfractionated PBMC in 3 of 51 (6%) patients with TB, 2 of 38 (5%) subjects with TBI, and 2 of 36 (6%) controls. In CD34− cells, only 1 of 31 (3%) subjects with TBI tested positive for Mtb DNA. ConclusionsMtb DNA was detected at low frequencies and levels in the PBMC of subjects with TBI and donors with TB living in a low-burden country. In particular, Mtb DNA was detected more frequently in CD34+ cells, supporting the hypothesis that these cells may represent a Mtb niche. This finding informs biological understanding of Mtb pathogenesis and may support the development of a microbial blood biomarker for Mtb infection.

Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study

Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

PET-CT guided characterisation of progressive, preclinical tuberculous infection and its association with low-level circulating Mycobacterium tuberculosis DNA in household contacts in Leicester, UK: a prospective cohort study

Incipient tuberculosis, a progressive state of Mycobacterium tuberculosis infection with an increased risk of developing into tuberculosis disease, remains poorly characterised. Animal models suggest an association of progressive infection with bacteraemia. Circulating M tuberculosis DNA has previously been detected in pulmonary tuberculosis by use of Actiphage, a bacteriophage-based real-time PCR assay. We aimed to investigate whether serial [18F]fluorodeoxyglucose ([18F]FDG)-PET-CT could be used to characterise the state and progressive trajectory of incipient tuberculosis, and examine whether these PET-CT findings are associated with Actiphage-based detection of circulating M tuberculosis DNA. We did a prospective 12-month cohort study in healthy, asymptomatic adults (aged ≥16 years) who were household contacts of patients with pulmonary tuberculosis, and who had a clinical phenotype of latent tuberculosis infection, in Leicester, UK. Actiphage testing of participants' blood samples was done at baseline, and [18F]FDG PET-CT at baseline and after 3 months. Baseline PET-CT features were classified as positive, indeterminate, or negative, on the basis of the quantitation (maximum standardised uptake value [SUVmax]) and distribution of [18F]FDG uptake. Microbiological sampling was done at amenable sites of [18F]FDG uptake. Changes in [18F]FDG uptake after 3 months were quantitatively categorised as progressive, stable, or resolving. Participants received treatment if features of incipient tuberculosis, defined as microbiological detection of Mtuberculosis or progressive PET-CT change, were identified. 20 contacts were recruited between Aug 5 and Nov 5, 2020; 16 of these participants had a positive result on IFNγ release assay (QuantiFERON-TB Gold Plus [QFT]) indicating tuberculosis infection. Baseline PET-CT scans were positive in ten contacts (all QFT positive), indeterminate in six contacts (three QFT positive), and negative in four contacts (three QFT positive). Four of eight PET-CT-positive contacts sampled had M tuberculosis identified (three through culture, one through Xpert MTB/RIF Ultra test) from intrathoracic lymph nodes or bronchial wash and received full antituberculosis treatment. Two further unsampled PET-CT-positive contacts were also treated: one with [18F]FDG uptake in the lung (SUVmax 9·4) received empirical antituberculosis treatment and one who showed progressive [18F]FDG uptake received preventive treatment. The ten untreated contacts with [18F]FDG uptake at baseline (seven QFT positive) had stable or resolving changes at follow-up and remained free of tuberculosis disease after 12 months. A positive baseline Actiphage test was associated with the presence of features of incipient tuberculosis requiring treatment (p=0·018). Microbiological and inflammatory features of incipient tuberculosis can be visualised on PET-CT and are associated with M tuberculosis detection in the blood, supporting the development of pathogen-directed blood biomarkers of tuberculosis risk. MRC Confidence in Concept.

The Xpert® MTB/RIF cycle threshold value predicts M. tuberculosis transmission to close contacts in a Brazilian prospective multicenter cohort.

The Xpert® MTB/RIF rapid molecular test provides a quantitative measure of Mycobacterium tuberculosis (Mtb) DNA in the form of cycle threshold (Ct) values. This information can be translated into mycobacterial load and used as a potential risk measure of bacterial spread for tuberculosis cases, which can impact infection control. However, the role of Ct values in assessing Mtb transmission to close contacts has not yet been demonstrated. A prospective study was performed to investigate the association between Xpert® MTB/RIF Ct values and Mtb transmission to close contacts of patients with culture-confirmed pulmonary TB in a multi-center Brazilian cohort. We evaluated clinical and laboratory data, such as age, sex, race, smoking habits, drug use, alcohol use, chest radiograph, Xpert® MTB/RIF results among pulmonary tuberculosis cases, and QuantiFERON(QFT)-Plus results at baseline and after six months for close contacts who had a negative result at baseline. A total of 1,055 close contacts of 382 pulmonary tuberculosis cases were included in the study. The median Ct values from pulmonary tuberculosis cases of QFT-Plus positive (at baseline or six months) close contacts were lower compared with those who were QFT-Plus negative. An adjusted logistic regression demonstrated that reduced Ct values from the index cases were independently associated with QFT-Plus conversion from negative to positive (OR: 1.61, 95% CI: 1.12-2.32) after adjusting for clinical characteristics. Close contacts of pulmonary TB index cases exhibiting low Xpert MTB/RIF Ct values displayed higher rates of TB infection, reflecting Mtb transmission.

Macromolecular crowding potently stimulates DNA supercoiling activity of Mycobacterium tuberculosis DNA gyrase

Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyethylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the KM of Mtb DNA gyrase, and simultaneously increased the Vmax or kcat of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of GyrB in a closed or semi-closed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of GyrB. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.

Transrenal Mycobacterium tuberculosis DNA in pulmonary tuberculosis patients during the first 14 days of treatment.

We assessed the performance of a novel real-time PCR-based transrenal DNA (trDNA) assay for the specific detection of Mycobacterium tuberculosis as a candidate marker of the early anti-tuberculosis therapy response. The study was performed on 288 urine samples from 72 tuberculosis patients collected at baseline and days 3, 7, and 14 of treatment with amoxicillin-clavulanic acid alone or in combination with meropenem, ertapenem, optimized-dose rifampicin, or standard treatment control in South Africa. trDNA was detected in one-third of the samples. The highest proportion of positive PCR results (cycle threshold < 36) was observed on days 3 and 7, reflecting the point in time when maximum bacterial killing and disintegration are expected. When analyzed by study arms, the trend was observed in groups treated with active antibiotics affecting cell wall integrity (meropenem, control) but not in inactive drugs (ertapenem, amoxicillin/clavulanic acid alone) or active drugs not affecting the cell wall (rifampicin). Overall, however, the trDNA assay did not correlate well with sputum culture-based decline of viable bacteria. This is possibly due to trDNA reflecting the killing of both culturable and non-culturable bacteria and should be explored further.IMPORTANCEThis study presents the results of the evaluation of a novel method for the detection of Mycobacterium tuberculosis, the causative agent of tuberculosis, in urine. Detecting parts of the mycobacteria in urine is of particular interest as it allows us to use a sample that is easy to obtain and that does not require uncomfortable procedures or safety precautions like obtaining sputum for culture, which is the most commonly used sample in the diagnosis of tuberculosis. In certain groups of individuals who cannot produce sputum, for example, children, non-sputum-based methods have particular importance. We found that the method tested was able to detect bacterial killing by active antibiotics that disrupt the cell wall and lead to fragmentation of bacteria. However, the assay can't detect inactive bacteria or bacteria that are active with an intact cell wall.

GeneXpert Ultra in Urine Samples for Diagnosis of Extra-Pulmonary Tuberculosis.

Extra-pulmonary tuberculosis (EPTB) continues to be difficult to diagnose. Novel biomarkers in biological specimens offer promise. Detection of Mycobacterium tuberculosis (Mtb) DNA in urine could prove useful in diagnosis of EPTB, possibly due to disseminated disease or micro-abscesses reported in kidneys. The current study was designed to detect Mtb DNA in stored urine samples from patients with EPTB. Diagnosis of EPTB was reached using Microbiological Reference Standards (MRS) on samples from the disease site using WHO Recommended Diagnostics (WRD), [smear microscopy, liquid culture (MGIT-960)] and GX (molecular WRD, mWRD) and Comprehensive reference standards [CRS, clinical presentation, microbiological reference standards, radiology, histopathology]. GX-Ultra was performed on urine samples stored in -80oC deep freezer, retrospectively. Of 70 patients, 51 (72.9%) were classified as confirmed TB, 11 (15.7%) unconfirmed TB, and 8 (11.4%) unlikely TB. GX-Ultra in urine samples demonstrated sensitivity of 52.9% and specificity of 57.9% against MRS, and higher sensitivity of 56.5% and specificity of 100% against CRS. The sensitivity and specificity of GX-Ultra in urine was 53.6% and 75% for pus sample subset and 52.2% and 53.3% for fluid sample subset. Urine being non-invasive and easy to collect, detection of Mtb DNA using mWRD in urine samples is promising for diagnosis of EPTB.

Polyheterocyclic peptidomimetics: Parallel solid phase synthesis of oligo cyclic guanidines and their inhibition activity against Mycobacterium tuberculosis DNA gyrase

Polyheterocycles are one of the most desired synthetic targets due to their numerous and valuable applications in various fields. We report the design and the parallel synthesis of novel linear oligocyclic guanidine peptidomimetics from predesigned reduced polyamides. A screening of these compounds identified active Mycobacterium tuberculosis DNA gyrase inhibitors which do not inhibit human DNA topoisomerase IIα and topoisomerase I.

Performance evaluation of QuantStudio 1 plus real-time PCR instrument for clinical laboratory analysis: A proof-of-concept study

ObjectiveThe real-time PCR system is one of the most powerful research tools available in the life sciences field. The aim of this study was to preliminarily evaluate the analytical performance of QuantStudio 1 Plus real-time PCR system (QS 1 plus) for clinical procedures. MethodsThe consistency of QS 1 plus with the reference system in terms of various clinical procedures was evaluated. For qualitative data, the Kappa test was used to analyze the agreement of the results. For the quantitative data, Passing-Bablok regression analysis and Bland-Altman plot analysis were used to assess the concordance between QS 1 plus and the reference instrument. ResultsPassing-Bablok regression showed an excellent agreement between the QS 1 plus and LC 480 systems for HBV DNA quantification (y = 0.928 + 0.970x), whereas Bland-Altman plot analysis showed very small mean deviations between the two systems. The QS 1 plus yielded perfectly consistent results with the reference instrument for methylenetetrahydrofolate reductase (MTHFR) C677T melting curve genotyping analysis, MTHFR C677T genotyping analysis, Norovirus RNA negative/positive analysis, influenza B virus (Flu B) RNA negative/positive analysis, Mycobacterium tuberculosis (MTB) DNA negative/positive analysis, Human Papillomavirus (HPV) genotyping analysis, epidermal growth factor receptor (EGFR) gene mutation analysis. Both the relative quantitative analysis and the relative quantitative analysis (standard curve) confirmed the satisfactory concordance between the QS 1 plus instrument and the ABI 7500 instrument by Passing-Bablok regression analysis (y = 0.180 + 0.817x and y = 0.012 + 1.000x, respectively) and Bland-Altman plot analysis. ConclusionsOur research has proven that QS 1 plus is adaptable to most test procedures in the clinical laboratory. This may provide the basis for its further application.

New Horizons in the Diagnosis of Tuberculosis of the Spine: The Role of Whole Genome Sequencing.

Prospective study. To evaluate the utility of whole genome sequencing (WGS) in drug resistance testing, lineage of the organisms, and organism- related factors responsible for bacilli settling in the spine. The workstream for the diagnosis of tuberculosis (TB) involves isolation and culture of the organism and drug resistance testing using phenotypic methods. Xpert MTB/RIF Ultra is a genetic-based method that detects for Mycobacterium tuberculosis DNA in the rpoB gene. Meanwhile, WGS is a newer genetic-based method that assesses the whole genome of the bacterium. Very few studies have reported the use of WGS for extrapulmonary TB. Herein, we used WGS to diagnose spinal TB. Tissues from 61 patients undergoing surgery for spinal TB underwent histologic examination, Xpert MTB/RIF Ultra, and culture and sensitivity testing. DNA from the cultured bacteria was sent for WGS. The test bacterial genome was compared to a reference strain of pulmonary TB. Acid-fast bacilli were observed in 9/58 specimens. Meanwhile, histology confirmed TB in all the patients. Bacilli were cultured in 28 patients (48.3%), and the average time to culture was 18.7 days. Xpert MTB/RIF Ultra was positive in 47 patients (85%). WGS was performed in 23 specimens. Overall, 45% of the strains belonged to lineage 2 (East Asian). There was one case of multidrug- resistant TB and two cases of non-tuberculous mycobacteria on WGS. We could not confirm any genomic difference between pulmonary and spinal TB strains. Xpert MTB/RIF Ultra of tissues or pus is the investigation of choice when diagnosing spinal TB. Meanwhile, WGS can diagnose multidrug-resistant TB and non-tuberculous mycobacteria more accurately. No mutations were identified in spinal and pulmonary TB bacteria.

The value of cell free DNA of Mycobacterium tuberculosis in the early diagnosis of tuberculous meningitis

Objective: To detect the cell-free DNA of Mycobacterium tuberculosis (Cf-TB) in the cerebrospinal fluid (CSF) of patients with tuberculous meningitis (TBM), and to assess the diagnostic value of this method for TBM. Methods: We prospectively included patients with suspected meningitis from the Department of Tuberculosis, Beijing Chest Hospital, Department of Neurology, Beijing Chaoyang Hospital and Department of Neurology, 263 Hospital of the People's Liberation Army from September 2019 to March 2022. A total of 189 patients were included in this study. Among them, 116 were male and 73 were female, aged from 7 to 85 years, with an average of (38.5±19.1) years. The CSF specimens of the patients were collected for Cf-TB, MTB culture and Xpert MTB/RIF. SPSS 20.0 was used for statistical analysis and the difference was statistically significant with P<0.05. Results: Among the 189 patients, there were 127 patients in the TBM group and 62 patients in the non-TBM group. The sensitivity of Cf-TB was 50.4% (95%CI 41.4%-59.3%), the specificity was 100% (95%CI 92.7%-100.0%), the positive predictive value was 100% (95%CI 92.9%-100.0%), and the negative predictive value was 49.6% (95%CI 40.6%-58.6%). Using clinical diagnosis as the gold standard, the sensitivity of Cf-TB was 50.4% (64/127), which was significantly higher than that of MTB culture (8.7%, 11/127) and Xpert MTB/RIF (15.7%,20/127) (all P<0.001). Using etiology as the gold standard, the sensitivity of Cf-TB was 72.7% (24/33), which was significantly higher than that of MTB culture [33.3%, 11/33, (χ2=10.28, P=0.001)] and was similar to Xpert MTB/RIF (60.6%, 20/33) (χ2=1.091, P=0.296). Conclusion: The sensitivity of the Cf-TB test was significantly higher than that of CSF MTB culture and Xpert MTB/RIF. Cf-TB may provide evidence for earlier diagnosis and treatment of TBM.

Atypical presentation of tuberculosis: a case report highlighting the challenges of diagnosis and management

Tuberculosis (TB) remains a significant public health issue worldwide and can present with atypical symptoms, making it difficult to diagnose. Atypical TB symptoms include non-specific symptoms and extrapulmonary TB, and patients with comorbidities may present with atypical TB due to impaired immune function. TB treatment includes antibiotics, which can cause hepatotoxicity, and require close monitoring for signs of liver injury. A 32-year-old female with intellectual developmental disorders was admitted to the hospital with suspected pneumonia and acute respiratory viral infection. Despite initial treatment with antibiotics, her oxygen saturation decreased, and she developed recurrent pneumothorax on the right side and neutrophilic pleuritis. Computed tomography (CT) scans showed increasing infiltrative changes, and bronchoalveolar lavage eventually revealed Mycobacterium tuberculosis DNA, leading to the diagnosis of tuberculosis pneumonia with pleurisy. This case report highlights the challenges of diagnosing TB and the importance of maintaining a high index of suspicion for TB in patients who do not fit the typical profile of the disease. The case also emphasizes the potential for drug-induced liver damage with TB treatment and the need for close monitoring. Early diagnosis and appropriate management are crucial to prevent further complications and improve outcomes in patients with TB.

Optimizing mycobacteria molecular diagnostics: No decontamination! Human DNA depletion? Greener storage at 4 °C!

Tuberculosis (TB) is an infectious disease caused by the group of bacterial pathogens Mycobacterium tuberculosis complex (MTBC) and is one of the leading causes of death worldwide. Timely diagnosis and treatment of drug-resistant TB is a key pillar of WHO's strategy to combat global TB. The time required to carry out drug susceptibility testing (DST) for MTBC via the classic culture method is in the range of weeks and such delays have a detrimental effect on treatment outcomes. Given that molecular testing is in the range of hours to 1 or 2 days its value in treating drug resistant TB cannot be overstated. When developing such tests, one wants to optimize each step so that tests are successful even when confronted with samples that have a low MTBC load or contain large amounts of host DNA. This could improve the performance of the popular rapid molecular tests, especially for samples with mycobacterial loads close to the limits of detection. Where optimizations could have a more significant impact is for tests based on targeted next generation sequencing (tNGS) which typically require higher quantities of DNA. This would be significant as tNGS can provide more comprehensive drug resistance profiles than the relatively limited resistance information provided by rapid tests. In this work we endeavor to optimize pre-treatment and extraction steps for molecular testing. We begin by choosing the best DNA extraction device by comparing the amount of DNA extracted by five commonly used devices from identical samples. Following this, the effect that decontamination and human DNA depletion have on extraction efficiency is explored. The best results were achieved (i.e., the lowest Ct values) when neither decontamination nor human DNA depletion were used. As expected, in all tested scenarios the addition of decontamination to our workflow substantially reduced the yield of DNA extracted. This illustrates that the standard TB laboratory practice of applying decontamination, although being vital for culture-based testing, can negatively impact the performance of molecular testing. As a complement to the above experiments, we also considered the best Mycobacterium tuberculosis DNA storage method to optimize molecular testing carried out in the near- to medium-term. Comparing Ct values following three-month storage at 4 °C and at -20 °C and showed little difference between the two. In summary, for molecular diagnostics aimed at mycobacteria this work highlights the importance of choosing the right DNA extraction device, indicates that decontamination causes significant loss of mycobacterial DNA, and shows that samples preserved for further molecular testing can be stored at 4 °C, just as well at -20 °C. Under our experimental settings, human DNA depletion gave no significant improvement in Ct values for the detection of MTBC.

Detection of antigen Ag85B expression is useful for the diagnosis of tuberculosis, especially for those with an antituberculosis treatment history.

The present study used immunohistochemistry (IHC) to detect antigen Ag85B in tissue sections and aimed to evaluate its validity in histopathologic diagnosis of tuberculosis (TB). In total, 204 patients with confirmed TB and 40 other diseases were included in the present study. Ziehl-Neelsen (Z-N) stains, IHC (anti-Ag85B), and quantitative fluorescence polymerase chain reaction were used to detect acid-fast bacilli, Mycobacterium tuberculosis (MTB) antigen, and MTB DNA. Immunohistochemistry was significantly more sensitive than Z-N stains (93.1% vs 67.2%; P < .001). The sensitivity of Z-N stains significantly correlated with anti-TB treatment history. The sensitivity of Z-N stains was lower in rifampicin (RIF)-resistant TB compared with RIF-sensitive TB (52.8% vs 69.0%; P = .091) and those without treatment history (52.8% vs 84.0%; P = .015). However, IHC was not significantly affected by treatment history (P = .410). Moreover, expression patterns of Ag85B were dependent on treatment history and commonly showed weak scattered spots in RIF-susceptible TB. Conversely, strong brown rods were often found in those with RIF-resistant TB. Immunohistochemistry is a simple, sensitive technique for the diagnosis of TB, especially for those patients with treatment history. The expression pattern of Ag85B is a potential marker for evaluating anti-TB treatment response.

Prevalence and Patterns of Gene Mutations Associated with &lt;i&gt;M. tuberculosis&lt;/i&gt; Resistance to Isoniazid and Rifampicin in Patients with Different Clinical Manifestations of Tuberculosis

The objective: to study prevalence and patterns of mutations in the katG, inhA, ahpC, rpoB genes associated with Mycobacterium tuberculosis (MTB) resistance to isoniazid (H) and rifampicin (R) in patients with various clinical manifestations of pulmonary tuberculosis (TB).Subjects and Methods. 441 sputum samples collected in tuberculosis patients were tested using biological microchips. Tests were carried out in Group 1 - patients with confirmed bacterial excretion (n = 256) and in Group 2 (n = 185) - patients without bacterial excretion. The same patients were enrolled in Group 3 - patients with acute progressing tuberculosis (n = 52) and Group 4 (n = 99) - patients with localized tuberculosis.Results. In Group 1, DNA of Mycobacterium tuberculosis was found in 79.3% of patients, in Group 2 - in 57.8%. Among all samples, mutations in the genes encoding resistance to isoniazid were detected in 15.5%, resistance to rifampicin - in 58.1%. Resistance to isoniazid was more often caused by mutations in the katG gene (49%) versus the inhA (29%) and ahpC (4.2%) genes. We found 13 most common types of mutations in the rpoB gene associated with resistance to rifampicin. The dominant mutations in both groups were Seu531-&gt;Leu mutations - 19.7% in Group 1 and 24.3% in Group 2. In Group 1, mutations in the katG gene (53.7%) were observed more often than mutations in the inhA gene (27.7%). In Group 3, mutations in the katG gene were registered in 30.8%, in the inhA gene - in 25%. There were no statistically significant differences in patterns of mutations in the katG, inhA, ahpC, rpoB genes between Groups 1, 2 and 3, 4. Thus, patients without bacterial excretion and patients with localized tuberculosis are a hidden dangerous reservoir of tuberculous mycobacteria with multiple drug resistance to rifampicin and drug resistance to isoniazid.