Search

Rapid and accurate diagnosis of tuberculosis (TB) is of great significance to control the spread of this devastating infectious disease. In this work, a sensitive and low-cost point-of-care testing (POCT) detection platform for TB was developed based on recombinase polymerase amplification (RPA)-catalytic hairpin assembly (CHA)-assisted dual signal amplification strategy. This platform could achieve homogeneous fluorescent and visual diagnosis of TB by using CdTe quantum dots (QDs) signal reporter. In the presence of target DNA (IS1081 gene fragment), RPA amplicons blocked by short oligonucleotide strands could trigger CHA signal amplification, leading to the Ag + releasing from C-Ag + -C structure and the fluorescence quenching of CdTe QDs by the released Ag + . Furthermore, the detection performance of CdTe QDs modified by 3-mercaptopropionic acid (MPA) or thiomalic acid (TMA) (MPA-capped QDs and TMA-capped QDs) was systematically compared. Experimental results demonstrated that TMA-capped QDs exhibited better detection sensitivity due to their stronger interaction with Ag + . The limits of detection (LODs) of fluorescence and visual analysis were as low as 0.13 amol L -1 and 0.33 amol L -1 . This method was successfully applied to the clinical sputum samples from 36 TB patients and 20 healthy individuals, and its quantitative results were highly consistent with those obtained by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The proposed approach has the advantages of high sensitivity and specificity, simple operation and low cost, and is expected to be applied in clinical TB screening and diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Extrapulmonary tuberculosis (TB) is a relatively rare form of tuberculosis infection, accounting for approximately 15% of all tuberculosis infections. Lymph nodes are the most commonly affected sites, while involvement of the parotid gland is extremely rare., Case Presentation: We present the case of a 65-year-old male patient with a one-month history of a left parotid mass. The patient has a history of diabetes and long-term smoking, and a chest X-ray revealed secondary fibrotic pulmonary tuberculosis, while sputum smears were culture-negative for Mycobacterium tuberculosis (Mtb). The parotid mass was surgically removed and subjected to routine HE staining, acid-fast staining, and PCR molecular testing for Mtb. The final diagnosis was Warthin's tumor of the parotid gland with concomitant tuberculosis. One month after removal of the parotid mass, the patient's chest CT showed cavitary tuberculosis. Subsequently, the patient received anti-tuberculosis treatment; however, due to severe gastrointestinal adverse effects, the patient stopped the medication in less than a month and did not receive regular treatment. Four months after stopping the medication, the patient's pulmonary tuberculosis progressed and worsened., Conclusion: Combined tuberculosis in Warthin's tumor is extremely rare, with only 14 cases reported to date. However, the specific pathogenesis of this condition is not yet fully understood, and the preliminary treatment and prognosis have not been conclusively determined. Early diagnosis of tuberculosis, standardized and effective use of anti-tuberculosis drugs, and personalized treatment are crucial in the management of tuberculosis. We have reviewed the treatment progress of this rare disease and analyzed the potential pathogenesis of the condition. Furthermore, we have summarized the current understanding of the pathogenesis of tuberculosis, drug resistance mechanisms, and the latest treatment advances. These studies have important clinical implications for better understanding and treating extrapulmonary tuberculosis and tuberculosis within Warthin's tumor of the parotid gland. This comprehensive analysis sheds light on the complexities of tuberculosis and provides valuable insights for improved management and care of affected individuals., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Written and informed consent to publish was obtained from the patient. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Effective control of tuberculosis (TB) depends on early diagnosis of disease, yet available tests are unable to perfectly detect infected individuals. In novel hosts diagnostic testing methods for TB are extrapolated from other species, with unknown accuracy. The primary challenge to evaluating the accuracy of TB tests is the lack of a perfect reference test. Here we use a Bayesian latent class analysis approach to evaluate five tests available for ante-mortem detection of pulmonary TB in captive sun bears and Asiatic black bears in Southeast Asia. Using retrospective results from screening of 344 bears at three rescue centres, we estimate accuracy parameters for thoracic radiography, a serological assay (DPP VetTB), and three microbiological tests (microscopy, PCR (Xpert MTB/RIF, Xpert MTB/RIF Ultra), mycobacterial culture) performed on bronchoalveolar lavage samples. While confirming the high specificities (≥ 0.99) of the three microbiological tests, our model demonstrated their sub-optimal sensitivities (<0.7). Thoracic radiography was the only diagnostic method with sensitivity (0.95, 95% BCI: 0.76, 0.998) and specificity (0.95, 95% BCI: 0.91, 0.98) estimated above 0.9. We recommend caution when interpreting DPP VetTB results, with the increased sensitivity resulting from treatment of weakly visible reactions as positive accompanied by a drop in specificity, and we illustrate how the diagnostic value of weak DPP VetTB reactions is particularly reduced if disease prevalence and/or clinical suspicion is low. Conversely, the reduced utility of negative microbiological tests on bronchoalveolar lavage fluid samples when prevalence and/or clinical suspicion is high is demonstrated. Taken together our results suggest multiple tests should be applied and accompanied by consideration of the testing context, to minimise the consequences of misclassification of disease status of bears at risk of TB in sanctuary settings., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Officer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Peritoneal tuberculosis (TB) is a rare extrapulmonary infection caused by Mycobacterium tuberculosis It is mainly found in countries with a high burden of TB, so travelling from an endemic area should raise suspicion. Although it is a well-recognised disease, it remains a challenge due to its difficult clinical and microbiological diagnosis. This can lead to a delay, both in diagnosis and treatment with serious prognostic implications. We describe the case of a woman in her mid-50s who presented with fever, abdominal pain, vomiting, breathlessness, anorexia and weight loss. On physical examination, she had decreased breath sounds at the right lung and clinical ascites. Blood tests revealed elevated inflammatory markers and anaemia. Imaging showed ascites, enhanced peritoneal thickening, densification of the mesenteric fat and right pleural effusion. After paracentesis, diagnostic thoracotomy and laparoscopy were undertaken. Based on a presumptive diagnosis, treatment was started. M. tuberculosis was lastly isolated in ascitic fluid culture after 6 weeks. The patient completed a 6-month course of anti-TB drugs with a favourable outcome., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Current tuberculosis (TB) treatment is typically effective against drug-susceptible Mycobacterium tuberculosis, but can fail due to acquired drug resistance or phenotypic resistance. M. tuberculosis persisters, a subpopulation of viable but non-replicating (VBNR) antibiotic-tolerant bacteria, are thought to contribute to poor TB treatment outcomes. In this exploratory study, we investigated treatment-naïve drug-susceptible clinical isolates collected from people with TB, who subsequently had unsuccessful treatment outcomes. These were compared to isolates from cured individuals in terms of their ability to form VBNR subpopulations. Clinical isolates from individuals with unfavorable treatment outcomes form larger subpopulations of VBNR M. tuberculosis (2.67-13.71%) than clinical isolates from cured cases (0- 1.63%) following infection of THP-1 macrophages. All isolates were drug susceptible based on phenotypic and genotypic analysis. Whole genome sequencing identified 23 non-synonymous genomic variants shared by treatment failure clinical isolates, that were not present in isolates from cured cases. This exploratory study highlights the ability of treatment-naïve clinical isolates to form heterogeneous populations containing VBNR M. tuberculosis. We also demonstrate that clinical isolates from individuals with unsuccessful treatment outcomes form higher percentages of VBNR M. tuberculosis. The findings of this exploratory study suggest that an increased propensity to form VBNR subpopulations may impact TB treatment outcome., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval Ethical approval was obtained from the Stellenbosch Human Research Ethics Committee (Registration Number N10/01/013) and the Biological and Environmental Safety committee (Registration Number BES-2023-13049)., (© 2024. The Author(s).)

Identifying individuals with tuberculosis (TB) with a high risk of onward transmission can guide disease prevention and public health strategies. Here, we train classification models to predict the first sampled isolates in Mycobacterium tuberculosis transmission clusters from demographic and disease data. We find that supervised learning, in particular balanced random forests, can be used to develop predictive models to identify people with TB that are more likely associated with TB cluster growth, with good model performance and AUCs of ≥ 0.75. We also identified the most important patient and disease characteristics in the best performing classification model, including host demographics, site of infection, TB lineage, and age at diagnosis. This framework can be used to develop predictive tools for the early assessment of potential cluster growth to prioritise individuals for enhanced follow-up with the aim of reducing transmission chains., Competing Interests: Competing interests The authors declare no competing interests. Ethics declarations Ethics were obtained from the University of British Columbia (certificate H12-00910) and informed consent for participation in the study was not required, as determined by institutional REB review., (© 2024. The Author(s).)

Objective: To 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., Design: We 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., Results: Several 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 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 CT, and the 3D microanatomy characterisation of human tuberculosis lung using μ 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., Conclusion: The 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., (© 2024. The Author(s).)

Brain abscesses caused by Mycobacterium tuberculosis are rarely reported and are typically found in immunocompromised patients. The challenges in identifying the pathogen and the frequent delays in initiating appropriate therapy often result in a poor prognosis for patients. We herein report a rare case of a brain abscess caused by M. tuberculosis in a 63-year-old patient with a history of hypertension but no history of pulmonary tuberculosis. The patient was hospitalized twice because of worsening right upper limb weakness and speech difficulties. Both a pus culture and cerebrospinal fluid culture were negative. Ultimately, the diagnosis of M. tuberculosis infection was confirmed through next-generation sequencing and the tuberculosis-specific T-cell test (T-SPOT). The patient's symptoms improved following anti-tuberculosis treatment. This case highlights the importance of considering M. tuberculosis as a potential cause of brain abscesses, even in immunocompetent individuals, and underscores the need for early diagnosis for effective treatment., Competing Interests: Declaration of conflicting interestsThe authors declare that they have no conflicts of interest.

Background: Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results., Methods: Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD., Results: The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile., Conclusions: Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias., (© 2024 John Wiley & Sons Ltd.)

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R 2  = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Context.—: Recent publications have featured immunohistochemistry (IHC) as a sensitive tool for detecting Mycobacterium tuberculosis and nontuberculous mycobacteria, but performance is limited to cases suspected to have mycobacterial infection., Objective.—: To examine cross-reactivity of a polyclonal antimycobacterial antibody with various types of pathogens, tissues, and inflammatory patterns., Design.—: Surgical pathology files during a period of 6 years were searched, and 40 cases representing a variety of pathogens, tissue types, and inflammatory responses were retrieved. Cases were stained with a rabbit polyclonal antimycobacterial antibody (Biocare Medical, Pacheco, California). The cases and associated histochemical stains, culture, and molecular results were reviewed by 3 pathologists., Results.—: All 8 cases of mycobacterial infection previously diagnosed by other methods were positive for mycobacteria by IHC. In addition, multiple bacterial and fungal organisms and 1 case of Leishmania amastigotes were also immunoreactive with the mycobacterial IHC., Conclusions.—: Although highly sensitive for mycobacteria, the polyclonal antibody shows significant cross-reactivity with other organisms. This is a sensitive but nonspecific stain that can be used as an alternative confirmation method for mycobacteria, but attention should be paid to inflammatory reaction and organism morphology when IHC is positive to avoid misdiagnosis., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Laboratory algorithms using Acid-Fast Bacilli staining and Mycobacterium tuberculosis (Mtb) polymerase chain reaction (PCR) are often used to remove isolation precautions. A retrospective case review of 52 patients with culture-confirmed pulmonary Mtb revealed 4 subjects with negative sputum Acid-Fast Bacilli smears and negative Mtb PCRs. All had significant risk factors for Mtb and had a positive interferon-gamma release assay. A negative PCR test result does not exclude an Mtb diagnosis., (Copyright © 2024 Association for Professionals in Infection Control and Epidemiology, Inc. All rights reserved.)

Introduction: Early and accurate diagnosis of tuberculosis (TB) is crucial for initiating timely treatment and preventing new infections. In this study, we introduced the iFIND TBR assay, an automated all-in-one tuberculosis detection approach that simultaneously detect Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance., Methods: The limits of detection (LOD), sensitivity, specificity, and RIF-R rpoB mutation detection of the iFIND TBR were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M.tuberculosis H37Rv. Frozen clinical samples from patients suspected of having TB were also tested., Results: The LOD of the iFIND TBR for MTB detection were 13.34 CFU/ml (95% CI, 11.71-16.47), and for RIF resistance was 109.79CFU/mL (95% CI, 95-138.19). The iFIND TBR assay accurately distinguish MTB strains from non-tuberculous mycobacteria (NTM) without any cross reactivity. Testing on 157 clinical sputum samples, compared with the bacteriologically TB standard, the overall sensitivity and specificity of the iFIND TBR was 100% (95%CI, 94.64, 100) and 85.29% (95% CI, 74.61, 92.72), respectively. When assessing RIF susceptibility, the iFIND TBR achieved a sensitivity of 98.15% (95% CI, 90.11-99.95) and a specificity of 85.71% (95% CI, 67.33-95.97), compared with phenotypic drug susceptibility testing. Discordant RIF susceptibility results were more frequently observed in samples exhibiting heteroresistance., Discussion: These findings demonstrate that iFIND TBR assay performs well in detecting TB and RIF resistance, and shows promise as a point-of-care tool in resource-limited areas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ou, Song, Xing, Zhao, Pei, Teng, Zhang, Sun, Liu, Xia, Zhou, Zheng, Song, Zhang, Wang, Anthony and Zhao.)

Background: Retrospective and descriptive molecular epidemiology studies have shown that Mycobacterium tuberculosis whole genome sequencing can identify outbreaks and disease transmission events with higher resolution than conventional epidemiological investigations. Those studies have strengthened our understanding of genomic polymorphisms correlating with person-to-person transmission and helped resolve putative transmission clusters. To date, systematic genomic surveillance programs implemented for M. tuberculosis were only implemented in low-incidence settings. The purpose of this study is to determine whether there is an impact of routine M. tuberculosis whole genome sequencing on tuberculosis case detection in a high-incidence setting., Methods: A cluster randomized controlled trial will be performed. Forty-eight rural village groups (or Fokontany) in the Vohibato district of Madagascar will be randomized to one of three interventions arms. Arm 1 (standard of care) involves healthcare facility-based passive case detection with smear microscopy testing. Arm 2 (best practice) consists of active case finding and Xpert MTB/RIF Ultra PCR testing followed by household contact investigations. Arm 3 (novel intervention) includes the same interventions as arm 2, with addition of sputum culture and M. tuberculosis whole genome sequencing for all newly diagnosed cases. In arm 3, molecular suggested putative outbreaks are investigated, and additional TB suspects are appropriately tested. The intervention observational period will be 2 years. The primary outcome will be the number of detected cases/100,000/year in each arm after 1 year of intervention., Discussion: This study is designed to determine whether there is an impact of prospective whole genome sequencing-based molecular typing on tuberculosis case detection in high-incidence settings. Investigating potential outbreaks and focusing active case finding in spatiotemporal settings where disease transmission is suggested by genomic typing is hypothesized to improve case detection in rural communities., Trial Registration: ClinicalTrials.gov NCT05406453 . Retrospectively registered on June 6, 2022., (© 2024. The Author(s).)

Background: Mono-resistance to rifampicin/isoniazid increases poor treatment outcomes and the risk of multi-drug resistance (MDR) in tuberculosis (TB) patients. Limited information exists about mono-resistance status of TB patients in Uttar Pradesh, North India. This study aimed to estimate the burden of rifampicin and isoniazid mono-resistance in Western Uttar Pradesh., Methods and Results: 153 sputum samples of suspected pulmonary tuberculosis patients were processed to isolate Mycobacterium tuberculosis using the Lowenstein-Jensen (L-J) culture medium. The isolates were identified using an immuno-chromatographic test and IS6110 PCR. The confirmed Mycobacterium tuberculosis isolates were tested for drug susceptibility testing against rifampicin and isoniazid anti-tuberculosis drugs. The results of the drug susceptibility testing were compared with demographic information and analyzed statistically. Out of 153 sputum samples, 83 (54.24%) samples were positive for growth on L-J medium, including 82 (98.79%) Mycobacterium tuberculosis isolates. Of the 82 Mycobacterium tuberculosis isolates, 16 (19.51%), 7 (8.54%), and 5 (6.10%) isolates were MDR, mono-resistant to rifampicin and isoniazid, respectively. The occurrence of RIF/INH mono-resistant-TB was higher in patients of male gender, age above 45 years, living in rural conditions, history of weight loss, and previous anti-TB treatment, but the effect was not statistically significant., Conclusions: The study reported the status of rifampicin and isoniazid mono-resistance among TB patients and highlighted the need for continuous monitoring and improved intervention for the initial detection of mono-drug-resistant cases. This will improve clinical treatment outcomes and decrease the rate of drug-resistant TB in Uttar Pradesh, North India., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: To analyze the clinical manifestations, diagnostic and therapeutic processes of perinatal tuberculosis in children, providing reference for clinicians in the diagnosis and treatment of this disease., Methods: A retrospective analysis was conducted on the epidemiological history, clinical manifestations, laboratory and imaging findings, and treatment follow-up of 20 cases of perinatal tuberculosis diagnosed in the Second Department of Infectious Disease, Kunming Children's Hospital, from February 2014 to September 2021., Results: Of the 20 cases, 13 were male (65.0%) and 7 were female (35.0%). The average age at onset was 35.35 ± 23.03days, with an average time from onset to diagnosis of 24.75 ± 15.55days. Tuberculin skin test (TST) was positive in 1 out of 4 cases (25.0%). Gamma interferon release assays (IGRAs) were positive in 9 out of 17 cases (52.9%).acid-fast staining was positive in 7 out of 16 cases (43.7%), and Mycobacterium tuberculosis nucleic acid polymerase-chain-reaction(PCR) was positive in 14 out of 20 cases (70.0%). Chest CT showed miliary changes in 4 out of 19 cases (21.0%), multiple nodular and patchy opacities in 6 out of 19 cases (31.6%), and pulmonary consolidation and atelectasis in 10 out of 19 cases (52.6%). After anti-tuberculosis treatment, 16 out of 20 cases (80.0%) improved, and no recurrence of tuberculosis was observed during follow-up periods ranging from 9 months to 3 years. The growth and development of these children were similar to those of healthy children., Conclusion: The clinical manifestations and chest imaging features of perinatal tuberculosis are nonspecific. In suspected cases, it is crucial to investigate the mother's condition thoroughly and complete etiological examinations to achieve early diagnosis and timely treatment, which can improve prognosis., (© 2024. The Author(s).)

Cost efficient and rapid detection tools to detect mutations especially those linked to drug-resistance are important to address concerns of the rising multi-drug resistance infections. Here we integrated dual probes, namely a calibrator probe and an indicator probe, into isothermal amplification detection system. These two probes are designed to bind distinct regions on the same amplicon to determine the presence or absence of mutation. The calibrator probe signal is used as an internal signal calibrator for indicator probe which detects the presence or absence of the mutation. As an illustrative example, we evaluated the applicability of this dual probe method for detecting mutations associated with rifampicin (RIF) drug resistance at codons 516, 526 and 531 of the rpoB gene in Mycobacterium tuberculosis. In this assessment, we examined 127 artificial samples comprising wild types and mutants with single or multiple mutations. Our results demonstrated 100% accuracy for both wild types and mutants for mutations at codons 526 and 531. As regards to mutations at codon 516, the wild type was identified with 100% accuracy, while the mutants were identified with 95% accuracy. Moreover, when we extended our evaluation to include clinical MTB strains and the Zeptometrix MTB Verification panel, our method achieved 100% accuracy (5 out of 5) in identifying wild-type strains. Additionally, we successfully detected a RIF-resistant strain with mutations at codon 531 of the rpoB gene in Zeptometrix verification panel. Our isothermal mutation detection system, relying on dual probes exhibits a versatile approach. With the capability to identify mutations without prior knowledge of their specific mutation direction, our dual-probe method shows significant promise for applications in drug resistance nucleic acid testing, particularly in resource-limited settings., Competing Interests: The proprietary subjects disclosed herein are property of Revvity, Inc. Although the authors are employed and funded by Revvity, Inc, this should not detract from the objectivity of data generation or interpretation., (Copyright: © 2024 Nandu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A paradoxical reaction (PR) during the treatment of tuberculosis was defined as the worsening of preexisting disease either clinically or radiologically or the appearance of a new tuberculous lesion. These reactions are frequently observed in patients coinfected with human immunodeficiency virus (HIV) upon the initiation of antiretroviral therapy (ART). Herein, we present a unique case of a paradoxical reaction in a previously healthy 19-year-old female who started anti-tuberculosis treatment for disseminated tuberculosis. Four weeks after treatment initiation, she developed two new swollen masses in her left dorsum of the hand, accompanied by fever and new right submandibular painful lymphadenopathy, with worsening of the preexisting left lower neck lymph node. The patient underwent needle aspiration from her new skin abscess on the dorsum of her left hand, which revealed positive polymerase chain reaction (PCR) for Mycobacterium tuberculosis. Anti-tuberculosis treatment was continued, and the patient fully recovered. We described an unusual presentation of paradoxical reaction manifested by a skin abscess at a site distant from her primary disease in an immunocompetent TB patient, which demonstrated the importance of considering paradoxical reactions in HIV-negative patients who present with worsening signs and symptoms after initial improvement following treatment initiation., (© 2024. The Author(s).)

Background: India, with the highest global burden of tuberculosis (TB) and drug-resistant TB, aims to eliminate TB by 2025. Yet, limited evidence exists on drug resistance patterns and retreatment among patients with silico-tuberculosis. This study explores these patterns and assesses the impact of silicosis on TB retreatment in India., Methods: This secondary data analysis stems from a larger retrospective cohort study conducted in Khambhat, Gujarat, between January 2006 and February 2022. It included 138 patients with silico-tuberculosis and 2,610 TB patients without silicosis. Data from the Nikshay TB information portal were linked with silicosis diagnosis reports from the Pneumoconiosis Board using the unique Nikshay ID as the linking variable. Drug-resistant TB was defined as resistance to any anti-TB drug recorded in Nikshay. Retreatment refers to TB patients who have previously undergone anti-TB treatment for one month or more and need further treatment. Recurrent TB denotes patients who were previously declared cured or had completed treatment but later tested positive for microbiologically confirmed TB. Multivariable logistic regression was used to determine the impact of co-prevalent silicosis on drug resistance and retreatment., Results: Patients with silico-tuberculosis showed a higher proportion of retreatment compared to those without silicosis (55% vs. 23%, p < 0.001). Notably, 28% of patients with silico-tuberculosis were recurrent TB cases, compared to 11% among those without silicosis. Regarding drug resistance, the silico-tuberculosis group exhibited a higher rate (6% vs. 3%), largely due to rifampicin resistance (5% vs. 2%, p = 0.022). Co-prevalent silicosis was associated with a 2.5 times greater risk of drug-resistant TB (adjusted OR 2.5, 95% CI, 1.1-5.3; p = 0.021). Additionally, patients with silico-tuberculosis had a fourfold increased risk of retreatment for TB (adjusted OR 4, 95% CI, 3-6; p < 0.001)., Conclusions: Co-prevalent silicosis significantly elevates the risk of drug resistance, recurrence, and retreatment among TB patients in India. This study indicates a need for improved treatment protocols and suggests that future research should focus on randomized controlled trials to evaluate appropriate anti-TB regimen and duration of therapy for this high-risk group. Given India's goal to eliminate TB by 2025, addressing the challenges posed by silico-tuberculosis is critical., (© 2024. The Author(s).)

In 2014, the World Health Organisation (WHO) published target product profiles (TPP) for development of novel tuberculosis (TB) diagnostics. One of the key highlights is the need for point-of-care non-sputum-based tests capable of detecting all forms of TB through identification of characteristic biomarkers or biosignatures. Compared to the limitations associated with sputum-based TB tests, non-sputum samples are easy to collect, non-invasive, with potential to improve TB diagnosis among children and among people living with HIV/AIDS (PLHIV). This review gives an overview of the existing evidence on TB diagnostic studies of non-sputum-based samples collected non-invasively from or through the oral-gastrointestinal tract (GI) and nasal pharynx regions of humans and the biomarkers detected. We further summarized evidence of these biomarkers and sample types from research done in paediatric and PLHIV. The review identified; saliva, cough aerosols, oral swabs, oral wash, dental plaque, tongue swabs, face mask sampling, exhaled breath, and stool, as the non-sputum samples investigated. These biomarkers can be categorized into Deoxyribose Nucleic Acid (DNA), Ribonucleic Acid (RNA), inflammatory, antigen-antibody, volatile and non-volatile compounds, microbiome and microbiota. The biomarkers identified were derived both from the host and pathogen. Similar biomarkers were identified in the general population, children and among PLHIV. These biomarkers have been detected by either already approved simple point of care or sophisticated devices. Differences in methodology and sample types investigated, small sample size of children and PLHIV populations, bias due to confounding factors, were some of the identified challenges in these studies. There is need to conduct larger and standardized multi centre studies to evaluate non-sputum-based biomarker-based tests in children and PLHIV., (© 2024. The Author(s).)

Background: Tuberculous meningitis (TBM) is a severe central nervous system (CNS) infection with a challenging diagnosis due to inadequate detection methods. This study evaluated current clinical detection methods and their applicability., Methods: A cohort of 514 CNS infection patients from 2018 to 2020 was studied. Data on general demographics, Cerebrospinal Fluid (CSF) analysis, epidemiology, and clinical outcomes were collected. TBM patients were identified, and the sensitivities of mmetagenomic next-generation sequencing (NGS), GeneXpert, and microbial culture were compared. Kappa statistic assessed the consistency between methods., Results: Among the patients involved, TBM (29%) and neurosyphilis (25%) were the two most prevalent CNS infections. CSF analysis indicated that 76% of patients had leukocytosis, suggesting a potential CNS inflammation. In TBM cases, 92.5% had elevated CSF protein and leukocyte counts. Moreover, the percentage of positive mNGS results was 55.6%. GeneXpert and MTB cultures alone had lower sensitivity, but combined use resulted in a 53.4% positive rate., Conclusions: This study highlights the high sensitivity of mNGS, comparable to GeneXpert and MTB culture. The combined methods are cost-effective and straightforward, and can partially substitute for mNGS, offering valuable alternatives for TBM diagnosis and providing insights into multiple diagnostic strategies in clinical practice., (© 2024. The Author(s).)

Despite advancements in detection and treatment, tuberculosis (TB), an infectious illness caused by the Mycobacterium TB bacteria, continues to pose a serious threat to world health. The TB diagnosis phase includes a patient's medical history, physical examination, chest X-rays, and laboratory procedures, such as molecular testing and sputum culture. In artificial intelligence (AI), machine learning (ML) is an advanced study of statistical algorithms that can learn from historical data and generalize the results to unseen data. There are not many studies done on the ML algorithm that enables the prediction of treatment success for patients with pulmonary TB (PTB). The objective of this study is to identify an effective and predictive ML algorithm to evaluate the detection of treatment success in PTB patients and to compare the predictive performance of the ML models. In this retrospective study, a total of 1236 PTB patients who were given treatment under a randomized controlled clinical trial at the ICMR-National Institute for Research in Tuberculosis, Chennai, India were considered for data analysis. The multiple ML models were developed and tested to identify the best algorithm to predict the sputum culture conversion of TB patients during the treatment period. In this study, decision tree (DT), random forest (RF), support vector machine (SVM) and naïve bayes (NB) models were validated with high performance by achieving an area under the curve (AUC) of receiver operating characteristic (ROC) greater than 80%. The salient finding of the study is that the DT model was produced as a better algorithm with the highest accuracy (92.72%), an AUC (0.909), precision (95.90%), recall (95.60%) and F1-score (95.75%) among the ML models. This methodology may be used to study the precise ML model classification for predicting the treatment success of TB patients during the treatment period., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ahamed Fayaz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Tuberculous meningitis (TBM) with adults Epstein-Barr (EB) virus encephalitis is a very rare infectious disease, with a high mortality and disability. Metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) is highly diagnostic. We report on a case of severe meningoencephalitis caused by co-infection with mycobacterium tuberculosis and EB virus. Brain MRI indicated a parenchyma lesion in the brain. mNGS of CSF indicated Mycobacterium tuberculosis and EB virus amplification, positive serum EB virus IgG antibodies, and improved symptoms after anti-tuberculosis and antiviral treatment. A re-examination of the brain MRI revealed that the significantly absorption of the lesions., Case Report: A 49-year-old male patient presented with a chief complaint of headache and fever with consciousness disturbance. The brain magnetic resonance imaging showed a lesions in the right parenchymal brain with uneven enhancement, accompanied by significantly increased intracranial pressure, elevated CSF cell count and protein levels, as well as notably decreased glucose and chloride levels. mNGS of CSF showed the coexistence of Mycobacterium tuberculosis and EBV. The patient was diagnosed as TBM with EBV encephalitis. The patient's symptoms gradually improved with the active administration of anti-tuberculosis combined with antiviral agents, the use of hormones to reduce inflammatory reaction, dehydration to lower intracranial pressure, and intrathecal injection. Subsequent follow-up brain magnetic resonance imaging indicated significant absorption of the lesions, along with a marked decrease in CSF count and protein levels, as well as obvious increase in glucose and chloride levels., Conclusion: TBM associated with adult EBV encephalitis is extremely rare. The disease's early stages are severe and have a high fatality rate. A prompt and accurate diagnosis is particularly important. NGS of CSF is of great value for early diagnosis., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Li, Zhu, Cheng and Kong.)

Background: Multidrug- or rifampicin-resistant tuberculosis (TB; MDR/RR-TB) is a significant public health threat. However, the mechanisms involved in its transmission in Sichuan, China are unclear. To provide a scientific basis for MDR/RR-TB control and prevention, we investigated the drug-resistance characteristics, genetic diversity, and transmission dynamics and analyzed the demographic and clinical characteristics of patients to identify risk factors for the acquisition of MDR/RR-TB in Sichuan, Western China., Methods: Whole-genome sequencing was performed using a sample comprised of all MDR/RR-TB strains isolated from patients with pulmonary TB (≥ 15 years) at the 22 surveillance sites in Sichuan province between January 2019 and December 2021, to analyze genotypic drug resistance and genetic diversity. Moreover, we performed statistical analyses of the epidemiological characteristics and risk factors associated with the transmission dynamics of MDR/RR-TB., Results: The final analysis included 278 MDR/RR TB strains. Lineage 2.2, the major sub-lineage, accounted for 82.01% (228/278) of isolates, followed by lineage 4.5 (9.72%, 27/278), lineage 4.4 (6.83%, 19/278), and lineage 4.2 (1.44%, 4/278). The drug resistance rates, ranging from high to low, were as follows: isoniazid (229 [82.37%]), streptomycin (177 [63.67%]), ethambutol (144 [51.80%]), pyrazinamide (PZA, 119 [42.81%]), fluoroquinolones (FQs, 93 [33.45%]). Further, the clofazimine, bedaquiline, and delamanid resistance rates were 2.88, 2.88, and 1.04%, respectively. The gene composition cluster rate was 32.37% (90/278). In addition, 83.81% (233/278) of MDR/RR-TB cases were determined to be likely caused by transmission. Finally, patients infected with lineage two strains and strains with the KatG S315T amino acid substitution presented a higher risk of MDR/RR-TB transmission., Conclusion: Transmission plays a significant role in the MDR/RR-TB burden in Sichuan province, and lineage 2 strains and strains harboring KatG S315T have a high probability of transmission. Further, high levels of FQ and PZA drug resistance suggest an urgent need for drug susceptibility testing prior to designing therapeutic regimens. New anti-TB drugs need to be used standardly and TB strains should be regularly monitored for resistance to these drugs., (© 2024. The Author(s).)

Objectives: This study aims to develop a novel diagnostic approach using the recombinase aided amplification-lateral flow dipstick(RAA-LFD) assay for the distinction of Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC), enabling rapid and convenient as well as accurate identification of them in clinical samples., Methods: Our study established a duplex RAA-LFD assay capable of discriminating between MTB and MAC. Based on the principles of RAA primer and probe design, specific primers and probes were developed targeting the MTB IS6110 and the MAC DT1 separately. Optimization of reaction time points and temperatures was conducted, followed by an evaluation of specificity, sensitivity, and reproducibility. The established detection method was then applied to clinical samples and compared with smear microscopy, liquid culture, LAMP, and Xpert/MTB RIF in terms of diagnostic performance., Results: The complete workflow allows for the effective amplification of the MTB IS6110 and MAC DT1 target sequences at constant 37°C within 20min, and the amplification products can be visually observed on the LFD test strip. This method exhibits high specificity, showing no cross-reactivity with nucleic acids from M. kansassi , M. abscessus, M. gordonae, M. chelonae, M. fortuitum, M. scrofulaceum, M. malmoense, M. chimaera, M. szulgai and common respiratory pathogens. It also demonstrates high sensitivity, with a detection limit as low as 10 2 CFU/mL. Additionally, the method's Coefficient of Variation (CV) is less than 5%, ensuring excellent repeatability and reliability. Furthermore, clinical performance evaluations, using Xpert/MTB RIF as the gold standard, demonstrated that the duplex RAA-LFD assay achieves a sensitivity of 92.86% and a specificity of 93.75%. It is also noteworthy that the assay exhibits considerable diagnostic efficacy in smear-negative patients., Conclusions: Our study introduces a rapid, specific, and sensitive duplex RAA-LFD assay for the discriminatory diagnosis of MTB and MAC. This method represents a significant advancement in the field of infectious disease diagnostics, offering a valuable tool for rapid detection and management of MTB and MAC infections. The implementation of this approach in point-of-care settings could greatly enhance TB control and prevention efforts, especially in resource-limited environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Zhang, Wang, Yi and Fu.)

Tuberculous meningitis (TBM) is a fatal tuberculosis caused by a large number of Mycobacterium tuberculosis (M. tuberculosis) spread by blood flow, with a case fatality rate of more than 50%. It is one of the most serious complications of miliary tuberculosis (MT), whose incidence is closely related to MT. If doctors can provide early diagnosis and active treatment for TBM, the case fatality rate will be significantly reduced. At present, there is a lack of methods to predict the progression of MT to TBM in clinic. To explore whether MT cases will experience TBM, we propose an early screening model of miliary tuberculosis with tuberculous meningitis (MT-TBM) based on few-shot learning with multiple windows and feature granularities (MWFG). This model aims to screen potential TBM cases through chest computerized tomography (CT) images of MT cases. Chest CT is a routine examination for MT cases. The MWFG module can extract more comprehensive features from a set of CT images of each MT case. The softmax classifier with adaptive regularization is trained on the cooperation of support set and query set, which can effectively prevent overfitting. Experiments on a dataset of 40 MT cases with chest CT images established by the medical records demonstrate that our proposed model achieves state-of-the-art performance in the early screening of MT-TBM. It can establish the connection between MT and MT-TBM through chest CT images of MT cases. The early screening model of MT-TBM based on few-shot learning with MWFG fills the research gap in computer-aided predicting TBM and has certain clinical effects. This research can provide some reference for clinicians in early diagnosis of MT-TBM and help clinicians in the early prevention and treatment of TBM for MT patients., (© 2024. The Author(s).)

Background: The World Health Organization predicted 10.6 million new tuberculosis cases and 1.5 million deaths in 2022. Tuberculous meningitis, affecting 1% of active TB cases, is challenging to diagnose due to sudden onset, vague symptoms, and limited laboratory tests. Nanopore-targeted sequencing (NTS) is an emerging third-generation sequencing technology known for its sequencing capabilities. We compared its detection efficiency with Xpert, MTB culture, PCR, and AFB smear in cerebrospinal fluid samples to highlight the substantial potential of NTS in detecting intracranial tuberculosis., Methods: This study included 122 patients suspected of having intracranial tuberculosis at the Second Hospital of Nanjing in Jiangsu Province, China, between January 2021 and January 2024. The Univariate logistic regression and random forest regression identified risk factors and clinical markers. A chi-square test evaluated diagnostic accuracy for different image types of intracranial tuberculosis., Results: The research involved 100 patients with intracranial tuberculosis. Among them, 41 had tuberculous meningitis, 27 had cerebral parenchymal tuberculosis, and 32 had mixed intracranial tuberculosis. Besides, 22 patients were diagnosed with other brain conditions. In diagnosing intracranial tuberculosis, NTS demonstrated a sensitivity of 60.0% (95% CI: 49.7-69.5%) and a specificity of 95.5% (95% CI:75.1-99.8%), with an AUC value of 0.78 (95% CI: 0.71 to 0.84), whose overall performance was significantly better than other detection methods. There was no notable difference (P > 0.05) in diagnostic accuracy between NTS and the final diagnosis for intracranial tuberculosis patients with varying imaging types. Furthermore, patients who tested positive had a 31.500 (95% CI: 6.205-575.913) times higher risk of having intracranial tuberculosis compared to those with negative results., Conclusion: Due to its convenience, efficiency, quick turnaround time, and real-time sequencing analysis, NTS might become a promising and reliable method for providing microbiological diagnoses for patients with intracranial tuberculosis and for screening populations at risk., (© 2024. The Author(s).)

Complete identification methods are critical for evaluating nontuberculous mycobacteria (NTM). Here, we describe a novel diagnostic method for identification of eight NTM, Mycobacterium tuberculosis complex, and three drug resistance markers using PCR/matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) from cultured organisms. With this technology, a multiplex end-point PCR is performed for targets of interest. Detection probes that are extended in the presence of a target are added. The extended probes have greater molecular weight and can be detected by MALDI-TOF MS. An AFB Primary Panel was designed to differentiate Mycobacterium avium ; Mycobacterium intracellulare subsp. chimaera ; Mycobacterium avium complex (other); Mycobacterium abscessus subsp. abscessus , bolletii , and massiliense ; Mycobacterium kansasii , and M. tuberculosis complex. This design should cover 90% (3,483/3,691) of mycobacteria seen onsite. A development set of unblinded isolates ( n = 217) was used to develop PCR primers, detection probes, and probe barcodes. It demonstrated 99.1% (215/217) agreement with reference methods. An evaluation set using blinded isolates ( n = 320) showed an overall sensitivity of 94.3% (range by target: 90.0-100%). Overall specificity from negative media, non-target mycobacteria, and bacteria was 99.1% (108/109; range by target: 94.4-100%). Three drug resistance markers erm (41), rrl, and rrs demonstrated 100%, 91%, and 100% sensitivity, respectively, and >99% specificity. Limit of detection per target ranged from 2.2 × 10 3 to 9.9 × 10 6 CFU/mL. The AFB Primary Panel allows for mycobacterial speciation, subspeciation, and resistance mutation detection, which is essential for diagnosis, appropriate therapy, identifying outbreaks, and managing treatment-refractory disease. It can perform with high-throughput and high specificity and sensitivity from isolates.IMPORTANCEEven closely related mycobacteria can have unique treatment patterns, but differentiating these organisms is a challenge. Here, we tested an innovative platform that combines two commonly used technologies and creates something new: matrix-assisted, laser-desorption ionization time-of flight mass spectrometry was performed on PCR amplicons instead of on proteins. This created a robust system with the advantages of PCR (high discriminatory power, high throughput, detection of resistance) with the advantages of mass spectrometry (more targets, lower operational cost) in order to identify closely related mycobacterial organisms., Competing Interests: Dr. Wang was a consultant for Agena from June 2022 to May 2023.

We evaluated the Xpert MTB/Rif Ultra assay performance for Mycobacterium tuberculosis (MTB) detection in formalin-fixed paraffin-embedded tissue (FFPET) compared to mycobacterial culture or laboratory-developed MTB PCR test (LDT). FFPET samples with histological features suggestive of tuberculosis from 2018 to 2023 were selected. Five hundred microlitres of tissue lysis buffer was added to FFPET scrolls and incubated at 75 °C for 5 min. After adding 50 µl of proteinase K and overnight incubation at 56 °C, sample aliquots were processed as per the manufacturer's instructions. MTB culture or LDT assay results were used as a reference for sensitivity and specificity calculations. Of 51 eligible FFPET, 32 were positive for MTB either by culture or LDT PCR on FFPET. Xpert MTB/Rif Ultra detected MTB in 23/32 positive specimens [71.9%, 95% confidence interval (CI) 54.6-84.4%]. Of nine discordant specimens, seven were MTB positive by culture and two were identified by LDT MTB PCR only, as no specimen was submitted for MTB culture. Of 19 negative samples, 100% specificity (95% CI 83.2-100.0%) was attained via Xpert MTB/Rif Ultra. Implementation of Xpert MTB/Rif Ultra on FFPET within clinical laboratories is promising, given its improved turnaround time compared to MTB culture and ability to detect MTB in cases where no tissue is available for culture.

Background: Tuberculous pleurisy (TP) is one of the most common types of extrapulmonary tuberculosis, often secondary to tuberculosis (TB). Clinical and imaging manifestations of non-tuberculous mycobacterial pulmonary diseases (NTM-PD) are usually similar to those of tuberculosis. Because of their similarity and the high incidence of tuberculosis, non-tuberculous mycobacterial infections are often overlooked for a long time. Especially in people without immunodeficiency., Methods: Mycobacterium tuberculosis (MTB) in pleural effusion was found by metagenomic next-generation sequencing (mNGS). During anti-tuberculosis treatment, mNGS of lung tissue by ultrasound-guided percutaneous lung puncture revealed that this patient had combined NTM-PD., Results: Mycobacterium chelonae (M. chelonae) was detected by mNGS, and after anti-NTM treatment, the patient's chest CT showed that the inflammation was absorbed more than before, and the patient's symptoms improved., Conclusions: When TB is poorly treated with standardized anti-tuberculosis therapy, comorbid non-tuberculous mycobacterial infections may be considered, and mNGS may complement traditional pathogenetic testing.

Objective: This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing (tNGS) of bronchoalveolar lavage fluid (BALF) in pulmonary mycobacterial infections., Methods: This retrospective study was conducted on patients who underwent bronchoscopy and tNGS, smear microscopy, and mycobacterial culture of BALF. Patients with positive Mycobacterium tuberculosis (MTB) culture or GeneXpert results were classified into the tuberculosis case group. Those diagnosed with nontuberculous mycobacteria (NTM)-pulmonary disease (NTM-PD) composed the case group of NTM-PD patients. The control group comprised patients without tuberculosis or NTM-PD. Sensitivity, specificity, and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance., Results: For tuberculosis patients with positive mycobacterial culture results, the areas under the ROC curves (AUCs) for tNGS, GeneXpert, and smear microscopy were 0.975 (95% CI: 0.935, 1.000), 0.925 (95% CI: 0.859, 0.991), and 0.675 (95% CI: 0.563, 0.787), respectively. For tuberculosis patients with positive GeneXpert results, the AUCs of tNGS, culture, and smear microscopy were 0.970 (95% CI: 0.931, 1.000), 0.850 (95% CI: 0.770, 0.930), and 0.680 (95% CI: 0.579, 0.781), respectively. For NTM-PD, the AUCs of tNGS, culture, and smear-positive but GeneXpert-negative results were 0.987 (95% CI: 0.967, 1.000), 0.750 (95% CI: 0.622, 0.878), and 0.615 (95% CI: 0.479, 0.752), respectively. The sensitivity and specificity of tNGS in NTM-PD patients were 100% and 97.5%, respectively., Conclusion: tNGS demonstrated superior diagnostic efficacy in mycobacterial infection, indicating its potential for clinical application., (© 2024. Huazhong University of Science and Technology.)

Bertiella studeri, a typical intestinal cestode of nonhuman primates, accidentally infects human beings. However, B. studeri infection in monkeys has been rarely reported in recent years. A case of B. studeri infection was identified in one captive rhesus macaque with natural infection of Mycobacterium tuberculosis. This study contributes to alerting the public of this helmintic zoonosis, though its infection rate is quite lower in recent years., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Until recently, multidrug-resistant TB (MDR-TB) was treated with lengthy and toxic regimens. New three-drug anti-TB regimens raise the question of whether they are sufficiently active for MDR-TB in Central Asia, an MDR-TB hotspot region.In a cohort of rifampicin-resistant (RR) and MDR-TB patients in the Kyrgyz Republic, we investigated the impact of the number of drugs that were tested susceptible by whole-genome sequencing (WGS) and conventional drug susceptibility testing (DST) and used for treatment on the treatment response, defined as 'matches'. Logistic regressions were performed to assess the effect of having ≥ 4 susceptible drugs in a regimen at baseline and at Month 2 on the treatment response.The study included 227 participants with RR/MDR-TB (30.8% female; median age 30.4 years). The age- and sex-adjusted analysis showed an association between a regimen with ≥ 4 WGS matches at baseline (adjusted odds ratio [aOR] 2.10, 95% CI 1.00-4.41). No association was found when using conventional DST to define matches.Our study confirms that the inclusion of four efficacious anti-TB drugs in an MDR-TB regimen increases the chances of a positive treatment response. Susceptibility of at least four drugs in WGS-DST predicts a positive treatment response..

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

Background: Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). The world is currently facing challenges due to the spread of anti-tuberculosis drug-resistant of M. tuberculosis. Isoniazid-resistant (INH), is one of the first-line anti-tuberculosis agents that has a high resistance case. This study used Multiplex allele-specific Polymerase Chain Reaction (MAS-PCR) to detect the most common mutations associated with isoniazid resistance on inhA, katG, and ahpC gene., Methods: This study used samples from clinical isolates of M. tuberculosis which had been tested for their antibiotic sensitivity of first-line anti-tuberculosis drugs. The DNA extraction process was carried out using the boiling method and then amplified with specific primers for inhA, katG, and ahpC genes using the MAS-PCR method. The results are then read on the electrophoretic gel with an interpretation of the mutation gene when the target gene DNA bands were absent according to the allele-specific fragments target., Results: A total of 200 isolates were tested in this study consisting of isoniazid-resistant and susceptible with the largest distribution of Multi-Drug Resistant (MDR) isolates with a total of 146 isolates (73%). The most significant gene mutation was on the ahpC gene in 61 isolates (30,5%) and the combination mutation of the katG + ahpC gene in 52 isolates (26%) with sensitivity and specificity of the test reaching 87% and 42% for the detection of INH-resistant., Conclusion: Mutation on the ahpC gene has the highest percentage in this study. AhpC gene can be considered one of the essential genes to be tested for the cause of isoniazid-resistant. Using MAS-PCR for detecting gene mutation in isoniazid-resistant was simple and easy, it has the potential to be widely used as a rapid screening molecular test., (Copyright © 2023. Published by Elsevier B.V.)

Context: Early intervention and treatment are key measures for tuberculosis (TB) prevention and control, making early, rapid, and accurate diagnostic methods crucial. The Liquid-solid (Biphasic) rapid cultures is a novel tool for the differential diagnosis of tuberculosis., Objective: The study intended to evaluate the value of the biphasic cultures by comparing it to the acid-fast staining and liquid cultures, which have been the traditional gold-standard technology, to determine its value in the diagnosis of TB., Design: The research team conducted an experimental study., Setting: The study took place at the Affiliated Wuxi Fifth Hospital of Jiangnan University in Wuxi, China., Participants: Participants were 221 patients with suspected pulmonary tuberculosis who had been admitted to the hospital between July 2020 and December 2021., Outcome Measures: Using three methods-liquid-solid (biphasic) culture, acid-fast staining, and mycobacterial growth indicator tube (MGIT) 960 liquid culture, the research team tested participants' sputum samples: (1) for sensitivity; (2) for time to positive culture results, and (3) for differential diagnosis., Results: The biphasic culture's sensitivity was significantly higher than that of acid-fast staining, (P = .0003), and no significant difference existed between it and the MGIT 960 liquid cultures. The biphasic cultures's mean time to positivity was significantly shorter than that of the MGIT 960 liquid culture at the intervals 11-20 d (P < .0001) and 21-35 days (P = .0001). Moreover, the biphasic cultures could preliminarily differentiate nontuberculous mycobacteria (NTM) from mycobacterium tuberculosis (MTB), which is a significant advantage in tuberculosis diagnosis., Conclusions: This study highlights the potential of a biphasic culture as a reliable tool for the rapid differential diagnosis of tuberculosis, with a faster detection cycle and a higher sensitivity than conventional methods. The biphasic cultures is a valuable addition to the tuberculosis diagnostic armamentarium and can help improve patients' outcomes by enabling earlier diagnosis and treatments.

Rationale: Isoniazid-resistant tuberculosis (Hr-TB) is often overlooked in diagnostic algorithms because of reliance on first-line molecular assays testing only for rifampicin resistance. Objectives: To determine the prevalence, outcomes, and molecular mechanisms associated with rifampin-susceptible, isoniazid-resistant TB (Hr-TB) in the Eastern Cape, South Africa. Methods: Between April 2016 and October 2017, sputum samples were collected from patients with rifampin-susceptible TB at baseline and at Weeks 7 and 23 of drug-susceptible TB treatment. We performed isoniazid phenotypic and genotypic drug susceptibility testing, including FluoroTypeMTBDR, Sanger sequencing, targeted next-generation sequencing, and whole-genome sequencing. Results: We analyzed baseline isolates from 766 patients with rifampin-susceptible TB. Of 89 patients (11.7%) who were found to have Hr-TB, 39 (44%) had canonical katG or inhA promoter mutations; 35 (39%) had noncanonical katG mutations (including 5 with underlying large deletions); 4 (5%) had mutations in other candidate genes associated with isoniazid resistance. For 11 (12.4%), no cause of resistance was found. Conclusions: Among patients with rifampin-susceptible TB who were diagnosed using first-line molecular TB assays, there is a high prevalence of Hr-TB. Phenotypic drug susceptibility testing remains the gold standard. To improve the performance of genetic-based phenotyping tests, all isoniazid resistance-associated regions should be included, and such tests should have the ability to identify underlying mutations.

This study explored Mycobacterium tuberculosis (MTB) growth from tongue swabs, both experimentally infected after sampling from healthy controls, or sampled from patients with smear-microscopy confirmed pulmonary tuberculosis (PTB). For both, we evaluated the performance of NALC-NaOH/MGIT960 (MGIT), Kudoh-Ogawa (KO), and cetylpyridinium chloride-Löwenstein-Jensen (CPC/LJ) culture processing methods. Experimentally spiked swabs from 20 participants exhibited 94.4% MTB growth when inoculated within 7 days of CPC exposure, declining significantly after 14-21 days (p<0.00001). KO-processed specimens showed 100% MTB growth, with a non-significant reduction after storage (94.1%; p=0.21), and all spiked swabs yielded growth in MGIT. In the field evaluation on 99 PTB patients, MGIT isolated MTB from 89% of tongue swabs, with an 8% contamination rate, compared to 99% MGIT positivity from sputum. Solid media had lower positivity, 62% for KO and 49% for CPC/LJ, suggesting MGIT as optimal for growing MTB from tongue swabs. Further testing of presumptive PTB patients is recommended., Competing Interests: Declaration of competing interest None of the authors have a competing interest in this research to declare., (Copyright © 2024. Published by Elsevier Inc.)

Background: Targeted next-generation sequencing (NGS) can rapidly and simultaneously detect mutations associated with resistance to tuberculosis drugs across multiple gene targets. The use of targeted NGS to diagnose drug-resistant tuberculosis, as described in publicly available data, has not been comprehensively reviewed. We aimed to identify targeted NGS assays that diagnose drug-resistant tuberculosis, determine how widely this technology has been used, and assess the diagnostic accuracy of these assays., Methods: In this systematic review and meta-analysis, we searched MEDLINE, Embase, Cochrane Library, Web of Science Core Collection, Global Index Medicus, Google Scholar, ClinicalTrials.gov, and the WHO International Clinical Trials Registry Platform for published and unpublished reports on targeted NGS for drug-resistant tuberculosis from Jan 1, 2005, to Oct 14, 2022, with updates to our search in Embase and Google Scholar until Feb 13, 2024. Studies eligible for the systematic review described targeted NGS approaches to predict drug resistance in Mycobacterium tuberculosis infections using primary samples, reference strain collections, or cultured isolates from individuals with presumed or confirmed tuberculosis. Our search had no limitations on study type or language, although only reports in English, German, and French were screened for eligibility. For the meta-analysis, we included test accuracy studies that used any reference standard, and we assessed risk of bias using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The primary outcomes for the meta-analysis were sensitivity and specificity of targeted NGS to diagnose drug-resistant tuberculosis compared to phenotypic and genotypic drug susceptibility testing. We used a Bayesian bivariate model to generate summary receiver operating characteristic plots and diagnostic accuracy measures, overall and stratified by drug and sample type. This study is registered with PROSPERO, CRD42022368707., Findings: We identified and screened 2920 reports, of which 124 were eligible for our systematic review, including 37 review articles and 87 reports of studies collecting samples for targeted NGS. Sequencing was mainly done in the USA (14 [16%] of 87), western Europe (ten [11%]), India (ten [11%]), and China (nine [10%]). We included 24 test accuracy studies in the meta-analysis, in which 23 different tuberculosis drugs or drug groups were assessed, covering first-line drugs, injectable drugs, and fluoroquinolones and predominantly comparing targeted NGS with phenotypic drug susceptibility testing. The combined sensitivity of targeted NGS across all drugs was 94·1% (95% credible interval [CrI] 90·9-96·3) and specificity was 98·1% (97·0-98·9). Sensitivity for individual drugs ranged from 76·5% (52·5-92·3) for capreomycin to 99·1% (98·3-99·7) for rifampicin; specificity ranged from 93·1% (88·0-96·3) for ethambutol to 99·4% (98·3-99·8) for amikacin. Diagnostic accuracy was similar for primary clinical samples and culture isolates overall and for rifampicin, isoniazid, ethambutol, streptomycin, and fluoroquinolones, and similar after excluding studies at high risk of bias (overall sensitivity 95·2% [95% CrI 91·7-97·1] and specificity 98·6% [97·4-99·3])., Interpretation: Targeted NGS is highly sensitive and specific for detecting drug resistance across panels of tuberculosis drugs and can be performed directly on clinical samples. There is a paucity of data on performance for some currently recommended drugs. The barriers preventing the use of targeted NGS to diagnose drug-resistant tuberculosis in high-burden countries need to be addressed., Funding: National Institutes of Allergy and Infectious Diseases and Swiss National Science Foundation., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Objective: To investigate the value of histopathological examination (HPE) and Xpert Mycobacterium tuberculosis bacilli/rifampicin (MTB/RIF) assay in diagnosis of cervical lymph node tuberculosis (LN TB) after coarse needle biopsy (CNB)., Methods: We retrospectively analyzed 612 samples obtained from October 2017 to August 2023 from patients suspected cervical LN TB with surgically pathological, microbial culture confirmed, and clinically confirmed cervical lymph node enlargement who received ultrasound-guided CNB assisted by contrast-enhanced ultrasound (CEUS) at our hospital. All specimens were assessed by HPE and the Xpert (MTB/RIF) assay. We analyzed the results to determine the diagnostic value of HPE and Xpert (MTB/RIF) assay in samples taken after CEUS-assisted CNB of LN TB, and to evaluate the safety of CNB., Results: Based on the comprehensive reference standard established in this study, 532 of 612 patients were diagnosed with cervical LN TB, of which 476 were CNB positive cases, the positive rate of diagnosis was 89.5%。The sensitivity, specificity, positive predictive value, negative and predictive value of HPE were 80.4%, 91.2%, 98.4%, 41.2% respectively, while those of the Xpert MTB/RIF assay were 75.7%, 98.7%, 99.7%, 38.0% respectively. No postoperative complications were noted, and the Clavien-Dindo grade was 2., Conclusion: CEUS-assisted CNB has high diagnostic value and is safe for cervical LN TB. The sensitivity of HPE is slightly higher than that of Xpert (MTB/RIF) assay, and the specificity of Xpert (MTB/RIF) assay is higher than that of HPE, so Xpert (MTB/RIF) assay can correct the cervical lymph node tuberculosis with negative HPE., (© 2024. The Author(s).)

Objectives: To find out the diagnostic accuracy of stool Cartridge-based nucleic acid amplification test (CBNAAT) as an alternate method as compared to CBNAAT in gastric aspirate (GA) samples in pediatric tuberculosis (TB)., Methods: This cross-sectional study was performed at Department of Pediatrics of a tertiary care hospital. Children aged 0-18 y diagnosed as presumptive tuberculosis were consecutively enrolled. Gastric aspirate and corresponding stool sample was subjected to CBNAAT and its performance was compared in both samples using appropriate statistical tests., Results: Total 100 patients were enrolled in the study. Diagnostic accuracy of CBNAAT was 81% and 80% in gastric aspirate and stool sample respectively. On comparing gastric aspirate with corresponding stool sample there was 97% agreement, with Cohen's kappa value of 0.94. There was a statistically significant association observed between gastric aspirate CBNAAT and stool CBNAAT p <0.001 using chi square test. Sensitivity of gastric aspirate CBNAAT and stool CBNAAT was 75% and 73% respectively and specificity was 100% for both the samples compared against Composite Reference Standard (CRS)., Conclusions: The diagnostic accuracy of stool CBNAAT is comparable to GA CBNAAT in children and can be used as a good alternative to gastric aspirate for diagnosis of pulmonary and disseminated tuberculosis in children., (© 2023. The Author(s), under exclusive licence to Dr. K C Chaudhuri Foundation.)

Background: Serpiginous-like choroiditis (SLC) denotes ocular tuberculosis (TB), in the presence of positive tuberculin skin test (TST) or interferon gamma release assay (IGRA)., Methods: Retrospective review of SLC patients from a TB-endemic country, with negative TST and IGRA tests, but responsive to anti-TB therapy., Results: Fifteen patients (13 bilateral) with active SLC  were included.   Eleven (73.3%) patients had received corticosteroids ± immunosuppressive therapy prior to presentation. Chest radiographic abnormalities were found in four (26.7%) patients. We treated all patients with a combination of anti-TB therapy (ATT) and corticosteroids. Paradoxical worsening was noted in nine (60%) patients, complete resolution of lesions in 12 (80%), persistent inflammation (post-ATT) in one, while two were yet to complete ATT. None had recurrence after complete resolution of lesions (median follow-up of 71 weeks [range 15-676 weeks])., Conclusions: TB-SLC may present with negative TST and IGRA tests but may still have clinical appearance, and treatment response, like test-positive disease.

Introduction: Very few studies have been devoted to extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (TB) in Gabon., Aim: The aim of the present study is to present the epidemiology of pre-XDR and XDR TB and the evolution over time of patients with multidrug-resistant TB., Methods: This retrospective study covered the activities from 2019 to 2022 of the Nkembo anti-tuberculosis center., Results: Fifteen patients were eligible, including 11 (73.3%) pre-XDR patients and 4 (26.7%) XDR-TB patients. Three (20.0%) patients had HIV/TB co-infection. The sample consisted of 7 men (46.7%) and 8 women (53.3%), a sex ratio (M/F) of 0.87. The average age was 35.6 years, and the median 34 years, with extremes of 23 and 60 years. Eight patients (53.3%) represented new cases of pre-XDR or XDR-TB tuberculosis. The majority (60%; n=9) came from deprived neighborhoods with widespread promiscuity. The therapeutic success rate among pre-XDR patients was 4 (36.4%) versus 2 (50.0%) among XDR-TB patients. Reported mortality occurred 5 (33.3%) patients during treatment, including 3 pre-XDR and 2 XDR-TB patients. In all cases, they died before the end of the first trimester of follow-up., Conclusion: The high frequency of primary pre-extensively drug-resistant tuberculosis underscores the pervasiveness of resistance to anti-tuberculosis drugs and underlines a pressing need for detection of contact cases and early treatment., (Copyright © 2024 SPLF. Published by Elsevier Masson SAS. All rights reserved.)

Failure in recognizing non-tuberculous mycobacteria (NTM) leads to misdiagnosis of multidrug-resistant Mycobacterium tuberculosis Complex (MTBC). There is an unmet need for diagnostic tools that can differentiate between NTMs and MTBC, and that are affordable for Low- and Middle-income Countries (LMIC). Earlier we developed a strip-based CrfA assay technology to detect the Carbapenem Resistance Factor A (CrfA) enzyme present only in MTBC. However, the strip-based CrfA assay had low turnaround time and lacked high-throughput capabilities. In this current research, we have developed a 96well-formatted CrfA assay for high-throughput detection of MTBC and differentiation with NTMs. This 96well-formatted CrfA assay displays a low turnaround time of 6-8 h with 100 % specificity and 93.75 % sensitivity on clinical samples. Based on these attributes, this 96well-formatted assay represents a valuable complementary tool to mitigate the misdiagnosis of chronic pulmonary tuberculosis with non-tuberculous mycobacteria in poorer nations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Drug-resistant tuberculosis is a severe global public health threat. Virulence factors and antibiotic resistance are generally considered to play a significant role in bacterial pathogenesis. However, the interaction between resistance and virulence in Mycobacterium tuberculosis (MTB) remains unclear., Methodology: Here, we used whole genome sequences from 667 MTB isolates from 14 countries to complete an in silico evaluation of the correlations between virulence gene mutations, drug resistance, and lineage classification. The chi-square (χ2) test was used to determine whether specific virulence gene mutations and drug resistance were related., Results: Our results showed that Mce1R&#95;G171R and Pks15&#95;V333A, were positively correlated with streptomycin and ethambutol resistance, respectively, and Pks15&#95;T46I was correlated with isoniazid, rifampin, ethambutol, pyrazinamide and streptomycin resistance. We also identified an additional 24 and 40 single nucleotide polymorphisms as well as 6 and 2 insertions or deletions in various virulence genes that are likely to be associated with changes in drug susceptibility in L2 and L4, respectively., Conclusions: Taken together our data suggest that there may be some degree of co-selection between virulence and resistance factors, which may help MTB more easily adapt to new environments., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Yu Zhang, Xinchang Chen, Shiyong Wang, Ning Jiang, Lingyun Shao, Jiazhen Chen.)

Background: Tuberculosis (TB) remains a major public health issue in Iran, especially smear-positive pulmonary tuberculosis (SPPTB), due to its high transmission rate. Examining the effective reproduction number( R t ) of SPPTB and patient characteristics is crucial for crafting targeted TB control measures. This study aimed to assess the R t of SPPTB in Iran from 2011 to 2021 and profile SPPTB patient demographics, initial smear bacilli density, diagnosis delays, and spatial distribution. Study Design: This is a historical cohort study., Methods: A time-dependent method was used to estimate R t , and monthly data from the national TB registry were scrutinized from 2011 to 2021., Results: A decline was observed in SPPTB incidence rates of 50909 SPPTB cases in Iran from 2011 to 2021. Approximately 29.1% of the cases were diagnosed within a month, while 44.5% experienced a one to three-month delay in diagnosis. The analysis revealed substantial heterogeneity in TB transmission dynamics across various provinces of Iran. Provinces such as Sistan and Baluchestan, Golestan, Guilan, Khuzestan, Tehran, and Khorasan Razavi exhibited the highest effective reproduction numbers. Additionally, there was a decreasing trend in the effective reproduction numbers across all provinces from 2011 to 2020., Conclusion: Effective reproduction numbers declined in most provinces from 2011 to 2020 but increased moderately after the COVID-19 pandemic, highlighting the need for targeted public health interventions. Although SPPTB incidence rates are declining nationally, elevated incidence rates and effective reproduction numbers in regions such as Sistan and Baluchestan, Golestan, Guilan, Khuzestan, Tehran, and Khorasan Razavi signify the need for persistent TB management efforts in Iran., (© 2024 The Author(s); Published by Hamadan University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

Tuberculosis (TB) is the top cause of death from a single infectious pathogen after COVID-19. Despite molecular diagnostic advances, two-thirds of the 10 million annual TB cases are still diagnosed using direct smear microscopy which has ~50% sensitivity. To increase the analytical performance of smear microscopy, we developed and characterized a novel polymer (Polydiallyldimethylammonium chloride [PDADMAC]) engraftment on inexpensive polystyrene (PS) specifically functionalized for mycobacterial capture. Engraftment is achieved via UV photopolymerization of DADMAC monomer on plasma-activated PS. The platform was tested on sputum from presumptive TB cases in Kampala, Uganda (n = 50), with an increased overall sensitivity of 81.8% (27/33) vs. fluorescent smear microscopy 57% (19/33) compared to a molecular (Cepheid GeneXpert MTB/RIF) gold standard. Frugal smear diagnostic innovation that is rapid and does not require dedicated instrumentation may offer an important solution to bridge the TB diagnostic gap., (© 2024. The Author(s).)