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

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 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.

Comparison of Nucleotide MALDI-TOF MS with Xpert MTB/RIF for Rifampicin Susceptibility Identification and Associated Risk Factors of Rifampicin Resistance Among Drug Resistant Mycobacterium tuberculosis.

Purpose: Nucleotide-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (nucleotide MALDI-TOF MS) is an emerging molecular technology used for the diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB)and its drug resistance. This study aimed to compare the ability of nucleotide MALDI-TOF MS to detect rifampicin (RIF) resistance in drug-resistant TB (DR-TB) patients with Xpert MTB/RIF and to analyze the disparate results individually. Additionally, potential factors associated with rifampicin resistance among DR-TB patients in Qingdao were investigated. Patients and Methods: A retrospective study was conducted at Qingdao Chest Hospital, and patients with DR-TB were enrolled. Corresponding frozen isolates were recovered and subjected to nucleotide MALDI-TOF MS, Xpert MTB/RIF, and phenotypic drug susceptibility testing (pDST). Sanger sequencing was performed for the discordant results of nucleotide MALDI-TOF MS and Xpert MTB/RIF. Univariate and multivariate logistic regression analyses were used to identify potential factors associated with rifampicin resistance among patients with DR-TB. Results: A total of 125 patients with DR-TB (18.8%, 125/668) were enrolled in this study from May 1 to July 31, 2023. Rifampicin-resistant (DR-TB/RR, 29) and rifampicin-sensitive (DR-TB/RS, 96) groups were divided according to the pDST results. Nucleotide MALDI-TOF MS performed better than Xpert MTB/RIF in terms of sensitivity, specificity, accuracy, and agreement with pDST. Only six cases had inconsistent results, and the sequencing results of five cases were identical to nucleotide MALDI-TOF MS. Furthermore, chest pain (aOR=12.84, 95% CI, 2.29-91.97, p=0.005), isoniazid sensitivity (aOR=0.14, 0.02-0.59, p=0.013), and ethambutol sensitivity (aOR=0.02, 0.00-0.10, p=0.000) were potential factors associated with rifampicin resistance among DR-TB patients in Qingdao. Conclusion: The overall concordance between nucleotide MALDI-TOF MS and Xpert MTB/RIF was 95.2%, with the former performing better in determining rifampicin susceptibility among DR-TB cases in Qingdao. Chest pain, isoniazid, and ethambutol resistance might be factors associated with RIF resistance among patients with DR-TB in Qingdao.

Case report: Tenosynovial giant cell tumor.

Tenosynovial giant cell tumor (TGCT) is a rare type of tumor that originates from the synovium of joints and tendon sheaths. It is characterized by recurring genetic abnormalities, often involving the CSF1 gene. Common symptoms include pain and swelling, which are not specific to TGCT, so MRI and a pathological biopsy are needed for an accurate diagnosis. We report the case of a 45-year-old man who experienced painful swelling in his right hip for six months. Initially, this was diagnosed as Erdheim-Chester disease. However, whole exome sequencing (WES) and RNA-Sequencing revealed a CSF1::GAPDHP64 fusion, leading to a revised diagnosis of TGCT. The patient was treated with pegylated interferon and imatinib, which resulted in stable disease after three months. Single-cell transcriptome analysis identified seven distinct cell clusters, revealing that neoplastic cells expressing CSF1 attract macrophages. Analysis of ligand-receptor interactions showed significant communication between neoplastic cells and macrophages mediated by CSF1 and CSF1R. Our findings emphasize the importance of comprehensive molecular analysis in diagnosing and treating rare malignancies like TGCT.

Diagnostic Value of Metagenomic next-generation sequencing and X-pert in Bronchoalveolar lavage fluid for pneumonia in HIV-infected and HIV-uninfected patients.

Background: The pathogens causing unexplained pneumonia in both HIV-infected or HIV-unfected patients are likely to be complex. This retrospective study aimed to characterize the etiology of pneumonia in HIV-infected and HIV-uninfected patients using bronchoalveolar lavage fluid (BALF) analysis with metagenomic next-generation sequencing (mNGS) and X-pert MTB/RIF. Methods: Between January 2022 and May2024, 141 HIV-infected and 104 HIV-uninfected patients admitted to Nanjing Second Hospital with pneumonia were included. BALF samples were collected and analyzed using mNGS to detect bacteria, fungi, viruses, tuberculosis (TB) and non-tuberculous mycobacteria (NTM), and X-pert for TB detection. Clinical data including CD4 T-cell counts, comorbidities, and ART status were collected and analyzed. Results: HIV-uninfected patients were found to be older and exhibited a higher prevalence of comorbidities compared to HIV-infected patients. Despite higher median CD4 T-cell counts in HIV-uninfected individuals (412 cells/µL vs. 31 cells/µL in HIV-infected), TB detection rates using X-pert and mNGS were lower than anticipated, particularly in HIV-infected patients. Mixed-pathogen infections were significantly more prevalent in HIV-infected patients, especially those with lower CD4 T-cell counts. ART use showed variable impacts on pathogen diversity, with longer treatment durations associated with reduced infection complexity but persistent immunodeficiency in some cases.In patients with pneumonia, whether HIV-infected or HIV-uninfected, pathogens often exhibit complexity, underscoring the critical role of timely mNGS and X-pert analysis of BALF for early pathogen detection.

Clinicopathological Profile in Patients with Tubercular Cervical Lymphadenitis and Its Treatment Outcome.

Tuberculosis is a common occurrence in developing countries. Drug resistance, co-morbidities, and limited availability of new rapid tests such as the GeneXpert/MTB Rif assay make diagnosis and treatment of extrapulmonary tuberculosis burdensome. A cross-sectional study was carried out at Employees' State Insurance Corporation Medical College and Hospital, Faridabad, Haryana of patients treated for tubercular cervical lymphadenopathy from December 2021 to March 2023 in the department of ENT. This study included 58 patients. The clinicopathological profile of patients and the outcome of treatment with antitubercular therapy were noted. The majority of patients had level V (39.6%) involvement. Incidental diagnosis of diabetes mellitus was seen in 2 cases (3.4%). Fever was the commonest constitutional symptom observed in 27.5% of cases. FNAC was suggestive of tubercular abscess in 48.2% and the GeneXpert MTB/RIF assay detected Mycobacterial tuberculosis in all the cases with rifampicin resistance in only one case. 56 cases (96.5%) had complete resolution after completion of antitubercular therapy including patients with rifampicin resistance and patients with diabetes mellitus. In the remaining two cases, treatment was prolonged for a few months before the resolution of the disease was observed. Timely diagnosis, patient compliance to antitubercular therapy, and adequate management of comorbidities lead to successful treatment outcomes in tubercular cervical lymphadenitis. Delayed response to treatment in a few cases needs further research into factors like immune status, nutrition, living conditions, and quality of drugs available to the public.

Paving the Way to Innovative, Child-Friendly Pediatric Diagnostic Methods for Tuberculosis: Introduction of Stool-Based Testing in Ukraine.

Like many countries, Ukraine faces challenges with diagnosing tuberculosis (TB) in children due to the paucibacillary nature of the disease and difficulty obtaining respiratory samples. To improve diagnostic efficiency, stool testing is being integrated into routine pediatric TB services. This started with a pilot introduction at 12 regional TB facilities, where stool was collected for children with a preliminary diagnosis of TB, based on clinical and/or radiological or laboratory findings, in addition to routine testing. For 168 children, a stool test was conducted between November 2021 and September 2022, with samples submitted in all 12 pilot regions. For 132 children, other samples were available in addition to stool. Mycobacterium tuberculosis (MTB) was bacteriologically confirmed in 37 children (in stool for 18 children). For 7 of the 18 children with MTB in stool, stool was the only sample in which MTB was detected. Rifampicin resistance was detected in seven children (in stool for three). This noninvasive TB diagnostic sample is especially beneficial for young children who cannot produce sputum. Early detection of TB and its drug-resistant strains in children will allow medical workers to provide safer and more effective treatment and save more lives. Based on the pilot implementation, Ukraine's national TB program began implementing stool testing throughout the country.

HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection.

Background: To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Methods: Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). Results: CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Conclusion: Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

The Impact of GeneXpert Cerebrospinal Fluid Testing on Tuberculous Meningitis Diagnosis in Routine Care in Botswana.

Background: Tuberculous meningitis (TBM) disproportionately impacts high-HIV prevalence, resource-limited settings where diagnosis is challenging. The GeneXpert platform has utility in TBM diagnosis, but uptake remains limited. In Botswana, before the introduction of GeneXpert, tuberculosis (TB) testing was only available through mycobacterial culture at the National TB Reference Laboratory. Data describing routine use of Xpert MTB/RIF for cerebrospinal fluid (CSF) testing in resource-limited settings are scarce. Methods: Electronic records for patients with CSF tested in government facilities in Botswana between 2016 and 2022 were obtained from a central online repository as part of ongoing national meningitis surveillance. Samples were excluded from 1 site where Xpert MTB/RIF is performed universally. The proportion receiving TB-specific investigation on CSF and the number positive for Mycobacterium tuberculosis following increased Xpert MTB/RIF capacity were determined. Results: The proportion of CSF samples receiving TB-specific investigation increased from 4.5% (58/1288) in 2016 to 29.0% (201/693) in 2022, primarily due to increased analysis with Xpert MTB/RIF from 0.9% (11/1288) to 23.2% (161/693). There was an overall decline in the annual number of CSF samples analyzed, but the proportion with microbiologically confirmed TBM increased from 0.4% to 1.2%. The proportion of samples tested for TB that were collected from health care facilities >100 km from the National TB Reference Laboratory increased with Xpert MTB/RIF rollout from 65.9% (87/132) to 78.0% (494/633). Conclusions: In Botswana, access to TB culture is challenging in remote populations; more accessible near-patient testing using Xpert MTB/RIF increased the number of patients receiving TB-specific testing on CSF and the number of confirmed TBM cases.

Isolated Tuberculosis of the Radius in an Immunocompetent Indian Male Without Pulmonary Involvement: A Report of a Rare Case.

Extrapulmonary tuberculosis is less commonly reported, and isolated tuberculous involvement of bones such as the radius, without any pulmonary lesions, is extremely rare. Diagnosing this condition can be challenging due to ambiguous clinical features and non-specific radiological findings in the early stages. The present case describes a rare instance of isolated tuberculosis of the radius in an immunocompetent Indian male with no pulmonary involvement. The diagnosis was achieved through a high index of suspicion in an endemic region, advanced radiometric investigations, and the isolation of Mycobacterium tuberculosis using the cartridge-based nucleic acid amplification test. The patient was started on a 12-month course of appropriate chemotherapy.

Silico-tuberculosis: An updated review.

Silico-Tuberculosis (silico-TB) is a severe combination of tuberculosis and silicosis, caused by occupational exposure to fine crystalline silica dust, which has become a global health concern. This comprehensive review compiles the updated knowledge regarding pathophysiology, clinical manifestations, important diagnostic techniques, treatment aspects, and challenges in understanding silico-TB. The review compiles the disease's history and epidemiology, highlighting a lack of data owing to poor monitoring and healthcare particularly in low- and middle-income countries like India. Further weak safety regulations, lack of preventative measures, and inadequate education increase the rates of silico-TB. The pathophysiology shows how silica particles impair the immune system and stimulate Th2 cells and M2 macrophages, which exacerbate TB, while inhibiting Th1 cells and M1 macrophages, which fight against the disease. Subsequently, it can be difficult to distinguish current TB from pre-existing silicosis. In cases where sputum and X-ray results are negative, chest CT scans may be helpful since radiographic screening identifies TB earlier than sputum assessment. Isoniazid, rifampicin, or both minimize the risk of active tuberculosis in people with silicosis. Consistent anti-tuberculosis drug therapy is recommended for 8-9 months to stop recurrence. The assessment recommends integrating silicosis and TB control initiatives to fight this combined health issue.

Intensive reprocessing of reusable bronchoscopes can reduce the false positive rate of Xpert MTB/RIF caused by nucleic acid residue.

Background/Purpose: Tuberculosis remains a leading cause of infectious death worldwide, The potential for nucleic acid residue on bronchoscopes to cause false positive results in molecular diagnostic methods and subsequently lead to tuberculosis misdiagnosis has long perplexed clinical. Methods: We utilized Xpert MTB/RIF to analyze the liquid collected after bronchoscope washing, employed by patients either with or without active pulmonary tuberculosis, and subjected to standard reprocessing (SR) or intensive reprocessing (IR) procedures. The IR procedure included specialized training and the provision of patient information to cleaning staff before the SR procedure, and repeated washing and suction of the bronchoscope with sterilized water post SR procedure. Results: 55 participants enrolled in the study were divided into three groups: SR group (n = 28), IR group(n = 14), and the control group(n = 13). Among the 55 enrolled patients, neither Mycobacterium tuberculosis nor contamination was detected by MIGT 960 liquid culture in the washing liquid. The positive rate of MTB/RIF in the SR group (12/28) was significantly higher than that in the IR group (1/14), with a statistically significant difference observed between them (42.86 % vs. 7.14 %, P=0.018). Conclusions: Nucleic acid residue on reusable bronchoscopes cleaned via the SR procedure was found to potentially cause false positives in MTB/RIF tests. Reprocessing bronchoscopes via the IR procedure was effective in significantly reducing nucleic acid residue, although complete elimination was not achieved.

Synergy of Xpert (MTB/RIF) and Line probe assay for detection of rifampicin resistant strains of Mycobacterium tuberculosis.

INTRODUCTION: Early diagnosis and successful treatment of drug-resistant tuberculosis (TB) demands rapid, precise, and consistent diagnostic methods to minimise the development of resistance. Therefore, this comparative study was designed to evaluate the diagnostic performance of Xpert (MTB/RIF) and Line probe assay (LPA) for detecting drug-resistant TB. METHODOLOGY: This study comprised 389 (279 pulmonary and 110 extrapulmonary) samples from patients suspected of having TB. All samples were subjected to Xpert (MTB/RIF), LPA, solid culture, and drug-susceptibility testing. Out of 320 samples, only 180 culture (gold standard) positive were included in the final evaluation. The diagnostic characteristics for methods used were determined by calculating diagnostic sensitivity, specificity, and predictive values. The agreement between all methods was determined by calculating the kappa coefficient. RESULTS: The sensitivity and specificity for Xpert (MTB/RIF) for detecting TB were 88.5% and 96.4%, respectively, against the solid culture. On the other hand, LPA showed sensitivity and specificity at 94.3% and 100%, respectively. Xpert (MTB/RIF) showed moderate agreement (kappa 0.65, p < 0.01) - (73.3% sensitivity; 97.6% specificity) for the detection of rifampicin resistance. However, LPA achieved better diagnostic accuracy (kappa 0.80, p < 0.01) - (84.6% sensitivity; 98.4% specificity) against drug-resistant TB. CONCLUSIONS: Xpert (MTB/RIF) and LPA have outstanding diagnostic sensitivity and specificity against RIF resistance with a shorter turnaround time, which could result in a substantial therapeutic outcome. Our findings showed LPA superiority over Xpert (MTB/RIF) for drug resistance. However, due to operational challenges, the requirement of technical expertise and infrastructure issues, LPA cannot be used as point-of-care testing in resource-limited countries.

Evaluation of molecular and bacteriological detection methods performed on the formalin-fixed paraffin-embedded biopsy samples collected from endometrial and lymph node tuberculosis suspected patients.

BACKGROUND: Endometrial Tuberculosis is one of the most common gynecological problems known to have serious implications for the quality of life like infertility. The commonly practiced histopathology solely relies on the suggestive feature of Tuberculosis (TB) with low specificity. Regarding the alternative bacteriological and molecular detection tools, little evidence was generated on their utility in the diagnosis of endometrial tuberculosis in Ethiopia. Therefore, we aim to investigate the detection rate of molecular and bacteriological detection methods on formalin-fixed paraffin-embedded biopsy samples for the diagnosis of endometrial and lymph node TB. METHODS: A retrospective cross-sectional study was conducted on 90 formalin fixed paraffin embedded biopsy samples from patients with gynecologic and lymph problems collected between 2018 and 2022 at St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia. SPSS version 26 was used for statistical analysis. The diagnostic performance was calculated using the histopathology method as the reference standard. Cohen's Kappa value was used to measure the level of agreement. A test with a P-value of < 0.05 was considered statistically significant. RESULTS: A total of 90 samples were analyzed in the current study. Auramine O, GeneXpert MTB/RIF assay, and Real-Time PCR tests have shown a detection rate of 32/90 (36%), 43/90 (47.8%), and 54/90 (60%) respectively (P ≤ 0.01). The sensitivity and specificity of AO were 38.1% and 95% respectively. RT PCR showed superior sensitivity followed by GeneXpert MTB/RIF assay, 70% and 58.6%. AO and molecular methods have shown a similarly low level of agreement with histopathology (Kappa value = 0.2). CONCLUSIONS: In a resource-limited setting, the selection of diagnostic tools needs careful attention. Putting the patients on anti-TB treatments based solely on histopathological findings may lead to undesired and adverse complications. Therefore, applying molecular and bacteriological detection methods along with histopathology, could help minimize inappropriate antimicrobial use.

Diagnostic value of the Xpert MTB/RIF assay combined with endobronchial ultrasonography with a guide sheath for peripheral nodular pulmonary tuberculosis.

BACKGROUND: The diagnosis of peripheral isolated nodular lesions that are suspected as pulmonary tuberculosis (PTB) is challenging, which are not easily accessible via conventional bronchoscopy. This study evaluated the combined use of Xpert MTB/RIF assay and endobronchial ultrasonography with a guide sheath (EBUS-GS) for detecting MTB infection in peripheral lung bands, for early detection of PTB. METHODS: The clinical data of 232 patients with suspected peripheral nodular PTB who underwent EBUS-GS between June 2020 and October 2023 were retrospectively reviewed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of acid-fast bacilli smear, culture, Xpert MTB/RIF assay, and pathological examination were calculated. To assess diagnostic accuracy, the results of the four methods were directly compared with the final clinical diagnosis. RESULTS: In total, 146 and 86 patients were clinically diagnosed with peripheral nodular PTB and non-PTB, respectively. The sensitivity, specificity, PPV, NPV, and AUC values of combined Xpert MTB/RIF assay and EBUS-GS were 47.26%, 100.0%, 100.0%, 52.76%, and 0.74; those of acid-fast bacilli smear were 8.22%, 97.67%, 85.71%, 38.53%, and 0.53; those of culture were 31.51%, 100.0%, 100.0%, 46.24%, and 0.66; and those of pathological examination were 23.97%, 97.67%, 94.59%, 43.08%, and 0.61, respectively. CONCLUSION: The diagnostic accuracy of the combined Xpert MTB/RIF assay and EBUS-GS was significantly better than that of other conventional tests. Hence, this novel technique can be routinely applied for diagnosing and managing peripheral nodular PTB.

TB burden and diagnostic challenges at Sandaun Provincial Hospital in West Sepik Province of PNG, 2016-2021.

SETTING: Bacteriological confirmation of TB diagnosis remains a key operational challenge in Papua New Guinea. Sandaun Provincial Hospital (SPH) is the main TB diagnostic and treatment centre of West Sepik Province. OBJECTIVE: To evaluate TB caseload, patient characteristics, and quality of diagnosis at SPH between 2016 and 2021. DESIGN: A retrospective descriptive study using TB treatment, laboratory, and presumptive TB registers to collect data on all TB patients. We used multivariable logistic regression to determine factors associated with bacteriological confirmation. RESULTS: Of 1,305 TB patients registered, 25% were children (<15 years) and 30% had extrapulmonary TB. The quality of sputum was associated with a positive smear microscopy result (P = 0.002). The proportion bacteriologically confirmed was low (37.3%), being higher in young adults 15-44 years (50.6%, 377/745) than in children <15 years (6.3%, 20/319) or older adults ≥45 years (37.6%, 68/181). Bacteriological confirmation was less likely in people travelling ≥3 hours to a health facility (adjusted OR 0.58, 95% CI 0.34-0.97) and extrapulmonary TB (aOR 0.01, 95% CI 0.00-0.03) but more likely for retreatment cases (aOR 1.59, 95% CI 1.00-2.51). CONCLUSION: Diagnostic services in West Sepik Province need strengthening to achieve a higher proportion of bacteriological confirmation in new pulmonary and extrapulmonary TB cases of all ages and improve access for the rural population.

CONTEXTE: La confirmation bactériologique du diagnostic de la TB reste un défi opérationnel majeur en Papouasie-Nouvelle-Guinée. L'hôpital provincial de Sandaun (SPH) est le principal centre de diagnostic et de traitement de la TB de la province du Sepik occidental. OBJECTIF: Évaluer le nombre de cas de TB, les caractéristiques des patients et la qualité du diagnostic à SPH entre 2016 et 2021. MÉTHODE: Une étude descriptive rétrospective utilisant le traitement de la TB, les registres de laboratoire et les registres de la TB présumée pour recueillir des données sur tous les patients atteints de TB. Nous avons utilisé la régression logistique multivariée pour déterminer les facteurs associés à la confirmation bactériologique. RÉSULTATS: Sur les 1 305 patients atteints de TB enregistrés, 25% étaient des enfants (<15 ans) et 30% avaient une TB extrapulmonaire. La qualité des expectorations a été associée à un résultat positif à la microscopie (P = 0,002). La proportion de bactéries confirmées était faible (37,3%), plus élevée chez les jeunes adultes de 15 à 44 ans (50,6% ; 377/745) que chez les enfants <15 ans (6,3% ; 20/319) ou les adultes plus âgés ≥45 ans (37,6% ; 68/181). La confirmation bactériologique était moins probable chez les personnes voyageant ≥3 h pour se rendre dans un établissement de santé (OR ajusté [ORa] 0,58 ; IC à 95% 0,34­0,97) et la TB extrapulmonaire (ORa 0,01 ; IC à 95% 0,00­0,03) mais plus probable pour les cas de retraitement (ORa 1,59 ; IC à 95% 1,00­2,51). CONCLUSION: Les services de diagnostic dans la province du Sepik occidental doivent être renforcés afin d'atteindre une proportion plus élevée de cas de TB pulmonaire et extrapulmonaire de tous âges et d'améliorer l'accès pour la population rurale.

Subtracting the background by reducing cell-free DNA's confounding effects on Mycobacterium tuberculosis quantitation and the sputum microbiome.

DNA characterisation in people with tuberculosis (TB) is critical for diagnostic and microbiome evaluations. However, extracellular DNA, more frequent in people on chemotherapy, confounds results. We evaluated whether nucleic acid dyes [propidium monoazide (PMA), PEMAX] and DNaseI could reduce this. PCR [16S Mycobacterium tuberculosis complex (Mtb) qPCR, Xpert MTB/RIF] was done on dilution series of untreated and treated (PMA, PEMAX, DNaseI) Mtb. Separately, 16S rRNA gene qPCR and sequencing were done on untreated and treated sputa before (Cohort A: 11 TB-negatives, 9 TB-positives; Cohort B: 19 TB-positives, PEMAX only) and 24-weeks after chemotherapy (Cohort B). PMA and PEMAX reduced PCR-detected Mtb DNA for dilution series and Cohort A sputum versus untreated controls, suggesting non-intact Mtb is present before treatment-start. PEMAX enabled sequencing-based Mycobacterium-detection in 7/12 (58%) TB-positive sputa where no such reads otherwise occurred. In Cohort A, PMA- and PEMAX-treated versus untreated sputa had decreased α- and increased ß-diversities. In Cohort B, ß-diversity differences between timepoints were only detected with PEMAX. DNaseI had negligible effects. PMA and PEMAX (but not DNaseI) reduced extracellular DNA in PCR and improved pathogen detection by sequencing. PEMAX additionally detected chemotherapy-associated taxonomic changes that would otherwise be missed. Dyes enhance microbiome evaluations especially during chemotherapy.

Diagnostic Dilemma: Tuberculosis Mastoiditis in an Immunocompromised Patient.

Tuberculosis (TB) is endemic in Malaysia but rarely affects the middle ear cleft. Common presentations of TB mastoiditis include unilateral, painless otorrhea, multiple small perforations of the tympanic membrane, and facial nerve palsy, although these symptoms can vary among patients. The diagnosis of TB mastoiditis is challenging due to its rarity and its similar presentation to common bacterial ear infections. This often leads to missed diagnoses, resulting in significant delays in treatment and potential complications. CT scans and histopathological examinations are crucial for diagnosing TB mastoiditis. Real-time polymerase chain reaction offers higher sensitivity and specificity compared to conventional methods for detecting Mycobacterium tuberculosis. TB infection should be considered in cases of otitis media that do not respond well to empirical antibiotic therapy. It is essential to send appropriate samples for TB testing to ensure timely diagnosis and treatment. This case report highlights the diagnostic challenges and complications encountered in a 22-year-old immunocompromised woman with TB mastoiditis.

Spina Ventosa of the Left Ring Finger With Tuberculous Pyomyositis in the Forearm Muscles in a Young Immunocompetent Indian Male With No Pulmonary Involvement: A First Report of Its Type.

Tuberculous infection of the extrapulmonary sites, especially the small bones, is a seldom reported entity even in endemic countries. Moreover, simultaneous involvement of the forearm muscles is a very rare presentation with no such case reported showing concurrent involvement of the two sites. The diagnosis is challenging due to the paucibacillary nature of the disease, a lack of awareness among primary clinicians, and ambiguity in clinical features with other musculoskeletal disorders, especially when there is no pulmonary involvement. Herein, we present a first-of-its-type case of spina ventosa of the left ring finger with a tuberculous abscess in the forearm in a 15-year-old Indian male with no pulmonary seeding. The diagnosis was achieved through a detailed diagnostic workup, which resulted in the detection of Mycobacterium tuberculosis. He was initiated on antituberculous treatment with a remarkable improvement.

The METHYLTRANSFERASE B-SERRATE interaction mediates the reciprocal regulation of microRNA biogenesis and RNA m6A modification.

In eukaryotes, RNA N6-methyladenosine (m6A) modification and microRNA (miRNA)-mediated RNA silencing represent two critical epigenetic regulatory mechanisms. The m6A methyltransferase complex (MTC) and the microprocessor complex both undergo liquid-liquid phase separation to form nuclear membraneless organelles. Although m6A methyltransferase has been shown to positively regulate miRNA biogenesis, a mechanism of reciprocal regulation between the MTC and the microprocessor complex has remained elusive. Here, we demonstrate that the MTC and the microprocessor complex associate with each other through the METHYLTRANSFERASE B (MTB)-SERRATE (SE) interacting module. Knockdown of MTB impaired miRNA biogenesis by diminishing microprocessor complex binding to primary miRNAs (pri-miRNAs) and their respective MIRNA loci. Additionally, loss of SE function led to disruptions in transcriptome-wide m6A modification. Further biochemical assays and fluorescence recovery after photobleaching (FRAP) assay indicated that SE enhances the liquid-liquid phase separation and solubility of the MTC. Moreover, the MTC exhibited enhanced retention on chromatin and diminished binding to its RNA substrates in the se mutant background. Collectively, our results reveal the substantial regulatory interplay between RNA m6A modification and miRNA biogenesis.