Mycobacterium Tuberculosis Culture Research Articles

Comparison of Metagenomic Second- and Third-Generation Sequencing by Diagnostic Sensitivity and Specificity in Tuberculosis Patients.

The third-generation sequencing (TGS) of Mycobacterium tuberculosis (MTB) is rarely reported in clinical practice. This study aims to compare the efficacy difference between next-generation sequencing (NGS) and TGS in the diagnosis of MTB and share the experience of NGS/TGS in the diagnosis of PTB. The past cases of suspected pulmonary tuberculosis (PTB) in our hospital were reviewed. Patients suspected of PTB who had undergone NGS/TGS detection, MTB culture, and molecular biology detection were included in the study. The clinical data of patients were reviewed, and the clinical comprehensive diagnosis of patients was used as the gold standard to analyze the efficacy of four methods for diagnosis of MTB infection, namely, metagenomic sequencing, MTB culture, molecular biology, and MTB culture + molecular biology. Then, we analyzed the results of NGS and TGS and compared the efficacy of the two metagenomic sequencing methods in the diagnosis of MTB infection. Ninety patients suspected of PTB were included in the study. 37 patients (41.11%) completed NGS and 53 patients (58.89%) completed TGS. Sixty-five patients (72.11%) had MTB infection in the lung or other parts of the body, and the remaining 25 patients (27.89%) had no MTB infection. Traditional laboratory tests such as MTB culture have low sensitivity and accuracy in diagnosing MTB. The sensitivity, specificity, and detection accuracy of metagenomic sequencing are significantly higher than those of other detection methods. The diagnostic accuracy of TGS reached 88.68%, which is significantly higher than that of NGS (64.86%). Compared with NGS, TGS has higher sensitivity and specificity in the diagnosis of MTB. In clinical practice, TGS is a better choice for patients who are suspected of MTB infection but cannot be diagnosed by traditional laboratory indicators.

Rapid Slide Culture Technique for Mycobacterium Tuberculosis and Predictors of Poor Outcomes among Pulmonary Tuberculosis Patients

Tuberculosis (TB), a dreadful disease known to mankind continues to be a problem in a developing country likeIndia. The incidence of people getting infected with TB is on the rise due to compounding factors like coinfectionwith the human immunodefiency virus and multidrug-resistant strains. There is a definitive need for earlydiagnosis and treatment of TB to curb transmission of the infection. Direct smear microscopy, though cheap andrapid, lacks sensitivity. Isolation of Mycobacterium tuberculosis in culture requires a long time, because of whichthere is a need for a rapid method which has good sensitivity and specificity for the detection of M. tuberculosis.Hence the present study was undertaken to determine Performance of rapid slide culture method for confirmingpulmonary tuberculosis in clinically suspected and smear positive cases and also to assess the factors responsiblefor poor outcomes in the study population.

A novel biosensor based on a bio-barcode for the detection of Mycobacterium tuberculosis.

Tuberculosis (TB), the second (after COVID-19) deadliest infectious killer, is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (M.T.), where early diagnosis and management are the key to containing the condition. Here, we report a novel biosensor for the detection of M.T. DNA based on magnetic separation, urease catalysis and silicon nanowire field effect transistor (SiNW FET) detection. M.T. DNA is sequence-specifically captured by magnetic nanoparticles and urease-labelled silica nanoparticles simultaneously to form a sandwich complex and urea is catalyzed into ammonium carbonate by urease modified on a sandwich complex. By using SiNW FET, the detection of M.T. DNA is realized indirectly by the detection of ammonium carbonate. The limit of detection (LOD) was determined to be 78.541 fM. The specificity of the biosensor was confirmed by detecting a panel of bacterial species. The utility of the biosensor was demonstrated in real-sample analysis and the recovery study of M.T. DNA was done in the genomic DNA extracted from cultured Mycobacterium tuberculosis. The biosensor holds promise to become a rapid, sensitive and accurate method for clinical diagnosis.

A retrospective observational study of presentation, diagnosis, and management of patients with genitourinary tuberculosis in a tertiary care hospital of India

Objectives: To study the clinical features, diagnostic methods, and management of genitourinary tuberculosis (GUTB). Methods: This retrospective study included 117 patients with GUTB admitted over a period of 10 years. The analysis of the patients was done in terms of presentation, organ involved, diagnostic methods, and management. The 6-month anti-tubercular treatment (ATT) was given to all patients, which included isoniazid, rifampicin, and ethambutol/pyrazinamide. Follow-up period was 1–10 years. Results: The mean age of the patients was 32.5 ± 6.4 years with a male: female ratio of 1:1.51. The majority of the patients presented with irritative voiding symptoms (66.47%) and hematuria (47.6%). The common infected organs were kidney (64.9%), ureter (27.35%), urinary bladder (17.09%), prostate (3.4%), and epididymis (5.19%). 25.6% of patients had positive chest X-ray, and 61.2% of the patients had positive Mantoux test. The diagnostic positivity rate for polymerase chain reaction, urine Mycobacteriumtuberculosis culture test, and urine acid-fast bacilli test was 67.7%, 35.4%, and 21.6%, respectively. 71 (60.13%) of the patients needed surgical intervention. After medical therapy, the adverse reactions noted were abnormal liver function in four cases, pruritus in three cases, and skin eruption in one case. Among the total of 117 patients followed over a period of 1 year to 10 years, none recurred. Conclusion: In a retrospective study of 117 patients with GUTB, majority had bladder symptoms, 60.13% needed surgery, and all were treated with 6-month course of ATT regimen consisting of isoniazid, rifampicin, and ethambutol/pyrazinamide, with none showing recurrence during the period of 1–10 years of follow-up.

Novel FujiLAM assay to detect tuberculosis in HIV-positive ambulatory patients in four African countries: a diagnostic accuracy study

Development of rapid biomarker-based tests that can diagnose tuberculosis using non-sputum samples is a priority for tuberculosis control. We aimed to compare the diagnostic accuracy of the novel Fujifilm SILVAMP TB LAM (FujiLAM) assay with the WHO-recommended Alere Determine TB-LAM Ag test (AlereLAM) using urine samples from HIV-positive patients. We did a diagnostic accuracy study at five outpatient public health facilities in Uganda, Kenya, Mozambique, and South Africa. Eligible patients were ambulatory HIV-positive individuals (aged ≥15 years) with symptoms of tuberculosis irrespective of their CD4 T-cell count (group 1), and asymptomatic patients with advanced HIV disease (CD4 count <200 cells per μL, or HIV clinical stage 3 or 4; group 2). All participants underwent clinical examination, chest x-ray, and blood sampling, and were requested to provide a fresh urine sample, and two sputum samples. FujiLAM and AlereLAM urine assays, Xpert MTB/RIF Ultra assay on sputum or urine, sputum culture for Mycobacterium tuberculosis, and CD4 count were systematically carried out for all patients. Sensitivity and specificity of FujiLAM and AlereLAM were evaluated against microbiological and composite reference standards. Between Aug 24, 2020 and Sept 21, 2021, 1575 patients (823 [52·3%] women) were included in the study: 1031 patients in group 1 and 544 patients in group 2. Tuberculosis was microbiologically confirmed in 96 (9·4%) of 1022 patients in group 1 and 18 (3·3%) of 542 patients in group 2. Using the microbiological reference standard, FujiLAM sensitivity was 60% (95% CI 51-69) and AlereLAM sensitivity was 40% (31-49; p<0·001). Among patients with CD4 counts of less than 200 cells per μL, FujiLAM sensitivity was 69% (57-79) and AlereLAM sensitivity was 52% (40-64; p=0·0218). Among patients with CD4 counts of 200 cells per μL or higher, FujiLAM sensitivity was 47% (34-61) and AlereLAM sensitivity was 24% (14-38; p=0·0116). Using the microbiological reference standard, FujiLAM specificity was 87% (95% CI 85-89) and AlereLAM specificity was 86% (95 CI 84-88; p=0·941). FujiLAM sensitivity varied by lot number from 48% (34-62) to 76% (57-89) and specificity from 77% (72-81) to 98% (93-99). Next-generation, higher sensitivity urine-lipoarabinomannan assays are potentially promising tests that allow rapid tuberculosis diagnosis at the point of care for HIV-positive patients. However, the variability in accuracy between FujiLAM lot numbers needs to be addressed before clinical use. ANRS and Médecins Sans Frontières.

Culture filtrate supplementation can be used to improve Mycobacterium tuberculosis culture positivity for spinal tuberculosis diagnosis.

Culture remains the gold standard to diagnose spinal tuberculosis (STB) despite the paucibacillary nature of the disease. Current methods can take up to 42 days to yield a result, delaying the ability to rapidly detect drug resistance. Studies have demonstrated the use of supplementation with culture filtrate (CF) from an axenic culture of Mycobacterium tuberculosis (Mtb) as a source of growth factors to improve culture rates. Our objective was to test a modified culture assay, utilizing CF supplemented media (CFSM), to improve culture positivity rates for suspected STB. Twelve patients with suspected STB were assessed by conventional culture (BACTEC™ MGIT 960), GeneXpert™ and standard histopathological examination. Spinal biopsies were taken from areas of diseased vertebral tissue or abscess, predetermined from MRI. Additional biopsies were obtained to assess CFSM for improved detection and faster culture of Mtb. All cases were diagnosed as STB and treated empirically for tuberculosis based on either bacteriological evidence (GeneXpert™, MGIT and/or CFSM positive), or based on clinical presentation. 5 specimens (45.45%) were positive for Mtb DNA as detected by GeneXpert™ and 1 specimen (8.33%) was cultured using MGIT (time to detection; 18 days). CFSM was able to culture 7 specimens (58.3%), with all CFSM positive specimens yielding a culture within 14 days. Two samples were positive only using the CFSM assay pointing to additional yield for diagnostic workup. Modification of standard culture can improve detection of Mtb and reduce time to positivity in individuals with STB where culture material is a requirement.

Current innovations in medical laboratory diagnosis of Tuberculosis

Tuberculosis (TB) is a major global health problem, ranking as the second highest cause of death from an infectious disease globally, after the human immunodeficiency virus (HIV). Nigeria ranked first in Africa with burden of TB. Tuberculosis is a major public health problem in Nigeria with about 407,000 people infected. The World Health Organization estimates that 120,000 people developed TB in 2019, with 154,000 death of whom, 13% were HIV positive individuals. Among the incident cases, 56% were from the South-East Asia and Western Pacific Regions and one quarter were from Africa. The African continent accounts for the highest rates of cases and deaths relative to population. Traditionally, tuberculosis is mostly being diagnosed by a combination of chest X-rays, the staining of sputum with special dyes followed by microscopy, the growth of Mycobacterium tuberculosis in culture and the Mantoux test. The sputum smear microscopy is easy to do and is very cheap and combined with chest X-rays has been used for a long time by TB control agencies worldwide. However, the sputum smear microscopy (sputum AFB) test has some problems in HIV-positive patients and children also in patients with low bacterial load and in latent TB infections. TB continues to be a complex disease to diagnose and manage due to the chronicity of the disease, the nature of the host-pathogen relationship, and the resulting diversity in its clinical manifestations. Therefore, this review considers recent innovations in TB laboratory diagnosis in conventional (traditional), non- conventional (molecular), serological (immunological) methods and the latest methods of TB diagnosis.

Characteristics of Drug-sensitive and Drug-resistant Tuberculosis Cases among Adults at Tuberculosis Referral Hospitals in Indonesia.

As Indonesia's rifampin resistance testing rates are lower than global testing rates per the 2020 WHO global tuberculosis (TB) report, prevalence of multidrug-resistant TB may be underestimated. Our study aimed to evaluate prevalence and patterns of TB drug resistance (DR) within Indonesia. We conducted a cross-sectional analysis of baseline data collected from 2017-2018 as part of a cohort study of adults with presumed pulmonary TB at 7 DR-TB referral hospitals in Indonesia. Bacteriological examinations (acid-fast bacilli, GeneXpert, sputum culture) and drug-susceptibility testing were performed following the guidelines of the National TB Program. Of 447 participants with complete bacteriological examinations, 312 (69.8%) had positive sputum cultures for Mycobacterium tuberculosis. The proportion of MDR and pre-extensively drug-resistant was higher in previously treated compared with newly diagnosed participants (52.5% [73/139] versus 15% [26/173]). Compared with drug-sensitive case, drug-resistant TB was associated with cavities. Given the difference between rates of DR in TB referral hospitals from our study compared with the WHO survey in 2019 that showed 17.7% and 3.3% DR among previously treated and newly diagnosed participants globally, further characterization of Indonesia's TB epidemiology in the general population is needed. Strategies, including public policies to optimize case finding, strengthen capacity for resistance testing, and prevent loss to follow-up will be critical to reduce the burden of TB in Indonesia.

Chinese expert consensus on diagnosis of pleural effusion

Pleural effusion(PE) is a common medical problem with various causes. The differential diagnosis for PE is often challenging. This consensus was generated by members of the academic group of the pleural and mediastinal diseases(preparatory) of Chinese Thoracic Society and some external experts. The members convened in virtual meetings and conducted an extensive literature investigation and assessed the quality of the evidence using a modified grading of recommendations assessment, development, and evaluation(GRADE) approach. This consensus included three chapters: the initial evaluation of PE, the diagnosis of PE with common causes, and the diagnosis of PE with uncommon causes.The main recommendations of Chapter Ⅰ were as follows:(1) For patients suspected of PE according to medical history and clinical manifestations, thoracic CT or ultrasound is recommended to confirm the presence or absence of PE.(2) Ultrasound-guided thoracentesis is recommended when available. Recommended tests for all sampled pleural effeusions include total protein, lactate dehydrogenase (LDH), adenosine deaminase (ADA), differential cell count, and cytological examination.(3) It is recommended to use Light's criteria to distinguish exudate and transudate. When PE is classified to be exudates with heart failure, it is recommended to detect N-terminal pro-brain natriuretic peptide of PE or serum-pleural fluid albumin gradient to assist the judgment.(4) Pleural biopsy is recommended for patients for whom the causes of PE cannot be identified by the detection of PE samples, and CT or ultrasound-guided pleural biopsy is more accurate. Thoracoscopy is recommended for patients whose etiology cannot be identified by laboratory tests of PE and/or pleural biopsy histopathology.The main recommendations of Chapter Ⅱ were as follows:(1)It is suggested to obtain more samples or use immunocytochemistry to assist the diagnosis and cell typing when initial cytopathology examination shows atypical cells, suspicious malignant or malignant cells. (2) Liquid medium for Mycobacterium tuberculosis culture is recommended to improve the positive rate. Molecular diagnosis (nucleic acid amplification or Xpert MTB/RIF) is recommended when tuberculous PE is suspected. For suspected tuberculous PE where the examination of PE is inconclusive. CT or ultrasound-guided pleural biopsy or thoracoscopy is recommended to obtain pleural tissue for acid-fast staining, Mycobacterium tuberculosis nucleic acid amplification and culture.(3)C-reactive protein (CRP) of PE is recommended to distinguish uncomplicated PPE from complicated PPE. It is suggested to inoculate pleural effusion into blood culture bottles or culturing specimens from ultrasound-guided pleural biopsy to increase the positive rate.The main recommendations of Chapter Ⅲ were as follows:(1) It is recommended to comprehensively analyze the patients' medical history, clinical manifestations, effusion characteristics, and biopsy pathological results to indentify uncommon causes.(2) It is recommended to detect the presence of chylomicrons or cholesterol crystals, with testing of the levels of triglyceride and cholesterol in PE for clinical suspicion of chylothorax or pseudochylothorax. (3) PE may be the result of a combination of various causes, and it is recommended to screen factors such as heart failure, hypoalbuminemia, and thoracic infection for critical patients.(4) For patients with PE whose cause has not been identified by thoracoscopic pleural biopsy, close follow-up for at least 2 years is recommended to exclude malignant diseases.

Clinical, laboratory, and radiographic aspects of patients with pulmonary tuberculosis and dysglycemia and tuberculosis treatment outcomes.

To analyze the association of dysglycemia with clinical, laboratory, and radiographic characteristics of patients with pulmonary tuberculosis (PTB), as well as with their tuberculosis treatment outcomes. This was a longitudinal study involving 140 patients diagnosed with PTB (positive cultures for Mycobacterium tuberculosis or positive Xpert MTB/RIF results from sputum samples). Patients were evaluated at diagnosis (M0), after completing the second month of treatment (M2), and at the end of treatment (MEND). At M0, the patients were classified into three groups: normoglycemia+PTB (NGTB); pre-diabetes mellitus+PTB (PDMTB), and diabetes mellitus+PTB (DMTB), in accordance with glycated hemoglobin levels (< 5.7%, 5.7%-6.4%, and ≥ 6.5%, respectively). Treatment outcomes were classified as favorable (cure or treatment completion) and unfavorable (death, loss to follow-up, or treatment failure). In our sample, 76 patients (61.4%) had dysglycemia, 20 of whom (14.3%) had DM at M0. The patients with dysglycemia, in comparison with those in the NGTB group, more frequently presented with positive sputum smear microscopy (94.2% vs. 75.9%; p = 0.003); cavities (80.2% vs. 63.0%; p = 0.03); bilateral lesions (67.4% vs. 46.0%; p = 0.02); and higher median of affected thirds of the lungs (3.0 vs. 2.0; p = 0.03) on chest radiography. No significant differences regarding outcomes were found among the groups, but tuberculosis lethality was higher in the DMTB group than in the PDMTB and NGTB groups (20% vs. 2.2%). PTB patients with dysglycemia had laboratory and radiographic manifestations indicative of more advanced disease, and the risk of death was higher in the DMTB group. These findings reinforce the recommendation for early screening for DM in patients with newly diagnosed tuberculosis in order to reduce the risk of death during treatment.

Evaluation of two short standardised regimens for the treatment of rifampicin-resistant tuberculosis (STREAM stage 2): an open-label, multicentre, randomised, non-inferiority trial

The STREAM stage 1 trial showed that a 9-month regimen for the treatment of rifampicin-resistant tuberculosis was non-inferior to the 20-month 2011 WHO-recommended regimen. In STREAM stage 2, we aimed to compare two bedaquiline-containing regimens with the 9-month STREAM stage 1 regimen. We did a randomised, phase 3, non-inferiority trial in 13 hospital clinics in seven countries, in individuals aged 15 years or older with rifampicin-resistant tuberculosis without fluoroquinolone or aminoglycoside resistance. Participants were randomly assigned 1:2:2:2 to the 2011 WHO regimen (terminated early), a 9-month control regimen, a 9-month oral regimen with bedaquiline (primary comparison), or a 6-month regimen with bedaquiline and 8 weeks of second-line injectable. Randomisations were stratified by site, HIV status, and CD4 count. Participants and clinicians were aware of treatment-group assignments, but laboratory staff were masked. The primary outcome was favourable status (negative cultures for Mycobacterium tuberculosis without a preceding unfavourable outcome) at 76 weeks; any death, bacteriological failure or recurrence, and major treatment change were considered unfavourable outcomes. All comparisons used groups of participants randomly assigned concurrently. For non-inferiority to be shown, the upper boundary of the 95% CI should be less than 10% in both modified intention-to-treat (mITT) and per-protocol analyses, with prespecified tests for superiority done if non-inferiority was shown. This trial is registered with ISRCTN, ISRCTN18148631. Between March 28, 2016, and Jan 28, 2020, 1436 participants were screened and 588 were randomly assigned. Of 517 participants in the mITT population, 133 (71%) of 187 on the control regimen and 162 (83%) of 196 on the oral regimen had a favourable outcome: a difference of 11·0% (95% CI 2·9-19·0), adjusted for HIV status and randomisation protocol (p<0·0001 for non-inferiority). By 76 weeks, 108 (53%) of 202 participants on the control regimen and 106 (50%) of 211 allocated to the oral regimen had an adverse event of grade 3 or 4; five (2%) participants on the control regimen and seven (3%) on the oral regimen had died. Hearing loss (Brock grade 3 or 4) was more frequent in participants on the control regimen than in those on the oral regimen (18 [9%] vs four [2%], p=0·0015). Of 134 participants in the mITT population who were allocated to the 6-month regimen, 122 (91%) had a favourable outcome compared with 87 (69%) of 127 participants randomly assigned concurrently to the control regimen (adjusted difference 22·2%, 95% CI 13·1-31·2); six (4%) of 143 participants on the 6-month regimen had grade 3 or 4 hearing loss. Both bedaquiline-containing regimens, a 9-month oral regimen and a 6-month regimen with 8 weeks of second-line injectable, had superior efficacy compared with a 9-month injectable-containing regimen, with fewer cases of hearing loss. USAID and Janssen Research & Development.

Rapid identification of Mycobacterium tuberculosis in cultures by molecular and immunological methods

Objective : The study aimed to evaluate molecular and immunological methods and to propose a workflow using them for tuberculosis (TB) diagnosis routine. Methods : A cross-sectional retrospective study was performed, including 121 liquid cultures from a TB laboratory located in the extreme south of Brazil. All cultures were positive for Mycobacterium tuberculosis complex (MTBC) by in-house Polymerase Chain Reaction (PCR) using DNA extracted by the CTAB method (PCR-CTAB) for IS6110 detection. These cultures were subjected to faster tests than this one, the immunological MPT64 assay and the PCR using DNA extracted by thermal lysis method (PCR-TL), and these were evaluated for MTBC identification using PCR- CTAB as a reference method. Results :The sensitivity of MPT64 assay and PCR-TL to identify MTBC in positive cultures by PCR-CTAB were 73.6% (89/121) and 98.3% (119/121), respectively. We proposed a workflow based on the use of MPT64 assay in liquid cultures suggestive of MTBC, and in case of a negative result, we suggest the performance of PCR-TL. The PCR-CTAB is suggested only if faster tests are negative. Conclusions : Methods capable of confirming MTBC in cultures should continue to be standardized, tested, and optimized to meet the ideal requirements of simplicity, quickness, and effectiveness. The molecular and immunological methods evaluated have differences in the execution and detection of MTBC in cultures, but they are rapid tools for laboratory TB diagnosis.

Empyema Necessitans Secondary to Tuberculosis: A Case Report

Empyema necessitans (EN) is a sporadic disease in the 21st century. It is a condition where the empyema has diffused and is loculated in the extrapleural space. It is usually caused by Tuberculosis (TB) infection or immunocompromised condition. Its manifestation, which is often vague in the beginning, leads to diagnostic dilemmas and delays in management. This has considerably affected patients adversely and incurred avoidable healthcare costs. We describe a rare case of empyema necessitans secondary to pulmonary tuberculosis in a 43-year-old male who presented to the emergency department with right anterior chest wall swelling, which was associated with pain, lethargy, malaise and significant loss of weight. Targeted ultrasonography of the anterior chest wall revealed a right anterior chest wall collection with intrathoracic extension. Contrast-enhanced computed tomography of the chest showed a multiloculated right chest wall collection with intrathoracic extension. Percutaneous drainage of the collection was performed, and the pus sent for Mycobacterium tuberculosis culture was positive. This case highlights the importance of a high index of suspicion and clinical awareness for this dangerous and silent disease.

Specific Profiles and Amplification of Resistance to Anti-tuberculosis Drugs in Tuberculosis Patients in the Republic of Karelia

The objective: to determine the frequency of resistance of Mycobacterium tuberculosis (MTB) to the most effective anti-tuberculosis drugs in tuberculosis patients in the Republic of Karelia, to characterize cases of amplification of MTB drug resistance during chemotherapy (CT).Subjects and Methods. MTB cultures of 1,137 tuberculosis patients were studied, the patients received chemotherapy from 2016 to 2021, among them 634 (55.8%) had multiple drug resistance (MDR). Among MBT cultures with MDR, there was a high frequency of MTB with extensive drug resistance (XDR) – 42.3% (268/634) including new cases. It provided a negative impact on the chemotherapy efficacy. Most often, amplification of MTB drug resistance was observed in patients treated as per regimen IV – 37/49 (75.5%) cases. MTB drug resistance amplified more often in the cases who were already resistant to 3 and more anti-tuberculosis drugs, 31/49 (63.3%). Most often, amplification was noted in aminoglycosides (Km, Am) and glycopeptide (Cm), (71.6% of cases). The high frequency of MTB drug resistance amplification from MDR up to XDR was due to the lack of rapid molecular genetic methods to test drug susceptibility to fluoroquinolones and aminoglycosides.

STUDY OF RESISTANCE TO FIRST-LINE DRUGS IN MULTIDRUG RESISTANCE TUBERCULOSIS SUSPECTS FROM SPUTUM SAMPLES BY GOLD STANDARD – STANDARD ECONOMIC VARIANT 1% PROPORTION METHOD IN NORTH COASTAL ANDHRA PRADESH

Objectives: The objectives of the study were to culture Mycobacterium tuberculosis from sputum samples of multidrug resistance (MDR) suspects on Lowenstein-Jensen (LJ) media, to test for susceptibility and resistance to first-line antitubercular drugs rifampicin (RMP), isoniazid (INH), streptomycin (SM), and ethambutol (EMB) by conventional DST on LJ media by standard economic variant proportion (1%) method and to calculate the rate of MDR and pattern of drug resistance in MDR tuberculosis (MDR-TB) suspects. Methods: A prospective, laboratory-based study was done on a total of 80 samples from cases of suspected pulmonary and extra-pulmonary TB. Identification of culture isolates as M. tuberculosis was done by susceptibility to p-nitrobenzoic acid (PNB), niacin test, and catalase activity at 68°C/pH 7. Drug sensitivity testing (DST) of M. tuberculosis cultures on LJ medium for drugs was performed by a standard economic variant of the 1% proportion method. Results: Among MDR suspects, the maximum is in the age group of 45–60 years (35%) with a mean age of 42.75±15.93 years. The sex ratio of male to female is 2:1. Out of 80 samples, 36 samples (45%) were graded 1+, 31 samples (38.7%) 2+, and 13 samples (16.3%) 3+. All 80 sputum samples showed growth on LJ media within 8 weeks. When colonies obtained on LJ medium were subjected to biochemical tests for identification, all were positive for M. tuberculosis. Out of 80 M. tuberculosis isolates tested for sensitivity against first-line drugs (except pyrazinamide), 45 (56.3%) were found to be sensitive to all four drugs (INH, RMP, EMB, and SM), and 35 (43.7%) were resistant to at least one drug. Nine isolates (11.3%) showed resistance to a single drug (monoresistance). The various MDR patterns were INH+RMP seen in 6 strains (7.5%) and INH+RMP+SM seen in 4 strains (5%). Conclusion: Drug resistance trends must be closely monitored to evaluate the efficiency of existing therapies and their effect on pulmonary tuberculosis epidemic. To design the new drug regulations, more extensive data on drug sensitivity with standardized testing protocols which is quality assured are required.

Evaluation of Xpert MTB/RIF Assay, MTB Culture and Line Probe Assay for the Detection of MDR Tuberculosis in AFB Smear Negative Specimens.

The global burden of tuberculosis (TB), particularly with multidrug resistance (MDR), is escalating and has become a major health challenge. It is well known that acid-fast bacilli (AFB) smear-negative TB patients are the major source of spreading TB to healthy individuals when left untreated. Early diagnosis of TB and rapid detection of drug resistance are important for the proper management of drug-resistant TB (DR-TB). Therefore, a laboratory based cross-sectional study was conducted from July to December 2019 at the National Tuberculosis Centre, Thimi, Nepal, with the objective of evaluating the diagnostic performance of Xpert MTB/RIF assay, Mycobacterium tuberculosis (MTB) culture and line probe assay (LPA) for the detection of MDR-TB in AFB smear-negative sputum samples. We evaluated a total of 222 AFB smear-negative sputum specimens, of which 21.6% (n = 48) showed MTB positive with Xpert MTB/RIF assay and, while culturing on Lowenstein-Jensen (LJ) media, 21.2% (n = 47) were MTB culture positive. The sensitivity, specificity, PPV and NPV at 95% confidence interval of Xpert MTB/RIF assay on diagnosing M. tuberculosis from smear-negative specimens were 73% (57-84), 92% (87-96), 71% (59-81) and 93% (89-95), respectively. In addition, the sensitivity of Xpert MTB/RIF assay and LPA in detecting rifampicin resistance was 75% (42-94, 95% CI) and 91.67% (62-99, 95% CI), respectively. The current study also assessed a significant association between the occurrence of pulmonary tuberculosis with different age group, TB history and alcohol consumption. These findings indicate that Xpert MTB/RIF assay and LPA are appropriate methods for early detection and accurate diagnosis of TB and RIF mono-resistant cases.

Clinical characteristics in 26 children with congenital tuberculosis in Central Southern China: a retrospective study

ABSTRACT Background Congenital tuberculosis (CTB) is relatively rare and most patients are described in case reports. Aim To investigate the clinical characteristics of CTB in 26 children. Methods A retrospective analysis of 26 children with CTB from January 2013 to December 2021 in Changsha Central Hospital in Central Southern China was undertaken. Results The median age at onset was 25 days (17–33) and within 4 weeks of age in approximately 73% of cases. Of 24 mothers (including two mothers of twins), 18 (75.0%) were asymptomatic during pregnancy, and four were diagnosed with tuberculosis prenatally. The numbers of tuberculous meningitis, tuberculous encephalitis and liver TB were 17 (65.4%), five (19.2%) and four (15.4%), respectively. The main symptoms were fever (n = 18, 69.2%) and cough (n = 16, 61.5%). Positive rates of T-SPOT.TB, acid-fast bacilli smear, culture of Mycobacterium tuberculosis and GeneXpert MTB/RIF test were, respectively, 84.2% (16/19), 42.3% (11/26), 43.5% (10/23) and 83.3% (5/6). Radiograph or computed tomography demonstrated typical pulmonary tuberculous lesions in all cases and the head magnetic resonance imaging (MRI) showed marked meningeal enhancement or parenchymal lesions in seven cases (26.9%). One case had drug-resistant TB. During follow-up, nine cases had varying degrees of liver injury, and one had delayed growth and development. Eight died and 18 recovered satisfactorily. Conclusion Maternal TB status during pregnancy, the epidemiological history, T-SPOT.TB and other TB-related aetiological tests and imaging are important for the early diagnosis and treatment of CTB, and are associated with a favourable outcome. Abbreviations AFB: acid-fast bacilli; Amk: amikacin; Cs: cycloserine; CT: computed tomography; E: ethambutol; GeneXpert MTB/RIF: GeneXpert Mycobacterium tuberculosis and rifampicin resistance; H: isoniazid; IVF-ET: in-vitro fertilization-embryo transfer; Lzd: linezolid; Mfx: moxifloxacin; MTB: Mycobacterium tuberculosis; mNGS: next generation sequencing; MTB-DNA: Mycobacterium tuberculosis-deoxyribonucleic acid; Pto: protionamide; R: rifampicin; TB: tuberculosis; T-SPOT.TB: spot test of mycobacterium TB infection T-lymphocytes; Z: pyrazinamide.

Mycobacterium tuberculosis Genotypes and Drug Susceptibility Test Results from Timor-Leste: A Pilot Study.

Tuberculosis (TB) is prevalent and a major public health problem in Timor-Leste. The government of Timor-Leste is prioritising the surveillance of TB and drug-susceptibility testing (DST) to understand the burden of TB and TB drug resistance in the country. Moreover, little is known about the origin of Mycobacterium tuberculosis (MTB) in Timor-Leste. This study reports MTB DST and sequencing for Timor-Leste. A pilot study was carried out in which a convenience sample of TB isolates from mucopurulent sputum collected from presumptive TB patients in the capital Dili between July and December 2016 was tested for phenotypic and genotypic evidence of drug resistance. Standard MTB culture was performed at the Timor-Leste National Health Laboratory (NHL). The MTB isolates were sent to the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Australia for DST and sequencing. Overall, 36 MTB isolates were detected at the NHL; 20 isolates were recovered during sub-culturing at VIDRL. All 20 isolates were susceptible to rifampicin, isoniazid, pyrazinamide, and ethambutol, with no genotypic markers of resistance identified. On sequencing, lineage 4 was the most common. The results of this study provide a small snapshot of MTB diversity and resistance in an under-sampled region with very high TB incidence. Future investment in whole-genome sequencing capacity in Timor-Leste will make it possible to undertake further, more representative analyses that may be used to evaluate transmission dynamics and epidemiology of genotypic markers of resistance.

Bronchial Washing Using a Thin Versus a Thick Bronchoscope to Diagnose Pulmonary Tuberculosis: A Randomized Trial.

This study was performed to evaluate the efficacy of using a thin bronchoscope for the diagnosis of pulmonary tuberculosis (PTB). Between March 2019 and November 2021, we prospectively enrolled participants with suspected PTB whose sputum acid-fast bacilli (AFB) smear and tuberculosis (TB) polymerase chain reaction (PCR) tests were negative or who could not produce self-expectorated sputum. Participants were randomized to a control group (bronchial washing [BW] using a 5.9-mm conventional bronchoscope guided by chest computed tomography) or an investigational group (BW using a 4.0-mm thin bronchoscope under virtual bronchoscopic navigation guidance). The primary outcome was detection of TB in BW fluid, defined as a positive result in the Xpert MTB/RIF assay. The secondary outcomes included AFB smear and Mycobacterium tuberculosis culture positivity, time to treatment initiation, and bronchoscopy-related complications. In total, 85 participants were included in the final analysis (43 in the control group and 42 in the investigational group). Twenty-three and 29, respectively, were finally diagnosed with PTB. The TB detection rate in BW fluid was higher in the investigational group (72.4% vs 43.5%, P = .035). Mycobacterium tuberculosis culture positivity was also higher in the investigational group (79.3% vs 52.2%, P = .038). No participants required premature bronchoscopy termination because of complications. Of the participants with PTB, the time to treatment initiation was shorter in the investigational group (median, 2.0 days vs 4.0 days, P = .001). BW using a thin bronchoscope increases the TB detection rate in patients with PTB compared to conventional bronchoscopy. Clinical Trials Registration.ȃNCT03802812.

The Clinical Experience of Mycobacterial Culture Yield of Pleural Tissue by Pleuroscopic Pleural Biopsy among Tuberculous Pleurisy Patients.

Background and Objectives: Tuberculous pleurisy is a common extrapulmonary TB that poses a health threat. However, diagnosis of TB pleurisy is challenging because of the low positivity rate of pleural effusion mycobacterial culture and difficulty in retrieval of optimal pleural tissue. This study aimed to investigate the efficacy of mycobacterial culture from pleural tissue, obtained by forceps biopsy through medical pleuroscopy, in the diagnosis of TB pleurisy. Materials and Methods: This study retrospectively enrolled 68 TB pleurisy patients. Among them, 46 patients received semi-rigid pleuroscopy from April 2016 to March 2021 in a tertiary hospital. We analyzed the mycobacterial culture from pleural tissue obtained by forceps biopsy. Results: The average age of the study participants was 62.8 years, and 64.7% of them were men. In the pleuroscopic group, the sensitivity of positive Mycobacterium tuberculosis (M. TB) cultures for sputum, pleural effusion, and pleural tissue were 35.7% (15/42), 34.8% (16/46), and 78.3% (18/23), respectively. High sensitivities of M. TB culture from pleural tissue were up to 94.4% and 91.7% when pleural characteristic patterns showed adhesion lesions and both adhesion lesions and presence of micronodules, respectively. Conclusions: M. TB culture from pleural tissue should be considered a routine test when facing unknown pleural effusion during pleuroscopic examination.