Mycobacterium Tuberculosis Culture Research Articles

Evaluation of a real-time PCR assay performance to detect Mycobacterium tuberculosis, rifampicin, and isoniazid resistance in sputum specimens: a multicenter study in two major cities of Indonesia.

Tuberculosis (TB) is one of the major global health issues due to its high mortality rate, especially in low- and middle-income countries. One of the key success points of the TB eradication program is early TB diagnosis, which requires rapid and accurate diagnostic testing. This study aimed to evaluate the performance of a newly developed RT-PCR kit (Indigen MTB/DR-TB RT-PCR) in a routine TB clinical setting. A multi-fluorescence RT-PCR assay was designed and developed to detect regions within IS6110, rpoB, katG, and inhA of the Mycobacterium tuberculosis (MTB) genes. Sputum specimens were obtained from suspected TB patients who visited TB healthcare facilities in two major cities of Indonesia from September 2022 to May 2023. Specimens were assessed using Indigen MTB/DR-TB RT-PCR, acid-fast bacillus (AFB) smear microscopy, MTB culture, and drug susceptibility testing (DST) methods. Fisher's exact test (χ2) was used to analyze the Indigen performance relative to culture methods. The performance of Indigen MTB/DR-TB RT-PCR to detect MTB was assessed using 610 sputum specimens obtained from suspected patients. The overall sensitivity and specificity were 94.12% (95% CI: 90.86-96.48%) and 98.32% (95% CI: 96.20-99.46%), respectively. When the analysis was performed on AFB smear-negative TB subjects (386 subjects), a lower sensitivity level was found at 78.57% (95% CI: 68.26-86.78%), while the specificity level remained similar at 98.34% (95% CI: 96.18-99.46%). The overall performance of Indigen MTB/DR-TB RT-PCR to detect MTB showed substantial agreement with the MTB culture method (kappa value 0.93). In comparison to DST, the sensitivity and specificity levels of Indigen to detect RIF resistance or INH resistance were 78.2% (95% CI: 61.8-90.2%) and 82.8% (95% CI: 64.2-94.2%), respectively, while the specificity level for both groups was at 100% (95% CI, 87.7-100%). Indigen MTB/DR-TB RT-PCR demonstrated reliable performance for TB molecular diagnostic testing and can be implemented in routine TB diagnostic settings.

Relative Value of Immunohistochemistry in Detection of Mycobacterial Antigen in Suspected Cases of Tuberculosis in Tissue Section.

Due to its infectious nature, complex immunological response, chronic progression, and the necessity for long-term treatment, tuberculosis has always been a major health burden. Immunohistochemistry (IHC) has the capacity to highlight the occurrence of mycobacterial antigens for tissue diagnosis. This study was conducted to understand the advantage of immunostaining over culture of Mycobacterium tuberculosis. A cross-sectional study was conducted on 30 samples of suspected cases of tuberculosis. Specimens received were fixed in 10% formalin and processed; 3-5 µm thick sections were made from paraffin block, stained with hematoxylin and eosin, Ziehl-Neelsen stain, and immunohistochemistry. Culture was done using Lowenstein-Jensen medium. Immunohistochemistry was interpreted as fine granular brownish cytoplasmic, coarse granular brownish cytoplasmic, and bacillus staining. Out of the 30 samples studied, 12 (40.0%) were culture positive while 20 (66.7%) of them were IHC positive. Immunohistochemistry showed 17 granulomatous lesions of which 11 (55.0%) were well-formed granulomas. The sensitivity and negative predictive value were found to be high with immunohistochemistry, while specificity and positive predictive value were found to be on the lower side. Among the 20 positive IHC cases, the degree of staining was fine granular cytoplasmic staining in 13 cases (65.0%) and coarse granular staining in 7 cases (35.0%). Immunohistochemistry is a reliable test with high sensitivity as well as high negative predictive value which can be done rapidly for establishing an etiological diagnosis of tuberculosis in histologic specimens.

Blood RNA biomarkers for tuberculosis screening in people living with HIV before antiretroviral therapy initiation: a diagnostic accuracy study

Undiagnosed tuberculosis remains a major threat for people living with HIV. Multiple blood transcriptomic biomarkers have shown promise for tuberculosis diagnosis. We sought to evaluate their diagnostic accuracy and clinical utility for systematic pre-antiretroviral therapy (ART) tuberculosis screening. We enrolled consecutive adults (age ≥18 years) referred to start ART at a community health centre in Cape Town, South Africa, irrespective of symptoms. Sputa were obtained (using induction if required) for two liquid cultures. Whole-blood RNA samples underwent transcriptional profiling using a custom Nanostring gene panel. We measured the diagnostic accuracy of seven candidate RNA signatures (one single gene biomarker [BATF2] and six multigene biomarkers) for the reference standard of Mycobacterium tuberculosis culture status, using area under the receiver-operating characteristic curve (AUROC) analysis, and sensitivity and specificity at prespecified thresholds (two standard scores above the mean of healthy controls; Z2). Clinical utility was assessed by calculating net benefit in decision curve analysis. We compared performance with C-reactive protein (CRP; threshold ≥5 mg/L), WHO four-symptom screen (W4SS), and the WHO target product profile for tuberculosis triage tests. A total of 707 people living with HIV (407 [58%] female and 300 [42%] male) were included, with median CD4 count 306 cells per mm3 (IQR 184-486). Of 676 participants with available sputum culture results, 89 (13%) had culture-confirmed tuberculosis. The seven RNA signatures were moderately to highly correlated (Spearman rank coefficients 0·42-0·93) and discriminated tuberculosis culture positivity with similar AUROCs (0·73-0·80), but none statistically better than CRP (AUROC 0·78, 95% CI 0·72-0·83). Diagnostic accuracy was similar across CD4 count strata, but lower among participants with negative W4SS (AUROCs 0·56-0·65) compared with positive (AUROCs 0·75-0·84). The RNA biomarker with the highest AUROC point estimate was a four-gene signature (Suliman4; AUROC 0·80, 95% CI 0·75-0·86), with sensitivity 83% (95% CI 74-90) and specificity 59% (55-63) at the Z2 threshold. In decision curve analysis, Suliman4 and CRP had similar clinical utility to guide confirmatory tuberculosis testing, but both had higher net benefit than W4SS. In exploratory analyses, an approach combining CRP (≥5 mg/L) and Suliman4 (≥Z2) had sensitivity of 80% (70-87), specificity of 70% (66-74), and higher net benefit than either biomarker alone. RNA biomarkers showed better clinical utility to guide confirmatory tuberculosis testing for people living with HIV before ART initiation than symptom-based screening, but their performance did not exceed that of CRP and fell short of WHO recommended targets. Interferon-independent approaches might be required to improve accuracy of host-response biomarkers to support tuberculosis screening before ART initiation. South African Medical Research Council, European and Developing Countries Clinical Trials Partnership 2, National Institutes of Health National Institute of Allergy and Infectious Diseases, The Wellcome Trust, National Institute for Health and Care Research, Royal College of Physicians London.

In vitro evaluation of the binding activity of novel mouse IgG1 opsonic monoclonal antibodies to Mycobacterium tuberculosis and other selected mycobacterial species

Antimicrobial resistance alongside other challenges in tuberculosis (TB) therapeutics have stirred renewed interest in host-directed interventions, including the role of antibodies as adjunct therapeutic agents. This study assessed the binding efficacy of two novel IgG1 opsonic monoclonal antibodies (MABs; GG9 & JG7) at 5, 10, and 25 µg/mL to live cultures of Mycobacterium tuberculosis, M. avium, M. bovis, M. fortuitum, M. intracellulare, and M. smegmatis American Type Culture Collection laboratory reference strains, as well as clinical susceptible, multi-drug resistant, and extensively drug resistant M. tuberculosis strains using indirect enzyme-linked immunosorbent assays. These three MAB concentrations were selected from a range of concentrations used in previous optimization (binding and functional) assays. Both MABs bound to all mycobacterial species and sub-types tested, albeit to varying degrees. Statistically significant differences in MAB binding activity were observed when comparing the highest and lowest MAB concentrations (p < 0.05) for both MABs GG9 and JG7, irrespective of the M. tuberculosis resistance profile. Binding affinity increased with an increase in MAB concentration, and optimal binding was observed at 25 µg/mL. JG7 showed better binding activity than GG9. Both MABs also bound to five MOTT species, albeit at varied levels. This non-selective binding to different mycobacterial species suggests a potential role for GG9 and JG7 as adjunctive agents in anti-TB chemotherapy with the aim to enhance bacterial killing.

The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia.

Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Evaluation of molecular diagnostic test for detection of adult pulmonary tuberculosis: A generic protocol.

Tuberculosis (TB) continues to be the second most-leading cause of death due to a single infectious agent as of 2022 after COVID-19. Many affordable new molecular diagnostic tools are being developed for early and more accurate diagnosis, especially for low-resource settings in low- and middle-income countries. In this context, there is a need to develop a standardized protocol for validation of new diagnostic tools. Here, we describe a generic protocol for multi-centric clinical evaluation of molecular diagnostic tests for adult pulmonary TB. This protocol describes a cross-sectional study in TB reference laboratories in India. Adults (>18 yr) visitng the chest clinics or outpatient departments with symptoms of TB need to be enrolled consecutively till the required sample size of 150 culture positives and 470 culture negatives are met. Mycobacterium tuberculosis (Mtb) culture (mycobacteria growth indicator tube liquid culture) to be used under this protocol as the gold standard and Xpert MTB/RIF molecular test will be used as the comparator. The sputum samples will be tested by smear microscopy, Mtb culture, Xpert MTB/RIF and index molecular test as per the proposed algorithm. The specificity sensitivity, and positive/ negative predictive values are to be calculated for the index test with reference to the gold standard. TB diagnosis poses many challenges as it differs with type of disease, age group, clinical settings and type of diagnostic tests/kits used. Globally, different protocols are used by several investigators. This protocol provides standard methods for the validation of molecular tests for diagnosis of adult pulmonary TB, which can be adopted by investigators.

Tubercular Meningitis Mimicking Acute Disseminated Encephalomyelitis (ADEM)- A Case Report

Tuberculosis remains endemic in India, and its manifestation in the central nervous system (CNS) poses significant challenges in diagnosis and management. We present a rare case of a 65-year-old male with tubercular meningitis exhibiting atypical features of acute disseminated encephalomyelitis (ADEM). The patient presented with a month-long history of fever, severe headache, altered sensorium, and neurological signs including brisk reflexes, neck rigidity, and bilateral motor weakness. Initial investigations revealed elevated inflammatory markers and abnormal cerebrospinal fluid (CSF) findings consistent with tubercular meningitis. Despite negative CSF culture and nucleic acid amplification tests (NAATs) for Mycobacterium tuberculosis, the patient displayed characteristic asymmetrical bilateral cerebral lesions on MRI suggestive of ADEM. Given the diagnostic ambiguity and a high suspicion of CNS tuberculosis with concurrent ADEM, the patient was treated with anti-tubercular therapy (ATT) and a course of intravenous methylprednisolone followed by oral prednisolone. Subsequent clinical improvement prompted gradual tapering of prednisolone, leading to a full recovery at discharge. This case highlights the complexity of diagnosing CNS tuberculosis coexisting with ADEM, emphasizing the importance of a comprehensive clinical assessment and tailored therapeutic interventions in managing such intricate neurological presentations.

Second Nationwide Tuberculosis Outbreak Caused by Bone Allografts Containing Live Cells - United States, 2023.

During July 7-11, 2023, CDC received reports of two patients in different states with a tuberculosis (TB) diagnosis following spinal surgical procedures that used bone allografts containing live cells from the same deceased donor. An outbreak associated with a similar product manufactured by the same tissue establishment (i.e., manufacturer) occurred in 2021. Because of concern that these cases represented a second outbreak, CDC and the Food and Drug Administration worked with the tissue establishment to determine that this product was obtained from a donor different from the one implicated in the 2021 outbreak and learned that the bone allograft product was distributed to 13 health care facilities in seven states. Notifications to all seven states occurred on July 12. As of December 20, 2023, five of 36 surgical bone allograft recipients received laboratory-confirmed TB disease diagnoses; two patients died of TB. Whole-genome sequencing demonstrated close genetic relatedness between positive Mycobacterium tuberculosis cultures from surgical recipients and unused product. Although the bone product had tested negative by nucleic acid amplification testing before distribution, M. tuberculosis culture of unused product was not performed until after the outbreak was recognized. The public health response prevented up to 53 additional surgical procedures using allografts from that donor; additional measures to protect patients from tissue-transmitted M. tuberculosis are urgently needed.

Tuberculous peritonitis: an analysis of case series of 49 consecutive patients

Objectives: The incidence of tuberculous peritonitis has been reported between 0.1% and 0.7% among all tuberculosis types. This study, it was aimed to evaluate the cases with tuberculous peritonitis, which has an important place in the differential diagnosis of patients with ascites, clinically, biochemically, microbiologically, and histopathologically. Methods: : Forty-nine patients with a definite clinical, radiological, and histopathological diagnosis of tuberculous peritonitis, which formed the basis of our study, were analyzed retrospectively. Results: The mean age of patients with tuberculous peritonitis was found to be 39.45 ± 19.02 years. Purified-protein derivative (PPD) was positive in 23 (72%) of 32 patients with tuberculous peritonitis whose PPD results were recorded, in 9 (28%) PPD results were evaluated as anergic or negative. QuantiFERON-TB Gold In-Tube (QFT-GIT) test was sent in 10 of the patients, the result was positive in 9 (90%) patients and negative in 1 (10%) patient. There were 32 patients in whom tuberculosis polymerase chain reaction (PCR) was studied from ascitic fluid, 7 (22%) of the patients were PCR positive and 25 (78%) negative. Mycobacterium tuberculosis culture positivity was found in 18 (69%) of 26 patients who were biopsied. In total, 29 (59%) of the patients had M. tuberculosis culture positivity. Conclusions: Tuberculous peritonitis constitutes a public health problem in endemic regions of the world and tuberculous peritonitis should be considered in patients presenting with ascites. Despite all diagnostic difficulties, necessary tests, especially peritoneal biopsy, should be performed for early diagnosis, and it should not be forgotten that early initiation of treatment is very important in terms of morbidity and mortality of the disease.

A rare case of isoniazid mono-resistant tuberculosis presenting as cardiac tamponade along with an anterior mediastinal mass in a 15-year-old immunocompetent patient.

A 15-year-old boy presented with a sudden onset of breathlessness for 7 days, gradual loss of weight of 17.6 lbs over the last month and progressive hoarseness of voice for 7 months. The contrast-enhanced computed tomography (CECT) scan revealed a heterogeneously enhancing lesion in the anterior mediastinum with multiple discrete lymph nodes in the cervical and mediastinal locations. The GeneXpert MTB/RIF assay performed on the CT-guided biopsy of the mass was negative, but the culture for Mycobacterium tuberculosis was positive at 7 weeks of incubation. There was a suboptimal radiological response after 6 months of treatment. First-line drug susceptibility testing (DST) performed by line probe assay (LPA) on the positive culture detected high-level resistance to isoniazid. The treatment was modified as per DST results to which the patient responded well.

Clinical Significance of Preoperative Pyrazinamide-Containing Therapy in Tuberculous Constrictive Pericarditis.

Tuberculous constrictive pericarditis (TCP) is recommended to be treated with anti-tuberculosis (TB) therapy before pericardiectomy. Whether different preoperative anti-TB regimens may lead to different outcomes is unclear. We retrospectively collected patients diagnosed as TCP and received pericardiectomy from April 2016 to June 2023. The study patients were assigned into the active TCP (A-TCP) group and the inactive TCP (IA-TCP) group according to the results of Mycobacterium tuberculosis (MTB) culture and MTB RNA assay. Baseline characteristics including anti-TB regimens and surgical outcomes were compared between the two groups. Logistic regression analysis and subgroup analysis were conducted to identify the protective factors of A-TCP. Of the 102 study patients, 24 was in the A-TCP group and 78 was in the IA-TCP group. The rate of preoperative anti-TB regimen containing pyrazinamide was 37.5% in the A-TCP group, as compared with 74.4% in the IA-TCP group (P = 0.001). Multivariate analysis showed that preoperative use of pyrazinamide was the protective factor of A-TCP (OR 0.194, 95% CI 0.053-0.703, P = 0.013). Subgroup analysis based on age also showed consistent findings. In the analyses of surgical outcomes, A-TCP was the independent risk factor of postoperative cardiac complications (OR 4.231, 95% CI 1.317-13.593, P = 0.015) and associated with longer hospital stay (P = 0.004) and higher hospitalization cost (P = 0.001). A strategy involving anti-TB regimen containing pyrazinamide before pericardiectomy was superior to that without pyrazinamide in the patients with TCP. The strategy was associated with lower risk of A-TCP and might lead to better postoperative recovery and cost-effectiveness.

Factors influencing histoplasmosis incidence in multidrug-resistant pulmonary tuberculosis patients: A cross-sectional study in Indonesia.

Histoplasmosis is an infectious disease caused by the dimorphic fungus Histoplasma capsulatum, which in chronic conditions, is generally difficult to distinguish from pulmonary tuberculosis (TB) based on its clinical appearance; therefore, diagnostic errors could occur. Meanwhile, the prevalence of multidrug-resistant pulmonary TB (MDR-TB) in Indonesia remains high. Study determining the incidence of histoplasmosis in MDR-TB is unavailable worldwide. The aim of this study was to determine the risk factors of histoplasmosis incidence in MDR-TB patients in Indonesia. A cross-sectional was conducted at H. Adam Malik General Hospital, Medan, Indonesia and the ELISA platform (semi-quantitative) was used to detect histoplasma antibodies. Factors associated with histoplasmosis incidence among MDR-TB were determined using a Chi-squared test. A total of 50 MDR-TB patients were included this study of which 14 of them (28%) had histoplasmosis. The majority of histoplasmosis occurred in males, in MDR-TB patients with a history of TB treatment and among who had chest x-rays with far-advanced lesions. However, statistical analyses indicated none of those factors (sex, TB treatment history, status of the lung) as well as age group, acid-fast bacillus result, Mycobacterium tuberculosis culture result, having pet, living in damp house, working in the field or plantation, having HIV infection and smoking status were associated with histoplasmosis incidence. This study highlights that the incidence of histoplasmosis is relatively high and therefore further studies are important to be conducted in Indonesia that has a high MDR-TB cases.

Using ultrasound guided needle biopsy in conjunction with GeneXpert MTB/RIF to diagnose epididymal tuberculosis

This study aimed to investigate the diagnostic utility of percutaneous ultrasound-guided needle biopsy conjunction with GeneXpert MTB/RIF for epididymal tuberculosis. A retrospective analysis was conducted on the pathological and laboratory examinations of 20 patients with epididymal lesions undergoing ultrasound guided biopsy at Shandong Public Health Clinical Center. Laboratory examination included acid-fast staining, Mycobacterium tuberculosis culture by BACTEC MGIT 960, and GeneXpert MTB/RIF test. Diagnosis and complications were comprehensively analyzed. Among the 20 patients, 15 had epididymal tuberculosis and 5 had non-epididymal tuberculosis. Ten patients had granulomatous inflammation and necrotic tissues. The sensitivity and specificity of acid-fast staining, Mycobacterium tuberculosis culture, and GeneXpert MTB/RIF for the diagnosis of epididymis tuberculosis were 26.67% and 100.00%, 33.33% and 100.00%, and 86.67% and 100.00%, respectively. The diagnostic value analysis of the 3 detection techniques indicated that the GeneXpert MTB/RIF technique (Kappa = 0.765, Area under the curve (AUC) = 0.933) was superior to Mycobacterium tuberculosis culture (Kappa = 0.200, AUC = 0.667) and acid-fast staining (Kappa = 0.154, AUC = 0.633). Ultrasound-guided percutaneous biopsy is a safe procedure. The GeneXpert MTB/RIF test has high sensitivity, specificity, and superior AUC value, which are of great value in the diagnosis of epididymal tuberculosis and rifampicin resistance detection.

Diagnostic value of bronchoscopy in sputum‑negative presumptive pulmonary tuberculosis patients and its clinicoradiological profile

Abstract: CONTEXT: Tuberculosis (TB) remains endemic in India. Little local data have been reported on bronchoscopic evaluation of sputum‑negative pulmonary TB patients, which poses a significant diagnostic and therapeutic challenge. AIMS: To determine the diagnostic value of bronchoscopy and bronchoalveolar lavage (BAL) and its clinical and radiological profile in sputum‑negative, pulmonary TB patients. METHODS: We performed an analysis of patients with probable pulmonary TB with overall negative (smear and/or polymerase chain reaction [PCR]) or scanty sputum that had undergone bronchoscopy with BAL over a period of 11 months. Patients’ symptoms, radiological features, lung lobe lavaged, BAL acid‑fast bacilli (AFB) stain, Mycobacterium TB (MTB)‑PCR, and mycobacterial cultures were analyzed. Mycobacterial culture was used as a reference standard. RESULTS: 54 patients underwent a diagnostic bronchoscopy. Dry cough and fever were the most common symptoms. Uncontrolled diabetes mellitus was the most frequent comorbidity identified in 15 (28%) patients. Fifty‑nine percent of the patients had diffuse lung infiltrates, with consolidation being the most common abnormality (41%), followed by cavitation (39%). Right upper lobe was the most frequent lung lobe lavaged (31%). BAL mycobacterial culture and MTB Polymerase Chain Reaction (PCR) were positive in 43 (80%) and 41 (76%) patients and BAL AFB was positive in 5 patients respectively. Using BAL culture as reference, male gender, predominant upper lobe disease, upper lobe lavage, and presence of cavities on chest imaging had the highest diagnosticyield (82%, 100%, 63%, and 89%, respectively) (P = 0.02,0.006, 0.03, and 0.02, respectively).There was 100% concordance among the three diagnostic modalities. CONCLUSIONS: An overall diagnostic yield of 80% was achieved with combined use of BAL MTB PCR, culture, and AFB staining. Keywords: Broncho alveolar lavage, bronchoscopy, Mycobacterium tuberculosis polymerase chain reaction, sputum negative, tuberculosis

Higher Sensitivity of Xpert MTB/RIF Ultra Over Tuberculosis Culture for the Diagnosis of Spinal Tuberculosis With Open or Computed Tomography-Guided Biopsies.

Diagnostic specimens for spinal tuberculosis (STB) are mostly collected via open surgery. Percutaneous computed tomography (CT)-guided biopsies are used in times of limited surgical availability. However, poor diagnostic accuracy of Mycobacterium tuberculosis (Mtb) culture has been reported with this method, due to limited sample volume and the paucibacillary nature of STB. We evaluated Xpert MTB/RIF Ultra on open and CT-guided biopsies as compared with the gold standard Mtb culture and histopathology. We conducted a prospective diagnostic accuracy study of Xpert Ultra, as compared with tuberculosis culture and histopathology, in adults with signs and symptoms of STB at a tertiary academic hospital in South Africa from November 2020 to December 2021. Diagnostic testing was performed on 31 patients with available samples. Xpert Ultra had a sensitivity of 94.7% (95% CI, 75.3%-99.7%) and specificity of 100% (95% CI, 75.7%-100.0%) against a reference standard of Mtb culture and histopathology. Xpert Ultra had high diagnostic accuracy in open and CT-guided biopsy samples with sensitivity and specificity of 100% and 100% (open) and 89% and 100% (CT), respectively. Mtb culture had limited specificity for CT-guided biopsies (43%; 95% CI, 15.8%-74.9%). HIV-1 coinfection did not affect Mtb abundance measures by Xpert Ultra or culture. Xpert Ultra was also superior to culture for STB diagnosis in patients concurrently treated for pulmonary tuberculosis. Xpert Ultra detected more STB cases than culture for CT-guided biopsy samples. There was also no difference in sensitivity for open biopsies, irrespective of HIV-1 status, making it an important tool for rapid diagnosis, especially during times or in locations where open surgery is not possible or concurrent pulmonary tuberculosis treatment is initiated.

Host factors associated with false negative results in an interferon‐γ release assay in adults with active tuberculosis

ObjectiveTo identify host factors associated with false-negative results of interferon-γ release tests in adults with active tuberculosis. MethodsThe clinical data of 943 patients with active tuberculosis diagnosed by acid-fast smear staining, Mycobacterium tuberculosis culture, Mycobacterium tuberculosis PCR and pathological examination at West China Hospital of Sichuan University were retrospectively analysed. According to the results of the interferon γ release test (IGRA), the patients were divided into the IGRA− group and IGRA+ group. Logistic regression was used to analyze the sociodemographic data and clinical characteristics of participants in the IGRA− group and IGRA+ group. ResultsAmong 943 patients with active tuberculosis, 174 (18.5 %) were IGRA negative (false negative), and 769 (81.5 %) were IGRA positive. Multivariate logistic regression analysis identified the following characteristics independently associated with IGRA negativity: age (OR: 1.02; 95 % CI: 1.01 1.03; p = 0.006), anti-tuberculosis treatment >1 month (OR: 1.68; 95 % CI: 1.12 2.52; p = 0.013), HIV infection (OR: 9.48; 95 % CI: 3.23 27.85; p = 0.000), combined with connective tissue diseases (OR: 2.78; 95 % CI: 1.30 5.94; p = 0.008) and low hemoglobin (OR: 0.99; 95 % CI: 0.98 1.00; p = 0.044) was associated with an increased false-negative probability of IGRA. ConclusionAge, anti-tuberculosis therapy >1 month, coinfection with HIV, coassociated connective tissue disease and decreased hemoglobin were identified as risk factors for false-negative results of IGRA. Our results suggest a careful interpretation of IGRA in adults with these characteristics.

Storage of Mycobacterium tuberculosis culture isolates in MicrobankTM beads at a South African laboratory.

Mycobacterium tuberculosis complex (MTBC) isolates are typically stored at -70 °C in cryovials containing 1 mL aliquots of a liquid medium, with or without 50% glycerol. Multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. Small culture aliquots may be more practical; however, storage capacity remains challenging. MicrobankTM beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing MTBC isolates is uninvestigated. The study aimed to determine the feasibility of using MicrobankTM beads for long-term storage of MTBC isolates at a laboratory in South Africa. In February 2020, 20 isolates in liquid culture were stored in MicrobankTM beads, following an in-house developed protocol, at -70 °C. At defined time points (16 months [15 June 2021] and 21 months [18 November 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. Stored liquid isolates demonstrated MTBC growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. Contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. MicrobankTM beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. MicrobankTM beads represent a storage solution for the medium-term storage of MTBC isolates. This study evaluated the use of MicrobankTM beads as an alternate method for storing MTBC culture isolates at -70 °C and provided a suitable option for medium-term storage of MTBC.

Histopathological features in the clinical specimens with tuberculosis diagnosis by BACTEC MGIT 960 culture

Diagnosis of extrapulmonary tuberculosis remains challenging in lower-middle income countries with high burden of tuberculosis (TB). This study aims to describe the histological characteristics in biopsy samples from patients with confirmed TB. This is a retrospective study of clinical biopsy specimens with positive liquid medium culture for Mycobacterium tuberculosis and histopathological examination in the National Lung Hospital in Vietnam. Among 1045 biopsy specimens with mycobacteria culture, the overall rate of growth of Mycobacteria tuberculosis in culture was 20.7% (216/1045). The positivity rates of MIGT culture among surgical biopsy specimens were 75% in bone specimen, followed by vertebral specimens (51.3%), and joint specimens (26.4%). For specimens obtained by the fine needle aspiration, the positivity rates of MIGT culture were 26.3% in lymph node and 25.3% in pleural specimen. Among specimens with culture confirmation of TB, the most common histopathoglogical suggestive finding of TB was the presence of epithelioid cell (83.3%), Langhans giant cells (75.9%), and caseous necrosis (75.5%). The high proportion of histological features suggestive of TB among the TB culture confirmed biopsy samples support for further evaluation of histological examination and its combination with other recommended rapid molecular assays in specimens with suspicion of TB.

Detection of drug-resistant Mycobacterium tuberculosis in pericardial fluid culture and its correlation with cartridge based nucleic acid amplification test and adenosine deaminase activity

BackgroundPericardial effusion is the accumulation of fluid in the pericardial cavity. In nations with high tuberculosis (TB) load, TB is the most common cause of pericardial effusion. 1–2% of patients with pulmonary TB develop Pericardial TB worldwide. Multi-drug-resistant (MDR) TB, including extrapulmonary TB (EPTB) cases, are rising in number. Adenosine Deaminase (ADA) is an enzyme in lymphocytes and myeloid cells, which has certain immune functions in the body. ADA levels are increased in inflammatory conditions, like pleural, pericardial, or joint effusions, of bacterial etiology, granulomatous conditions, neoplasms, and autoimmune pathologies. TB is the only lymphocytosis involving disease with increased ADA levels. MDR EPTB is rare, but cases are on the rise, and tuberculous pericardial effusion is one such example. Hence, it is important to know the percentage of cases detected by a culture that can be identified by cartridge-based nucleic acid amplification test (CBNAAT), their resistance patterns, and to identify potential markers like ADA, which can help in early identification of cases. The objectives of this study were to identify the Mycobacterium tuberculosis (MTB) bacilli in culture, and correlate them with cartridge-based nucleic acid amplification test (CBNAAT) results and their drug-resistance, in the Pericardial tubercular effusion, and to find if Adenosine Deaminase (ADA) levels can be used as a predictor of the presence of MTB in pericardial fluid. MethodologyWe enrolled 52 patients with moderate to large tuberculous pericardial effusion, based on pericardial fluid analysis, CBNAAT, and culture methods, between January 2021 and December 2021. ResultsThe mean age of the patients was 41.85 + 17.88 years, with a median of 38 years. Males made up 57.7% of the total patients. MTB was detected in 16 (30.8%) patients in the CBNAAT evaluations. 14 (87.5%) of the CBNAAT-positive TB patients were sensitive to Rifampicin, whereas the remaining 2 (12.5%) were resistant to Rifampicin on CBNAAT. MTB was found to be growing in 8 (15.38%) drug sensitivity test cultures. Out of these 8, 6 were sensitive to first-line drugs, whereas 2 were resistant to both Isoniazid and Rifampicin. The presence of cough was found to have a significant difference between CBNAAT-detected MTB positive and negative patients (p = 0.020), whereas an insignificant difference was found for the presence of hypertension, diabetes mellitus, obesity, dyspnea, or fever. There was also an insignificant difference between the number of patients positive for the Tuberculin skin test, between the two groups. ADA was significantly higher in the MTB-detected CBNAAT group (85.91 + 37.60U/L vs 39.78 + 24.31U/L, p = 0.005), whereas the total leukocyte count, lymphocytes, neutrophils, random blood sugar levels, and serum protein levels had no significant difference. The area under the Receiver Operator Curve (CBNAAT positive: dependent variable; ADA: test result variable) was 0.854 (null hypothesis rejected), with a standard error of 0.078. ConclusionsCulture is the gold standard method to diagnose tuberculosis. Detection of MTB on pericardial fluid culture is very uncommon, though in our study, culture came out positive in 16% of patients, and 4% were resistant to rifampicin and isoniazid. Higher ADA levels in pericardial fluid are an indicator of tuberculous pericardial effusion.

Diagnostic value of nucleic acid amplification tests for tuberculous pleural effusion.

Diagnosing tuberculous pleural effusion (TPE) is challenging for pulmonologists and laboratory scientists. The gold standards for TPE diagnosis are pleural fluid Ziehl-Neelsen staining, Mycobacterium tuberculosis (Mtb) culture and pleural biopsy. These tools have limitations, including low sensitivity, long turnaround time and invasiveness. The nucleic acid amplification test (NAAT) is a rapid and minimally invasive tool for diagnosing TPE. This review summarizes the diagnostic accuracy of available NAATs for TPE, with a focus on the evidence from systematic reviews and meta-analyses. The NAATs summarized in this review include in-house NAATs, GeneXpert-MTB/RIF, GeneXpert-MTB/RIF Ultra, simultaneous amplification and testing-tuberculosis, FluoroType MTB and loop-mediated isothermal amplification.