Search Results (15)

Background: Accurate differentiation between Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) is essential for proper diagnosis and treatment. This study compares the diagnostic performance of two commercial real-time PCR kits, AdvanSure^TM TB/NTM and Kogene PowerChek^TM MTB/NTM, for detecting MTB, NTM, and negative (no growth, NG) clinical specimens. Methods: A total of 390 clinical residual specimens were collected from patients between December 2022 and June 2023. The samples, including sputum, bronchoalveolar lavage, tracheal aspirate and body fluid, were initially tested with MGIT culture and then analyzed using both PCR kits. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy were evaluated. Discrepant results between the two PCR assays were further investigated using sequencing to identify the detected mycobacterial species, and final diagnoses were verified by culture results and review of electronic medical records. Results: Of the 390 specimens, both AdvanSure^TM and PowerChek^TM real-time PCR assays demonstrated 100% sensitivity for both MTB and NTM detection. For MTB detection, AdvanSure^TM demonstrated a specificity of 100%, with a PPV, NPV, and overall accuracy all reaching 100%. In comparison, PowerChek^TM showed a specificity of 98.62%, a PPV of 96.15%, an NPV of 100%, and an overall accuracy of 98.97%. For NTM detection, both AdvanSure^TM and PowerChek^TM exhibited identical performance metrics. The specificity was 99.58% for both assays, with a PPV of 99.34%, NPV of 100%, and an overall accuracy of 99.74%. Five discrepant results were finally confirmed as four NTM detection cases and one negative case by culture and clinical diagnosis which showed four cases of PowerChek^TM MTB+NTM detection and one case of NTM detection, respectively. Conclusions: The PowerChek^TM MTB/NTM real-time PCR kit demonstrated excellent diagnostic performance for the detection of MTB and NTM, with high sensitivity, specificity, and accuracy. Minor discrepancies, particularly in detecting MTB+NTM mixed infections, highlight the importance of complementary sequencing analysis for resolving uncertain results. These findings support the clinical utility of both PCR assays as reliable tools for rapid diagnosis of mycobacterial infections. PowerChek^TM showed occasional false positives, suggesting that optimizing the assay’s cutoff threshold or amplification parameters could enhance its specificity and reduce false-positive results in clinically ambiguous cases. Full article

Background and Objectives: Tuberculosis (TB) is one of the most common infectious diseases in developing countries. The resistance of the causative agent, Mycobacterium tuberculosis, to two or more first-line anti-TB drugs results in multidrug-resistant (MDR) TB, posing a serious challenge to the control of TB worldwide. This study was designed to determine the changes in drug resistance over time in TB strains isolated from patients in all departments of Uludağ University Hospital in western Türkiye. Materials and Methods: We retrospectively analyzed 104,598 clinical samples sent to our laboratory for the investigation of the presence of TB between 1996 and 2023. BACTEC 460 TB, BACTEC MGIT 960 culture systems and Löwenstein–Jensen medium were used for the culture of these samples. The susceptibility of M. tuberculosis complex strains grown in culture to isoniazid (INH) (0.1 μg/mL), rifampicin (RIF) (1.0 μg/mL), ethambutol (ETB) (5.0 μg/mL) and streptomycin (SM) (1.0 μg/mL) antibiotics was studied according to the manufacturer’s recommendation. Results: Out of 104,598 patient samples, 2752 (2.6%) were culture-positive, and the susceptibility test results of 1869 of these were analyzed. Of the isolates, 358 (19.2%) were found to be resistant to at least one first-line drug, i.e., INH, RIF, ETB, or SM. In addition, 2.9% were resistant to two or more first-line drugs. Conclusions: Drug susceptibility testing is essential to ensure the optimal treatment and control of drug-resistant TB strains. This study highlights the value of ongoing efforts to control tuberculosis drug resistance in the fight against this disease. Full article

Since 2013, the World Health Organization has recommended the use of rapid molecular tests as the initial diagnostic step for Mycobacterium tuberculosis (MTB) infection to enhance the control of tuberculosis. In recent years, the prevalence of infections by non-tuberculous mycobacteria (NTM) in humans has also risen, particularly in countries with low tuberculosis incidence, such as Italy. Therefore, the rapid differentiation between NTM and Mycobacterium tuberculosis complex is crucial for timely therapeutic decisions. This study evaluates a new rapid molecular assay, Standard M10 MTB/NTM, designed to detect MTB, NTM, or co-detection in Mycobacteria Growth Indicator Tube cultures from different biological matrices. The assay was validated using 100 positive and 50 negative liquid mycobacteria cultures, already confirmed by specific real-time PCR and Sanger sequencing. Following optimization of assay conditions for culture sample processing and assessment of potential interference, Standard M10 demonstrated excellent sample stability, high specificity, and good sensitivity, identifying all 50 MTB and 49 NTM samples. Some limitations included the non-detection of M. celatum in one case and false positive results (MTB co-infection) in two NTB cases. Nevertheless, overall, the adoption of this test could be considered for laboratory management to enable rapid and effective sample targeting for subsequent diagnostic evaluation and treatment decision-making. Full article

Background: Extrapulmonary tuberculosis (TB) presents significant diagnostic challenges, particularly in the context of multidrug-resistant (MDR) strains. This study assessed the utility of the WHO-recommended rapid molecular assays, originally validated for pulmonary TB, in diagnosing extrapulmonary TB and detecting the MDR Mycobacterium tuberculosis complex (MTBC). Materials and Methods: A total of 6274 clinical samples, including 4891 pulmonary and 1383 extrapulmonary samples, were analyzed between 2019 and 2022 using the BD MAX™ MDR-TB assay (BD MAX), the Xpert^® MTB/RIF assay (Xpert MTB/RIF), the Xpert^® MTB/XDR assay (Xpert MTB/XDR), FluoroType MTB, and phenotypic drug susceptibility testing (DST). Results: MTBC was detected in 426 samples using BD MAX (376 pulmonary and 50 extrapulmonary), of which 277 were culture-confirmed. Phenotypic testing confirmed 299 positive cultures on Löwenstein–Jensen (LJ) medium and 347 in BD BACTEC™ MGIT™ (BACTEC MGIT) mycobacterial growth indicator tube (BBL) liquid culture. BD MAX showed high sensitivity and specificity for extrapulmonary TB detection (93.1% and 98.4%, respectively). Resistance to isoniazid or rifampicin was identified in 11% of MTBC-positive cases, whereas 3.69% were confirmed as MDR-TB. The molecular assays effectively detected resistance-associated mutations (katG, inhA, and rpoB), with high concordance to phenotypic tests (DST) (κ = 0.69–0.89). Conclusions: This study demonstrates that molecular assays, although validated for pulmonary TB, are also reliable for extrapulmonary TB detection and drug resistance profiling. Their rapid turnaround and robust accuracy support broader implementation in routine diagnostics, especially for challenging extrapulmonary specimens where early detection is critical for targeted therapy. Full article

In Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and gold standard phenotypic drug sensitivity testing (DST) questions the significance of embB406 mutations used in molecular DST. This study tabulates embB mutations found in Canadian M. tuberculosis isolates and evaluates the impact of specific mutations on ethambutol resistance. The National Reference Centre for Mycobacteriology culture collection (n = 2796) was screened for isolates with embB mutations. Phenotypic DST was performed on the BACTEC™ MGIT™ 960 at ethambutol concentrations of 2–5 μg/mL. Whole genome sequencing was used for drug resistance predictions, phylogenomics and single nucleotide polymorphism analysis. Detection of resistance-associated embB mutations corresponded to a positive predictive value of 64.3%, negative predictive value of 99.2%, 98.7% specificity, and 73.3% sensitivity compared to phenotypic DST. Two embB406 mutation subtypes (Gly406Asp, Gly406Ala) were found among 16 isolates, of which 12 were sensitive at 5 µg/mL ethambutol with variable resistance between 2–4 µg/mL. A novel frameshift mutation in regulator embR (Gln258fs) was found in nine isolates. Mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 μg/mL). These novel mutations may exacerbate variability in ethambutol resistance. Full article

In several low-income countries, the transport of sputa could take up to one week to reach the laboratories, resulting in increased contamination rates and a loss of growth. The aim of this study was to evaluate the effect of the OMNIgene-SPUTUM in preserving Mycobacterium tuberculosis on sputum samples simulating three hypothetical scenarios for conservation and/or decontamination: (1) sputum was mixed with OMN and conserved at room temperature for five days and then processed for culture (OMN); (2) sputum cultures followed the routine standing operating procedure at day 0 (STD); and (3) sputum samples were kept at room temperature for five days and mixed with the standard decontamination reagent (SDT5) and then processed for culture. The positivity rate based on smear microscopy was 36.4%, 29.1%, and 27.3% for STD, STD5, and OMN, respectively. The proportion of positive results by liquid culture (MGIT) was 39.1% (43/110) for STD, 26.4% (29/110) for STD5, and 20.0% for OMN (22/110). The overall concordance of liquid culture results was 51.8% (57/110): 37.3% (41/110) for negative results, 11.8% (13/110) for MTBC growth, and 2.7% (3/110) for contaminated results. The OMN arm showed better performance in solid culture than in liquid culture, with a notable reduction in contaminated results. Full article

The study sought to determine the rate of discordant results between genotypic and phenotypic tests for the diagnosis of drug-resistant tuberculosis (DR-TB). Sputum samples and cultured isolates from suspected DR-TB patients were, respectively, analyzed for Mycobacterium tuberculosis by Xpert^® MTB/RIF (Cepheid, Sunnyvale, CA, USA) and line probe assays (LPA) (Hain, Nehren, Germany). Discrepant rifampicin (RMP)-resistant results were confirmed using BACTEC MGIT960 (BD, New York, NY, USA). Of the 224 RMP-resistant results obtained by Xpert MTB/RIF, 5.4% were susceptible to RMP by LPA. MGIT960 showed a 75% agreement with LPA. The discrepancy was attributed to either heteroresistance or DNA contamination during LPA testing in 58.3% of cases. In 25% of the samples showing agreement in RMP resistance between Xpert MTB/RIF and MGIT960, the discrepancy was attributed to laboratory errors causing false RMP susceptible results with LPA. In 16.7% of the cases, the discrepancy was attributed to false RMP susceptible results with Xpert MTB/RIF. Out of the 224 isolates, susceptibility to isoniazid (INH) by LPA was performed in 73.7% RMP-resistant isolates, of which, 80.6% were resistant. All RMP-resistant isolates by Xpert MTB/RIF were confirmed in 98.5% by LPA if TB isolates were resistant to INH, but were only confirmed in 81.3% if TB isolates were susceptible to INH (p < 0.001). In conclusion, laboratory errors should be considered when investigating discordant results. Full article

Alternative tools are needed to improve the detection of M. tuberculosis (M. tb) in HIV co-infections. We evaluated the utility of Tuberculosis Molecular Bacterial Load Assay (TB-MBLA) compared to lipoarabinomannan (LAM) to detect M. tb in urine. Sputum Xpert MTB/RIF-positive patients were consented to provide urine at baseline, weeks 2, 8, 16, and 24 of treatment for TB-MBLA, culture, and LAM. Results were compared with sputum cultures and microscopy. Initial M. tb. H37Rv spiking experiments were performed to validate the tests. A total of 63 urine samples from 47 patients were analyzed. The median age (IQR) was 38 (30–41) years; 25 (53.2%) were male, 3 (6.5%) had urine for all visits, 45 (95.7%) were HIV positive, of whom 18 (40%) had CD4 cell counts below 200 cells/µL, and 33 (73.3%) were on ART at enrollment. Overall urine LAM positivity was 14.3% compared to 4.8% with TB-MBLA. Culture and microscopy of their sputum counterparts were positive in 20.6% and 12.7% of patients, respectively. Of the three patients with urine and sputum at baseline, one (33.33%) had urine TB-MBLA and LAM positive compared to 100% with sputum MGIT culture positive. Spearman’s rank correction coefficient (r) between TB-MBLA and MGIT was −0.85 and 0.89 with a solid culture, p > 0.05. TB-MBLA has the promising potential to improve M. tb detection in urine of HIV-co-infected patients and complement current TB diagnostics. Full article

Mycobacterial culture remains the gold standard for the diagnosis of active tuberculosis. However, an appropriate digestion and decontamination method is essential for the effective recovery of tubercle bacilli in culture. The study was designed to compare the efficacy of sputum treated with power ultrasound (PU) and routine NALC-NaOH methods for mycobacterial culture from clinically suspected cases of pulmonary tuberculosis. To evaluate the PU and routine NALC-NaOH methods, sputum specimens (n = 597) were studied (culturing on MGIT 960), and the performances were compared. Of the 597 samples, 89 (14.91%) sputum samples treated with the NaOH-NALC method were mycobacterial culture positive, including Mycobacterium tuberculosis (M.TB; n = 77, 12.90%) and nontuberculous mycobacteria (NTM; n = 12, 2.01%). One hundred and ten (18.43%) sputum samples treated with the PU method were culture positive, including M.TB (n = 87, 14.57%) and NTM (n = 23, 3.85%). The PU method detected 10 additional cases of M.TB and 11 additional cases of NTM when compared to the NALC-NaOH method. Statistical analysis showed that a significant difference was found in the culture-positive ratio of M.TB and NTM between the two method groups (p < 0.05). Compared with that of the NALC-NaOH method (8.04%), sputum treated with PU method (4.86%) had a significantly lower contamination rate (p < 0.05). In conclusion, our data indicate that, compared with the NALC-NaOH method, the PU method is a rapid and effective approach for mycobacterial culture when detecting active TB. However, its accurate mechanism has not been well addressed, and further investigation is still required. Full article

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed to successfully diagnose, treat, and manage infections caused by NTM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS, was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The present study aims to develop a new extraction protocol to yield rapid and accurate identification of NTM from primary MGIT cultures by MALDI-TOF MS. A total of 60 positive MGIT broths were examined by the Bruker Biotyper system with Mycobacteria Library v. 2.0 (Bruker Daltonics GmbH & Co. KG., Bremen, Germany). The results were compared with those obtained by the molecular method, line probe assay GenoType Mycobacterium CM/AS/NTM-DR. All samples were concordantly identified by MALDI-TOF MS and the molecular test for all the tested mycobacteria. Fifty-seven (95%) MGIT positive cultures for NTM from clinical samples had a MALDI-TOF MS analysis score S ≥ 1.8. Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results suggest that MALDI-TOF MS could represent a promising routine diagnostic tool for identifying mycobacterial species directly from primary liquid culture. Full article

Background: Scientific evidence is scarce for the antimicrobial effect of copper on bacteria characterized as more resistant. Using Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant microorganism, as a pathogen model, copper ion treatment has shown a significant bactericidal effect; however, the sustainability of MAP against copper toxicity was also reported in several studies. Accordingly, the present study aimed to evaluate the impacts of copper on MAP. Methodology: This study considered physicochemical properties and copper concentration in a buffer since it could modulate MAP response during the application of copper treatment. Results: Despite the efficacy of copper ions in significantly reducing the MAP load in Phosphate Buffered Saline, some MAP cells were able to survive. The copper concentration generated by the copper ion treatment device increased significantly with increasing exposure times. MAP bacterial load decreased significantly when treated with copper ions as the exposure times increased. An increase in pH decreased oxygen consumption, and an increase in conductivity was reported after treatment application. Conclusions: Even with higher concentrations of copper, the efficacy of MAP control was not complete. The concentration of copper must be a key element in achieving control of highly resistant microorganisms. Full article

Mycobacterium avium subspecies paratuberculosis (MAP) has long been suspected to be involved in the etiology of Crohn’s disease (CD). An obligate intracellular pathogen, MAP persists and influences host macrophages. The primary goals of this study were to test new rapid culture methods for MAP in human subjects and to assess the degree of viable culturable MAP bacteremia in CD patients compared to controls. A secondary goal was to compare the efficacy of three culture methods plus a phage assay and four antibody assays performed in separate laboratories, to detect MAP from the parallel samples. Culture and serological MAP testing was performed blind on whole blood samples obtained from 201 subjects including 61 CD patients (two of the patients with CD had concurrent ulcerative colitis (UC)) and 140 non-CD controls (14 patients in this group had UC only). Viable MAP bacteremia was detected in a significant number of study subjects across all groups. This included Pozzato culture (124/201 or 62% of all subjects, 35/61 or 57% of CD patients), Phage assay (113/201 or 56% of all subjects, 28/61 or 46% of CD patients), TiKa culture (64/201 or 32% of all subjects, 22/61 or 36% of CD patients) and MGIT culture (36/201 or 18% of all subjects, 15/61 or 25% of CD patients). A link between MAP detection and CD was observed with MGIT culture and one of the antibody methods (Hsp65) confirming previous studies. Other detection methods showed no association between any of the groups tested. Nine subjects with a positive Phage assay (4/9) or MAP culture (5/9) were again positive with the Phage assay one year later. This study highlights viable MAP bacteremia is widespread in the study population including CD patients, those with other autoimmune conditions and asymptomatic healthy subjects. Full article

(1) Background: Present methods for drug susceptibility tests (DST) rely on culture methods that are sophisticated and relatively faster, or a slow and cheaper option. These methods frustrate disease control; therefore, there is a need for methods that incorporate key functions of microscopy and culture, with reduced cost burden and sophistry. Thus, the purpose of this study was to identify which, among the most commonly used (in Ghana) methods, can conveniently be used at health centers located in rural areas for effective DST determination of Mycobacterium tuberculosis (MTB). (2) Methods: Mycobacterium tuberculosis isolates were tested for their susceptibility to streptomycin, isoniazid, rifampicin, ethambutol (SIRE), and pyrazinamide by microscopic observation drug susceptibility (MODS) and BACTEC MGIT 960 methods. Evaluations were based on shorter turnaround periods, rapidity, ease of use, cost, etc. A comparative analysis was statistically expressed as kappa values. (3) Results: Endpoints for drug susceptibilities by MODS averaged 13 days (7–32), whilst that for BACTEC MGIT 960 was 10 days with a further 12 days to detect resistance. Therefore, a turnaround period of 22 days was needed for DST by BACTEC MGIT 960, compared to 13 days for MODS. There were differences in correlation levels between the two methods, as determined by their kappa values. (4) Conclusion: The MODS assay was found to be less costly, more user-friendly, and still able to be conveniently used at health centers located in rural areas known to be endemic for TB, particularly in Ghana. Full article

Introduction: There are more than 10 million prisoners in the world. Tuberculosis incidence is 10−100 times higher in prisoners than in the general population. Inmates have close contact with other prisoners and with prison workers and visitors, so tubercle bacilli may be easily spread. Most of the inmates come back to normal life and contact with the general population. The aim of the study was to assess active tuberculosis incidence among prisoners and homeless persons in the Silesia region. Materials and Methods: In total 897 people entered the study, of whom 720 were Silesian penitentiary system inmates, and 177 were homeless. BACTEC MGIT fast TB detection system and GenoType Mycobacteria Direct test were used. Drug susceptibility testing was done using SIRE KIT and PZA KIT. Results: Tuberculosis was diagnosed in 13 out of 897 persons (1.45%): in 11 out of 720 inmates (1.53%) and in 2 out of 177 homeless persons (1.13%). Data concerning drug susceptibility were obtained for 11 persons. M. tuberculosis strains isolated from eight persons were susceptible to four first-line antituberculosis drugs (streptomycin, isoniazid, rifampin, ethambutol), while M. tuberculosis strains isolated from three persons were drug-resistant. One out of three isolated strains was resistant to ethambutol, but susceptible to streptomycin, isoniazid, rifampin, and pirazynamide. The second strain was resistant to streptomycin and pyrazinamide but susceptible to isoniazid, rifampin, and ethambutol. The third strain was susceptible to rifampin but resistant to the other four tested drugs. According to the obtained data, culture-positive pulmonary tuberculosis was 100 times more frequent in the examined population than in the general population of the Silesia region in the same period of time. Conclusions: The health project enabled effective detection of tuberculosis in risk groups and should be continued in the following years. The set of the applied diagnostic methods allowed the detection of in the studied subpopulations people suffering from tuberculosis. Patients were treated with antituberculosis drugs that would stop them from spreading the disease to other people. Full article

The aim of this study was to evaluate the utility of the quantitative analysis of mycolic acids by HPLC technique in drug susceptibility testing of the M. tuberculosis isolates to the first-line antituberculous drugs: isoniazid and rifampicin. Drug susceptibility of the 30 clinical M.tbc isolates was examined by the mycolic acids analysis with HPLC technique and results were compared to the proportion method on solid L-J medium and liquid medium in MGIT system. In HPLC method drug susceptibility of M. tuberculosis strains was described by TAMA index defined as the ratio of thetotal area under mycolic acids peaks (TAMA) from cultures with drug to the TAMA of control. At critical concentrations of drugs, TAMA indexes of resistant strains were >0.5, and TAMA indexes of susceptible strains were <0.05. The average error of the TAMA analysis was ±9.5% The quantitative analysis of mycolic acids by HPLC gives results compatible with standard proportion method and is a reliable method for determination of drug susceptibility of M. tuberculosis. Full article