Indicator Tube Research Articles

Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis Isolates by Multiplex Allelic Specific PCR

Mycobacterium tuberculosis is a type of bacteria causing infectious desease called tuberculosis. According to the Makassar City Health Office, total tuberculosis (TB) patients in 2019 were 5,412 with 83% cure rate. The number of TB cases had dropped to 3,250 in 2020 with 85% cure rate before it increased again to 3,911 in 2021. This study aimed at identifying Mycobacterium TB resistance to INH (isonicotinyl hydrazide) specifically in probable tuberculosis case in Makassar. Patients with suspected tuberculosis who have not taken medication were selected as study subject. They were examined through several stages including sample decontamination, microscopic examination (ZN Method), isolation on LJ media, culture on liquid medium MGIT (Mycobacteria Growth Indicator Tube), test on.DST (Drug Susceptible Test) MGIT, and DNA extraction using specific multiple allelic (MAS) techniques. According to the results of the culture, every sample (100%) was positive. With the use of the MGIT approach, we followed up on the encouraging culture results. In this instance, we used two MGIT tubes for each sample, one for the isoniazid antibiotic control and one for the subsequent sample. From the third day following vaccination through the twelfth, observations were made. On the third day, if the control tube is positive, then the second tube is observed until the fifth day (observation of the tube containing antibiotics is carried out two days after the control tube is positive. We also use isolates from positive culture results for Multiplex Allelic Specific PCR (MAS-PCR) ).

Discordance in Phenotypic and Genotypic Susceptibility Testing for Streptomycin Due to Nonsynonymous/Nonsense/Deletion Frame-Shift Mutations in gidB among Clinical Mycobacterium tuberculosis Isolates in Kuwait

Objective: Increasing reports of resistance to newer antituberculosis drugs have prompted the search for other alternative drugs. Streptomycin (STR) could be used for the treatment of drug-resistant tuberculosis if susceptibility of Mycobacterium tuberculosis isolate to STR could be accurately detected. We performed phenotypic and genotypic drug susceptibility testing (DST) of 118 M. tuberculosis isolates for STR. Materials and Methods: Fifty pansusceptible and 68 multidrug-resistant M. tuberculosis (MDR-TB) isolates were used. Phenotypic DST for STR, rifampicin, isoniazid, and ethambutol was performed by mycobacteria growth indicator tube 960 System. Genotypic DST was done by GenoTypeMTBDRplus assay for rifampicin and isoniazid and by PCR-sequencing of rpsL, rrs, and gidB genes for STR. MDR-TB isolates were genotyped by spoligotyping. Results: Phenotypic DST identified 50 isolates susceptible to all four drugs (pansusceptible). Sixty-one of 68 MDR-TB isolates were resistant to STR. Genotypic testing for rifampicin and isoniazid yielded expected results. Fifty pansusceptible and 7 STR-susceptible MDR-TB isolates contained no mutation in rpsL or rrs, while 47, 2, and 1 STR-resistant isolate contained rpsL, rrs, and rpsL + rrs mutations, respectively. Of the remaining 11 STR-resistant MDR-TB, 9 isolates contained deletion frame-shift/nonsynonymous mutations in gidB. Surprisingly, 13 pansusceptible isolates also contained deletion frame-shift/nonsense/nonsynonymous mutations in gidB. Also, 30 of 68 MDR-TB but only 2 of 50 pansusceptible isolates belonged to the Beijing genotype. Conclusions: Our data show that, like rifampicin, ethambutol, and pyrazinamide, STR also exhibits discordant phenotypic and genotypic DST results for some M. tuberculosis isolates. Hence, STR should be included in therapy regimens only if both phenotypic and genotypic resistance testing indicate susceptibility to avoid amplification of resistance and drug toxicity.

Xpert MTB/RIF Ultra versus mycobacterial growth indicator tube liquid culture for detection of Mycobacterium tuberculosis in symptomatic adults: a diagnostic accuracy study

Xpert MTB/RIF Ultra (Ultra) is an automated molecular test for the detection of Mycobacterium tuberculosis in sputum. We compared the sensitivity of Ultra to that of mycobacterial growth indicator tube (MGIT) liquid culture, considered the most sensitive assay in routine clinical use. In this prospective, multicentre, cross-sectional diagnostic accuracy study, we used a non-inferiority design to assess whether the sensitivity of a single Ultra test was non-inferior to that of a single liquid culture for detection of Mtuberculosis in sputum. We enrolled adults (age ≥18 years) with pulmonary tuberculosis symptoms in 11 countries and each adult provided three sputum specimens with a minimum volume of 2 mL over 2 days. Ultra was done directly on sputum 1, and Ultra and MGIT liquid culture were done on resuspended pellet from sputum 2. Results of MGIT and solid media cultures done on sputum 3 were considered the reference standard. The pre-defined non-inferiority margin was 5·0%. Between Feb 18, 2016, and Dec 4, 2019, we enrolled 2906 participants. 2600 (89%) participants were analysed, including 639 (25%) of 2600 who were positive for tuberculosis by the reference standard. Of the 2357 included in the non-inferiority analysis, 877 (37%) were HIV-positive and 984 (42%) were female. Sensitivity of Ultra performed directly on sputum 1 was non-inferior to that of sputum 2 MGIT culture (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; 95% CI -2·8 to 1·1). Sensitivity of Ultra performed on sputum 2 pellet was also non-inferior to that of sputum 2 MGIT (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; -2·7 to 1·0). For the detection of M tuberculosis in sputum from adults with respiratory symptoms, there was no difference in sensitivity of a single Ultra test to that of a single MGIT culture. Highly sensitive, rapid molecular approaches for M tuberculosis detection, combined with advances in genotypic methods for drug resistance detection, have potential to replace culture. US National Institute of Allergy and Infectious Diseases.

Evaluation of Loop-Mediated Isothermal Amplification Assay in Diagnosing Tuberculous Meningitis.

Resource-limited settings like India need a simple, quick, and temperature-independent point-of-care diagnostic test that can diagnose tuberculous meningitis (TBM) at the earliest. A prospective study was carried out at a tertiary care center in North India wherein 50 subjects suspected of TBM were recruited and followed up for six months between January 2019 and December 2020. The aim was to evaluate the performance of loop-mediated isothermal amplification (TB-LAMP) in diagnosing TBM as compared to a composite reference standard (CRS), mycobacteria growth indicator tube 960 (MGIT 960) culture, and GeneXpert®. Out of 50 patients, 32 were TBM cases (64%), and 18 were non-TBM cases (36%). The sensitivity of TB-LAMP and GeneXpert® for TBM diagnosis against CRS was 53% (17/32) for both, and the specificity was 78% (14/18) and 89% (16/18), respectively. On comparing TB-LAMP against GeneXpert® for TBM diagnosis, the two methods had almost perfect agreement (Cohen's kappa=0.83) with statistical significance (p<0.001). The performance of TB-LAMP assay is comparable to GeneXpert® in diagnosing TBM, and it may be used as a substitute for CSF GeneXpert® in resource-limited settings.

Study of Culture Conversion in Drug-Resistant Pulmonary Tuberculosis on All-Oral Longer Regimen at IGIMS, Patna

ABSTRACT Background: Multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that resists at least two primary drugs, namely isoniazid and rifampicin. To assess the management of MDR-TB, sputum culture conversion is performed. This study aimed to determine the culture conversion status of MDR-TB patients undergoing an all-oral longer regimen. Methods: This research constitutes an observational and prospective study conducted within a hospital setting. The study was done at the Department of Microbiology, IGIMS, Patna, from October 2020 to March 2022. Culture conversion in multidrug resistance pulmonary tuberculosis on all-oral longer regimens took one spot and one morning sample of sputum as per standard protocol after completing two months of all-oral longer regimens and culturing it in liquid broth using Mycobacterium Growth Indicator Tube (MGIT) 960 System at two, four then six months till we got a negative result. Results: Maximum number of the cases, 77 (74.8%), belonged to 19–35 years of age group. Males were 68 (66.1%) and females were 35 (33.9%), respectively, with male to female ratio of 1.9:1. After 2 months of oral longer regimen treatment, out of 103 cases, we found 98 (95.1%) patients had sputum for culture positive and only five (4.2%) patients had sputum for culture negative. After 6 months of oral longer regimen treatment, out of 101 cases, we found 16 (15.8%) patients had sputum for culture positive and 85 (85.2%) patients had sputum for culture negative. Conclusion: In patients with multidrug-resistant pulmonary tuberculosis (MDR-TB) who received an all-oral longer regimen, the introduction of bedaquiline led to positive outcomes as evidenced by a greater number of negative sputum cultures, a decrease in culture reversions, and a reduced risk of developing a more resistant form of MDR-TB.

The prevalence of pulmonary and extrapulmonary tuberculosis in forensic autopsies in a teaching hospital in South India.

An autopsy is a valuable tool for finding the cause of death, exploring the clinical diagnosis, documenting unexpected findings, and resolving diagnostic questions. However, this may subject the forensic pathologist and other workers to a wide variety of blood-borne and aerosolized pathogens. Tuberculosis (TB) is the most common cause of illness and death, resulting in infection transmission in the autopsy room. Our objective in this study was to estimate the prevalence of pulmonary and extrapulmonary tuberculosis among forensic autopsies in a tertiary care hospital in South India. We identified positive TB cases from acid-fast bacteria staining and culture (Mycobacterium Growth Indicator Tube and Lowenstein-Jensen medium) out of 380 autopsy cases. The prevalence of tuberculosis was 2.4% (n = 9), among which 2.1% of cases were positive for only pulmonary tuberculosis (n = 8), and 0.3% of cases had both pulmonary and extrapulmonary involvement (n = 1). In the bivariate analysis of TB cases, sex, occupation, family history of TB, habit of smoking, BCG vaccine scar, period of hospital stay, and cause of death were potentially significant. The prevalence of TB in forensic autopsy cases were similar to forensic autopsy-based studies, and it was less as compared to the prevalence of TB in the general population.

TB LAMP assay, a beneficial tool for the diagnosis of Tubercular meningitis in resource-limited settings.

Tubercular meningitis (TBM) is a serious public health problem in developing countries as it leads to significant mortality and residual neurological sequelae. The estimated mortality due to TBM in India is 1.5 per 100,000 population. In resource-limited settings, only the Ziehl-Neelsen (ZN) stain, which has very little sensitivity, is available. The World Health Organization recommended the Loop Mediated Isothermal Amplification (TB LAMP) assay for pulmonary tuberculosis only. We evaluated this test for tubercular meningitis as well. In a cross-sectional study of 2-year duration, we have taken 239 cerebrospinal fluid samples from suspected cases of tubercular meningitis patients. ZN staining along with Mycobacteria Growth Indicator Tube (MGIT) TB culture, Xpert MTB/RIF Ultra assay, and commercial TB LAMP assay were performed for each sample. Out of 239 samples, 40 samples (16.73%) were found TB LAMP assay positive, 48 samples (20.08%) were found Xpert ultra-assay positive, 12 samples (5.02%) were MGIT TB culture positive and acid-fast bacillus smear positive in ten samples (4.18 %). Out of 12 MGIT-positive samples, all samples (100%) were TB LAMP and Xpert ultra positive and one sample (8.33%) was ZN smear positive. In 199 negative samples from the TB LAMP assay, eight samples were positive by Xpert, none by MGIT TB culture and AFB smear. Sensitivity and specificity were found as 100% and 87.66%, respectively, for the TB LAMP assay. TB LAMP assay is a rapid, cost-effective, sensitive, and specific test for tubercular meningitis infection in resource-limited settings.

Accuracy of the tuberculosis molecular bacterial load assay to diagnose and monitor response to anti-tuberculosis therapy: a longitudinal comparative study with standard-of-care smear microscopy, Xpert MTB/RIF Ultra, and culture in Uganda

In 2018, the tuberculosis molecular bacterial load assay (TB-MBLA), a ribosomal RNA-based test, was acknowledged by WHO as a molecular assay that could replace smear microscopy and culture for monitoring tuberculosis treatment response. In this study, we evaluated the accuracy of TB-MBLA for diagnosis and monitoring of treatment response in comparison with standard-of-care tests. For this longitudinal prospective study, patients aged 18 years or older with presumptive tuberculosis (coughing for at least 2 weeks, night sweats, and weight loss) were enrolled at China-Uganda Friendship Hospital Naguru (Kampala, Uganda). Participants were evaluated for tuberculosis by TB-MBLA in comparison with Xpert MTB/RIF Ultra (Xpert-Ultra) and smear microscopy, with Mycobacteria Growth Indicator Tube (MGIT) culture as a reference test. Participants who were positive on Xpert-Ultra were enrolled on a standard 6-month anti-tuberculosis regimen, and monitored for treatment response at weeks 2, 8, 17, and 26 after initiation of treatment and then 3 months after treatment. Between Nov 15, 2019, and June 15, 2022, 210 participants (median age 35 years [IQR 27-44]) were enrolled. 135 (64%) participants were male and 72 (34%) were HIV positive. The pretreatment diagnostic sensitivities of TB-MBLA and Xpert-Ultra were similar (both 99% [95% CI 95-100]) but the specificity was higher for TB-MBLA (90%[83-96]) than for Xpert-Ultra (78% [68-86]). Ten participants were Xpert-Ultra trace positive, eight (80%) of whom were negative by TB-MBLA and MGIT culture. Smear microscopy had lower diagnostic sensitivity (75%[65-83]) but higher specificity (98% [93-100]) than TB-MBLA and Xpert-Ultra. Among participants who were smear microscopy negative, the sensitivity of TB-MBLA was 96% (95 CI 80-100) and was 100% (95% CI 86-100) in those who were HIV positive. 129 (61%) participants were identified as tuberculosis positive by Xpert-Ultra and these individuals were enrolled in the treatment group and monitored for treatment response. According to TB-MBLA, 19 of these patients cleared bacillary load to zero by week 2 of treatment and remained negative throughout the 6-month treatment follow-up. Positivity for tuberculosis decreased with treatment as measured by all tests, but therate was slower with Xpert-Ultra. Consequently, 31 (33%) of 95 participants were still Xpert-Ultra positive at the end of treatment but were clinically well and negative on TB-MBLA and culture at 6 months of treatment. Two patients were still Xpert-Ultra positive with a further 3 months of post-treatment follow-up. The rate of conversion to negative of the DNA-based Xpert-Ultra was 3·3-times slower than that of the rRNA-based TB-MBLA. Consequently for the same patient, it would take 13 weeks and 52 weeks to reach complete tuberculosis negativity by TB-MBLA and Xpert-Ultra, respectively. Participants who were positive on smear microscopy at 8weeks, who received an extra month of intensive treatment, had a similar TB-MBLA-measured bacillary load at 8weeks to those who were smear microscopy negative. TB-MBLA has a similar performance to Xpert-Ultra for pretreatment diagnosis of tuberculosis, but is more accurate at detecting and characterising the response to treatment than Xpert-Ultra and standard-of-care smear microscopy. European and Developing Countries Clinical Trials Partnership, Makerere University Research and Innovation Fund, US National Institutes of Health.

A modified decontamination and storage method for sputum from patients with tuberculosis.

Collecting and storing large number of sputum samples with a view to culturing these in the future requires an efficient initial handling method. We devised a modified sputum digestion and decontamination method that maximised storage capacity and Mycobacterium tuberculosis (M.tb) recovery from culture while minimising laboratory workload and risk of contamination. We collected smear microscopy positive sputum samples from patients with pulmonary tuberculosis (TB). The sputum samples were split and processed using both the standard N-Acetyl-L-cysteine and sodium hydroxide (NALC-NaOH) method and our modified method before freezing and later culturing in BD BACTEC 960 Mycobacterium Growth Indicator Tubes (MGIT) system. We assessed the Time to Positivity (TPP) and Growth Unit (GU) data. We selected 22 sputum samples to compare two digestion and decontamination methods. The samples that underwent the modified method had longer TTP (p < 0.05) but similar GU in comparison to standard method. Overall, 1/22 samples failed to grow in MGIT after being processed by the modified method. We then applied the modified method to 348 sputum samples with Rifampicin resistance detected by GeneXpert MTB/RIF assay, which were frozen for between 1-25 months. The overall MGIT positive, negative, and contamination rate was 90.5%, 7.8%, and 1.7%, respectively. There was no significant difference in MGIT result when samples were grouped by duration of storage or positive smear grade. Our modified method yielded acceptable M.tb recovery rate and low contamination risk while allowing us to collect and store thousands of sputum samples over a long period of time for future tests.

Co-resistance to isoniazid and second-line anti-tuberculosis drugs in isoniazid-resistant tuberculosis at a tertiary care hospital in Thailand.

The prevalence of isoniazid-resistant tuberculosis (Hr-TB) is the highest among other types of drug-resistant tuberculosis. Currently, the World Health Organization (WHO) guidelines recommend the treatment of Hr-TB with rifampicin, ethambutol, pyrazinamide, and levofloxacin for 6 months. The susceptibility profiles of Hr-TB clinical isolates, especially when they are co-resistant to second-line drugs, are critical in the selection of the appropriate treatment regimen to prevent treatment failure. This study highlights the susceptibility profiles of the WHO-recommended treatment regimen in Hr-TB clinical isolates from a tertiary care hospital in Thailand and the concordance and importance of using the phenotypic drug susceptibility testing or genotypic drug susceptibility testing for accurate and comprehensive interpretation of results.

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.

Quabodepistat in combination with delamanid and bedaquiline in participants with drug-susceptible pulmonary tuberculosis: protocol for a multicenter, phase 2b/c, open-label, randomized, dose-finding trial to evaluate safety and efficacy

BackgroundDelamanid and bedaquiline are two of the most recently developed antituberculosis (TB) drugs that have been extensively studied in patients with multidrug-resistant TB. There is currently a need for more potent, less-toxic drugs with novel mechanisms of action that can be used in combination with these newer agents to shorten the duration of treatment as well as prevent the development of drug resistance. Quabodepistat (QBS) is a newly discovered inhibitor of decaprenylphosphoryl-β-D-ribose-2′-oxidase, an essential enzyme for Mycobacterium tuberculosis to synthesize key components of its cell wall. The objective of this study is to evaluate the safety, efficacy, and appropriate dosing of a 4-month regimen of QBS in combination with delamanid and bedaquiline in participants with drug-susceptible pulmonary TB in comparison with the 6-month standard treatment (i.e., rifampicin, isoniazid, ethambutol, and pyrazinamide).MethodsThis phase 2b/c, open-label, randomized, parallel group, dose-finding trial will enroll approximately 120 participants (including no more than 15% with human immunodeficiency virus [HIV] coinfection) aged ≥ 18 to ≤ 65 years at screening with newly diagnosed pulmonary drug-sensitive TB from ~8 sites in South Africa. Following a screening period of up to 14 days, eligible participants will be randomized in a ratio of 1:2:2:1 to one of four arms. Randomization will be stratified by HIV status and the presence of bilateral cavitation on a screening chest x-ray. After the end of the treatment period, participants will be followed until 12 months post randomization. The primary efficacy endpoint is the proportion of participants achieving sputum culture conversion in Mycobacteria Growth Indicator Tube by the end of the treatment period. The safety endpoints consist of adverse events, clinical laboratory tests, vital signs, physical examination findings, and electrocardiographic changes.DiscussionQBS’s potent bactericidal activity and distinct mechanism of action (compared with other TB drugs currently available for human use) may make it an ideal candidate for inclusion in a novel treatment regimen to improve efficacy and potentially prevent resistance to concomitant TB drugs. This trial will assess the effectiveness, safety, and dosing of a new, shorter, QBS-based, combination anti-TB treatment regimen.Trial statusClinicalTrials.gov NCT05221502. Registered on February 3, 2022

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Evaluation of Induced Sputum against Gastric Juice Aspirate in the Diagnosis of Tuberculosis in Children: A Cross-sectional Study

Introduction: Diagnosing Tuberculosis (TB) in children is difficult as they do not expectorate sputum on their own, and the sample is usually paucibacillary. Hence, alternative sampling methods like Gastric juice Aspiration (GA), which is the widely accepted method, and Induced Sputum (IS) collection, a more novel approach, are used. The IS method has several advantages, such as being less invasive, not requiring inpatient admission, causing less discomfort, and not necessitating overnight fasting, compared to the GA method. Aim: To evaluate IS against GA for diagnosing TB using XpertMTB/ RIF assay, as well as mycobacterial culture, in children aged between 2 and 15 years. Materials and Methods: This cross-sectional study was conducted in the Department of Paediatrics at Christian Medical College and Hospital, Vellore, Tamil Nadu, India from June 2019 to March 2020, involving 138 children aged between 2 and 15 years who were being evaluated for TB. GA samples were collected after an overnight fast, and on the same day, atleast two hours later, IS samples were collected by trained staff. Both samples underwent mycobacterial smear and culture using the Mycobacteria Growth Indicator Tube (MGIT) method and Xpert MTB/RIF assay. Confirmation of pulmonary TB was based on atleast one of these tests being positive. The ‘Wong-Baker’ Visual Analogue Scale (VAS) was individually administered to each patient to compare the discomfort associated with GA and IS procedures. The differences in yield between IS and GA were tested for significance using the Two-sample test of proportions with a significance level set at 5%. McNemar's χ2 test was employed to compare matched observations. The MannWhitney test was used for comparing continuous variables, and the Chi-Square test for categorical variables. Cohen’s Kappa (κ) was used to assess interobserver agreement between the sampling methods using the different tests. Results: Out of the 138 cases recruited with suspected pulmonary TB, the diagnosis was microbiologically confirmed in 13 cases (9.4%). The overall diagnostic yield was 12/138 (8.7%) for GA and 10/138 (7.2%) for IS. In children under 10 years, GA outperformed IS with all three cases being positive by GA and none by IS. For those aged 10 years and above, 10 children (100%) tested positive with IS, while nine children (90%) were positive with GA. According to the Wong-Baker VAS measuring discomfort during the procedure, IS was favoured over GA (p-value&lt;0.0001). Conclusion: IS performs similarly or better than GA in children over 10 years, while GA performs better than IS in children under 10 years of age. IS is reported to cause less discomfort than GA on the Wong-Baker VAS.

Epidemiology of multi-drug resistant Tuberculosis in the western region of Kenya.

Multidrug-resistant tuberculosis (TB) (MDR-TB), or TB that is simultaneously resistant to both isoniazid (INH) and rifampicin (RIF), is a barrier to successful TB control and treatment. Stratified data on MDR-TB, particularly in the high-burden western Kenya region, remain unknown. This data is important to monitor the efficacy of TB control and treatment efforts. Herein, we determined the molecular epidemiology of drug-resistant TB and associated risk factors in western Kenya. This was a non-experimental, population-based, cross-sectional study conducted between January and August 2018. Morning sputum samples of individuals suspected of pulmonary TB were collected, processed, and screened for Mycobacterium tuberculosis (Mtb) and drug resistance using line probe assay (LPA) and Mycobacterium growth indicator tubes (MGIT) culture. MGIT-positive samples were cultured on brain heart infusion (BHII) agar media, and the presence of Mtb was validated using Immunochromatographic assay (ICA). Drug sensitivity was performed on MGIT and ICA-positive but BHI-negative samples. Statistical significance was set at P < 0.05. Of the 622 Mtb isolates, 536 (86.2%) were susceptible to RIF and INH. The rest, 86 (13.83%), were resistant to either drugs or both. A two-sample proportional equality test revealed that the MDR-TB prevalence in western Kenya (5%) did not vary significantly from the global MDR-TB estimate (3.9%) (P = 0.196). Men comprised the majority of susceptible and resistant TB (75.9% and 77.4%%, respectively). Also, compared with healthy individuals, the prevalence of HIV was significantly higher in MDR-TB patients (35.9% vs 5.6%). Finally, TB prevalence was highest in individuals aged 25-44 years, who accounted for 58.4% of the total TB cases. Evidently, the prevalence of MDRTB in western Kenya is high. Particular attention should be paid to men, young adults, and those with HIV, who bear the greatest burden of resistant TB. Overall, there is a need to refine TB control and treatment programs in the region to yield better outcomes.

Promising Ursolic Acid as a Novel Antituberculosis Agent: Current Progress and Challenges.

Tuberculosis (TB) stands as the second most prevalent cause of global human mortality from infectious diseases. In 2022, the World Health Organization documented an estimated number of global TB cases reaching 7.5 million, which causes death for 1.13 million patients. The continuous growth of drug-resistant TB cases due to various mutations in the Mycobacterium tuberculosis (MTB) strain, raises the urgency of the exploration of novel anti-TB treatments. Ursolic acid (UA) is a natural pentacyclic triterpene found in various plants that has shown potential as a novel anti-TB agent. This review aims to provide an overview of the therapeutic prospects of UA against MTB, with a particular emphasis on in silico, in vitro, and in vivo studies. Various mechanisms of action of UA against MTB are briefly recapped from in silico studies, such as enoyl acyl carrier protein reductase inhibitors, FadA5 (Acetyl-CoA acetyltransferase) inhibitors, tuberculosinyl adenosine transferase inhibitors, and small heat shock protein 16.3 inhibitor. The potential of UA to overcome drug resistance and its synergistic effects with existing antituberculosis drugs are briefly explained from in vitro studies using a variety of methods, such as Microplate Alamar Blue Assay, Mycobacteria Growth Indicator Tube 960 and Resazurin Assays, morphological change evaluation using transmission electron microscopy, and in vivo studies using BALB/C infected with multi drug resistant clinical isolates. Besides its promising mechanism as an antituberculosis drug, its complex chemical composition, limited availability and supply, and lack of intellectual property are also reviewed as those are the most frequently occurring challenges that need to be addressed for the successful development of UA as novel anti-TB agent.

Omadacycline pharmacokinetics/pharmacodynamics and efficacy against multidrug-resistant Mycobacterium tuberculosis in the hollow fiber system model.

Seventy-five years ago, first-generation tetracyclines demonstrated limited efficacy in the treatment of tuberculosis but were more toxic than efficacious. We performed a series of pharmacokinetic/pharmacodynamic (PK/PD) experiments with a potentially safer third-generation tetracycline, omadacycline, for the treatment of multidrug-resistant tuberculosis (MDR-TB). Mycobacterium tuberculosis (Mtb) H37Rv and an MDR-TB clinical strain (16D) were used in the minimum inhibitory concentration (MIC) and static concentration-response studies in test tubes, followed by a PK/PD study using the hollow fiber system model of TB (HFS-TB) that examined six human-like omadacycline doses. The inhibitory sigmoid maximal effect (Emax) model and Monte Carlo experiments (MCEs) were used for data analysis and clinical dose-finding, respectively. The omadacycline MIC for both Mtb H37Rv and MDR-TB clinical strain was 16 mg/L but dropped to 4 mg/L with daily drug supplementation to account for omadacycline degradation. The Mycobacteria Growth Indicator Tube MIC was 2 mg/L. In the test tubes, omadacycline killed 4.39 log10 CFU/mL in 7 days. On Day 28 of the HFS-TB study, the Emax was 4.64 log10 CFU/mL, while exposure mediating 50% of Emax (EC50) was an area under the concentration-time curve to MIC (AUC0-24/MIC) ratio of 22.86. This translates to PK/PD optimal exposure or EC80 as AUC0-24/MIC of 26.93. The target attainment probability of the 300-mg daily oral dose was 90% but fell at MIC ≧4 mg/L. Omadacycline demonstrated efficacy and potency against both drug-susceptible and MDR-TB. Further studies are needed to identify the omadacycline effect in combination therapy for the treatment of both drug-susceptible and MDR-TB.

Discordance between genotypic and phenotypic methods for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis and the correlation with patient treatment outcomes

BackgroundAccurate drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) is essential for proper patient management. We investigated discordance between genotypic (Xpert MTB/RIF and MTBDRplus) and phenotypic (MGIT 960) methods for the detection of rifampicin (RIF) and isoniazid (INH) resistant MTB and its correlation with patient treatment outcomes in Jimma, Southwest Oromia, Ethiopia. MethodsA retrospective study was conducted on 57 stored MTB isolates with known Xpert RIF resistance status (45 RIF resistant and 12 RIF susceptible) at Jimma University Mycobacteriology Research Center from November 2, 2021, to December 28, 2022. We did MTBDRplus and phenotypic DST (using the Mycobacterial Growth Indicator Tube (MGIT) system). The Xpert and MTBDRplus results were compared using phenotypic DST as a reference standard method. The treatment outcome was determined as per national guideline. The discordance between the genotypic and phenotypic DST was calculated using GraphPad software. ResultsAmong the 57 MTB isolates, six (10.5 %) had discordant results between the two DST methods. Xpert yielded five discordant results for RIF when compared with phenotypic DST (kappa coefficient (κ) = 0.76, 95 % confidence interval 0.56–0.96). The MTBDRplus compared with phenotypic DST gave three discordant results for RIF (κ = 0.86, 95 % confidence interval 0.71–1.00) and three for INH (κ = 0.86, 95 % confidence interval 0.70–1.00). Compared with Xpert, MTBDRplus yielded lower discordance with phenotypic DST for RIF. Out of six patients with discordant results, three had unfavorable outcomes while the other three were cured. Of the three patients with unfavorable outcomes, only one patient has received an inappropriate treatment regimen. There was no correlation between unfavorable outcomes and incorrect treatment regimens due to discordant results (Χ2 = 0.404; P = 0.525). ConclusionsDiscordance between genotypic and phenotypic DST for RIF or INH occurred in 10.5 % of isolates. Only one patient with discordant results has received an inappropriate treatment regimen, resulting in an unfavorable outcome. The impact of parallel use of rapid molecular assay with phenotypic DST on patient treatment outcomes requires further study.

Early Microbiologic Markers of Pulmonary Tuberculosis Treatment Outcomes.

Rationale: Earlier biomarkers of pulmonary tuberculosis (PTB) treatment outcomes are critical to monitor shortened anti-TB treatment (ATT). Objectives: To identify early microbiologic markers of unfavorable TB treatment outcomes. Methods: We performed a subanalysis of 2 prospective TB cohort studies conducted from 2013 to 2019 in India. We included participants aged ⩾18 years who initiated 6-month ATT for clinically or microbiologically diagnosed drug-sensitive PTB and completed at least one follow-up visit. Sputum specimens were subjected to a baseline Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay, acid-fast bacilli (AFB) microscopy and liquid and solid cultures, and serial AFB microscopy and liquid and solid cultures at weeks 2, 4, and 8. Poisson regression was used to assess the impact of available microbiologic markers (test positivity, smear grade, time to detection, and time to conversion) on a composite outcome of failure, recurrence, or death by 18 months after the end of treatment. Models were adjusted for age, sex, nutritional status, diabetes, smoking, alcohol consumption, and regimen type. Results: Among 1,098 eligible cases, there were 251 (22%) adverse TB treatment outcomes: 127 (51%) treatment failures, 73 (29%) recurrences, and 51 (20%) deaths. The primary outcome was independently associated with the Xpert MTB/RIF assay (medium-positive adjusted incidence rate ratio [aIRR], 1.91; 95% confidence interval [CI], 1.07-3.40; high-positive aIRR, 2.51; 95% CI, 1.41-4.46), positive AFB smear (aIRR, 1.48; 95% CI, 1.06-2.06), and positive liquid culture (aIRR, 1.98; 95% CI, 1.21-3.23) at baseline; Week 2 positive liquid culture (aIRR, 1.47; 95% CI, 1.04-2.09); and Week 8 positive AFB smear (aIRR, 1.63; 95% CI, 1.06-2.50) and positive liquid culture (aIRR, 1.54; 95% CI, 1.07-2.22). There was no evidence of Mycobacterium tuberculosis growth in the Mycobacterium Growth Indicator Tube at Week 4 conferring a higher risk of adverse outcomes (aIRR, 1.25; 95% CI, 0.89-1.75). Conclusions: Our analysis identifies Week 2 respiratory mycobacterial culture as the earliest microbiologic marker of unfavorable PTB treatment outcomes.

Diagnostic performance of two commercial MPT64 assays for the identification of Mycobacterium tuberculosis complex from liquid culture

BackgroundTuberculosis (TB) is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC). Early diagnosis of TB is the most essential component to control TB. In this study, we evaluated the diagnostic performance of SD Bioline TB Ag MPT64 (SD Bioline) and BD MGIT TBc Identification (BD TBc ID) kits for the detection of MTBC from MGIT (Mycobacteria Growth Indicator Tube) 960 culture tube, positive for acid-fast bacilli (AFB). MethodsA total of 198 AFB positive MGIT cultures [MTBC, 150 and non-tuberculous mycobacteria (NTM), 48] were collected during the study period (April 2021 to March 2023) and subjected to SD Bioline and BD TBc ID assays, respectively. All the 150 MTBC positive cultures were confirmed by multiplex PCR targeting MPB64, protein B and IS6110 genes, and the 48 NTM cultures were speciated by using line probe assays technique- Genotype Mycobacterium CM assay. ResultsSD Bioline and BD TBc ID kits detected all 150 MTBC strains correctly from MGIT positive cultures and no false negative was observed. However, there was one false positive for M. abscessus by SD Bioline and two false positive results for M. abscessus and M. intracellulare each by BD TBc ID. Overall, the sensitivity of both SD Bioline and BD TBc ID tests was 100 % and specificity was 97.9 % and 95.8 %, respectively. ConclusionOur results highlighted that SD Bioline and BD TBc ID assays played a promising role in the earlier identification of MTBC present in liquid culture and may act as a potential alternative to biochemical and expensive molecular techniques.