Resistant Tuberculosis Research Articles

Antibiotic Resistance in Mycobacterium Tuberculosis and Non-Tuberculous Mycobacteria

Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) antibiotic resistance presents an important challenge to the treatment of mycobacterial infections. The therapeutic approaches are complicated by the resistance of both MTB and NTM to a variety of antibiotics. Resistance to first-line drugs such as isoniazid, rifampicin, ethambutol, and streptomycin has been consistently increasing in MTB, underscoring the necessity of effective treatment strategies. Conversely, the necessity of species-specific treatment regimens is underscored by the high resistance rates of NTM species, such as Mycobacterium avium complex, M. kansasii, and M. abscessus complex, to commonly used anti-tuberculosis pharmaceuticals. A combination of intrinsic and acquired factors are involved in the mechanisms of antibiotic resistance in these mycobacteria. Features such as biofilm formation, thick cell walls, and reduced drug uptake are responsible for intrinsic resistance in NTM, whereas acquired resistance can develop as a result of protracted antibiotic exposure. Understanding these resistance mechanisms is essential for the development of new therapies and the prevention of the increasing prevalence of drug resistance in mycobacterial infections. The significance of continuous surveillance, species-specific treatment protocols, and the development of novel antimicrobial agents to effectively manage mycobacterial diseases is emphasized by the prevalence of antibiotic resistance in MTB and NTM. This review article focuses on the molecular mechanisms that have resulted in the development of resistance in both MTB and NTMs, as well as the extent to which various classes of antimycobacterial drugs act.

Time to Cure and Predictive Factors Among Patients with Multi-Drug Resistance Tuberculosis in Puntland, Somalia: A Retrospective Cohort Study

Background: Multi-drug-resistant tuberculosis, also known as MDR-TB, is caused by bacteria that are resistant to the most effective first-line anti-tuberculosis medications, which are rifampicin and isoniazid. MDR-TB is an increasing global concern, and its spread has varying cure times for patients affected. Thus, this study was intended to investigate the median time to cure and identify predictive factors for patients with MDR-TB in Puntland, Somalia. Methods: A retrospective cohort study was carried out at the MDR-TB center in Galkayo, Puntland, Somalia between the years of July 2017 and August 2023. The study utilized data that were collected from the inquiry form and monitoring cards of a randomly selected sample of 130 patients over a 6-year period. The data were entered into Epi-Data version 4.3 and analyzed using R Programming version 4.4.0. Non-parametric methods were used to estimate the median time to cure for patients with MDR-TB. Semi-parametric and parametric models were employed to determine predictive factors that influence the time to cure for MDR-TB patients. The Akaike Information Criteria (AIC) and Bayesian Information Criteria (BIC) of different survival models were compared to select the model that offers the best fit. Results: Out of the total of MDR-TB patients, 109(83.85%) were cured and 21(16.15%) were censored during the follow-up period. The median time to cure from MDR-TB was found to be 619 days which is approximately 20 months. In accordance with the AIC and BIC, the Log-logistic accelerated failure time model was the best fit for the data as compared to the other AFT models. The result of the Log-logistic AFT model revealed that age, sex, disease site, and comorbidities significantly affect the time to cure for MDR-TB. According to this study, older age with MDR-TB patients, male MDR-TB patients, MDR-TB patients with comorbid conditions, and extrapulmonary MDR-TB patients had a longer time to cure than their reference categories. Conclusion: Based on the findings of the study, the median time to cure for patients with MDR-TB in Puntland was found to be 20 months. Sex, disease site, comorbidities and age were predictive factors of time to cure from MDR-TB.

Small Molecule Inhibitors of Mycobacterium tuberculosis Topoisomerase I Identified by Machine Learning and In Vitro Assays

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a leading infectious cause of death globally. The treatment of patients becomes much more difficult for the increasingly common multi-drug resistant TB. Topoisomerase I is essential for the viability of M. tuberculosis and has been validated as a new target for the discovery of novel treatment against TB resistant to the currently available drugs. Virtual high-throughput screening based on machine learning was used in this study to identify small molecules that target the binding site of divalent ion near the catalytic tyrosine of M. tuberculosis topoisomerase I. From the virtual screening of more than 2 million commercially available compounds, 96 compounds were selected for testing in topoisomerase I relaxation activity assay. The top hit that has IC50 of 7 µM was further investigated. Commercially available analogs of the top hit were purchased and tested with the in vitro enzyme assay to gain further insights into the molecular scaffold required for topoisomerase inhibition. Results from this project demonstrated that novel small molecule inhibitors of bacterial topoisomerase I can be identified starting with the machine-learning-based virtual screening approach.

Quantitative MODS-Wayne assay for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis from sputum samples.

PZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.

Algorithm of surgical treatment of resistant forms of pulmonary tuberculosis

Objective. To improve the effectiveness of surgical treatment of multidrug–resistant pulmonary tuberculosis and extensively drug–resistant pulmonary tuberculosis. Materials and Methods. The work is based on the analysis of the results of treatment of 316 patients. The sequence of actions to determine the optimal type and extent of surgical intervention is regulated. Results. An algorithm for the surgical treatment of multidrug–resistant pulmonary tuberculosis and extensively drug–resistant pulmonary tuberculosis was developed and implemented, based on the results of a study of this complex category of patients who underwent preoperative preparation, a sequence of actions for surgical intervention, and postoperative management technology, taking into account current specific therapy regimens. Conclusions. The application of the developed algorithm of surgical treatment allowed to reduce the incidence of pulmonary tuberculosis recurrence and postoperative complications from (14.4 ± 2.3) to (7.0 ± 1.3)% and cure 92.5% of patients.

Distinct gene expression patterns of mono-isoniazid resistant Mycobacterium tuberculosis uncover divergent responses to isoniazid in host-mimicked condition.

Isoniazid stands as a frontline antibiotic utilized in the treatment of tuberculosis (TB), predominantly impacting the mycolic acid component within the cell wall of Mycobacterium tuberculosis (Mtb). It also affects the formation of lipoarabinomannan (LAM), an essential glycolipid in the cell envelope of Mtb. Despite the effectiveness of antibiotics for TB treatment, drug tolerance development in mycobacteria frequently stems from their adaptation to the hostile environment within the host, leading to treatment failure. Herein, we investigate mycobacterial adaptation to the isoniazid exposure in the host-mimicked conditions by focusing on the stress response genes (virS, icl1, whiB3, tgs1) and LAM-related genes (lprG, p55, lmeA, mptA, embC). Mtb H37Rv and mono-isoniazid resistant (INH-R) were cultivated in the host-mimicked multi-stress condition (MS) with or without isoniazid and the relative expressions of these gene candidates were measured using real-time PCR. In the INH-R strain, treatment with isoniazid in multi-stress conditions caused significant upregulation of tgs1, and LAM precursor-lipomannan (LM) synthesis and its transport genes (lprG, p55, lmeA, embC). In the case of H37Rv, all LAM-related genes and tgs1 were downregulated whereas other stress response genes were upregulated, remarkably in icl1 and whiB3. These findings highlight differences in gene expression patterns between drug-sensitive and resistant strains in multi-stress environments with drug pressure. Notably, stress response genes, particularly tgs1, may play a crucial role in regulating LAM production in the INH-R strain in response to isoniazid exposure. This study enhances our understanding of the mechanisms underlying drug resistance, offering valuable insights that could contribute to the development of new strategies for treating and eliminating TB.

Case Report: Multi drug resistant tuberculosis in an 18-month-old boy with seizure, vomiting, and loss of vision

Multi drug resistant tuberculosis (MDR-TB) in children is rare and requires tremendous skill and knowledge of clinicians for early detection and treatment. This case highlights the complexities in diagnosing and treating MDR-TB in young children. An 18-month-old immunocompetent boy presented with prolonged fever, sudden onset of vision loss, vomiting, and seizures, which led to a suspicion of meningitis. Given his family history of treated pulmonary TB (TB) in the father and uncle one year ago, the initial treatment followed the Nepali national protocol for non-MDR tuberculous meningitis (TBM). However, the child's lack of response to this treatment raised concerns about MDR-TB, particularly after discovering on close questioning that the uncle, who lived with the boy’s family, had actually been diagnosed with MDR-TB. The diagnosis was confirmed by molecular tests of cerebrospinal fluid (CSF) and customized treatment for MDR TBM administered. The boy then slowly improved and became less irritable, afebrile, seizure-free, developed spontaneous movements of all four limbs, and was discharged after one month. Delays in suspicion, confirmation, and treatment of MDR TBM led to complications of leptomeningitis with non-communicating hydrocephalus and bilateral optic atrophy, which was confirmed by magnetic resonance imaging (MRI) scan of the brain. This case highlights the importance of taking a very thorough and proper history and considering the possibility of MDR, especially in countries like Nepal where TB in general is rampant so that timely diagnosis and treatment is possible and complications are avoided.

Comparative Proteomic Analysis of Cell Wall Proteins of Aminoglycosides Resistant and Sensitive Mycobacterium tuberculosis Clinical Isolates.

The rising prevalence of Mycobacterium tuberculosis (M.tb) strains resistant to aminoglycosides (amikacin and kanamycin) challenges effective TB control and treatment. Understanding the mechanisms behind this resistance is crucial since aminoglycosides are a mainstay of TB therapy. The study aimed to analyze the cell wall proteins overexpressed in aminoglycoside-resistant isolates of Mycobacterium tuberculosis using proteomics approaches. We used two-dimensional electrophoresis and mass spectrometry to compare the cell wall proteomes of aminoglycosides-resistant and susceptible clinical isolates. The overexpressed protein spots were excised and identified using liquid chromatography-mass spectrometry (LC/MS). The identified proteins were subsequently analyzed for molecular docking, pupylation site identification, and STRING analysis. We found a total of nine significantly upregulated proteins in aminoglycosides-resistant isolates. Three of these proteins were the same (isoform), resulting in the identification of seven unique proteins. Specifically, Rv3841 and Rv1308 belonged to intermediary metabolism and respiration; Rv2115c to the cell wall and cell processes; Rv2501c, Rv2247 and Rv0295c to lipid metabolism; and Rv2416c to virulence, detoxification/adaptation. Notably, variations in these proteins support cell wall integrity, aiding mycobacteria's establishment and proliferation. Molecular docking study revealed that both drugs bind strongly to the proteins' active site regions. Additionally, the GPS-PUP algorithm successfully identified possible pupylation sites within these proteins, except Rv0295c. Based on interactome analysis using the STRING 12.0 database, we have identified potential interactive partners suggesting their role in aminoglycosides resistance. Overexpressed proteins not only act to counteract or regulate drug effects but also have a role in protein dynamics that allow for resistance. Some of these identified proteins may serve as innovative drug targets and biomarkers for the early detection of drug-specific resistance in M.tb. Further research is needed to elucidate the mechanisms by which these potential protein targets contribute to resistance in AK and KM M.tb isolates.

Incidence, types and predictors of adverse events and their impact on treatment outcomes in multidrug/rifampicin resistant tuberculosis patients receiving all oral treatment regimens.

Patients suffering from multidrug/rifampicin resistant tuberculosis (MDR/RR-TB) are treated for prolonged periods with a complex regimen comprised of relatively less effective and more toxic anti-TB drugs, consequently resulting in high incidence of adverse events (AEs). The current study evaluates the incidence, types, management and predictors of AEs, and their impact on treatment outcomes in MDR/RR-TB patients receiving all oral treatment regimens. A total of 242 eligible MDR/RR-TB patients treated at two different study sites from June 2019 to December 2021 were included in this study. Patients' sociodemographic, microbiological, clinical characteristics, reported AEs and treatment outcomes were retrospectively abstracted from their medical records. Chi-square, and Fisher exact tests (wherever applicable) were used to find the association between the variable and the occurrence of AEs. Majority of the patients were suffered from MDR-TB (71.9%) and were treated with longer treatment regimen (77.7%). Overall 206/242 patients (85%) experienced at least one AE during their treatment. Gastrointestinal disturbance was the most common AE (49.6%), followed by arthralgia (49.2%), psychiatric disturbances (39.3%), dermatological reactions (27.7%), body/headache (24.8%) and hyperuricemia (19%). Due to AEs, treatment modification was noted in 55 (22.72%) patients. Level of modification in the treatment regimen was higher in optic neuritis (100%) followed by neuropathies (80%) and myelosuppression (59%). Similarly, hepatotoxicity was the most serious AE in which the whole treatment regimen was terminated in 27% of patients. Furthermore, the results revealed that only patients' education status had statistically significant association with the incidence of AEs (p = 0.02). The treatment success rate was 80.6% whereas the ratio of died and LTFU patients were 15.3% and 4.1% respectively. Although patients who experienced AEs were more likely to develop successful treatment outcomes (82%) than their counterparts (72.2%), though this difference was not statistically significant. Although AEs were highly present in the current cohort, but they were successfully managed mostly by nonpharmacological interventions or symptomatic treatment. Besides, the incidence of AEs did not have a negative impact on treatment outcomes. High-risk patients for AEs must receive special attention and enhanced clinical management.

Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of Mycobacterium tuberculosis

Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. Methods: Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method, GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit, with some modifications. DNA quality was assessed based on concentration, purity, and integrity. Results: Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.

Discovery of potent dihydro-oxazinoquinolinone inhibitors of GuaB for the treatment of tuberculosis

Tuberculosis is the leading cause of death from an infectious disease, and is caused by Mycobacterium tuberculosis (M.tb). More than 1 billion people worldwide are thought to harbor an M.tb infection. The multidrug therapy that represents the current standard of care requires a minimum of four months of dosing and drug resistant Mycobacterium tuberculosis treatment regimens are significantly longer. Inosine-5′-monophosphate dehydrogenase (GuaB) is the enzyme that performs the rate-limiting step in de novo guanine nucleotide biosynthesis that is critical for growth and viability of bacteria including M.tb. The development of a novel antibiotic that inhibits GuaB could combine with existing therapies in novel ways and thereby contribute to effective therapeutic regimens for the treatment of tuberculosis. Here we describe the discovery of structurally distinct small molecule GuaB inhibitors that are potent against M.tb H37Ra and H37Rv strains and have desirable safety and AMDE profiles.

Identifying Predictors of Unfavorable Treatment Outcomes in Tuberculosis Patients

Objectives: In South Korea, there has been a continuous decrease in the incidence of tuberculosis (TB) attributable to a national TB elimination program; however, TB still poses a significant socioeconomic burden. This study aimed to analyze factors associated with successful TB treatment and to identify refractory patient groups with unfavorable outcomes. Methods: We analyzed anonymized data on 89,150 patients with TB provided by the Korea Disease Control and Prevention Agency. Specifically, we collected independent variables, which were categorized as individual, regional, and medical facility factors. Individual factors included age, sex, nationality, TB type, drug-resistant status, category of TB, housing type, underlying disease status, number of referrals, and smoking status. Regional factors referred to the region where the TB case was reported. Medical facility factors included the first visit to a medical facility, categorized by hospital type and the distinction between private–public mix (PPM) and non-PPM depending on the presence or absence of dedicated TB nurses. These factors were analyzed in relation to treatment success to identify refractory patient groups with unfavorable outcomes. Results: Multivariable logistic regression analysis revealed the following significant factors associated with successful TB treatment: sex, nationality, status of drug-resistant TB, category of TB, number of referrals, region of TB registry, underlying diseases, and smoking status. Specifically, compared with their relevant counterparts, male patients had a lower rate of successful treatment (adjusted odds ratio [aOR]: 0.66, reference [Ref.]: women); Korean nationals had a higher rate of treatment success (aOR: 7.20, Ref. foreign residents in Korea); resistant TB was associated with a lower rate of treatment success (aOR: 0.35, Ref.: non-resistant TB status); newly treated patients had a higher rate of treatment success (aOR: 1.75, Ref.: retreatment patient); switching hospitals once (aOR: 1.78), never (aOR: 1.41), or twice (aOR: 1.37) was associated with increased treatment success (Ref.: three or more times); having zero (aOR: 1.45), one (aOR: 1.31), or two (aOR: 1.24) underlying diseases was associated with a higher rate of treatment success (Ref. three or more underlying diseases); and past smokers (aOR: 1.40) and non-smokers (aOR: 1.35) had a higher rate of treatment success (Ref.: current smokers). Conclusions: Our study identified several factors contributing to unfavorable treatment outcomes in tuberculosis patients, including male patients, foreign residents in Korea, drug-resistant TB, retreatment patients, frequent hospital switching, multiple underlying diseases, and current smoking status. These research findings could inform the development of efficient management strategies and policies for improving the treatment success rate among patients with TB.

Development and preliminary assessment of the iFIND TBR: all-in- one molecular diagnostic assay for rapid detection of Mycobacterium tuberculosis and rifampicin resistance.

Early and accurate diagnosis of tuberculosis (TB) is crucial for initiating timely treatment and preventing new infections. In this study, we introduced the iFIND TBR assay, an automated all-in-one tuberculosis detection approach that simultaneously detect Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. The limits of detection (LOD), sensitivity, specificity, and RIF-R rpoB mutation detection of the iFIND TBR were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M.tuberculosis H37Rv. Frozen clinical samples from patients suspected of having TB were also tested. The LOD of the iFIND TBR for MTB detection were 13.34 CFU/ml (95% CI, 11.71-16.47), and for RIF resistance was 109.79CFU/mL (95% CI, 95-138.19). The iFIND TBR assay accurately distinguish MTB strains from non-tuberculous mycobacteria (NTM) without any cross reactivity. Testing on 157 clinical sputum samples, compared with the bacteriologically TB standard, the overall sensitivity and specificity of the iFIND TBR was 100% (95%CI, 94.64, 100) and 85.29% (95% CI, 74.61, 92.72), respectively. When assessing RIF susceptibility, the iFIND TBR achieved a sensitivity of 98.15% (95% CI, 90.11-99.95) and a specificity of 85.71% (95% CI, 67.33-95.97), compared with phenotypic drug susceptibility testing. Discordant RIF susceptibility results were more frequently observed in samples exhibiting heteroresistance. These findings demonstrate that iFIND TBR assay performs well in detecting TB and RIF resistance, and shows promise as a point-of-care tool in resource-limited areas.

A Rare Case of Tremor Induces by Cycloserine in Drug-Resistant Tuberculosis Patient

Background: Tuberculosis is a contagious disease that generated by Mycobacterium tuberculosis. Drug-Resistant tuberculosis (DR-TB) is inculpated the use of the second-line anti-tubercular treatment which is associated with many drug side effects or so called, Adverse Drug Reactions (ADR). Cycloserine (Cs) is an important drug against drug resistant tuberculosis (DR-TB). Cycloserine has been used in tuberculosis therapy since the late 1950s. Identical with most drug, Cycloserine can cause many Adverse Drug Reactions (ADR). Case Illustration: A 23-year-old woman diagnosed with drug-resistant tuberculosis is undergoing long-term treatment. The patient received treatment for DR-TB with the Bdq-Lfx-Cfz-Cs-E regimen. After the patient underwent the 10th month of advanced phase treatment, the patient complained of shaking in both hands (tremors). The tremor is felt to be more severe in the right hand and the patient cannot grip objects tightly. Discussion: Cycloserine (Cs) play an important role in second-line drug management of DR-TB. Cs-Induced psychosis and other neurological side-effects can be detrimental towards patients yet they are rarely reports in DR-TB cases. Cs is correlated with severe psychiatric cases and Central Nervous System related ADRs. Cs-associated ADR is most likely because of production of gamma-aminobutyric acid as a result of inhibition of glutamic decarboxylase. Study Shown that among 132 patients who reported side effects in the cycloserine group, 2 (1.4%) experienced major side effects, namely tremors. Side effects possibly or probably related to Cs appeared after a median of 71 days (range 10–331 days) of Cs treatment. Conclusion: the drug side effects such as tremors are very rare in drug-resistant tuberculosis patients. In this case, the patient's complaint of tremors could be caused by cycloserine as an anti-tuberculosis drug.

Prevalence of rifampicin and isoniazid mono-resistance among cases of pulmonary tuberculosis from Western Uttar Pradesh, North India.

Mono-resistance to rifampicin/isoniazid increases poor treatment outcomes and the risk of multi-drug resistance (MDR) in tuberculosis (TB) patients. Limited information exists about mono-resistance status of TB patients in Uttar Pradesh, North India. This study aimed to estimate the burden of rifampicin and isoniazid mono-resistance in Western Uttar Pradesh. 153 sputum samples of suspected pulmonary tuberculosis patients were processed to isolate Mycobacterium tuberculosis using the Lowenstein-Jensen (L-J) culture medium. The isolates were identified using an immuno-chromatographic test and IS6110 PCR. The confirmed Mycobacterium tuberculosis isolates were tested for drug susceptibility testing against rifampicin and isoniazid anti-tuberculosis drugs. The results of the drug susceptibility testing were compared with demographic information and analyzed statistically. Out of 153 sputum samples, 83 (54.24%) samples were positive for growth on L-J medium, including 82 (98.79%) Mycobacterium tuberculosis isolates. Of the 82 Mycobacterium tuberculosis isolates, 16 (19.51%), 7 (8.54%), and 5 (6.10%) isolates were MDR, mono-resistant to rifampicin and isoniazid, respectively. The occurrence of RIF/INH mono-resistant-TB was higher in patients of male gender, age above 45 years, living in rural conditions, history of weight loss, and previous anti-TB treatment, but the effect was not statistically significant. The study reported the status of rifampicin and isoniazid mono-resistance among TB patients and highlighted the need for continuous monitoring and improved intervention for the initial detection of mono-drug-resistant cases. This will improve clinical treatment outcomes and decrease the rate of drug-resistant TB in Uttar Pradesh, North India.

Exploring gene mutations and multidrug resistance in Mycobacterium tuberculosis: a study from the Lung Hospital in Vietnam.

Drug-resistant tuberculosis not only diminishes treatment efficacy but also heightens the risk of transmission and mortality. Investigating Mycobacterium tuberculosis resistance to first-line antituberculosis drugs is essential to tackle a major global health challenge. Using Sanger sequencing, this study investigates gene mutations associated with multidrug resistance in drug-resistant M. tuberculosis strains. Among 30 samples, mutations were found in genes linked to first-line anti-tuberculosis drug resistance. Rifampicin resistance was observed in 46.67% of the samples, with the most frequent mutation in the rpoB gene at codon 450 (S450L) occurring in 23.33% of cases. Similarly, isoniazid resistance was found in 86.67% of samples, with 33.33% of cases indicating the katG gene mutation at codon 315 (S315T). Additionally, streptomycin resistance was present in 76.67% of samples, and 30% of these cases were mainly linked to the rpsL gene mutation at codon 43 (K43R). These findings illuminate the genetic mechanisms behind drug resistance in M. tuberculosis. By identifying specific genetic markers, this research enhances our ability to diagnose and treat drug-resistant Tuberculosis more accurately and efficiently.

Formation of treatment regimens in newly diagnosed patients before the diagnosis of pulmonary tuberculosis verification

Abstract. One of the basic principles of treatment of tuberculosis patients is the appointment of treatment taking into account the drug resistance of Mycobacterium tuberculosis (MBT). However, during treatment, it may be necessary to change the intake regimen when new data on drug resistance become available. The search for ways to choose the optimal treatment method seems relevant.Aim: To analyze the prescribed regimens and the reason for their correction in newly identified patients before verifying the diagnosis of tuberculosis.Material and Methods. The following methods were used to detect MBT: luminescent microscopy, molecular genetic methods and seeding on liquid and solid media. Tests for drug sensitivity were conducted in parallel. The study group consisted of patients with established bacterial excretion (n = 79), whose diagnosis was confirmed by cultural methods. A retrospective method was used in the analysis.Results. At the beginning of treatment with bacterioscopy and polymerase chain reaction methods MBT was detected in 65/79 (82.2%) cases, the remaining 14 cases were detected only by culture methods. Forced change of the therapy regimen during treatment after receiving the results was in 25/79 (31.6%) cases, of which 1/25 (4%) with isoniazid resistance, 7/25 (28%) with multidrug-resistant tuberculosis, 17/25 (68%) with the pre-extensive drug resistance regimen. The latter was prescribed only after receiving results of tests for drug sensitivity on liquid and solid media after 1–3 months. The multidrug-resistant tuberculosis treatment regimen decreased after receiving data on resistance to fluoroquinolones in accordance with the seeding data, but the largest number of patients still remained on this regimen – 41/79 (51.9%). Among these patients a change in chemotherapy regimen from multidrug-resistant tuberculosis to pre-extensive drug resistance during the intensive phase of treatment occurred in 17/25 (68%).Conclusion. When prescribing a regimen, it is recommended to assess the risk of changing the regimen and, if there are predictors, apply a chemotherapy regimen before treatment.

Targeted next-generation sequencing - a promising approach in the diagnosis of Mycobacterium tuberculosis and drug resistance.

Targeted next-generation sequencing (tNGS) offers a high-throughput, culture-independent approach that delivers a comprehensive resistance profile in a significantly shorter turn-around time, making it promising in enhancing tuberculosis (TB) diagnosis and informing treatment decisions. This study aims to evaluate the performance of tNGS in the TB diagnosis and drug resistance detection of Mycobacterium tuberculosis (MTB) using MTB clinical isolates and bronchoalveolar lavage fluid (BALF) samples. A total of 143 MTB clinical isolates were assessed, tNGS, phenotypic antimicrobial susceptibility testing (AST), and AST based on whole genome sequencing (WGS) exhibited high concordance rates, averaging 95.10% and 97.05%. Among 158 BALF samples, culture, Xpert MTB/RIF, and tNGS reported 29, 70 and 111 positives, respectively. In the confirmed cases with etiological evidence (smears, cultures, or molecular test), the positive rate of tNGS (73/83, 87.95%) was higher than that of Xpert MTB (67/83, 80.72%). Additionally, 45% (27/60) of clinically diagnosed cases (with imaging or immunological evidence) were positive for tNGS. Further validation on the discrepant results between tNGS and Xpert MTB/RIF with droplet digital PCR (ddPCR) yielded 35 positives, tNGS detected all, and Xpert MTB/RIF only identified 6 positives. In conclusion, tNGS demonstrates robust and rapid performance in the identification of MTB and its associated drug resistance, and can be directly applied to clinical samples, positioning it as a promising approach for laboratory testing of tuberculosis.

Levofloxacin activity at increasing doses in a murine model of fluoroquinolone-susceptible and -resistant tuberculosis.

High-dose levofloxacin was explored in a clinical trial against multidrug-resistant tuberculosis and failed to show increased efficacy. In this study, we used a murine model to explore the efficacy of a dose increase in levofloxacin monotherapy beyond the maximum dose evaluated in humans. A total of 120 4-week-old female BALB/c mice were intravenously infected with 106 CFU of Mycobacterium tuberculosis H37Rv wild-type (WT) or isogenic H37Rv mutants harboring GyrA A90V or D94G substitutions; the MICs were 0.25, 4, and 6 µg/mL, respectively. Levofloxacin 250 and 500 mg/kg were given every 12 h (q12h) orally for 4 weeks. Pharmacokinetic parameters were determined after five doses. These two regimens decreased lung bacillary load in mice infected with H37Rv WT but not in mice infected with the A90V and D94G mutants. Levofloxacin 250 mg/kg q12h in mice generated pharmacokinetic parameters equivalent to 1,000 mg/d in humans, whereas 500 mg/kg q12h generated a twofold greater exposure than the highest equivalent dose tested in humans (1,500 mg/d). In our dose-response model, the effective concentration at 50% (EC50) produced an AUC/MIC (AUC0-24h/MIC) ratio of 167.9 ± 27.5, and at EC80 it was 281.2 ± 97.3. Based on this model, high-dose levofloxacin regimens above 1,000 mg/d are not expected to cause a significant increase in bactericidal activity. This study suggests no benefit of high-dose levofloxacin above 1,000 mg/d in the treatment of fluoroquinolone-susceptible or -resistant tuberculosis.

Comparison of Molecular Testing Methods in Detecting Drug Resistance Tuberculosis

Tuberculosis (TB) is the leading cause of infectious disease-related deaths in Indonesia, and the COVID-19 pandemic has led to a decline in TB reporting and an increase in Drug-Resistant Tuberculosis (DR-TB). While traditional culture and Drug Susceptibility Testing (DST) takes 2-8 weeks, therefore, genotypic assays such as GeneXpert, Line Probe Assay (LPA), and sequencing offer faster and more precise diagnosis and drug resistance information.