Isoniazid Resistance In Mycobacterium Tuberculosis Research Articles

Genetic mechanisms of co-emergence of INH-resistant Mycobacterium tuberculosis strains during the standard course of antituberculosis therapy.

The incidence of isoniazid (INH) resistant Mycobacterium tuberculosis is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in M. tuberculosis strains collected from the same patients during the standard course of treatment. Three M. tuberculosis strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated katG genes were electroporated into a KatG-deleted M. tuberculosis strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the katG of the two post-treatment strains, respectively. Transformation experiments showed that katG of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated katG genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two katG mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.IMPORTANCEThe evolution of drug-resistant strains of Mycobacterium tuberculosis within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel katG mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.

Whole Genome Sequence Dataset of Mycobacterium tuberculosis Strains from Patients of Campania Region

Tuberculosis (TB) is one of the deadliest infectious disorders in the world. To effectively TB manage, an essential step is to gain insight into the lineage of Mycobacterium tuberculosis (MTB) and the distribution of drug resistance. Although the Campania region is declared a cluster area for the infection, to contribute to the effort to understand TB evolution and transmission, still poorly known, we have generated a dataset of 159 genomes of MTB strains, from Campania region collected during 2018–2021, obtained from the analysis of whole genome sequence. The results show that the most frequent MTB lineage is the 4 according for 129 strains (81.11%). Regarding drug resistance, 139 strains (87.4%) were classified as multi susceptible, while the remaining 20 (12.58%) showed drug resistance. Among the drug-resistance strains, 8 were isoniazid-resistant MTB, 4 multidrug-resistant MTB, while only one was classified as pre-extensively drug-resistant MTB. This dataset expands the existing available knowledge on drug resistance and evolution of MTB, contributing to further TB-related genomics studies to improve the management of this disease.

Mutation Characteristics of inhA and katG Genes in Isoniazid-Resistant Mycobacterium Tuberculosis Patients in Xinjiang

Objective: To analyze the mutation characteristics of inhA and katG genes in isoniazid-resistant Mycobacterium tuberculosis in Xinjiang. Methods: The katG and inhA in 148 strains of isoniazid-resistant Mycobacterium tuberculosis were amplified through fluorescence quantitative PCR, and the amplified products were sequenced and compared. Results:The inhA gene mutation rate of 148 strains of isoniazid-resistant mycobacterium tuberculosis was 13.51% (20/148), among which the inhA gene mutation rate among patients of Han, Uygur, and Kazakh ethnicity were 15.87%, 13.21%, and 17.65%, respectively. There was no significant difference in the inhA mutation rate among nationalities (c2 = 2.897, P > 0.05). The mutation rate of the katG gene was 84.46% (125/148), among which the mutation rates of patients of Han, Uyghur, and Kazak ethnicities were 82.54%, 84.91%, and 76.47%, respectively. The Hui and other ethnic groups were all affected by the katG gene mutation. There was no significant difference in the mutation rate of the katG gene among different ethnicities (c2 = 3.772, P > 0.05). The mutation rates of the inhA gene in southern Xinjiang, northern Xinjiang, and other provinces were 18.60%, 9.28%, and 37.50%, respectively. The mutation rates of the inhA gene in different regions were statistically different (c2 = 6.381, P < 0.05). There was no significant difference in the inhA mutation rate between patients from southern and northern Xinjiang (c2 = 2.214, P > 0.05) and between southern Xinjiang and other provinces (c2 =1.424, P > 0.05). However, the mutation rate of the inhA gene in patients from other provinces was higher than that in northern Xinjiang (c2 = 5.539, P < 0.05). The mutation rates of the katG gene in southern Xinjiang, northern Xinjiang, and other provinces were 81.40%, 87.63%, and 62.50%, respectively. There was no significant difference in the mutation rates of the katG gene among different regions (c2 = 3.989, P > 0.05). Conclusion: katG gene mutation was predominant in isoniazid-resistant tuberculosis patients in Xinjiang Uygur Autonomous Region, and inhA and katG gene mutation were no different among different ethnic groups.

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.

Primer design and optimization of loop-mediated isothermal amplification (LAMP) for specific detection of genes isoniazid-resistant Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains one of the leading causes of death in Indonesia. The limited diagnostic tools for TB, including Multi-Drug Resistant Tuberculosis (MDR-TB), contribute to the increasing number of cases. MDR-TB refers to TB strains resistant to Anti-Tuberculosis Drugs such as Isoniazid (INH). In response, the WHO has recommended the use of a rapid, equipment-free method called loop-mediated isothermal amplification (LAMP). This study aims to design primers and optimize the LAMP method for the detection of Isoniazid-resistant M. tuberculosis. The research integrates bioinformatics testing, laboratory experiments, and validation methods. The study successfully designed LAMP primers for detecting Isoniazid-resistant comprising three pairs of primers: F3, B3; FIP, BIP; and LF, LB. The optimization of the LAMP method involved temperature and time parameters. Five temperature treatments were tested, revealing an optimum temperature of 70°C. For time optimization, eight treatments were conducted, with an optimum time range of 25-30 minutes. The study concludes that the LAMP method differs from the PCR method due to the higher number of primers employed and LAMP requires only a single isothermal temperature. Additionally, the study achieved optimization of temperature and time for the LAMP method, facilitating the detection of Isoniazid-resistant MTB.

Isoniazid-Resistant Tuberculosis - A Series of Clinical Cases

Abstract The emergence of M. tuberculosis strains resistant to Isoniazid (INH) and Rifampicin (RIF), the two most potent drugs of first-line anti-TB treatment is termed multidrug drug-resistant TB (MDR-TB). Multidrug-resistant tuberculosis has been a severe medical and epidemic problem worldwide. We present a series of clinical cases consisting of two patients diagnosed with isoniazid-resistant tuberculosis. Histopathological examination supports the diagnosis of tuberculous granulomas. The pathology molecular examination revealed the presence of Isoniazid-resistant Mycobacterium tuberculosis 1, 2 via the following mutation c.947G>A; p. Gly316Asp. Isoniazid-resistance is associated with mutations in the furA-katG and fabG1-inhA operons and in the ahpC gene. 64% of the isoniazid-resistance phenotypes were associated with the kat G 315 mutation worldwide. The second most common mutation is inhA-15, reported in 19% of the Isoniazid-resistant isolates. The significant association between the two mutations, inhA c-15 and kat G 315 respectively, and the high-level resistance is of interest in the interpretation of current and future molecular diagnostic testing, as an early prediction of the level of Isoniazid-resistance is essential to decide the benefit of high-dose Isoniazid use.

Drug resistance and the genotypic characteristics of rpoB and katG in rifampicin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates in central Vietnam.

Tuberculosis (TB) and drug-resistant TB (DR-TB) are national health burdens in Vietnam. In this study, we investigated the prevalence of rifampicin (RIF) and/or isoniazid (isonicotinic acid hydrazide, INH) resistance in patients with suspected TB, and applied appropriate techniques to help rapidly target DR-TB. In total, 1,547 clinical specimens were collected and cultured using the BACTEC MGIT system (Becton Dickinson and Co.). A resazurin microtiter assay (REMA) was used to determine the proportions of RIF and/or INH resistance. A real-time polymerase chain reaction panel with TaqMan probes was employed to identify the mutations of rpoB and katG associated with DR-TB in clinical isolates. Genotyping of the identified mutations was also performed. A total of 468 Mycobacterium tuberculosis isolates were identified using the REMA. Of these isolates, 106 (22.6%) were found to be resistant to 1 or both antibiotics. Of the resistant isolates, 74 isolates (69.8%) were resistant to isoniazid (INH) only, while 1 isolate (0.94%) was resistant to RIF only. Notably, 31 isolates (29.24%) were resistant to both antibiotics. Of the 41 phenotypically INH-resistant isolates, 19 (46.3%) had the Ser315Thr mutation. There were 8 different rpoB mutations in 22 (68.8%) of the RIF-resistant isolates. The most frequently detected mutations were at codons 531 (37.5%), 526 (18.8%), and 516 (6.3%). To help prevent new cases of DR-TB in Vietnam, it is crucial to gain a comprehensive understanding of the genotypic DR-TB isolates.

Design, Synthesis, and Evaluation of Novel Δ2-Thiazolino 2-Pyridone Derivatives That Potentiate Isoniazid Activity in an Isoniazid-Resistant Mycobacterium tuberculosis Mutant.

Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.

Application of the real-time PCR Taqman allelic discrimination assay for the detection of Isoniazid and/or Rifampicin resistant Mycobacterium Tuberculosis from clinical samples

Background: Drug-resistant Tuberculosis (DR-TB) is challenging public health problem in countries with high tuberculosis ​​prevalence and limited resources. Developing and applying the most appropriate and effective methods for diagnosing DR-TB from clinical samples is necessary, allowing a more rapid detection method for large-scale screening. Methods: Applying real-time PCR Taqman allelic discrimination with a PCR Taqman probes panel to identifying the DR-TB associated mutations in rpoB and katG of Mycobacterium Tuberculosis from isolates and clinical samples. Results: Comparing results of the real-time PCR allelic and DNA sequencing results, the sensitivity and specificity for Isoniazid resistance detection by analysing katG were found 95%(75.1 - 99.8) and 100%, Rifampicin resistance determining region (RRDR) of rpoB were found 95.5(77.16 - 99.88) and 100%, respectively. The real-time PCR TaqMan allelic discrimination also showed the sensitivities 100% for both katG and rpoB, and the specificities were 93.55% (78.58 - 99.21) for the rpoB and 93.94% (79.77 - 99.26) for the katG from clinical samples. Conclusions: This study showed that the real-time PCR taqman allelic discrimination assay is useful for detection of TB and DR-TB because of an accurate and rapid diagnosis in the early stages. Key words: rug-resistant, Tuberculosis, clinical samples, real-time PCR taqman allelic discrimination assay, Mycobacterium tuberculosis.

Diagnostic performance of the GenoType MTBDRplus VER 2.0 line probe assay for the detection of isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

Isoniazid (INH) resistant Mycobacterium tuberculosis (Hr-TB) is the most common type of drug resistant TB, and is defined as M tuberculosis complex (MTBC) strains resistant to INH but susceptible to rifampicin (RIF). Resistance to INH precedes RIF resistance in almost all multidrug resistant TB (MDR-TB) cases, across all MTBC lineages and in all settings. Therefore, early detection of Hr-TB is critical to ensure rapid initiation of appropriate treatment, and to prevent progression to MDR-TB. We assessed the performance of the GenoType MTBDRplus VER 2.0 line probe assay (LPA) in detecting isoniazid resistance among MTBC clinical isolates. A retrospective study was conducted among M. tuberculosis complex (MTBC) clinical isolates obtained from the third-round Ethiopian national drug resistance survey (DRS) conducted between August 2017 and December 2019. The sensitivity, specificity, positive predictive value, and negative predictive value of the GenoType MTBDRplus VER 2.0 LPA in detecting INH resistance were assessed and compared to phenotypic drug susceptibility testing (DST) using the Mycobacteria Growth Indicator Tube (MGIT) system. Fisher's exact test was performed to compare the performance of LPA between Hr-TB and MDR-TB isolates. A total of 137 MTBC isolates were included, of those 62 were Hr-TB, 35 were MDR-TB and 40 were INH susceptible. The sensitivity of the GenoType MTBDRplus VER 2.0 for detecting INH resistance was 77.4% (95% CI: 65.5-86.2) among Hr-TB isolates and 94.3% (95% CI: 80.4-99.4) among MDR-TB isolates (P = 0.04). The specificity of the GenoType MTBDRplus VER 2.0 for detecting INH resistance was 100% (95% CI: 89.6-100). The katG 315 mutation was observed in 71% (n = 44) of Hr-TB phenotypes and 94.3% (n = 33) of MDR-TB phenotypes. Mutation at position-15 of the inhA promoter region alone was detected in four (6.5%) Hr-TB isolates, and concomitantly with katG 315 mutation in one (2.9%) MDR-TB isolate. GenoType MTBDRplus VER 2.0 LPA demonstrated improved performance in detecting INH resistance among MDR-TB cases compared to Hr-TB cases. The katG315 mutation is the most common INH resistance conferring gene among Hr-TB and MDR-TB isolates. Additional INH resistance conferring mutations should be evaluated to improve the sensitivity of the GenoType MTBDRplus VER 2.0 for the detection of INH resistance among Hr-TB cases.

Green synthesis, antimycobacterial evaluation and molecular docking studies of novel 2,3-dihydro-1H-pyrazol-4-ylnaphthalene-1,4-diones

A novel series of 2,3-dihydro-1H-pyrazol-4-ylnaphthalene-1,4-diones is synthesized via one-pot, multi-component condensation of ethyl acetoacetate, 4-(trifluoromethyl)phenylhydrazine, aromatic aldehydes and 7-fluoro-2-hydroxynaphthalene-1,4‑dione in aqueous p-toluenesulfonic acid (15 mol%) at 100 °C. The synthesized compounds (5a-j) were evaluated for their in vitro antimycobacterial efficacy against drug-sensitive and isoniazid-resistant Mycobacterium tuberculosis (Mtb) H37Rv strains using the broth based method. All compounds displayed good to better antimycobacterial activity with MIC values ranging from 0.49 to 5.16 µM. Compound 5i (MIC = 0.49±0.08 μM) demonstrated equivalent antimycobacterial activity as that of the reference drug isoniazid (MIC = 0.35±0.06 μM). Molecular docking of the synthesized compounds 5a-j with an active site of membrane transporter MmpL3 protein (PDB ID: 6AJJ) was performed to justify the results of in vitro antimycobacterial activity and offer insight into the possible mode of action and binding interaction of compounds with the receptor protein. Molecular descriptors and drug-like features of novel 2,3-dihydro-1H-pyrazol-4-ylnaphthalene-1,4-diones were also examined.

Detection of katG, inhA and ahpC gene mutation in clinical isolates of isoniazid-resistant Mycobacterium tuberculosis in Makassar City, South Sulawesi, Indonesia

BackgroundTuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). The world is currently facing challenges due to the spread of anti-tuberculosis drug-resistant of M. tuberculosis. Isoniazid-resistant (INH), is one of the first-line anti-tuberculosis agents that has a high resistance case. This study used Multiplex allele-specific Polymerase Chain Reaction (MAS-PCR) to detect the most common mutations associated with isoniazid resistance on inhA, katG, and ahpC gene. MethodsThis study used samples from clinical isolates of M. tuberculosis which had been tested for their antibiotic sensitivity of first-line anti-tuberculosis drugs. The DNA extraction process was carried out using the boiling method and then amplified with specific primers for inhA, katG, and ahpC genes using the MAS-PCR method. The results are then read on the electrophoretic gel with an interpretation of the mutation gene when the target gene DNA bands were absent according to the allele-specific fragments target. ResultsA total of 200 isolates were tested in this study consisting of isoniazid-resistant and susceptible with the largest distribution of Multi-Drug Resistant (MDR) isolates with a total of 146 isolates (73%). The most significant gene mutation was on the ahpC gene in 61 isolates (30,5%) and the combination mutation of the katG + ahpC gene in 52 isolates (26%) with sensitivity and specificity of the test reaching 87% and 42% for the detection of INH-resistant. ConclusionMutation on the ahpC gene has the highest percentage in this study. AhpC gene can be considered one of the essential genes to be tested for the cause of isoniazid-resistant. Using MAS-PCR for detecting gene mutation in isoniazid-resistant was simple and easy, it has the potential to be widely used as a rapid screening molecular test.

Genetic mutations underlying isoniazid-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa, Pakistan

Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health issue in Pakistan. Isoniazid is a first-line pro-drug that requires activation through an enzyme called catalase peroxidase, but is subject to widespread resistance, driven by mutations in katG and inhA genes and other loci with compensatory effects (e.g., ahpC). Here, we used whole genome sequencing data from 51 M. tuberculosis isolates collected from Khyber Pakhtunkhwa province (years 2016–2019; all isoniazid phenotypically resistant) to investigate the genetic diversity of mutations in isoniazid candidate genes. The most common mutations underlying resistance were katG S315T (37/51), fabG1 -15C>T (13/51; inhA promoter), and inhA −154G>A (7/51). Other less common mutations (n < 5) were also identified in katG (R128Q, V1A, W505*, A109T, D311G) and candidate compensatory genes ahpC (−54C>T, −51G>A) and oxyS (M249T). Using DynaMut2 software, the mutants exhibited various degrees of stability and flexibility on protein structures, with some katG mutations leading to a decrease in KatG protein flexibility. Overall, the characterisation of circulating isoniazid resistant-linked mutations will assist in drug resistant TB management and control activities in a highly endemic area of Pakistan.

Modified gold nanoparticle colorimetric probe-based biosensor coupled with allele-specific PCR for rapid detection of G944C mutation associated with isoniazid resistance

The emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) makes tuberculosis (TB) difficult to control and treat. Therefore, rapid and reliable detection of MDR-TB within the community plays a significant role. The aim of this study was to investigate the use of modified gold nanoparticle (AuNP) colorimetric probe-based biosensor coupled with allele-specific PCR for the rapid detection of G944C mutation associated with isoniazid resistance in MTB. Based on the citrate reduction method, the AuNPs were synthesized and functionalized using thiol-modified oligonucleotides (AuNP-biosensor). The AuNP-biosensor method was compared to the gold standard in terms of analytical and clinical sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy. The gold standard was defined as a positive result in sequencing.The AuNP-biosensor had 100% sensitivity and specificity for detection of G944C mutation. After allele-specific PCR amplification the results were carried out for <15 min with ready-to-use AuNP-biosensor. PPV, NPV, DOR and accuracy of this method were 100%, 100%, 309 and 100%, respectively. The method designed in this study can identify the desired mutation with high efficiency after initial amplification, either from the sputum sample or from isolated bacteria. By designing a panel of this method, different MTB resistance-associated mutations could be detected more rapidly.

Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

Traditionally, single critical concentrations of drugs are utilized for Mycobacterium tuberculosis (Mtb) drug susceptibility testing (DST); however, the level of drug resistance can impact treatment choices and outcomes. Mutations at the katG gene are the major genetic mutations in multidrug resistant (MDR) Mtb and usually associated with high level resistance. We assessed the minimum inhibitory concentrations (MICs) of MDR or rifampin resistant (RR) and isoniazid (INH) resistant Mtb isolates to determine the quantification of drug resistance among key anti-tuberculosis drugs. The study was conducted on stored Mtb isolates collected as part of a national drug resistance survey in Ethiopia. MIC values were determined using Sensititre™ MYCOTB plates. A line probe assay (MTBDRplus) was also performed to identify genetic determinants of resistance for all isolates. MIC testing was performed on 74 Mtb isolates including 46 MDR, 2 RR and 26 INH phenotypically resistant isolates as determined by the Löwenstein Jensen (LJ) method. Four (15%) INH resistant Mtb isolates were detected as borderline rifampin resistance (MIC = 1 μg/ml) using MYCOTB MIC plates and no rifampin resistance mutations were detected by LPA. Among the 48 MDR/RR TB cases, 9 (19%) were rifabutin susceptible (MIC was between ≤0.25 and 0.5μg/ml). Additionally, the MIC for isoniazid was between 2-4 μg/ml (moderate resistance) for 58% of MDR TB isolates and 95.6% (n = 25) of the isolates had mutations at the katG gene. Our findings suggest a role for rifabutin treatment in a subset of RR TB patients, thus potentially preserving an important drug class. The high proportion of moderate level INH resistant among MDR Mtb isolates indicates the potential benefit of high dose isoniazid treatment in a high proportion of katG gene harboring MDR Mtb isolates.

Prevalence of Isoniazid Resistance in Mycobacterium tuberculosis in Western Rajasthan

Drug resistance in MTB is alarming because of the limited choice of drugs available for treatment. First line drugs are safe, effective and cheaper than second line drugs. Any resistance in rifampicin or isoniazid will increase the chances of sensitive MTB strain to shift toward MDR or Pre-XDR strains. The male population (70.80%) was more frequently involved than the females (29.2%). The rural population was exposed at higher proportion accounted for 69.8% of cases. Comorbidity tobacco and alcohol consumption was associated in 31.2% and 25.8% of tuberculosis cases. In our study mono resistance in isoniazid was 14.20% which was higher than mono rifampicin resistance of 9.20%. Our findings suggest high resistance to isoniazid alone is alarming as these strains may shift towards MDR, Pre-XDR or XDR strains therefore, furthermore studies should be conducted at a large scale at the community level to estimate the prevalence and prevent this shift of resistant strains.

Identification of S315T mutation in katG gene using probe-free exclusive mismatch primers for a rapid diagnosis of isoniazid-resistant Mycobacterium tuberculosis by real-time loop-mediated isothermal amplification

We developed a LAMP-based technique that employs primers that carry base mismatches to specifically detect the katG (S315T) SNP associated with isoniazid resistance in tuberculosis (TB). Antibiotic resistance in TB is still predominantly determined by the standard drug susceptibility testing that lacks sensitivity, and often requires a weeks-long effort of manual labor and extensive laboratory resources. Advanced real-time PCR techniques are highly efficient for detecting these drug-resistant SNPs, yet their accessibility is limited in TB-endemic regions due to prohibitive instrumental costs and a lack of highly-trained personnel. The isothermal assay described herein was optimized for fluorescence detection on the real-time PCR system. Its ability to discriminate the S315T mutant from wild-type katG without using the melting curve analysis has granted its adaptation into an end-point detection format performed on a hand-held potentiostat device through rapid electrochemical detection. Our technique exhibited the statistical sensitivity and specificity of 100% (95% CI: 98% − 100%) and 96% (95% CI: 87% − 99%), respectively, based on the validation with 218 tuberculosis isolates collected from patients performed under real-time detection mode. The versatility of analysis of this assay has unprecedently allowed the determination of a key INH-resistant associated SNP to be diagnosed without relying on detection probes or the melting curve analysis as often required by standard real-time PCR.

Prevalences of levofloxacin resistance and pncA mutation in isoniazid-resistant Mycobacterium tuberculosis in Hong Kong.

In 2018, the World Health Organization recommended a 6-month treatment regimen that included levofloxacin and pyrazinamide for isoniazid-resistant Mycobacterium tuberculosis without rifampicin resistance (Hr-TB). Susceptibility testing for both drugs is not routinely performed for Hr-TB in Hong Kong. This study examined the prevalences of levofloxacin and pyrazinamide resistances in Hr-TB and explored associated risk factors. All Hr-TB isolates archived during 2018 were retrieved. Isolates were de-duplicated to identify unique cases. Levofloxacin susceptibility testing was performed using the MGIT 960 System; pncA gene sequencing was used as a surrogate indicator of pyrazinamide susceptibility. Previous laboratory records for each case were analysed. In total, 160 phenotypic Hr-TB cases were identified from among 3411 patients with tuberculosis (4.7%). Among these, 157 were analysed, revealing 0.6% (n=1) levofloxacin resistance and 4.5% (n=7) pyrazinamide resistance, respectively. Independent risk factors associated with pncA mutations included history of tuberculosis in the affected patient and isoniazid poly-resistance (ie, double and triple resistances), but not mono-resistance. For Hr-TB in Hong Kong, levofloxacin resistance is rare and pyrazinamide resistance-associated pncA mutations are uncommon. Routine susceptibility testing for these drugs is not indicated unless related risk factors are identified.

An evolutionary functional genomics approach identifies novel candidate regions involved in isoniazid resistance in Mycobacterium tuberculosis

Efforts to eradicate tuberculosis are hampered by the rise and spread of antibiotic resistance. Several large-scale projects have aimed to specifically link clinical mutations to resistance phenotypes, but they were limited in both their explanatory and predictive powers. Here, we combine functional genomics and phylogenetic associations using clinical strain genomes to decipher the architecture of isoniazid resistance and search for new resistance determinants. This approach has allowed us to confirm the main target route of the antibiotic, determine the clinical relevance of redox metabolism as an isoniazid resistance mechanism and identify novel candidate genes harboring resistance mutations in strains with previously unexplained isoniazid resistance. This approach can be useful for characterizing how the tuberculosis bacilli acquire resistance to new antibiotics and how to forestall them.

Diagnostic performance of DNA microarray for detecting rifampicin and isoniazid resistance in Mycobacterium tuberculosis

BackgroundWhile rifampicin (RFP) and isoniazid (INH) are the most commonly used first-line antituberculosis drugs, multidrug resistance in Mycobacterium tuberculosis poses a threat to the success of tuberculosis (TB) control programs. Clinical practice guidelines and expert consensuses recommend drug susceptibility testing (DST) before the initiation of antituberculosis treatment. However, traditional DST is time-consuming and has high requirements for laboratory conditions. The recently developed molecular diagnostic techniques, such as DNA microarray, offer new options. We thus investigated the diagnostic value of DNA microarray in detecting RFP + INH-resistant TB, with an attempt to identify simple, efficient, and accurate drug-resistant TB testing methods.MethodsThe clinical features and DST results of patients diagnosed with pulmonary tuberculosis by Bactec MGIT 960 liquid culture system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) who received DNA microarray analysis in our center from July 2019 to July 2020 were retrospectively analyzed. Level of agreement between liquid culture and DNA microarray technology was assessed by using the Cohen kappa coefficient. With the results of liquid culture as the gold standard, the sensitivity and specificity of the DNA microarray were calculated, and the receiver operating characteristic (ROC) curves were used to assess the diagnostic values of the DNA microarray in detecting RFP + INH-resistant TB.ResultsA total of 825 patients were enrolled. The sensitivity and specificity of DNA microarray were 0.84 and 0.94, respectively, in the detection of RFP resistance, with an area under the curve (AUC) of 0.89 [95% confidence interval (CI): 0.87–0.91)] and a Cohen kappa coefficient of 0.78 (95% CI: 0.72–0.83). For INH resistance, the sensitivity and specificity of the DNA microarray were 0.73 and 0.97, respectively, with an AUC of 0.85 (95% CI: 0.82–0.87) and a Cohen kappa coefficient of 0.75 (95% CI: 0.70–0.80).ConclusionsThe DNA microarray had high specificity and sensitivity in detecting RFP + INH-resistant TB. As a rapid, accurate, and practical technique, it can be routinely performed in clinical laboratories.