Lowenstein-Jensen Research Articles

A STUDY OF IMPACT OF CBNAAT IN THE DIAGNOSIS OF TUBERCULOSIS AT A RURAL BASED TERTIARY CARE TEACHING CENTRE IN INDIA

Introduction: India has the highest number of TB cases in the world and it kills more adults than any other infectious disease. The Whole genome sequencing (WGS) and its utility product the GeneXpert MTB/RIF was adopted as integral part of NTEP for quick diagnosis of MTB with resistance to rifampine. Aim & Objective: To assess the impact of CBNAAT on the other modalities i.e. smear microscopy and conventional culture and sensitivity. Material Method: This is a prospective observational, cohort study conducted at RDGMC Ujjain, MP with an enrollment of 200 cases (150 PTB and 50 EPTB) that underwent all the three investigations to fulfill inclusion criteria after a prior approval of the ethic committee and patients consent. Result: Most the cases were in productive age group of and about 66% cases had low BMI and required nutritional support. The Microscopic sensitivity and specificity were 43% and 100% as against 97% and 90% that of CBNAAT. Discussion: The yield of CBNAAT is superior and promising in smear negative, EPTB and immuno-compromised cases being of pauci bacilli status. A meticulous investigation is required for those cases that are smear positive but CBNAAT negative cases for the presence of NTM infection. A CBNAAT reporting that of very low and indeterminate is to be repeated and supportive/ or additional clinical and or radiologic findings should be incorporated. Conclusion: CBNAAT should be mandatory for the entire suspect TB cases especially for EPTB, children and immune-compromised individuals. CBNAAT has certain limitations i.e. it cannot detect drug resistance other than rifampin and non tubercular mycobacterium (NTM). The best approach for diagnosis of EPTB is to combined CBNAAT, histopathology and AFB culture. However the conventional culture and sensitivity with LJ media is gold standard cannot be over looked.

Evaluation of extracts from used Xpert MTB/RIF cartridges for detection of resistance to second-line anti-tuberculosis drugs in patients with multidrug-resistant tuberculosis in Ethiopia

BackgroundEarly and accurate diagnosis of drug resistance, including resistance to second-line anti-tuberculosis (TB) drugs, is crucial for the effective control and management of pre-extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB). The Xpert MTB/XDR assay is the WHO recommended method for detecting resistance to isoniazid and second-line anti-TB drugs when rifampicin resistance is detected. Currently, the Xpert MTB/XDR assay is not yet implemented in Ethiopia, thus the MTBDRsl assay continues to be used. However, the MTBDRsl assay requires additional patient visits and specimen collection, which can lead to delays in diagnosis and treatment initiation.ObjectiveThis study aimed to evaluate the feasibility of using extracts from used Xpert MTB/RIF cartridges for detecting resistance to second-line anti-TB drugs by MTBDRsl assay in patients with rifampicin resistant-TB (RR-TB) in Eastern and Western Oromia, Ethiopia.MethodsA cross-sectional diagnostic evaluation study was conducted from June 2020 to May 2021 at two TB Referral Laboratories in Eastern and Western Oromia, Ethiopia. Sputum samples from RR-TB patients were split, with one aliquot being subjected for Xpert testing and the other being cultured on Lowenstein-Jensen media. DNA extracted from the used Xpert cartridges was amplified by PCR and tested by MTBDRsl assay, and the results were compared to those obtained from culture isolates. To establish the detection limits, the MTBDRsl assay was performed on cartridge extracts (CEs) from a series of dilutions of drug-susceptible and multidrug-resistant TB strains.ResultsThe MTBDRsl on CEs from dilutions at ≥ 102 CFU/mL (CT ≤ 22) accurately identified susceptibility and resistance patterns for fluoroquinolones (FQL) and second-line injectable drugs (SLIDs). The MTBDRsl on rifampicin-resistant CEs from sputum samples (n = 40) yielded 100% interpretable results for FQL and 90% (4 indeterminate) interpretable results for SLIDs. All interpretable CE results showed complete agreement with the MTBDRsl results from the culture isolates.ConclusionThis study demonstrated the feasibility of using extracts from used Xpert MTB/RIF cartridges for detecting resistance to second-line anti-TB drugs using the MTBDRsl assay. This approach could mitigate the need for additional specimen collection and allow for earlier treatment initiation, potentially improving patient outcomes and reducing the transmission of drug-resistant TB strains.

Genotypes and drug resistance pattern of Mycobacterium tuberculosis complex among clinically diagnosed pulmonary tuberculosis patients.

Clinically diagnosed pulmonary tuberculosis (TB) (CDPTB) patients account for a huge proportion of TB. However, little is known about the genetic diversity and drug resistance profile of Mycobacterium tuberculosis Complex (MTBC) strains in this group of patients. Unmatched case-control study was conducted among 313 PTB patients to compare the genetic diversity of MTBC and their drug resistance profiles among CDPTB (n = 173) and bacteriologically confirmed pulmonary TB (BCPTB) (n = 140) patients. Lowenstein-Jensen (LJ) culture, geneXpert and acid fast staining were performed on sputum specimen collected from both CDPTB and BCPTB patients. Spoligotyping, whole genome sequencing (WGS) and phenotypic drug resistance testing (DST) were done for a subset of LJ grown MTBC isolates. Data was analyzed by STATA version 17 software and a p-value <0.05 were considered statistically significant. The proportion of lineage 3 was larger among CDPTB patients (31%, 13/42) compared to BCPTB patients (15%, 11/74) (p-value <0.05). A higher proportion of MTBC isolates from CDPTB 16.6% (3/18) were phenotypically resistant to one or more anti-TB drugs than BCPTB 12% (4/33) (p-value >0.05). A single lineage 3 strain resistant to all the primary anti-TB drugs was detected in one CDPTB by both DST methods. The observed differences in the genotypes of MTBC isolates between CDPTB and BCPTB patients may be attributed to challenges in the identification of CDPTB that requires further investigation on sequenced genome of the MTBC strains for better understanding and recommendation based on the current finding. There was also primary drug resistant TB among culture positive CDPTB patients which would be otherwise missed by current national protocols.

Clonal Diversity and Spatial Dissemination of Drug-resistant Mycobacterium tuberculosis Complex among HIV Seropositive Patients in Southwest Nigeria

Mycobacterium tuberculosis complex display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. Therefore, this study was undertaken to investigate the clonal diversity and spatial dissemination of drug-resistant M. tuberculosis complex among HIV seropositive clients in Southwest Nigeria. A total of two-hundred and seventeen (217) consented HIV positive (case subjects) and fifty (50) non-HIV positive controls were recruited for this study. Two Sputum samples were collected from each participant in two consecutive days. The samples were divided into two parts: one part was decontaminated using NAOH-NALC method and aseptically cultured for Mycobacteria on Lowenstein-Jensen (LJ) medium with and without pyruvate. Mycobacterial isolates were characterized using conventional and molecular methods. Anti-TB drug sensitivity test was done using 1% proportion conventional method on LJ. MTBC isolates were sequenced using Genetic Analyzer 3130xl sequencer. Genetic relatedness and clonal diversity (phylogenetic analysis) of MTBC isolates were done using Bio- Edit software and MEGA 6. Out of 217 HIV positive participants, 105(48.4%) were male, while 112(51.6%) were female. Twenty-six (26(52.0%)) of the control subjects were male, while 24(48.0%) were females. The overall prevalence of TB among PLHIV was found to be 19.1%. The prevalence of NTM among PLHIV was found to be 2.8% with M. intracellulare being the predominant organism isolated. Participants’ age was found to be significantly associated with TB (P&lt;0.05), however there was no significant association between TB and sex of the participants (P&lt;0.05). The prevalence of culture confirmed drug-resistant TB (DR-TB) among PLHIV was found to be 11.9%, with Rifampicin having the highest monoresistance rate of 5.5%. The overall prevalence of MDR-TB among PLHIV was found to be 9.2%. The phylogenetic analysis of M. tuberculosis strains revealed same clone of all isolates. All PLHIV should be screened for drug-resistant TB especially MDR-TB as the prevalence of primary MDR-TB is high among them. Further study like 3R gene-based studies of adaptation and evolution is needed to facilitate further epidemiological studies of these bacteria.

An Etiology of Sputum Culture Contamination and their Drugs Susceptibility Patterns among TB-suspected Patients at Epicentre Mbarara Research Centre, Uganda

Aims: Contamination in TB cultures is problematic as it allows the growth of non-target bacterial or fungal species in sputum samples, potentially concealing M. tuberculosis. This study aimed to determine the prevalence, characterization, and drug susceptibility patterns of contaminants in sputum cultures from TB-suspected patients. Methods: A laboratory-based cross-sectional study was conducted from October 2023 to June 2024 at the Epicentre Mbarara Research Centre, involving 81 sputum samples from TB-suspected patients. Conventional decontamination procedures were used, and both solid Lowenstein Jensen (LJ) and liquid Mycobacterium Growth Indicator Tube (MGIT) culture methods were applied. Standard culture techniques and physicochemical analyses were employed. Bacteria were incubated at 37°C in O2 and Co2 incubators, with growth observed within 24 to 48 hours and up to three days for yeast contaminants. Sabouraud Dextrose Agar cultures were monitored for two weeks for filamentous fungi. Antibiograms for bacterial isolates were determined using the Kirby-Bauer, while broth microdilution was used for fungal isolates. Results: Of 81 samples, 14(17.28%) were contaminated. The most common bacterial contaminants were Staphylococcus aureus, Bacillus subtilis, Streptococcus viridans, Staphylococcus epidermidis, and Escherichia coli. Fungal contaminants included Aspergillus flavus, Penicillium species, Candida albicans, Cryptococcus neoformans, and non-albican Candida species. High resistance to erythromycin (73%) and tetracycline (50%) was noted among bacterial isolates, with sensitivity to vancomycin (67%), gentamycin (60%), and chloramphenicol (58%). Yeast isolates showed (87.5%) susceptibility to itraconazole and (75%) to amphotericin B but (50%) resistance to 5-flucytosine. Mold isolates exhibited (100%) susceptibility to itraconazole, amphotericin B, and 5-flucytosine but complete resistance to caspofungin. Conclusion: The level of contamination in TB cultures was noted, and Staphylococcus aureus and Candida albicans were observed as the most common contaminants. Bacterial isolates resisted erythromycin and tetracycline, while fungal isolates were generally susceptible to itraconazole and amphotericin B. We recommend Strengthening decontamination and monitoring common resistant contaminants.

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.

Genome sequencing analysis of the pncA, rpsA and panD genes responsible for pyrazinamide resistance of Mycobacterium tuberculosis from Indonesian isolates.

Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results. Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD. The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile. Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias.

Multicenter study of the performance of NTM Elite agar for the detection of nontuberculous mycobacteria from patients with cystic fibrosis.

The performance of a novel selective agar was evaluated against the performance of conventional mycobacterial cultures, i.e., a combination of the mycobacterial growth indicator tube (MGIT) with Löwenstein-Jensen (LJ), for the detection of nontuberculous mycobacteria (NTM) in sputum samples from people with cystic fibrosis (pwCF). Two hundred eighty-three sputum samples (231 fresh sputum and 52 spiked sputum) from 143 pwCF were collected. They were inoculated without prior decontamination on NTM Elite agar (30°C ± 2°C for 28 days) and inoculated on both MGIT and LJ (35°C-37°C for 6-8 weeks) after N-acetyl-L-cysteine-2% sodium hydroxide decontamination. NTM were identified by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry and/or PCR, and whole-genome sequencing. A total of 67 NTM were recovered overall by the combination of all culture media. NTM Elite agar allowed the recovery of 65 NTM (97%), compared to 22 for the conventional MGIT and LJ media combination (32.8%), including 22 NTM for MGIT (32.8%) and 3 NTM with the LJ medium (4.5%). For Mycobacterium abscessus complex, the sensitivity of NTM Elite agar was 95% compared with a sensitivity of 30% for the conventional MGIT and LJ media combination. Overall, 17.3% of cultures on NTM Elite agar were contaminated with other micro-organisms vs 46.3% on MGIT and 77% on LJ. This study shows that the novel selective agar (NTM Elite agar) significantly outperforms the conventional MGIT and LJ media combination in terms of sensitivity, selectivity, and ease of culture, without the requirement of an L3 laboratory.IMPORTANCENontuberculous mycobacteria (NTM) are significant pulmonary pathogens in patients with pre-existing structural lung conditions such as cystic fibrosis, bronchiectasis, or chronic obstructive pulmonary disease. Mycobacterium avium complex and Mycobacterium abscessus complex (MABSC) are the most frequently isolated organisms. Compared to the recommended culture method for NTM, which combines solid and liquid culture media, NTM Elite agar enables a faster/easier diagnosis and speeds up identification and susceptibility testing as the final reading is at 28 days instead of 6-8 weeks for the conventional mycobacterial cultures. In addition, for the NTM Elite agar, no decontamination stage before inoculation is necessary, unlike the conventional mycobacterial cultures. NTM Elite agar is derived from a formulation of medium adapted to rapidly growing mycobacteria (RGM). The medium enables the growth of RGM while suppressing other flora. It is supported with published clinical data showing the benefits of this medium.

Distribution of nontuberculous mycobacteria in dental unit waterlines: A potential health hazard in the dental office

BackgroundIt is essential to control the microbiology of dental unit water lines (DUWs) to prevent the spread of nontuberculous mycobacteria (NTM) and associated oral diseases. Therefore, the objective of this study was to quantify the presence of NTM in the water of 112 DUWs from dental centers and 57 DUWs from individual dental offices in Tehran, Iran. MethodsA total of 169 water samples were collected from DUWs. After filtration through a 0.45 μm membrane, the samples were decontaminated with 0.005 % cetylpyridinium chloride and then cultured on two Lowenstein-Jensen media, incubated at 25 °C and 37 °C for 8 weeks. Positive cultures for mycobacteria were analyzed using phenotypic tests, and the NTM species were identified through 16S rDNA, rpoB, and hsp65 genes analysis. Drug resistance was also assessed. ResultsOf the total isolates, 38 (34.5 %) were classified as slow-growing mycobacteria (SGM), while 72 (65.5 %) were categorized as rapid-growing mycobacteria (RGM). NTM isolates were identified using molecular tests, including M. chelonae, M. abscessus, M. lentiflavum, M. mucogenicum, M. fortuitum, M. kansasii, M. simiae, M. gordonae, M. conceptionense, M. phocaicum, M. porcinum, and M. aurum. The NTM counts ranged from 50 to >500 CFU/500 mL across these 188 samples, with a median of 350 CFU/500 mL. Additionally, we reported two cases of intraoral infection caused by M. abscessus and M. chelonae, where the source of infection was traced to NTM-contaminated DUWs. ConclusionsThe study found that most DUWs contained water contaminated with NTM, posing a potential health risk to humans. This research underscores the necessity of stringent quality control and certification of DUW water, with particular emphasis on ensuring the absence of NTM.

Establishment of cross-priming amplification for point-of-care detection of Mycobacterium tuberculosis and non-tuberculosis mycobacteria

ObjectiveTo simplify sputum sample preparation steps and achieve point-of-care testing (POCT) of Mycobacterium tuberculosis (MTB) and non-tuberculosis mycobacteria (NTM) using Cross-Priming Amplification (CPA) technology on portable devices, overcoming the challenges of existing nucleic acid detection technologies that cannot be widely promoted in grassroots settings in China. MethodsEvaluate the liquefying ability of high-concentration guanidine thiocyanate (GTC) for sputum and the effectiveness of MTB inactivation; establish a rapid detection system for MTB and NTM based on CPA technology using EasyNAT integrated detection tubes, with the left amplification zone specific to MTB CPA amplification and the right amplification zone specific to both MTB and NTM CPA amplification. Suspected pulmonary tuberculosis (PTB) patients or patients diagnosed as suspected NTM pulmonary infections specimens collected from the Second Hospital of Longyan, Fujian Province, from September 2022 to September 2023, acid-fast bacilli (AFB) smear, quantitative real-time PCR (RT-PCR) and CPA-POCT were performed. The kappa coefficients was used to evaluate the consistency between the RT-PCR and CPA-POCT. ResultsThe liquefaction effect of 6M GTC on sputum was equivalent to 4 % NaOH, and no MTB growth was observed in the Lowenstein-Jensen medium of sputum samples treated with 6M GTC incorporating the H37Rv strain. The newly established CPA-POCT method showed good agreement with RT-PCR with a positive compliance rate of 86.27 %, a negative compliance rate of 89.36 %, an overall compliance rate of 87.75 %, and a Kappa coefficients of 0.786 (P < 0.05). Conclusion6M GTC can liquefy sputum and render MTB non-viable, eliminating the need for Mycobacterium nucleic acid testing in BSL-2 laboratories; the newly established CPA method can rapidly and accurately distinguish MTB and NTM in the form of POCT, with simple and fast operation, suitable for promotion and application in grassroots medical institutions and remote rural areas.

Evaluation of mycobacterial microscopy and culture results from clinical samples: A five-year analysis.

Ehrlich-Ziehl-Neelsen (EZN) staining and culture methods are often used to diagnose tuberculosis. This study aimed to determine the acidfast bacteria (AFS) positivity rates in various clinical samples sent to our laboratory over five years and the growth and resistance rates in two different (solid and liquid) cultures and compare them with the data from Türkiye and the world. A total of 62.456 clinic samples were accepted in the microbiology laboratory between 2019 and 2024. The mycobacterial culture was performed by searching for acid-resistant bacilli microscopically and parallel inoculation media [solid Löwenstein-Jensen (L-J) and MGIT 960 liquid]. Those growing in the MGIT 960 system were identified using BD MGIT TBC Identification test kits that detect the MPT64 antigen. AFS and MPT64 antigenpositive samples were identified as Mycobacterium tuberculosis complex (MTBC) while AFS-positive samples and MPT64 antigen-negative results were classified as non-tuberculous mycobacteria (NTM). Drug susceptibility testing was performed with the BACTEC MGIT 960 SIRE kit. Susceptibility to NTM samples was not performed. Out of a total of 120.829 samples, 95.101 were lung samples and 25.728 were extrapulmonary samples. AFS positivity was detected in 2961 (2.4%) samples. MTBC grew in 6854 (5.6%) samples, and NTM grew in 1506 (1.24%) samples. Contamination was detected in 7171 (5.9%) media. Two thousand one hundred and sixty-nine susceptibility tests were performed. Considering antibiotic resistance rates, isoniazid resistance was detected in 154 (7%), rifampicin resistance in 140 (6.4%), ethambutol resistance in 18 (0.8%), and streptomycin resistance in 120 (0.5%) samples. All four-drug resistance was observed in 91 (4.1%) samples. AFP positivity and resistance rates for rifampicin have decreased significantly, while there have been no significant changes in NTM rates over the years. When our data was determined, the sensitivity of microscopy was low. It is understood that mycobacterial culture and microscopy must be evaluated together to exclude tuberculosis infection. The high mycobacterial culture positivity rate, which is 5.6%, is due to the high number of follow-up patients and new referrals. It is seen that the change in sensitivity rates is due to the period of the COVID-19 epidemic, and it is similar to World Health Organization (WHO) data.

Evaluation of Newly Modified Potassium Nitrate Containing Media in Detection and Antibiogram of Mycobacterium tuberculosis

Background: Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and the classic Hansen's strain of leprosy (Mycobacterium leprae) [1]. Tuberculosis is the most common life-threatening opportunistic infection (OI) and AIDS defining illness in developing countries [2]. The routine identification methods of tuberculosis includes, Tuberculin skin test, Chest X-ray, Growth and physical characteristics of the bacteria, automated based system such as BACTEC 460 TB method, Mycobacterium growth indicator tube (MGIT) method, MB/BacT system, as well as molecular based techniques as in Amplification of nucleic acid for detection of Mycobacterium tuberculosis [3-6]. Objectives: To evaluate the validity of newly modified potassium nitrate containing medium (NMK) for rapid detection and antibiogram of M tuberculosis. Material and method: This was a health facility based observational diagnostics retrospective study. 125 sputum specimens were investigated to evaluate the validity of newly modified potassium nitrate containing medium for rapid detection and antibiogram of M tuberculosis. Results: 83/125 (66.4%) showed growth after 5-9 days with a mean of 6.58± 0.977 days NMK - containing medium. Conclusions: The NMK - containing medium showed promising results, it showed a positive growth sings and susceptibility results within 5-9 days of inoculation showing much superior than the golden standard LJ medium.

Unveiling the complexity of rifampicin drug susceptibility testing in Mycobacterium tuberculosis: comparative analysis with next-generation sequencing.

Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment.Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms.Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding.Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role.Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB, including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases.Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.

Xpert MTB/RIF assay in the diagnosis of pulmonary tuberculosis in children in tertiary care setting in South India.

Xpert MTB/RIF is recommended for the diagnosis of tuberculosis (TB) in children. We determined the performance of Xpert MTB/RIF in the diagnosis of pulmonary TB in children. The characteristics of children influencing Xpert MTB/RIF positivity were explored. Children aged <15 years with symptoms suggestive of pulmonary TB were prospectively enrolled from 2013 to 2019. Two sputum/early morning gastric aspirate specimens were collected for examination by smear (fluorescence microscopy), Xpert MTB/RIF, and culture [Mycobacteria growth indicator tube (MGIT)/Lowenstein-Jensen (LJ) medium]. Diagnostic performance of Xpert MTB/RIF was evaluated using LJ and or MGIT culture positivity as the reference standard. Sensitivity, specificity with 95% confidence interval (CI) were calculated. Stratified analysis was done; P < .05 was considered statistically significant. Of the total 1727 enrolled children, 1674 (97%) with complete results for at least one sputum/gastric aspirate sample were analyzed. The sensitivity of Xpert MTB/RIF was 68.5% in sputum and 53.6% in gastric aspirate while the specificity was 99% for both. The sensitivity compared to smear was 68.5% vs. 33.7% (P < .001) and 53.6% vs. 14.5%; (P < .001) in sputum and gastric aspirate, respectively. The sensitivity of Xpert MTB/RIF was 23.9% with decision to treat as reference standard. Xpert MTB/RIF positivity was significantly influenced by sex, age, nutritional status, chest X-ray abnormality, TB infection status, and symptoms suggestive of TB. Xpert MTB/RIF as an upfront test compared to smear improves diagnosis of pulmonary TB in children yet the sensitivity is suboptimal. Newer TB diagnostic tools with improved sensitivity is warranted in children.

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

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

Mycobacterium tuberculosis Drug Resistance and Evaluation of the Current Situation

Aim: We aimed to determine the antituberculosis drug susceptibility status in Mycobacterium tuberculosis complex isolates, considering that current drug resistance rates will be an important indicator of the prevalence of primary drug resistance in the future. Material and Methods: All cases whose culture sample was taken with clinical suspicion of tuberculosis at Samsun Training and Research Hospital in the period between January 2018 and December 2023 and who had a positive result in at least one of the Acid-fast stain (AFS) and culture methods were included in the study. Results: The average age of the patients was 54.5±18.5 (range: 17-93) and 398 (74.1%) were male. In the study, 77.3% of the samples were sputum and 15.8% were bronchoalveolar lavage. 474 (88.3%) of the isolates were M. tuberculosis complex. 49% of the samples were positive with the AFS method, 96.5% with Mycobacteria Growth Indicator Tube (MGIT), and 84.5% with Löwenstein-Jensen (LJ) medium. The resistance rates in the isolates were 10.6%, 2.8%, 1.1% and 7.0% for isoniazid, rifampicin, ethambutol and streptomycin, respectively. All isolates resistant to rifampicin were also resistant to isoniazid. The rate of multidrug-resistant isolates was found to be 2.8%. The single drug resistance rate was found to be 14.0%. It was determined that the resistance rates before the pandemic were significantly higher than during the pandemic period. Conclusion: The resistance rates to isoniazid and streptomycin in M. tuberculosis complex isolates were around 10% and that the general resistance rates to primary anti-tuberculosis drugs decreased significantly during the pandemic period.

Comparison of Isolation of Mycobacterium tuberculosis from various clinical samples using liquidculture (MGIT) and PCR targeting IS6110 gene

Tuberculosis, one of the oldest recorded human afflictions, still one of the biggest killers among the infectious diseases, despite the worldwide use of a live attenuated vaccine and several antibiotics. The Aim of this study was to isolate Mycobacterium tuberculosis from various clinical samples using liquid culture (MGIT) and confirm the growth as MTB using Polymerase chain reaction (PCR).Material and Methods: All 85 samples were received from clinically suspected cases of pulmonary and extrapulmonary tuberculosis from the various clinical departments of SGRRIM&amp;HS, Dehradun over a period of one year. All samples were processed by ZN staining, culture in LJ medium &amp; MGIT, rapid card (BD MGIT TBcID) test and PCR done by using IS6110gene.Result: Out of total 85 samples, 35 (41%) came out to be positive by PCR and 50 (59%) cases were found to be negative. Correlation of ZN smear with MGIT culture: 2 cases (2%) were positive in both ZN stain and MGIT culture but 24 (28%) cases showed positivity in MGIT culture. Correlation of MGIT culture with PCR: 24 cases (28%) showed positivity in both MGIT culture and PCR.11 cases (13%) showed positivity in PCR but negative in MGIT culture. The positivity rate was 28% (24/85) in MGIT culture &amp; 41% (35/85) in PCR.Conclusion: Our study clearly reflects that PCR finding are more positive as compared to MGIT and ZN staining. Because of high specificity and sensitivity of PCR few MTB DNA can be easily detected which is not possible in MGIT and ZN staining. Techniques like PCR is highly valuable for detection of Mycobacterium tuberculosis but the limitation is that it can’t differentiate between live and dead bacteria.

Risk factors associated with multidrug-resistant tuberculosis in areas with a moderate tuberculosis burden.

The emergence of multidrug-resistant tuberculosis (MDR-TB) or rifampicin-resistant (RR) TB poses a significant challenge for TB control initiatives on a global scale. This study's aim was to estimate the incidence of MDR-/RR-TB and identify the risk factors associated with their incidence in four provinces in northern Iran. Drug susceptibility testing was conducted using the proportion method on Lowenstein-Jensen media. The demographic and clinical data were collected from the Iranian TB registry. Among 1083 individuals diagnosed with TB, 27 (2.5%) were identified as having MDR-/RR-TB, while 73 cases (6.7%) were any drug resistant (ADR). The statistical analysis revealed a significant association between marital status and MDR-/RR-TB (p=0.003). In addition, significant associations were observed between ADR-TB and gender (p=0.035) and previous treatment for TB (p=0.02). Our findings provide important information on the drug resistance pattern of Mycobacterium tuberculosis strains, as well as risk factors in northern Iran. Given the identified risk factors, creative approaches to promote treatment adherence in TB patients, particularly divorced/widowed women and individuals with a previous history of TB treatment, are required.

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) ).

QMAC-DST for Rapid Detection of Drug Resistance in Pulmonary Tuberculosis Patients: A Multicenter Pre-Post Comparative Study.

Background/Objectives: This study explores the impact of QMAC-DST, a rapid, fully automated phenotypic drug susceptibility test (pDST), on the treatment of tuberculosis (TB) patients. Methods: This pre-post comparative study, respectively, included pulmonary TB patients who began TB treatment between 1 December 2020 and 31 October 2021 (pre-period; pDST using the Löwenstein-Jensen (LJ) DST (M-kit DST)) and between 1 November 2021 and 30 September 2022 (post-period; pDST using the QMAC-DST) in five university-affiliated tertiary care hospitals in South Korea. We compared the turnaround times (TATs) of pDSTs and the time to appropriate treatment for patients whose anti-TB drugs were changed based on these tests between the groups. All patients were permitted to use molecular DSTs (mDSTs). Results: A total of 182 patients (135 in the M-kit DST group and 47 in the QMAC-DST group) were included. The median TAT was 36 days for M-kit DST (interquartile range (IQR), 30-39) and 12 days for QMAC-DST (IQR, 9-15), with the latter being significantly shorter (p < 0.001). Of the total patients, 10 (5.5%) changed their anti-TB drugs based on the mDST or pDST results after initiating TB treatment (8 in the M-kit DST group and 2 in the QMAC-DST group). In the M-kit DST group, three (37.5%) patients changed anti-TB drugs based on the pDST results. In the QMAC-DST group, all changes were due to mDST results; therefore, calculating the time to appropriate treatment for patients whose anti-TB drugs were changed based on pDST results was not feasible. In the QMAC-DST group, 46.8% of patients underwent the first-line line probe assay compared to 100.0% in the M-kit DST group (p < 0.001), indicating that rapid QMAC-DST results provide quicker assurance of the ongoing treatment by confirming susceptibility to the current anti-TB drugs. Conclusions: QMAC-DST delivers pDST results more rapidly than LJ-DST, ensuring faster confirmation for the current treatment regimen.