MGIT System Research Articles

Use of BD BACTEC™ MGIT™ for the detection of non-tuberculous mycobacteria in sanitary water samples.

The most commonly used method for the detection of non-tuberculous mycobacteria (NTM) is culture in BD BACTEC™ MGIT™ Mycobacteria Growth Indicator Tubes incubated in an automated growth detection reader BD BACTEC™ MGIT™ 960 Instrument. The system is currently validated for the detection of mycobacteria from clinical specimens but not environmental matrices. From November 2018 to December 2023, 1,369 sanitary water samples from 92 heater-cooler units (HCUs) and 747 sanitary water samples from 489 haemodialysis instruments (dialysis) were concentrated, decontaminated, and cultured on MGIT and solid Lowenstein-Jensen media to evaluate the presence of NTM. NTM-positive cultures (n = 261 HCUs and n = 20 dialysis) were purified by Middlebrook 7H11 agar plate subcultures and identified by MALDI-TOF mass spectrometry technology. The purpose of this study was to evaluate the accuracy and reproducibility of the MGIT system on sanitary water from HCU and dialysis, using the two strains most frequently isolated on these devices as sources of NTM during the Emilia- Romagna surveillance programme: M. chimaera (79%) and M. saskatchewanense (100%), respectively. To evaluate the accuracy, sanitary water was spiked with M. chimaera and M. saskatchewanense at the theoretical concentrations of 100 and 10 CFU/mL, and all resulted positive in MGIT tubes. No significant changes in time to positivity were observed when MGIT tubes were inoculated with NTM at the theoretical concentrations of 10 and 100 CFU/mL on 3 consecutive days, indicating that the detection method is reproducible. The MGIT system is suitable for detecting the presence of NTM in sanitary water samples as it was capable of detecting up to 4 CFU/mL for both M. chimaera and M. saskatchewanense. Our results indicate that the MGIT system can be used for NTM detection not only for clinical samples but also for environmental matrices.

The impact of physiological state and environmental stress on bacterial load estimation methodologies for Mycobacterium tuberculosis

When processed in solid or liquid medium, tuberculosis patient samples yield different proportions of a heterogenous bacterial community over the duration of treatment. We aimed to derive a relationship between methodologies for bacterial load determination and assess the effect of the growth phase of the parent culture and its exposure to stress on the results. Mycobacterium tuberculosis H37Rv was grown with and without antibiotic (isoniazid or rifampicin) and sampled on day 0, 3, 11 and 21 of growth in broth culture. The bacterial load was estimated by colony counts and the BD BACTEC MGIT system. Linear and nonlinear mixed-effects models were used to describe the relationship between time-to-positivity (TTP) and time-to-growth (TTG) versus colony forming units (CFU), and growth units (GU) versus incubation time in MGIT. For samples with the same CFU, antibiotic-treated and stationary phase cells had a shorter TTP than antibiotic-free controls and early-logarithmic phase cells, respectively. Similarly, stationary phase samples reached higher GUs and had shorter TTG than early-log phase ones. This suggests that there is a population of bacterial cells that can be differentially recovered in liquid medium, giving us insight into the physiological states of the original culture, aiding the interpretation of clinical trial outputs.

A stochastic model for the bacterial growth exhibiting staged growth, desynchronization, saturation and persistence

We consider a model of population growth based on the stochastic variation of the population size-controlled duplication of bacterial cells. It is shown that the proper choice of the control function allows for reproducing a variety of regimes: a logistic growth with saturation, a hindered growth typical for persistent bacterial systems, and a linear population growth detected for some mycobacterial populations. When supplied with the rectangular function having the width equal to the generation time, this approach represents the solution generalizing Rubinow’s age-maturity model reproducing systems with desynchronisation and saturation. The model’s plausibility is confirmed by the direct comparison with real data for the growth of M. tuberculosis populations obtained with the BACTEC MGIT system under different conditions of growth synchronisation.

Rapid lateral flow test for Mycobacterium tuberculosis complex and non-tuberculous mycobacteria differentiation

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases.Key points• Panel of mAbs for differentiating MTB versus NTM• IC strips for diagnosing MTBC and NTM after the BACTEC MGIT• Combined detection of MTP64 and Ag85B enhances diagnostic accuracy

Evaluating the efficacy of whole genome sequencing in predicting susceptibility profiles for first-line antituberculosis drugs

ObjectivesThis study aimed to examine the efficacy of whole genome sequencing (WGS) in accurately predicting susceptibility profiles, potentially eliminating the need for conventional phenotypic drug susceptibility testing (pDST) for first-line antituberculosis drugs in routine tuberculosis diagnosis. MethodsOver the period of 2017 to 2020, 1114 Mycobacterium tuberculosis complex isolates were collected with drug susceptibility testing conducted using the MGIT960 system and WGS performed for predicting drug resistance profiles. In addition, we implemented a new algorithm with an updated WGS workflow, omitting pan-susceptible strains from pDST. ResultsResults showed that out of 1075 analysed isolates, WGS-based genotypic sensitivity predictions for isoniazid, rifampicin, ethambutol, and pyrazinamide were 100% (95% CI, 99.6–100%), 100% (95% CI, 99.62–100%), 99.8% (95% CI, 99.26–99.94%), and 100% (95% CI, 99.63–100%), respectively. In contrast, the WGS-based genotypic resistance prediction, was 98.85% (95% CI, 93.77–99.79%) for isoniazid, 94.74% (95% CI, 82.71–98.54%) for rifampicin, 86.96% (95% CI, 67.87–95.46%) for ethambutol, and 75.7% (95% CI, 59.9–86.63%) for pyrazinamide. Moreover, WGS enabled the implementation of a new testing algorithm that made it unnecessary to perform pDST in 954 of all 1075 samples (88.7%) and in 890 of 901 pan-susceptible samples (98.8%). DiscussionIntegrating WGS into tuberculosis management offers significant potential to replace phenotypic drug susceptibility testing, especially for problematic drugs like pyrazinamide and ethambutol, potentially improving treatment outcomes.

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.

Analysis of Mycobacterium tuberculosis Complex Strains Isolated from the Samples of Patients Living in Northern Syria

Tuberculosis causes serious mortality and morbidity worldwide each year. A lot of effort and money is spent for the diagnosis and treatment of tuberculosis all over the world. The importance that countries give to health policies and public health is inversely proportional to the incidence of tuberculosis and multidrug resistant tuberculosis. The aim of our study was to evaluate the resistance profiles of Mycobacterium tuberculosis complex strains which were isolated from sputum samples, collected by World Health Organisation from patients living in the northern region of Syria, where health services were disrupted due to the civil war. According to the protocol signed between the World Health Organization and our hospital; sputum samples taken from tuberculosis patients living in Afrin, Azez and Idlib regions or suspected of being resistant to anti-tuberculosis drugs were studied in our hospital. The cultivation process was performed in our laboratory using Löwenstein Jensen media and MGIT-960 system. The susceptibility tests for primary anti-tuberculosis drugs were performed using MGIT-960 system for M.tuberculosis complex isolates. The isolates identified as MDR/RD-TB (multi-drug-resistant-rifampicin-resistant tuberculosis) were sent to National Tuberculosis Reference Laboratory of Public Health Institution of Türkiye for susceptibility testing to first and second line drugs. Mutation and wild-type determination were studied by "Line Probe Assay (LPA)" method to investigate the susceptibility of the isolates to isoniazid, rifampicin, fluoroquinolone and aminoglycoside/cyclic peptide. The results obtained from the patients were collected and evaluated retrospectively from the records. Growth was observed in 18 samples out of 171 sputum samples from 67 patients; 13 isolates were detected as MDR-TB while one isolate was detected as mono RR-TB. The rate of mono RR-TB was 1.5% and the rate of MDR-TB was 19.4%. MUT3 causing rifampicin resistance was detected in 17.9% of the patients, katG/MUT1 causing isoniazid resistance in 17.9% and WT loss causing aminoglycoside/cyclic peptide resistance were detected in 19.4% of the patients. Neither fluoroquinolone resistance nor a mutation leading to fluoroquinolone resistance was detected in the study. When the sputum samples taken from the patients living in Northern Syria were examined, the frequency of MDR-TB was found to be quite high. MDR-TB, which is an important public health problem, was found at high rates due to the internal turmoil in the region and poor accessibility to health services. Since the gene mutations causing drug resistance with the LPA method differ with the conducted studies, it is important to evaluate the dominant gene mutations for determining the TB treatment strategies in the region.

ГЕНОТИПІЧНА ТА ФЕНОТИПІЧНА ХАРАКТЕРИСТИКА СТІЙКОСТІ ДО АНТИМІКОБАКТЕРІАЛЬНИХ ПРЕПАРАТІВ 8 ШТАМІВ M. TUBERCULOSIS, ВИДІЛЕНИХ ВІД ХВОРИХ З ВПЕРШЕ ДІАГНОСТОВАНИМ ТУБЕРКУЛЬОЗОМ ЛЕГЕНЬ

The article presents the experience of whole-genome sequencing of M. tuberculosis (MBT) strains. Materials and methods. 8 MBT isolates were tested. The strains were obtained from patients with newly detected pulmonary tuberculosis (NDPTB). Critical concentrations of anti-mycobacterial drugs (AMBD): H; R; E; Z; Lfx; Mfx; Am; cm; Lzd; Bdq; Dlm and Cfz were determined using the BACTEC MGIT system. Sequencing of MBT strains was performed on Illumina MiSeq equipment at the laboratory of microbiology and biochemistry of the National Institute of phthisiology and pulmonology (NIPP). The obtained whole-genome sequences of MBT strains were deposited in fastq files and analyzed using the PhyResSE program (https://bioinf.fz-borstel.de/mchips/phyresse/) with the bioanalytical solution package. The panel of this package includes gene targets involved in the development of MBT to AMBP. Phylogenetic information and a certain spoligotype make it possible to obtain neutral single nucleotide polymorphisms. The genetic profile of the drug was performed according to the WHO mutation catalog (https://www.who.int/publications/i/item/9789240028173). The results. The dominant in Ukraine Beijing genotype was represented by 7 strains. The non-beijing group included 1 strain: Ural. 7 MBT strains belonged to the Beijing family, Lineage 2 phylogenetic line with the same spoligotype indices. The analysis of mutations according to the “Catalogue of mutations of the M. tuberculosis complex and their association with drug resistance”, WHO, 2021, allowed all detected mutations to be divided into categories - associated with resistance, associated with resistance - intermediate, not associated with resistance, with uncertain significance. Comparison of the resistance profile with the corresponding genotype showed that isolates, belonging to the Beijing genotype, usually carried MDR/pre-XDR mutations. Comparison of the resistance profile with the corresponding genotype showed that isolates belonging to the Beijing genotype usually carried MDR/pre-XDR mutations. All MBT strains with resistance to R had mutations found in the rpoB gene. The canonical amino acid substitution of rpoB S450L, associated with a high level of resistance to R with a non-significant decrease in the viability of the strains, was identified in 6 isolates. Another clinically significant mutation in rpoB (S435Y) was found in 2 cases and only in representatives of the Beijing family. Mutations of the categories: not associated with resistance (rpoC (E1092D)) and of uncertain significance: rpoC (I491V), rpoB (H674N), rpoB (V496A), were detected only in combination with “strictly associated” positions in phenotypic-resistant isolates of the genotype Beijing. The canonical single amino acid substitution katG S315T was detected in all isolates selected for sequencing. All 8 strains resistant to H showed mutations in genes associated with resistance to the drug. Mutations in the key Bdq resistance genes - atpE and mmpR - were not detected in any genome. In 1 strain of MBT, a C154R mutation associated with Lzd resistance was detected in the rplC gene. Similarly, in 1 strain of MBT, a mutation in ddn (D30S0) was detected with an as yet undetermined significance of association with resistance to Dlm. Conclusions. The study provided information on the genetic diversity of modern MBT isolates in TB patients. The predominance of the Beijing genotype testified to the stable population structure of the TB pathogen and the preservation of trends of high levels of the spread of TB forms of infection on the territory of the country. The combination of phenotype and molecular genetic data made it possible to give a more complete assessment of DR isolates. A large share of MDR was caused by mutations that did not have a negative effect on the viability and transmissibility of the pathogen, indicating a large epidemic reservoir of MBT strains with MDR/pre-XDR. The highly transmissible strains of Beijing carried mostly single uncombined R and H mutations, which were associated with a high level of resistance. Key words: genetic diagnostics, sequencing, pulmonary tuberculosis.

Correlating clinical breakpoint concentration of moxifloxacin with gyrA mutations using the GenoType MTBDRsl assay Version 2.0

IntroductionWidespread use of Fluoroquinolones (FQs) has led to the development of its resistance in clinical isolates of Mycobacterium tuberculosis. However, in Mycobacterium tuberculosis, phenotypic resistance to FQs has been shown to be heterogeneous, ranging from low-level resistance to high-level resistance. This stratification in resistance has important implications for the inclusion of moxifloxacin (Mfx) in the treatment regimen. The World Health Organization recommends the use of GenoType MTBDRsl assay as the initial test for detecting resistance conferring mutations (both high and low) to FQs in patients with confirmed MDR-RR TB. The present study was conducted to explore the relationship of MTBDRsl Version 2.0 detected mutations in gyrA gene and genotypic DST of Mfx at WHO defined Clinical Breakpoint (CB). Materials and methodsA total of 200 sputum samples from Confirmed MDR/RR TB patients were included in this study. All of these samples had mutations conferring resistance to FQ confirmed by GenoType MTBDRsl assay. These samples were further subjected to Phenotypic DST against moxifloxacin using the Bactec MGIT-960 system. ResultsAll of the 200 representative FQ resistant isolates had mutations in gyrA gene only with no detectable mutation in gyrB gene. 109 (54.5%) of the isolates had mutations associated with high-level increase in MIC while 91 (45.5%) isolates had mutations associated with low-level increase in MIC. Phenotypic DST of these 200 isolates against Mfx at CB (1.0μg/ml) revealed that of the 109 isolates with mutations associated with high-level increase in MIC and expected to be resistant at CB, only 34 (31.2%) were resistant and the remaining 75 (68.8%) were sensitive. ConclusionMoxifloxacin is an important drug in the regimen for treating Drug-resistant TB and the decision to exclude this drug from the regimen should not be taken merely on the basis of mutational patterns. It should rather be taken after considering the combined results of mutational analysis and phenotypic DST.

Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium.

The Mycobacterium avium (Mav) complex accounts for more than 80% of all pulmonary diseases caused by non-tuberculous mycobacteria (NTM) infections, which have an alarming increase in prevalence and vary in different regions, currently reaching 0.3–9.8 per 100,000 individuals. Poor clinical outcomes, as a result of increasing microbial drug resistance and low treatment adherence due to drug-toxicities, emphasize the need for more effective treatments. Identification of more effective treatments, however, appears to be difficult, which may be due to the intracellular life of NTM and concomitant altered drug sensitivity that is not taken into account using traditional drug susceptibility testing screenings. We therefore developed human cell-based in vitro Mav infection models using the human MelJuSo cell line as well as primary human macrophages and a fluorescently labeled Mav strain. By testing a range of multiplicity of infection (MOI) and using flow cytometry and colony-forming unit (CFU) analysis, we found that an MOI of 10 was the most suitable for Mav infection in primary human macrophages, whereas an MOI of 50 was required to achieve similar results in MelJuSo cells. Moreover, by monitoring intracellular bacterial loads over time, the macrophages were shown to be capable of controlling the infection, while MelJuSo cells failed to do so. When comparing the MGIT system with the classical CFU counting assay to determine intracellular bacterial loads, MGIT appeared as a less labor-intensive, more precise, and more objective alternative. Next, using our macrophage Mav infection models, the drug efficacy of the first-line drug rifampicin and the more recently discovered bedaquiline on intracellular bacteria was compared to the activity on extracellular bacteria. The efficacy of the antibiotics inhibiting bacterial growth was significantly lower against intracellular bacteria compared to extracellular bacteria. This finding emphasizes the crucial role of the host cell during infection and drug susceptibility and highlights the usefulness of the models. Taken together, the human cell-based Mav infection models are reliable tools to determine the intracellular loads of Mav, which will enable researchers to investigate host–pathogen interactions and to evaluate the efficacy of (host-directed) therapeutic strategies against Mav.

Minimum inhibitory concentration of cycloserine against Mycobacterium tuberculosis using the MGIT 960 system and a proposed critical concentration

We aimed to determine the breakpoint of cycloserine (CS) susceptibility in MGIT and to describe the molecular characteristics of CS-resistant Mycobacterium tuberculosis (MTB) isolates. A total of 124 MTB isolates were recruited in our analysis. Minimum inhibitory concentration (MIC) was determined using the MGIT system. The mutations of MTB isolates within alr, ddl, ald, and cycA, potentially conferring CS resistance were analyzed by the whole-genome sequencing. In vitro drug susceptibility testing of isolates with doubling concentrations of CS revealed that the modal MIC values was 4 mg/L for MGIT, accounting for 35.5% (44/124) of isolates tested. Seven isolates harbored mutations conferring CS resistance, consisting of five with alr mutations and two with ald mutations. On the basis of the MIC distributions of wild-type and resistotype populations, we proposed a tentative epidemiologic cut-off value of 16 mg/l. The proportion of CS resistance in extensively drug-resistant TB was significantly higher than that of multidrug-resistant TB. In conclusion, we propose critical concentration for MGIT 960 to properly diagnose CS-resistant MTB and demonstrate that mutations in alr and ald genes are the major mechanism conferring CS resistance in clinical isolates.

ПОРІВНЯННЯ СИСТЕМ GENEXPERT MTB/RIF І GENOTYPE ЗІ СТРИПАМИ MTBDRPLUS ДЛЯ ВИЯВЛЕННЯ МУТАЦІЙ, ПОВ’ЯЗАНИХ З РЕЗИСТЕНТНІСТЮ M. TUBERCULOSIS ДО РИФАМПІЦИНУ ПРИ ТУБЕРКУЛЬОЗІ

COMPARISON OF GENEXPERT MTB/RIF AND GENOTYPE SYSTEMS WITH MTBDRPLUS STRIPS FOR DETECTION OF MUTATIONS, ASSOCIATED WITH M. TUBERCULOSIS RESISTANCE TO RIFAMPICIN IN TUBERCULOSIS A. A. Zhurilo, A. I. Barbova, Yu. A. Cherednik, P. S. Trofi mova, S. V. Mironchenko, O. V. Pavlova, A. V. Chernov, L. M. Sladkova Summary The aim was to analyze the level of compliance of two molecular genetic methods GeneXpert MTB/RIF and GenoTypeDRplus in determining the drug resistance of M. tuberculosis to rifampicin when detecting mutations in the RRDR region associated with drug resistance. Object and methods. We studied strains of M. tuberculosis with resistance to rifampicin, which was detected by any of the studied molecular genetic methods. 96 sputum samples were taken. Sputum smears were examined for the presence of acid-fast bacteria by microscopy after staining with the Ziehl-Neelsen method. The material was inoculated into Middlebrook 7H9 broth and on Lowenstein-Jensen medium. The liquid medium was incubated in the BACTEC MGIT system. An immunochromatographic test was used to identify the strains. The drug susceptibility test of M. tuberculosis to rifampicin was performed using the BACTEC MGIT system. The GeneXpert MTB/RIF test was performed according to the manufacturer’s instructions. The GenoTypeDRplus assay was performed on decontaminated and concentrated sputum samples. The process was carried out in three stages: DNA extraction from the processed sputum sample; amplification of the RRDR region by PCR; hybridization of the PCR product to specific oligonucleotide probes immobilized on the test strip. For sequencing, M. tuberculosis DNA isolation was performed using the QIAamp® DNA Mini Kit. The DNA concentration was measured on a Denovix Quantus spectrophotometer. Targeted panel amplification was performed using the Deeplex Myc-TB kit. Amplicon purification was performed using Agencourt AMPure XP magnetic beads. Quantitative analysis of the purified amplification products was performed using a Qubit fluorometer. The M. tuberculosis DNA library was prepared for sample sequencing using the Nextera XT DNA library preparation kit. The library used 5.0 μl of input DNA at a concentration of 0.2 ng/μl. Sequencing was performed on MiSeq equipment with the library normalization and denaturation protocol. Results and discussion. The GeneXpert MTB/RIF and MTBDRplus systems target the same 81 bp rifampicin resistance domain. (RRDR) subunits of bacterial RNA polymerase (rpoB) for mutation detection using DNA probes, i.e. there is a correspondence of probes to each other and an expected similarity of probe binding. We analyzed all sputum samples using GeneXpert MTB/RIF and GenoType MTBDRplus and phenotypic BACCTEC MGIT methods. The level of agreement between two molecular genetic methods for the detection of rifampicin-associated mutations in the RRDR region has been established. The RRDR 81bp region of the rpoB gene of mismatched cases was studied by sequencing. GeneXpert MTB/RIF and GenoType DRplus matched the phenotypic method in 92.7% and 89.6% of cases of M. tuberculosis resistance, respectively. Complete agreement between the results of GeneXpert MTB/RIF and GenoTypeMTBDRplus was observed in 92.7% of cases. GeneXpert MTB/RIF and GenoType DRplus showed a similar pattern of binding failure of wild type probes (WT-probes) when scanning the 81 bp region (RRDRdomain), which leads to stability diagnostics through probe failure software. Sequencing of the RRDR region of “mismatched” strains showed that GeneXpert probes detected seven “mismatched” cases correctly, and GenoTypeDRplus was erroneous in all cases. GeneXpert has demonstrated greater accuracy in R-resistance detection for mismatched isolates compared to GenoTypeDRplus. GeneXpert MTB/RIF has a number of other benefits over GenoTypeDRplus. GeneXpert MTB/RIF is relatively easier to implement, biosafety requirements are minimal, study times are shorter, and the study process is more automated, resulting in less human error and more reproducible results. Given these facts and the results of GeneXpert MTB/RIF found in the study, it is recommended that GeneXpert MTB/RIF be used to detect MDR-TB. Conclusions. Sequencing of the 81bp RRDR region of mismatched M. tuberculosis strains showed that GeneXpert MTB/RIF performed more accurately than GenoTypeDRplus in detecting mutations associated with rifampicin resistance. GeneXpert MTB/RIF is relatively easier to perform, biosafety requirements are minimal, time to study is shorter, and the study process is more automated, resulting in less human error and greater reproducibility of results, so it is reasonable to use it for the detection of multidrug-resistant tuberculosis. The Deeplex Myc-TB analytical solution delivers a wealth of insightful information from antimycobacterial drug resistance markers and speeds up data analysis with easy-to-use software. Key words: tuberculosis, Mycobacterium tuberculosis, drug resistance, GeneXpert MTB/RIF and GenoTypeDRplus molecular genetic systems, BACTEC MGIT phenotypic system, sequencing. Ukr. Pulmonol. J

Discordant results between genotypic and phenotypic assays (Xpert MTB/RIF vs. BACTEC MGIT 960 system) for detection of RIF-resistant Mycobacterium tuberculosis isolates in a high burden region.

Clinical isolates with discordant phenotypic and genotypic results were submitted to DNA sequencing to identify which were genuinely resistant to rifampin and determine the frequency of silent and disputed mutations in our region. We present the retrospective analysis of all the culture-proven TB cases tested with the Xpert®MTB/RIF assay at the Tuberculosis Clinic and Laboratory of the Tijuana General Hospital, Mexico. Clinical isolates showing a discrepancy between phenotypic and molecular tests were analyzed by DNA sequencing. Thirteen isolates tested as rifampin susceptible on the MGIT system were rifampin-resistant according to Xpert®MTB/RIF assay. DNA sequencing showed that seven (53.8%) isolates had a silent (P514P) mutation; three isolates showed different missense (L511P, D516Y, and S531L) mutations. Three isolates showed no mutations. The existence of heteroresistance and silent or disputed mutations warrants that all rifampin-resistance cases diagnosed with the Xpert®MTB/RIF should be referred to specialized centers for DNA sequencing.

Primary Bedaquiline Resistance Among Cases of Drug-Resistant Tuberculosis in Taiwan.

Bedaquiline (BDQ), which is recommended for the treatment of drug-resistant tuberculosis (DR-TB), was introduced in Taiwan in 2014. Due to the alarming emergence of BDQ resistance, we conducted BDQ resistance analyses to strengthen our DR-TB management program. This retrospective population-based study included initial Mycobacterium tuberculosis isolates from 898 rifampicin-resistant (RR) or multidrug-resistant (MDR) TB cases never exposed to BDQ during 2008–2019. We randomly selected 65 isolates and identified 28 isolates with BDQ MIC<0.25μg/ml and MIC≥0.25μg/ml as the control and study groups, respectively. BDQ drug susceptibility testing (DST) using the MGIT960 system and Sanger sequencing of the atpE, Rv0678, and pepQ genes was conducted. Notably, 18 isolates with BDQ MIC=0.25μg/ml, 38.9% (7/18), and 61.1% (11/18) isolates were MGIT-BDQ resistant and susceptible, respectively. Consequently, we recommended redefining MIC=0.25μg/ml as an intermediate-susceptible category to resolve discordance between different DST methods. Of the 93 isolates, 22 isolates were MGIT-BDQ-resistant and 77.3% (17/22) of MGIT-BDQ-resistant isolates harbored Rv0678 mutations. After excluding 2 MGIT-BDQ-resistant isolates with borderline resistance (GU400growth control-GU100BDQ≤1day), 100% (15/15) harbored Rv0678 gene mutations, including seven novel mutations [g-14a, Ile80Ser (N=2), Phe100Tyr, Ala102Val, Ins g 181–182 frameshift mutation (N=2), Del 11–63 frameshift mutation, and whole gene deletion (N=2)]. Since the other 22.7% (5/22) MGIT-BDQ-resistant isolates with borderline resistance (GU400growth control-GU100BDQ≤1day) had no mutation in three analyzed genes. For isolates with phenotypic MGIT-BDQ borderline resistance, checking for GU differences or conducting genotypic analyses are suggested for ruling out BDQ resistance. In addition, we observed favorable outcomes among patients with BDQ-resistant isolates who received BDQ-containing regimens regardless of Rv0678 mutations. We concluded that based on MIC≥0.25μg/ml, 3.1% (28/898) of drug-resistant TB cases without BDQ exposure showed BDQ resistance, Rv0678 was not a robust marker of BDQ resistance, and its mutations were not associated with treatment outcomes.

The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells.

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a 'challenge' experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as 'Moderate' in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

ROLE OF GENE XPERT AND LIQUID CULTURE IN DIAGNOSIS OF EXTRA PULMONAR TUBERCULOSIS

INTRODUCTION: Extrapulmonary Tuberculosis (EPTB) accounts for 15- 25% of all TB cases. It is more difficult to diagnose than Pulmonary tuberculosis and often requires invasive procedures to obtain tissue and or fluid samples. Histology is time-consuming and establishing a diagnosis of TB with high specificity remains difficult. Tissue smear microscopy after special staining is often negative. Tissue culture often leads to considerable delays compromising patient care and outcomes. AIMS AND OBJECTIVES:1. To diagnose Extra Pulmonary Tuberculosis by Gene Xpert(Xpert MTB/Rif assay or CBNAAT) and Liquid Cultures. 2. To evaluate the Sensitivity and Specificity of Gene Xpert in Extra Pulmonary Tuberculosis in comparison with Liquid Culture MGIT960 system. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out by reviewing all suspected extra pulmonary tuberculosis samples of 430 patients attending OPD at Institute of Respiratory Diseases, Jaipur from April 2020 to March 2021.The extrapulmonary samples (pleural fluid,CSF,pus,BAL,Ascitic fluid,Synovial fluid,Gastric aspirate,Liver aspirate) were subjected to GeneXpert and Liquid culture MGIT960 system. RESULTS: Of the 430 Extra Pulmonary Samples, The Sensitivity and Specificity of CBNAAT was 79.77% and 95.30% respectively in comparison with Liquid Culture. Out of the 430 Samples CBNAAT was Positive in 87 samples of which 71(81.60%) were Rifampicin sensitive and 16(18.39%) were Rifampicin Resistant.Out of the 430 Samples,Liquid cultures was Positive in 89 samples. CONCLUSION: Gene Xpert has a notable advantage of detecting tuberculosis within two hours which is acceptable to all clinicians to institute early treatment.CBNAAT is one of the rapid diagnostic tests available in the country and it should be routinely used under the public and private health sector effectively to detect early tuberculosis in Extra Pulmonary Samples.

The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells.

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a 'challenge' experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as 'Moderate' in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

A comparative study between microscopy and culture in detection of M.tb among smear negative pulmonary and extra pulmonary tuberculosis

A comparative study between microscopy and culture in detection of M.tb among smear negative pulmonary and extra pulmonary tuberculosis - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/2394-5478.2018.0066, Indian Journal of Microbiology Research-Indian J Microbiol Res

Transmission of Mycobacterium tuberculosis presenting unusually high discordance between genotypic and phenotypic resistance to rifampicin in an endemic tuberculosis setting

BackgroundSince the implementation of the Xpert MTB/RIF in Sao Paulo, Brazil, numerous Mycobacterium tuberculosis isolates presenting “rifampicin-resistant genotype with rifampicin-susceptible phenotype” were observed. ObjectiveTo evaluate the prevalence, rpoB mutations and transmission of M. tuberculosis resistant to rifampicin on Xpert MTB/RIF but susceptible on BACTEC MGIT system, in Sao Paulo state. MethodsPatients’ isolates with this pattern of rifampicin discordance, collected from 2014 to 2017, had their rpoB predominant rifampicin-resistance-determining region sequenced and were genotyped by IS6110 restriction fragment-length polymorphism. FindingsThe prevalence of rifampicin-discordant M. tuberculosis with genotypic resistance was 55.1% (156/283). Among the sequenced and genotyped isolates, 75.5% (111/147) were in clusters, largely associated with the type of rpoB mutation. Most isolates (98.6%; 72/73) harbouring the predominant mutation, His445Asn, were pooled into the two largest clusters, SP2ga (42/72; 58.3%) and SP5o (12/72; 16.7%). Ranking second, isolates carrying the silent mutation Phe433Phe were mostly (92.3%; 24/26) gathered into four groups of the family SP25. ConclusionThese findings suggest that this unusual high rifampicin discrepancy proportion was greatly influenced by few actively circulating clusters. Further studies on many of the rpoB mutations identified in our setting are needed to elucidate their association with phenotypic rifampicin resistance.

Mycobacteriophage-based test system for phenotypic drug sensitivity of clinical isolates of tuberculous mycobacteria

The issue of rapid phenotypic drug sensitivity testing of clinical isolates of tuberculous mycobacteria remains relevant.The objective of the study is to develop a new test system for rapid phenotypic drug sensitivity testing of MTB clinical isolates based on lytic mycobacteriophages, capable of testing resistance to first and second line drugs.Subjects and methods. Cultures of MTB (108 clinical isolates after primary culturing in Bactec MGIT) were incubated for 48 hours with addition of anti-tuberculosis drugs, then for 48 hours more after adding lytic mycobateriophage D29. The subsequent multiplex reaction of the polymerase chain reaction in real time allowed performing quantitative analysis of MTB DNA and mycobacteriophage DNA. The drug sensitivity of the tested sample was assessed by ranges of the differences in fluorescence threshold levels corresponding to the amount of mycobacteriophages between the control and test sample. At the same time, the drug sensitivity/resistance of all MTB clinical isolates after repeated culture was tested by Bactec MGIT which was adopted as a reference method.Results. Lytic mycobacteriophage-based drug sensitivity testing of 108 MTB isolated to four first line drugs and 90 isolates to six second line anti-tuberculosis drugs demonstrated a high level of concordance with the results of the Bactec MGIT system. It provided 99.5% sensitivity and 100% specificity of the method for 3 first-line drugs; it was slightly lower for ethambutol (86 and 96.9%, respectively), while for second line drugs, its sensitivity made 94.83% and specificity - 98.85%.