Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management

Mycobacterium intracellulare among TB suspected patients in Bulgaria – microbiological aspects

Introduction: Nontuberculous mycobacteria (NTM) are representatives of the genus Mycobacterium with a worldwide distribution, associated mainly with water, soil, and biofilms. Some of NTMs, such as Mycobacterium avium complex (MAC), are etiological agents of human diseases – disseminated or with different localization, most often pulmonary. Aim: In the present study, we analyzed Mycobacterium intracellulare isolates recovered from clinical specimens of tuberculosis (TB) suspected patients in Bulgaria, 2018-2020. Materials and methods: The cultures were grown on solid and liquid media. For species identification, we used immune chromatographic (TB Ag MPT64) test and Line Probe Assay (LPA) from the positive cultures. Results: M. intracellulare was identified in 32 patients from 82,780 specimens. It was predominantly isolated in females – 62.5% vs. 37.5% in males. The most affected age group was 65 years and over (38%). The distribution of the isolates in Bulgaria was uneven. Most of them (65.6%) were concentrated in two districts of the country: Plovdiv and Sofia-city. All strains were sensitive to macrolides and aminoglycosides except one with macrolide resistance. NTM pulmonary disease was confirmed in 16 patients with M. intracellulare isolate. Conclusions: Analysing the 32 M. intracellulare isolates identified among TB suspected patients in Bulgaria between 2018 and 2020, we found that only half of them met the American Thoracic Society (ATS) diagnostic criteria for NTM pulmonary disease. For the remaining patients with M. intracellulare isolates we did not have sufficient data to support this diagnosis. Efforts by Bulgarian respiratory and microbiological societies are needed for adherence to the international guidelines

First Etiologically Confirmed Cases of Mycobacterium Marinum Infection in Bulgaria

This study aimed to describe the first two microbiologically confirmed cases of cutaneous and soft tissue Mycobacterium marinum infection in Bulgaria. The isolation of the Nontuberculous Mycobacteria (NTM) strains and their species identification was performed at NRL TB, NCIPD using specific media and cultivation conditions, and PCR based Line Probe Assay (LPA) from the positive cultures. The two patients had closely related jobs to fishes and water reservoirs and both of them had a similar clinical manifestation of M. mari­num infection known as “swimming pool” or “fish tank” granuloma. The prolonged specific treatment with at least two-drug combina­tion of rifampicin plus ethambutol and some complications were a big challenge for clinicians as well as the patients

Eicosanoid and cytokine levels differentiate between stages of MTB infection

Abstract Introduction: The need for biomarkers predicting the course of MTB infection and the necessity of specific therapy are well recognized. Recent data point to the role of cytokines and lipid mediators in protective immunity against tuberculosis. Aim: We evaluated the balance between cytokines, and eikosanoids as a possible prognostic indicator in MTB infection. Material and methods: The induced expression of effector and regulatory cytokines IFN-γ, TNF-α, IL-2, IL-17, IL-6, and IL-10 was measured in relation to the lipid mediators PGE2 and LXA4 in active TB infection (ATB, n=15) before and after therapy (ATB-T, n=6), established latent infection (LTBI, n=22), recent contacts of ATB (RC, n=12), and healthy controls (n=11) A flow cytometry microarray (CBA, BD Biosciences) and quantitative ELISA (SunRed Tech) were employed. Results: The regulatory cytokines (RC) were characterized by a high potential for IL-17 and Th1 cytokine secretion, combined with low IL-6 expression, while ATB donors had a partially preserved TNF-α potential, and higher IL-6 expression. The PGE2-to-LXA4 ratio discriminated between situations with high bacterial load (ATB), and contained infection (LTBI, ATB-T), and defined clearly cut subgroups among RC and ATB donors. Conclusions: Our results suggest that increased PGE2/LXA4 ratio coupled with high induced IL-10 level indicates infection after a recent contact. In the settings of ATB, increased ratio and low TNF-α level point to inefficient granuloma formation in the settings of ATB

Factors associated with treatment success and death in cases with multidrug-resistant tuberculosis in Bulgaria, 2009–2010

Objective: To analyze determinants of success and death in multidrug-resistant tuberculosis patients (MDR-TB; resistance to, at least, isoniazid and rifampicin) placed on treatment in Bulgaria during the period September 2009 to March 2010 using logistic regression. Results: Fifty MDR-TB patients started treatment. Male:Female ratio was 2.3:1; mean age 43 years (range: 18–77); 19 patients (38%) were new; median duration of disease before treatment was 5 years (range: 1–13). All patients tested negative for HIV. Eight cases had XDR-TB (MDR-TB plus resistance to any fluoroquinolone and any second-line injectable). Twenty-four months after starting treatment, 24 patients (48%) had a successful outcome, in 6 (12%) treatment failed, 19 (38%) died, and one (2%) interrupted treatment. XDR-TB cases experienced higher mortality than others (75% vs. 30.9%, respectively, P < 0.05). Sputum smear positivity at start of treatment and weight loss or no weight gain were positively associated with death (adjusted Odds ratio: 5.16; 95% confidence interval: 1.16–22.84 and 5.61; 1.48–21.20, respectively) and negatively with success (0.13; 0.02–0.94 and 0.02; 0.00–0.19). No previous TB treatment increased likelihood of success (7.82; 1.09–56.15). Discussion and conclusions: Most MDR-TB patients in this first treatment cohort using WHO-recommended norms had advanced disease explaining the high mortality and low success. Early, adequate treatment of MDR-TB patients can improve outcomes and avert transmission

Whole genome sequencing of Bulgarian rifampicin resistant Mycobacterium tuberculosis strains

Introduction: The transmission of drug-resistant tuberculosis is one of the greatest challenges facing the global tuberculosis control. Aim: The aim of the study was to investigate the resent transmission of rifampicin resistant tuberculosis in Bulgaria and to describe the mutations related to the antimicrobials’ resistance using whole genome sequencing. Materials and methods: As part of an ECDC funded pilot study for evaluation of the systematic use of whole genome sequencing (WGS) of Mycobacterium tuberculosis (MTB) surveillance (EUSeqMyTB), Bulgaria provided 65 rifampicin resistant isolates over a three years’ timeframe (2017-2019) representing 87.5% of the notified rifampicin resistant cases. Drug resistance prediction and relatedness analysis of the resistant isolates was performed in collaboration with San Raffaele Scientific Institute, Milan, Italy. Results: Almost all of the isolates were identified as Euro-American lineage (96.9%); 18.5% of the isolates were found to be resistant to fluoroquinolones, but no mutations conferring resistance to bedaquiline or linezolid could be identified. Less than half (43.3%) of the isolates were clustered (<5 SNPs distance) into a total of seven national SNP-based clusters, while a total of six isolates were found to be part of different cross-border clusters. All clustered cases originated from Bulgaria. Conclusions: WGS has proven to be a reliable tool for surveillance and tracing of recent transmission of tuberculosis and has the potential for resistance prediction for most of the antituberculosis drugs

Whole genome sequencing of Bulgarian rifampicin resistant Mycobacterium tuberculosis strains

Introduction: The transmission of drug-resistant tuberculosis is one of the greatest challenges facing the global tuberculosis control. Aim: The aim of the study was to investigate the resent transmission of rifampicin resistant tuberculosis in Bulgaria and to describe the mutations related to the antimicrobials’ resistance using whole genome sequencing. Materials and methods: As part of an ECDC funded pilot study for evaluation of the systematic use of whole genome sequencing (WGS) of Mycobacterium tuberculosis (MTB) surveillance (EUSeqMyTB), Bulgaria provided 65 rifampicin resistant isolates over a three years’ timeframe (2017-2019) representing 87.5% of the notified rifampicin resistant cases. Drug resistance prediction and relatedness analysis of the resistant isolates was performed in collaboration with San Raffaele Scientific Institute, Milan, Italy. Results: Almost all of the isolates were identified as Euro-American lineage (96.9%); 18.5% of the isolates were found to be resistant to fluoroquinolones, but no mutations conferring resistance to bedaquiline or linezolid could be identified. Less than half (43.3%) of the isolates were clustered (<5 SNPs distance) into a total of seven national SNP-based clusters, while a total of six isolates were found to be part of different cross-border clusters. All clustered cases originated from Bulgaria. Conclusions: WGS has proven to be a reliable tool for surveillance and tracing of recent transmission of tuberculosis and has the potential for resistance prediction for most of the antituberculosis drugs

Beijing Lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007-2011

To assess the spread of the Mycobacterium tuberculosis Beijing genotype among patients with multidrug-resistant and extensively resistant tuberculosis in Bulgaria, we genotyped 188 (72%) of 261 microbiologically confirmed resistant isolates obtained during 2007-2011. The estimated prevalence of the Beijing genotype among these patients was 3.2

Tuberculosis-spoligo-rifampin-isoniazid typing: an all-in-one assay technique for surveillance and control of multidrug-resistant tuberculosis on Luminex devices

As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere