Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains. RESULTS: To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal. CONCLUSION: Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

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%.

Disseminated Mycobacterium avium Infection with Different Clinical Presentation in Two Human Immunodeficiency Virus-positive Patients.

ABSTRACT: Microorganisms belonging to the Mycobacterium avium complex (MAC) are ubiquitous in the environment, but only a minority of infected persons develop disease. An underlying lung disease or immune deficiency is a prerequisite for clinical manifestation. However, disseminated MAC disease primarily manifests in people living with human immunodeficiency virus (HIV) in the severe immunodeficiency stage with a whole host of clinical symptoms. We present two cases of disseminated M. avium infection in people living with HIV in the stage of severe immunodeficiency. Both patients exhibited distinct disease progression, with the absence of pulmonary symptoms being a common characteristic. The first patient predominantly experienced high fever, accompanied by diarrhea and severe anemia. The normothermia in the second patient was incongruent with the presence of marked cachexia, severe abdominal pain, and magnetic resonance imaging evidence of abdominal lymph node involvement. The causative agent was isolated from both sputum and stools. The patients underwent treatment that comprised aminoglycoside, macrolide, ethambutol, and rifampicin. Although both patients achieved optimal viral suppression of HIV, the immunologic response to antiretroviral therapy was suboptimal. The first patient died in the setting of severe immunodeficiency due to the development of decompensated liver cirrhosis, while the second patient demonstrated a slight reverse course of the disease.

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.

Eicosanoid and cytokine levels differentiate between stages of MTB infection.

Abstract.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

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.

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.

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. mari-num infection known as "swimming pool" or "fish tank" granuloma. The prolonged specific treatment with at least two-drug combina-tion of rifampicin plus ethambutol and some complications were a big challenge for clinicians as well as the patients.

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.

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.