Tuberculosis Complex Strains Research Articles

Personalized Approach as a Basis for the Future Diagnosis of Tuberculosis (Literature Review)

The global spread of tuberculosis remains one of actual problems of public health despite of introduction of public health safety programs. Early, rapid and accurate identification of M. tuberculosis and determination of drug susceptibility are essential for treatment and management of this disease. Delay in delivering results prolongs potentially inappropriate antituberculosis therapy, contributing to emergence of drug resistance, reducing treatment options and increasing treatment duration and associated costs, resulting in increased mortality and morbidity. Faster, more comprehensive diagnostics will enable earlier use of the most appropriate drug regimen, thus improving patient outcomes and reducing overall healthcare costs. The treatment of infection based on the using of massive antimicrobial therapy with analysis of bacterial strains resistance to first line drugs (FLD) isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), ethambutol (EMB) and streptomycin (SM). However, the public health practitioners pay no attention to functional activity of human immune system genes. The interaction of bacterial genomes and immune system genes plays the major role in infection progress. There is growing evidence that, together with human and environmental factors, Mycobacterium tuberculosis complex strain diversity contributes to the variable outcome of infection and disease in human TB. We suppose that the future of diagnosis and treatment of tuberculosis lies in the field of personal medicine with comprehensive analysis of host and pathogen genes.

Longitudinal Outbreak of Multidrug-Resistant Tuberculosis in a Hospital Setting, Serbia.

A retrospective population-based molecular epidemiologic study of multidrug-resistant Mycobacterium tuberculosis complex strains in Serbia (2008–2014) revealed an outbreak of TUR genotype strains in a psychiatric hospital starting around 1990. Drug unavailability, poor infection control, and schizophrenia likely fueled acquisition of additional resistance and bacterial fitness–related mutations over 2 decades.

Clonality and genetic profiles of drug-resistant Mycobacterium tuberculosis in the Eastern Cape Province, South Africa.

In this study, we investigated the diversity of drug‐resistant Mycobacterium tuberculosis isolates from families who own cattle in the Eastern Cape Province of South Africa using spoligotyping and mycobacterial interspersed repetitive‐unit‐variable number tandem repeat (MIRU‐VNTR) typing. The Mycobacterium tuberculosis was investigated using MIRU‐VNTR and the Mycobacterium tuberculosis families were evaluated using spoligotyping. Spoligotyping grouped 91% of the isolates into seven clusters, while 9% of the deoxyribonucleic acid (DNA) from TB isolates were unclustered from a total of 154 DNA used. Previously described shared types were observed in 89.6% of the isolates, with the Beijing family, SIT1, the principal genotype in the province, while the families T, SIT53 and X1, SIT1329 were the least detected genotypes. MIRU‐VNTR grouped 81% of the isolates in 23 clusters while 19% were unclustered. A combination of the VNTR and spoligotyping grouped 79% of the isolates into 23 clusters with 21% unclustered. The low level of diversity and the clonal spread of drug‐resistant Mycobacterium tuberculosis isolates advocate that the spread of TB in this study may be instigated by the clonal spread of Beijing genotype. The results from this study provide vital information about the lack of TB control and distribution of Mycobacterium tuberculosis complex strain types in the Eastern Cape Province of South Africa.

Genetic diversity of Mycobacterium tuberculosis complex strains isolated from livestock workers and cattle in Nigeria

Molecular typing techniques are useful in understanding tuberculosis epidemiology; yet, they have been under-utilised at the human-animal interface in Nigeria. Sixty-four Mycobacterium tuberculosis complex (MTBC) isolates including 42 M. tuberculosis, 13 M. bovis and nine M. africanum obtained from livestock workers (LW, n = 47) and their cattle (n = 17) in three geographical zones of Nigeria were genotyped to identify and evaluate the genetic diversity of the circulating MTBC using spoligotyping. Distribution into clades of M. tuberculosis revealed; 45.3% Uganda I- [SIT46- cattle: 1; LW: 28], 14.1% Latin American Mediterranean- [SIT61, cattle: 1; LW: 8], and 1.6% T- [SIT53—LW: 1]. The M. bovis strains were 6.3% SB0944 [cattle: 4] and 1.6% each of SB0300, SB1026, SB1027 and SB1439 [cattle: 4]. Seventeen MTBC isolates [cattle: 7; LW: 10] yielded 14 new spoligotype patterns including three M. tuberculosis strains (three isolates), five M. bovis strains (five isolates) and six M. africanum strains (nine isolates), two of which belonged to MAF1. Only few families namely, the not previously described Uganda I-, LAM and SB0944 are predominant among the LW and cattle, with other types in lower prevalences. The strain population structure indicates an intriguing diversity and possible zoonotic linkage with consequences for TB control in the country. The need to employ newer molecular techniques such as Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats and whole genome sequence to decipher circulating MTBC strains in Nigeria is advocated.

Transmission of tuberculosis among people living in the border areas of Poland, the Czech Republic, and Slovakia.

In 2007, Poland, the Czech Republic, and Slovakia joined the Schengen Agreement, abolishing restrictions on people crossing the borders. Currently, these areas are places of population movements for economic, family, and touristic reasons. This favors the transmission of infectious diseases, including tuberculosis, and requires enhanced control over the spread of the source of infection in the population of patients living in the border areas. The aim of this study was to investigate the genetic relatedness among Mycobacterium tuberculosis complex strains isolated from patients living in 3 border areas: Poland, the Czech Republic, and Slovakia. PATIENTS AND METHODS The study group consisted of 209 patients with tuberculosis diagnosed and treated between 2007 and 2011 in health care facilities in the Silesia Province in Poland (121 patients [58%]), Žilina in Slovakia (57 [27%]), and the Moravian-Silesian Region in the Czech Republic (31 [15%]). Genotyping of strains was performed using spoligotyping and IS6110-Mtb1-Mtb2 polymerase chain reaction. Among 209 strains, 23 molecular families (clusters) were identified. Seventeen clusters were identified as national. Six international clusters consisted of 30 strains isolated from patients of various nationalities. We identified 6 potential outbreaks of tuberculosis transmission between patients of different nationalities. The circumstances favorable to potential contacts of patients included mainly travelling to the neighboring countries, hospital stays, and addictions. However, there was no evidence of an epidemiological link between these patients, so it may be assumed that if they had come in contact with one another, it was accidental. We observed that the greater incidence of tuberculosis on the Polish territory did not affect the incidence in the Czech Republic or Slovakia over the analysis period.

Detection of multidrug resistant Mycobacterium tuberculosis in Tabuk, Saudi Arabia, using Genotype MTBDRplus.

The global increase in the rates of multidrug-resistant (MDR) tuberculosis (TB) has made the timely identification of resistant Mycobacterium tuberculosis complex strains an important emergence to achieve effective disease management and to prevent their spread in the community. The present study aimed to determine the MDR-TB in Tabuk province, north of the Kingdom of Saudi Arabia. The GenoType MTBDRplus assay was used to determine the mutations associated with isoniazid (INH) and rifampicin (RIF) resistances. A total number of 61 confirmed M. tuberculosis positive-sputum samples were scanned for the mutation in the rpoB, inhA, and katG genes. The present study revealed that 67.2% of the samples were susceptible, 29.5% were monoresistant, and 3.3% were MDR. The monoresistant showed 26.2% for INH and 3.3% for RIF. The early detection of MDR could guide the starting of appropriate regimen of treatment.

Drug-resistance profiling and transmission dynamics of multidrug-resistant Mycobacterium tuberculosis in Saudi Arabia revealed by whole genome sequencing.

BackgroundIn Saudi Arabia, cross-border transmission of multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains might be particularly fostered by high immigration rates. Herein, we aimed to elucidate the transmission dynamics of MDR-MTBC strains and reveal a detailed prediction of all resistance-conferring mutations for the first- and second-line drugs.MethodsWe investigated all MDR-MTBC strains collected between 2015 and 2017 from provincial mycobacteria referral laboratories and compared demographic and clinical parameters to a cohort of non-MDR-TB patients using a whole genome sequencing approach. Clusters were defined based on a maximum strain-to-strain genetic distance of five single-nucleotide polymorphisms (SNPs) as surrogate marker for recent transmission, and then investigated molecular drug-resistance markers (37 genes).ResultsForty-eight (67.6%) MDR-MTBC strains were grouped in 14 different clusters, ranging in size from two to six strains; 22.5% (16/71) of all MDR-MTBC isolates were predicted to be fully resistant to all five first-line drugs, and five strains (7.0%) exhibited fluoroquinolone resistance. Moreover, we revealed the presence of 12 compensatory mutations as well as 26 non-synonymous SNPs in the rpoC gene and non-hotspot region in rpoB, respectivelyConclusionOptimized TB molecular surveillance, diagnosis, and patient management are urgently needed to contain MDR-MTBC transmission and prevent the development of additional drug resistance.

Antimicrobial Resistance Pattern of Mycobacterium Tuberculosis Complex Isolated from Extrapulmonary Tuberculosis Patientsin Sohag Governorate

As the number of drug resistant pulmonary TB is increasing around the world, the number of drug resistant TB with extrapulmonary manifestations are also on rise. However, there is surprisingly scant information in medical literatures on prevalence and impact of extrapulmonary drug-resistant TB Aim:This study aimed to detect and isolate Mycobacterium Tuberculosis Complex (MTC or MTBC) strains from extrapulmonary samples then determine the drug resistance pattern of these strains to antituberculus drugs. Materials and Methods: A total of 100 clinical specimens were collected during the study period from September 2016 to January 2018 at Medial Microbiology & Immunology Department, Sohag Faculty of Medicine from patients suspected to have EPTB. All samples subjected to ZN staining &cultures on LJ media then all positive culture were subjected for identification of the type of mycobacteria by two methods, phenotypically by biochemical tests and genotypically by conventional PCR fordetection of MTC.Antimicrobial susceptibility pattern was done for MTC strains. Results:Out of100 collected extrapulmonary samples, 66 samples were positive for mycobacterial culture. By using biochemical tests and PCR for identification of species, 53 isolates were identified as MTC and 13 isolates were identified as NTM. Antimicrobial susceptibility pattern was done for MTCstrains and detected 5 strains were MDR and one strain was XDR. Conclusion:Extrapulmonary TB infection rate in Sohag governorate is high and the problem of drug resistance in extrapulmonary tuberculosis (EPTB) cannot be overlooked.

Detection of a streptomycin-resistant Mycobacterium bovis strain through antitubercular drug susceptibility testing of Tunisian Mycobacterium tuberculosis complex isolates from cattle

BackgroundA rising isolation trend of drug-resistant M. bovis from human clinical cases is documented in the literature. Here we assessed Mycobacterium tuberculosis complex isolates from cattle for drug susceptibility by the gold standard agar proportion method and a simplified resazurin microtitre assay (d-REMA). A total of 38 M. tuberculosis complex strains, including M. bovis (n = 36) and M. caprae (n = 2) isolates, from cattle in Tunisia were tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and pyrazinamide.ResultsM. caprae isolates were found to be susceptible to all test drugs. All M. bovis strains were resistant to pyrazinamide, as expected. In addition, one M. bovis isolate showed high-level resistance to streptomycin (MIC > 500.0 μg/ml). Concordant results with the two methods were found. The most common target genes associated with streptomycin resistance, namely the rrs, rpsL and gidB genes, were DNA sequenced. A non-synonymous mutation at codon 43 (K43R) was found in the rpsL gene. To the best of our knowledge, this is the first report describing the isolation of a streptomycin-resistant M. bovis isolate from animal origin.ConclusionsAntitubercular drug susceptibility testing of M. bovis isolates from animals should be performed in settings where bTB is endemic in order to estimate the magnitude of the risk of drug-resistant tuberculosis transmission to humans.

Phenotypic and genotypic drug sensitivity of <em>Mycobacterium tuberculosis</em> complex isolated from South Omo Zone, Southern Ethiopia

BackgroundKnowledge of drug-sensitivity patterns of Mycobacterium tuberculosis complex (MTBC) strains isolated from patients is an important aspect of TB control strategy. This study was conducted to evaluate the drug sensitivity of MTBC isolates in South Omo, southern Ethiopia.Materials and methodsA total of 161 MTBC isolates (153 from new cases and eight re-treatment TB cases) were isolated using Lowenstein Jensen medium of which 126 isolates were able to be tested for drug sensitivity by BACTEC™MGIT™ 960 system, while all the 161 isolates were tested by GenoType® MTBDRplus VER 2.0. Descriptive statistics and logistic regression were used to express and present results.ResultsOn the basis of MGIT 960 system, the prevalence of mono-resistance was 9.2% (11/119) in the new cases, although neither poly-resistance nor multidrug resistance (MDR) was recorded in these cases. On the basis of GenoType MTBDRplus assay, two of the 153 isolates (1.3%) of the new cases were mono-resistant for rifampicin (RIF) and one of these isolates had known rpoB gene mutation (H526D). One of the eight (12.5%) isolates obtained from the re-treatment cases was MDR with rpoB gene mutation (D516V) and katG gene mutation (S315T2). Taking MGIT 960 system as a gold standard, the sensitivities of the MTBDRplus assay were 33.3%, 100% and 100% for detection of resistance to isoniazid, RIF and MDR, respectively. On the other hand, its specificities were 99.2%, 100% and 100% for detection of resistance to RIF, isoniazid and MDR, respectively.ConclusionThe magnitude of drug resistance was relatively low in the new TB cases of South Omo as compared to the reports from the other regions of the country. This is encouraging and hence the TB Control Program in the Zone should strengthen its program so that the emergence of drug resistance is inhibited.

Harmonized Genome Wide Typing of Tubercle Bacilli Using a Web-Based Gene-By-Gene Nomenclature System.

BackgroundGlobal tuberculosis (TB) control is challenged by uncontrolled transmission of Mycobacterium tuberculosis complex (Mtbc) strains, esp. of multidrug (MDR) or extensively resistant (XDR) variants. Precise analysis of transmission networks is the basis to trace outbreak M/XDR clones and improve TB control. However, classical genotyping tools lack discriminatory power due to the high similarity of strains of particular successful lineages, e.g. Beijing or outbreak strains. This can be overcome by whole genome sequencing (WGS) approaches, but these are not yet standardized to facilitate larger investigations encompassing different laboratories or outbreak tracing across borders.MethodsWe established and improved a whole genome gene-by-gene multi locus sequence typing approach encompassing a stable set of core genome genes (cgMLST) and linked it to a web-based nomenclature server (cgMLST.org) facilitating assignment and storage of allele numbers.FindingsWe evaluated and refined a previously suggested cgMLST schema by using a reference strain set (n = 251) reflecting the global diversity of the Mtbc. A set of 2891 genes showed excellent performance with at least 97% of the genes reliably identified in strains of all Mtbc lineages and in discriminating outbreak strains. cgMLST allele numbers were automatically retrieved from and stored at cgMLST.org.InterpretationThe refined cgMLST schema provides high resolution genome-based typing of clinical strains of all Mtbc lineages. Combined with a web-based nomenclature server, it facilitates rapid, high-resolution, and harmonized tracing of clinical Mtbc strains needed for prospective local and global surveillance.

Development of a New Genome-Wide MLST Scheme for High-Resolution Typing of Diverse Mycobacterium tuberculosis Complex Strains.

In this issue of EBioMedicine, Kohl and colleagues describe the development of a new core genome MLST scheme (cgMLST) for Mycobacterium tuberculosis complex strains based on a set of 2891 genes. Here, the application of the scheme to a number of tuberculosis surveillance studies is examined.

Genotyping of Mycobacterium tuberculosis complex strains isolated from clinical samples using pulsed-field gel electrophoresis

Genotyping of Mycobacterium tuberculosis complex strains isolated from clinical samples using pulsed-field gel electrophoresis

Fluoromycobacteriophages Can Detect Viable Mycobacterium tuberculosis and Determine Phenotypic Rifampicin Resistance in 3-5 Days From Sputum Collection.

The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybombϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested mCherrybombΦ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. The many attributes of mCherrybombΦ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

In vivo virulence of Mycobacterium tuberculosis depends on a single homologue of the LytR-CpsA-Psr proteins

LytR-cpsA-Psr (LCP) domain containing proteins fulfil important functions in bacterial cell wall synthesis. In Mycobacterium tuberculosis complex (Mtbc) strains, the causative agents of tuberculosis (TB), the genes Rv3484 and Rv3267 encode for LCP proteins which are putatively involved in arabinogalactan transfer to peptidoglycan. To evaluate the significance of Rv3484 for Mtbc virulence, we generated a deletion mutant in the Mtbc strain H37Rv and studied its survival in mice upon aerosol infection. The deletion mutant failed to establish infection demonstrating that Rv3484 is essential for growth in mice. Following an initial phase of marginal replication in the lungs until day 21, the Rv3484 deletion mutant was almost eliminated by day 180 post-infectionem. Interestingly, the mutant also showed higher levels of resistance to meropenem/clavulanate and lysozyme, both targeting peptidoglycan structure. We conclude that Rv3484 is essential for Mtbc virulence in vivo where its loss of function cannot be compensated by Rv3267.

Klinik Örneklerden İzole Edilen Mycobacterium tuberculosis Kompleks İzolatlarının Birinci ve İkinci Seçenek Anti-Tüberküloz İlaçlara Karşı Duyarlılığın Sensititre MycoTB Plate Yöntemi ile Araştırılması

Klinik Örneklerden İzole Edilen Mycobacterium tuberculosis Kompleks İzolatlarının Birinci ve İkinci Seçenek Anti-Tüberküloz İlaçlara Karşı Duyarlılığın Sensititre MycoTB Plate Yöntemi ile Araştırılması

Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease causing major mortality worldwide. As part of a systematic methodology for studying M. tuberculosis surface proteins which might be involved in host-pathogen interactions, our group found that LpqG surface protein (Rv3623) found in M. tuberculosis complex strains was located on the mycobacterial envelope and that peptide 16661 (21SGCDSHNSGSLGADPRQVTVY40) had high specific binding to U937 monocyte-derived macrophages and inhibited mycobacterial entry to such cells in a concentration-dependent way. A region having high specific binding to A549 alveolar epithelial cells was found which had low mycobacterial entry inhibition. As suggested in previous studies, relevant sequences in the host-pathogen interaction do not induce an immune response and peptides characterised as HABPs are poorly recognised by sera from individuals regardless of whether they have been in contact with M. tuberculosis. Our approach to designing a synthetic, multi-epitope anti-tuberculosis vaccine has been based on identifying sequences involved in different proteins’ mycobacteria-target cell interaction and modifying their sequence to improve their immunogenic characteristics, meaning that peptide 16661 sequence should be considered in such design.

Use of Ultrasonication as a Rapid Pretreatment Method for MALDI-TOF MS of Mycobacterial Samples

Background: In Japan, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used as a simple and accurate method for mycobacterial identification, but existing pretreatment techniques are time-consuming and laborious.Objective: We characterized a new pretreatment technique that extracts proteins using ultrasonic disruption.Methods: We compared this new technique with the current pretreatment method and examined the usefulness of ultrasonication, including its use in mycobacterial inactivation. A total of 174 mycobacterial isolates were tested, including 50 Mycobacterium tuberculosis complex strains, 57 Mycobacterium avium strains, 55 Mycobacterium intracellulare strains, and 12 Mycobacterium kansasii strains. The ultrasonic pretreatment method was performed with or without heat-pretreatment at 95°C for 30 minutes, in parallel with the current conventional method. For all tested strains, the mycobacterial identification agreed when comparing the new and conventional methods.Results: Samples prepared by ultrasonication without heat pretreatment exhibited multiple significant differences when compared with samples prepared by ultrasonication with heat pretreatment or by conventional methods. However, most scores were over 2.0, and the lowest score exceeded 1.7. Furthermore, the new techniques could be performed in only 10 minutes for diagnosis. In addition, we confirmed that 1 minute of ultrasonic pretreatment yielded complete inactivation of M. tuberculosis complex strains.Conclusion: The more rapid technique of protein extraction/inactivation by ultrasonication without heat pretreatment is expected to be highly useful in clinical laboratory settings.

Molecular characterization of Mycobacterium tuberculosis complex strains isolated from cattle in Masovian Voivodeship in 2008-2012

Since 2009, Poland has had a TB-free status, although over the last seven years 12-34 cases of bovine TB have been recorded annually. In 2009-2012 the largest number of cattle infected with Mycobacterium bovis and Mycobacterium caprae were culled in Masovian Voivodeship. Likewise, the largest number of sources of this zoonosis were recorded in that voivodeship. The vicinity of farms where bTB was found indicated that it could have been transmitted between their herds. The aim of this study was to characterise the molecular patterns of bovine bacillus strains isolated from cattle in Masovian Voivodeship and the molecular relationships between them. The material for microbiological examination came from 38 cattle (Bos taurus) located in 7 counties of Masovian Voivodeship. These 38 strains of MTBC were further identified as M. bovis (24 isolates; 63%) and M. caprae (14 isolates; 37%). A two-step genotyping analysis of the 38 MTBC strains identified 24 molecular patterns, closely related phylogenetically, which were assigned to 8 clusters of 2-6 strains. Sources of transmission were identified in 8 out of 13 herds examined in the 7 counties of Masovian Voivodeship. The results of the genotyping analysis excluded the possibility of TB transmission between different herds in Masovian Voivodeship. It was proved, however, that TB had been transmitted between animals bred on one of the farms..

Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis.

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M.bovis in a farmer underlines the importance of M.bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.