Genotype mtbc

Manufactured by Hain Lifescience

Sourced in Germany

The GenoType MTBC is a molecular diagnostic test designed to identify and differentiate members of the Mycobacterium tuberculosis complex (MTBC) from other mycobacterial species. The test utilizes DNA-based technology to provide accurate and reliable identification of MTBC organisms.

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22 protocols using genotype mtbc

Mycobacterial Species Identification using LPAs

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The mycobacterial species characterization was initially done on the extracted DNA samples using LPAs from Hain Lifescience GmbH, Germany: GenoType MTBC (for speciation of members of the MTBC) and GenoType Mycobacterium CM (for speciation of common NTM). The assays were performed using the reagents provided and in accordance with the manufacturer's instructions. Each assay is made up of two steps: an amplification step, which is a multiplex PCR, followed by a reverse hybridization.
A positive control sample containing the Control DNA (C+) provided, which is M. kansasii DNA, was used as the positive control, and sterile nuclease-free water was used as the negative control in place of the templates. Hybridization was performed on a TwinCubator and the resulting banding patterns were compared to the reference chart provided by the manufacturer. The species of the mycobacteria were then determined based on the interpretation or reference charts of Mycobacterium GenoType MTBC and CM, as provided by the manufacturer.

Tingan T.K., Mensah G.I., Agyekum E.B., Amanor I.B., Addo S.O., Ayamdoo Y.I., Duah M.S., Mosi L, & Addo K.K. (2022). Non-tuberculous mycobacteria, not Mycobacterium bovis, are a significant cause of TB-like lesions observed in slaughtered cattle in Ghana. IJID Regions, 3, 8-14.

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Identification and Drug Susceptibility of Mycobacteria

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Samples for mycobacteria identification were digested, decontaminated by the NALC-NaOH method [19 , 20 ], and inoculated in both Löwenstein-Jensen medium and MGIT tubes (Becton-Dickinson, Sparks, MA), according to the manufacturer’s specifications. Samples labelled as biopsies were additionally inoculated in Stonebrink culture medium. All positive cultures were further identified by DNA probe (Accuprobe, San Diego, CA). During the study period, all M. tuberculosis complex isolates were identified to the species level. We defined M. bovis as a positive culture with dysgonic growth and biochemical tests results (niacin production, nitrate reduction, thiophen-2-carboxylic acid anhydride susceptibility, and pyrazinamidase deamidation) typical of this species, and M. tuberculosis as a positive culture with eugonic growth and compatible biochemical test results (niacin production, nitrate reductase positive test and heat-stable catalase activity) [21 ]. Additionally, both M. bovis and M. tuberculosis identification was confirmed by spoligotyping from 2000 to 2013 and by the GenoType MTBC (Hain Lifescience GmbH, Nehren, Germany) test from 2013 to 2015 [22 (link), 23 (link)]. Susceptibility testing for rifampicin, streptomycin, isoniazid, and ethambutol was performed using the radiometric BACTEC 460 or the BACTEC 960 culture system (Becton-Dickinson, Sparks, MA) [24 (link)].

Torres-Gonzalez P., Cervera-Hernandez M.E., Martinez-Gamboa A., Garcia-Garcia L., Cruz-Hervert L.P., Bobadilla-del Valle M., Ponce-de Leon A, & Sifuentes-Osornio J. (2016). Human tuberculosis caused by Mycobacterium bovis: a retrospective comparison with Mycobacterium tuberculosis in a Mexican tertiary care centre, 2000–2015. BMC Infectious Diseases, 16, 657.

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Rapid Mycobacterial Detection and Identification

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Biological samples were taken as part of routine diagnostics. Samples were decontaminated using the modified Petroff method [16 ]. An aliquot was inoculated into Middlebrook 7H9 broth in the BACTEC™ MGIT™ automated mycobacterial detection system (BD, Sparks, MD, USA). Blood and bone marrow samples were not decontaminated and were directly inoculated into the BD BACTEC Myco/F Lytic culture vials (BD, Sparks, MD, USA), which were incubated in the BD BACTEC™ FX blood culture system (BD, Sparks, MD, USA). DNA was extracted and evaluated with the GenoType MTBC (HAIN Lifescience, Nehren, Germany), following the manufacturer’s instructions, to ensure reliable identification of MTb.

Valencia-Trujillo D., Avila-Trejo A.M., García-Reyes R.L., Narváez-Díaz L., Segura del Pilar M., Mújica-Sánchez M.A., Becerril-Vargas E., León-Juárez M., Mata-Miranda M.M., Rivera-Gutiérrez S, & Cerna-Cortés J.F. (2024). Genetic Diversity of Mycobacterium tuberculosis Strains Isolated from HIV-Infected Patients in Mexico. Pathogens, 13(5), 428.

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Isolation and Identification of Mycobacterium from Deer and Cattle

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All red deer and cattle that were tested MTC-positive or equivocal by real-time PCR after DNA extraction protocol 1 were further processed by culture under BSL3 conditions at the Bavarian Health and Food Safety Authority, Oberschleißheim, as described by Rettinger et al. [17 ]. As mentioned earlier, not all 11 tissue samples per animal were cultivated, but only samples with visible lesions/granulomas and all the tissue samples tested positive and equivocal by real-time PCR were chosen for further investigation. Single colonies of the culture material were used for species identification using GenoType MTBC (Hain Lifescience GmbH, Nehren, Germany).

Fell S., Bröckl S., Büttner M., Rettinger A., Zimmermann P, & Straubinger R.K. (2016). Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples. BMC Microbiology, 16, 213.

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National TB Laboratory Network Diagnostics

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The national TB laboratory network (Fig 1) entitled to carry out mycobacterial laboratory diagnostics for all public healthcare facilities dealing with diagnosis, treatment, and follow-up of the patients with acute and chronic pulmonary disease (81 outpatient pulmonology departments, 29 general and special hospitals, four clinical centers, three military healthcare institutions, and one center providing healthcare in correctional facilities) participated in the study. The laboratories in the network performed preliminary identification of mycobacterial cultures based on their phenotypic traits, i.e. microscopical and cultural characteristics. The cultures were then sent to the NRL for molecular identification by line probe assays GenoType MTBC [12 (link)] and GenoType Mycobacterium CM [13 (link)] (Hain Lifescience, Nehren, Germany). All strains were tested by the GenoType MTBC assay which differentiates MTBC species, and the isolates not recognized as members of the complex were further tested by the GenoType CM. The version of the GenoType CM used during the study period (VER 1.0) enabled identification of 14 most relevant NTM species. In accordance with the manufacturer’s instructions, the isolates not identifiable to the species level but recognized as members of the genus Mycobacterium were designated Mycobacterium sp.

Dakić I., Arandjelović I., Savić B., Jovanović S., Tošić M., Kurucin T, & Vuković D. (2018). Pulmonary isolation and clinical relevance of nontuberculous mycobacteria during nationwide survey in Serbia, 2010-2015. PLoS ONE, 13(11), e0207751.

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Analysis of Mycobacterium bovis BCG in Tissue

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The tissue samples listed in the outline of Experiment A above were analyzed at the International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark, for the presence of live Mycobacterium bovis BCG. Specimens were disintegrated with a scalpel and in a sample homogenizer (GentleMACS, Dissociator, Miltenyi Biotec, Bergisch Gladbach, Germany) and then pre-treated with MycoPrep (NALC-NaOH, Becton Dickinson, Sparks, MD). The pre-treated material was analyzed with microscopy, PCR specific for Mycobacterium tuberculosis complex (Cobas TaqMan MTB, Roche Diagnostics, Rotkreutz, Switzerland), and culture in MGIT (Becton Dickinson) and Löwenstein-Jensen (SSI Diagnostika) media. The growth of M. bovis BCG was verified with GenoType MTBC (Hain Lifescience, Nehren, Germany).

Jensen K.J., Hansen M.S., Skovgaard K., Svensson E., Larsen L.E., Heegaard P.M., Benn C.S, & Jungersen G. (2023). Immunogenicity of Bacillus Calmette-Guérin in pigs: potential as a translational model of non-specific effects of BCG. Frontiers in Immunology, 14, 1219006.

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Identification of Non-Tuberculous Mycobacterium

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Isolates were maintained on Lowenstein Jensen slants and modified Middle Brook 7H9 medium (Becton Dickinson, USA). The genomic DNA was extracted by using the QIAamp DNA Mini kit (Qiagen, Germany). The primary screening to identify the NTM’s was carried out by commercially available line probe assay kit- Genotype MTBC (Hain Life science, Germany). The non-MTBC isolates were initially identified with Genotype Mycobacterium CM kit (Hain Life science, Nehren, Germany) and unidentified isolates were further tested with Genotype Mycobacterium AS kit (Hain Lifescience, Nehren Germany).
Isolates which were detected by the Genotype Mycobacterium AS assay up to genus level (Mycobacterium species) only were included in the study as “unidentified” species. This study has been reviewed and approved by the Office of Research Affairs in King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.

Varghese B., Enani M., Shoukri M., AlThawadi S., AlJohani S, & Al- Hajoj S. (2017). Emergence of Rare Species of Nontuberculous Mycobacteria as Potential Pathogens in Saudi Arabian Clinical Setting. PLoS Neglected Tropical Diseases, 11(1), e0005288.

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Molecular Identification of Mycobacterium Strains

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The initial molecular identification of the strains was based on analysis of the hsp65 sequence [49 (link)]. The investigated mycobacterium species was identified using the PRASITE database [50 ]. As this method does not differentiate species within the Mycobacterium tuberculosis complex (MTBC), further identification of MTBC strains was then performed using the GenoType MTBC (Hain Lifescience, Germany) molecular test, combined with spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) analysis.
The GenoType MTBC assay was performed in accordance with the manufacturer’s instructions. Spoligotyping was performed as described previously by Kamerbeek et al. (1997) [51 (link)]. To allow comparison with other databases, spoligotype patterns were entered in binary format into the SpolDB4 proprietary database of the Pasteur Institute of Guadeloupe [52 (link)]. Spoligotypes were also assigned according to international spoligotype nomenclature [7 (link)]. The MIRU-VNTR typing was performed using a public protocol [15 ]. The MIRU-VNTR-type was defined after combining the results for the 15 loci in the following order: MIRU 4, MIRU 10, MIRU 16, MIRU 26, MIRU 31, MIRU 40, VNTR 424, VNTR 577, VNTR 2165, VNTR 2401, VNTR 3690, VNTR 4156, VNTR 2163b, VNTR 1955 and VNTR 4052.

Orłowska B., Krajewska-Wędzina M., Augustynowicz-Kopeć E., Kozińska M., Brzezińska S., Zabost A., Didkowska A., Welz M., Kaczor S., Żmuda P, & Anusz K. (2020). Epidemiological characterization of Mycobacterium caprae strains isolated from wildlife in the Bieszczady Mountains, on the border of Southeast Poland. BMC Veterinary Research, 16, 362.

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Mycobacterium caprae Identification Procedure

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For bacteriological cultivation, two to three grams of pathologically suspicious tissue material was homogenized in five ml NaCl solution (0.9%) using the IKA Ultra-Turaxx^® Tube Drive Workstation (IKA^®, Staufen, Germany). The suspension was decontaminated with 1% N-Acetyl-L-Cystein-NaOH solution and neutralized with 20 ml phosphate buffer (pH 6.8) as recommended by the World Organization for Animal Health (OIE) (2012) Manual [20 ]. The solution was centrifuged for 20 min at 3300 x g and the suspension discarded. The suspension pellet was plated on two growth media—Lowenstein-Jensen with Glycerin and PACT (polymyxin B, amphotericin B, carbenicillin and trimethoprim) and Stonebrink with PACT (both purchased from Heipha) using swabs. Incubation was performed at 37°C for 12 weeks and bacterial growth was frequently screened. M. caprae species identification was performed by reversed line blotting (Geno Type® MTBC, HAIN Life Science, Germany).

Leth C., Varadharajan A., Mester P., Fischaleck M., Rossmanith P., Schmoll F, & Fink M. (2017). Matrixlysis, an improved sample preparation method for recovery of Mycobacteria from animal tissue material. PLoS ONE, 12(7), e0181157.

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Household Tuberculosis Screening Protocol

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Sputum samples were collected from all household contacts at 0, 2, 6, and 12 months (two samples on two consecutive days at baseline, one sample at the remaining time points) were evaluated by smear microscopy for acid-fast bacilli (AFB) and cultured on both liquid (BACTEC MGIT 960™ [Becton and Dickinson, USA]) and solid (Lowenstein-Jensen) media as described previously (32 (link)). Positive cultures were confirmed using the HAIN kit (GenoType MTBC, Hain Lifescience GmbH, Nehren, Germany). CXR was repeated at the end of the study for all participants. Peripheral blood (~2.5 ml) was drawn for biomarker analysis at 0, 2, 6, and 12 months in the PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) and stored at -80°C until RNA extraction (PAXgene Blood RNA kit; PreAnalytiX, Hilden, Germany). Subsequently, 3 ml of blood was collected (1 ml each collected in Nil, TB antigen, and Mitogen tubes) for the QuantiFERON^® -TB Gold In-Tube (QFT-GIT) test (Cellestis, Australia) at 0, 2, 6, and 12 months.

Sivakumaran D., Jenum S., Srivastava A., Steen V.M., Vaz M., Doherty T.M., Ritz C, & Grewal H.M. (2023). Host blood-based biosignatures for subclinical TB and incipient TB: A prospective study of adult TB household contacts in Southern India. Frontiers in Immunology, 13, 1051963.

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