Culture isolates with a negative results for SD Bioline assay were processed first for primary identification using reverse hybridization-based line probe assay, GenoType Mycobacterium CM (Hain Lifescience, Nehren Germany), then the GenoType Mycobacterium AS for culture isolates which were negative to GenoType Mycobacterium CM. The process was performed as recommended by the manufacturer and was consisted of an extraction, amplification and hybridization [12 ].
Genotype mycobacterium cm
Manufactured by Hain Lifescience
Sourced in Germany
The GenoType Mycobacterium CM is a molecular-based laboratory test that identifies the presence of Mycobacterium species in a clinical sample. It utilizes a DNA-based method to detect and differentiate Mycobacterium species. The core function of this product is to provide an accurate and reliable identification of Mycobacterium species from patient specimens.
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Lab products found in correlation
Genotype mycobacterium as, by Hain Lifescience (3 mentions)
Genotype mtbc, by Hain Lifescience (2 mentions)
Bactec mgit 960, by BD (1 mentions)
Mgit 960, by BD (1 mentions)
Atcc 27294, by American Type Culture Collection (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Miseq sequencing platform, by Illumina (1 mentions)
11 protocols using genotype mycobacterium cm
1
Mycobacterium Identification Protocol
2
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.
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.
3
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.
4
Mycobacterium abscessus Strain Characterization
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The bacterial strains used in the present study were M. abscessus type strain DSMZ 44196T, M. abscessus 390, M. abscessus BE37R, and M. abscessus BE48R (Table 1 ). The original M. abscessus type strain (DSMZ 44196T) displayed S colonies on agar medium (called 44196S), but after a few passages on agar medium, natural R colony mutants appeared (44196R). M. abscessus 390 was isolated in pure culture from an ileal granuloma in a patient with Crohn’s disease (Byrd and Lyons, 1999 (link)). M. abscessus 390 displayed R colonies on agar medium (390R), but after serial passages on agar medium, an S morphotype was isolated (390S) (Byrd and Lyons, 1999 (link)). M. abscessus BE37R and BE48R were isolated from pulmonary disease patients hospitalized at Bellvitge University Hospital in Barcelona, Spain. BE37R and BE48R were identified using polymerase chain reaction and reverse hybridization (GenoType Mycobacterium CM®, Hain Lifescience, Germany) and the partial sequencing of 16S rRNA. All strains were grown on trypticase soy agar (TSA; Scharlau, Spain) medium for 2 weeks at 37°C.
5
Identification of Non-Tuberculous Mycobacteria
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The isolated strains were identified using a GenoType® Mycobacterium CM (“Common Mycobacteria”) test (Hain Lifescience, Nehren, Germany) performed according to the manufacturer’s instructions. Non-tuberculous mycobacteria were further identified as described below.
6
Mycobacterial Analysis Pipeline in Cameroon
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The study was conducted at the TB-NRL for Bacteriological analysis of samples and JHY for clinical evaluation of confirmed NTM patients.
The TB-NRL is located at the Mycobacteriology Unit of the Centre Pasteur du Cameroun (CPC). The TB-NRL have a technical platform for TB bacteriological analysis using WHO recommended rapid diagnosis (WRD), TB-LAMP and Xpert MTB/ RIF; BACTEC MGIT 960 for Culture and drug susceptibility testing (DST) and NTM bacteriological analysis using reverse hybridization-based LPA, GenoType Mycobacterium CM and GenoType Mycobacterium AS (Hain Lifescience, Nehren Germany). This laboratory received samples from presumptive TB and NTM cases coming from about 93 Diagnosis and Treatment Centers (DTC) from 03 regions of Cameroon (Centre, Est and South) for Culture and DST analysis.
The JHY is a reference hospital for pneumology and a DTC of Yaounde and its surrounding areas. JHY is also specialized in the management of patients infected with multi-drug-resistant M. tuberculosis. The hospital provides diagnostic tests for TB using sputum smear microscopy and TB-LAMP tests.
The CPC and Jamot hospital are in close vicinity and have a long history of collaborative research.
The TB-NRL is located at the Mycobacteriology Unit of the Centre Pasteur du Cameroun (CPC). The TB-NRL have a technical platform for TB bacteriological analysis using WHO recommended rapid diagnosis (WRD), TB-LAMP and Xpert MTB/ RIF; BACTEC MGIT 960 for Culture and drug susceptibility testing (DST) and NTM bacteriological analysis using reverse hybridization-based LPA, GenoType Mycobacterium CM and GenoType Mycobacterium AS (Hain Lifescience, Nehren Germany). This laboratory received samples from presumptive TB and NTM cases coming from about 93 Diagnosis and Treatment Centers (DTC) from 03 regions of Cameroon (Centre, Est and South) for Culture and DST analysis.
The JHY is a reference hospital for pneumology and a DTC of Yaounde and its surrounding areas. JHY is also specialized in the management of patients infected with multi-drug-resistant M. tuberculosis. The hospital provides diagnostic tests for TB using sputum smear microscopy and TB-LAMP tests.
The CPC and Jamot hospital are in close vicinity and have a long history of collaborative research.
7
Identifying Mycobacterial Species from Respiratory Samples
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Written informed consent was provided by all patients. Mycobacterial cultures were grown from respiratory samples of all five patients in mycobacterial growth indicator tubes (MGIT 960, Becton Dickinson, Heidelberg, Germany). Positive cultures were further analyzed using a Ziehl-Neelsen and auramine-rhodamine staining. After detection of acid-fast rods in microscopy, we used the GenoType Mycobacterium CM (Hain Lifescience, Nehren, Germany) for further species determination. Cell morphology was analyzed after auramine-rhodamine staining. All isolation work was carried out in an ISO 17025 accredited clinical microbiology laboratory using internal controls for growth (ATCC® 27294) and microscopy (Axonlab, Luzern, Switzerland) as standard. Laboratory contamination is highly unlikely, especially as isolates were found sporadically over four years, and also in one case within samples from the same patient.
8
Mycobacterium Species Identification
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The isolated strains were identified to species level using GenoType®Mycobacterium CM (Hain Lifescience, Nahren, Germany). The test was performed according to the manufacturer’s instructions.
9
Sputum TB Diagnosis and HIV Testing
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Sputum specimens underwent fluorochrome microscopy and liquid culture, at the National Health Laboratory Services of South Africa. Ten percent of all microscopy slides were double read, for quality control purposes. Positive cultures underwent speciation using GenoType® Mycobacterium CM (Hain Lifescience, Nehren, Germany) and drug susceptibility testing for isoniazid and rifampicin.
Anonymised urine samples were tested for HIV antibodies using the MAXIM HIV-1 urine EIA (Maxim Biomedical Inc, MD, USA). The urine EIA has a reported sensitivity of 99% and specificity of 90% among high HIV risk populations [29] .
Anonymised urine samples were tested for HIV antibodies using the MAXIM HIV-1 urine EIA (Maxim Biomedical Inc, MD, USA). The urine EIA has a reported sensitivity of 99% and specificity of 90% among high HIV risk populations [29] .
10
DNA Hybridization and Mycobacterium Identification
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DNA hybridization was performed on a GeneAmp 2720 DNA amplifier (Thermo Fisher Scientific, USA) and a GeneAmp PCR System 9700 electric thermostat for PCR amplification of DNA (Applied Biosystems Inc., USA), using GenoType Mycobacterium CM and GenoType Mycobacterium AS kits (Hain Lifescience GmbH, Germany).
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