scholarly journals Comparison of the Three Molecular Diagnostic Assays for Molecular Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria Species in Sputum Samples

2020 ◽  
Vol 26 (3) ◽  
pp. 170-178
Author(s):  
Jinyoung Bae ◽  
Sung-Bae Park ◽  
Ji-Hoi Kim ◽  
Mi Ran Kang ◽  
Kyung Eun Lee ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Identification ◽  
Nontuberculous Mycobacteria ◽  
Molecular Diagnostic ◽  
Diagnostic Assays

scholarly journals Validation of novel Mycobacterium tuberculosis isoniazid resistance mutations not detectable by common molecular tests

2018 ◽  
Author(s):  
Justin L. Kandler ◽  
Alexandra D. Mercante ◽  
Tracy L. Dalton ◽  
Matthew N. Ezewudo ◽  
Lauren S. Cowan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
Resistance Mutations ◽  
Isoniazid Resistance ◽  
Diagnostic Assays ◽  
Molecular Tests ◽  
The Common ◽  
Promoter Mutations

AbstractResistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of ≥0.25 μg/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R MTBC compared to current targeted molecular testing methods and could provide earlier diagnosis of drug-resistant TB.

scholarly journals Molecular Detection of Mycobacterium tuberculosis in Oral Mucosa from Patients with Presumptive Tuberculosis

2020 ◽  
Vol 9 (12) ◽  
pp. 4124
Author(s):  
Barbara Molina-Moya ◽  
Nelly Ciobanu ◽  
Marta Hernandez ◽  
Cristina Prat-Aymerich ◽  
Valeriu Crudu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Oral Mucosa ◽  
The Elderly ◽  
Storage Conditions ◽  
Molecular Diagnostic ◽  
Smear Microscopy ◽  
X Rays ◽  
Rt Pcr ◽  
Tuberculosis Complex ◽  
Tb 34

Tuberculosis (TB) diagnosis is increasingly based on the detection of Mycobacterium tuberculosis complex (MTBC) DNA in sputum using molecular diagnostic tests as the first test for diagnosis. However, sputum can be difficult to obtain in children, patients without productive cough, and the elderly and approaches testing non-sputum samples are needed. We evaluated whether TB can be detected from the oral mucosa of patients with TB. Adults with presumptive TB were examined using culture, Xpert MTB/RIF, smear microscopy and X-Rays. Oral mucosa swabs collected on PrimeStore-MTM, stored at room temperature if tested within 30 days or at −20 °C if examined at a later time. RT-PCR was performed to detect M. tuberculosis DNA. Eighty patients had bacteriologically-confirmed TB, 34 had bacteriologically-negative TB (negative tests but abnormal X-rays) and 152 were considered not to have TB (not TB). Oral swabs RT-PCR were positive in 29/80 (36.3%) bacteriologically-confirmed, 9/34 (26.5%) bacteriologically-negative and 29/152 (19.1%) not TB. The yield varied among samples stored for less and more than 30 days (p = 0.013) from 61% (11/18) and 29% (18/62) among bacteriologically confirmed, and 30.8% (4/13) and 23.8% (5/21) among bacteriologically-negative participants. Among not TB patients, the specificity was 80.9% (123/152), being 78.3% (18/23) among samples stored less than 30 days and 81.4% (105/129) among samples stored for more than 30 days (p = 0.46). The detection of M. tuberculosis in oral mucosa samples is feasible, but storage conditions may affect the yield.

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

scholarly journals Assessment of the BD MGIT TBc Identification Test for the Detection ofMycobacterium tuberculosisComplex in a Network of Mycobacteriology Laboratories

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Diana Machado ◽  
Jorge Ramos ◽  
Isabel Couto ◽  
Nureisha Cadir ◽  
Inácio Narciso ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sample Preparation ◽  
Nontuberculous Mycobacteria ◽  
Molecular Methods ◽  
Predictive Values ◽  
Acid Fast Bacilli ◽  
Negative Results ◽  
Resource Limited ◽  
Identification Test

We evaluate the performance of the TBcID assay in a panel of 100 acid-fast bacilli cultures. Sixty-four isolates were TBcID positive forMycobacterium tuberculosiscomplex (MTBC), whereas 36 gave negative results. These included 28 nontuberculous mycobacteria, one nonmycobacterial isolate, oneM. tuberculosis, and sixM. bovisBCG strains. This corresponds to a sensitivity of 90.14%, specificity of 100%, and positive and negative predictive values of 100% and 80.55%, respectively. The test is rapid, easy to perform and interpret, and does not require sample preparation or instrumentation. However, a negative result does not exclude the presence of a strain belonging to MTBC, especially when mutations inmpb64gene are present or someM. bovisBCG strains are isolated. The TBcID showed potential to assist in the identification of MTBC when the implementation and usage of molecular methods are often not possible, principally in resource-limited countries.

scholarly journals Use of Quantitative Molecular Diagnostic Assays to Investigate Fusarium Dry Rot in Potato Stocks and Soil

2005 ◽  
Vol 95 (12) ◽  
pp. 1462-1471 ◽  
Author(s):  
D. W. Cullen ◽  
I. K. Toth ◽  
Y. Pitkin ◽  
N. Boonham ◽  
K. Walsh ◽  
...  
Keyword(s):  
Control Strategies ◽  
Disease Incidence ◽  
Harvest Date ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Seed ◽  
Fusarium Spp ◽  
Dry Rot ◽  
Diagnostic Assays ◽  
Final Harvest

Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 104, 105, and 106 conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 104 conidia/ml for F. sulphureum and 105 conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.

scholarly journals Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study

1970 ◽  
Vol 1 (1) ◽  
pp. 14-21 ◽  
Author(s):  
Raunak Shrestha ◽  
Rubin Narayan Joshi ◽  
Kriti Joshi ◽  
Bal Hari Poudel ◽  
Bhupal Govinda Shrestha
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Blood ◽  
Diagnostic Technique ◽  
Molecular Diagnostic ◽  
Multi Drug Resistance ◽  
Base Line ◽  
Blood Samples ◽  
Pcr Rflp ◽  
Development Region ◽  
Whole Blood Samples

Ser315Thr mutations in genes encoding the mycobacteria catalase-peroxidase (KatG) has been associated with the major resistance to isoniazid (INH) in Mycobacterium tuberculosis (MTB). Also G/C polymorphisms in INT4 region of the solute carrier family 11 member 1 gene (SLC11A1) and susceptibility towards tuberculosis (TB) has been demonstrated worldwide. 24 drug resistant MTB culture positive samples and 24 whole?blood samples were collected from different TB patients of Central Development Region of Nepal in 2009. A Polymerase Chain Reaction (PCR) - Restriction Fragment Length Polymorphism (RFLP) assay was carried out in order to investigate Ser315Thr KatG mutation and G/C polymorphism in INT4 region. 4 (16.67%) samples out of 24 MTB culture samples demonstrated the Ser315Thr KatG mutation whereas none of the 24 whole blood samples were found to contain G/C polymorphism in INT4. Though no significant correlation could be found between INT4 polymorphism and TB susceptibility, overall scenario of Nepal cannot be drawn from this data. Molecular diagnostic technique such as PCR-RFLP can be used in a robust scale to carry out base line studies in the TB population of Nepal. Key words: Multi?drug resistance; Tuberculosis; PCR; RFLP Nepal Journal of Biotechnology. Jan. 2011, Vol. 1, No. 1 : 14-21

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