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 Validation of NovelMycobacterium tuberculosisIsoniazid Resistance Mutations Not Detectable by Common Molecular Tests

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Justin L. Kandler ◽  
Alexandra D. Mercante ◽  
Tracy L. Dalton ◽  
Matthew N. Ezewudo ◽  
Lauren S. Cowan ◽  
...  
Keyword(s):  
Reference Strain ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
Resistance Mutations ◽  
Content Type ◽  
Diagnostic Assays ◽  
Molecular Tests ◽  
The Common ◽  
Promoter Mutations

ABSTRACTResistance to the first-line antituberculosis (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-RMycobacterium tuberculosiscomplex (MTBC) clinical isolates that lacked the commonkatGS315T orinhApromoter 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 clinicalkatGmutations 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 recombineeredkatGmutations conferred resistance to INH at a MIC 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 useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.

scholarly journals Isoniazid Resistance in Mycobacterium tuberculosis Is a Heterogeneous Phenotype Composed of Overlapping MIC Distributions with Different Underlying Resistance Mechanisms

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Arash Ghodousi ◽  
Elisa Tagliani ◽  
Eranga Karunaratne ◽  
Stefan Niemann ◽  
Jennifer Perera ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanisms ◽  
Whole Genome ◽  
Tube System ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Indicator Tube ◽  
Promoter Mutations ◽  
Level Resistance ◽  
Growth Indicator

ABSTRACT MIC testing using the Bactec mycobacteria growth indicator tube system 960 of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing explained most of the levels of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration endorsed by the Clinical and Laboratory Standards Institute to detect low-level resistance to isoniazid and is, consequently, likely not optimally set.

scholarly journals Molecular Diagnostic Testing for Aspergillus

2016 ◽  
Vol 54 (11) ◽  
pp. 2655-2660 ◽  
Author(s):  
Margaret V. Powers-Fletcher ◽  
Kimberly E. Hanson
Keyword(s):  
Direct Detection ◽  
Diagnostic Testing ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
Content Type ◽  
Clinical Specimens ◽  
Molecular Tests ◽  
Assay Performance ◽  
Clinical Mycology ◽  
Invasive Infections

The direct detection ofAspergillusnucleic acid in clinical specimens has the potential to improve the diagnosis of aspergillosis by offering more rapid and sensitive identification of invasive infections than is possible with traditional techniques, such as culture or histopathology. Molecular tests forAspergillushave been limited historically by lack of standardization and variable sensitivities and specificities. Recent efforts have been directed at addressing these limitations and optimizing assay performance using a variety of specimen types. This review provides a summary of standardization efforts and outlines the complexities of molecular testing forAspergillusin clinical mycology.

scholarly journals Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
S. Battaglia ◽  
A. Spitaleri ◽  
A. M. Cabibbe ◽  
C. J. Meehan ◽  
C. Utpatel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genetic Variants ◽  
Protein Structures ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Clear Understanding ◽  
Data Set ◽  
Gene Target ◽  
Genomic Regions

ABSTRACT The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants’ role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Overview of Molecular Testing of Cytology Specimens

2019 ◽  
Vol 64 (1-2) ◽  
pp. 136-146 ◽  
Author(s):  
Min Huang ◽  
Shuanzeng Wei
Keyword(s):  
Personalized Medicine ◽  
Clinical Laboratory ◽  
Molecular Testing ◽  
Molecular Tests ◽  
Dna And Rna ◽  
Tissue Degradation ◽  
Pertinent Information ◽  
The Common ◽  
Short Time

Objective: Utilizing cytology specimens for molecular testing has attracted increasing attention in the era of personalized medicine. Cytology specimens are clinically easier to access. The samples can be quickly and completely fixed in a very short time of fixation before tissue degradation occurs, compared to hours or days of fixation in surgical pathology specimens. In addition, cytology specimens can be fixed without formalin, which can significantly damage DNA and RNA. All these factors contribute to the superb quality of DNA and RNA in cytology specimens for molecular tests. Study Design: We summarize the most pertinent information in the literature regarding molecular testing in the field of cytopathology. Results: The first part focuses on the types of cytological specimens that can be used for molecular testing, including the advantages and limitations. The second section describes the common molecular tests and their clinical application. Conclusion: Various types of cytology specimens are suitable for many molecular tests, which may require additional clinical laboratory validation.

scholarly journals Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Paolo Miotto ◽  
Andrea M. Cabibbe ◽  
Silke Feuerriegel ◽  
Nicola Casali ◽  
Francis Drobniewski ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multicenter Study ◽  
Genetic Variants ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Resistance Mutations ◽  
High Confidence ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Very High

ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. IMPORTANCE Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

scholarly journals Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis

2006 ◽  
Vol 50 (8) ◽  
pp. 2640-2649 ◽  
Author(s):  
Manzour Hernando Hazbón ◽  
Michael Brimacombe ◽  
Miriam Bobadilla del Valle ◽  
Magali Cavatore ◽  
Marta Inírida Guerrero ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
Sample Selection ◽  
Multidrug Resistant ◽  
Small Sample ◽  
Inverse Association ◽  
Resistance Mutations ◽  
Isoniazid Resistance ◽  
Large Scale Analysis ◽  
Small Sample Sizes

ABSTRACT The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

scholarly journals Validation of the FluoroType MTBDR Assay for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates

2018 ◽  
Vol 56 (6) ◽  
pp. e00072-18 ◽  
Author(s):  
Doris Hillemann ◽  
Carsten Haasis ◽  
Sönke Andres ◽  
Tobias Behn ◽  
Katharina Kranzer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sanger Sequencing ◽  
High Sensitivity ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Molecular Diagnostic ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Specificity And Sensitivity ◽  
Genotype Mtbdrplus

ABSTRACT For Mycobacterium tuberculosis complex (MTBC), the rapid and accurate diagnosis of drug resistance is crucial to ensure early initiation of appropriate therapy. Recently, a new molecular diagnostic test, the FluoroType MTBDR, aimed at detecting rifampin and isoniazid resistance has become available. This study aimed to evaluate the FluoroType MTBDR in comparison to phenotypic drug susceptibility testing (DST) using M. tuberculosis complex isolates. MTBC isolates underwent phenotypic DST and were tested using the FluoroType MTBDR and Genotype MTBDRplus. Sanger sequencing of the key regions of rpoB, katG, inhA, and aphC was performed for isolates with discordant phenotypic and molecular results. Furthermore, isolates with specific wild-type bands missing in the Genotype MTBDRplus, indicating the presence of a mutation, were investigated by Sanger sequencing. Specificity and sensitivity, defined as the proportions of isolates correctly determined as susceptible and resistant by the FluoroType MTBDR compared to phenotypic DST, were calculated. A total of 180 culture isolates were included; phenotypic DST showed 85 isolates susceptible to isoniazid and rifampin, 7 with isoniazid monoresistance, 7 with rifampin monoresistance, and 81 with multidrug resistance. The specificity of the FluoroType MTBDR was 100% (95% confidence interval [CI], 96.0 to 100%) for both rifampin and isoniazid. The sensitivity was 91.7% (95% CI, 83.6 to 96.6%) for isoniazid and 98.9% (95% CI, 93.8 to 100.0%) for rifampin. The FluoroType MTBDR has a high sensitivity and specificity for the detection of rifampin and isoniazid resistance when using culture isolates.

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