Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during Mycobacterial sequencing programs

ABSTRACTIntroductionDetecting laboratory cross-contamination and mixed tuberculosis infection are important goals of clinical Mycobacteriology laboratories.ObjectivesTo develop a method detecting mixtures of different M. tuberculosis lineages in laboratories performing Mycobacterial next generation sequencing (NGS).SettingPublic Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive Mycobacterial Growth Indicator tubes.MethodsWe analysed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, obtained during development and production use of a diagnostic process using NGS. Counts of the most common (major) variant, and all other variants (non-major variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development.ResultsFor each sample we determined the non-major variant proportions at 55 sets of lineage defining positions. The non-major variant proportion in the two most mixed lineage defining sets (F2 metric) was compared with that in the 47 least mixed lineage defining sets (F47 metric). Three patterns were observed: (i) not mixed by either metric, (ii) high F47 metric suggesting mixtures of multiple lineages, and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevation relative to F47. Pattern (ii) was observed in batches, with similar patterns in the H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in each pattern were 97%, 2.8%, and 0.001%, respectively.ConclusionThe F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis.

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Identifying Mixed Mycobacterium tuberculosis Infection and Laboratory Cross-Contamination during Mycobacterial Sequencing Programs

Journal of Clinical Microbiology ◽

10.1128/jcm.00923-18 ◽

2018 ◽

Vol 56 (11) ◽

Cited By ~ 9

Author(s):

David H. Wyllie ◽

Esther Robinson ◽

Tim Peto ◽

Derrick W. Crook ◽

Adebisi Ajileye ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Process Development ◽

Diagnostic Process ◽

Cross Contamination ◽

Mycobacterium Tuberculosis Infection ◽

Content Type ◽

The Public ◽

Next Generation Sequencing Ngs ◽

Mycobacterial Growth ◽

Defining Sets

ABSTRACT The detection of laboratory cross-contamination and mixed tuberculosis infections is an important goal of clinical mycobacteriology laboratories. The objective of this study was to develop a method to detect mixtures of different Mycobacterium tuberculosis lineages in laboratories performing mycobacterial next-generation sequencing (NGS). The setting was the Public Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive mycobacterial growth indicator tubes. We analyzed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, which were obtained during development and production use of a diagnostic process using NGS. The counts of the most common (major) variant and all other variants (nonmajor variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development. For each sample, we determined the nonmajor variant proportions at 55 sets of lineage-defining positions. The nonmajor variant proportion in the two most mixed lineage-defining sets (F2 metric) was compared with that of the 47 least-mixed lineage-defining sets (F47 metric). The following three patterns were observed: (i) not mixed by either metric; (ii) high F47 metric, suggesting mixtures of multiple lineages; and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevations relative to F47. Pattern ii was observed in batches, with similar patterns in the M. tuberculosis H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in the patterns were 97%, 2.8%, and 0.001%, respectively. The F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis genomes.

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An Atypical Manifestation of Mycobacterium tuberculosis Infection

European Journal of Case Reports in Internal Medicine ◽

10.12890/2021_002598 ◽

2021 ◽

Author(s):

Iván Fernández Castro ◽

María Jesús Isorno-Porto ◽

Ignacio Novo-Veleiro ◽

Clara Casar-Cocheteux ◽

Lucía Barrera-López ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Imaging Techniques ◽

Extrapulmonary Tuberculosis ◽

Tuberculosis Infection ◽

Diagnostic Process ◽

Mycobacterium Tuberculosis Infection ◽

The Past ◽

Aortic Aneurysm And Dissection ◽

Serious Disease ◽

Atypical Manifestation

Introduction: Aortitis is seen in a wide variety of diseases. It was rarely found in the past but this is changing because of new imaging techniques. Case description: We present the case of a 45-year-old man who was found on thyroid ultrasound to have infrarenal aortitis and pathological lymphadenopathies in different locations. After an exhaustive diagnostic process, tuberculous aortitis, an infrequent manifestation of extrapulmonary tuberculosis, was diagnosed. The condition resolved after a 6-month course of antibiotics and a 6-week course of corticosteroids. Conclusion: Tuberculous aortitis is an atypical manifestation of Mycobacterium tuberculosis infection. The absence of typical symptoms and the difficulty of isolating the microorganism makes its diagnosis difficult. Therefore, clinical suspicion, microbiological tests and imaging are key for reaching the diagnosis and starting treatment for a serious disease that can cause aortic aneurysm and dissection.

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The utility of reverse phenotyping: a case of lysinuric protein intolerance presented with childhood osteoporosis

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2021-0018 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Enise Avci Durmusalioglu ◽

Esra Isik ◽

Durdugul Ayyildiz Emecen ◽

Damla Goksen ◽

Samim Ozen ◽

...

Keyword(s):

Autosomal Recessive ◽

Autosomal Recessive Disorder ◽

Diagnostic Process ◽

Homozygous Mutation ◽

Lysinuric Protein Intolerance ◽

Case Presentation ◽

Lysinuric Protein ◽

Next Generation Sequencing Ngs ◽

Protein Intolerance ◽

Generation Sequencing

Abstract Objectives Childhood osteoporosis is often a consequence of a chronic disease or its treatment. Lysinuric protein intolerance (LPI), a rare secondary cause of the osteoporosis, is an autosomal recessive disorder with clinical features ranging from minimal protein intolerance to severe multisystemic involvement. We report a case diagnosed to have LPI using a Next Generation Sequencing (NGS) panel and evaluate the utility of reverse phenotyping. Case presentation A fifteen-year-old-boy with an initial diagnosis of osteogenesis imperfecta, was referred due to a number of atypical findings accompanying to osteoporosis such as splenomegaly and bicytopenia. A NGS panel (TruSight One Sequencing Panel) was performed and a novel homozygous mutation of c.257G>A (p.Gly86Glu) in the SLC7A7 gene (NM_001126106.2), responsible for LPI, was detected. The diagnosis was confirmed via reverse phenotyping. Conclusions Reverse phenotyping using a multigene panel shortens the diagnostic process.

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One Multilocus Genomic Variation Is Responsible for a Severe Charcot–Marie–Tooth Axonal Form

Brain Sciences ◽

10.3390/brainsci10120986 ◽

2020 ◽

Vol 10 (12) ◽

pp. 986

Author(s):

Federica Miressi ◽

Corinne Magdelaine ◽

Pascal Cintas ◽

Sylvie Bourthoumieux ◽

Angélique Nizou ◽

...

Keyword(s):

Genetic Diseases ◽

Trna Synthetase ◽

Genomic Variation ◽

Causative Mutation ◽

Diagnostic Process ◽

Inherited Disorders ◽

Mitofusin 2 ◽

Charcot Marie Tooth ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Charcot–Marie–Tooth (CMT) disease is a heterogeneous group of inherited disorders affecting the peripheral nervous system, with a prevalence of 1/2500. So far, mutations in more than 80 genes have been identified causing either demyelinating forms (CMT1) or axonal forms (CMT2). Consequentially, the genotype–phenotype correlation is not always easy to assess. Diagnosis could require multiple analysis before the correct causative mutation is detected. Moreover, it seems that approximately 5% of overall diagnoses for genetic diseases involves multiple genomic loci, although they are often underestimated or underreported. In particular, the combination of multiple variants is rarely described in CMT pathology and often neglected during the diagnostic process. Here, we present the complex genetic analysis of a family including two CMT cases with various severities. Interestingly, next generation sequencing (NGS) associated with Cov’Cop analysis, allowing structural variants (SV) detection, highlighted variations in MORC2 (microrchidia family CW-type zinc-finger 2) and AARS1 (alanyl-tRNA-synthetase) genes for one patient and an additional mutation in MFN2 (Mitofusin 2) in the more affected patient.

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