Clinical Evaluation of Diagnosis Efficacy of Active Mycobacterium tuberculosis Complex Infection via Metagenomic Next-Generation Sequencing of Direct Clinical Samples

Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.

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Laboratory implementation of Next Generation Sequencing for medium and high throughput whole genome analysis of clinical M. tuberculosis complex strains

10.26226/morressier.5991c409d462b80292388d15 ◽

2017 ◽

Author(s):

Christian Utpatel

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Genome Analysis ◽

Whole Genome ◽

Next Generation ◽

Tuberculosis Complex ◽

Whole Genome Analysis ◽

Generation Sequencing

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Pulmonary coinfection of Mycobacterium tuberculosis and Tropheryma whipplei: a case report

Journal of Medical Case Reports ◽

10.1186/s13256-021-02899-y ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Binghua Zhu ◽

Jing Tang ◽

Rong Fang ◽

Xuejie Fei ◽

Qing Wang ◽

...

Keyword(s):

Computed Tomography ◽

Mycobacterium Tuberculosis ◽

Next Generation Sequencing ◽

Tropheryma Whipplei ◽

Diagnostic Methods ◽

Next Generation ◽

Computed Tomography Scan ◽

Chest Computed Tomography ◽

Tomography Scan ◽

Generation Sequencing

Abstract Background We diagnosed a clinical case of pulmonary infection involving Mycobacterium tuberculosis and Tropheryma whipplei in a patient with acute respiratory distress syndrome. The diagnosis was assisted by metagenomic next-generation sequencing of bronchoalveolar lavage fluid. Case presentation A 44-year-old Han Chinese inmate was transferred to the emergency department because of dry cough, chest tightness, and shortness of breath. The patient’s body temperature rose to 39.3 °C following empirical cephalosporin treatment for 1 week. The blood CD4+/CD8+ ratio was 0.7, suggesting immunodeficiency. Routine microbiological tests were performed, and tuberculosis interferon gamma release assays were positive. Mycobacterium tuberculosis polymerase chain reaction was also positive. Chest computed tomography scan revealed miliary nodules and ground-glass opacifications, which were in accordance with tuberculosis. To fully examine the etiology, we performed routine laboratory tests and metagenomic sequencing, the results of which indicated the presence of Mycobacterium tuberculosis and Tropheryma whipplei. We administered anti-tuberculosis regimen in combination with trimethoprim/sulfamethoxazole. The patient recovered, with chest computed tomography scan showing absorption of lesions. Conclusions Compared with traditional diagnostic methods such as culture and serology, metagenomic next-generation sequencing has the advantage of detecting a wide array of microorganisms in a single test and therefore can be used for clinical diagnosis of rare pathogens and microbial coinfections. It is particularly useful for immunocompromised patients as they are more prone to infection by opportunistic microorganisms.

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Clinical evaluation of a dedicated next generation sequencing panel for routine glioma diagnostics

Acta Neuropathologica Communications ◽

10.1186/s40478-018-0633-y ◽

2018 ◽

Vol 6 (1) ◽

Cited By ~ 11

Author(s):

Nathalie E. Synhaeve ◽

Martin J. van den Bent ◽

Pim J. French ◽

Winand N. M. Dinjens ◽

Peggy N. Atmodimedjo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Clinical Evaluation ◽

Next Generation ◽

Generation Sequencing

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Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009779 ◽

2021 ◽

Vol 15 (10) ◽

pp. e0009779

Author(s):

Fakhriddin Sarzhanov ◽

Funda Dogruman-Al ◽

Monica Santin ◽

Jenny G. Maloney ◽

Ayse Semra Gureser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gastrointestinal Symptoms ◽

Molecular Methods ◽

Clinical Samples ◽

Rrna Gene ◽

Next Generation ◽

Dientamoeba Fragilis ◽

Significant Difference ◽

Generation Sequencing ◽

Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

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