Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens
ABSTRACT Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients’ sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-02-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.01358-21 |
| _version_ | 1819279622095765504 |
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| author | Jing Song Weili Du Zichen Liu Jialu Che Kun Li Nanying Che |
| author_facet | Jing Song Weili Du Zichen Liu Jialu Che Kun Li Nanying Che |
| author_sort | Jing Song |
| collection | DOAJ |
| description | ABSTRACT Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients’ sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains unclear. An amplicon-based targeted NGS sequencing panel was developed to predict susceptibility to 9 antituberculosis drugs, including 3 first-line drugs, by directly detecting FFPE tissues. A total of 178 tissue samples from TB patients who underwent phenotypic drug susceptibility test were retrospectively tested from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. Phenotypic drug susceptibility test results were used as the reference standard. We identified 22 high-quality mutations from 178 FFPE tissue samples, including 15 high+moderate+minimal confidence-level mutations associated with drug resistance (rpoB D435V, S450F/L; KatG S315T; inhA-fabG promoter c-15t; embB G406S, M306V; rpsL K43R, K88R, rrs a1401g, a514c; gyrA D94G/Y/A, A90V), 6 mutations not associated with resistance (rpoB D435Y, H445S, L430P, L452P; embB G406A/D), and one mutation site embB M306I defined as indeterminate. Compared to the phenotypic method, sensitivities (95% CI) for rifampicin, isoniazid, and ethambutol were 96% (79.65–99.90%), 93.55% (78.58–99.21%), and 71.43% (35.24–92.44%), respectively; while for second-line drugs, it varied from 23.53% (9.05–47.77%) for capreomycin to 86.84% (72.20–94.72%) for streptomycin. Specificities for all drugs were satisfactory (>94.51%). Therefore, important pathological FFPE tissue samples, despite partially degraded DNA, can be used as essential specimens for molecular diagnosis of drug resistant TB by amplicon-based targeted NGS technology. IMPORTANCE Amplicon-based targeted NGS technology focuses on a set of gene mutations of known or suspected associations with drug susceptibility in Mycobacterium tuberculosis (MTB). This method offers many benefits, such as low sequencing cost, easy customization, high throughput, shorter testing time and not culture dependent. Formalin-fixed and paraffin-embedded (FFPE) tissues are important pathological specimen in diagnosing tuberculous disease because they are noninfectious and provide excellent preservation of tissue morphology with low storage cost. However, the performance of amplicon-based targeted NGS method on FFPE samples has not been reported yet. Therefore, we evaluated the performance of this method using FFPE samples collected from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. We demonstrate that the amplicon-based targeted NGS method performs excellent on FFPE samples, and it can be applied to pathological diagnosis of drug resistant tuberculosis. |
| first_indexed | 2024-12-24T00:30:49Z |
| format | Article |
| id | doaj.art-c687f5bac152483e96dacf8481a9c53a |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-12-24T00:30:49Z |
| publishDate | 2022-02-01 |
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| series | Microbiology Spectrum |
| spelling | doaj.art-c687f5bac152483e96dacf8481a9c53a2022-12-21T17:24:16ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-02-0110110.1128/spectrum.01358-21Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE SpecimensJing Song0Weili Du1Zichen Liu2Jialu Che3Kun Li4Nanying Che5Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, ChinaDepartment of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, ChinaDepartment of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, ChinaDepartment of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, ChinaDepartment of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, ChinaDepartment of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, ChinaABSTRACT Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients’ sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains unclear. An amplicon-based targeted NGS sequencing panel was developed to predict susceptibility to 9 antituberculosis drugs, including 3 first-line drugs, by directly detecting FFPE tissues. A total of 178 tissue samples from TB patients who underwent phenotypic drug susceptibility test were retrospectively tested from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. Phenotypic drug susceptibility test results were used as the reference standard. We identified 22 high-quality mutations from 178 FFPE tissue samples, including 15 high+moderate+minimal confidence-level mutations associated with drug resistance (rpoB D435V, S450F/L; KatG S315T; inhA-fabG promoter c-15t; embB G406S, M306V; rpsL K43R, K88R, rrs a1401g, a514c; gyrA D94G/Y/A, A90V), 6 mutations not associated with resistance (rpoB D435Y, H445S, L430P, L452P; embB G406A/D), and one mutation site embB M306I defined as indeterminate. Compared to the phenotypic method, sensitivities (95% CI) for rifampicin, isoniazid, and ethambutol were 96% (79.65–99.90%), 93.55% (78.58–99.21%), and 71.43% (35.24–92.44%), respectively; while for second-line drugs, it varied from 23.53% (9.05–47.77%) for capreomycin to 86.84% (72.20–94.72%) for streptomycin. Specificities for all drugs were satisfactory (>94.51%). Therefore, important pathological FFPE tissue samples, despite partially degraded DNA, can be used as essential specimens for molecular diagnosis of drug resistant TB by amplicon-based targeted NGS technology. IMPORTANCE Amplicon-based targeted NGS technology focuses on a set of gene mutations of known or suspected associations with drug susceptibility in Mycobacterium tuberculosis (MTB). This method offers many benefits, such as low sequencing cost, easy customization, high throughput, shorter testing time and not culture dependent. Formalin-fixed and paraffin-embedded (FFPE) tissues are important pathological specimen in diagnosing tuberculous disease because they are noninfectious and provide excellent preservation of tissue morphology with low storage cost. However, the performance of amplicon-based targeted NGS method on FFPE samples has not been reported yet. Therefore, we evaluated the performance of this method using FFPE samples collected from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. We demonstrate that the amplicon-based targeted NGS method performs excellent on FFPE samples, and it can be applied to pathological diagnosis of drug resistant tuberculosis.https://journals.asm.org/doi/10.1128/spectrum.01358-21amplicon-based targeted NGStuberculosisdrug resistant mutationsFFPE tissue samples |
| spellingShingle | Jing Song Weili Du Zichen Liu Jialu Che Kun Li Nanying Che Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens Microbiology Spectrum amplicon-based targeted NGS tuberculosis drug resistant mutations FFPE tissue samples |
| title | Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens |
| title_full | Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens |
| title_fullStr | Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens |
| title_full_unstemmed | Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens |
| title_short | Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens |
| title_sort | application of amplicon based targeted ngs technology for diagnosis of drug resistant tuberculosis using ffpe specimens |
| topic | amplicon-based targeted NGS tuberculosis drug resistant mutations FFPE tissue samples |
| url | https://journals.asm.org/doi/10.1128/spectrum.01358-21 |
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