Diagnosis of pleural tuberculosis by multi-targeted loop-mediated isothermal amplification assay based on SYBR Green I reaction: comparison with GeneXpert® MTB/RIF assay

ABSTRACT

Background

Diagnosis of pleural tuberculosis (TB) is tedious owing to its close resemblance with malignant pleural effusion and sparse bacterial load in clinical specimens. There is an immediate need to design a rapid and dependable diagnostic test to prevent unnecessary morbidity/mortality.

Research design and methods

A multi-targeted loop-mediated isothermal amplification (MT-LAMP) was deliberated using mpt64 and IS6110 to diagnose pleural TB within pleural fluids/biopsies. MT-LAMP products were analyzed by gel-based and visual detection methods, viz. SYBR Green I, SYBR Green I+deoxyuridine triphosphate uracil-N-glycosylase (dUTP-UNG), and dry methyl green reactions.

Results

In a pilot study, while assessing pleural TB/non-TB control subjects (n = 40), both SYBR Green I+dUTP-UNG/gel-based MT-LAMP assays exhibited better sensitivity/specificity than SYBR Green I and dry methyl green MT-LAMP. Since it is facile to work with SYBR Green I+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green I+dUTP-UNG in a higher number of specimens (n = 97), which revealed somewhat higher sensitivity (85.2 vs. 81.5%) and specificity (97.7 vs. 90.7%) than SYBR Green I MT-LAMP. Furthermore, the sensitivity attained by SYBR Green I+dUTP-UNG MT-LAMP was significantly higher (p < 0.001) than GeneXpert.

Conclusions

Our SYBR Green I+dUTP-UNG MT-LAMP is a simple and reliable method to diagnose pleural TB, which may translate into a point-of-care test.

KEYWORDS:

• Diagnosis
• dry methyl green
• GeneXpert
• multi-targeted LAMP
• Mtb
• pleural TB
• SYBR Green I
• SYBR Green I+dUTP-UNG assay

Acknowledgments

Thanks are due to Mandira Varma-Basil, Microbiology Dept, Vallabhbhai Patel Chest Institute, University of Delhi for providing us with the purified Mtb H₃₇Rv DNA. We are grateful to Sanjay Kumar, Microbiology Dept, UHS, Rohtak for performing GeneXpert assays. We are also thankful to Director, Centre for Biotechnology, MDU, Rohtak for providing the laboratory facilities. Part of this work was presented in the ‘63^rd Annual Conference of Association of Microbiologists of India-2023’ at the MDU, Rohtak (Haryana), India on 2-4 February. 2023.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Author contributions

PK Mehta conceived the study and designed the experiments, while A Soni, PK Mehta, B Dahiya, K Nehra, V Hooda, A Yadav and N Kumar carried out majority of the experiments. A Sheoran, A Soni, B Dahiya, V Hooda, K Nehra and PK Mehta participated in data analysis. A Guliani and V Kumar provided clinical samples and assisted in the interpretation of clinical data, whereas PK Mehta, A Soni and B Dahiya prepared the final draft of the manuscript. All the authors read the manuscript, participated in editing the manuscript and approved the final version of the manuscript.

Reviewer disclosures

A peer reviewer on this manuscript received an honorarium from Expert Review of Respiratory Medicine for their review work, but have no other relevant financial relationships to disclose.

Additional information

Funding

This work is financially supported by the Indian Council of Medical Research, New Delhi ((5/8/25/ITRC/Diag/2022/ECD-1). A. Soni acknowledges the University Grants Commission (Ref. No 221610106016) and DCRUST, Murthal for awarding the Junior Research Fellowship and University Research Scholarship, respectively.