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

ABSTRACTBackground 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+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green+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 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: Diagnosisdry methyl greenGeneXpertmulti-targeted LAMPMtbpleural TBSYBR Green ISYBR Green I+dUTP-UNGDisclaimerAs a service to authors and researchers we are providing this version of an accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proofs will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to these versions also. Author contributionsPK 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 Sheoran 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.Declaration of interestThe 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.Reviewer disclosuresA 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 discloseAcknowledgementsThanks are due to Mandira Varma-Basil, Microbiology Dept, Vallabhbhai Patel Chest Institute, the 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 '63rd Annual Conference of Association of Microbiologists of India-2023' at the MDU, Rohtak (Haryana), India on 2-4 February. 2023.Figure 1. Grouping of the study participants. (A) The flowchart of the study participants categorized into different groups to diagnose pleural TB by gel-based, SYBR Green I, SYBR Green I+dUTP-UNG and dry methyl green MT-LAMP assays. (B) The flowchart of the study participants categorized into different groups to diagnose pleural TB by SYBR Green I+dUTP-UNG MT-LAMP and SYBR Green I MT-LAMP assays. (C) The flowchart of the study participants categorized into different groups to diagnose pleural TB by SYBR Green I+dUTP-UNG MT-LAMP and GeneXpert assays.Display full sizeFigure 2. LOD determination for purified Mtb H37Rv DNA by gel-based (A) mpt64 LAMP and (B) IS6110 LAMP. Lane M, molecular marker (100 bp DNA ladder), Lanes 1-6, serial tenfold dilutions of purified DNA (ranging from 104 fg/μL to 0.1 fg/μL), Lane 7, negative control (no template DNA).Display full sizeFigure 3. LOD determination for purified Mtb H37Rv DNA by (A) mpt64 SYBR Green I and (B) mpt64 SYBR Green I+dUTP-UNG LAMP showing fluorescence under UV-transilluminator: Tubes 1-6, serial tenfold dilutions of purified Mtb DNA (ranging from 104 fg/μL to 0.1 fg/μL), where Tubes 1-4 revealed a green fluorescence, indicating a positive LAMP reaction, whereas Tubes 5-6 revealed an orange color, indicating a negative LAMP reaction; Tube 7, negative control (no template DNA). (C) mpt64 dry methyl green LAMP showing a blue-green color by a naked eye. Tubes 1-6, serial tenfold dilutions of purified Mtb DNA (ranging from 104 fg/μL to 0.1 fg/μL), where Tubes 1-4 revealed a blue-green color, indicating a positive LAMP reaction, while Tube 5-6 revealed no color change, indicating a negative LAMP reaction; Tube 7, negative control (no template DNA).Display full sizeFigure 4. LOD determination for purified Mtb H37Rv DNA by mpt64 LAMP in spiked pleural fluids of non-TB controls analyzed by (A) Agarose gel electrophoresis: Lane M, molecular marker (100 bp DNA ladder); lane 1-6, serial tenfold dilutions of pleural fluid of non-TB control spiked with purified Mtb DNA (ranging from 104 fg/μL to 0.1 fg/μL); lane 7, negative control (no template DNA), (B) SYBR Green I reaction: Tubes 1-6, serial tenfold dilutions of purified MtbDNA (ranging from 104 fg/μL to 0.1 fg/μL), where Tubes 1-3 showed a green fluorescence under UV-transilluminator, indicating a positive LAMP reaction; Tube 7, negative control (no template DNA), (C) dry methyl green reaction: Tubes 1-6, serial tenfold dilutions of purified Mtb DNA (ranging from 104 fg/μL to 0.1 fg/μL), wherein Tubes 1-3 showed a blue-green color by a naked eye, indicating a positive LAMP reaction; Tube 7, negative control (no template DNA) and (D) SYBR Green I+dUTP-UNG reaction: Tubes 1-6, serial tenfold dilutions of purified Mtb DNA (ranging from 50 pg/μL to 0.5 fg/μL), wherein Tubes 1-3 showed a green fluorescence under UV-transilluminator, indicating a positive LAMP reaction; Tube 7, negative control (no template DNA). The experiment was performed with six pleural fluid specimens obtained from non-TB controls and one of the representative figures has been shown.Display full sizeFigure 5. Representatives of pleural TB/non-TB specimens analyzed by mpt64 SYBR Green I+dUTP-UNG LAMP under UV-transilluminator, wherein Tube 1, positive control (purified Mtb DNA, 10 ng/mL); Tube 2, negative control (no template DNA); Tube 3-5 and 8, positive pleural TB specimens (showing a green fluorescence); Tube 6-7, non-TB controls.Display full sizeAdditional informationFundingThis 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.