Study settings

In this study, we collected sputum samples at the State Tuberculosis Demonstration and Training Centre (State TB Cell) in Thiruvananthapuram, Kerala, India. The State TB Cell has TB testing facilities for culture, Xpert MTB/RIF, and smear test methods. The study was conducted from October 2019 to March 2020 and January 2023 to March 2024, during which we collected sputum samples from presumptive TB patients. The gap in the study was due to the SARS-CoV-2 pandemic. The samples were tested using MGIT Culture as the microbiological reference standard (hereafter named MRS), sputum smear microscopy, Xpert MTB/RIF, and rt-LAMP assays.

Sample size calculation

Buderer’s formula was utilized to determine the required sample size to evaluate the assay’s diagnostic parameters to achieve the desired level of accuracy in sensitivity and specificity²⁰. Assuming a sensitivity and specificity of 90% (0.90) for rt-LAMP and a 10% prevalence of TB among suspected cases being tested at the State TB cell in Trivandrum²¹ we calculated a sample size of 346. Therefore, we included 350 patients in the study to ensure a representative sample.

Recruitment and inclusion criteria

All patients suspected of having TB and visiting the State TB Cell were referred for CB-NAAT or culture tests by a chest physician at peripheral, secondary, or tertiary hospitals. The State TB Center offers free TB diagnostic testing for suspected patients. This study included patients over 15 years old who were suspected of pulmonary TB.

Real-time LAMP (rt-LAMP) kit

rt-LAMP primers for the mpt64 gene (GenBank accession number NC_000962.3) were designed based on melting temperature (Tm) critical for the LAMP assay using LAMP designer 1.16 software (Premier Biosoft Interpairs, USA). The primer details, including its positions in the mpt64 gene and specificity to the M. tuberculosis complex, are provided in Supplementary Table S1.

The rt-LAMP kit consisted of a 200 µL master mix containing 1.6 µM Forward and Backward Inner Primers (FIP and BIP), 0.4 µM Forward and Backward outer primers (F3 and B3), 0.2 µM Loop primers (LF and LB), 1X WarmStart LAMP Master Mix (NEB, USA), and 2 µM SYTO 16, with nuclease-free water added to reach a final volume for 10 reactions. For each reaction, 20 µL of the master mix was aliquoted into a PCR tube, and 5 µL (1.5 ng/µL) of Mycobacterium tuberculosis H37Rv genomic DNA (ATCC 27294) was added, making the final reaction volume to 25 µL. The rt-LAMP assay was carried out at 65⁰C for 40 min in a QuantStudio 5 qPCR machine (ThermoFisher Scientific, USA) programmed in isothermal cycles. The fluorescence levels were recorded every minute for 40 min using QuantStudio Design and Analysis Software v1.5.1.

Development of proof of concept of the rt-LAMP

Recombinant mpt64 plasmid as positive control

For rt-LAMP, we selected the mpt64 gene, which is a single-copy gene per genome of M. tuberculosis. The primary reason for this selection was to make the assay more quantitative and to avoid false negatives, especially for the South Asian population, where Mycobacterium tuberculosis complex (MTB complex) may lack the IS6110 gene²².

The mpt64 gene construct was cloned using the Gibson assembly protocol²³. The primers were designed to amplify the mpt64 gene fragment and 20 base pairs of homology to the linearized (digested with Xba1 and HindIII) cloning vector pL4440 (Addgene #1654) using the software NEBuilder Assembly tool, v 2.7.1. The primer sequences are given below:

Forward 5′ cgaggtcgacggtatcgatATCGTTTTGCTCTGTTGTTC 3′.

Reverse 5′ ccaccgcggtggcggccgctACGTGGGACCAATACCTG 3′.

The amplified mpt64 gene fragment (666 bp) was cloned into the XbaI and HindIII digested pL4440 (2712 bp) vector. The recombinant plasmid was further transformed into DH5α E.coli strain and isolated on a large scale. The plasmid containing mpt64 was named pL-mpt64 and was used as the positive control for both assay standardisation and for the limit of detection assay.

Limit of detection

To assess the quantitative sensitivity of the rt-LAMP assay, serial titration of pL-mpt64 DNA in deionized water was prepared and used as templates. The pL-mpt64 plasmid concentration measured was converted into copy numbers by following the equation²⁴: Number of DNA copies/µl = (M x 6.022×10²³x10^-9)/ (n x660)

M is the amount of DNA in ng/µl, n is the total base pair of DNA target (3334 bp for pLmpt-64) plasmid, Avogadro’s number 6.022 × 10²³ and 660 is the average molecular weight of the DNA base pair.

The amplification time of rt-LAMP was plotted against the log values of copies of DNA/µl for the analysis. The assays were performed with DNA copies ranging from 1.91 × 10⁸ to 1.91 × 10⁰, with each dilution assessed in triplicate.

Validation of the rt-LAMP using clinical samples

In this study, 350 presumptive pulmonary TB sputum samples were collected and subjected to smear microscopy, Xpert MTB/RIF, MRS, and rt-LAMP assays. Patients’ written informed consent was obtained to collect the epidemiological, demographic and clinical data (Table S2).

Sample collection

For the study, the morning and spot sputum samples were collected from 350 presumptive TB patients in sterile sputum collection tubes. The morning sputum sample was used for MRS and the spot sputum sample was further divided into 1 ml each for smear microscopy testing, Xpert MTB/RIF and rt-LAMP assay.

The flow chart below illustrates the collection and allocation of samples for the specified tests (Fig. 1).

Workflow of the study. 350 presumptive TB patients were included in the study.

Sputum Smear test

The smear test was carried out, using NALC-NaOH processed sputum samples, following the Ziehl–Neelsen (ZN) staining procedure. The results were graded according to RNTCP guidelines²⁵.

MRS: MGIT culture

The sputum samples were decontaminated, adding an equal volume of NALC-NaOH solution (containing 2% NaOH).

The culturing of MTB was performed using MGIT protocol in tubes containing Middle brook 7H9 broth²⁶. The tubes were incubated at 37 °C for 42 days and monitored for fluorescence using the MGIT 960 instrument (BD, USA) to detect mycobacterial growth. All MGIT-positive cultures were confirmed as Mycobacterium tuberculosis complex (MTBc) using the MPT64 antigen-based test (SD Bioline TB Ag MPT64 Rapid, Abbott, USA)²⁷.

MGIT culture results are taken as microbiological research standard (MRS).

Xpert MTB/RIF

The Xpert MTB/RIF assay was performed by following the kit protocol (Cepheid, CA, USA)²⁸. The results were interpreted semi-quantitatively using cycle threshold (Ct) values as follows: very low (Ct above 28), low (Ct 22–28), medium (Ct 16–22), and high (Ct below 16) using GeneXpert software (see Supplementary Table S2).

DNA extraction from sputum samples for rt-LAMP

From sputum samples treated with NALC-NaOH, the DNA was extracted using the standard protocol with modifications²⁹. Briefly, around 2 ml of patients’ sputum samples were treated with an equal volume of NALC-NaOH solution. The sample was centrifuged at 800 × g for 10 min, and the supernatant was discarded. 100 µl Lysis buffer (0.4 M NaCl, 2 mM EDTA, 10 mM Tris–Cl, and 0.5% Triton X-100, pH 8) was added to the pellet and resuspended by vortexing. The tubes were incubated at 95 °C for 20 min before adding 100 µl neutralization buffer (10 mM Tris–Cl, pH 7.5). The tubes were sealed and stored at 4 °C till use. The samples were used for rt-LAMP assay without further purification.

Evaluation of rt-LAMP assay

A volume of 5 µl of the extracted DNA sample was added to a 20 µL single-tube rt-LAMP kit containing 1X WarmStart LAMP mix, six primers, and SYTO 16 dye. The rt-LAMP assay was performed at 65 °C for 40 min using a QuantStudio™ 5 qPCR machine (ThermoFisher Scientific, USA). Fluorescence readings were recorded every minute for 40 min using QuantStudio™ Design and Analysis Software v1.5.1.

Quality control (QC)

QC was ensured in the form of sterility and performance testing of each batch of rt-LAMP. Performance testing of reagents was carried out using positive (pL-mpt64 DNA) and negative control (without DNA) in each set of tests.

Data analysis

rt-LAMP kit was evaluated against the reference Standards, MRS, Xpert MTB/RIF, and smear test. The discriminative ability of the rt-LAMP test was quantified by measures of diagnostic accuracy, including sensitivity and specificity, positive and negative predictive values (PPV, NPV), likelihood ratio, Youden index, ROC curve, and accuracy compared to culture, Xpert MTB/RIF and smear tests.

Ethical considerations

Ethical clearance for the research study was obtained from the SCTIMST Institutional Ethics Committee (IEC/1230). Patients provided written informed consent to collect their epidemiological, demographic, and clinical data, which was available at the State TB Center when the sample was submitted. The study was conducted in accordance with the Declaration of Helsinki.