Operational Feasibility of Using Loop-Mediated Isothermal Amplification for Diagnosis of Pulmonary Tuberculosis in Microscopy Centers of Developing Countries

Abstract

The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified manual DNA extraction was evaluated for accuracy and ease of use. The sensitivity of LAMP in smear-and culture-positive sputum specimens was 97.7% (173/177 specimens; 95% confidence interval [CI], 95.5 to 99.9%), and the sensitivity in smear-negative, culture-positive specimens was 48.8% (21/43 specimens; CI, 33.9 to 63.7%). The specificity in culture-negative samples was 99% (500/505 specimens; CI, 98.1 to 99.9%). The average hands-on time for testing six samples and two controls was 54 min, similar to that of sputum smear microscopy. The optimal amplification time was 40 min. No indeterminate results were reported, and the interreader variability was 0.4%. Despite the use of a single room without biosafety cabinets for all procedures, no DNA contamination was observed. The assay was robust, with high end-point stability and low rates of test failure. Technicians with no prior molecular experience easily performed the assay after 1 week of training, and opportunities for further simplification of the assay were identified.

“Paper Machine” for Molecular Diagnostics

“…22−25 This robust tolerance toward inhibitors further streamlines assays, because it simplifies sample preparation and complexity/expense of equipment. 26 Although devices integrating LAMP amplification with detection have entered the market (for example, the IllumiGene system; Meridian Bioscience, Cincinnati, OH), they have not directly incorporated the steps necessary for sample preparation into the instrument. 27 Further, as products targeted toward developed world settings, they employ complex optical systems and user interfaces that increase cost and, especially, the requirements for maintenance and repair.…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…Three main technical aspects of the test are (a) amplification of DNA with LAMP, (b) ultra-rapid extraction (PURE) of DNA that can remove inhibitors of the test without significant loss of genetic material and (c) straightforward visual detection of the result using fluorescence under ultra-violet light. 98,99 The Loopamp TM LF-160 machine does not need maintenance or calibration which makes the overall process simple and easy to pick up; dismissing the need for extensively trained personnel. The sensitivity of TB LAMP is 100 times higher than sputum smear microscopy.…”

Rapid Identification of Mycobacterium tuberculosis in BACTEC MGIT960 Cultures by In-House Loop-Medicated Isothermal Amplification

“…LAMP generates a large amount of DNA with high specificity, allowing amplification to be detected by the naked eye as fluorescence or turbidity (13)(14)(15)(16)(17). LAMP has been used to detect a number of infectious organisms, including M. tuberculosis (18,19), and has been proposed as a useful tool for the rapid and accurate diagnosis of M. tuberculosis in resource-limited laboratories (20,21). In this study, we used an in-house LAMP assay to rapidly identify MTBC from MGIT culture in Thailand.…”