RLEP LAMP for the laboratory confirmation of leprosy: towards a point-of-care test

Abstract

Background Nucleic acid-based amplification tests (NAAT), above all (q)PCR, have been applied for the detection of Mycobacterium leprae in leprosy cases and household contacts with subclinical infection. However, their application in the field poses a range of technical challenges. Loop-mediated isothermal amplification (LAMP), as a promising point-of-care NAAT does not require sophisticated laboratory equipment, is easy to perform, and is applicable for decentralized diagnosis at the primary health care level. Among a range of gene targets, the M. leprae specific repetitive element RLEP is regarded as highly sensitive and specific for diagnostic applications. Methods Our group developed and validated a dry-reagent-based (DRB) RLEP LAMP, provided product specifications for customization of a ready-to-use kit (intended for commercial production) and compared it against the in-house prototype. The assays were optimized for application on a Genie® III portable fluorometer. For technical validation, 40 “must not detect RLEP” samples derived from RLEP qPCR negative exposed and non-exposed individuals, as well as from patients with other conditions and a set of closely related mycobacterial cultures, were tested together with 25 “must detect RLEP” samples derived from qPCR confirmed leprosy patients. For clinical validation, 150 RLEP qPCR tested samples were analyzed, consisting of the following categories: high-positive samples of multibacillary (MB) leprosy patients (> 10.000 bacilli/extract), medium-positive samples of MB leprosy patients (1.001–10.000 bacilli/extract), low-positive samples of MB leprosy patients (1–1.000 bacilli/extract), endemic controls and healthy non-exposed controls; each n = 30. Results Technical validation: both LAMP formats had a limit of detection of 1.000 RLEP copies, i.e. 43–27 bacilli, a sensitivity of 92% (in-house protocol)/100% (ready-to-use protocol) and a specificity of 100%. Reagents were stable for at least 1 year at 22 °C. Clinical validation: Both formats showed a negativity rate of 100% and a positivity rate of 100% for high-positive samples and 93–100% for medium positive samples, together with a positive predictive value of 100% and semi-quantitative results. The positivity rate for low-positive samples was 77% (in-house protocol)/43% (ready-to-use protocol) and differed significantly between both formats. Conclusions The ready-to-use RLEP DRB LAMP assay constitutes an ASSURED test ready for field-based evaluation trials aiming for routine diagnosis of leprosy at the primary health care level.

Biosensor-Based Multiple Cross Displacement Amplification for the Rapid Detection of Mycobacterium leprae

“…23−25 Four different RLEP sequences have been previously reported, namely, RLEP1, RLEP2, RLEP3, and RLEP4 (National Center for Biotechnology Information accession numbers FM211192.1, X17151.1, X17153.1, and X17152.1, respectively). 13 Additionally, RLEP genes have a high specificity and are widely used for leprosy diagnosis. 9,11− 13 The current study used RLEP2 as the target sequence for M. leprae detection, which is the core of the RLEP element.…”

“…A reaction temperature of 65 °C is commonly used for Bst DNA polymerase. ,, Considering the high GC content of the target RLEP gene, we examined amplification temperatures in the range of 63–70 °C with 1 °C intervals to determine the optimal reaction temperature of the MCDA assay. Additionally, M.…”

“…12 Although PCR methods demonstrate outstanding analytical capabilities, their limited availability, the need for thermal cyclers, and time-consuming detection procedures restrict their application in point-of-care and field laboratories. 13 To overcome the disadvantages of PCR techniques, isothermal amplification methods have been developed to detect pathogens, including LAMP, recombinase polymerase amplification, and multiple cross displacement amplification (MCDA). 14−16 Currently, nucleic acid-based isothermal amplification products are validated using conventional agarose gel electrophoresis, real-time fluorescence detection, visualization reagents such as malachite green (MG), real-time turbidity measurements, and nanoparticlebased lateral flow biosensors (LFBs).…”

“…RT-PCR involves a special apparatus for detecting fluorophores . Although PCR methods demonstrate outstanding analytical capabilities, their limited availability, the need for thermal cyclers, and time-consuming detection procedures restrict their application in point-of-care and field laboratories . To overcome the disadvantages of PCR techniques, isothermal amplification methods have been developed to detect pathogens, including LAMP, recombinase polymerase amplification, and multiple cross displacement amplification (MCDA). – Currently, nucleic acid-based isothermal amplification products are validated using conventional agarose gel electrophoresis, real-time fluorescence detection, visualization reagents such as malachite green (MG), real-time turbidity measurements, and nanoparticle-based lateral flow biosensors (LFBs). ,– MCDA is an isothermal single enzyme-promoting nucleic acid amplification technique that achieves a high specificity by recognizing and amplifying ten distinct regions of the target gene. ,, MCDA can also robustly amplify trace amounts of DNA with a higher sensitivity and in less time compared with LAMP .…”

Challenges and advances in serological and molecular tests to aid leprosy diagnosis

“…The qPCR, 85 nested PCR 84 and variations such as multiplex, 87,91 duplex, 92 and droplet digital (ddPCR) 93 have been reported for different M. leprae target sequences, with RLEP and 16S rRNA representing the most used ones. [94][95][96] A study showed that multiplex PCR helps detect early leprosy cases and household contact surveillance for leprosy. 97 Nested PCR is useful for amplifying genes from samples with low bacillary load in PB patients, as this technique amplifies sequences within the amplicon generated in the first amplification round.…”