Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis

Abstract

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.

Advances in the Diagnosis of Leprosy

“…The Brazilian Health Regulatory Agency (Anvisa) approved the NAT-HANS test for people suspected of leprosy in 2021. The test showed high sensitivity and specificity values comparable to Lateral Flow Assays with a limit of detection of 2.29 copies of the M. leprae genome (33,34).…”

Assessment of esxA, hsp18, and 16S transcript expression as a measure of Mycobacterium leprae viability: A comparison with the mouse footpad assay

“…16 However, most of the in vitro viability assays, while simplified and quicker, require large numbers of freshly prepared bacilli and are therefore unsuitable for clinical applications. With the sequencing of the M. leprae genome 17 and investigations of the functional transcriptome, [18][19][20][21] molecular assays are gaining ground 22,23 and will undoubtedly come to support clinical and histopathological diagnostics as a standard of care.…”

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

“…1 Leprosy occurs mostly in developing countries and is often a neglected infectious disease despite 140,546 new leprosy cases having been reported in 135 member states of the World Health Organization (WHO) in 2021. 3,5 The burden of leprosy can be significantly reduced through early detection and prompt treatment with multidrug therapy (MDT). 3 Evidence suggests that early diagnosis could contribute to epidemiologic control and prevent transmission.…”

“…3 Evidence suggests that early diagnosis could contribute to epidemiologic control and prevent transmission. 5,6 Therefore, an effective ML detection technique is essential for early diagnosis, timely treatment, and reduction of leprosy disability.…”

“…leprae damages the skin, nerves, limbs, and eyes and may cause deformities and disabilities if not treated early. – It is also usually cultivated in animal models as it cannot be cultured in vitro or in a laboratory . Leprosy occurs mostly in developing countries and is often a neglected infectious disease despite 140,546 new leprosy cases having been reported in 135 member states of the World Health Organization (WHO) in 2021. , The burden of leprosy can be significantly reduced through early detection and prompt treatment with multidrug therapy (MDT) . Evidence suggests that early diagnosis could contribute to epidemiologic control and prevent transmission. , Therefore, an effective ML detection technique is essential for early diagnosis, timely treatment, and reduction of leprosy disability.…”