Molecular epidemiological characteristics of Mycobacterium leprae in highly endemic areas of China during the COVID-19 epidemic.

Objectives: The present study analyzed the impact of the COVID-19 pandemic on the prevalence and incidence of new leprosy cases, as well as the diversity, distribution, and temporal transmission of Mycobacterium leprae strains at the county level in leprae-endemic provinces in Southwest China. Methods: A total of 219 new leprosy cases during two periods, 2018-2019 and 2020-2021, were compared. We genetically characterized 83 clinical isolates of M. leprae in Guizhou using variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). The obtained genetic profiles and cluster consequences of M. leprae were compared between the two periods. Results: There was an 18.97% decrease in the number of counties and districts reporting cases. Considering the initial months (January-March) of virus emergence, the number of new cases in 2021 increased by 167% compared to 2020. The number of patients with a delay of >12 months before COVID-19 (63.56%) was significantly higher than that during COVID-19 (48.51%). Eighty-one clinical isolates (97.60%) were positive for all 17 VNTR types, whereas two (2.40%) clinical isolates were positive for 16 VNTR types. The (GTA)9, (TA)18, (TTC)21 and (TA)10 loci showed higher polymorphism than the other loci. The VNTR profile of these clinical isolates generated five clusters, among which the counties where the patients were located were adjacent or relatively close to each other. SNP typing revealed that all clinical isolates possessed the single SNP3K. Conclusion: COVID-19 may have a negative/imbalanced impact on the prevention and control measures of leprosy, which could be a considerable fact for official health departments. Isolates formed clusters among counties in Guizhou, indicating that the transmission chain remained during the epidemic and was less influenced by COVID-19 preventative policies.

Highly sensitive and rapid determination of Mycobacterium leprae based on real-time multiple cross displacement amplification.

BACKGROUND: Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed. RESULTS: In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor. CONCLUSION: The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

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

Leprosy is an ancient disease caused by Mycobacterium leprae (ML) that remains a public health problem in poverty-stricken areas worldwide. Although many ML detection techniques have been used, a rapid and sensitive tool is essential for the early detection and treatment of leprosy. Herein, we developed a rapid ML detection technique by combining multiple cross displacement amplification (MCDA) with a nanoparticle-based lateral flow biosensor (LFB), termed ML-MCDA-LFB. MCDA induced a rapid isothermal reaction using specific primers targeting the RLEP gene, and the LFB enabled instant visual amplicon detection. The pure genomic DNA of ML and nucleic acids from various pathogens were employed to evaluate and optimize the ML-MCDA-LFB assay. The optimal conditions for ML-MCDA-LFB were 68 °C and 35 min, respectively. The limit of detection for pure ML genomic DNA was 150 fg per vessel, and the specificity of detection was 100% for the experimental strains. Additionally, the entire detection process could be performed within 40 min, including the isothermal amplification (35 min) and result confirmation (1-2 min). Hence, the ML-MCDA-LFB assay was shown to be a rapid, sensitive, and visual method for detecting ML and could be used as a potential tool for early clinical diagnosis and field screening of leprosy.