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.

Single-Dose Rifapentine in Household Contacts of Patients with Leprosy.

BACKGROUND: Previous studies have suggested that a single dose of rifampin has protective effects against leprosy in close contacts of patients with the disease. Rifapentine was shown to have greater bactericidal activity against Mycobacterium leprae than rifampin in murine models of leprosy, but data regarding its effectiveness in preventing leprosy are lacking. METHODS: We conducted a cluster-randomized, controlled trial to investigate whether single-dose rifapentine is effective in preventing leprosy in household contacts of patients with leprosy. The clusters (counties or districts in Southwest China) were assigned to one of three trial groups: single-dose rifapentine, single-dose rifampin, or control (no intervention). The primary outcome was the 4-year cumulative incidence of leprosy among household contacts. RESULTS: A total of 207 clusters comprising 7450 household contacts underwent randomization; 68 clusters (2331 household contacts) were assigned to the rifapentine group, 71 (2760) to the rifampin group, and 68 (2359) to the control group. A total of 24 new cases of leprosy occurred over the 4-year follow-up, for a cumulative incidence of 0.09% (95% confidence interval [CI], 0.02 to 0.34) with rifapentine (2 cases), 0.33% (95% CI, 0.17 to 0.63) with rifampin (9 cases), and 0.55% (95% CI, 0.32 to 0.95) with no intervention (13 cases). In an intention-to-treat analysis, the cumulative incidence in the rifapentine group was 84% lower than that in the control group (cumulative incidence ratio, 0.16; multiplicity-adjusted 95% CI, 0.03 to 0.87; P = 0.02); the cumulative incidence did not differ significantly between the rifampin group and the control group (cumulative incidence ratio, 0.59; multiplicity-adjusted 95% CI, 0.22 to 1.57; P = 0.23). In a per-protocol analysis, the cumulative incidence was 0.05% with rifapentine, 0.19% with rifampin, and 0.63% with no intervention. No severe adverse events were observed. CONCLUSIONS: The incidence of leprosy among household contacts over 4 years was lower with single-dose rifapentine than with no intervention. (Funded by the Ministry of Health of China and the Chinese Academy of Medical Sciences; Chinese Clinical Trial Registry number, ChiCTR-IPR-15007075.).

Genotyping of Mycobacterium leprae for understanding the distribution and transmission of leprosy in endemic provinces of China.

OBJECTIVES: Understanding the nature of Mycobacterium leprae transmission is vital to implement better control strategies for leprosy elimination. The present study expands the knowledge of county-level strain diversity, distribution, and transmission patterns of leprosy in endemic provinces of China. METHODS: We genetically characterized 290 clinical isolates of M. leprae from four endemic provinces using variable number tandem repeats (VNTR) and single nucleotide polymorphisms (SNPs). Attained genetic profiles and cluster consequences were contrasted with geographical and migration features of leprosy at county levels. RESULTS: Considering the allelic variability of 17 VNTR loci by the discriminatory index, (GTA)9, (AT)17, (AT)15, (TA)18, (TTC)21, and (TA)10 are reported to be more highly polymorphic than other loci. The VNTR profile generated the low-density clustering pattern in the counties of Sichuan and Yunnan, whereas clusters have been observed from the isolates from Huayuan (N = 6), Yongding (N = 3), Zixing (N = 3), Chenxi (N = 2) and Zhongfang (N = 2) counties of Hunan, and Zhijin (N = 3), Anlong (N = 2), Zhenning (N = 2), and Xixiu (N = 2) counties of Guizhou. In some clusters, people's social relations have been observed between villages. From the 290 clinical isolates, the most predominantly reported SNP was 3K (278, 95.8%), followed by SNP 1D (10, 3.4%), which are typically observed to be predominant in China. We also detected the novel SNP 3J (2, 0.8%), which has not yet been reported in China. CONCLUSION: The clustering pattern of M. leprae indicates the transmission of leprosy still persists at county levels, suggesting that there is a need to implement better approaches for tracing the close contacts of leprosy patients.

Molecular surveillance of antimicrobial resistance and transmission pattern of Mycobacterium leprae in Chinese leprosy patients.

Reports on antimicrobial resistance (AMR) of Mycobacterium leprae, relationship with bacteriological index (BI), and transmission in China are limited. We investigated the emergence of AMR mutations, the relationship between BI and AMR in complete, moderate and lack of BI decline cases, and molecular epidemiological features of AMR cases by enrolling 290 leprosy cases from four endemic provinces. Seven (2.41%), one (0.34%), five (1.72%), one (0.34%), and one (0.34%) strains had single mutations in folP1, rpoC, gyrA, gyrB, and 23S rRNA, respectively. Double mutations in folP1 and gyrA, rpoB and gyrA, and gyrA and 23S rRNA were observed in one (0.34%) strain each. Mutated strains occurred in three out of 81 (95% CI-0.005-0.079, p = 0.083) cases with complete BI decline, in seven out of 103 (95% CI 0.018-0.117, p = 0.008) cases with moderate BI decline, and in four out of 34 (95% CI 0.003-0.231, p = 0.044) cases with lack of BI decline. Most of these mutated strains were geographically separated and diverged genotypically. AMR mutations may not be the main cause of the lack of BI decline. The low transmission of AMR strains at the county level indicates an ongoing transmission at close contact levels.

Evaluation of Prospective HLA-B*13:01 Screening to Prevent Dapsone Hypersensitivity Syndrome in Patients With Leprosy.

Importance: Dapsone hypersensitivity syndrome (DHS) is the most serious adverse reaction associated with dapsone administration and one of the major causes of death in patients with leprosy, whose standard treatment includes multidrug therapy (MDT) with dapsone, rifampicin, and clofazimine. Although the HLA-B*13:01 polymorphism has been identified as the genetic determinant of DHS in the Chinese population, no studies to date have been done to evaluate whether prospective HLA-B*13:01 screening could prevent DHS by identifying patients who should not receive dapsone. Objective: To evaluate the clinical use of prospective HLA-B*13:01 screening for reduction of the incidence of DHS by excluding dapsone from the treatment for patients with HLA-B*13:01-positive leprosy. Design, Setting, and Participants: A prospective cohort study was conducted from February 15, 2015, to April 30, 2018, in 21 provinces throughout China. A total of 1539 patients with newly diagnosed leprosy were enrolled who had not received dapsone previously. After excluding patients who had a history of allergy to sulfones or glucose-6-phosphate dehydrogenase deficiency, 1512 individuals underwent HLA-B*13:01 genotyping. All of the patients were followed up weekly for the first 8 weeks after treatment to monitor for adverse events. Exposures: Patients who were HLA-B*13:01 carriers were instructed to eliminate dapsone from their treatment regimens, and noncarrier patients received standard MDT. Main Outcomes and Measures: The primary outcome was the incidence of DHS. The historical incidence rate of DHS (1.0%) was used as a control. Results: Among 1512 patients (1026 [67.9%] men, 486 [32.1%] women; mean [SD] age, 43.1 [16.2] years), 261 (17.3%) were identified as carriers of the HLA-B*13:01 allele. A total of 714 adverse events in 384 patients were observed during the follow-up period. Dapsone hypersensitivity syndrome did not develop in any of the 1251 patients who were HLA-B*13:01-negative who received dapsone, while approximately 13 patients would be expected to experience DHS, based on the historical incidence rate of 1.0% per year (P = 2.05 × 10-5). No significant correlation was found between other adverse events, including dermatologic or other events, and HLA-B*13:01 status. Conclusions and Relevance: Prospective HLA-B*13:01 screening and subsequent elimination of dapsone from MDT for patients with HLA-B*13:01-positive leprosy may significantly reduce the incidence of DHS in the Chinese population.

Evaluation of Immunodiagnostic Tests for Leprosy in Brazil, China and Ethiopia.

Leprosy remains persistently endemic in several low- or middle income countries. Transmission is still ongoing as indicated by the unabated rate of leprosy new case detection, illustrating the insufficiency of current prevention methods. Therefore, low-complexity tools suitable for large scale screening efforts to specifically detect M. leprae infection and diagnose disease are required. Previously, we showed that combined detection of cellular and humoral markers, using field-friendly lateral flow assays (LFAs), increased diagnostic potential for detecting leprosy in Bangladesh compared to antibody serology alone. In the current study we assessed the diagnostic performance of similar LFAs in three other geographical settings in Asia, Africa and South-America with different leprosy endemicity. Levels of anti-PGL-I IgM antibody (humoral immunity), IP-10, CCL4 and CRP (cellular immunity) were measured in blood collected from leprosy patients, household contacts and healthy controls from each area. Combined detection of these biomarkers significantly improved the diagnostic potential, particularly for paucibacillary leprosy in all three regions, in line with data obtained in Bangladesh. These data hold promise for the use of low-complexity, multibiomarker LFAs as universal tools for more accurate detection of M. leprae infection and different phenotypes of clinical leprosy.

Genome-Wide Analysis of Protein-Coding Variants in Leprosy.

Although genome-wide association studies have greatly advanced our understanding of the contribution of common noncoding variants to leprosy susceptibility, protein-coding variants have not been systematically investigated. We carried out a three-stage genome-wide association study of protein-coding variants in Han Chinese, of whom were 7,048 leprosy patients and 14,398 were healthy control subjects. Seven coding variants of exome-wide significance were discovered, including two rare variants: rs145562243 in NCKIPSD (P = 1.71 × 10-9, odds ratio [OR] = 4.35) and rs149308743 in CARD9 (P = 2.09 × 10-8, OR = 4.75); three low-frequency variants: rs76418789 in IL23R (P = 1.03 × 10-10, OR = 1.36), rs146466242 in FLG (P = 3.39 × 10-12, OR = 1.45), and rs55882956 in TYK2 (P = 1.04 × 10-6, OR = 1.30); and two common variants: rs780668 in SLC29A3 (P = 2.17 × 10-9, OR = 1.14) and rs181206 in IL27 (P = 1.08 × 10-7, OR = 0.83). Discovered protein-coding variants, particularly low-frequency and rare ones, showed involvement of skin barrier and endocytosis/phagocytosis/autophagy, in addition to known innate and adaptive immunity, in the pathogenesis of leprosy, highlighting the merits of protein-coding variant studies for complex diseases.

How to improve early case detection in low endemic areas with pockets of leprosy: a study of newly detected leprosy patients in Guizhou Province, People's Republic of China.

Although leprosy in China is controlled at a low endemic level, the number of new cases in Guizhou province has shown no significant decrease over the past 20 years. Guizhou remains the province with the second highest prevalence in China. The authors conducted a study in which the characteristics of newly detected leprosy cases, found between 2008 and 2012 in Guizhou, were analysed. These cases represented people from pocket areas of leprosy in a generally low endemic environment. The purpose of the study was to understand characters of newly detected cases, strong points and weakness of routine detection approaches for improving the effectiveness of early case detection in the future. The analysis considered data that was collected from a 'Leprosy Management Information' report system and also from annual statistical reports of leprosy that reflect the situation throughout the province. 1274 new patients were detected in Guizhou from 2008 to 2012. That number included 58 (4.6%) children (0-14 years old). The average age of patients at diagnosis was 42.6 ± 16.5 years. The proportion of people with WHO Grade 2 disability (WHO DG2) among new patients was 35.7% and the proportion of people with Grade 1 disability (DG1) constituted 10.1%. The average delay before diagnosis after the onset of symptoms of leprosy was 41.7 ± 49.8 months. Suspect survey was a major method by which most cases were detected. Trough this method 790 (62.0%) new patients were detected. It was also in this group that the highest proportion of people with WHO DG2 359 of 790 (45.4%) was reported. Self- reporting, diagnosis at a general skin clinic, household contact examination, and spot surveys accounted for 13.0%, 11.8%, 11.5% and 1.7% of other cases detected respectively. It was generally found that cases detected through household contact examinations were earlier cases (delay to diagnosis < 24 months = 70.7%). It was also recorded that fewer of these had WHO DG2 (12.9%). The proportion of men with WHO DG2 was higher than that of females (38.2% compared with 28.8%). The proportion of Han Chinese new cases with WHO DG2 was significantly higher than that of the main minority group (41.5% compared with 29.2%). The proportion of new cases among the main minority group who self-reported (50%) was significantly higher than those detected through other detection approaches. Detecting leprosy early in low endemic situations where pockets persist was difficult to achieve. The authors suggest that if more early patients are to be detected earlier, the quality of suspect surveys and household contact examination should be improved. Professional training and supervision might affect that result. Greater emphasis should be given to the role of general skin clinics as surveillance sites and advocacy for new health policy that will enhance the detection leprosy should be sustained.

Six years' follow-up of multibacillary leprosy patients treated with uniform multi-drug therapy in China.

OBJECTIVE: This study was conducted to evaluate the effectiveness of uniform multi-drug therapy (UMDT) in patients with multibacillary (MB) leprosy. METHODS: Newly detected MB leprosy patients were treated with six months of UMDT as recommended by the World Health Organization (WHO). The effectiveness of treatment was evaluated by clinical status and skin smear tests. RESULTS: At the start, 114 patients were recruited, examined, and treated. These patients were re-examined and followed annually for up to six years. A total of 75 (65.8%) patients completed six years of follow-up. Dropouts were attributable to death, severe drug reactions, and other reasons. The mean ± standard deviation bacteriological index (BI) of all patients decreased from 3.01 ± 1.50 before treatment to 0.02 ± 1.84 at the end of year 6, reflecting a mean annual decrease of 0.50. The rate of smear negativity in all patients was 98.7% at the end of year 6 of follow-up. A total of 53 leprosy reactions were observed. One patient relapsed 13 months after the cessation of treatment. CONCLUSIONS: A 6-month administration of UMDT is effective in MB leprosy patients. The changes in BI values and the frequency of leprosy reactions were similar to those cited in reports in the literature of patients treated with 1- or 2-year regimens of MDT. However, further research should be conducted to confirm the present results.

Government health workers as implementers of prevention of disability measures: an assessment of a prevention of disability project in selected counties of Guizhou Province, Peoples' Republic of China.

OBJECTIVE: The purpose of this study was to assess the effectiveness of government health workers as agents for the prevention of disability. DESIGN: A prevention of disability (POD) project for people affected by leprosy was conducted in nine counties of Guizhou Province, Peoples' Republic of China. The project was implemented by government health workers. In accordance with the principles and national criteria of the National Centre for Leprosy Control (NCLC) POD Pilot programme, 1215 people affected by leprosy were selected, followed up and assessed with the use of impairment summary forms through which essential indicators were routinely collected. RESULTS: Most improvements of disabilities occurred in the 1st year of the POD project. Fifty five people with neuritis were detected and treated with prednisolone out of 262 new patients; 47 of these improved; 1130 people completed a 3-year self-care programme; 88.5% of red eyes, 83.9% of hand ulcers and 62.8% of simple foot ulcer cases healed during that period. One hundred and ninety six people who presented with complicated ulcers were treated; of these 73 (37.2%) people presented with feet free of ulcers at the end of the project period. CONCLUSION: The POD project was a cost-effective method of preventing further disability occurrence among people affected by leprosy. Government health workers were generally able to implement and monitor the project effectively. Most of people affected by leprosy were satisfied that the improvements in their disabilities had been due to self-care. The programme had helped them to increase their confidence to implement self-care activities.

The sustainability of self-care in two counties of Guizhou Province, Peoples' Republic of China.

This paper presents the findings of a follow-up survey conducted in two rural counties of Guizhou Province, PRC where a programme to prevent disabilities amongst leprosy affected people had been conducted. An initial 3-year programme had been conducted. One year after the final evaluation of the programme, a team was deployed to conduct a survey in the area. The objective of the survey was to establish the level of adherence to self-care. It was found that 87% of the sample of people living in leprosy villages that were surveyed (n = 31) and 50% of the sample of people living in general communities (n = 50) had continued to apply self-care. Interviews with family members suggested that 18 of the 27 self-care practising subjects living in the leprosy villages received encouragement or active support from family members (9 were single people). Twenty three of the 25 self-care practising subjects living in the communities also received family support (2 were single people). Family support was a highly significant factor influencing adherence in the community (OR = 15.8, CI = 3.0 to 83) but it may not have been the primary motivating factor in the leprosy villages where single people were just as likely to have adhered to self-care than people who were living in families (OR 0.5, CI = 0.06 to 4.2). The prevalence of foot ulceration among that population was recorded but a hypothetical association between the prevalence of foot ulceration and self-care adherence could not be investigated due to insufficient data to address the potential effects of confounding variables. Thirty-eight percent of subjects who did not practice self-care presented with ulceration or foot cracks (n = 29) compared with only 25% of people who did (n = 52).

Risk of relapse in leprosy after fixed-duration multidrug therapy

Between 1986 and 1995, 8307 leprosy patients have completed fixed-duration multidrug therapy (FD-MDT) and were followed annually for possible relapse. The mean relapse rate for multibacillary (MB) leprosy is 0.15/1000 person-years (py) and for paucibacillary (PB) 0.55/1000 py. There is no difference in the relapse rates between patients with or without chemotherapy before FD-MDT. In MB patients, the five relapses occurred between 4 and 7 years; in PB patients, five relapses occurred at 4-5 years after FD-MDT. Six additional PB relapses self-reported 1-4 years after the 5-year surveillance period and were not included in the relapse rates. Most PB patients relapsed into MB due to wrong classification and insufficient therapy. For the known 62 irregular MB patients the cumulative relapse rate is 6.5%.