Polymorphisms in mitochondrial ribosomal protein S5 (MRPS5) are associated with leprosy risk in Chinese.

Leprosy is an infectious disease caused by Mycobacterium leprae (M. leprae), with about 210,000 new cases per year worldwide. Although numerous risk loci have been uncovered by genome-wide association studies, the effects of common genetic variants are relatively modest. To identify possible new genetic locus involved in susceptibility to leprosy, whole exome sequencing was performed for 28 subjects including 14 patients and 12 unaffected members from 8 leprosy-affected families as well as another case and an unrelated control, and then the follow-up SNP genotyping of the candidate variants was studied in case-control sample sets. A rare missense variant in mitochondrial ribosomal protein S5 (MRPS5), rs200730619 (c. 95108402T>C [p. Tyr137Cys]) was identified and validated in 369 cases and 270 controls of Chinese descent (Padjusted = 0.006, odds ratio [OR] = 2.74) as a contributing factor to leprosy risk. Moreover, the mRNA level of MRPS5 was downregulated in M. leprae sonicate-stimulated peripheral blood mononuclear cells. Our results indicated that MRPS5 may be involved in leprosy pathogenesis. Further studies are needed to determine if defective MRPS5 could lead to impairment of energy metabolism of host immune cells, which could further cause defect in clearing M. leprae and increase susceptibility to infection.

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.

Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.

[Genotyping of Mycobacterium leprae in Guangdong province].

OBJECTIVE: To understand the genotypes of Mycobacterium leprae collected from Guangdong province, China and to analyze the routes of leprosy transmission both inside and outside of Guangdong. The impact of emigrant leprosy patients to the endemic nature of the disease in Guangdong was also studied. METHODS: Typing on strains with variable number of tandem repeats (VNTR) and single nucleotide polymorphism (SNP) were performed on the local cases and emigrant cases based on skin biopsy. RESULTS: Most isolates from local patients belong to SNP type 1 and SNP type 3 isolates were found just in a small part of local isolates. However, all the emigrants were carrying SNP type 3. Within the SNP type 1 strain from Guangdong, alleles at the 18-8, 12-5, ML-1, (TA) 10 and (GGT) 5 differed from SNP 3 strains collected from other areas in China. However, all the SNP type 1 and SNP type 3 local isolates identified from Guangdong were having close VNTR profiles and the main differences appeared in the alleles at ML-1, (TA) 10 and (GGT) 5. CONCLUSION: The transmission of strain with SNP type 1 seemed to be associated to the "Silk Road on the Sea", calling for monitoring and confirming the transmission of patients with SNP type 3 in Guangdong were from the secondary transmission, by the emigrant patients. Further study on the historic spread and phylogenetic relationships between SNP type 1 and novel SNP type 3 in Guangdong is needed.

Molecular, ethno-spatial epidemiology of leprosy in China: novel insights for tracing leprosy in endemic and non endemic provinces.

Leprosy continues to be detected at near stable rates in China even with established control programs, necessitating new knowledge and alternative methods to interrupt transmission. A molecular epidemiology investigation of 190 patients was undertaken to define Mycobacterium leprae strain types and discern genetic relationships and clusters in endemic and non-endemic regions spanning seventeen provinces and two autonomous regions. The findings support multiple locus variable number of tandem repeat (VNTR) analysis as a useful tool in uncovering characteristic patterns across the multiethnic and divergent geographic landscape of China. Several scenarios of clustering of leprosy from township to provincial to regional levels were recognized, while recent occupational or remote migration showed geographical separation of certain strains. First, prior studies indicated that of the four major M. leprae subtypes defined by single nucleotide polymorphisms (SNPs), only type 3 was present in China, purportedly entering from Europe/West/Central Asia via the Silk Road. However, this study revealed VNTR linked strains that are of type 1 in Guangdong, Fujian and Guangxi in southern China. Second, a subset of VNTR distinguishable strains of type 3, co-exist in these provinces. Third, type 3 strains with rpoT VNTR allele of 4, detected in Japan and Korea were discovered in Jiangsu and Anhui in the east and in western Sichuan bordering Tibet. Fourth, considering the overall genetic diversity, strains of endemic counties of Qiubei, Yunnan; Xing Yi, Guizhou; and across Sichuan in southwest were related. However, closer inspection showed distinct local strains and clusters. Altogether, these insights, primarily derived from VNTR typing, reveal multiple and overlooked paths for spread of leprosy into, within and out of China and invoke attention to historic maritime routes in the South and East China Sea. More importantly, new concepts and approaches for prospective case finding and tracking of leprosy from county to national level have been introduced.

[Study on the factors influencing steady transmission of leprosy in Qiubei county, China].

OBJECTIVE: To explore the factors influencing the steady transmission of leprosy as indicated by new case detection rate in Qiubei county, Yunnan province, China despite the implementation of MDT for the last 25 years. METHODS: Information related to case-finding was collected. ELISA and PCR were applied to detect anti-PGL-1 antibody in sera and Mycobacterium leprae in nasal secretions respectively, in leprosy patients, their household contacts and the general population. M. leprae by PCR was also detected from water in the highly endemic villages. VNTR typing was performed to explore the mode and chain of transmission of M. leprae. RESULTS: Prior to 2001, the proportion of new cases detected from the examination of household contacts of leprosy patients was low (number, compared to), while the proportion of patients whose identification was delayed by more than 2 years, was high (number, compared to). Qualities of these two indicators has been improved, along with the improvement of leprosy control program since 2001, but the detection rates has been steady at 4-5/100 000 during 1986 - 2010. The PGL-1 seropositivity rate was 20% - 30% in general population, with the peak rate (30%) detected in the teenage population in the endemic villages. In addition to the fact that M. leprae was detected in nasal secretion from patients, their contacts and from water, the M. leprae VNTR genotypes were found to be highly similar between skin biopsy and nasal secretion in untreated cases. Families with multi-cases were clustered and located in the Northern part of the County, and the genotypes of M. leprae were identical within those families. The percentage of clusters was considerably higher in Northern rather than Southern parts of the County. CONCLUSION: Results from this molecular study demonstrated evidence that transmission of leprosy within the families and in the endemic-villages was severe. M. leprae were detected in waters from the endemic villages and others areas which might have a relation to the continued transmission of leprosy.

Transmission of leprosy in Qiubei County, Yunnan, China: insights from an 8-year molecular epidemiology investigation.

Leprosy continues to be endemic in parts of China. To track the occurrence of leprosy and determine at risk communities, molecular strain typing based on variable number of tandem repeats (VNTRs) was applied in Qiubei County, Wenshan Prefecture, Yunnan Province of the People's Republic of China, a multiethnic region that is home to four predominant ethnic minorities. A previous study, conducted between 2002 and 2005, provided the first descriptions of Mycobacterium leprae strains in the region. M. leprae strains in Qiubei are highly conserved, so only sufficiently polymorphic loci can distinguish strains. A balance between mutation rate and loci stability is needed, so that secondary transmissions can be identified as genotypic matches. The long incubation period of leprosy necessitated an extension of the study to assess the validity of VNTR typing and observe allelic shifts in the same multiethnic population. From 2006 to early 2010 the extension was performed to yield a cumulative total of 164 enrolled patients and 130 skin samples suitable for VNTR typing. Patient demographic information revealed that the case detection rate among certain minority populations in the county is considerably higher than the national rate. Cluster analysis of allele frequencies showed similar strain types within family groups and neighboring townships. Allele frequencies were not found to significantly differ between genders or clinical presentations. The percentage of cases showing near-matching genotypes varied with geography; showing a considerably higher rate in the northern townships. The northern townships continue to show strain types falling into the groups previously defined. Southern genotypes were distinct from those in the north, but clonal genetic relationships were indiscernible in the south. Social interactions and the physical, residential and occupational environments may be more conducive to transmission of community strains in the north.

VNTR typing studies of Mycobacterium leprae in China: assessment of methods and stability of markers during treatment.

OBJECTIVE: To evaluate the reliability and feasibility of two methods of multilocus variable number of tandem repeat analysis (MLVA) for strain typing of M. leprae, and to study whether short tandem repeat loci are stable and suitable for epidemiological study of leprosy. METHODS: Total DNA was extracted from skin biopsies of 20 new multibacillary (MB) patients from China diagnosed in 2006. To determine the copy numbers of short tandem repeats (STRs) for 13 loci, we amplified each locus individually by PCR, followed by sequence analysis of the amplicons. Separately, the same loci, plus four others were amplified by Multiplex PCRs (MP) using fluorescent primers and the copy number was identified by fragment length analysis (MP-FLA). MLVA was also performed at different times during treatment for a subset of the patients. RESULTS AND CONCLUSIONS: Genetic variability of M. leprae in China can be assessed in microsatellite loci. (GTA)9 and (TTC)21 loci are hypervariable, with array sizes of 25 repeat units or more. The expansion of the (GTA)9 locus is a characteristic of some M. leprae isolates in China. A high level of allele concordance was observed between PCR-sequencing and MP-FLA methods. However, MP-FLA method was cost-effective, rapid, high throughput and suitable for strain typing. Five of the 20 isolates of M. leprae were from patients residing in the same township in Qiubei County, Yunnan, and matched closely by MLVA. Three of these patients are family contacts of previously diagnosed patients, with intra-familial strain types being similar, suggesting infections from common sources and transmission chain(s). The VNTR patterns were highly similar in biopsy and slit skin smears (SSS) before treatment, and in the SSS collected at various time points during treatment. Taken together, VNTR strain typing is a useful tool for study of short range transmission in leprosy.

[Study on genotyping of Mycobacterium leprae and families with multi-cases].

OBJECTIVE: Multiple locus variable number-tandem repeat (VNTR) analysis (MLVA) had been proposed as a means of strain typing for tracking of source and studying the transmission chain of pathogens. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. METHODS: MLVA on 7 VNTR loci was applied to the strain typing on prevalent Mycobacterium leprae isolates collected from Qiubei county, Yunnan province during 2002-2006 in the study on the relationship between geographic distribution and genotypes of M. leprae. The strain typing, combined with conventional epidemiological investigation was performed to trace the transmission chain. RESULTS: (1) Phylogenetic analyses through application of PAUP 4.0, The M. leprae were grouped into A, B, C, D and E strains according to the allelic range 9, 11-13, 15-26 and > 26 on the GTA9 locus. The strains with 9 copies on GTA9 locus, was named A. (2) Genotypes of strains from the five multi-case families located at North and North-West parts were similar and belonged to A strains. VNTR patterns of intra-family were identical or similar but not identical inter-family. (3) Not only A cluster appeared higher proportion in total isolates but also distributes cluster, indicating ongoing transmission from recent findings. CONCLUSION: VNTR strain typing was suitable to trace the short chain of transmission in both small area and intra-families. Multi-case families might constitute epidemic foci and source of M. leprae in villages, causing the predominant strain or cluster which tends to be those identified in multi-case families and resulted in the spreading of leprosy. A long-term study was underway to reveal whether A strain was predominant strain and to observe the evolution of M. leprae in this spatially and temporally defined endemic population.

Identification and distribution of Mycobacterium leprae genotypes in a region of high leprosy prevalence in China: a 3-year molecular epidemiological study.

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been proposed as a means of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population are lacking. To this end, a study was initiated to assess the diversity and distribution of prevalent Mycobacterium leprae strains in Qiubei County, Yunnan Province, People's Republic of China, where the annual detection rate of leprosy is 10-fold higher than the national average rate. Sixty-eight newly diagnosed leprosy patients were included in the study. MLVA at eight M. leprae loci was applied using DNA extracts from skin biopsies. The number of alleles per locus ranged from 4 to 24, providing adequate strain discrimination. MLVA strain typing identified several clusters of patients whose M. leprae specimens shared similar VNTR profiles. Two of these clusters were comprised of patients who resided predominantly in the north and northwest parts of Qiubei County. Furthermore, it was found that multicase families are common in this county: 23 of the 68 patients were from 11 families. Intrafamilial VNTR profiles closely matched within six families, although they were different between the families. Moreover, VNTR patterns related to those found in some multicase families were also detected in patients in the same or adjacent townships, indicating the utility of VNTR strain typing to identify and detect short-range transmission events. Social contact through village markets is proposed as a means of transmission.

[Preliminary study on the genotyping of Mycobacterium leprae on 50 isolates from China].

OBJECTIVE: To understand the genotypic mapping of Mycobacterium leprae identified in China and to compare with those from other countries to select suitable alleles for epidemiological investigation in the transmission chain of leprosy. METHODS: Various number of tandem repeat(VNTR) in genomic DNA of Mycobacterium leprae was used in the present genotyping study. 33 skin biopsies from Wenshan prefecture,Yunnan province and 17 from other parts of China were studied. DNA extracted from skin biopsies of leprosy patients was subjected to PCR followed by agarose gel analysis and DNA sequencing to determine the number of repeats. RESULTS: Loci GGT-5,12-5,21-3 and 23-3 were as highly homogenous as 100%; The homogeneity of loci AC-8, 18-8, 27-5 and rpoT were 97%, 94%, 97% and 85% respectively. Loci GTA-9, AC-9 and 6-7 showed significant allelic diversity in isolates and the diversity of GTA-9 in Mycobacterium leprae isolated from China was also different from those identified other countries. We had subjected loci GTA-9 and the ten loci to phylogenetic tree analysis respectively. CONCLUSION: The present study revealed that the genotype of Mycobacterium leprae identified from China was close to the strains from the Philippines and India although a few loci were somehow differentiate. Locus 12-5 manifested as only 3 copies in China whereas 4-5 copies predominating in other countries. 12-5 locus might serve as a useful marker to diffrentiate Chinese strains from those in other countries. However, further study on the diversity of GTA-9 was needed in China. The molecular typing of Mycobacterium leprae from different geographic areas might be useful in studying the transmission of leprosy.

Immuno-histopathology in the diagnosis of early leprosy

The present study of 45 early leprosy cases in an endemic area in China indicates: a) Sensitivity of acid-fast bacilli (AFB) detection can be significantly improved by examining approximately 30 serial sections. AFB and/or phenolic glycolipid-I (PGL-I) were mostly detected in the infiltrates in the subepidermal zone, intraneurium, perineurium and around blood vessels. b) PGL-I antigen was positive in 10 clinically suspected, single lesion leprosy cases and AFB positive in 7 patients, AFB and/or PGL-I in nerve in 6 patients. c) Nonspecific chronic inflammation in indeterminate leprosy presented as selective perineural and/or intraneural infiltration with lymphocytes predominating. In the infiltrating mass, fragments of neural tissue were demonstrated with anti-S-100 protein staining. d) Except for 3 cases with unknown numbers of lesions, the present positive immunohistopathological findings are in direct correlation with the number of lesions at first diagnosis, namely: 41.6% (10/24) for single lesion, 66.6% (6/9) for 2 lesions, and 88.8% (8/9) for patients with > or = 3 lesions. e) Typical epithelioid or macrophage granuloma formations were not seen in early leprosy with a single lesion. In testing the immunological inclination of these patients with CD68 or tumor necrosis factor-alpha (TNF-alpha) a positive test is likely to be of prognostic value since TNF-alpha is involved in granuloma formation and nerve damage.