Transcriptomic Analysis of Mycobacterium leprae-Stimulated Response in Peripheral Blood Mononuclear Cells Reveal Potential Biomarkers for Early Diagnosis of Leprosy.

We aimed to identify an unique host transcriptional signature in peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium leprae antigens to distinguish between patients with leprosy and non-leprosy controls for early diagnosis of the disease. Sixteen individuals were enrolled in the discovery cohort [eight patients with leprosy, comprising four multibacillary (MB) and four paucibacillary (PB); and eight non-leprosy controls, comprising four healthy house contacts (HHCs) and four endemic controls (ECs)]. The differences in the transcriptome response of PBMCs to M. leprae sonicate antigen were evaluated between leprosy patients and non-leprosy controls, and 12 differentially expressed genes (CCL2/MCP-1, IL-8, JAKM, ATP, ND1, SERP, FLJ10489, LINC00659, LOC34487, LOC101928143, MIR22, and NCF1C) were identified. The accuracy of the 12 differentially expressed genes was further validated for the diagnosis of leprosy using real-time quantitative PCR in 82 individuals (13 MB, 10 PB, 37 HHCs, and 22 ECs) in the validation cohort. We found that a 5 gene signature set IL-8, CCL2/MCP-1, SERP, LINC00659 and FLJ10489 had a suitable performance in discriminating leprosy from ECs. In addition, elevated expression of IL-8, CCL2/MCP-1, SERP and LINC00659 was associated with MB diagnosis compared with ECs, whereas increased expression of IL-8, CCL2/MCP-1, SERP and FLJ10489 was found to be useful biomarkers for PB diagnosis from ECs. Moreover, we found decreased expression of NCF1C among leprosy patients could distinguish leprosy from HHCs, whereas higher expression of CCL2 among MB than PB could distinguish different leprosy patients. In conclusion, among the 12 candidate host genes identified, a three gene signature IL-8, CCL2/MCP-1, and SERP showed the best performance in distinguishing leprosy patients from healthy controls. These findings may have implications for developing a rapid blood-based test for early diagnosis of leprosy.

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 novel tools to facilitate the detection and characterization of leprosy patients in China.

Leprosy is the disabling outcome of chronic infection with Mycobacterium leprae. The disease often evades early detection, particularly now that fewer clinicians are able to confidently diagnose the disease following the integration of leprosy control measures within general health services in many countries. Although leprosy is officially eliminated in China, endemic regions remain in some difficult-to-reach, underdeveloped areas in Southwest China. In order to better understand the extent of M. leprae infection and identify new leprosy cases in a timely manner, simple tools that can detect infection and the early disease are required. In this report we evaluated the performance of antigen-specific ELISA, the NDO-LID rapid diagnostic test, and antigen-specific whole blood assays (WBA) as potential diagnostic tools. Our data support the use of antibody detection tests and WBA to facilitate the diagnosis of multibacillary and paucibacillary leprosy, respectively. These tools could be invaluable for increased, but simplified, monitoring of individuals in order to provide referrals for clinical exam and early leprosy diagnosis.

Application of RLEP real-time PCR for detection of M. leprae DNA in paraffin-embedded skin biopsy specimens for diagnosis of paucibacillary leprosy.

The TaqMan real-time polymerase chain reaction (PCR) assay was evaluated systematically with respect to the standard curve, linear range, and used for detecting Mycobacterium leprae DNA in paraffin-embedded skin biopsy specimens from 60 confirmed leprosy patients and three healthy individuals and 29 other dermatoses and bacterial DNA from 21 different species. The test was further evaluated with 51 paucibacillary (PB) patients. The results showed that the test had good sensitivity (8 fg) and good specificity with no cross-reactivity with 21 other bacterial species and the control specimens, except one with Xanthomatosis. The real-time PCR detection rate for the 51 PB specimens was 74.5% (38 of 51). We conclude that the real-time PCR test is a useful adjunct test for diagnosing early stage or PB leprosy cases.

Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy.

We evaluated the sensitivity and specificity of a nested-polymerase chain reaction (PCR) method for detection of Mycobacterium leprae DNA from whole blood. Whole-blood specimens were subjected to nested-PCR amplification of M. leprae repeat DNA sequences in 49 multibacillary (MB) and 30 paucibacillary (PB) leprosy patients, 96 household contacts (HHCs), 18 tuberculosis (TB) patients, and 35 normal healthy individuals. M. leprae DNA was detected in 95.92% (47/49) of MB, 70% (21/30) of PB, and 6.25% (6/96) of HHC, but it was not detected in 18 TB or 35 normal controls. The sensitivities of the anti-bovine serum albumin (ND-O-BSA) immunoglobulin M (IgM) and antifusion protein of ML0405-ML2331 IgG for MB were 97.96% and 89.8%, and these values for PB were 70% and 53.33%. However, the ND-O-BSA enzyme-linked immunosorbent assay (ELISA) had lower specificity, with relatively high false-positive results for TB patients (16.67%) and normal healthy controls (10%). Based on these promising findings, we propose the use of nested PCR of whole-blood samples along with ELISA test for early detection of leprosy cases.

[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.

[Epidemiological investigation of welder's pneumoconiosis in two container manufacturing industries in Guangdong province].

OBJECTIVE: To investigate the incidence of welder's pneumoconiosis in container manufacturing industries in Guangdong province and present some preventive and curative strategy. METHODS: Occupational epidemiology study methods were used to study the incidence of welder's pneumoconiosis in two container manufacturing enterprises (enterprise A and enterprise B). RESULTS: Before 2004, the rate of up-to-standard of weld fume concentration in the workplaces was relatively low (< or = 40%), and the maximum value of time weighted average (TWA) was 26.7 mg/m(3). After 2004, with the system of dust control, the rate of up-to-standard was 85% in the enterprise A. Of the 813 weld workers examined, 19 were diagnosed as welder's pneumoconiosis (stage I was 15, while I (+) was 4 ) and the incidence was 2.34% (19/813). The age of electric welders suffering from welder's pneumoconiosis and the duration of dust exposure were (33.45 + 4.64) and (8.04 + 1.97) years respectively. Chest radiographic examination showed mainly small round opacities "p". The value of MVV, FEF25%, FEF50% and FEF75% in lung function tests were significantly lower than those of the control group (P < 0.05 or P < 0.01). CONCLUSION: The main features of welder's pneumoconiosis in container manufacturing industries are the short duration of dust exposure, and small round opacities "p" in the chest radiographs. Therefore, it should be the key point of the prevention and treatment of occupational diseases in this industry to control the harmful weld fume.

[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.

[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.

[Studies on the microwave leakage of the interphone].

OBJECTIVE: To assay and study the microwave leakage of 4 types of interphones. METHODS: The radiation intensities of four types of 199 interphones were determined by the microwave leakage measure instrument of model ML-91 made in China. RESULTS: The average intensities of microwave leakage at a distance of 5 cm from aerial part and other parts of interphones during launching [(1 316.0 +/- 144.3), (971.0 +/- 131.6) microW/cm(2) respectively] were significantly higher than during waiting [(14.4 +/- 5.3), (13.2 +/- 4.9) microW/cm(2) respectively] (P < 0.01). The average intensities of microwave leakage at a distance of 50 cm from different parts were (357.3 +/- 27.8) microW/cm(2). The daily average intensity of microwave leakage to which the head, chest and abdomen exposed was (945.5 +/- 447.1) microW.h/cm(2) in total, that exceeded the hygienic standard of microwave in China (400 microW.h/cm(2)), during the normal communication by interphones. CONCLUSION: The microwave leakage was higher during launching than during waiting, and was the highest at the aerial part of the interphones. The microwave radiation of most interphones was higher than the current national standard. It may lead to potential effects on the owner of interphone, so protection against it should be made.

[Epidemiological study of gem worker's silicosis].

OBJECTIVE: To study the hazards of gem dusts and the epidemiological features of the occurrence of gem worker's silicosis (GWS). METHODS: Epidemiological investigation in a gem factory in Guangdong province was carried out. RESULTS: The average dusts concentration in workshop was 2.3 mg/m(3), average free silica content was 94.64%. There were 47 patients diagnosed as silicosis. The prevalence of GWS was 4.58% (47/ 1,027). The age of patients suffering from silicosis and the duration of dust exposure were (27.04 +/- 3.25) years and (4.67 +/- 1.17) years respectively. Chest radiographic examination showed mainly small round opacities "q". The incidences of GWS complicated by pulmonary tuberculosis, spontaneous pneumothorax and pulmonary emphysema were 10.64%, 10.64%, 44.68% respectively. CONCLUSION: The hazards of gem dust to workers might be serious. Higher incidence, younger age suffered from silicosis, shorter duration of dust exposure, more complications, and poorer prognosis in gem workers appeared to be the main epidemiological features of GWS in Guangdong.

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.

Fixed-duration multidrug therapy in multibacillary leprosy

Six-hundred-fifty-seven active multibacillary (MB) leprosy patients were put on fixed-duration multidrug therapy (FD-MDT) between 1985 and 1992 (190 had had no and 235 had had previous treatment with dapsone) and were followed for 5 years after therapy. Two relapses occurred during year 5 of surveillance and both had received dapsone prior to chemotherapy, giving an overall relapse rate of 0.08/100 person-years (py). Excluding the two relapses, 99.4% of the MB patients converted to smear negativity at year 6 after a regular course of FD-MDT. The relapse rate for 35 MB patients with an initial bacterial index (BI) of > 4 with 5 years of surveillance was 0.24/100 py. Reactions occurred more frequently during the first 6 months of MDT, decreasing gradually thereafter, and reaching 0 in year 4 of surveillance. The deformity rate at intake was 22.7% and only 1.8% of MB patients developed new deformities or an increased grade in deformity during therapy.

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%.