Association of the LRRK2 genetic polymorphisms with leprosy in Han Chinese from Southwest China.

Leprosy is a chronic infectious and neurological disease that is caused by infection of Mycobacterium leprae (M. leprae). A recent genome-wide association study indicated a suggestive association of LRRK2 genetic variant rs1873613 with leprosy in Chinese population. To validate this association and further identify potential causal variants of LRRK2 with leprosy, we genotyped 13 LRRK2 variants in 548 leprosy patients and 1078 healthy individuals from Yunnan Province and (re-)analyzed 3225 Han Chinese across China. Variants rs1427267, rs3761863, rs1873613, rs732374 and rs7298930 were significantly associated with leprosy per se and/or paucibacillary leprosy (PB). Haplotype A-G-A-C-A was significantly associated with leprosy per se (P=0.018) and PB (P=0.020). Overexpression of the protective allele (Thr2397) of rs3761863 in HEK293 cells led to a significantly increased nuclear factor of activated T-cells' activity compared with allele Met2397 after lipopolysaccharides stimulation. Allele Thr2397 could attenuate 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced autophagic activity in U251 cells. These data suggest that the protective effect of LRRK2 variant p.M2397T on leprosy might be mediated by increasing immune response and decreasing neurotoxicity after M. leprae loading. Our findings confirm that LRRK2 is a susceptible gene to leprosy in Han Chinese population.

A study on the methods for early serological diagnosis of leprosy and their potential use.

This is a serial study. In this series we have established 12 methods for the early serological diagnosis of leprosy, including the FLA-ABS test, ELISAs with artificial products (ND-O-, ND-P-, NT-O-, NT-P-BSA; PGL-I, whole M. leprae and M. smegmatis), monoclonal antibody specific binding assay (McAb/SBA), latex agglutination test (LAT), and MLPA. These methods were compared with each other on a large scale in leprosy patients and in the field. The results indicate that 1) Excellent results were obtained when ELISAs were conducted with skim milk or egg albumin as the blocking agent and by using blood from earlobes instead of from venipuncture. 2) According to the four "S" standard (sensitivity, specificity, simplicity and speed), among the 12 methods the ND-O-BSA-ELISA (ND-ELISA) is the best and the MLPA is more suitable for use in the field because it is simple and rapid. 3) In the ND-ELISA, the increase or decrease of the OD value has a positive correlation with the BI, and the order of positive rates was a) in various types of leprosy: LL > BL > BB > BT > TT; b) in household contacts (HC), random population (RP), normal controls in endemic areas (ENC) and normal controls in nonendemic areas (NNC): HC > RP > ENC > NNC. 4) In a population with subclinical M. leprae infection, the highest risk group was between the ages of 15 and 25 and had an increase or a persistence of high OD values prior to onset of disease. 5) OD values gradually decreased over time following treatment and these declines paralleled declines in the BI. 6) In cases cured with dapsone therapy, there was an increase or a persistence of high OD values in ND-ELISA prior to the onset of a leprosy relapse. In conclusion, we have compared and evaluated 12 immuno-assays and have shown that the ND-ELISA is the most practical one for use in investigating sero-immunological epidemiology, subclinical infection with M. leprae, early detection of disease, monitoring of antimicrobial therapy, and even for the prediction of leprosy relapse.

Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9-grown M. bovis BCG cells and determination of the N-terminal amino-acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP-utilizing enzymes was explored. External ATP is important in the activation of macrophage surface-associated P2Z receptors, whose activation has been postulated to allow phagosome-lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosome fusion or macrophage apoptotic death.

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%.

Integration of traditional and modern methods in the identification of AFB cultures isolated from clinical specimens of patients with skin diseases.

This article reports the identification of 57 AFB cultures isolated from clinical specimens by using traditional methods (TM, including biochemical and cultural methods) and modern ELISA with monoclonal antibody (McAb-ELISA) and nested primer gene amplification assay (NPGAA). The representive AFB culture M. A1, A7, A19, A21 and A22) isolated from human lepromas were identified as new species by TM and it was shown that they were not identical to M. leprae by McAb-ELISA and NPGAA. Among another set of samples (M. S17, S1, S2, S2R, S7, S29), M. S17 was identical to M. scrofulaceum as assessed by TM only, while the others were found to be similar to M. tuberculosis and different from M. leprae using TM and McAb-ELISA, and identical to M. tuberculosis with NPGAA. The authors conclude that TM and MM are very useful for identifying mycobacteria, while MM was much more sensitive and specific than TM. The selection and use of these methods depends on practical need.

Rapid serodiagnosis for leprosy--a preliminary study on latex agglutination test.

In this study, we have developed two latex agglutination tests (LATs) with phenolic glycolipid-I (PGL-I) and natural disaccharide-octyl-bovine serum albumin (ND-O-BSA) as antigens in 110 leprosy patients (LL = 30, BL = 30, BT = 30, and TT = 20), 50 tuberculosis cases, and 30 normal controls. These two LATs were compared with corresponding ELISAs (ND-O-BSA ELISA and PGL-I ELISA) and analyzed by the chi-squared test. There were no significant differences between the two LATs (PGL-I LAT and ND-O-BSA LAT) and their corresponding ELISAs. There was an increase in the proportion of positive cases detectable which coincided with the clinical classification of leprosy, i.e., lepromatous cases were more likely to be positive than tuberculoid cases. LATs are more simple and rapid than ELISAs and have high sensitivity (77% in ND-O-BSA LAT, 80.5% in PGL-I LAT) and specificity (99% in both LATs). LATs may become useful tools for the immunodiagnosis of leprosy in the field. The stability and repeatability of LATs are discussed in detail.

Evaluation of fluorescent leprosy antibody absorption test versus enzyme-linked immunosorbent assay with phenolic glycolipid 1 and their use in immuno-epidemiological studies on leprosy.

We systematically conducted comparative studies on the validity, reliability and practicality of FLA-ABS.T/PGI-ELISA in large samples. Namely, 284 leprosy patients, 20 tuberculosis patients, 172 normal controls (from nonendemic area of leprosy), 425 leprosy household contacts (HC) and 2573 random samples from the general population (RS) were involved. The results indicated that FLA-ABS.T/PGI-ELISA are highly sensitive and specific for detecting antibodies against M. leprae. Their Youden's indexes (YI) are greater than 90%, and the positive predicative and negative values are 90%. The test results agreed with immuno-epidemiological studies: 1. The positive rates using FLA-ABS.T/PGI-ELISA increased gradually from TT to LL leprosy patients (in HC, the positive rates of PGI-ELISA were much higher in contacts of multibacillary patients than in contacts of paucibacillary patients); 2. The positive rates detected by FLA-ABS.T were identical to those of PGI-ELISA both in HC and in RS; 3. Among RS, the positive rates detected by FLA-ABS. T/PGI-ELISA were similar in each district and were in concordance with the general prevalence rates. Thus, both FLA-ABS.T and PGI-ELISA are useful tests in diagnosing leprosy and detecting subclinical infection with M. leprae. However, because the PGI-ELISA is simple, it will be more practical than FLA-ABS.T in the future. The authors emphasize that the methodology of obtaining dried blood from ear lobes is important for the immuno-epidemiological study of leprosy on a large scale.

Serological activity of natural disaccharide octyl bovine serum albumin (ND-O-BSA) in sera from patients with leprosy, tuberculosis, and normal controls.

We studied the natural disaccharide-octyl-bovine serum albumin (ND-O-BSA) enzyme-linked immunosorbent assay (ELISA) in sera from 151 leprosy patients, 20 tuberculosis patients, and 42 normal persons from a nonendemic area. The three ELISAs, whole Mycobacterium leprae (WML), phenolic glycolipid-I (PGL-I), and ND-O-BSA, are all highly sensitive for detecting antibodies against M. leprae. The results indicate that the serological activity has highly significant, positive correlations among the three types of antigens used. Their positivity rates are 100% with PGL-I and 97.4% with WML and ND-O-BSA in leprosy patients, and 0% with any antigen used in normal persons at NV-a (a supposed theoretical normal value). However, all three antigens show crossreactivity with tuberculosis patients at different levels. At NV-c (a supposed practical normal value, PNV), this crossreaction significantly decreased in the WML ELISA (PNV = 0.28) and the PGL-I ELISA (PNV = 0.16), and disappeared in the ND-O-BSA ELISA (PNV = 0.20). Under the same conditions, the positivity rates did not decrease significantly in leprosy patients, especially in multibacillary patients. Therefore, we suggest that the PGL-I ELISA in combination with the ND-O-BSA ELISA may be very useful for clinical applications, serodiagnosis, and for the study of subclinical infection in leprosy.

Determination of antibodies in dried blood from earlobes of leprosy patients by enzyme-linked immunosorbent assay--a preliminary report.

An enzyme-linked immunosorbent assay (ELISA) was used with soluble antigens of Mycobacterium leprae. All blood samples collected from the earlobes of 109 leprosy patients and 100 healthy controls (from a non-endemic area of leprosy) were absorbed with M. vaccae, BCG, cardiolipin, and lecithin according to the technology of the FLA-ABS test before being tested in the ELISA. The results (at a 1:200 blood dilution) showed that antibody activity gradually increased from TT to LL (mean OD values: TT = 0.43, BT = 0.62, BB = 0.72, BL = 0.84, LL = 0.89), and the rates of positive reactions were 100% in all classifications of patients except TT (66.6%). Antibody activity in the controls was less pronounced than in leprosy patients, their mean OD value being only 0.15. We suggest that the ELISA is highly sensitive and specific for the determination of anti-M. leprae antibodies, and is useful for clinical serodiagnosis and for the study of subclinical infections in leprosy.