A study of leprosy in relation with HBsAg

In the present study presence of HBsAg in the serum of 130 leprosy patients (LL 50; ENL 30; TT 50) and its relationship with various complications has been studied. The HBsAg positivity in controls and various types of leprosy did not show any significant difference. Similarly complication also had no relation with HBsAg positivity. So we conclude that leprosy has no relation with HBsAg.     

Serological pattern of hepatitis B virus markers (HBsAg, anti-HBs, IgM anti-HBc and HBV specific DNA polymerase) in leprosy patients

Sera of 134 lepromatous (LL/BL) and 57 tuberculoid (TT/BT) leprosy patients were analysed for four HBV markers. HBsAg was detected in 6.71% of lepromatous and 3.5% of tuberculoid sera. The per cent positivity of lepromatous and tuberculoid sera for anti-HBs antibodies was 30.59% and 35.08%, respectively. The positivity of normal sera for HBsAg and anti-HBs was 3.60% and 21.69%, respectively. The difference in the positivity of three groups of sera (lepromatous, tuberculoid and normal) for HBsAg or anti-HBs was not statistically significant. Anti-HBc (IgM) antibodies were detected in 6% of lepromatous sera. HBV-specific DNA-polymerase activity was found in 22.22% of HBsAg positive (but anti-HBc negative) sera, and 66.66% of anti-HBc positive (but HBsAg negative) sera. The pattern of acute HBV infection in leprosy patients followed the typical pattern prevalent in the normal population.     

An appraisal of enzyme linked immunosorbent assay (ELISA) and serum antibody competition test (SACT) in leprosy.

Seventy-eight untreated leprosy patients, 104 treated patients and 105 healthy contacts were tested using two serological tests, SACT (serum antibody competition test based on competitive inhibition of monoclonal antibody binding to the MY2a determinant of M. leprae) and ELISA (measurement of IgM antibodies to the neoglycoproteins D-BSA and ND-BSA representing the phenolic-glycolipid antigen of M. leprae). The controls included normal healthy individuals, patients with sputum positive pulmonary tuberculosis, and active cases of rheumatoid arthritis from the department of rheumatology. The specificity of SACT was found to be very high. ELISA was found to be positive in two patients with rheumatoid arthritis, one each for D-BSA and ND-BSA ELISA. Both tests had a high sensitivity in BL and lepromatous patients. The sensitivity to both tests was considerably lower in tuberculoid and BT patients i.e., below 40%. Therefore the diagnostic value of a negative test in suspected cases of leprosy was very low employing either of the two tests. A proportion of patients with paucibacillary tuberculoid and BT leprosy were positive after six months or longer after therapy. Similarly a large number of BL and lepromatous patients were positive after considerably longer periods of treatment. The use of either tests for determining the duration of therapy is therefore limited. SACT appears to be more sensitive than ELISA with ND-BSA in detecting subclinical infection. The cumulative positivity of the two tests may be used as a measure of the infectivity of the disease in the community and for evaluating disease control methods.     

Certain aspects of dapsone metabolism in leprosy patients as studied by high performance liquid chromatography (HPLC) and qualitative screening tests

Dapsone (DDS) in urine of 250 leprosy patients collected on surprise visits were screened by simple paper spot, tile tests and sensitive Enzyme linked immunosorbent assay (ELISA) and Haemagglutination inhibition (HI) tests. The urinary DDS concentration as well as DDS/C ratios were also studied. Simultaneously, 50 microliter of blood was collected from each of these patients and its dapsone content was estimated by HPLC. Urine samples with means of 25 to 30 micrograms/ml DDS and 55-64 micrograms/mg DDS/C ratios were found to give positive tests by any of the above screening procedures, while their mean blood DDS concentration was found to be 0.91 microgram/ml. The corresponding values for those specimens giving negative tests were 3.8 to 5.7 micrograms DDS per ml and 9 to 13 micrograms/mg DDS/C ratio. The blood DDS concentration in this group was ranging from 0.16 to 0.18 micrograms/ml. The findings are discussed in relation to their metabolic significance and their application in a leprosy control programme.     

鼻分泌物及皮肤组织中麻风菌及其PGL-1抗原的检测

为了更好地理解麻风菌鼻携带在麻风病传播、维持中的作用，以及运用鼻携带检测来评价麻风病防治效果，比较了PCR和Dot-ELISA／ECL平行检测32例活动性麻风患者、13例愈后者和143名麻风家内接触者鼻分泌物及皮肤组织中的麻风菌及其PGL-1抗原。结果显示，Dot-ELISA／ECL具有较好的敏感性、特异性，是一项适用于现场研究的简便、快速、经济的麻风流行病学工具。此外，用于免疫学试验，GVHP是一种吸附性好，适合于检测粘膜分泌物抗原的载体。     

Detection of a Mycobacterium leprae cell wall antigen in the urine of untreated and treated patients.

A total of 90 leprosy patients, 12 household contacts and 10 normal subjects were studied for the detection of Mycobacterium leprae cell wall antigen in urine using monoclonal antibody (ML30A2 IgG). In untreated multibacillary leprosy (BL-LL) the M. leprae cell wall antigen could be demonstrated in the urine of 14 (64%) patients by immunofluorescence (IF) and 22 (100%) by ELISA. In untreated paucibacillary leprosy (TT-BT), it could be demonstrated in 3 (11.5%) and in 13 (50%) patients by IF and ELISA methods respectively. All but 1 household contact (later confirmed to have BL leprosy) and all 10 normal subjects' urine was negative for M. leprae cell wall antigen by both methods. The same antigen was, however, demonstrated in urine of 50% paucibacillary patients who had received 6 months of treatment and in 68% multibacillary patients who had received 24 months of WHO recommended multidrug therapy. M. leprae cell wall antigen assays in urine will not be useful in the follow-up of leprosy patients on multidrug therapy.     

High Incidence of IgG Antibodies to Phenolic Glycolipid in Non-Leprosy Patients in India

Purified phenolic glycolipid (PGL-1) from Mycobacterium leprae was used to detect IgG antibodies against PGL-1 in leprosy patients in an enzyme-linked immunosorbent assay (ELISA). A total of 698 sera were screened; they came from patients suffering from leprosy, autoimmune disease, myeloma, tuberculosis and sexually transmitted diseases (STDs). Cases with miscellaneous diseases and persons undergoing AIDS screening were also included. Sera from lepromatous and tuberculoid leprosy patients gave positivity rates of 60.5% and 41.7%, respectively. In non-leprosy cases, the PGL-1 ELISA showed an overall positivity rate of 6.9%; this was greatest in patients with tuberculosis (43.8%) followed by autoimmune diseases (40.9%) and miscellaneous cases including liver diseases (37.9%). This study emphasizes that PGL-1 ELISA has a low predictive value for diagnosis of active infection by Mycobacterium leprae. Positive reactions in a significant percentage of patients with autoimmune disease are intriguing and need indepth study.     

Serum samples from patients with mycobacterial infections cross-react with HIV structural proteins Gp41, p55 and p18

BACKGROUND: Infection with Mycobacterium leprae is associated with a high frequency of false positive results in a variety of serological assays. Our studies have found cross-reactivity to HIV structural proteins in serum samples from leprosy patients, irrespective of the type of disease, treatment duration, age and gender and from a few patients with active TB disease. METHODS: Western blot (WB) analysis revealed that sera from HIV negative leprosy patients across the spectrum showed high reactivity with p18, Gp41 and p55 and lower reactivity with other HIV proteins. The reactivity appeared to be specific; western blot-positive samples were negative in ELISA and in several rapid tests for HIV. Cross-reactivity was not found in sera from patients with leishmaniasis or from normal healthy individuals. RESULTS: None of the WB reactive leprosy patients seroconverted to HIV positivity within 6 months to 1 year after Western blot testing. BLAST analysis revealed that envelope antigens of HIV (Gp41, Gp120 and Gp160) contained amino acid sequences similar to M. leprae ML0470, putative integral membrane protein, Rv0740, mmpL9 (M. tuberculosis). Core (gag) antigens (p18) had similarities to ML0406, but polymerase antigens (p52) had similarities to PE_PGRS (M. tuberculosis, H37Rv). Nucleotide sequence analysis, on the other hand, did not reveal any significant homology between M. leprae or M. tuberculosis and HIV. CONCLUSIONS: The occurrence of these high false-positive rates in M. leprae-infected individuals suggests a possible complication of serodiagnosis of HIV in regions where mycobacterial infections are endemic. There is a need for caution in reporting HIV infection among leprosy patients. Our observations emphasise the value of the various rapid assay kits for HIV, where this false positivity is not observed.     

早期麻风血清学诊断方法的评价及应用研究

目的：评价现有的早期麻风菌感染检测方法的实用性，探讨它们的潜在应用前景。方法：共比较了１２种血清学检测方法，将之按４个“Ｓ”标准和疾病筛检标准评价，并动态观察在亚临床感染及疾病检测中的实用意义。结果：结果表明各方法间的结果是可以比较的。方法敏感性高，特异性强，且简便，廉价，实用。     

Serodiagnosis of leprosy in patients' contacts by enzyme-linked immunosorbent assay

Serum samples from 3336 contacts of leprosy patients were tested for antiphenolic glycolipid I antibodies by enzyme-linked immunosorbent assay with the albumin coupled synthetic disaccharide antigen. The overall positivity rate was 9.3%. No significant differences were seen between a group of household contacts of lepromatous patients and those of the other types of the disease. The proportion of ELISA positives was slightly higher in the relatives as compared to workplace contacts and neighbours but significantly different only between the two former (p less than 0.05). Among those contacts with absorbance values higher than 0.100, 5 new leprosy patients were diagnosed, 2 of them with positive skin smears. A sixth contact was detected with a very high absorbance value in whom no single skin lesion was found but whose lepromin reaction was 0 mm and the skin smear showed a bacteriological index of 3+.     

Leprosy, liver and jaundice

Nine patients of leprosy, 5 BL and 4 LL who developed jaundice during the course of disease were investigated. Two LL patients developed jaundice during ENL reaction. There was slight hepatomegaly in 5 patients and moderate splenomegaly in 3 only. There was significant alterations in liver enzymes and serum bilirubin in all patients. The abnormalities of the enzymes levels persisted for abnormally long periods even when the serum bilirubin had come down and the patients had become asymptomatic. Blood for HBsAg and anti-HAV IgM was negative in all the patients except one in whom HBsAg was positive. Drugs could not be implicated as the cause of jaundice, all patients maintained recovery even after restarting antileprosy drugs. The possibility of non A, non B viruses producing hepatitis during the course of disease is brought out. Course of prolonged jaundice in leprosy is compared with other diseases which could result in a similar situation.     

Increased incidence of cytoplasmic ANCA (cANCA) and other autoantibodies in leprosy patients from western India

The prevalence of various autoantibodies was studied in 75 leprosy patients comprising eight patients with lepromatous leprosy (LL), 36 patients with borderline lepromatous leprosy (BL) and 31 patients with borderline tuberculoid leprosy (BT), along with 100 normal controls. Certain autoantibodies such as anti-nuclear antibodies (ANA), anti-single stranded DNA (anti-ssDNA) and anti-neutrophil cytoplasmic antibodies (ANCA) were raised among leprosy patients. When ANCA specificities to anti-myeloperoxidase (anti-MPO), anti-proteinase3 (anti-PR3) and anti-lactoferrin (anti-LF) were studied, it was found that the patterns of immunofluorescence such as perinuclear (p-ANCA), cytoplasmic (c-ANCA) and atypical (X-ANCA) and specificity by ELISA to anti-MPO, anti-PR3 and anti-LF varied in the LL, BL and BT groups. However, a higher amount of c-ANCA was observed in 62.5% of leprosy cases, while the incidences of p-ANCA and X-ANCA were lower. The LL group showed a higher incidence of autoantibodies as compared with the BL and BT groups, along with a male preponderance for autoantibody development. Some unusual antibody profiles such as 'X'-ANCA were also observed. The study suggests that autoantibody formation could be quite prevalent and also variable in the spectrum of leprosy cases, and there seems to be a serological overlap among leprosy and autoimmune disease, which could have pathogenetic importance in the leprosy patients developing complications.     

Desmoglein 3-ELISA: a pemphigus vulgaris-specific diagnostic tool

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune-blistering disease of the skin and mucous membranes caused by autoantibodies against desmoglein 3 (Dsg3), an epidermal desmosomal adhesion protein of the cadherin family. Cloning of the Dsg3 gene and expression of the protein in a native conformation enabled the recent development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of PV autoantibodies. OBJECTIVES: To evaluate serum samples from patients with PV and other dermatologic diseases for anti-Dsg3 antibodies. To compare ELISA values with autoantibody titers obtained by classic indirect immunofluorescence (IIF). DESIGN: Serum samples from patients with PV and various other bullous and nonbullous skin diseases were tested for anti-Dsg3 reactivity by ELISA. SETTING: Ambulatory and hospitalized patients from a university hospital. PATIENTS: Fifty-two serum samples from 11 patients with PV, and serum samples from 11 patients with bullous pemphigoid, 12 patients with other bullous diseases, 22 patients with various nonbullous skin disorders, and 10 healthy individuals were tested. RESULTS: Forty-seven (98%) of 48 serum samples from patients with PV that were positive by IIF on monkey esophagus were also reactive by Dsg3-ELISA, whereas 4 of 4 IIF-negative PV serum samples showed no reactivity by ELISA. In addition, negative ELISA results were obtained from 11 of 11 serum samples from patients with bullous pemphigoid, 10 of 12 serum samples from patients with other bullous skin disorders, 7 of 9 serum samples from patients with autoimmune-connective tissue diseases, and 13 of 13 serum samples from patients with other nonbullous skin diseases. Interestingly, 1 patient with paraneoplastic pemphigus had positive ELISA results. There was a positive correlation (r = 0.654) between ELISA values and IIF titers within the whole population with PV. In addition, when multiple serum samples from 1 patient with PV sampled over a 2-year period were tested, ELISA reactivity paralleled both the IIF titers and the clinical course. CONCLUSION: The Dsg3-ELISA is a sensitive, objective, and PV-specific test that should be considered as an adjunct test for the management of patients with PV.     

性病与HIV感染者中HSV2、HBsAg和HCV的检测

目的 比较性病患者和HIV感染者血清中单纯疱疹病毒(HSV)、乙型肝炎病毒(HBV)及丙型肝炎病毒(HCV)的感染情况,为采取综合性的防治方案提供参考依据。方法 对经蛋白印迹法确证的HIV(+)/AIDS库存血清标本和梅毒、淋病及衣原体感染患者血清标本,用ELISA方法同时检测HSV2-IgG、HSV2-IgM、HBsAg和HCV-IgG4项指标,并对2组标本的检测结果进行比较。结果 在76份性病样品中,检出HSV2-IgG24例占31.58%;HSV2-IgM1例占1.32%;HBsAg8例占10.53%和HCV1例占1.32%。在另外分组的14例HIV阳性标本中,检出HSV2-IgG7例占50.00%;HSV2-IgM5例占35.71%;HBsAg8例占57.14%和HCV3例占21.43%。在总共90份标本中,有6例标本同时检测到HSV和HBV;2例标本同时检出HSV-IgM和HBV;2例同时检出上述4项指标。统计分析发现,HIV(+)/AIDS组中HSV、HBV和HCV的检出率明显高于性病组(P<0.05)。结论 HIV(+)/AIDS患者中合并HSV、HBV及HCV的感染率明显高于其他性病患者。     

Serological detection of leprosy employing Mycobacterium leprae derived serine-rich 45 kDa, ESAT-6, CFP-10 and PGL-I: a compilation of data from studies in Indian populations

This article is a compilation of our findings recorded in the recent past where we have investigated the serological performance of Mycobacterium leprae antigens like-serine-rich 45 kDa protein (45 kD), early secretary antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) and phenolic glycolipid-I (PGL-I) for detection (employing antibody detecting ELISA) of leprosy patients, particularly those belonging to the paucibacillary (PB) group. All of these antigens were capable of detecting, by themselves the majority (82-100%) of multibacillary (MB) patients. However, with respect to PB patients, only 18-47% (i.e. less than half) of the cases could be detected. Based on the results of serological assays for each of the four antigens separately a combinatorial approach was performed for these antigens, which increased the sensitivity for detection of PB patients to 73%, giving 36% improvement over conventional PGL-I based ELISA. Thus, the multi-antigenic serological approach is worthwhile for its establishment for detection of leprosy patients. Since ESAT-6 and CFP-10 are secreted proteins by nature, antibodies against them are worth exploring for detection of early infections and for monitoring of treatment efficiency. Nevertheless, efforts towards identification of more new antigens with serological potential are still desirable in order to further improve the detection rate of leprosy.     

Utility of serodiagnostic tests for leprosy: a study in an endemic population in South India

In order to evaluate the usefulness of natural disaccharide (PGL1) and 35 kDa antigens based serology in diagnosis of leprosy and in detecting high risk groups for leprosy, this study was conducted in an endemic population in South India. Out of 3346 cases and their households and neighbouring household contacts, serum samples from 2994 and 2875 individuals were screened for antibodies against PGL1 and 35kDa antigens respectively. While the overall positivity for contacts and leprosy cases was 3.3% for PGL1 antibody, the positivity for 35 kDa antibody was 6.3%. The positivity for contact population was 2.7% and 5.4% for PGL1 and 35 kDa antibodies, respectively. Lepromatous and borderline lepromatous patients showed positivity of 35.1% for PGL1 antibody and 45.7% for 35 kDa antibody. Follow-up of contacts showed that the majority (>90%) remained seronegative for both the antibodies and most of the new cases emerged from the seronegative group. The study clearly indicates that specific serological assays are not sensitive enough for application, both for diagnosis and for identifying any individual at risk for leprosy in the south Indian endemic population.     

麻风患者治疗前后血清麻风杆菌特异性抗原和抗体的变化

用免疫斑点试验改良法（Ｍ－Ｄｏｔ－ＥＬＩＳＡ）和酶联免疫吸附试验（ＥＬＩＳＡ）对１０例多菌型麻风患者治疗前后的９９份血清进行了ＰＧＬ－Ｉ抗原和抗体检测。结果表明治疗前后及治疗中各段时间的抗原下降速度远快于抗体，二者之间有非常显著性差异（Ｕ＝９．０５，＞２．５８，Ｐ＜０，０１）。抗原量的下降以治疗后第１个月最快（７９．１４％），治疗第３个月时已下降９９％以上，这提示抗原的检测可用于监测多菌型麻风的早期疗效，在发现耐药和抗麻风新药筛选方面亦有应用价值。     

IgM and IgG antibodies to phenolic glycolipid I from Mycobacterium leprae in leprosy: insight into patient monitoring, erythema nodosum leprosum, and bacillary persistence

Serum IgM and IgG antibodies against Mycobacterium leprae-derived phenolic glycolipid I (PG) were determined in leprosy patients, contacts, and controls by enzyme-linked immunosorbent assay (ELISA). Anti-PG IgM levels increased from the tuberculoid (TT) to the lepromatous (LL) pole of the disease spectrum. There was a positive linear correlation between anti-PG IgM and bacillary index (BI). Patients with erythema nodosum leprosum (ENL) had lower levels of serum anti-PG IgM than non-ENL patients of comparable BI, suggesting that anti-PG IgM is involved in the pathogenesis of ENL. Initial observations indicate that high anti-PG IgM levels in bacillary-negative patients might reflect bacillary persistence. A study of 2 different substrate reagents in the ELISA [2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 0.1 mM H2O2, serum diluted 1:20, and o-phenylenediamine (OPD), 5 mM H2O2, serum diluted 1:300] showed generally good correlation in detection of anti-PG IgM. However the OPD system detected more paucibacillary disease (BT), while the ABTS system detected the significant effect of ENL on the relationship between BI and anti-PG IgM. Anti-PG IgM was clearly dominant over anti-PG IgG. However, certain patients, including several patients who had upgraded from LL and borderline lepromatous leprosy (BL), showed high levels of anti-PG IgG. Since studies have shown that LL patients are selectively deficient in cell-mediated immunity, T-cell products may be required for the IgM to IgG isotype switch. We conclude that anti-PG IgM is useful for monitoring the bacillary load in individual patients and should prove useful for leprosy control strategies.     

DETECTION OF MYCOBACTERIUM LEPRAE DNA IN FORMALIN-FIXED, PARAFFIN-EMBEDDED SAMPLES FROM MULTIBACILLARY AND PAUCIBACILLARY LEPROSY PATIENTS BY POLYMERASE CHAIN REACTION

Background. Diagnosis of paucibacillary leprosy is often difficult. A method that could confirm the diagnosis is the polymerase chain reaction (PCR) of M. leprae DNA. This reaction was applied to biopsied tissues of leprotic patients to determine the suitability and sensitivity of the reaction. Methods. Biopsy samples were taken from previously untreated patients with multibacillary (5 patients) and paucibacillary (3 patients) leprosy, fixed in formalin, and embedded in paraffin, DNA was extracted from paraffin blocks and PCR applied. The sensitivity of the PCR method was tested by using the serially diluted DNA sample as the template. Results. All eight patients showed a positive PCR for M. leprae DNA. The sensitivity was such that a single organism of M. leprae, as counted by staining of the acid-fast bacilli was identified by the PCR. Conclusions. The PCR method is simple, sensitive, specific, and does not require the use of radioisotopes. It can be applied to the unequivocal diagnosis of paucibacillary leprosy which is difficult by other means. The diagnosis can be obtained within 10 hours. Int J Dermatol 1993; 32:710–713     

Use of non-conventional antigen: M. habana in detecting M. leprae antibodies from leprosy patients and contacts in FLA-ABS test

An indirect immunofluorescent (FLA-ABS) test has been developed to detect M. leprae specific antibodies in the active and subclinical cases of leprosy. An antigenically related mycobacterium, M. habana, was used as an antigen to detect M. leprae specific antibodies in the sera samples of leprosy patients. A comparison was made with M. leprae antigen using same set of sera samples. M. habana is capable of detecting anti-M. leprae antibodies in the serum samples of leprosy patients, previously absorbed with various mycobacterial antigens, cardiolipin and lecithin, almost to the same percentage as M. leprae. Possible use of M. habana antigen as an alternative to M. leprae, in the serodiagnosis of leprosy, has been discussed.