Study of serum lipids in leprosy

Fifty fresh and untreated patients of leprosy constituted the study group. Fifty, age and sex matched healthy individuals formed the controls. Ridly and Jopling system of classification was used in the study. Majority i.e. 21 cases were of BT group, 12 of BB, 7 of BL, 9 of LL and one case was of TT leprosy. The serum triglyceride level was lower than normal in TT, showed no alteration in BT or BB and was insignificantly increased in bL and LL patients. The total cholesterol was lower than normal in TT, showed no alteration in BT or BB and was insignificantly increased in Bland LL patients. The total cholesterol was lower than normal in TT, whereas in BT, BB, BL and LL groups the levels were statistically decreased. The HDL cholesterol was within normal range in TT, significantly decreased in BT and LL patients, showed no significant alteration in BB and was insignificantly decreased in BL group. The LDL cholesterol in TT was low but was not so low statistically when compared with the controls, whereas in BT, BB, BL and LL groups the levels were statistically decreased. The VLDL cholesterol was within normal range in TT and BT, was raised insignificantly in 3 of 12 cases of BB, was within normal range in BL and in LL leprosy it was raised in one out of 9 cases. In the absence of any derangement of liver function tests, it can be concluded that leprosy per se leads to alterations in lipid metabolism. However, no correlation could be established between the group/type of leprosy, bacterial indices and levels of different lipid fractions in the present study.     

Erythrocyte superoxide dismutase, catalase activities and hydrogen peroxide induced lipid peroxidation in leprosy

OBJECTIVES: To assess erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities and hydrogen peroxide induced lipid peroxidation in leprosy. DESIGN: One hundred leprosy patients and 50 normal healthy controls were studied for the parameters. The data was analysed by grouping the patients into Ridley-Jopling (RJ) types [Tuberculoid leprosy (TT, n = 22), Borderline tuberculoid leprosy (BT, n = 28), Borderline leprosy (BB, n = 13), Borderline lepromatous leprosy (BL, n = 16) and Lepromatous leprosy (LL, n = 21)] and into different levels of Bacteriological Index (BI) [bacteriologically negative (n = 32), BI = 0.1-1 (n = 22), BI = 1.1-2 (n = 16), BI = 2.1-3 (n = 14), BI = 3.1-4 (n = 10) and BI = 4.1-6 (n = 06)]. RESULTS: The induced peroxidation was significantly high and the enzyme activities were significantly low in leprosy (total patients) as compared to controls. A progressive increase in peroxidation was detected along the leprosy spectrum from TT to LL and the increase was significant in BB, BL and LL groups as compared to controls. Induced peroxidation in LL group as compared to TT, BT and BB and in the BL group as compared to TT and BT were significantly different. A concomitant progressive decline in enzyme activity was detected along the leprosy spectrum from TT to LL. The SOD activity in BB, BL and LL and the CAT activity in BL and LL were significantly low as compared to controls. SOD activity in BB, BL and LL groups as compared to TT and in the LL group as compared to BT were significantly different. A progressive trend of increasing peroxidation and decreasing SOD and CAT activity were also detected along the leprosy groups with advancing level of BI. Induced peroxidation and SOD activity were significantly different in bacteriologically positive groups as compared to controls and in the BI levels 1.1-2, 2.1-3, 3.1-4 and 4.1-6 as compared to bacteriologically negative group. The peroxidation was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to BI level 0.1-1. The CAT activity was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to controls. CONCLUSION: The study findings suggest oxidative stressful state associated with reduced antioxidant defence potential in erythrocytes of leprosy patients. The study implicates association of erythrocyte oxidative stress with bacterial load and type of leprosy.     

Dermatoglyphics in leprosy (II Metric analysis of palms)

A group of 100 leprosy patients consisting of 50 lepromatous (BL/LL) and 50 tuberculoid (BT/TT) were investigated for metric analysis of the patterns present on their palms. Hundred normal persons were also selected from the families of patients to serve as controls. BT/TT patients and controls did not show any significant difference in their palmar patterns. On the other hand, significant differences were observed in the patterns between BL/LL patients and controls.     

Lymphocytotoxins in leprosy

Lymphocytotoxic antibodies (LCAs) were assayed in serum samples from 60 patients of leprosy spectrum before starting multi-drug therapy (MDT). Seventeen healthy volunteers without any history of viral infection provided control samples. Post treatment follow up samples were also included in the study. In all pretreatment sera of LL, BL and BT/TT patients the levels of LCAs were elevated, the figures were 39.05 +/- 23.87, 44.89 +/- 20.74 and 34.16 +/- 17.50 respectively. With treatment a significant fall in LcAs production was observed in all types of leprosy patients.     

Detection of Mycobacterium leprae DNA in skin lesions of leprosy patients by PCR may be affected by amplicon size

Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group.     

Correlation of serum lipids and lipoproteins in leprosy.

Serum lipids and lipoproteins were assessed in sixty leprosy and forty age and sex matched healthy controls. The study subjects included cases of LL with reactions, LL without reactions, BL with reactions, BL without reactions, BT and TT types of leprosy. The levels of serum phospholipids, triglycerides, total cholesterol, LDL and VLDL fractions were significantly decreased in leprosy patients compared to control subjects. The levels of serum HDL cholesterol and HDL fraction were significantly elevated in leprosy patients. Maximum elevation in serum HDL cholesterol level and HDL fraction and maximum reduction in the levels of serum phospholipids, triglycerides, total cholesterol and LDL and VLDL fractions were observed in lepromatous leprosy (LL) patients with reactions.     

Characterization of circulating immune complexes in leprosy patients and their correlation with specific antibodies against Mycobacterium leprae

Circulating immune complex (CIC) levels and their antibody and antigenic composition were evaluated in patients with leprosy as well as in any individuals living with them; they were precipitated with 3.5% polyethylene glycol (PEG) and, after affinity chromatography isolation and purification, analysed by sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with monoclonal antibodies (Mabs). The presence of CICs was demonstrated throughout the clinical and immunopathological leprosy spectrum at levels related to bacterial load, and in leprosy patients they showed a positive correlation with specific anti-PGL and anti-65 kDa antibodies. The isolation and analysis, however, failed to identity any Mycobacterium leprae antigenic components; although two specific antibodies anti-PGL-1 and anti-65kDa were identified as possible CIC constituents and may be potentially useful in the follow-up of leprosy patients, especially to chirk bacterial load evolution, PG1.-1 being an authentic antigen of this mycobacterium. Also, the involvement of 65 kDa in CICs, being homologous with the human heat shock protein (HSP) 60 kDa family, suggests an autoimmune mechanism in leprosy pathogenesis, Furthermore, those results support the inclusion of CIC anti-body reactivity studies to enhance the sensitivity of serology.     

HLA和麻风相关的研究Ⅱ.L型麻风与HLA-DR2相关

本文对69名江苏汉族麻风病人(L型32名.T型37名》进行了HLA-DR分型,弃分别与65名健康对照相比较.观察到全部病人组和L型病人组DR2抗原频率显著增高,校正后仍有显著性(两组分别为X²=9.16,P=0.003,Pc=0.024,RR=3.15;X²=11.69,P=0.0006,Pc=0:005).T型缪观察到DR:低度增加一(频率差为0.163),但无显著意叉.恨据Porta-McHugh公式,按常巢色体隐性模型(A.Rmodel)用HLA-SA-B程序计算216个HLA-ADR,HLA-B,DR单语型筒假设的麻风易感基因.三位点连锁不平衡参数相对值,发现了四个相对易感麻风的单倍型,All-DR2为相对易感L型麻风的单倍型A9-DR2,All-DRs,Bwso-DR3是相对易感T型麻风的单倍型.本文结果进一步提示;假设的麻风易威基因是同HLA区域相连锁的,向接说明疾病易感基因可能在DR位点侧两型麻风均司能呈多抗原弱相关.两型麻风具有不同的遗传背景一可能是尸组异质性疾病.     

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.     

Alpha-1-antitrypsin in leprosy

Estimation of Alpha-1-antitrypsin (AAT) levels was carried out in 52 patients of various types of leprosy. Fifty age and sex matched healthy individuals served as controls. The mean level of AAT in controls was 290.12 +/- 59.56 mg/dl. In patients of tuberculoid leprosy (TT), borderline tuberculoid leprosy (BT) and borderline leprosy (BB), the AAT levels were found to be 284 +/- 47.03, 314.37 +/- 31.56 and 324.44 +/- 32.05 mg/dl respectively. These were statistically insignificantly raised when compared with controls. In borderline lepromatous leprosy (BL), lepromatous leprosy without erythema nodosum leprosum (LL without ENL) and in LL with ENL there was a statistically significant rise in AAT levels. The maximum levels of AAT were observed in patients of LL with ENL (mean 500.8 +/- 93.44 mg/dl. P less than 0.001).     

麻风病人外周血中性粒细胞和淋巴细胞产生氧自由基功能的研究

用化学发光法测定麻风病人外周血中性粒细胞（ＰＭＮ）和淋巴细胞（ＬＣ）受刺激后释放氧自由基，引发化学发光的情况。与正常人比较，ＬＬ、ＢＬ活动期病人的ＰＭＮ发光值均弱（Ｐ＜０．０５）；ＴＴ、ＢＴ活动期病人的ＰＭＮ发光值稍差，但其ＬＣ发光值增高（Ｐ＜０．０５）；各型麻风愈复期病人的ＰＭＮ和ＬＣ发光值与正常人近似。说明活动期病人ＰＭＮ产生氧自由基功能低下，是麻风发病的主要因素。     

Evaluation of diagnostic role of in situ PCR on slit-skin smears in pediatric leprosy

A large proportion of early cases of leprosy in children remain AFB negative in skin smears. Such cases required additional techniques to confirm the diagnosis. In situ PCR on slit- skin smears is minimally invasive and less cumbersome as compared to skin biopsies. This study was initiated in our institute with the objective to evaluate the diagnostic value of in situ PCR on slit- skin smears in pediatric leprosy. A total of 25 cases of leprosy below 16 years of age were included in the study. After detailed history and thorough clinical examination, informed consent was obtained from the parents of children for slit- skin smears from lesion sites for AFB staining and for in situ PCR technique. Cases were clinically categorized according to IAL classification into indeterminate (I), tuberculoid tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) and lepromatous (LL). Most of the patients (76%) were between 9-16 years of age and 64% of the cases had history of contact with leprosy patients within the family. Skin smears were positive for AFB in only 20% of the cases. On applying in situ PCR, it was observed that 62.5% cases of I/TT/BT/BB category and 88.8% of BL/LL category gave positive signals. Overall in situ PCR confirmed the diagnosis in 72% cases while by slit smears diagnosis was confirmed in only 20% of cases. Further, out of 20 skin smear negative cases, 13 were positive by in situ PCR. Specificity of the signals of in situ PCR was established by demonstrating the absence of signals in nonleprosy dermatological conditions of vitiligo and P.alba. This study supports the potential usefulness of in situ PCR on slit- skin smears of early pediatric leprosy cases. This strategy will be especially useful in cases where skin smears are negative and in those cases where skin biopsy can not be done either because of unusual locations of lesions or because of sensitive age of the patients.     

透明质酸及其受体CD44在麻风患者愈后表皮中的表达

目的研究透明质酸及其受体CD44在麻风患者皮损愈后者表皮的表达是否发生改变。方法用免疫组化方法比较透明质酸及其受体CD44在不同类型麻风患者愈后及正常表皮中的表达。结果与正常表皮比较,透明质酸在LL、BL及BT型麻风患者皮损愈后表皮的表达明显增强;而在TT型表皮的表达没有明显的改变;在BB型基底层的表达也轻微减弱。CD44在BB,BT及TT类型麻风患者愈后表皮的表达均增强,尤其是TT型。而在LL型的表达则轻微减弱。CD44在BL型的表达没有明显改变。结论麻风患者愈后表皮透明质酸及CD44的表达均有改变。     

Specificity of lymphocytotoxic autoantibodies (LCAbs) found in the serum of leprosy patients: class I MHC antigens

Lymphocytotoxic autoantibodies (LCAbs) of the IgM class have been identified in patients with borderline tuberculoid (BT) and borderline lepromatous (BL) leprosy with Type I reactions (I) as well as lepromatous leprosy (LL) patients with erythema nodosum leprosum reactions (ENL). The observation that lymphocytotoxic activity (LCA) was reduced in the presence of platelets led us to determine whether LCAbs had specificities for Class I Major Histocompatibility Complex (MHC) determinants. Absorption of LCA positive sera with platelets, classically used to deplete Class I specific lymphocytotoxic antibodies, reduced LCA towards autologous as well as allogeneic target cells. This was true for LCA positive sera from all patient classifications (group BT in the autologous system, p less than 0.01; in all other patient groups, p less than 0.001). Introducing B-2m to cytotoxicity assays only marginally reduced LCA when added at high concentrations (5 mg/ml). An anti-Class I MHC antiserum which blocked the lytic activity. The data indicate that LCAbs while absorbed by platelets, are not specific for the Class I MHC antigens. The autoantigen recognized by these autoantibodies therefore remains to be identified.     

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.     

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.     

Correlation of clinical, histological and immunological features across the leprosy spectrum

The Ridley-Jopling system of classification of the variegated clinical pattern of leprosy is based on the specific cell-mediated immunity observed in the histopathology of skin lesions conforming to a spectrum from TT at one end to LL at the other. In this study a fairly large sample of 90 patients was classified on clinical grounds; the histopathology of the skin lesions was studied blind. There was an overall concordance of 90% between the clinical and histological classifications. In addition, the systemic cell-mediated and humoral immune responses were studied. The in vivo cell-mediated immune response, namely the Mitsuda skin response, mostly conformed to the clinical classification. While the in vitro lymphoproliferative responses to BCG and its sonicate were high, the lymphoproliferative responses to Dharmendra lepromin were surprisingly poor. Humoral responses to 35 kDA protein of M. leprae and PGL-1 were good in most LL, BL patients and tapered off towards TT. IgG antibodies to recombinant ML 65 kDa proteins denoted mycobacterial presence.     

51例麻风患者临床及皮肤病理分析

回顾性分析2013年1月至2020年6月我科收治的51例麻风患者临床资料,其中男34例,女17例;35例首诊考虑麻风,16例误诊。皮损主要位于面颈部、躯干、四肢,表现为弥漫性浸润性斑片、斑块,其中伴眉毛脱落(21例)、尺神经粗大(1例)、感觉异常(13例)、畸形(1例),皮疹处瘙痒(2例)。临床分型:结核样型(TT)2例、界线类偏结核样型(BT)2例、中间界线类(BB)2例、界线类偏瘤型(BL)10例、瘤型(LL)34例、未定类麻风(I)1例。I型麻风反应5例(9.8%)、II型麻风反应12例(23.52%)。组织病理示表皮萎缩、变薄33例(64.70%)、有无浸润带30例(58.82%),病变累及真皮全层8例(15.60%);抗酸染色(Wade-Fite染色)阳性率49例(96%)。     

麻风皮损石蜡切片中酚糖脂抗原表达

用免疫组化染色技术检测了６７例各型麻风患者皮肤活检石蜡切片中酚糖脂（ＰＧＬ－Ｉ）抗原的表达，除２例结核样型外有６５例标本ＰＧＬ－Ｉ抗原呈阳性表达。麻风杆菌和有关的可溶性抗原染色在形态学上呈５种染色模式。在瘤型、偏瘤型和中间界线类麻风中，ＰＧＬ－Ｉ阳性与抗酸染色阳性基本一致。而在结核样型和偏结核样型麻风中，ＰＧＬ－Ｉ阳性高于抗酸染色。本研究结果表明免疫组化技术有较高的特异性和敏感性，为麻风病理学诊断和病因学研究提供了新方法。     

Immunogenicity of Mycobacterium leprae unique antigens in leprosy endemic populations in Asia and Africa

Ongoing transmission of leprosy is evident from the stable disease incidence in high burden areas. Tools for early detection of Mycobacterium leprae (M. leprae) infection, particularly in sub-clinically infected individuals, are urgently required to reduce transmission. Following the sequencing of the M. leprae genome, many M. leprae-unique candidate proteins have been identified, several of which have been tested for induction of M. leprae specific T cell responses in different leprosy endemic areas. In this study, 21 M. leprae-unique proteins and 10 peptide pools covering the complete sequence of five M. leprae-unique proteins (ML0576, ML1989, ML1990, ML2283, and ML2567) were evaluated in 160 individuals in Nepal and Ethiopia. These included: tuberculoid and borderline tuberculoid (TT/BT), borderline borderline and borderline lepromatous (BB/BL) leprosy patients; healthy household contacts (HHC); tuberculosis (TB) patients and endemic controls (EC). Immunogenicity of the proteins was determined by IFN-gamma secretion via stimulation of PBMC in 6 days lymphocyte stimulation tests (LST) or in whole blood assays (WBA). In LST, BB/BL patients (40%) responded to ML0573 and ML1601 whereas ML1604 was most immunogenic in TT/BT (35%) and HHC (36%). Additionally, significant numbers of EC displayed IFN-gamma production in response to ML0573 (54%), ML1601 (50%) and ML1604 (54%). TB patients on the other hand, hardly responded to any of the proteins except for ML1989. Comparison of IFN-gamma responses to ML0121, ML0141 and ML0188 for TT/BT patients showed specific increase in diluted 6 days WBA compared to the undiluted 24 hours WBA, whereas EC showed a reduced response in the diluted WBA, which may indicate detection of disease-specific responses in the 6 days WBA. In summary, identification of multiple M. leprae proteins inducing M. leprae-specific T cell responses in groups at high risk of developing leprosy may contribute to improve early detection for M. leprae infection.