Detection of lepromatous leprosy patients shedding M. leprae in nasal droppings by enzyme immunoassay

Enzyme immunoassays (EIAs) for detection of lepromatous leprosy (LL) patients harbouring M. leprae in nasal mucosa are described. One EIA measures IgM antibodies against the synthetic disaccharide (ND-BSA) residue of phenolic glycolipid I of M. leprae, whereas the other titrates primarily IgG antibodies against sonicate supernatant antigens of Mycobacterium w. (M.w.). Fifty coded leprosy sera were analysed by EIAs under a double blind code. Amongst the 20 LL patients with positive nasal smear, 18 (90%) were positive in EIA based on ND-BSA, in comparison to 19 (95%) in EIA using M.w. antigens. The assays can be performed on fresh serum samples or on blood samples collected on filter paper discs. These assays can be useful for leprosy control programmes.     

上转换发光技术侧流免疫夹心法检测59例麻风患者和87例家庭接触者PGL-I抗体结果分析

目的：评估上转换发光技术侧流免疫夹心法（UCP-LFA）检测PGL-I抗体对麻风接触者发病风险预测的价值。方法：采用UCP-LFA 方法检测贵州59例麻风患者、87例家庭接触者和55例健康对照进行PGL-I抗体水平。数据统计采用SPSS 20.0进行卡方分析。根据ROC曲线确定临界值。结果：麻风患者中PGL-I抗体阳性率为81.4%、麻风接触者为5.7%、健康对照人群为1.8%,三组人群PGL-I抗体阳性率差异有统计学意义（χ2 =126.47,P<0.005）。少菌型和多菌型PGL-I抗体阳性率分别为40% 和89%,差异有统计学意义（χ2=13.386,P<0.005）。BI+、BI-患者PGL-I抗体阳性率分别为83%和0%,差异有统计学意义（χ2 =17.560,P<0.005）。结论：麻风患者PGL-1抗体阳性率>麻风病接触者>正常对照,多菌型PGL-1抗体阳性率高于少菌型,提示PGL-I IgM抗体水平对麻风发病有一定的预测作用。     

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.     

Evaluation of Phenolic Glycolipid-I (PGL-I) Antibody as a Multidrug Therapy (MDT) Monitor

Since phenolic glycolipid-I (PGL-I) is an unequivocal marker for Mycobacterium leprae, this antigen has been a good candidate for the serodiagnosis and monitoring of the effectiveness of leprosy chemotherapy. The present study, a continuation of an earlier report, was undertaken to estimate PGL-I antibody titers in 40 leprosy patients 3 and 6 months after starting MDT. All the leprosy groups showed significant declines in anti PGL-I reactivity after 6 months. There was a good correlation between bacteriological indices (BI) and anti PGL-I antibody levels. Thus, PGL-I based serology may be useful in monitoring the response to multidrug therapy.     

A dot enzyme immunoassay for detection of IgM antibodies against phenolic glycolipid-I in sera from leprosy patients

A visual dipstick dot enzyme immunoassay (EIA) for diagnosis of leprosy is described. The assay is based on detection of IgM antibodies against phenolic glycolipid (PGL-I) in sera from leprosy patients. The antigen (PGL-I or synthetic disaccharide of PGL-I) was dotted on a nitrocellulose pad stuck on a plastic strip (dipstick). Sera were used at a dilution of 1:200. Peroxidase coupled mouse anti-human IgM monoclonal antibodies were used as the conjugate. A positive test gave a blue dot against a white background. The test was highly specific for leprosy, and was quite sensitive for detection of bacilliferous (BL/LL) leprosy. The antigen dotted and preblocked dipsticks stored at room temperature upto 4 months of observation period, were unable in the assay.     

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。     

Significance of antibodies to phenolic glycolipid-I in leprosy diagnosis.

A gelatin particle agglutination assay for the detection of anti PGL-I antibodies in 40 clinically diagnosed and variously classified groups of leprosy cases revealed elevated PGL-I antibody titers in 85% of cases. In contrast, the slit-skin smear examination was positive in only 30% of cases. It was further observed that, out of 28 cases with Bacteriological Index (B.I.) zero, 22 cases (78.5%) had significant levels of PGL-I antibodies. There was no case in which the slit skin smear was positive and the PGL-I antibody titer was not significant. The elevated titers of PGL-I antibody better correlated (84%) with histopathological findings than did B.I. Thus it was concluded that estimation of PGL-I antibody titer is a better supplement to clinical diagnosis than B.I. Significant levels of PGL-I antibody were seen in 85% of cases who had no earlier chemotherapy or were treated for less than 2 months. Similar findings were observed in 12 patients who were on MDT for more than 5 months but for less than 2 years. In order to determine the significance of anti PGL-I antibodies in monitoring the response of patients to chemotherapy, a longer follow up with a greater number of cases should be contemplated.     

Gelatin particle agglutination assay to detect anti-PGL-I antibodies in leprosy patients and in household contacts: a preliminary study.

A preliminary study of anti-phenolic glycolipid-I (PGL-I) IgM antibody detection using M. leprae gelatin particle agglutination (MLPA) test kit is described. Antibodies were demonstrated in 70% of our leprosy patients taking antileprosy treatment. The percentage of positivity of multibacillary cases was 86.0, whereas that of paucibacillary cases was 30.0. Good correlation was found between bacteriological index and the presence of antibodies. Antibodies were detected in 28% of our patients released from treatment. Fourteen out of 27 household contacts were found to have antibodies but none of the normal controls were seropositive. These preliminary data demonstrate that MLPA test is not applicable as sero-diagnostic test or as a test of cure, but may be useful for epidemiological studies and as a research tool.     

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.     

The role of Mycobacterium leprae phenolic glycolipid I (PGL-I) in serodiagnosis and in the pathogenesis of leprosy

PGL-I (phenolic glycolipid I) emerged in the early 1980s on the one hand as part of intensive efforts to define the typing antigens of a host of Mycobacterium spp. and also from characterisation of the lipids of skin biopsies from highly bacillary positive lepromatous leprosy patients. PGL-I, despite its extreme lipophilicity due to its inherent phthiocerol dimycocerosyl component, is highly antigenic evoking high titre IgM antibodies in lepromatous leprosy patients, attributable largely to the unique 3,6-di-O-methyl-beta-D-glucosyl entity at the non-reducing terminus of its trisaccharide. PGL-I itself or in the form of semisynthetic neoglycoproteins containing the synthetic terminal disaccharide or the whole trisaccharide chemically conjugated to such as bovine or human serum albumin, has found its greatest utility in the serological diagnosis, confirmation and management of lepromatous leprosy. PGL-I has also been implicated in the tropism of M. leprae for Schwann cells, through specific binding to laminin, and to play an important role in downregulation of the inflammatory immune response and inhibition of dendritic cell maturation and activation, thereby facilitating the persistence of M. leprae/leprosy.     

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.     

M-Dot-ELISA检测麻风家内接触者血清中麻风杆菌特异性抗原

用改良酶联免疫斑点１５试验（简称 Ｍ－Ｄｏｔ－ＥＬＩＳＡ）对麻风家内接触者（ＨＣ）血清进行了麻风杆菌特异性酚酚糖脂Ｉ（ＰＧＬ－Ｉ）抗原检测。７５例ＨＣ均经麻风杆菌明胶微粒凝集试验（ＭＬＰＡ）和酶联免疫吸附试验（ＥＬＩＳＡ）证实为麻风杆菌特异性抗体阳性者。结果表明：①７５例ＨＣ中抗原阳性者１６例，阳性率为２１％。接触多菌型麻风（ＭＢ）者的抗原阳性率（２８％）明显高于接触少菌型麻风（ＰＢ）者（０％），两者之间有极显著性差异（ Ｐ ＜０．０１ ）。②抗体弱、强阳性组的抗原阳性率和抗原量之间亦有极显著差异（Ｐ＜０．０１）。③４０例正常人及１０例经ＭＬＰＡ和ＥＬＩＳＡ检测证实特异性抗体阳性的非麻风患者血清标本，抗原检测均阴性。     

Antibody-based enzyme-linked immunosorbent assay for determination of anti-PGL-I specific circulating immune complex in leprosy patients

A serological study was performed in 122 individuals: 75 leprosy patients and 47 healthy controls. The ELISA test was performed for IgG and IgM using the glycolipid PGL-I antigen from Mycobacterium leprae. Circulating immune complexes (CIC) were isolated by PEG 6000 precipitation method and after dissociation with an acid solution, the IgG and IgM specific against PGL-I were tested with the ELISA test. The multibacillary patients had high levels of antibodies, compared with paucibacillary patients and controls. The antibodies isolated from the CIC presented a similar spectrum spectral distribution as the serology. A positive correlation between the levels of free and CIC bound antibodies was observed. In contrast with tuberculosis patients, specific antibodies present in CIC were not responsible for false-negative results found in some multibacillary patients' serology, since no or very low levels of specific antibodies were found in PEG precipitated serum of these patients. No relation was observed with specific antibody levels detected in CIC during leprosy reactions.     

Comparative study of anti-PGL-1, anti-35 kDa and anti-lipoarabinomannan assays for serodiagnosis of leprosy

Three antibody assays (anti-PGL-1, anti-35 kDa and anti-LAM) were used to determine the levels of antibodies in the sera of untreated leprosy patients. All the three assays showed higher levels of antibodies in BL/LL patients as compared to I and TT/BT patients, as well as healthy controls. BL/LL patients showed positivity of 100%, 84.2% and 78.9% by anti-PGL-1, anti-35 kDa and anti-LAM assays respectively. All the three assays were negative for leprosy in healthy controls. Anti-PGL-1 assay was positive in 20% of TT/BT patients and 17.9% of I patients. Anti-35 kDa assay was negative in all the TT/BT patients and positive in 7.14% of I patients. Anti-LAM assay was positive in 13.3% of TT/BT patients and in 10.7% of I patients. Hence, while these assays are valuable in diagnosing BL/LL patients, their usefulness in diagnosing I, BT or TT leprosy is limited.     

酚糖酯-免疫球蛋白M和鼻分泌物中麻风菌PCR检测在麻风流行病学中的应用

目的通过对流行乡村(同烘和南丘)麻风病患者、家内接触者及普通人群麻风菌感染的检测,评估实验流行病学对预测麻风病传播的意义。方法采用酚糖酯-酶联免疫吸附试验(PGL-ELISA)和检测鼻携带麻风菌的PCR方法,开展流行病学调查。结果(1)麻风病家内接触者的酚糖酯-免疫球蛋白M(PGL-IgM)阳性率和PCR检测的麻风菌鼻携带率分别为30.4%和23.1%;但PGL抗体阳性率在家内接触者和普通村民之间却无显著性差异。(2)两村普通村民的PGL-IgM阳性率,在统计学上无显著差异。然而,在<20岁的年龄组中,同烘村的PGL-IgM阳性率却显著高于南丘村。无论同烘或南丘村,PGL-IgM阳性率高峰均在<20岁的年龄组。随年龄的增加,阳性率逐渐下降。此外,女性的PGL-IgM阳性率高于男性。结论两村的新发现病人主要为年轻人,这与两村PGL-IgM阳性高峰位于<20岁年龄组相关。在<20岁的年龄组中,同烘村的PGL-IgM阳性率显著高于南丘村,除与同烘村患病率和发现率均高于南丘村相关,也与消除麻风病运动(LEC)后,同烘村仍有新病人出现有关。这一现象似乎支持麻风患病率与PGL-IgM阳性率相关。为评价麻风病的传播是否得到控制,以PGL的血清学仍是一种有用的方法。     

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.     

Early infection with M. leprae and antibodies to phenolic glycolipid-I in the nine-banded armadillo

Nine-banded armadillos were intravenously infected with 10(9) M. leprae. IgM antibodies to PGL-I were evaluated three times during the six months before and every two months after the infection. A thorough autopsy examination was done on animals that died or were sacrificed at intervals of 3, 4, 6, 12, 15 and 18 months after the infection. Three animals which had acquired the infection in the wild and one experimentally infected animal showed significant increases in antibody levels corresponding to their high bacterial load. In the other five experimentally infected animals, M. leprae infection was established in the cells of the reticulo endothelial system (RES) long before the IgM antibody levels to PGL-I became positive. It is possible that in human leprosy also M. leprae may enter and multiply in the RES initiating antibody production during the incubation period before clinical disease with neuritis becomes manifest.     

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。     

Effect of unique Mycobacterium leprae phenolic glycolipid-I (PGL-I) on tumour necrosis factor production by human mononuclear cells

Mycobacterium leprae cell wall-associated components are found in large amounts in the tissues of leprosy patients, particularly those at the lepromatous pole. Among these molecules, the phenolic glycolipid-I (PGL-I), unique to M. leprae, has been involved in the selective anergy observed in the lepromatous patients. Armadillo-derived M. leprae retains only a small proportion of the total PGL-I found in infected tissues. Therefore, the addition of PGL-I to M. leprae in vitro is important for a better understanding of M. leprae effects in vivo. We have studied the influence of PGL-I on TNF production by normal human peripheral blood mononuclear cells (PBMC) and by a human monocytic leukaemia cell line (THP-1) following stimulation with killed M. leprae. PGL-I alone did not induce TNF secretion by PBMC, but when associated with a sub-optimal dose of armadillo-derived M. leprae increased the release of this cytokine. In agreement with these results, M. leprae-exposed THP-1 cells did not secrete detectable levels of TNF unless PGL-I was simultaneously added to the culture. This increase in TNF production suggests that PGL-I plays a role in the induction of TNF during the natural infection. In addition, the modulatory effect of PGL-I on TNF release by THP-1 cells reinforces that monocytes are one of the possible targets of this molecule.     

Association of mycobacterial-specific and Mycobacterium leprae specific antibody levels with clinical activity in tuberculoid leprosy: a comparative study of three serological enzyme-immunoassays

The ELISAs for polyclonal antibodies against Mycobacterium leprae (ML-ELISA) and specific antibodies against epitopes on 35 kDa protein (SACT-ELISA) and phenolic glycolipid I (PG-ELISA) of M. leprae were evaluated comparatively in a group of 88 tuberculoid leprosy patients. The overall seropositivity rate with a battery of 3 tests (68%) was not significantly higher than that obtained with ML-ELISA alone (55%) for IgG class of antibodies. Seropositivities for SACT-ELISA and PG-ELISA were, respectively, 38% and 26%. ML-ELISA for IgM class of antibodies was least sensitive, showing only 8% positivity. A significant correlation was noted between individual values of the three assays, but the positive proportions overlapped maximally in the case of ML-ELISA (IgG) and SACT-ELISA. Further, positivity for the latter two assays, particularly SACT-ELISA, showed significant associations with the extent of 'active' (largely untreated) infection. Immunoblotting revealed that the main antibody response was directed towards M. leprae antigens in the molecular weight range of 20-40 kDa and the densitometry results of this zone correlated significantly with corresponding SACT-ELISA and ML-ELISA (IgG) values.