Prevalence of IgM antibodies to phenolic glycolipid I among household contacts and controls in Korea and the Philippines.

Phenolic glycolipid I (PGL-I) is a Mycobacterium leprae-specific antigen and the antibodies to the antigen may suggest an M. leprae infection. To compare the M. leprae transmission among the populations, we compared the prevalence of anti-PGL-I IgM antibodies among household contacts and controls between Korea and the Philippines. In Korea (prevalence of leprosy--0.04: 1000), the prevalence of anti-PGL-I antibodies were 4.8% among controls and 8.0% among contacts, respectively. On the other hand, the seroprevalence rate was 10.8% among controls and 13.4% among contacts in the Philippines (prevalence of leprosy--0.70: 1000). Interestingly, a marked difference was noted in the prevalance of anti-PGL-I antibodies among children between the countries; 10-14% among children under 10 years old and 15-18% among those aged between 10 and 19 in the Philippines compared to 0% and 2.9-6.4% in Korea, respectively. This study, therefore suggests that a high prevalance of anti-PGL-I IgM antibodies among children may indicate an active transmission of M. leprae, resulting in a higher incidence of leprosy in the population.     

The evolution of antibody response in armadillos inoculated with Mycobacterium leprae

Plasma from 30 armadillos (Dasypus novemcinctus) was collected prior to inoculation and at approximately 3-month intervals for a period of 1-3 years. These animals were inoculated intravenously with 6.1 x 10(8) +/- 2 x 10(8) (x +/- SD) armadillo-derived Mycobacterium leprae. These samples were analysed for antibodies of IgM and IgG class to phenolic glycolipid-I (PGL-I) and to sonicated M. leprae components using ELISA and immunoblotting techniques, respectively. We had previously observed among a group of 11 armadillos, that some animals produced and maintained a high IgG antibody response to PGL-I. In this study, an animal's ability to produce and maintain an elevated IgG anti-PGL-I response was significantly correlated with their ability to delay dissemination of the infection and their ability to survive longer. When the animals were moribund, a significant decrease in the IgG anti-PGL-I absorbance value was observed. The detection of PGL-I in the plasma samples collected from moribund armadillos suggested that high concentrations of PGL-I in the plasma may have contributed to a drop in absorbance values by the formation of non-lattice-type immune complexes in vivo. As detected by immunoblotting, the IgM and IgG response to antigens derived from sonically disrupted M. leprae was directed towards molecules with broad bands of immunoreactivity ranging from 21- to 45-kDa. There were no distinguishing features of these antibody responses among armadillos as was evident with the IgG anti-PGL-I responses.     

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.     

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.     

Microscopic leprosy skin lesions in primary neuritic leprosy

The histologic diagnosis of primary neuritic leprosy (PNL) remains a public health care concern, especially when nerve biopsies cannot be performed. As such, some authors emphasize the importance of performing a skin biopsy of a hypoesthetic area even without clinically visible lesions. In this study, an attempt was made to define the histologic changes in the sensory altered skin of 42 clinically diagnosed PNL patients. Histologic alterations caused by leprosy were seen in 31% of these patients: 6 were classified as borderline tuberculoid and 7 as indeterminate. In addition, 33% showed mild, non-specific, mononuclear cell infiltrates around the blood vessels within the papillary and reticular dermis that probably reflected an early inflammatory reaction to Mycobacterium leprae infection. Only 36% of those biopsied had no significant lesions. Our results suggested that, while not all PNL patients are similar, histologic skin examination can contribute to early leprosy detection and commencement of adequate treatment.     

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。     

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。     

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.     

早期麻风分子生物学和免疫学诊断

传统的麻风实验技术对早期麻风的诊断有一定的局限性。新的分子生物学和免疫学技术为提高早期麻风诊断率,包括抗酸染色阴性的少菌型与纯神经炎麻风患者的诊断均提供了新的方法。本文综述近年来国内外采用PCR技术检测麻风菌基因、运用新发现抗原血清学与T细胞试验对早期麻风诊断的研究进展,阐述其目前存在的问题与发展前景。     

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.     

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.     

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.     

Primary neuritic leprosy: a reappraisal at a tertiary care hospital

AIM: To study the clinico-epidemiological profile of primary neuritic leprosy. METHODS: Retrospective analysis of the leprosy records of clinically diagnosed patients of PNL who attended the Leprosy Clinic from 2000 to 2004 was carried out for details of presenting complaints, age and sex distribution, duration, number of nerves involved and pattern of nerve enlargement, BI (skin), nerve abscess, and deformities. RESULTS: There were 32 (4.6%) patients of PNL out of a total of 686. Majority (56.2%) had complaints for less than a year. There were 29 (90.6%) males, and 3 (9.4%) females, with 15-30 years as the commonest age-group (65.6%) involved. Paraesthesia and numbness were the presenting complaints in 20 (62.5%), and motor deficit (paresis) in 11 (34.4%); deformities (claw hand, foot drop, trophic changes) were seen in 16 (50%) cases. Polyneuritic pattern was noted in 21 (63.56%) patients and mononeuritic in 11 (16.5%) with ulnar nerve as the most commonly enlarged nerve (63.6%). Nerve abscess was noted in 4 (12.5%) cases. Slit-skin smear was positive in 2 (6.2%) cases only. DISCUSSION: PNL continues to be common in India. Sensory complaints are early and more common. The disease is more common in males. Polyneuritic pattern was predominant, and the ulnar nerve was the most commonly involved nerve. Majority of the cases belong to the tuberculoid spectrum. Early diagnosis depends on complete neurological examination in order to reduce the sequelae of the disease.     

A comparative evaluation of skin and nerve histopathology in single skin lesion leprosy

BACKGROUND: In spite of leprosy being a disease of nerves, ROM therapy for single skin lesion leprosy was based on clinical trials without much evidence-based studies of nerve pathology. The present study was undertaken to compare the histology of skin and nerve in single skin lesion leprosy, and to assess the scientific rationale and justification of single dose ROM therapy. METHODS: Twenty-seven untreated patients with single skin lesion without significantly thickened peripheral nerves were selected. Skin and nearby pure cutaneous nerve biopsies were studied under both H&E and Fite's stain. RESULTS: All the skin biopsies were negative for AFB and clinico-pathological correlation was positive in 51.85% of skin biopsy specimens. Histopathological diagnosis of leprosy was evident in 55.5% of clinically normal looking nerves, with AFB positivity in 29.6% of nerve biopsy specimens. Correlation between clinical diagnosis and nerve histopathology was poor (26%). CONCLUSIONS: Single skin lesion without thickened peripheral nerves as criteria for single dose ROM therapy is not logical, since the histological diagnosis of leprosy in normal looking nerves with presence of AFB is revealed in this study. Pure cutaneous nerve biopsy is a simple outpatient procedure, without complications. This study emphasizes the need to consider nerve pathology as an important tool for further therapeutic recommendations, than just clinical trials and skin pathology alone. Though single dose ROM therapy has been withdrawn recently, the principle holds good for any future therapeutic recommendations.     

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.     

Generation and characterization of a human monoclonal antibody against phenolic glycolipid-I of M. leprae

The development of an Epstein-Barr virus transformed human B-cell line secreting a monoclonal antibody (MoAb), KR2/B5 is described. KR2/B5 is an IgM type of antibody and is highly specific for phenolic glycolipid-I (PGL-I) a component unique to M. leprae. The MoAb appears to be directed against the terminal sugar residue of the immunodominant trisaccharide component of PGL-I.     

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 INFIR Cohort Study: investigating prediction, detection and pathogenesis of neuropathy and reactions in leprosy. Methods and baseline results of a cohort of multibacillary leprosy patients in north India

The aim of this study was to find predictors of neuropathy and reactions, determine the most sensitive methods for detecting peripheral neuropathy, study the pathogenesis of neuropathy and reactions and create a bank of specimen, backed up by detailed clinical documentation. A multi-centre cohort study of 303 multibacillary leprosy patients in Northern India was followed for 2 years. All newly registered MB patients requiring a full course of MDT, who were smear positive and/or had six or more skin lesions and/or had two or more nerve trunks involved, were eligible. A detailed history was taken and physical and neurological examinations were performed. Nerve function was assessed at each visit with nerve conduction testing, warm and cold detection thresholds, vibrometry, dynamometry, monofilaments and voluntary muscle testing. Because the latter two are widely used in leprosy clinics, they were used as 'gold standard' for sensory and motor impairment. Other outcome events were type 1 and 2 reactions and neuritis. All subjects had a skin biopsy at registration, repeated at the time of an outcome event, along with a nerve biopsy. These were examined using a variety of immunohistological techniques. Blood sampling for serological testing was done at every 4-weekly clinic visit. At diagnosis, 115 patients had an outcome event of recent onset. Many people had skin lesions overlying a major nerve trunk, which were shown to be significantly associated with an increased of sensory or motor impairment. The most important adjusted odds ratios for motor impairment were, facial 4.5 (1.3-16) and ulnar 3.5 (1.0-8.5); for sensory impairment they were, ulnar 2.9 (1.3-6.5), median 3.6 (1.1-12) and posterior tibial 4.0 (1.8-8.7). Nerve enlargement was found in 94% of patients, while only 24% and 3% had paraesthesia and nerve tenderness on palpation, respectively. These increased the risk of reactions only marginally. Seven subjects had abnormal tendon reflexes and seven abnormal joint position sense. In all but one case, these impairments were accompanied by abnormalities in two or more other nerve function tests and thus seemed to indicate more severe neuropathy. At diagnosis, 38% of a cohort of newly diagnosed MB leprosy patients had recent or new reactions or nerve damage at the time of intake into the study. The main risk factor for neuropathy found in this baseline analysis was the presence of skin lesions overlying nerve trunks. They increased the risk of sensory or motor impairment in the concerned nerve by 3-4 times. For some nerves, reactional signs in the lesions further increased this risk to 6-8 times the risk of those without such lesions. Patients with skin lesions overlying peripheral nerve trunks should be carefully monitored for development of sensory or motor impairment.     

Giant nerve abscesses in leprosy

Two leprosy patients with neuritis caused by giant abscesses involving almost the entire ulnar nerve are described. One patient, who also had skin lesions, was diagnosed histopathologically as having borderline tuberculoid leprosy both on skin and nerve biopsy, and the other, with only nerve involvement, belonged to the pure neuritic group. The lepromin test was strongly positive (with a vesicular reaction in one patient) and lymphocyte transformation to Mycobacterium leprae antigen was raised. These lesions can be easily mistaken for a peripheral nerve tumour in places where leprosy is uncommon. A brief account of the management of nerve abscess in leprosy is given.     

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。