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.     

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.     

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.     

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。     

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.     

Role of PGL-I antibody detection in the diagnosis of pure neural leprosy

Pure neural leprosy (PNL) is difficult to diagnose because skin lesions and acid-fast bacilli (AFB) in slit smears are absent. At present, the gold standard for PNL diagnosis is the histopathological examination of a peripheral nerve biopsy. Even so, detection of bacteria is difficult and histological findings may be non-specific. Furthermore, nerve biopsy is an invasive procedure that is only possible in specialized centres. Therefore, there is a need for additional diagnostic methods that may help to confirm the clinical diagnosis of PNL. In the present study, an additional laboratory test, the ELISA for anti-phenolic glycolipid I (PGL-I) IgM antibodies, was performed on 103 individuals with clinical and neurophysiological signs of peripheral neuropathy, of which 67 were diagnosed as PNL patients and 36 remained as 'not diagnosed as PNL', as well as on a control group of 34 patients with other neurological diseases. An antibody response was present in 14/67 (21%) of the patients diagnosed as PNL as compared with 3/34 (9%) of controls. Anti-PGL-I positivity was observed in 5/8 (63%) of the AFB positive cases. Patients whose diagnosis was confirmed solely by Mycobacterium leprae PCR on the nerve sample had 4/25 (16%) seropositivity. In addition, anti-PGL-I antibodies were detected in 9/40 (23%) of the PNL patients who were PCR negative for M. leprae DNA. Moreover, two patients who showed clinical and eletrophysiological manifestations suggestive of PNL were diagnosed with the help of their positive test results in the anti-PGL-I ELISA. In conclusion, detection of antibodies against PGL-I in patients with peripheral neuropathy is useful as an additional laboratory test to help PNL diagnosis.     

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.     

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.     

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.     

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.     

Bullous pemphigoid: a correlative study of autoantibodies, circulating immune complexes and dermo-epidermal deposits

Twenty bullous pemphigoid (BP) patients were studied to establish any correlation between free anti-basement membrane zone (BMZ) antibodies, circulating immune complexes (CIC) and dermo-epidermal junction deposits. CIC levels were evaluated by 2% polyethylene glycol (PEG) precipitation. The twenty patients were found to have IgG and/or C3 deposited in the BMZ. Eight of the twelve patients who had no free anti-BMZ antibodies displayed a positive in vivo C4 and/or C_Iq staining and high levels of CIC. Moreover, CIC were detected in only one patient with positive circulating free anti-BMZ antibodies. The presence of free anti-BMZ antibodies was generally found to correlate with the absence of cutaneous deposits of C1q and/or C4 and with negative CIC; on the other hand, the absence of free anti-BMZ antibodies was generally found to correlate with high levels of CIC and with deposits of C3 and C_Iq and/or C4. The absence of circulating free anti-BMZ antibodies in BP patients, could be explained by the formation of CIC. It is possible that BMZ antigens released from damaged tissue could combine with free antibodies and form complexes in the blood. The release could involve locally formed immune complexes. Elevated CIC levels were generally found to correlate with the presence of active disease.     

Serum immune complexes in erythema nodosum leprosum reactions of leprosy

Serum estimations of immunoglobulins, complement components and their presence in circulating immune complexes were carried out in 39 Lepromatous, 44 ENL and 22 Post ENL leprosy patients. Serum IgG, IgA, IgM, C3 and C4 levels were determined by single radial immunodiffusion. Serum immune complexes were precipitated with Polyethylene Glycol (PEG) and IgG, IgA, IgM, C3 and C4 were estimated by single radial immunodiffusion and expressed as % of precipitation of their serum level. Decreased IgG, IgM; increased IgA and C3; and no change in C4 levels are observed in ENL than Lepromatous and Post ENL patients. However, a gradual insignificant reduction of IgG, IgA, and IgM was found from Lepromatous to ENL and Post ENL patients in the PEG-precipitates. Similarly, C3 and C4 was found reduced insignificantly in ENL than Lepromatous and Post ENL patients. The significance of these estimations in relation to immune status of ENL reactions are discussed.     

Serologic studies of erythema chronicum migrans Afzelius and acrodermatitis chronica atrophicans with indirect immunofluorescence and enzyme-linked immunosorbent assays

To determine whether antibodies to Borrelia spirochetes were present, sera from 88 patients with uncomplicated erythema chronicum migrans Afzelius (ECMA), from 9 patients with ECMA-related extracutaneous complications and from 26 patients with acrodermatitis chronica atrophicans (ACA) were submitted to an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence (IF) assay. The assays were calculated to be 95% specific. There was good correlation between the IF test with a polyvalent conjugate and IgG ELISA. Of patients with uncomplicated ECMA, 18% were seropositive by IgG ELISA and 11% by IgM ELISA, and 15% showed elevated IF titers. Elevated serum antibody levels of IgG as measured by ELISA and elevated IF titers were found in all patients with extracutaneous complications and in the patients with ACA. Declining IgG titers were observed at follow-up 6-12 months after therapy, but the majority of the patients with ACA were still seropositive.     

The ML flow test as a point of care test for leprosy control programmes: potential effects on classification of leprosy patients

OBJECTIVE: To evaluate the use of the ML Flow test as an additional, serological, tool for the classification of new leprosy patients. DESIGN: In Brazil, Nepal and Nigeria, 2632 leprosy patients were classified by three METHODS:: (1) as multibacillary (MB) or paucibacillary (PB) according to the number of skin lesions (WHO classification), (2) by slit skin smear examination, and (3) by serology using the ML Flow test detecting IgM antibodies to Mycobacterium leprae-specific phenolic glycolipid-I. RESULTS: The proportion of MB leprosy patients was 39.5, 35.6 and 19.4% in Brazil, Nepal and Nigeria, respectively. The highest seropositivity in patients was observed in Nigeria (62.9%), followed by Brazil (50.8%) and Nepal (35.6%). ML Flow test results and smears were negative in 69.1 and 82.7% of PB patients, while smears were positive in 58.6% of MB patients in Brazil and 28.3% in Nepal. In MB patients, both smears and ML Flow tests were negative in 15.6% in Brazil and 38.3%, in Nepal. Testing all PB patients with the ML Flow test to prevent under-treatment would increase the MB group by 18, 11 and 46.2% for Brazil, Nepal and Nigeria, respectively. Using the ML Flow test as the sole criterion for classification would result in an increase of 11.3 and 43.5% of patients requiring treatment for MB leprosy in Brazil and Nigeria, respectively, and a decrease of 3.7% for Nepal. CONCLUSIONS: The ML Flow test could be used to strengthen classification, reduce the risk of under-treatment and minimize the need for slit skin smears.     

Immune complexes in the pathogenesis of hypergammaglobulinemic purpura

Direct immunofluorescence (DIF) was performed and circulating immune complex (CIC) levels evaluated in three patients with hypergammaglobulinemia purpura (HGP) of Waldenstr?m. The Raji cell immunoradiometric assay was used to detect complexes of both IgG and IgM type. IgM and C3 were detected in blood vessel walls of all three patients. Elevated levels of IgG complexes were detected in two patients, and elevated levels of IgM complexes were detected in all three patients. Clinical improvement after plasmapheresis was noted in one patient. A cause-and-effect relationship between CIC and the inflammation of the superficial dermal blood vessels is postulated.     

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.     

Comparative evaluation of enzyme immunoassays based on synthetic glycoconjugates and phenolic glycolipid-I for immunodiagnosis of leprosy

Enzyme immunoassays (EIAs) based on synthetic glycoconjugates containing the terminal monosaccharide (M-BGG) or disaccharide (ND-BSA) residue of the trisaccharide component of phenolic glycolipid-I (PGL-I), for immunodiagnosis of leprosy are described. The results of the assays were compared with that of the EIA using PGL-I. All the three assays were highly specific for leprosy. The per cent positivity of active lepromatous leprosy (LL) patients with M-BGG was 78.05 in comparison to 85.36 with ND-BSA and 82.11 with PGL-I. Similarly, the positivity of tuberculoid (TT) leprosy patients in M-BGG assay was lower than that in EIAs using ND-BSA or PGL-I. However, the difference in the positivity of individual category of leprosy patients in the three EIAs was not statistically significant. The correlation between absorbance values of leprosy sera in EIAs based on M-BGG and PGL-I, as well as that in assays using ND-BSA and PGL-I was statistically significant.     

Cutaneo-systemic necrotizing vasculitis occurring during desensitization

The authors report a case of cutaneo-systemic necrotizing vasculitis, predominantly affecting the skin and digestive tract, observed in an asthmatic patient undergoing desensitization with Graminaceae pollen extract and mite-enriched house dust. Vasculitis developed 22 months after the first injections, became autonomous after the end of treatment and responded well to systemic corticosteroid therapy in daily doses of 1 mg/kg. The patient was followed up for 8 months during which the vasculitis did not recur, despite reduction in steroid dosage. Reports of necrotizing vasculitis occurring during desensitization seem to be very rare. They raise the problem of whether specific immunotherapy plays a role in the pathogenesis of vasculitis. Most of the characteristics that emerged from the cases previously published are concordant with those found in our patient: the interval between the first injections and the initial symptoms varied from 45 days to 8 years; in every case the condition followed its own course after the injections were discontinued; most patients were being desensitized to several allergens; the cutaneous symptoms were pronounced, eosinophilia was moderate and hepatitis B serology was constantly negative. The few prospective studies aimed at determining, by different methods, the levels of circulating immune complexes (CIC) have given conflicting results. Atopic subjects have high CIC levels, but these do not seem to be influenced by desensitization. The nature of the CIC remains unknown, and the various suggestions put forward (cross-reaction between blocking IgG and auto-antibodies, CIC [blocking IgG, anti-idiotype antibodies]) require confirmation, although the hypothesis of pathogenic CIC (allergen, blocking IgG) is unlikely to be correct.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of ML Flow serology and slit skin smears to assess the bacterial load in newly diagnosed leprosy patients in Brazil

INTRODUCTION: The ML Flow test is an immunochromatographic assay that detects IgM antibodies against M. leprae-specific anti-phenolic glycolipid I (PGL-I). In addition to slit skin smears stained by the Ziehl-Neelsen technique, it can be helpful in the operational classification of leprosy patients for treatment purposes. OBJECTIVE: This work studied the relationship between antibody levels as detected by semi-quantitative ML Flow serologic test and bacterial load as quantified by slit skin smear. PATIENTS AND METHODS: 135 patients with newly detected leprosy at the reference service in Sanitary Dermatology in Brazil had slit skin smears (registered as bacillary index - BI) and an ML Flow test (registered qualitatively and semi-quantitatively) performed at admission. A logistic regression and agreement measures (kappa index) were calculated. RESULTS: Slit skin smears were positive in 35.9% of patients and 57% of patients were seropositive for PGL-1 antibodies. Among the seropositive patients, 416% had five or fewer skin lesions, and 65.8% had more than one peripheral nerve involved. Slit skin smears were positive in only three seronegative patients (5.6%), and negative in 41.9% of seropositive patients. Patients with a BI of 4 + had an OR of 33 for being seropositive in comparison to those with a low BI. CONCLUSIONS: There is a correlation between serologic test and slit skin smear results. Therefore, an ML Flow test may become a useful tool in the clinical classification of leprosy, besides slit skin smears, which require a proper laboratory infrastructure and experienced personnel.