Salivary anti-PGL-1 IgM may indicate active transmission of Mycobacterium leprae among young people under 16 years of age

Abstract Considering that the main route of Mycobacterium leprae transmission is the upper respiratory tract, detection of salivary antibodies can be a useful tool for diagnosing early infection. The study aimed to analyze salivary anti-PGL-1 IgA and IgM antibodies in 169 children aged 4-16 years old, who lived nearby or inside the house of multibacillary or paucibacillary leprosy patients in two endemic cities in Alagoas State - Brazil. Salivary anti-PGL-1 antibodies were quantified by modified ELISA method. The frequency of contact and clinical form of the index case were significantly associated with salivary antibody levels. High frequency of IgM positivity strongly suggests active transmission of M. leprae in these communities. We suggest in the present work that salivary anti-PGL IgA and IgM are important biomarkers to be used for identifying communities with probable active transmission of M. leprae.

Salivary anti-PGL-1 IgM may indicate active transmission of Mycobacterium leprae among young people under 16 years of age.

Considering that the main route of Mycobacterium leprae transmission is the upper respiratory tract, detection of salivary antibodies can be a useful tool for diagnosing early infection. The study aimed to analyze salivary anti-PGL-1 IgA and IgM antibodies in 169 children aged 4-16 years old, who lived nearby or inside the house of multibacillary or paucibacillary leprosy patients in two endemic cities in Alagoas State - Brazil. Salivary anti-PGL-1 antibodies were quantified by modified ELISA method. The frequency of contact and clinical form of the index case were significantly associated with salivary antibody levels. High frequency of IgM positivity strongly suggests active transmission of M. leprae in these communities. We suggest in the present work that salivary anti-PGL IgA and IgM are important biomarkers to be used for identifying communities with probable active transmission of M. leprae.

Lateral flow assay for simultaneous detection of cellular- and humoral immune responses.

OBJECTIVE: The development of a cytokine detection assay suitable for detection of multiple biomarkers for improved diagnosis of mycobacterial diseases. DESIGN AND METHODS: A lateral flow (LF) assay to detect IL-10 was developed utilizing the up-converting phosphor (UCP) reporter-technology. The assay was evaluated using blood samples of leprosy patients. Multiplex applications were explored targeting: 1) IL-10 and IFN-γ in assay buffer; 2) IL-10 and anti-phenolic glycolipid (PGL-I) antibodies in serum from leprosy patients. RESULTS: Detection of IL-10 below the targeted level of 100pg/mL in serum was shown. Comparison with ELISA showed a quantitative correlation with R(2) value of 0.92. Multiplexing of cytokines and simultaneous detection of cytokine and antibody was demonstrated. CONCLUSIONS: The UCP-LF IL-10 assay is a user-friendly, rapid alternative for IL-10 ELISAs, suitable for multiplex detection of different cytokines and can be merged with antibody-detection assays to simultaneously detect cellular- and humoral immunity.

Nasal mucosa study of leprosy contacts with positive serology for the phenolic glycolipid 1 antigen.

UNLABELLED: Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The disease more frequently affects the nasal mucosa and can occur independently of its clinical form or even before lesions on the skin or on other parts of the body. It is necessary to employ epidemiological surveillance of household contacts with new leprosy cases for early disease diagnosis. AIM: identify specific and early leprosy lesions through endoscopic, baciloscopy, histopathology exams, and real time polymerase chain reaction of the nasal cavity mucosa on household and peridomiciliary contacts with positive serology for the phenolic glycolipid 1 antigen. METHODOLOGY: Between 2003 at 2006 there was a prospective cross-sectional clinical study with 31 contacts with patients with leprosy with positive serology against PGL-1, 05 negative controls and 01 positive control. RESULTS: Between seropositive contacts, real-time PCR was positive for M. leprae DNA in 06 (19.35%) of them and the higher number of genome copies were found in contacts who became sick. CONCLUSION: Nasal mucosa tests alone did not enable the early diagnosis of Leprosy. However, through the combination of various methods, tests on the contacts can help identify subclinical infection and monitor the contacts that could be responsible for spreading the disease.

Estudo da mucosa nasal de contatos de hanseníase, com positividade para o antígeno glicolipídio fenólico 1 / Nasal mucosa study of leprosy contacts with positive serology for the phenolic glycolipid 1 antigen

A hanseníase é uma doença infecciosa de evolução crônica causada pelo Mycobacterium leprae que acomete com maior frequência a mucosa nasal. Esse acometimento independe da forma clínica da doença e pode ocorrer mesmo antes do aparecimento de lesões na pele ou em outras partes do corpo. Faz-se necessário a vigilância epidemiológica dos contatos de casos novos de hanseníase para o diagnóstico precoce da doença. OBJETIVOS: Identificar lesões específicas e precoces de hanseníase por meio de exame endoscópico, baciloscópico, histopatológico e da reação em cadeia da polimerase em Tempo Real da mucosa das cavidades nasais dos contatos domiciliares e peridomiciliares com sorologia positiva para o antígeno glicolipídio fenólico. MATERIAL E MÉTODOS: Estudo prospectivo transversal em 31 contatos de pacientes de hanseníase com sorologia positiva (PGL-1), 05 controles negativos e 01 positivo no período de 2003 a 2006. RESULTADOS: Entre os contatos soropositivos a PCR-RT foi positiva para a presença de DNA de M. leprae em 06 (19,35 por cento) destes e o maior número de cópias do genoma do bacilo foi encontrado no contato que adoeceu. CONCLUSÃO: Isoladamente os exames da mucosa nasal não permitiram o diagnóstico precoce da hanseníase, mas com a combinação de vários métodos, o exame dos contatos pôde ajudar na identificação da infecção subclínica e monitoramento daqueles que poderiam ter papel importante na transmissão da doença.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The disease more frequently affects the nasal mucosa and can occur independently of its clinical form or even before lesions on the skin or on other parts of the body. It is necessary to employ epidemiological surveillance of household contacts with new leprosy cases for early disease diagnosis. AIM: identify specific and early leprosy lesions through endoscopic, baciloscopy, histopathology exams, and real time polymerase chain reaction of the nasal cavity mucosa on household and peridomiciliary contacts with positive serology for the phenolic glycolipid 1 antigen. METHODOLOGY: Between 2003 at 2006 there was a prospective cross-sectional clinical study with 31 contacts with patients with leprosy with positive serology against PGL-1, 05 negative controls and 01 positive control. RESULTS: Between seropositive contacts, real-time PCR was positive for M. leprae DNA in 06 (19.35 percent) of them and the higher number of genome copies were found in contacts who became sick. CONCLUSION: Nasal mucosa tests alone did not enable the early diagnosis of Leprosy. However, through the combination of various methods, tests on the contacts can help identify subclinical infection and monitor the contacts that could be responsible for spreading the disease.

An immunohistochemical, clinical and electroneuromyographic correlative study of the neural markers in the neuritic form of leprosy.

The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100% of the neuritic biopsies. NGFr positivity was also reduced in 81.8%, PGP staining in 100% of the affected nerves, S100 positivity in 90.9%, and myelin basic protein immunoreactivity in 90.9%. Hypoesthesia was associated with decreased NGFr (81.8%) and PGP staining (90.9%). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6%) and nerve fiber neurofilament staining (45.4%) by immunohistochemistry and with loss of myelinated fibers (100%) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40% of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.

An immunohistochemical, clinical and electroneuromyographic correlative study of the neural markers in the neuritic form of leprosy

The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100 percent of the neuritic biopsies. NGFr positivity was also reduced in 81.8 percent, PGP staining in 100 percent of the affected nerves, S100 positivity in 90.9 percent, and myelin basic protein immunoreactivity in 90.9 percent. Hypoesthesia was associated with decreased NGFr (81.8 percent) and PGP staining (90.9 percent). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6 percent) and nerve fiber neurofilament staining (45.4 percent) by immunohistochemistry and with loss of myelinated fibers (100 percent) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40 percent of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.

Lipids of 'Mycobacterium habana', a synonym of Mycobacterium simiae with vaccine potential.

'Mycobacterium habana' was proposed as a distinct species within the genus Mycobacterium; however, it is actually a synonym of Mycobacterium simiae and included in the serotype I of this species. The potential use of 'M. habana' as a vaccine in both leprosy and tuberculosis has led to the analysis of its lipid composition in an attempt to define distinctive markers that could be used in the quality control of true strains of this bacterium. Lipids of taxonomic value (fatty and mycolic acids) are similar in 'M. habana' and M. simiae; nevertheless, they clearly differ on the basis of glycopeptidolipid (GPL) composition. Thus, contrary to M. simiae, most strains of 'M. habana' can be defined by the presence of three polar compounds, designated GPL-I, GPL-II and GPL-III, easily determined by thin-layer chromatography, and characterized, respectively, by the content of l-fucose, 2,4-di-O-Me-d-glucuronic acid, and 4-O-Me-d-glucuronic acid, as epitopes.

Immuno-histopathology in the diagnosis of early leprosy.

The present study of 45 early leprosy cases in an endemic area in China indicates: a) Sensitivity of acid-fast bacilli (AFB) detection can be significantly improved by examining approximately 30 serial sections. AFB and/or phenolic glycolipid-I (PGL-I) were mostly detected in the infiltrates in the subepidermal zone, intraneurium, perineurium and around blood vessels. b) PGL-I antigen was positive in 10 clinically suspected, single lesion leprosy cases and AFB positive in 7 patients, AFB and/or PGL-I in nerve in 6 patients. c) Nonspecific chronic inflammation in indeterminate leprosy presented as selective perineural and/or intraneural infiltration with lymphocytes predominating. In the infiltrating mass, fragments of neural tissue were demonstrated with anti-S-100 protein staining. d) Except for 3 cases with unknown numbers of lesions, the present positive immunohistopathological findings are in direct correlation with the number of lesions at first diagnosis, namely: 41.6% (10/24) for single lesion, 66.6% (6/9) for 2 lesions, and 88.8% (8/9) for patients with > or = 3 lesions. e) Typical epithelioid or macrophage granuloma formations were not seen in early leprosy with a single lesion. In testing the immunological inclination of these patients with CD68 or tumor necrosis factor-alpha (TNF-alpha) a positive test is likely to be of prognostic value since TNF-alpha is involved in granuloma formation and nerve damage.

Recognition of phenolic glycolipid-I (Mycobacterium leprae) and sulfolipid-I (M. tuberculosis) by serum from Mexican patients with leprosy or tuberculosis.

SETTING: Differential diagnosis of leprosy and tuberculosis in regions where both illnesses are endemic is a prerequisite for proper identification and treatment. OBJECTIVE: To evaluate the recognition of phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and sulfolipid-I (SL-I) of M. tuberculosis by serum from patients with leprosy (LL) or pulmonary tuberculosis (PTB). DESIGN: Purified PGL-I and SL-I were used as antigens in an ELISA test set up to assess recognition of these lipids by serum from 43 LL patients, 44 PTB patients and 38 healthy individuals. RESULTS: Leprosy patients gave higher IgM than IgG responses to PGL-I and had comparable IgM and IgG responses to SL-I. A similar situation was observed with PTB serum. Some healthy individuals were found to contain significant levels of antibodies to both lipids. CONCLUSION: There is no specific recognition of either of the two lipid antigens tested by serum from both leprosy and tuberculosis patients; this rules out the possibility of using PGL-I and SL-I as tools for the differential diagnosis of these two mycobacterial diseases.

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.

Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections.

A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid was not detected in bacteriologically negative tissue but lipoarabinomanan (LAM) and protein antigens were detected. Staining with LAM was strongest and gave least background. The transfer of this immunohistochemical technique to paraffin embedded material will allow examination of tissue with better morphology and from clinics without access to tissue freezing facilities.

Rapid detection of Mycobacterium bovis on its lipid profile by thin layer chromatography.

Sixteen Mycobacterium bovis (M. bovis) strains isolated from bovine tissues and one standard reference strain of M. bovis AN5 alongwith other species of mycobacteria for comparison were investigated for the presence of phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) for rapid identification of M. bovis by thin-layer chromatography (TLC). The study indicated presence of PGL with an Rf value of 0.75 in chloroform-methanol solvent in all 17 M. bovis strains. The dimycocerostate A corresponding to spot A was the major constituent among all the three spots in M. bovis strains. TLC appeared to be a promising alternative to conventional biochemical methods for identification of M. bovis taking into consideration both PGL and PDIM lipids.

[Characterization of phenolic glycolipid-I (PGL-I) like antigen in renal cell carcinoma].

BACKGROUND: We investigated whether phenolic glycolipid-I (PGL-I) produced by Mycobacterium leprae (M. leprae), can be used as a marker of renal tumors. METHODS: Using a preparted anti-PGL-I monoclonal antibody (SF-I), anti-PGL-I group antibodies and PGL-I group antigens were detected and PGL-I immunohistologic staining were performed. RESULTS: The titer of anti-PGL-I antibody in patients with renal cell carcinoma was 0.283 +/- 0.103, showing a trend of rising titer of anti-PGL-I group antibody with higher cytologic atypia. When compared by the extent of tumor infiltration, a high antibody titer was observed in stage 4 infiltration group. Regarding the PGL-I group antigen that was detected using SF-1 in the urine of patients with renal cell carcinoma, no relationships between the positive rte of the antigens with common antigenicity to PGL-I and the grade of carcinoma were observed. However, in terms of the extent of tumor infiltration, a trend that the positive rate for PGL-I group antigen increased with the progression of cancer stage was seen. The Western blot assay of the urine of patients with renal cell carcinoma following electrophoresis revealed a change around 40 KD. The immunohistologic stains with SF-1 disclosed a predominance in the proximal kidney tubule. CONCLUSION: Using PGL-I group antigen and PGL-I group antibody as markers of renal cell carcinoma, a rapid and precise measurement may be provided.

Is PGL-1 also present in Leishmania donovani promastigotes?

A soluble antigen complex (SAC) derived from the ruptured promastigotes of Leishmania donovani parasites (LD-SAC) was used for complement fixation test (CFT) in leprosy Cases of tuberculoid and borderline tuberculoid leprosy, post-kala azar dermal leishmaniasis (TT, BT, PKDL) and control sera gave negative CFT. Smear-positive cases of borderline (BB, BL) and lepromatous (LL) leprosy and drug-resisting cases of pulmonary tuberculosis gave positive CFT; smear-negative cases of LL leprosy sera also gave positive CFT. Sera of smear-negative inactive LL patients contained only PGL-1 and PDIM antigens for a long time after they become inactive. Therefore, the positive CFT in inactive LL makes us suspect whether PGL-1 is present in LD promastigotes.

[Biochemistry and bioactivities of mycobacterial components].

The most characteristic pathological change in mycobacterial infection is caseous necrosis followed by tuberculous cavity formation due to the cellular immunity induced by antigenic proteins and adjuvant active cell wall components. Mycobacterial cell well contains unique hydrophobic compounds possessing mycolic acids (a long branched-chain high molecular weight fatty acid) and shows distinctive properties such as acid-fastness and wax-like hydrophobicity. Mycobacteria do not produce exotoxin and the virulence cannot be determined by a single toxic substance, but the cell wall components to contact with the host cells are the most important surface molecule at the early stage of infection. Cord factor (trehalose 6,6'-dimycolate) is the most classical virulence factor which is lethally toxic for mice. However, cord factor exists among various species of mycobacteria and even in Nocardia and Rhodococcus. Furthermore, cord factor can produce granulomas without protein antigen in mice and it shows antitumor or non-specific prevention promotion of infection and induction promotion of various cytokines. Sulfolipid (tetracyl trehalose sulfate) also plays a role as a virulence factor by phagocytic process inhibition. Glycopeptidolipid (GPL) from M. avium and phenolglycolipid (PGL) from M. leprae also appeared to be immunomodulatory molecules which inhibit the developing of cellular immunity. Lipoarabinomannan (LAM) is also unique amphipatic molecule, like gram-negative endotoxin. Here, we discuss the involvement of various surface molecutes to contribute to pathogenesis in mycobacterial infection and immunity.

Distribution of surface-exposed antigenic glycolipids in recent clinical isolates of Mycobacterium tuberculosis.

The distribution of surface-exposed antigenic glycolipids in seven recent clinical isolates of Mycobacterium tuberculosis was established. Thin-layer and liquid chromatographies revealed a uniformity in the glycolipid pattern. Chemical analysis of the individual glycolipids of a selected strain enabled the identification of glycolipids of serological interest in all the other clinical isolates. Phenolic glycolipid-Tb1 (PGL-Tb1) was lacking in all strains, but appreciable amounts of a partially deglycosylated version (PGL-Tb1D) were present in the seven isolates. Diacyltrehaloses (DATs) were detected in all strains, showing themselves to be major glycolipids. Lipooligosaccharides (LOS-II) were present in the seven strains studied though only in trace amounts. These results shed new light on the open debate on the distribution of these interesting glycolipids in typical clinical isolates of M. tuberculosis. In the search for a serological test for tuberculosis, and in accordance with our observations, we believe that PGL-Tb1 and LOS-II should not be the target molecules for serology and that it is worthwhile to continue investigating the value of DATs as antigens. We also believe that it would be of interest to undertake research to assess the usefulness of PGL-Tb1D as an antigen.

Experimental leprosy in monkeys. II. Longitudinal serological observations in sooty mangabey monkeys.

In this study, 11 SMM were grouped and inoculated with differing doses of SMM-origin Mycobacterium leprae (ML) between 4.5 x 10(8) and 1 x 10(9) by either combined IV/IC routes or by IV or IC route alone. The combined route was the most effective in eliciting progressive, disseminated LL leprosy. In all, 6 of 7 SMM inoculated by the combined routes developed leprosy requiring treatment at some point. Only 1 of 4 inoculated by a single route developed persisting leprosy requiring chemotherapy. Either no disease or spontaneous regression of initial disease occurred in the other 3 animals inoculated by a single route. Doses in excess of 1 x 10(9) ML were more effective than lesser doses. An association was observed between the development of IgG anti-PGL-I ELISA OD values and resistance to leprosy and between IgM anti-PGL-I and leprosy progression or susceptibility. Serum PGL-I antigen levels, determined by dot ELISA, paralleled disease severity longitudinally. High positive OD values of anti-LAM IgG prior to ML inoculation were observed in the majority of leprosy-susceptible SMM in contrast to negative levels in more resistant animals. Anti-LAM IgG OD values exceeded the positive cut-off point after inoculation in 5 of 11 SMM; 3 of these 5 had concurrent detectable serum levels of PGL-I antigen.

Minocycline in lepromatous leprosy.

Twelve patients were treated with three dose levels of minocycline for 30 days, primarily to detect the dose-related effects on Mycobacterium leprae viability, followed by another 5 months of daily minocycline for overall efficacy and persistence of clinical and antibacterial effects. Subsequently, the patients were given standard WHO/MDT chemotherapy for multibacillary leprosy. Clinical improvement was recognizable during the first month, occurring much earlier among those on minocycline 200 mg daily than those who received minocycline 100 mg daily. A similar change also was observed in one patient 11 days after three daily doses of 100 mg of minocycline. At the end of 6 months, all patients were clinically improved with a slight reduction in the average bacterial index (BI) and logarithmic index of bacilli in biopsy (LIB). The effects of minocycline on viability by mouse foot pad inoculation and palmitic acid oxidation assays were noted beginning at 10 to 14 days of daily dosing and becoming more definite after 30 days of treatment. Both tests correlated fairly well. Doses of 200 mg daily did not appear to be more efficient than minocycline 100 daily. Phenolic glycolipid-I (PGL-I) antigen determinations done on some patients during the first month remained positive and did not correlate with changes in viability results. At the end of 6 months, after 5 months of 100 mg of minocycline monotherapy, no viable organisms could be demonstrated by mouse foot pad inoculation and palmitic acid oxidation assays; assays for PGL-I antigen were all negative.(ABSTRACT TRUNCATED AT 250 WORDS)