Evaluation of antigen-specific immune responses for leprosy diagnosis in a hyperendemic area in China.

OBJECTIVE: To evaluate antigen-specific immune responses for leprosy diagnosis in a hyperendemic area in China. METHODS: Eighty-three leprosy patients and 161 non-leprosy controls were enrolled from Hani-yi Autonomous Prefecture of Honghe, Yunnan Province, China. Leprosy patients were divided into multibacillary (MB, n = 38), paucibacillary (PB, n = 23), and post-multi-drug therapy (MDT, n = 22) groups. Controls were divided into the following groups: healthy household contacts (HHC, n = 119), tuberculosis (TB, n = 11), and endemic controls (EC, n = 31). The NDO-LID Rapid Test, M. leprae antigen-specific ELISA and antigen-specific IFN-γ secretion in a whole blood assay (WBA) were used to evaluate these subjects. RESULTS: The NDO-LID Rapid Test achieved higher positive response rates in MB than in PB patients[94.7%(36/38) vs 65.2%(15/23)], and these rates were higher than those observed by ELISA using anti-LID-1[92.1%(35/38) vs 52.2%(12/23)], anti-NDO-LID[92.1%(35/38) vs 47.8% (11/23)], and anti-ND-O-BSA[89.5%(34/38) vs 60.9%(14/23)]. However, the NDO-LID Rapid Test also showed a higher positive response rate in the EC group (33.3%,10/31), which was higher than the rates observed for anti-NDO-LID (12.9%,4/31) and anti-ND-O-BSA (16.1%,5/31). M. leprae antigen-specific ELISA demonstrated relatively high specificity (86.84-97.37%) but low sensitivity (15.97-72.73%) in discriminating between leprosy patients and non-leprosy controls by ROC curve analysis. In contrast, M. leprae antigen-specific IFN-γ secretion detection achieved higher positive response rates in PB than in MB patients (positive ratio of MB vs PB: 40% vs 56% for LID-1, 28.6% vs 47.8% for ML89, 31.4% vs 60.7% for ML2044, and 31.4 vs 47.8% for ML2028) and could distinguish MB from EC when stimulated with ML89(AUC = 0.6664) and PB fromTB when stimulated with ML2044 and ML2028(AUC = 0.7549 and 0.7372, respectively). CONCLUSION: The NDO-LID Rapid Test and M. leprae antigen-specific ELISA are useful tools to assist in the diagnosis of leprosy patients, especially MB patients, although the former had higher sensitivity but lower specificity than the latter. M. leprae antigen-specific IFN-γ release assessed by WBA has diagnostic value for distinguishing PB from TB but not for distinguishing PB from HHC or EC. Screening novel M. leprae-specific antigens, combining different M. leprae antigens and a multi-cytokine analyte model may be needed for more effective diagnosis of leprosy.

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.

Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR.

Attempts were made to enhance the sensitivity of immuno-PCR assay based on the detection of cocktail of mycobacterial antigen 85B (Rv1886c), ESAT-6 (Rv3875) and cord factor (trehalose 6,6'-dimycolate) in pulmonary and extrapulmonary TB patients. Detection of Ag85B was found to be superior to the detection of cocktail in TB patients.

Immunocytochemical detection of mycobacterial antigen in extrapulmonary tuberculosis.

The aim of the study is to determine whether immunostaining for mycobacterial antigen can contribute to the cytological diagnosis of extrapulmonary tuberculosis (EPTB). The study was carried out on aspirated material of lymph nodes, and other accessible sites, from 65 patients with clinical diagnosis of tuberculosis (TB). Twenty patients, diagnosed by fine-needle aspiration, with non-tuberculous granulomas served as controls. The diagnosis of TB was based on the demonstration of acid-fast bacilli (AFB), culture positivity for Mycobacterium tuberculosis (M. tuberculosis), or response to treatment with standard anti-tubercular therapy. Immunostaining was done using polyclonal antibody to mycobacteria. AFB positivity by Ziehl Neelsen (ZN) staining was 21%, 65.38%, and 68% respectively in Pattern 1 (granulomas alone), in Pattern 2 (granulomas with necrosis), and in Pattern 3 (necrosis alone). Overall AFB positivity was 56.92%. Twenty-eight of 65 cases were negative for AFB on direct smear. Culture was positive in 46% (13/28). Sensitivity and specificity of immunostaining were 96.92% (63/65) and 95%, respectively. Immunoreactivity was seen in 26 (92.8%) of 28 cases which were negative by ZN staining. Except in the case of leprosy, in which cross reactivity was seen, there was no immunoreactivity in the control group. Immunocytochemistry (ICC) had high sensitivity (96.2%) and specificity (95%) in the diagnosis of EPTB. ICC may be a useful adjunct to evaluation of cytomorphology and ZN staining.

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

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.

A quantitative view on Mycobacterium leprae antigens by proteomics.

Leprosy is an ancient disease and the focus of the researchers' scrutiny for more than a century. However, many of the molecular aspects related to transmission, virulence, antigens and immune responses are far from known. Initially, the implementation of recombinant DNA library screens raised interesting antigen candidates. Finally, the availability of Mycobacterium leprae genomic information showed an intriguing genome reduction which is now largely used in comparative genomics. While predictive in silico tools are commonly used to identify possible antigens, proteomic approaches have not yet been explored fully to study M. leprae biology. Quantitative information obtained at the protein level, and its analysis as part of a complex system, would be a key feature to be used to help researchers to validate and understand many of such in silico predictions. Through a re-analysis of data from a previous publication of our group, we could easily tackle many questions regarding antigen prediction and pseudogene expression. Several well known antigens are among the quantitatively dominant proteins, while several major proteins have not been explored as antigens. We argue that combining proteomic approaches together with bioinformatic workflows is a required step in the characterization of important pathogens.

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.

Leprosy-specific B-cells within cellular infiltrates in active leprosy lesions.

Leprosy is a spectral disease with polar lepromatous and tuberculoid forms correlating with enhanced humoral and cell-mediated immunity, respectively, against Mycobacterium leprae and the borderline forms, borderline lepromatous, midborderline, and borderline tuberculoid showing in-between clinical and immunological characteristics. Histopathologically, the cellular infiltrates of leprosy lesions show predominantly the presence of interacting T-cells and antigen presenting cells like macrophages, whereas the presence of B-cells has only been sporadically reported. The present study demonstrates by immunohistochemical techniques the presence of B-cells, including plasma cells, in active lesions from lepromatous leprosy, skin smear negative borderline lepromatous, and paucibacillary borderline tuberculoid leprosy. Furthermore, the study demonstrates the in situ production of M leprae-specific antibodies from BT lesions using an organotypic skin explant culture model. Finally, analysis of the cytokine release profile in supernatants of lesional organotypic skin cultures showed a microenvironment conducive to the differentiation and maturation of B-cells. The results demonstrate the presence of different functionally active B-cell stages within lesions of patients with leprosy, including borderline tuberculoid patients, which could secrete anti-M leprae-specific antibodies. However, their role in leprosy pathology remains to be elucidated.

Semi-quantitative detection of Mycobacterium leprae antigens in skin scrapings: suitability as a laboratory aid for field diagnosis of leprosy.

We describe here a method, potentially suitable for field applications, for semi-quantitative detection of Mycobacterium leprae antigens in skin scrapings, which are taken normally for smear microscopy. Thirty acid-fast bacilli-negative paucibacillary (PB) leprosy patients comprised the main study group; eight acid-fast bacilli-positive multibacillary (MB) patients and five healthy laboratory workers served as controls. Samples in saline were spotted on nitrocellulose paper and probed with mycobacterium-specific polyclonal or M. leprae-specific mAbs against 12, 35 and 65kDa protein antigens, using a dot-ELISA format. Spot densities were read through a densitometer and also graded visually. The polyclonal antibody produced the best sensitivity, resulting in densitometric detection of mycobacterial antigen in 100% MB, 76% multiple-lesion PB and 62% single-lesion PB patients. None of the healthy volunteers showed antigen positivity. A correlation was noted between the densitometric and visual estimates of the antigen. Determination of antigen in the lesion and an apparently uninvolved area of skin in a subset of PB patients provided clues to the state of the underlying infection. Serological positivity of PB patients for M. leprae-specific antibodies against the 35kDa and phenolic glycolipid-I antigens was too low (<20%) for any diagnostic significance.

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.

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy.

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.