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。     

Leprosy: review of the epidemiological,clinical, and etiopathogenic aspects - Part 1

Leprosy is caused by Mycobacterium leprae and has been known since biblical times. It is still endemic in many regions of the world and a public health problem in Brazil. The prevalence rate in 2011 reached 1.54 cases per 10,000 inhabitants in Brazil. The mechanism of transmission of leprosy consists of prolonged close contact between susceptible and genetically predisposed individuals and untreated multibacillary patients. Transmission occurs through inhalation of bacilli present in upper airway secretion. The nasal mucosa is the main entry or exit route of M. leprae. The deeper understanding of the structural and biological characteristics of M. leprae, the sequencing of its genome, along with the advances in understanding the mechanisms of host immune response against the bacilli, dependent on genetic susceptibility, have contributed to the understanding of the pathogenesis, variations in the clinical characteristics, and progression of the disease. This article aims to update dermatologist on epidemiological, clinical, and etiopathogenic leprosy aspects.     

Development of a mouse food pad model for detection of sub clinical leprosy

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.     

An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique.

One of the most important unsolved questions in epidemiology of leprosy is the highly uneven geographic distribution of the disease. There are many hyperendemic "pockets" in endemic countries. Little is known about the reasons why leprosy is hyperendemic in these areas. We conducted, therefore, a series of epidemiological studies on Mycobacterium leprae infection and prevalence of leprosy in North Maluku district, Maluku Province, Indonesia where leprosy is highly endemic. It was found that considerable number of general inhabitants are seropositive to various mycobacterial antigens and 27% of the villagers were carrying leprosy bacilli on their surface of nasal cavity. These results suggested the importance of M. leprae in the residential environment in infection of the leprosy bacillus and the resulting transmission of the disease. Based on these observations, we conclude that new preventive measures are essential for global elimination of leprosy in addition to early diagnosis and multidrug therapy (MDT).     

Recent advances in immunodiagnosis of leprosy

Although prevalence of leprosy is considerably reduced, the unabated emergence of about 300,000 cases worldwide indicates that the source of infection and transmission are not being addressed. Early diagnosis and treatment still remain the cornerstone of leprosy control. Many diagnostic issues hinder the correct and timely diagnosis and classification of leprosy. Delayed and missed diagnosis of infectious leprosy patients and the lack of tests to measure asymptomatic M. leprae infection in contacts also hamper the assessment of transmission of M. leprae infection. An important goal would be the development of improved diagnostic tools to diagnose difficult cases and to detect M. leprae infection before clinical manifestation. The search for an ideal immunodiagnostic tool for leprosy had gone through various phases and development over the years, with inherent limitations in the sensitivity and specificity of the immunodiagnostic tests for leprosy. With improvement in technology many modifications of previously used PGL-1 assay in the form of rapid and less expensive techniques, such as dipstick, ELISA, ML flow test, have been introduced. Many new skin test antigens with potential for improving their efficiency, such as MLSA LAM, MLCwA and their fractionates, have been studied. After the completion of genome sequencing of M. leprae in 2000, many genes that were studied in M. tuberculosis and found potential for the immunodiagnosis of tuberculosis, such as CFP-10 and ESAT-6 proteins, have been investigated in M. leprae also. Genes that are unique to M. leprae with no homologous in M. tuberculosis have been explored for novel M. leprae-specific antigens. In order to overcome the problem of cross-reactivity, a number of workers have synthesized overlapping short peptides of different M. leprae recombinant proteins and studied their sequence divergence and attempted to identify M. leprae-specific B- and T-cell epitopes. This review makes an effort to present an overview of all these developments in the field of immunodiagnosis of leprosy.     

Evaluation of polymerase chain reaction-based detection of Mycobacterium leprae for the diagnosis of leprosy

Because Mycobacterium leprae cannot be cultivated in vitro , laboratory diagnosis of leprosy is generally made by microscopic and histopathological examination. The objective of the present study was to evaluate the sensitivity and utility of polymerase chain reaction (PCR) to detect M. leprae in comparison with other conventional methods for diagnosis such as split skin smears, histopathology and serodiagnosis. PCR amplification of the M. leprae -specific 16S ribosomal RNA was compared to other methods. Samples included 37 multibacillary (MB) patients with a positive bacteriological index (BI), 32 newly diagnosed paucibacillary (PB) patients whose BI were negative and 30 plaque psoriasis patients not residing in leprosy endemic areas as controls. The sensitivity of PCR was 30 fg of M. leprae DNA, which is equivalent to the DNA from 8.3 bacilli. The detection rate in MB and PB were 100% and 50%, respectively; the specificity was 100%. Semiquantitative evaluation of PCR correlated well with BI, but not with the morphological index (MI) nor with the serum antibody against phenolic glycolipid-1 (PGL-1). PCR detection of M. leprae targeting 16S ribosomal RNA was specific and more sensitive than conventional methods, and can contribute to early and accurate diagnosis of leprosy.     

Comparison of PCR mediated amplification of DNA and the classical methods for detection of Mycobacterium leprae in different types of clinical samples in leprosy patients and contacts

Traditional staining and microscopic examination techniques for the detection of Mycobacterium leprae, DNA amplification by polymerase chain reaction (PCR) of a 531-bp fragment of the M. leprae specific gene encoding the 36-kDa antigen, and serodiagnosis with M. leprae specific antigens (PGL-1 and D-BSA) were compared on different clinical specimens (serum samples, slit-skin smears, biopsies and swabs) from 60 leprosy patients attending the Sanatorium of Fontilles. Patients were divided into groups; (i) 20 multibacillary patients (MB) with positive bacteriological index (BI) by conventional methods and on WHO multidrug therapy (MDT); (ii) 30 MB patients with negative BI and completed minimum 2 years treatment MDT; (iii) 10 paucibacillary (PB) patients who had completed 6 months MDT at least 8 years ago. Control groups included four non-leprosy patients for PCR methods and 40 health control patients and 10 tuberculosis patients for serological methods. In the multibacillary BI positive group, there was a good correlation between all methods. All tests were negative in the paucibacillary group, although only a few patients were tested and all had been treated many years ago. One must be cautious concerning the diagnostic potential of these techniques in this type of leprosy. We also studied different combinations of leprosy diagnosis methods to determine the potential risk in a leprosy contact individuals group. The prevalence of antibodies to M. leprae antigens in serum was measured, together with the presence of M. leprae DNA in the nose and lepromin status in a group of 43 contacts of leprosy patients (12 household and 31 occupational) to evaluate the maintenance of infection reservoirs and transmission of the disease. Only two individuals were found to form a potential high risk group.     

Diagnostic methods to cutaneous leishmaniasis detection in domestic dogs and cats

Cutaneous leishmaniasis is caused by different species of Leishmania. In domestic animals such as dogs and cats, the diagnostic consists of clinical, epidemiological and serological tests, which changes among countries all around the world. Because of this diversity in the methods selected, we propose this systematic literature review to identify the methods of laboratory diagnosis used to detect cutaneous leishmaniasis in domestic dogs and cats in the Americas. Articles published in the last 5 years were searched in PubMed, ISI Web of Science, LILACS and Scielo, and we selected 10 papers about cutaneous leishmaniasis in dogs and cats in the Americas. In Brazil, often the indirect immunofluorescence and enzyme immunoassay (ELISA) have been applied. Other countries like United States and Mexico have been using antigenic fractions for antibodies detections by Western blot. ELISA and Western blot showed a higher sensitivity and efficacy in the detection of leishmaniasis. Analysis of sensibility and specificity of the methods was rarely used. Although confirmatory to leishmaniasis, direct methods for parasites detection and polymerase chain reaction showed low positivity in disease detection. We suggested that more than one method should be used for the detection of feline and canine leishmaniasis. Serological methods such as Western blot and enzyme immunoassay have a high efficacy in the diagnosis of this disease.     

Studies on lepromin and soluble antigens of M.leprae: their classification standardization and use.

Before the discovery of armadillo as a susceptible animal the source of M.leprae was limited and hence the use of lepromin was not common in the field. In recent times, the soluble antigens of armadillo-derived M.leprae have been used extensively in the field. Although the results of the study show that these antigens do not differentiate always a susceptible form from the resistant form, they are able to segregate the polar forms of leprosy. In a given field situation the criteria for diagnosis is so stressed that leprosy is overdiagnosed and within one year of follow up nearly half the number of cases are noted as not leprosy. Hence, in such situations lepromin reaction would be definitely a poor correlate with the type of leprosy. However, in hospital based studies the lepromin reaction has always been and would remain useful in confirming the classification (Sengupta et al 1984). Lepromins and M.leprae soluble antigens have gone through extensive standardization procedures. As these antigens contain mostly common mycobacterial antigens along with the M.leprae-specific antigens, these antigens are unable to specifically diagnose M.leprae infection. After purification of M.leprae from infected armadillo tissue, it was expected that the soluble antigen of M.leprae would probably be as useful as tuberculin. However, this was not found to be true in case of lepromin. Specificity for M.leprae has been noted in the epitopes (antigenic sites) on cross reacting molecules (12 kd, 18 kd, 28 kd, 35 kd, 36 kd) of mycobacteria (Ivanyi et al 1983; Watson 1989). These specific epitopes, if synthesized, could be of use as skin test antigens for determining M.leprae infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythema nodosum leprosum: reactional leprosy

The different clinical forms of leprosy are mainly related to the variety of immunological responses to the infection. Thus, lepromatous leprosy occurs in patients with a poor cell-mediated immunity to Mycobacterium leprae, whereas tuberculoid leprosy is associated with a high resistance to leprosy bacillus. Intermediate forms, including borderline tuberculoid leprosy, borderline lepromatous leprosy, and borderline leprosy, are a continuous and unstable spectrum of the disease. Leprosy reactions are rare and not well-known states that interrupt the usual chronic course and clinical stability of patients with leprosy. They are expressions of immunological perturbations. Attending to the clinical and histopathological manifestations, leprosy reactions may be separated in 2 or 3 different variants: reverse reaction (type I), erythema nodosum leprosum (type II), erythema polymorphous (type II) and Lucio's phenomenon, mainly considered a type II reaction, but sometimes designated type III. Type I leprosy reaction, also named "upgrading reaction," occurs in borderline leprosy states and is associated with a shift toward the tuberculoid pole. Type II reaction usually occurs in lepromatous leprosy, and there are 3 different clinical variants, including erythema nudosum leprosum, erythema polymorphous-like reaction, and Lucio's phenomenon.     

Postgenomic Mycobacterium leprae antigens for cellular and serological diagnosis of M. leprae exposure, infection and leprosy disease

Due to changes in leprosy control programs and the special expertise required for diagnosis, the need for simple rapid diagnostic tests that could be applied in non-expert settings may now be greater than ever before. Since the sequencing of the M. leprae genome, many research groups have investigated the potential of M. leprae antigens in either serologic or cell mediated assays. Here we provide an overview of the nearly 200 recombinant single proteins that were investigated during the last decade for their potential to be applied in field-friendly tests for the early diagnosis of leprosy.     

Epidemiological situation of leprosy in Salvador from 2001 to 2009

Mycobacterium leprae was first described as the bacillus that causes leprosy, a chronic granulomatous infectious disease, in 1873 by Amauer Hansen. Leprosy is part of a group of 10 neglected diseases and Bahia has endemic levels of this illness, varying between high and very high. The detection of 52 new cases of leprosy in children under 15 years old in Salvador in 2006 is alarming, and suggests an early contact with the disease. The aim of this review is to analyze the epidemiological situation, the detection rate and evaluate the clinical and epidemiological profile of leprosy in Salvador, in the period 2001-2009. A retrospective cross-sectional study was performed using secondary data collected at Notifiable Diseases Information System Database (SINAN) through the notification of patients with leprosy. Over these nine years 3,226 patients were reported, with a predominance of: females (51.5%), and clinical multibacillary forms in the general population (51.7%), but when we analyze those under 15 years old, paucibacillary forms (tuberculoid + indeterminate) prevailed. The tuberculoid form was the most diagnosed type of presentation. The annual detection rate in Salvador remained at a very high level of endemicity during the studied period and for those under 15 years old it ranged between high and very high. Grade 2 disabilities both at the time of diagnosis and at discharge after cure, varied between low and medium. Based on these data we conclude that the high levels of leprosy detection rates in the general population, plus the variation between high and very high levels in those under 15 years old, associated with the medium level of grade 2 disabilities at the time of diagnosis and discharge, demonstrate the need for improvement on the existing services, investment in active case finding and training of the healthcare professionals in Salvador.     

Phenotypic,molecular and antimicrobial susceptibility assessment in isolates from chronic ulcers of cured leprosy patients: a case study in Southern Brazil

BACKGROUND

One of the most stigmatizing physical sequelaeof leprosy in cured patients is the development of chronic lower extremity ulcers. The bacterial diversity present in ulcers is considered one of the factors that can delay the healing process, as well as serve as a focus for severe secondary infections.

OBJECTIVE

To identify the microbiota and antimicrobial resistance profile of bacteria isolated from skin ulcers in patients cured of leprosy.

METHODS

After obtaining informed consent, material was collected from ulcers of 16 patients treated at the Outpatient Public Health Dermatology Clinic of Rio Grande do Sul and Hospital Colônia Itapuã. Sampleswere collected during dressing, and the material sent to the Microbiology Laboratory of the Federal University of Health Sciences of Porto Alegre for microbiological culture. Methicillin-resistant Staphylococcus aureus (MRSA) was characterized by two molecular methods, including detection of the mecA gene by PCR and SCCmecgene typing.

RESULTS

Cultures revealed microorganisms in all ulcers: Gram-negative bacilli in 80%, Gram-positive cocci in 63%, and mixed microflora in 36%. Staphylococcus aureus and Pseudomonas aeruginosa were the most prevalent bacteria. Assessment of the antimicrobial resistance profile was notable for the presence of MRSA. Molecular analysis of this isolate revealed presence of the mecA gene contained in a type IV staphylococcal cassette chromosome mec (SCCmec).

CONCLUSIONS

In patients with leprosy, laboratory culture of skin ulcers is essential for correct antibiotic selection and to control emerging pathogens, such as MRSA carrying SCCmec type IV.     

Leprosy

Leprosy is a slowly progressive, chronic infectious disease caused by the bacillus Mycobacterium leprae. It is a very serious, mutilating and stigmatizing disease in many parts of the world and early diagnosis and therapy is the most important strategy for its control. The skin and peripheral nerves are the most affected organs. It is highly infective, but has low pathogenicity and low virulence with a long incubation period. The geographical distribution of leprosy has varied greatly with time and it is now endemic only in tropical and subtropical regions such as India and Brazil. The diagnosis of leprosy is made from the clinical picture, but must be complimented by skin bacilloscopy and histopathology. Leprosy has a number of distinct clinical presentations. Indeterminate leprosy is frequently the initial form consisting of a few lesions that either evolves into the other forms or resolves spontaneously. Lepromatous leprosy is the more contagious form and affects mainly the skin. In addition, some peripheral nerves may be thickened and other symptoms maybe present. The tuberculid form affects the skin and nerves, although usually there are few lesions. There is also a form borderline between the lepromatous and tuberculoid forms. Current treatment of leprosy involves use of 3 drugs: rifampicin (rifampin); clofazimine; and dapsone. Multidrug therapy aims to effectively eliminate M. leprae in the shortest possible time to prevent resistance from occurring. The duration of therapy was recently reduced from 24 to 12 months. Other treatment options are under evaluation in both preclinical and clinical trials and a number show promise. The combination of rifampicin, ofloxacin and minocycline given as a single dose has been recommended for the treatment of paucibacillar leprosy. Only when physicians, other health workers, and the population in endemic countries become fully aware of, and able to recognize, the disease in its initial phase, will it be possible for therapy to be instituted at the very beginning with either the standard scheme or the newer ones. Intervention at such an early stage will avoid the onset of the more serious signs and symptoms, meaning that leprosy will eventually become a less important public health problem. Therefore, efforts must be made to alert populations at risk and all health workers of the importance of an early diagnosis and treatment in leprosy infection.     

Colorimetric microtitre plate hybridization assay for the detection of Mycobacterium leprae 16S rRNA in clinical specimens

We have developed a colorimetric microtitre plate hybridization assay in order to simplify detection of Mycobacterium leprae in clinical specimens. This system detects the products amplified by a sensitive RT-PCR assay targeting a species-specific sequence of the bacterial 16S rRNA. The assay detected as few as 10 bacilli isolated from infected nude mouse lymph nodes or human skin biopsies. Sensitivity for diagnosis of clinical specimens was assessed for 58 tissue biopsies from untreated leprosy patients. The assay detected M. leprae RT-PCR products in 100% of biopsies from patients with multibacillary disease and 80% of biopsies from patients with paucibacillary disease, for an overall sensitivity of 91.3%. The test was highly specific as no RT-PCR products were amplified from skin biopsies of normal individuals or patients with skin diseases other than leprosy. The colorimetric assay is faster, more sensitive, and simplifies detection of RT-PCR products compared to Southern blot analysis. It may be useful for diagnosis of difficult cases of leprosy, and, since RNA is rapidly degraded after cell death, it may be appropriate for assessing response to therapy and for distinguishing relapse from reaction.     

Genomics and the chemotherapy of leprosy

The information deduced from the genome sequence of Mycobacterium leprae is of immense value for the chemotherapy of leprosy. Knowing the complete set of genes, enzymes and proteins allows us to understand why some drugs are without effect whereas others are fully active. It may also enable better use to be made of existing drugs, such as beta-lactams, and opens new avenues for the development of novel compounds. M. leprae is relatively susceptible to a wide range of drugs, unlike the highly related tubercle bacillus, and several new multidrug regimens are in clinical trials. Genomics provides a number of possible explanations for this broader susceptibility as some of the genes encoding enzymes involved in antibiotic inactivation have decayed whereas the number of transporters available to contribute to drug efflux is considerably lower than in Mycobacterium tuberculosis. Several leads for new drug targets have been uncovered.     

Case for diagnosis. Infiltrated areas on the trunk

Leprosy is an infectious disease with chronic evolution, caused by Mycobacterium leprae, an acid-fast bacillus that mainly affects the skin and peripheral nervous tissue. Many of the clinical manifestations of leprosy can mimic connective tissue diseases. The authors present the case of a 49-year-old woman who had been treated for four years for systemic lupus erythematosus in a rheumatological service. Skin biopsy of a plaque on the inguinal region was compatible with borderline lepromatous leprosy associated with a type 1 lepra reaction. The patient is undergoing treatment with multibacillary multidrug therapy, showing clinical improvement.     

麻风免疫诊断标记物研究进展

麻风是麻风分枝杆菌感染所致的慢性传染性疾病,体液免疫及细胞免疫参与麻风的发病机制。目前血清抗体及辅助性T细胞2(Th2)相关细胞因子检测主要应用于诊断多菌型麻风(MB),辅助性T细胞1(Th1)相关细胞因子检测主要应用于诊断少菌型麻风(PB)。microRNAs可用于区分瘤型及结核型麻风。但上述诊断试验的敏感性及特异性不一,何种标记物诊断效能更佳仍存在争议。本文主要从血清抗体、细胞因子及microRNA标记物检测及其临床应用等方面进行综述,以期为新型早期诊断麻风方法的优化提供依据。     

Recent histopathological studies in leprosy, with particular reference to early diagnosis and leprous neuropathy

In histopathological studies in leprosy, two important areas were identified in recently published work. They are early diagnosis and neuropathy. In histopathological examination, finding of M. leprae in tissues and/or granulomatous destruction of nerves are the two important findings to confirm the diagnosis. Immunopathological staining of M. leprae, PCR amplification of M. leprae antigen and S100 staining of Schwaann cells have considerably enhanced the sensitivity of histopathological diagnosis. If the two clinical findings such as hypopigmented patches with impaired sensation and thickened nerves accompanied by loss of sensation are the only ones that are taken into account for diagnosis, then a significant number of early patients will be missed. It is pointed out that biopsy examination of skin and nerves, when necessary, and skin-smear studies are indispensable diagnostic procedures. In the study of leprous neuropathy, there are several studies trying to decipher the entry of M. leprae into Schwann cells. The sharing of antigens between M. leprae and surface membrane of Schwann cells may be an important factor. However, there is much more to be learned in this area. In the control and prevention of neuritis, although corticosteroids administered along with multi-drug therapy was helpful, the benefit was not sustained.