Two Rapidly Growing Mycobacterial Species Isolated from a Brain Abscess: First Whole-Genome Sequences of Mycobacterium immunogenum and Mycobacterium llatzerense

Rapidly growing mycobacteria are rarely found in central nervous system infections. We describe a case of polymicrobial infection in a brain abscess including two rapidly growing Mycobacterium species, M. immunogenum and M. llatzerense. The Mycobacterium isolates were distinguishable by molecular methods, and whole-genome sequencing showed <60% pairwise nucleotide identity.     

In Vitro Lymphocyte Response to Purified Protein Derivative, BCG, and Mycobacterium leprae in a Population Not Exposed to Leprosy

Lymphocytes from 14 BCG-vaccinated donors, seven tuberculin positive and seven tuberculin negative by skin testing, were stimulated in vitro with four mycobacterial antigens, purified protein derivative (PPD), PPD/BCG, whole BCG bacilli, and whole Mycobacterium leprae and also with Candida antigen and phytohemagglutinin. The response was measured by incorporation of ³H-labeled thymidine. The response to PPD, PPD/BCG, and BCG was found to correlate with the result of skin testing with turberculin. The turberculin-positive group also responded more strongly to M. leprae, whereas the two groups did not differ significantly in their response to Candida antigen or phytohemagglutinin. These findings indicate a certain degree of cross-reactivity between BCG and M. leprae. The use of the lymphocyte transformation test to measure antigenic cross-reactivity is discussed.     

Rapid Quantitative Serological Test for Detection of Infection with Mycobacterium leprae,the Causative Agent of Leprosy

Leprosy remains an important health problem in a number of regions. Early detection of infection, followed by effective treatment, is critical to reduce disease progression. New sensitive and specific tools for early detection of infection will be a critical component of an effective leprosy elimination campaign. Diagnosis is made by recognizing clinical signs and symptoms, but few clinicians are able to confidently identify these. Simple tests to facilitate referral to leprosy experts are not widely available, and the correct diagnosis of leprosy is often delayed. In this report, we evaluate the performance of a new leprosy serological test (NDO-LID). As expected, the test readily detected clinically confirmed samples from patients with multibacillary (MB) leprosy, and the rate of positive results declined with bacterial burden. NDO-LID detected larger proportions of MB and paucibacillary (PB) leprosy than the alternative, the Standard Diagnostics leprosy test (87.0% versus 81.7% and 32.3% versus 6.5%, respectively), while also demonstrating improved specificity (97.4% versus 90.4%). Coupled with a new cell phone-based test reader platform (Smart Reader), the NDO-LID test provided consistent, objective test interpretation that could facilitate wider use in nonspecialized settings. In addition, results obtained from sera at the time of diagnosis, versus at the end of treatment, indicated that the quantifiable nature of this system can also be used to monitor treatment efficacy. Taken together, these data indicate that the NDO-LID/Smart Reader system can assist in the diagnosis and monitoring of MB leprosy and can detect a significant number of earlier-stage infections.     

Methods for the Detection of a Specific Mycobacterium leprae Antigen in the Urine of Leprosy Patients

Two methods for detecting the phenolic glycolipid, PGL-1, a Mycobacterium leprae-specific molecule, in the urine of leprosy patients are described. Both methods rely on the 100-fold preconcentration of the urine, which can be accomplished by a single-step ultrafiltration procedure. The equivalent of approximately 2.5 micrograms of PGL-1/ml was detected in the urine of LL patients with an inhibition ELISA. The second method, a direct dot-blot assay on nitrocellulose paper, was much simpler and more sensitive. As little as 3 ng of antigen was detected by the dot-blot technique. PGL-1 was detected in the urine of LL patients.     

T-Cell Stimulation with Purified Mycobacterial Antigens in Patients and Healthy Subjects infected with Mycobacterium leprae: Secreted Antigen 85 is Another Immunodominant Antigen

Peripheral blood leucocytes from 9 paucibacillary and 12 multibacillary leprosy patients, from 18 healthy controls and from 34 healthy leprosy contacts were stimulated with three mycobacterial heat shock proteins with respective molecular weights of 70,65 and 18 kDa and with the secreted 30–32 kDa protein, also called antigen 85. Antigen 85 was found to be the most powerful T-cell antigen (as measured by lymphoproliferation and IFN-γ secretion), eliciting a positive response in all (100%) paucibacillary patients and in all lepromin-positive controls and contacts. The three heat shock proteins (hsp) were less active T-cell stimuli. Reactivity to the 70 kDa hsp was found in only 44% of the paucibacillary patients, in 80% of the lepromin-positive controls and in 60% of the lepromin-positive leprosy contacts. The 65 kDa hsp stimulated T cells in 89% of the paucibacillary patients and in 80% of the lepromin-positive controls and contacts. Responsiveness to the 18 kDa hsp, finally, was clearly more frequent in tuberculoid leprosy patients (78%) than in lepromin-positive controls (40%) or lepromin-positive leprosy contacts (4%). T-cell reactivity of 8 lepromin-negative controls, of 9 lepromin-negative contacts and of 12 multibacillary leprosy patients was low to all the antigens tested. Although proliferative and IFN-γ responses were generally closely related, some subjects demonstrated a dissociation of these two immune parameters. Our data confirm previous findings on the powerful T-cell stimulatory properties of antigen 85 during M. leprae infection and suggest that this antigen is indeed a potentially protective T-cell immunogen.     

Serological activity of a characteristic phenolic glycolipid from Mycobacterium leprae in sera from patients with leprosy and tuberculosis

Serological activity against a purified phenolic glycolipid from Mycobacterium leprae, which may be obtained in large amounts from M. leprae infected armadillo liver, was investigated using immunodiffusion and an enzyme linked immunosorbent assay (ELISA). Generally a good correlation was obtained between these techniques, but the ELISA was more sensitive and convenient. Relatively high IgG and IgM anti-glycolipid antibody levels were found in lepromatous leprosy patients. The antibody titres to the glycolipid were, however, low when compared with antibody titres to crude sonicates. Since the glycolipid is present in large quantities, this suggests that it is not very immunogenic. Antibody against the glycolipid especially of the IgM class, was demonstrable in some tuberculoid leprosy patients, although at much lower titres than in the lepromatous leprosy sera. In lepromatous leprosy patients that were skin smear negative after more than 5 years of treatment the IgG anti-M. leprae derived glycolipid activity had decreased markedly. The anti-IgG and IgM glycolipid antibody levels in tuberculosis patients did not differ significantly from the levels in appropriate normal healthy subjects. The glycolipid antibody levels in patients infected with M. kansasii, M. avium or M. intracellulare also fell within the range of normal healthy individuals.     

Potential Serological Use of a Recombinant Protein That Is a Replica of a Mycobacterium tuberculosis Protein Found in the Urine of Infected Mice

The recent availability of numerous well-characterized Mycobacterium tuberculosis recombinant proteins has revived interest in the serological diagnosis of tuberculosis. Several promising results have been reported, particularly when more than one antigen is used in the test. However, thus far these antigens have not been used in routine diagnostic tests because they lack sufficient sensitivity. In addition, with the exception of one antigen, most recombinant M. tuberculosis proteins do not identify the majority of tuberculosis patients coinfected with human immunodeficiency virus (HIV). Here, we report a newer M. tuberculosis protein that is a promising candidate for increasing the sensitivity of the serological tests, in particular for patients coinfected with HIV. The protein was found in the urine of mice during the early stages of infection with M. tuberculosis (10 to 14 days), thus suggesting that the antigen is abundantly released during the in vivo growth of the mycobacterium. Reverse genetics was used to produce the recombinant protein, which we named U1 (for urine protein 1). Using a conventional enzyme-linked immunosorbent assay (ELISA), antibody to U1 could be detected in 60% of patients with pulmonary tuberculosis with no signs of coinfection with HIV (n = 83). Conversely, anti-U1 antibody was detected in 87% of the sera from tuberculosis patients coinfected with HIV (n = 47). Out of 12 HIV-infected nontuberculosis patients' sera, 9 did not react with U1 and three sera gave borderline ELISA signals (signal/cutoff of ≤1.75). These results suggest that the high efficiency of U1 in identifying tuberculosis patients coinfected with HIV may be related to abundant release of this protein during the initial phase of the HIV coinfection. The immediate availability of the antigen at a time point in which the patient's immune system is still competent would lead to a secondary immune response to U1 that persists for months in the patient's serum.     

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.     

Use of Recombinant BP26 Protein in Serological Diagnosis of Brucella melitensis Infection in Sheep

Previously a Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and B. melitensis Rev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in Escherichia coli in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (n = 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (n = 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (n = 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (n = 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with B. melitensis H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and B. melitensis Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that a B. melitensis Rev.1 bp26 gene deletion mutant should be constructed to ensure this differentiation.     

IgA and IgM Antibodies against Mycobacterium leprae in Cord Sera and in Patients with Leprosy: An Indicator of Intrauterine Infection in Leprosy

A solid-phase radioimmunoassay was developed for demonstration and quantification of IgA and IgM anti- M. leprae antibodies. IgA and IgM anti- M. leprae antibodies were demonstrated in a lepromatous serum pool, in various amounts in individual patients with lepromatous leprosy, and in lower concentration in tuberculoid leprosy and non-leprosy controls. IgA and IgM anti- M. leprae antibodies were demonstrated in cord sera from babies of mothers with leprosy. The reliability of fetal IgA and IgM antibody synthesis as an indicator of intrauterine infection in leprosy is discussed.     

Inhibition of the Multiplication of Mycobacterium leprae by Vaccination with a Recombinant M. bovis BCG Strain That Secretes Major Membrane Protein II in Mice

The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-γ) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8⁺ T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-γ on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.Leprosy, which is caused by Mycobacterium leprae, is an infectious disease that still affects thousands of people worldwide. According to WHO''s weekly epidemiological report, 254,525 new cases were detected in 2007 (25). One reason why leprosy is still prevalent may be due to the inherent characteristics of M. leprae, i.e., slow growth and weak pathogenicity. It takes 12 to 14 days for M. leprae to replicate, so it is predicted that 2 to 5 years are necessary for the clinical manifestations to appear after an infection (1, 18). Likewise, it takes 6 to 8 months for the recognizable swelling of the footpad to appear in nude mice (22).Leprosy is clinically divided into two major categories: multibacillary (MB) leprosy and paucibacillary (PB) leprosy. In the lesions of patients with PB leprosy, dendritic cells (DCs) and activated T cells are involved with confining M. leprae to a localized area. These pathological observations indicate that cell-mediated reactions are triggered and that the activation of both CD4⁺ and CD8⁺ T cells is closely associated with inhibition of the spread of the bacilli. In contrast, abundant foamy macrophages loaded with bacilli but not DCs appear in the lesions of MB patients (11). It can be speculated that antigen (Ag)-presenting cells such as DCs recognize the immunodominant Ags of M. leprae and express those derivatives on their surfaces, thereby activating T cells. Previously, using T cells from patients with PB leprosy, we have identified major membrane protein II (MMP-II), also known as bacterioferritin (ML2038), as one of the immunodominant Ags (8). We found that MMP-II activates DCs through Toll-like receptor 2, leading to higher levels of expression of major histocompatibility complex class I and class II, CD86, and CD83 Ags and increased levels of production of interleukin-12 p70. Furthermore, MMP-II-pulsed DCs derived from patients with PB leprosy activated both autologous CD4⁺ T cells and CD8⁺ T cells to produce gamma interferon (IFN-γ) in amounts larger than the amounts produced by T cells from patients with MB leprosy and M. bovis BCG-vaccinated healthy individuals, indicating that T cells from patients with PB leprosy may be primed with MMP-II in vivo.The BCG vaccine has been used for the prevention of tuberculosis, although its role in the prevention of leprosy is still being debated. The protective efficacy of BCG against leprosy has been tested in several trials, including studies in the Karonga District of northern Malawi, in which 50% protection was observed (17). Through combined systematic analyses of experimental studies, Setia et al. found that the BCG vaccine had an overall level of protective efficacy of 26% against human leprosy (19). Their observational studies overestimated the protective effect at 61%. In another review of 29 studies, Zodpey reported that 44.8% of the reports indicated that the BCG vaccine had a level of efficacy of 50% or more (26). These observations indicate that improvements to the BCG vaccine are necessary to increase its protective effect. Recently, we produced a recombinant BCG strain that secretes MMP-II (strain BCG-SM, where SM indicates secreting MMP-II). Since MMP-II has the ability to ligate Toll-like receptor 2, we expected BCG-SM to highly activate human T cells. In fact, BCG-SM activated not only naïve CD4⁺ T cells but also naïve CD8⁺ T cells through DCs (9). The fact that BCG-SM was more efficient than the parental BCG strain at the activation of both subsets of naïve T cells led us to seek further insights into the protective activity of BCG-SM. In the present study, we investigated the effect of vaccination of BCG-SM on the multiplication of M. leprae in mice.     

Identification of Primary Drug Resistance to Rifampin in Mycobacterium leprae Strains from Leprosy Patients in Amazonas State,Brazil

The aim of this study was to identify polymorphisms in the folp1, gyrA, and rpoB genes in leprosy patients treated in Amazonas State, Brazil. Among 197 slit-skin smear samples from untreated or relapsed patients, we found three cases of primary resistance to rifampin and one confirmed case of multidrug resistance.     

Detection of Phenolic Glycolipid I of Mycobacterium leprae in Sera from Leprosy Patients before and after Start of Multidrug Therapy

A total of 100 untreated new leprosy patients were recruited prospectively and examined for the presence of phenolic glycolipid I (PGL-I) antigen in their serum specimens by dot enzyme-linked immunosorbent assay (ELISA) using rabbit anti-PGL-I antiserum. The presence of circulating PGL-I antigen was closely related to the bacterial indices (BI) of the patients. The PGL-I antigen was detectable in 27 (93.1%) of 29 patients with a BI of 4.0 or above and in 15 (68.2%) of 22 patients with a BI of 3.0 to 3.9. However, none of the 37 patients with a BI of less than 1.9 had detectable PGL-I antigen by the methods used in this study. The level of PGL-I in serum declined rapidly by about 90% 1 month after the start of multidrug therapy. This study showed clearly that anti-PGL-I IgM antibodies and circulating PGL-I antigen levels reflect the bacterial loads in untreated leprosy patients. The serological parameters based on the PGL-I antigen may therefore be useful in the assessment of leprosy patients at the time of diagnosis and possibly in monitoring patients following chemotherapy.     

Specific Lymphoproliferation, Gamma Interferon Production, and Serum Immunoglobulin G Directed against a Purified 32 kDa Mycobacterial Protein Antigen (P32) in Patients with Active Tuberculosis

Twenty-one patients treated for active tuberculosis were examined for immune reactivity to purified protein derivative (PPD) and to a purified 32-kDa protein antigen (P32) from Mycobacterium bovis , strain BCG. Lymphoproliferation of peripheral blood leucocytes to PPD and P32 was positive in 95% and 71% of the patients respectively. A positive IFN-γ response was detected in 62% against PPD and in 48% against P32. Low blastogenesis and IFN-γ production were observed, especially in patients with poor general health and advanced tuberculous lesions. Twelve out of twelve (100%) of the tuberculin-positive healthy volunteers responded to PPD and P32 with mean lymphoproliferation and IFN-γ values that were higher than in the patient group. Twelve tuberculin-negative control subjects were completely unreactive to PPD and P32 antigen. On the other hand, IgG antibodies in the serum were detected in 95% of the patients against PPD, in 77% of the patients against P32 but in none of the tuberculin-positive or negative healthy volunteers. The highest IgG levels against PPD were found in those patients with the lowest in vitro lymphoproliferation and IFN-γ production (r=-0.54; P <0.05). Nonspecific interferon production following induction with Newcastle disease virus, Corynebacterium parvum , or phytohaemagglutinin was comparable in the control and patient groups. Finally, low IFN-α titres were detected in the serum of about 50% of the patients.     

Serological monitoring of previously treated lepromatous patients during a course of multiple immunotherapy treatments with heat-killed Mycobacterium leprae and BCG.

Two-hundred and seventy lepromatous patients who had completed treatment received multiple treatments with heat-killed M. leprae and BCG and were monitored for changes in humoral responses to M. leprae-specific antigens. These patients were divided into four treatment groups: placebo (n = 69); BCG (n = 68); M. leprae only (n = 71); and BCG + M. leprae (n = 62). They were monitored for 15 months, receiving five inoculations for each treatment regimen. Two ELISA systems, one measuring antibodies to M. leprae-specific epitopes of the phenolic glycolipid I (NDO-ELISA) and the other of 36-kD protein antigens (INH-ELISA) were used to measure serological changes during this period of immunotherapy. We found no significant increase in serological reactivity with the different treatments, as measured by NDO-ELISA. INH-ELISA similarly showed no significant changes, with the exception of increased values in a small group 13% (36/270) which became skin test-positive during the course of the study. The NDO-ELISA results indicate that use of heat-killed M. leprae or BCG + heat-killed M. leprae did not stimulate the humoral response to the semi-synthetic PG-I antigens of M. leprae. Thus, the NDO-ELISA may be useful in monitoring the outcome of vaccine trials in which killed M. leprae or M. leprae fractions are used, since seroconversion may indicate disease, rather than a response to the vaccine material.     

Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+ T Cells of the Mycobacterium leprae Memory Repertoire

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4(+) T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.     

Rational Design and Evaluation of a Multiepitope Chimeric Fusion Protein with the Potential for Leprosy Diagnosis

Despite the reduction in the number of leprosy cases registered worldwide as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains stable in many countries. This indicates that Mycobacterium leprae, the causative agent of leprosy, is still being transmitted and that, without an earlier diagnosis, transmission will continue and infection will remain a health problem. The current means of diagnosis of leprosy is based on the appearance of clinical symptoms, which in many cases occur after significant and irreversible nerve damage has occurred. Our recent work identified several recombinant antigens that are specifically recognized by leprosy patients. The goal of the present study was to produce and validate the reactivity of a chimeric fusion protein that possesses the antibody binding properties of several of these proteins. The availability of such a chimeric fusion protein will simplify future test development and reduce production costs. We first identified the antibody binding regions within our top five antigen candidates by performing enzyme-linked immunosorbent assays with overlapping peptides representing the amino acid sequences of each protein. Having identified these regions, we generated a fusion construct of these components (protein advances diagnostic of leprosy [PADL]) and demonstrated that the PADL protein retains the antibody reactivity of the component antigens. PADL was able to complement a protein that we previously produced (the leprosy IDRI [Infectious Disease Research Institute] diagnostic 1 [LID-1] protein) to permit the improved diagnosis of multibacillary leprosy and that had a good ability to discriminate patients with multibacillary leprosy from control individuals. A serological diagnostic test consisting of these antigens could be applied within leprosy control programs to reduce transmission and to limit the appearance of leprosy-associated disabilities and stigmatizing deformities by directing treatment.Leprosy is a devastating disease caused by infection with the bacterium Mycobacterium leprae. The clinical symptoms, underlying immunological responses, bacterial burden, and pathology vary widely between individuals. In 1966, Ridley and Jopling proposed a now widely used classification scheme that allows reasonable consistency in clinical and research practice by dividing the leprosy spectrum into five parts (18). Patients with the true tuberculoid (TT) form of leprosy present with few skin lesions and little nerve damage, granulomas that are histologically similar to those found in tuberculosis, and a T-helper-1-skewed immune response such that very few, if any, bacteria can be detected in biopsy specimens. At the other end of the spectrum, patients with the lepromatous leprosy (LL) form present with multiple skin lesions and a loss of sensation caused by nerve damage. Patients with LL leprosy have diffuse rather than well-organized granulomas mainly consisting of foamy macrophage influxes, and the immune response is biased toward a T-helper-2 type of response. Consequently, large numbers of bacteria and high antibody titers to components of M. leprae can be observed in LL patients. The borderline tuberculoid (BT), the borderline borderline (BB), and borderline lepromatous (BL) forms are distinguishable between the polar extremes of the TT and LL forms, according to the number lesions and the organization of the granuloma (22).Leprosy is typically classified on the basis of clinical manifestations and skin smear results. Simple guidelines have been suggested by the World Health Organization (WHO), with diagnostic criteria listed as being one or more of the following: hypopigmented or reddish skin patches with a definite loss of sensation, thickened peripheral nerves, and the appearance of acid-fast bacilli on analysis of skin smears and biopsy specimens (25a). In the classification based on skin smears, patients showing negative smears with samples from all sites are grouped as having paucibacillary (PB) leprosy, while those showing positive smears with samples from any site are grouped as having multibacillary (MB) leprosy. In practice, however, because skin-smear services are absent or unreliable, most programs use clinical criteria to classify individual patients and select their treatment regimens. The clinical system uses the number of skin lesions and the number of nerves involved to group leprosy patients into one of two simplified categories: MB leprosy (five or more lesions) and PB leprosy (less than five lesions). Thus, TT patients and most BT patients are categorized as having PB leprosy, while LL, BL, BB, and some BT patients are categorized as having MB leprosy.Leprosy treatment involves a prolonged regimen of antibiotics in the form of multidrug therapy (MDT). For MB leprosy, a combination therapy employing rifampin, dapsone, and clofazimine is recommended for 12 months, while for PB leprosy a regimen with only rifampin and dapsone given over 6 months is recommended. It is particularly important to ensure that patients with MB disease are not undertreated with the regimen for the PB form of the disease. Thus, determination of an appropriate treatment regimen requires the accurate and differential diagnosis of the MB and PB forms.A rapid, easy-to-use test that would simplify leprosy diagnosis could greatly assist with the prompt initiation of treatment. Tests based on IgM recognition of phenolic glycolipid I (PGL-I) have been used in a confirmatory role in the clinic (3, 4, 6, 14, 19, 20, 26). Testing for the presence of PGL-I-specific IgM gives a fairly high false-positive rate (>10%) in areas where leprosy is endemic, and while positive responses are indicated to be risk factors in the development of disease, the fact that many people with antibodies against PGL-I do not develop leprosy has hindered the widespread adoption of these tests in screening programs (4, 5, 14). For these reasons, additional antigens have been produced by our group and others with the goal of providing a clear, accurate, and rapid means of diagnosis of leprosy (8, 9, 15, 23).In the study described here, we have extended our previous observations by creating a new polyepitope chimeric fusion protein with the potential to bind to serum antibodies of leprosy patients to provide a leprosy diagnosis. We refined our previous observations by determining antibody-reactive regions within select antigens in order to produce a synthetic protein that combines these reactive portions within a single product. Our results indicate that all portions contained within the synthetic protein retain their antibody binding activity and that this protein has utility for leprosy diagnosis.     

结核分枝杆菌相关IFN-γ释放试验在结核病诊断及疗效监测中的应用

目的：目的：探讨结核分枝杆菌相关γ-干扰素释放试验（TB-IGRA）在结核病诊断及临床抗结核治疗效果评价中的应用价值。方法：对196例临床确诊结核患者进行相关病史收集,同时进行TB-IGRA、结核菌素皮肤试验、结核杆菌培养、涂片镜检、结核抗体检测,结核患者的治疗过程中按用药疗程（初治患者第2、5、6个月,复治患者第2、5、8个月）对其进行随访,在治疗结束后6个月和1年后各随访一次,比较TB-IGRA与结核菌素皮肤试验在治疗过程中的变化。结果：确诊结核病例196例,其中肺结核158例,肺外结核38例,TB-IGRA在结核诊断中灵敏度为82．1％,特异性为91．7％,阳性预测值为93．1％,阴性预测值为79．3％,与传统实验室检查方法相比有显著性差异（P〈0．01）。在结核患者的抗结核治疗过程中,TB-IGRA阳性率及浓度水平治疗前与治疗后2、6月以及复治后2、5、8月比较显著下降（P〈0．01）,TB—IGRA水平与结核分枝杆菌数量存在较好相关性,与传统的TST方法评估疗效相比有显著性差异（P〈0．01）。结论：TB-IGRA在结核诊断中敏感性和阳性预测值较高,可作为较好的结核诊断的依据,同时TB-IGRA对肺外结核的灵敏度较高,为肺外结核患者的诊断提供了较为可靠的依据,通过治疗过程TB—IGRA检测分析,外周血TB-IGRA的变化随着抗结核治疗的进程下降的趋势明显,与结核涂片镜检结果符合率较高,是临床疗效评估较为理想的指标。     

Identification of a Novel 27-kDa Protein from Mycobacterium tuberculosis Culture Fluid by a Monoclonal Antibody Specific for the Mycobacterium tuberculosis Complex

Mycobacterium tuberculosis antigens inducing species-specific immune responses are likely to be particularly important for serodiagnosis or for skin testing of tuberculosis. In the present study, we describe the characterization of two novel monoclonal antibodies (MoAbs) A3h4 (IgG2a) and B5gl (IgM) that are directed to M. tuberculosis 27-kDa and 25-kDa proteins respectively. Specificity analysis by immunoblotting using 20 different species of mycobacterial sonicates revealed that MoAb A3h4 was specific for M. tuberculosis complex alone while MoAb B5gl showed a limited cross-reactivity. Direct comparison with previously characterized MoAbs revealed that these MoAbs A3h4 and B5gl defined new antigenic determinants of M. tuberculosis. By using M. tuberculosis complex-specific MoAb A3h4 we have identified a distinct 27-kDa protein in the M. tuberculosis H37Rv culture fluid. Since this MoAb did not bind to the previously characterized MPT44, MPT59, MPT45, MPT51 and MPT64 proteins as well as the 23-kDa superoxide dismutase (SOD) protein of M. tuberculosis, we conclude that MoAb A3h4 recognizes a novel protein in the M. fuberculosis H37Rv culture fluid. Studies of the subccllular distribution of these MoAb-reaetive proteins indicate that the MoAb A3h4-reactive 27-kDa protein is present not only in the culture fluid bnt also in the cytosol and the cell wall of M. tuberculosis. By contrast. B5gl-reactive protein is mainly a cytosolic protein. When these MoAbs were tested in a previously established ELISA with intact mycobacteria derived from early cultures, only MoAb A3h4 showed the positive reactivity to mycobacteria belonging to the M. tuberculosis complex. In addition, during the present comparative studies of MoAbs we have also found that the previously described MoAb F116-5, which is known to recognize the mycobacterial 23-kDa SOD protein [17], cross-reacted with the MPT44, MPT59, MPT45 and MPT51 secreted proteins but not with MPT64 and MPB70. These findings indicate that the family of four secreted proteins of M. tuberculosis share a common epitope with M. tuberculosis SOD protein.     

Characterization of a 34-Kilodalton Protein of Mycobacterium leprae That Is Isologous to the Immunodominant 34-Kilodalton Antigen of Mycobacterium paratuberculosis

During DNA sequence analysis of cosmid L373 from the Mycobacterium leprae genome, an open reading frame of 1.4 kb encoding a protein with some homology to the immunodominant 34-kDa protein of Mycobacterium paratuberculosis, but lacking significant serological activity, was detected. The DNA sequence predicted a signal peptide with a modified lipoprotein consensus sequence, but the protein proved to be devoid of lipid attachment.