High Intraspecific Genetic Diversity of Nocardia brasiliensis, a Pathogen Responsible for Cutaneous Nocardiosis Found in France: Phylogenetic Relationships by Using sod and hsp65 Genes.

This study aims at genetic characterization and phylogenetic relationships of Nocardia brasiliensis focusing by using housekeeping rrs, hsp65, and sodA genes. N. brasiliensis is the species responsible for 80% of cases of actinomycetoma, one form of cutaneous nocardiosis which occurs mainly in tropical regions reaching immunocompetent patients in which the disease can lead to amputation. We analyze 36 indigenous cases of N. brasiliensis that happened in France. Phylogenetic analysis targeting rrs gene showed no robustness at phylogenetic nodes level. However, the use of a concatenation of hsp65 and sodA genes showed that the tested strains surprisingly ranked in 3 well-defined genotypes. Genotypes 2 and 3 were phylogenetically closer to each other and both diverged from genotype 1 sustained by a high bootstrap of 81%. This last genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, excepting one of them, all the strains belonging to this group present a susceptibility to imipenem which is not the case in the other genotypes that rarely count among them strains being susceptible to this drug. The haplotype diversity (Hd) of hsp65 (0.927) and sodA (0.885) genes was higher than that of rrs (0.824). For this gene, we obtained 16 polymorphic sites whereas, for hsp65 and sodA genes, up to 27 and 29 were identified, respectively. This study reveals that these two genes have an important genetic discriminatory power for the evaluation of the intraspecies genetic variability of N. brasiliensis and they may be useful for identification purposes at species level. This study also reveals the possible existence of a new species harbored by genotype 1.

Cyclosporin a. Inhibition of experimental autoimmune uveitis in Lewis rats.

Cyclosporin A (CS-A), a selective inhibitor of T lymphocytes, is reported here to prevent S antigen (S-Ag) induced uveitis in Lewis rats. The S-Ag, found in all mammalian retinas, is uveitogenic under experimental conditions and patients with certain uveitic entities demonstrate cell mediated responses to this antigen. Daily treatment with CS-A (10 mg/kg) begun on the same day as S-Ag immunization totally inhibited the development of the uveitis in this experimental autoimmune model. Moreover a greater CS-A dose (40 mg/kg) efficiently prevented the disease process when therapy was started 7 d after S-Ag immunization. Anti-S-Ag antibody titers were observed to be similar in rats either protected or not protected with CS-A. Our data support strongly the need for T cell participation in this disease model. Since ocular inflammatory disease is an important cause of visual impairment, the data further suggest that CS-A may be useful in the treatment of patients with intractable uveitis.

Nocardia brasiliensis: from microbe to human and experimental infections.

Nocardia brasiliensis is a Gram-positive bacterium that lives as a saprophyte in soil. In this article the physical properties, chemical composition and taxonomic position of this species is reviewed. Human infections and an experimental model of actinomycetoma in BALB/c mice as well as the host-immune response is described.

Modulation of experimental autoimmune uveitis with cyclosporin A.

Cyclosporin A has been shown to be an effective inhibitor of T cell-mediated diseases. We show here that cyclosporin A was capable of totally preventing the clinical appearance of experimental autoimmune uveitis in Lewis rats, even when administered on an every-other-day schedule (10 mg/kg) or when begun seven days after immunization (40 mg/kg). At lower doses of the drug, a modulation of the disease was seen with evidence of a more chronic, granulomatous process. A long-lasting unresponsive state to the immunizing antigen was not uniformly induced with cyclosporin A if therapy was begun seven days after S antigen immunization. Because of cyclosporin A's effective control of this experimental model that is induced by an antigen to which certain patients with uveitis demonstrate cell-mediated immune responses, cyclosporin A may be an effective mode of therapy for T cell-mediated intraocular inflammatory disease.

[Immunoelectroblotting in Mexican subjects at high risk for infection with human immunodeficiency virus (HIV)]. / La inmunoelectrotransferencia en sujetos mexicanos de alto riesgo para la infección por el virus de la inmunodeficiencia humana (VIH).

Sera from 124 persons in high risk groups were analyzed including homosexuals, blood recipients, and spouses or siblings from AIDS patients. In this study, 118 individuals had a positive ELISA for anti-HIV antibodies. Six persons had a complete immunodeficiency syndrome and a negative ELISA test. In the Western blot, 111 sera were positive, four negative, and nine scored indeterminate; four of the latter converted to positive when retested three months later. Antibodies present in the positive sera were directed against the HIV gp 41 kD in 100% of the cases and against the gp 120 kD in 82%. Frequency of recognition of p55 kD was 96% but p18 kD was only 42%.

[Antibodies against human T-cell lymphotropic viruses in subjects at high risk for HIV in Monterrey]. / Anticuerpos anti-virus linfotrópicos de células T humanas en sujetos de alto riesgo para la infección por VIH en Monterrey.

HIV and HTLV-1 are retrovirus that can produce human disease. It is known that HTLV-1 is associated to the adult T cell leukemia and to the spastic tropical paraparesis. AIDS is now a pandemic infection and HTLV-1 has a high endemicity in the Caribbean region and Japan, whereas the south of the United States has a low endemicity. In Mexico there is little information on HTLV-1 incidence. In the present work we looked for anti HTLV-1 antibodies in one hundred persons that belong to the high risks AIDS population in the city of Monterrey, Mexico. We found that 93 sera were positive for anti HIV antibodies in a ELISA test and seven were negative. All 93 sera were also positive in the Western Blot assay. In the confirmatory test two out of the seven negative sera were classified as indeterminate and five as negative. We also included in this study 50 sera from healthy control volunteers that did not belong to the high risk AIDS population and resulted negative in the HIV and HTLV-1 test. Anti HTLV-1 antibodies were determined by using an agglutination test with gelatin particles covered with HTLV-1 and confirmed by a Western Blot assay. We found that only three sera resulted positive in this agglutination test, but were negative by the Western Blot technique.

[Antigens of the human immunodeficiency virus in serum of high risk people in the city of Monterrey]. / Antígenos del virus de inmunodeficiencia humana en el suero de personas del grupo de alto riesgo de la ciudad de Monterrey.

One hundred forty human sera distributed in 2 groups were analyzed. In group I, 50 serum samples from healthy individuals that did not belong to the high-risk acquired immune deficiency syndrome (AIDS) population were included. In group II, there were 90 individuals, most of whom were apparently healthy but were at high risk of getting AIDS through their life styles or by transfusion. Of the 90 persons, 5 had a clinical picture of AIDS. All sera were analyzed by the enzyme - linked immunosorbent assay (ELISA) for the anti VIH antibodies. The positive cases were confirmed by the Western blot assay. In all samples the presence of the human immune deficiency virus antigens was sought. The results showed that the 50 healthy individuals (control group) were negative for both HIV antigens and antibodies. Of the 90 sera for the high-risk group, 50 were negative for antibodies, and 2 of them (4%) were positive for HIV antigens. Forty sera were positive for anti HIV antibody and among them, 5 patients were diagnosed as AIDS, and showed positive for antigen and antibody. The other 35 patients were all positive for HIV antibody and in 8 of them HIV antigen was also present.

[Anti-Nocardia brasiliensis antibodies in patients with actinomycetoma and their clinical usefulness]. / Anticuerpos anti-Nocardia brasiliensis en pacientes con actinomicetoma y su utilidad clínica.

Anti-Nocardia brasiliensis antibodies quantification and its clinical utility was confirmed in this study. A protein cellular extract from a N. brasiliensis strain named HUJEG-1 and registered at the ATCC # 700358 was used in a western blot assay to identify the immunodominant antigens. The protein P24 was selected to set up an ELISA test because it exhibit no cross-reaction with sera from tuberculosis and leprosy patients. A purified protease was also used as antigen in the ELISA test to compare its utility. Sera from N. brasiliensis mycetoma persons gave absorbance values above 0.3 when the disease was active using the P24 as antigen, these values decreased after patients completed their medical treatment. Anti-protease antibodies showed great variation and absorbance values similar to the healthy controls. We confirmed the clinical usefulness of the ELISA test both in serodiagnosis and in assessing the response to medical treatment. This is the first sensitive and specific serologic test for routine clinical laboratory.

[Purpura fulminans. Report of a case]. / Púrpura fulminante. Aportación de un caso.

A case of fatal purpura fulminans is reported. The etiology and the pathogenesis is discussed. We stress the need for an early diagnosis and treatment based on the use of heparin, fresh frozen plasma and platelets.

[Chronic mucocutaneous candidiasis. Cutaneous expression of immunologic anomalies. Report of a case]. / Candidiasis mucocutánea crónica. Expresión cutánea de anomalías inmunológicas. Reporte de un caso.

A case of an 8 year old girl with granulomatous candidiasis and impaired cell mediated immunity, is presented. The pathogenesis, associated diseases and the treatment of chronic mucocutaneous candidiasis are discussed.

In situ detection and distribution of inflammatory cytokines during the course of infection with Nocardia brasiliensis.

Actinomycetoma, caused by the intracellular bacterium Nocardia brasiliensis, is characterized by an infiltration of several inflammatory cell populations. To explore aspects of the immune response in the pathogenesis of these bacteria we injected 10(6) CFU in footpads of BALB/c mice. After 1, 2, 3, 4, 7, 30 and 90 days immunohistochemistry was performed to compare presence and distribution of the inflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IFN-gamma, IL-4, IL-10, and TGF-beta. Analysis of serial paraffin tissue sections showed strong participation and differences in distribution of cytokine-producing cells during the course of infection. Several TNF-alpha immunoreactive lymphocytes of the dermis were present during the course of the infection, but absent in the site of inflammation. During the first 4 days, IL-1 beta immunoreactivity was observed in dendritic epidermal cells and in cells surrounding the neutrophils around the grain. In later stages of infection, immunoreactive cells to this cytokine were mainly in the periphery of the microabscesses. Strong immunoreactivity was observed with IL-6 during the course of infection. Some cells in the epidermis and dermis, as well as muscle cells and several cells at the periphery of the microabscesses, showed strong IL-6 immunoreactivity. Cells immunoreactive to IL-4, IL-10, IFN-gamma and TGF-beta were present at the site of infection and, in later stages, in cells at the periphery of the microabscesses. In conclusion a mix of proinflammatory and antiinflammatory cytokines are produced at the same time by host cells. According to their distribution, inflammatory cytokines seems to have different functions during the course of infection with the intracellular bacterium N. brasiliensis.

Experimental autoimmune uveitis in the athymic nude rat.

A single injection of the retinal soluble antigen (S-Ag, 30 micrograms) to the Lewis or the heterozygous (rnu/+) rats induces a severe bilateral uveitis, characterized initially by infiltration of the retina with inflammatory cells. The athymic nude rat (homozygous rnu/rnu), which lacks the thymus gland and T cell-mediated functions, does not develop ocular inflammatory disease, clinically of histologically, after repeated challenges with S-Ag. Circulating anti-S-Ag antibodies were found in S-Ag-immunized Lewis rats and in the heterozygous, but not the athymic nude rats. Good proliferative responses to concanavalin A, S-Ag and purified protein derivitive of tuberculin (PPD) were found in lymphocyte cultures prepared from the draining lymph nodes of immunized heterozygous rats, but not when lymphocytes from the athymic nude rats were used. Uveitis could be induced in the athymic nude rat when lymphocytes from S-Ag-immunized heterozygous rats were transferred to them. By stimulating the donor lymphocytes in vitro with S-Ag before transfer, the number of recipients that developed uveitis was increased. On the other hand, it was impossible to transfer disease with hyperimmune serum alone. The possible role of T lymphocytes in the induction of experimental autoimmune uveitis with S-Ag is discussed.

Enzyme-linked immunosorbent assay for serological diagnosis of Nocardia brasiliensis and clinical correlation with mycetoma infections.

We previously identified three immunodominant antigens obtained from a Nocardia brasiliensis cell extract and recognized by sera from mycetoma patients (M. C. Salinas-Carmona, L. Vera, O. Welsh, and M. Rodríguez, Zentralbl. Bakteriol. 276:390-397, 1992). In the present work, we obtained a crude extract from a mass culture of N. brasiliensis HUJEG-1 and purified two immunodominant antigens, the 26- and 24-kDa proteins, by using simple physiochemical techniques. With these antigens, we developed a conventional solid-phase enzyme-linked immunosorbent assay and tested 30 serum samples from mycetoma patients, 29 from tuberculosis patients, 24 from a leprosy group, and 31 from healthy individuals. Our results show for the first time statistically significant differences in serology among these groups. All mycetoma patients with a positive culture for N. brasiliensis had absorbance values higher than 0.3. On the other hand, the mycobacterium-infected patients as well as the healthy individuals all had absorbance values below that level. Moreover, we found a close correlation between the clinical condition of the mycetoma patients and the anti-26- and anti-24-kDa protein antibody concentrations. We therefore propose the use of this assay in routine clinical laboratories to confirm the diagnosis of N. brasiliensis infection in human mycetoma cases. In addition, the possible application of this assay in the serodiagnosis of Nocardia asteroides infection is also discussed.

Isolation and purification of two immunodominant antigens from Nocardia brasiliensis.

Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed.

Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis.

We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.

Antibody response to Nocardia brasiliensis antigens in man.

A crude extract from N. brasiliensis cells grown in brain heart infusion culture was analyzed. It showed a complex mixture of at least 37 bands when resolved with the discontinuous buffer system of Laemmli in a gradient SDS-PAGE. Western blot analysis of 16 sera from N. brasiliensis-infected individuals always showed the recognition of six bands of 61, 49, 45, 42, 26, and 24 kilodaltons (kDa). Some other bands also reacted but with less intensity. Sera from tuberculosis and leprosy patients reacted strongly with the 49, 45, and 42 kDa bands but weakly or not at all with the 61, 26, and 24 kDa. Sera from healthy control volunteers reacted with some bands but little or not at all with those three identified by the sera from mycetoma patients. These three immunodominant antigens (61, 26 and 24 kDa) may be of clinical value in the serodiagnosis of mycetoma by N. brasiliensis.

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

Immune response to Nocardia brasiliensis antigens in an experimental model of actinomycetoma in BALB/c mice.

Nine- to twelve-week-old BALB/c mice were injected in footpads with 10(7) CFU of a Nocardia brasiliensis cell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-gamma increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.