Evaluation of the Combined Therapy of DA-7218, a New Oxazolidinone, and Trimethoprim/ Sulfamethoxazole in the Treatment of Experimental Actinomycetoma by Nocardia brasiliensis.

OBJECTIVES: Currently, for actinomycetoma, combined antimicrobial therapy is preferred to the use of a single compound. This is in order to provide a broader-spectrum coverage due to a combinatory or synergistic effect between the drugs, and to decrease the possibility of emergence of natural resistant strains. A new oxazolidinone pro-drug, DA-7218 [(R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-2-oxo-5-oxazolidinyl) methyl-disodium-phosphate] (recently re-named TR-701), has shown very good in vitro and in vivo activities against several gram-positive bacteria including Nocardia spp. METHODS: In the present work we evaluated the effect of DA-7218 at two different doses, alone and combined with trimethoprim/ sulfamethoxazole (SXT), in an experimental Nocardia brasiliensis actinomycetoma murine model. We also included a negative and a positive control group (linezolid and saline solution respectively). RESULTS: At the end of the treatment period, we observed a clinically and statistically significant difference among the drug receiving groups (combined, alone and linezolid) and the control group (P=0.004). The difference was higher (P= 0.004) between the groups receiving DA-7218 (25mg/kg) alone or combined with SXT, and the control group (saline solution). CONCLUSIONS: In this work we proved that DA-7218 alone and combined with SXT is effective in the treatment of experimental actinomycetoma by Nocardia brasiliensis and that it could be potentially useful in the treatment of human actinomycetoma.

In vitro activity of PNU-100766 (linezolid), a new oxazolidinone antimicrobial, against Nocardia brasiliensis.

The in vitro activity of a novel oxazolidinone, linezolid, was studied by comparing the activity of linezolid with those of amikacin, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid against 25 strains of Nocardia brasiliensis isolated from patients with mycetoma. All N. brasiliensis strains tested were sensitive to linezolid (MIC at which 90% of strains are inhibited [MIC(90)], 2 microg/ml; MIC(50), 1 microg/ml). This antimicrobial might constitute a good alternative for treatment of actinomycetoma.

Phospholipase region of Mycobacterium tuberculosis is a preferential locus for IS6110 transposition.

Enzymes with phospholipase C activity in Mycobacterium tuberculosis have been recently described. The three genes encoding these proteins, plcA, plcB, and plcC, are located at position 2351 of the genomic map of M. tuberculosis H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the plcA and plcB genes in M. tuberculosis clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic M. tuberculosis clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the plcA and plcB genes and part of the plcC gene. In the M. tuberculosis strains studied the production of an amplicon approximately 1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS6110 element. We observed insertion elements in the plcA, plcB, and plcC genes. One site in plcB had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in plcA in the same nucleotide position. In all the cases, IS6110 was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS6110 elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included plcA and most of plcB. This can explain the negative results obtained by some authors when detecting the mtp40 sequence (plcA) by PCR. Given the high polymorphism in this region, the use of the mtp40 sequence as a genetic marker for M. tuberculosis sensu stricto is very restricted.

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.

A new therapeutic approach to type II leprosy reaction.

BACKGROUND: An increase in tumor necrosis factor (TNF) has been implicated in type II leprosy reaction. Thalidomide, which inhibits TNF, is an effective drug, but has severe side-effects in pregnant women. Other therapeutic drugs are required. METHODS: Clofazimine and pentoxifylline were evaluated for their efficacy against severe type II leprosy reaction in four patients (three men and one woman). RESULTS: All four patients showed a similar fast response to treatment. CONCLUSIONS: The results obtained in this study are promising; however, clofazimine and pentoxifylline must be evaluated in a larger group of patients in order to determine their value in controlling type II leprosy reaction.

Treatment of eumycetoma and actinomycetoma.

Mycetoma is a chronic disease caused by aerobic actinomycetes and eumycetes which mainly affects the lower extremities. It predominates among farm workers in tropical, subtropical and adjacent zones. Clinically it is characterized by a firm swelling with abscesses and fistulae discharging pus that contains granules or grains of the causal agent. Their color, size, consistency and histopathology contribute to their identification. Cultures and metabolic studies determine the disease's etiology. Eumycete and actinomycete antigens can be used serologically to diagnose and predict prognosis of the disease. Many different antimicrobials and antifungal drugs have been used with varying degrees of success. Trimethoprim-sulfamethoxazole alone or together with diamino-diphenyl-sulfone is the treatment of choice for actinomycetoma. Amikacin is used for severe cases, unresponsive to previous treatment, and for those in danger of dissemination to adjacent organs. Surgery is seldom used for actinomycetoma. In eumycetoma a combination of medical treatment and surgery is advised. Small eumycetomas are easily surgically removed. Ketoconazole at a dosage of 400 mg/day is the medical treatment of choice for eumycetoma caused by M. mycetomatis. The therapeutic response to itraconazole varies. Fluconazole has been unsuccessful in the treatment of eumycetoma but amphotericin B has shown good to poor therapeutic response.

Mycetoma.

Mycetoma is a chronic granulomatous disease caused by bacteria or true fungi. It affects the skin the underlying tissue and sometimes subjacent bones and organs. The diagnosis of the disease is confirmed by the microscopic identification and by isolating the infecting agent. Actinomycetoma are treated with trimethoprim-sulfamethoxazole and/or diamino-diphenyl-sulfona (DDS). In patients resistant to these treatments adding amikacin cures about 95% of the resistant cases. In true fungi mycetoma, amphotericin B, ketoconazole, itraconazole, and in some cases these combined with surgery is the treatment of choice.

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.

Once-weekly oral doses of fluconazole 150 mg in the treatment of tinea corporis/cruris and cutaneous candidiasis.

Ninety-five adult out-patients with tinea corporis and/or tinea cruris participated in a multicentre open non-comparative study investigating the safety and efficacy of 1-4 once-weekly doses of oral fluconazole 150 mg. Trichophyton rubrum was isolated most frequently (67 of 86 mycologically evaluable patients). A mean of 2.6 doses of fluconazole was administered; patients infected with Candida albicans or Epidermophyton floccosum required an average of 2 doses compared to 3-4 doses in patients infected with other organisms. Clinical cure was obtained in 85 of 92 (92%) patients at the last post-treatment evaluation, with the remaining seven patients being substantially improved. At long-term follow-up, 28-30 days after the last dose, 80 of 91 (88%) patients were assessed as clinically cured, three (3%) patients were improved and eight (9%) patients failed. Among the long-term clinical failures, there was one diagnosis of tinea corporis (3% failure rate) and seven diagnoses of tinea cruris (12% failure rate). Mycological evidence of infection occurred in only 1 of 86 patients assessed at the last post-treatment follow-up. Mycological relapse occurred in nine (11%) patients at long-term follow-up; one patient was infected with Trichophyton mentagrophytes and eight patients were infected with T. rubrum. Relapse occurred in 2 of 29 (7%) patients with tinea corporis and eight of 57 (14%) patients with tinea cruris (one patient who relapsed had both tinea corporis and cruris). There was no correlation between the number of doses received and the mycological response or relapse rates at long-term follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

[Epidemiology of mycetoma in Mexico: study of 2105 cases]. / Epidemiología del micetoma en México: estudio de 2105 casos.

A survey was carried out in Mexico to determine the incidence and epidemiological characteristics of mycetoma. Data was collected from a total of 2105 cases of mycetoma throughout a 30 year period (1956-1985), with an average incidence of 70 cases per year. Results showed a sex distribution of 76.1% male and 23.9% females. Age distribution indicated a 35% between 16 to 30 and 23% between 31 to 40 year old population. Most cases occurred in land-workers (60.2%) and in housewives with rural residence (21.3%). Lesions occurred most frequently in lower limbs (64.1%), trunk (17.4%) and upper limbs (13.6%). The geographic distribution within Mexico revealed that the States with the highest incidence were: Jalisco, Nuevo León, San Luis Potosi, Morelos and Guerrero The predominant etiologic agents found 97.8% corresponded to actinomycetes, from which Nocardia brasiliensis (86.6%) and Actinomadura madurae (10.2%) showed the higher frequency. Eumycetoma (2.2%) was due to Madurella grisea and M. mycetomatis in most cases.

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.

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.

Doses unicas semanais orais de fluconazol 150 mg no tratamento de tinea corporis/cruris e candidiase cutanea / Doses weekly only you pray of fluconazol 150 mg in the treatment of cutaneous tinea corporis/cruris and candidiasis

Noventa e cinco pacientes ambulatoriais com tinea corporis e/ou tinea cruris participaram de um estudo multicentrico nao comparativo aberto para investigar a seguranca e eficacia de 1-4 doses unicas semanais de fluconazol oral na dose de 150 mg. O trichophyton rabrum foi o organismo mais frequentemente isolado (67 de 86 pacientes avaliados micologicamente). Uma media de 2,6 doses de fluconazol foi administrada; pacientes infectados com Candida albicans ou Epidermophyton floccosum necessitaram, em media, de 2 doses enquanto foram necessarias 3-4 doses em pacientes infectados com outros organismos. A cura clinica foi obtida em 85 de 92 (92%) dos pacientes na ultima avaliacao depois do tratamento, tendo os sete pacientes restantes melhorado substancialmente. No seguimento a longo prazo, 28-30 dias apos a ultima dose, 80 de 91 (88%) pacientes foram considerados clinicamente curados, tres (3%) apresentaram melhora e oito (9%) tiveram insucesso terapeutico. Dentre os fracassos clinicos a longo prazo, houve um diagnostico de tinea corporis (3%) de taxa de insucesso) e sete diagnosticos de tinea cruris (12% de taxa de insucesso). Evidencias micologicas de infeccao ocorreram em apenas 1 de 86 pacientes seguidos ate o final do seguimento a longo prazo. Recidiva micologica ocorreu em nove (11%) dos pacientes do seguimeto a longo prazo; um paciente estava infectado pelo Trichophyton mentagrphytes e oito pacientes, pelo T. rubrum. Houve recidiva em 2 de 29 (7%) pacientes com tinea corporis e oito de 57 (14%) com tines cruris (um paciente que recidivou tinha tinea corporis e cruris). Nao se verificou correlacao entre o numero de doses recebidas e a resposta micologica ou as taxas de recidiva a longo prazo. O fluconazol foi bem tolerado; somente 5 de 95 pacientes tratados com fluconazol referiram efeitos adversos, um dos quais resultou em descontinuacao da terapia (urticaria moderada). A boa tolerancia comparada a dos outros antifugicos orais e a conveniencia de um esquema de dose unica semanal oral em comparacao aos tratamentos topicos e orais existentes tornam a dose unica oral semanal de fluconazol uma elternativa valiosa no tratamento da tinea corporis/cruris

Amikacin alone and in combination with trimethoprim-sulfamethoxazole in the treatment of actinomycotic mycetoma.

We report the excellent therapeutic response obtained with amikacin alone and in combination with trimethoprim-sulfamethoxazole in the treatment of 15 patients with actinomycotic mycetoma who had a poor response to the traditional pharmacologic agents and/or in whom important organs such as lungs, spinal cord, and bone were involved. We evaluated the results by clinical, radiologic, and laboratory tests. No important side effects were detected during or after the therapy.