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A qPCR identification scheme to detect the most common causative agents of actinomycetoma in Africa.

Mycetoma is a neglected tropical disease characterized by mutilating tumorous lesions in the subcutaneous tissue. The causative agents are found embedded in granules called grains. Mycetoma is either caused by bacteria (actinomycetoma) or fungi (eumycetoma). To initiate the appropriate treatment, it is important to identify the causative agent rapidly and molecular identification for eumycetoma revolutionized the time to identification. For actinomycetoma this was not possible yet. Here we developed a multiplex qPCR identification scheme for the most common causative agents of actinomycetoma in Africa. Whole genome sequencing was used to identify species-specific gene families for Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis. qPCR primers and probes were developed on these species and validated against DNA isolated from mycetoma strains and grains. Each probe was unique with no cross-reactivity with other tested species. The limit of detection ranged from 0.000013 to 0.00067 ng bacterial DNA. When the qPCRs were validated against 28 grain samples, all fungal grains remained negative and 11 out of 12 Actinomadura grains were correctly identified. This resulted in a sensitivity of 85.7% for the A. pelletieri probe and a specificity of 100%. For the A. madurae probe, a sensitivity and specificity of 100% was obtained. The actinomycetoma qPCR developed in this study can be used to identify the most common causative agents of actinomycetoma in Africa.

Mycetoma↗

Perianal actinomycetoma experience of 20 cases.

BACKGROUND: Actinomycetoma is a chronic infection resulting from aerobic Actinomycetes. The major agents are Nocardia brasiliensis, Actinomadura madurae, and Streptomyces somaliensis. The most frequent topographies are the lower and upper limbs. The prognosis of this disease is determined by several factors, such as etiologic agent, clinical topography, and depth of disease (degree of involvement, visceral, and bone affection). The purpose of this paper was to present our experience with actinomycetoma of the perianal region. METHODS: This study comprises 20 cases of perianal actinomycetoma, all of which were clinically and microbiologically proven by direct examinations, cultures, and biopsies. Clinical responses to the two principal treatment regimes used [combination of trimethoprim-sulfamethoxazole (TMS/SMX) and diaminodiphenylsulfone (DDS) or amikacine plus TMS/SMX] are reported. RESULTS: Most of the cases were male (17/20, 85%), the mean age was 42.1 years, and the farmers predominated (90%). The principal etiologic agent isolated was N. brasiliensis (85%). CONCLUSIONS: Perianal actinomycetoma is a rare entity. Differential diagnosis with anal sinuses, hydroadenitis, and cutaneous tuberculosis must be made in endemic areas by performing mycologic tests and biopsies. Treatment depends on the etiologic agent involved and the patient's condition.

Actinomycetales Infections↗

Actinomycetoma in the United States.

Actinomycetoma (Madura foot) caused by Actinomadura madurae occurred in an Indiana factory worker. Previous cases of culture-proven actinomycetoma from the United States and reports of drug therapy were reviewed. Treatment with sulfonamides, streptomycin, dapsone, and other antimicrobial agents has been effective. Trimethoprim-sulfamethoxazole (TMP-SMZ) therapy was effective in our case, but sulfadiazine was not. In vitro, SMZ was 16 times more active against the infecting strain of A. madurae than was sulfadiazine, and TMP was inactive, suggesting that our patient's satisfactory treatment might have been due to SMZ alone. Prolonged therapy is usually necessary. Relatively simple immunologic procedures and antimicrobiol susceptibility tests have been useful in the diagnosis and management of actinomycetoma. With appropriate antimicrobial therapy, surgical excision or amputation usually can be avoided. Recommendations for the medical management of actinomycetoma are summarized.

Actinomycetaceae↗

Treatment of actinomycetoma due to Nocardia spp. with amoxicillin-clavulanate.

BACKGROUND: Actinomycetoma is a chronic occupational condition that occurs frequently in tropical regions. In Mexico 85% of cases are caused by Nocardia brasiliensis. There are two treatments of choice for these cases: a regimen of dapsone plus trimethoprim-sulfamethoxazole (co-trimoxazole) and, recently, amikacin, either alone or combined. However, not all cases respond properly to these therapies. OBJECTIVES: To report a retrospective, 11-year study of cases of actinomycetomas caused by Nocardia spp., treated with amoxicillin-clavulanate (co-amoxiclav). METHODS: All cases were identified clinically and microbiologically and had previously failed standard therapies. Oral co-amoxiclav 875/125 mg was administered every 12 h. Clinical, microbiological and laboratory follow up was performed every 2 months during the treatment period. RESULTS: Twenty-one cases of actinomycetoma were included, 19 caused by N. brasiliensis and one each by N. asteroides and N. otitidiscaviarum. Clinical and microbiological cure occurred in 15 of 21 cases (71%); two cases improved (10%) and four failed (19%). Mean treatment period was 9.6 months, during which neither side-effects nor laboratory test alterations were reported. CONCLUSIONS: Treatment with co-amoxiclav represents an alternative or rescue treatment for cases that have previously failed standard therapies.

Adult↗

[Immunoglobulins in patients with Nocardia brasiliensis actinomycetoma].

Considering that some authors have reported an increasing of some immunoglobulins in actinomycetoma patients, in this study we propose to determine differential production of IgG1, IgG2, IgG3, IgG4 and IgGM in 25 patients with actinomycetoma and 25 healthy individuals from a mycetoma endemic area. Immunoglobulins were determined by ELISA technique. To sensibilize the plates, six Nocardia brasiliensis antigens were used: a crude antigen denominated NB and five derivatives (NB2, NB4, NB6, NB8 and NB10) obtained by their isoelectric point. Results showed that all IgG subclasses were higher in the patients' sera than in control sera, with a maximal difference to IgG3 and IgG4. To the latter subclass, six antigens were highly reactives. IgM levels were similar in both groups. As it occurs in other infections, in the actinomycetoma pathogenesis probably participate the increase or deficiency of a determined immunoglobulin class, as well as the relationship between different subclasses.

Adult↗

Cytokine production and lymphocyte proliferation in patients with Nocardia brasiliensis actinomycetoma.

IFN-gamma, TNF-alpha, IL-4, IL-10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensis and in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensis crude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosis were used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-gamma production was higher in the healthy controls than in the patients, whereas TNF-alpha secretion was slightly higher in the patients' cultures. IL-4 was detected in the patients' cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-gamma production, and higher concentrations of IL-4, IL-10 and TNF-alpha in the patients' PBMC cultures, indicating that they probably have a Th2 type of immune response.

Adolescent↗

Experimental production of actinomycetoma in BALB/c mice.

Chronic actinomycetoma associated with grain production was induced in BALB/c mice by subcutaneous inoculation of live Nocardia brasiliensis in Freund incomplete adjuvant into the hind footpads. Similar inoculation of N. asteroides and N. caviae resulted in local tumor formation which healed spontaneously after 5 months, the disease disseminating into the peritoneum, where masses or organisms could be detected. Grains were recovered from superficial skin lesions of N. caviae, but not from the N. asteroides-infected mice. Mycetoma lesions, appearing as early as 1 month after inoculation of 1.2 X 10(7) colony-forming units of N. brasiliensis per ml or as late as 3 months with inoculation of 1.0 X 10(5) colony-forming units per ml, became persistent and were readily detectable even 6 months after inoculation. No spontaneous healing occurred, and grains were recovered at different stages of the disease. Saline suspensions of N. brasiliensis also produced typical mycetoma lesions, although the incubation period was ca. 6 months. Adjuvant addition appeared to accelerate the onset of the disease. Experimental production of actinomycetoma in laboratory animals allows the study of many unanswered aspects of the disease and also provides a suitable model for therapeutic trials in the search for new and more effective chemotherapeutic agents.

Animals↗

[Combined cefotaxime and amikacin for immunomodulation in the treatment of actinomycetoma resistant to conventional treatment].

Actinomycetoma is a chronic disease that affects subcutaneous tissue. We present a case of a patient with abdominal actinomycetoma caused by Nocardia brasiliensis resistant to different treatments over several years, who also presented phagocyte immunodeficiency. He received two cycles (23 day cycles) of cefotaxime, 1 g every 8-h, and amikacin, 500 mg every 12 hours. Immunomodulation was carried out with levamisole 300 mg per week, during 4 weeks and bacterial antigen (at a concentration of 600,000,000 bacteria per mL), twice for a week during 20 months. The importance of susceptibility testing and immunological function investigation in this type of patients is discussed.

Abdominal Muscles↗

[Actinomycetomas in Senegal: study of 90 cases].

Mycetoma is a pathological process during which eumycetomic (fungal) or actinomycotic causative agents from exogenous source produce grains. Medical treatment is available for actinomycetomas and surgery is still the main treatment for eumycetoma. We report 90 cases of actinomycetoma occuring in male adult patients coming mainly from central Senegal. Patients living far from health structures consult after a long evolution period for tumors and pains which prevent them from carrying out their activities. The three etiological agents in our patients were Actinomadura pelletieri (60 cases), Actinomadura madurae (25 cases) and Streptomyces somaliensis (5 cases). The three clinical features are inflammatory forms (75 cases) mainly due to Actinomadura pelletieri, tumoral forms (13 cases) and cystic forms (2 cases). Lesions are localized on the foot in 50% of cases and in other part of the body for the other half. Bone damage was observed in 55% of cases. 83% of the patients were cured after a one-year treatment of sulfametoxazole adminstered orally. Two patients died of visceral involvement.

Actinomycosis↗

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.

Actinomycetales↗

Investigation of the temporal humoral immune response in a murine model of actinomycetoma.

The murine model of actinomycetoma offers the potential of studying many unknown aspects of this infection. In this work, the model was used to investigate the temporal humoral immune response to actinomycetoma agents. Groups of 7- to 9-week-old female BALB/c mice were inoculated in one of the hind footpads with one of four different Nocardia strains. To mimic the constant exposure of infected humans to the virulent soil inhabiting agents, a second injection consisting of live nocardiae in incomplete Freund's adjuvant was administered five months after the first one. Murine serum samples were collected throughout the study and their IgM and IgM titers were determined by ELISA and the Western blot assay. The results obtained indicate that the ELISA titers increased as the infection progressed and this correlated with a greater number of antigen bands being recognized in the blots. Overall, however, the ELISA titers were lower for the N. brasiliensis infected mice than those of the N. asteroides ones. This observation may be indicative of an immunosuppressive state and is worthy of further investigation.

Animals↗

Lymphocyte subsets, macrophages and Langerhans cells in actinomycetoma and eumycetoma tissue reaction.

The aim of this work was to demonstrate, quantify and compare cell elements in the inflammatory infiltrate of 23 skin lesions of actinomycetoma (ACM) and 17 of eumycetoma (EUM). Epidermal Langerhans cells (LC) population was also analyzed in 18 ACM, 13 EUM and ten normal skin samples as control group. Tissue response in both groups of mycetoma showed CD4 and CD8 T lymphocytes surrounding the neutrophils aggregates with macrophages, revealed by CD68 antibody, among them. B lymphocytes were not identified. ACM lesions showed a higher number of CD8+ lymphocytes (P=0.02) and macrophages (P=0.01) when compared with EUM lesions. As well as morphologically altered, displaying irregular and short dendritic processes, LC were depleted both in ACM and EUM lesions (P=0.0004) when compared with normal skin but no difference between both types of mycetoma (P>0.05) was found. Results suggest that cellular mediated immunity may play a role in mycetoma pathogenesis. The morphological alterations and marked reduction of LC in mycetoma lesions might reflect a depressed cellular immune response, partially explaining the chronic course and unresponsiveness to treatment of this group of diseases.

Adult↗

The immunopathology of actinomycetoma lesions caused by Streptomyces somaliensis.

The immune responses in actinomycetoma lesions caused by Streptomyces somaliensis in Sudan were characterized by immunohistochemistry during 1997-1998. In sections stained with haematoxylin and eosin, the inflammatory reaction around the grain was of 2 types. In type I there were 3 zones: a neutrophil zone immediately around the grain, an intermediate zone containing mainly macrophages, and a peripheral zone consisting of lymphocytes and plasma cells. Zone 1 stained positively for CD15 (neutrophils), zone 2 for CD68 (macrophages) and CD3 (T lymphocytes), and zone 3 for CD20 (B lymphocytes). In the type II reaction, there was no neutrophil zone, the grains being surrounded only by macrophages and giant cells. This was confirmed by immunohistochemistry, which demonstrated the presence of CD3 positive cells. Immunoglobulins G and M and complement were demonstrated on the surface of the grain and on filaments inside the grain. Neutrophils and macrophages were recruited into the lesion by complement and were involved in the fragmentation of the grain. The cytokine profile in the lesion and regional lymph nodes was of a dominant Th2 pattern (interleukins-10 and 4).

Actinomycetales Infections↗

Actinomycosis, nocardiosis, and actinomycetoma.

Cases of actinomycosis, nocardiosis, and actinomycetoma are seen infrequently but consistently in the United States. When they are found, diagnosis can be difficult because of their resemblance to other bacterial, mycobacterial, and fungal infections. This article reviews the clinical presentation, therapy, and laboratory diagnosis of these actinomycetales.

Actinomyces↗

Comparison of antigens from agents of actinomycetoma by immunodiffusion and electrophoresis procedures.

Antigenic extracts were prepared from culture filtrates of the principal agents known to cause actinomycetoma, namely Actinomadura madurae, Actinomadura pelletieri, Nocardia asteroides, Nocardia brasiliensis, Nocardia otitidis-caviarum, and Streptomyces somaliensis. These antigenic preparations were compared by immunodiffusion (ID), counterimmunoelectrophoresis (CIE), line immunoelectrophoresis (LIE) and rocket line immunoelectrophoresis (RLIE), with rabbit antisera prepared against each of the extracts. Cross-reactivity between antigenic extracts from the different actinomycetes, measured by determining the number of precipitin lines in homologous and heterologous systems, was common. Reactions were always stronger and more precipitin lines were present when antigenic extracts were tested against homologous antisera. Similarities between A. madurae and A. pelletieri antigens were of a low order and cross-reactivity did not exceed 33%. A. pelletieri resembled N. asteroides more closely than A. madurae, with 44% of detectable antigenic components cross-reacting. The three species of Nocardia had common antigenic epitopes, but the overall degree of similarity was of a low order (between 12 and 27% by LIE and RLIE). Antigenic extracts of S. somaliensis had few components in common with the other species tested and only one of the 34 lines present in the RLIE system for N. asteroides showed any reaction of identity with an antigenic component in the S. somaliensis extract. Single cross-reacting lines were also present in the CIE and LIE systems.

Actinomycetales Infections↗

Amoxicillin and clavulanic acid in the treatment of actinomycetoma.

BACKGROUND: Mycetoma is a relatively frequent disease in tropical countries. Drugs commonly used need a long period of treatment, and some cases are resistant to these drugs, especially those with bone or visceral involvement. The combination of amoxicillin-clavulanic acid has shown effectiveness against strains of Nocardia brasiliensis in vitro. METHODS: We have used this combination in two cases of mycetoma caused by N. brasiliensis, both with bone involvement and resistance to the drugs usually used. A dose of three tablets a day for 5 to 6 months was given (each tablet contains 500 mg of amoxicillin and 125 mg of clavulanic acid). RESULTS: Clinical and mycologic amelioration was observed in the two cases after completing the treatment. There were no side effects or relapses after 3 to 6 months of follow-up after treatment. CONCLUSIONS: We propose this antibiotic as a new option for the treatment of some special cases of actinomycetoma with bone or visceral involvement or resistance to the drugs commonly used in this disease.

Amoxicillin↗

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.

Animals↗