Paracoccidiodes brasiliensis (Paracoccidiomycosis)

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Microbiology

Paracoccidioides brasiliensis is a dimorphic fungus. The yeast-like form of P. brasiliensis grows well in brain heart agar at 37°C. It appears as spherical cells of 5 to more than 30 µm in diameter, with a thick and well-defined refringent cell wall and several peripheral buds (the pilot well). The mycelial form of P. brasiliensis grows in Sabouraud dextrose-agar at 25°C. Microscopically, these colonies exhibit branched, septate, hyaline hyphae 2 to 3 µm in diameter, with numerous intercalated or terminal chlamiconidia. (12, 73, 89). Ultrastructural studies have shown that yeast cells of P. brasiliensis are multinucleated.

Cells are aploid and six different patterns of chromosomas banding have been found. Septa in the mycelial form are single pore, similar to those of the Ascomycotina division, but the sexual state of P. brasiliensis is unknown. Differences in pathogenicity and immunogenicity among P. brasiliensis strains have been detected. (16, 26, 28, 45, 73, 87, 90, 98).

Epidemiology

Paracoccidioidomycosis has a restricted geographic distribution. It is endemic in humid subtropical areas of Latin America, from Mexico to Argentina and Uruguay; (12, 30, 48, 69, 73). It has a high prevalence in Brazil, which accounts for 80% of reported cases, but is also observed in Colombia, Venezuela, Argentina and Paraguay. The habitat of P. brasiliensis is not well known; it probably lives in soil adjacent to rivers or lakes. Based on the results of paracoccidioidin skin tests and the distribution of paracoccidioidomycosis in children, the habitat of P. brasiliensis is presumed to be near the natural aquatic bodies and armadillo burrows, especially where the indigenous forest were replaced by Pimus and Eucalyptus (91, 92). P. brasiliensis has been isolated from nature: soil samples, bat and penguin feces, and dog food contaminated with soil (1, 7, 27, 34, 36, 65). Spontaneous infections of armadillo (62) and squirrel monkey (39) have been reported. The geographic distributions of armadillos is similar to paracoccidioidomycosis, but its role in the transmission of this mycosis is not clear (19, 97, 105). Animal to human and human to human transmission has not been confirmed (45). Human infections outside the endemic areas have been reported but all patients had visited or lived previously in an endemic area (12, 66).

Primary infection is probably acquired by inhalation of conidiae; skin and mucous membranes seem to be uncommon portals of entry (73). The primary pulmonary infection is usually asymptomatic or mild and is self-limited (paracoccidioidomycosis-infection). Subclinical infections occur frequently in healthy inhabitants of endemic areas as shown by skin reactivity to paracoccidioidin (80). Surveys of reactors to paracoccidioidin skin tests show that infections are acquired at an early age (2, 43, 107). P. brasiliensis has been found in fibrous or calcified nodules in the lungs or lymph nodes (3, 78). Outbreaks of primary infections, similar to those reported in histoplasmosis, have not been observed in paracoccidioidomycosis (91). Reactivation of latent pulmonary or lymph node foci leads to progressive disease that is usually severe. Paracoccidiomycosis is frequently observed in adult males, between 30 to 60 years of age and is more common in rural workers. The juvenile form of paracoccidioidomycosis is less prevalent, representing only 3 % of progressive cases, and attacks both sexes (48).

Clinical Manifestations

Paracoccidioidomycosis Infection

Paracoccidioidomycosis infection is defined by a positive paracoccidioidin skin tests. It is often asymptomatic or subclinical and self-limited. Few cases of acute pulmonary primary infection with spontaneous resolution have been described (73). Calcifications of the lungs and lymph nodes are infrequent. The asymptomatic latency period between infection and clinical symptoms may last several years (48).

Paracoccidioidomycosis Disease

Paracoccidiomycosis is a granulomatous and suppurative disorder that primary involves lungs and then disseminates to other organs via lymphatics and blood streams (12, 66). Clinical cases that do not show a tendency to spontaneous regression are included under this classification (29).

Subacute Juvenile Form

This clinical entity affects children and adolescents of both sexes and probably results from rapid dissemination of a primary infection due to severe immunologic failure. Its course is subacute with marked deterioration of the overall general condition, with fever, anorexia, loss of body weight, subcutaneous abscesses generalized lymphadenopathy and hepatosplenomegaly. Skin, bone and gastrointestinal tract can be involved. Mucous membrane and lung involvement are uncommon (32, 48, 53).

Chronic Adult Form

The chronic adult form affects adults above 30 years of age, mainly men. The infection is chronic with a predominance of respiratory symptoms. In 25% of cases infections is localized to the lung, with manifestation of a chronic pulmonary disease (45, 84). Multifocal presentation is observed in more than 70% of patients with skin, mucous membranes, larynx, lungs, lymph nodes, adrenal glands, liver, spleen, bones and central nervous system involvement. (30, 32, 45, 46, 69, 76, 101).

Laboratory Diagnosis

P. brasiliensis antigens elicit a specific immune response that determines antibody production as well as delayed type of hypersensitivity (14,69, 90). Over 90% of patients with the progressive forms of paracoccidioidomycosis have specific antibodies that may be detected by routine serologic tests: immunodiffusion in agar gel, counter immunoelectrophoresis and complement fixation. Titers in these tests are proportional to the severity of disease and generally decline after clinical remission. The enzyme-linked immosorbent assay (ELISA) discriminates better between progressive and remittent lesions (64, 83). Agar immunodiffusion and agarose counter immunoelectrophoresis are more specific than ELISA and immunoblotting (4, 24).

Paracoccidioidin skin test is a delayed hypersensitivity reaction to P. brasiliensis antigens. Positive results occur in healthy individuals who live in endemic areas, but more than 50% of patients with severe paracoccidioidomycosis disease have negative skin tests. These negative reactions revert to positive after treatment when clinical remission is achieved (69, 94).

The diagnosis of paracoccidioidomycosis is confirmed by finding typical yeast-like elements of P. brasiliensis in microscopic study of wet-mount preparations of pus, sputum or tissue specimens. Cultures should be grown in yeast extract-agar, Sabouraud dextrose-agar, brain-heart infusion-agar with antibacterial antibiotics; these cultures are incubated at 25°C and 37°C, for 4 weeks. P. brasiliensis grows slowly and its isolation is often difficult; thus as a diagnosis method, cultures are less sensitive than microscopic examination (45,69, 73). Clinical specimens can be inoculated intratesticularly in guinea-pigs and hamsters; after 2 or 3 weeks, pus from experimental lesions reveals characteristic multiple budding cells of P. brasiliensis.

Various specific primers have been used in PCR for the identification of P. brasilisensis by molecular biology techniques and have been applied for cultures and for the identification of this fungus in infected tissues (8, 10, 22, 35,38, 58).

In histologic sections P. brasiliensis is better visualized when special stains, such as P.A.S. or Grocott methenamine-silver, are used. Direct immunofluorescence with a specific conjugate is not used routinely (69). In severe cases, extensive suppurative areas with necrosis and soft granulomas are observed; conversely, in mild forms, typical compact epithelioid cell granuloma with giant cells and few fungi is seen (67, 99).

Pathogenesis

P. brasiliensis conidiae enter the lungs by inhalation and are engulfed by pulmonary macrophages. Inside the alveolar macrophages, conidiae turn into yeast-like form that causes a non specific inflammatory response. A primary inoculation complex is produced. Hematogenous spread occurs at this time with the formation of multiple metastatic foci, usually without clinical manifestations. A positive skin test to paracoccidioidin can be documented (30, 32, 59). Approximately two weeks after infection, granulomas are formed in the lung or lymph nodes with giant cells, plasmocytes and lymphocytes. Microabscesses are common and caseous necrosis can be observed in lymph nodes. Tissue repair occurs via collagen fibrosis (18, 95).

Two polar immunoclinical forms of paracoccidioidomycosis have been accepted (45, 61, 67, 109). The correlation between these two polar immunoclinical forms and the Th 1 and Th 2 types of cells mediated immunity response, has been demonstrated in murine experimental models of paracoccidioidomycosis (13, 14, 15, 17, 20, 40).

SUSCEPTIBILITY IN VITRO AND IN VIVO

It should be noted that in vitro susceptibility testing for P. brasiliensishas not been standardized.

Single Drug

Sulfonamide derivatives, amphotericin B, and azoles are active against P. brasiliensis. Sulfonamides were introduced in the treatment of paracoccidioidomycosis by Oliveira Ribeiro in 1940. These compounds exert a fungiostatic effect against P. brasiliensis in vitro (41). Sulfadoxine exhibited a good in vitro activity with MICs between 6.5 µg/ml and 12.5 µg/ml of culture medium (44). Sulfonamides were also effective in treated experimental paracoccidioidomycosis in guinea pig, but definitive cure depended on the immunologic response of the host (81). Sulfonamide’s serum concentrations should be maintained at about 50 µg/ml during treatment (57). When sulfonamides are combined with trimethoprim, serum concentration of free sulfa should also be monitored and doses should be adjusted to keep these levels above 50 µg/ml.

Amphotericin B has been used in treating paracoccidioidomycosis since 1958. This polyenic antibiotic is active in vitro against P. brasiliensis with MIC of 0.06 µg/ml to 0.2 µg/ml (44).

All azoles including miconazole, econazole, ketoconazole, itraconazole, fluconazole, saperconazole, Sch 39.304 and posaconazole have been effective in the treatment of experimental paracoccidioidomycosis in animal models (47, 54, 69, 70, 71, 72, 74, 93, 102). P. brasiliensis is highly susceptible to ketoconazole. Its MIC range from 0.01 and 0.001 µg/ml and its minimum fungicidal concentration (MFC) between 0.1 and 0.01 µg/ml; these results are not affected by the addition of 10% bovine serum (88). Antagonism of its antifungal action is observed when 40 µg/ml of rifampin is added; conversely, addition of amphotericin B is synergistic in vitro (88).

Itraconazole is very active both in vivo and in vitro against P. brasiliensis; MIC for the yeast form of the fungus varies from 0.01 to 0.001 µg/ml and the mycelial form is 10 fold less susceptible (102). Electron microscope studies have shown that when yeast cells of P. brasiliensis are exposed to 70 ng/ml of itraconazole for 24 hours, destruction of organelles is observed and development of mycelia is prevented. This triazole is 10 to 100 times more active in vitro than ketoconazole (11).

Fluconazole is minimally active in vitro, even against fungi for which itsin vivo antifungal action is very marked. However, both MIC and MFC were approximately 1.6 µg/ml in Sabouraud dextrose medium at pH 7 (71).

Espinel-Ingroff et al., used the standard susceptibility techniques for yeast-like fungi to determine MICs for saperconazole, itraconazole, fluconazole, ketoconazole and amphotericin B in two culture media: buffered (MOPS) RPMI-1640 and synthetic amino acid medium-fungal (SAAMF) (25). A macrodilution test was performed with both the yeast and mycelial forms of 10 isolates of P. brasiliensis and the fungi were incubated at 35°C (yeast form) and 30°C (mycelial form) for 72 hours to 7 days. Saperconazole and itraconazole were the most active antimicrobial agents in vitro, showing MIC90s below 0.03 µg/ml for both forms of this fungus; amphotericin B was less active than the azoles-MIC90 between 0.12 and 4.0 µg/ml. A third generation triazole, posaconazole, is also very active in vitro against P. brasiliensis with MIC50and MIC90 lower than 0.125 µg/mL and 1 µg/mL respectively (47).

Combination Drugs

The combination of amphotericin B and rifampin increases the antifungal efficacy of amphotericin B in vitro (108), while rifampin decreases the antifungal activity in vitro of azoles against P. brasiliensis.

In Vivo (Animal Models)

The effectiveness of ketoconazole in the treatment of experimental paracoccidioidomycosis has been demonstrated in mice, rats, hamsters and guinea pigs (70). In all of these animal models, ketoconazole (80-120 mg/kg/day for approximately 4 weeks) strikingly reduces the number of lesions. Ketoconazole is more effective in guinea-pigs and rats than in hamsters (60).

In experimental paracoccidioidomycosis, the efficacy of itraconazole has been shown in mice, rats, and guinea pigs (74, 89). In these two latter animals, biological cure was reached with doses of 8-10 mg/kg/day for 3 weeks (71). Itraconazole proved to be significantly more active than fluconazole in a comparative study of the antifungal action of triazoles in experimental paracoccidioidomycosis in Wistar rats. Fluconazole only decreased lesions markedly at doses of 25 mg/kg/day (54, 71). In experimental trials with mice, fluconazole was effective at doses of 100 mg/kg/day (100).

Sch 39, 304 and saperconazole have been effective in the treatment of experimental animal models of paracoccidioidomycosis (93, 103). Saperconazole has also been used effectively in humans (31). Unfortunately, research with these triazoles have been abandoned because of tumor formation in rats.

ANTIMICROBIAL THERAPY

Drug of Choice

The progressive forms of paracoccidioidomycosis are fatal without specific treatment (66). Treatment of paracoccidioidomycosis usually involves two stages: initial intense antifungal therapy and maintenance treatment to prevent relapses. Azole derivatives, amphotericin B and sulfonamides have been used for initial therapy (57). Itraconazole, first used in 1982 for paracoccidioidomycosis, is now the drug of choice (49, 63, 72, 104). The standard regimen is 100 mg/day, orally for 6 months. This drug should by administered once daily after a meal. In 278 patients treated with itraconazole, all showed clinical improvement, and only 2.1% experienced clinical relapses. Patients with relapses responded favorably to repeat treatment with itraconazole. Patients with the acute juvenile form showed therapeutic responses similar to those observed in the chronic adult form (49, 50, 56, 86, 96). Itraconazole seems to be remarkably nontoxic; only subclinical increases in hepatic enzymes were recorded in 14.2% of cases. Failures with itraconazole are rare, usually due to the failure of gastrointestinal absorption in patients with malabsorption. Malabsorption is more frequent in the acute juvenile form (48). Maintenance treatment is not necessary in patients who receive itraconazole (57).

The main problem in paracoccidioidomycosis treatment is that many patients fail to comply with the full course of therapy. These compliance failures are attributable to the cost of treatment, the low socioeconomic status of the patient, and cultural factors (57, 74).

Combination therapy is not usually employed when itraconazole is administered.

Special Situations

Malabsorption

If malabsorption is problematic, amphotericin B and cotrimoxazole are alternative antimicrobial agents that can be administered intravenously. Good results have been observed with both drugs. Intestinal lesions occasionally worsen after treatment because of the brosis the inflammatory process. These patients should be managed with fractionated oral diet or parental nutrition. Patients with intestinal malabsorption usually improve on a low-fat diet (57, 74).

Central Nervous System Involvement

Itraconazole is 99.8% protein bound, which impairs penetration across the blood-brain barrier. Concentrations of the drug in cerebrospinal fluid are negligible. However, itraconazole has been shown to be very effective in some mycoses of the CNS, both experimentally and in humans, probably because of its high affinity for the cerebral parenchyma (57, 74). Abscessed granulomas are the most frequent clinical manifestations of neuroparacoccidioidomycosis, and in these lesions, itraconazole is effective (106). In meningoencephalitis, amphotericin B (0.8 mg/kg/day IV), cotrimoxazole (2.400 mg/480 mg/day IV), or fluconazole (400 mg/day IV), have been used successfully. Sulfonamides have also been used sucessfully.Nobrega and Spina-Franca consider sulfonamides to be the first option in the treatment of lesions in the CNS, with amphotericin B reserved for those resistant or intolerant to sulfa drugs (76). Neurosurgical procedures are occasionally indicated, but antifungal drugs must always be used.

Adrenal Insuffiency

Adrenal insufficiency occurs in more than 30% of patients with disseminated paracoccidioidomycosis. These patients should receive 30 mg/day of hydrocortisone which is usually administered lifelong (57,70).

Underlying Diseases

Tuberculosis

Association with tuberculosis is observed in 10-12% of patients; frequency varies by geographic region (45, 67, 73). Rifampin and the azoles may be antagonistic in vitro so azoles are not given. Instead, cotrimoxazole oramphotericin B can be administered to these patients.

AIDS Patients

In Brazil only 44 patients with AIDS related paracoccidioidomycosis had been reported up to December 1999. The incidence of paracoccidioidomycosis in these patients was 0.02%, while the estimated incidence of histoplasmosis was 1.4% in Brazil; i. e, an estimated ratio of 52.7 cases of AIDS-associated histoplasmosis for one case of AIDS-related paracoccidioidomycosis. The relatively low frequency of paraccidioidomycosis in HIV-positive patients might be related to the routine chemoprophylaxis ofPneumocystis carinii pneumonia with cotrimoxazole and the frequent use of azoles for candidiasis and other fungal infections. In 74.3% of cases occurring in HIV patients, paracoccidioidomycosis was the first life-threatening complication. The clinical picture resembled the acute or subacute form of disseminated paracoccidiodomycosis: lung involvement (58.9%); lymphadenopathy (51.3 %); skin lesions (46.1%); hepatosplenomegaly (25.6 %) and mucous membrane involvement (23.1%).

Due to the severity of clinical manifestations and malabsorption in these patients, the majority of them were treated with amphotericin B alone or in combination with sulfadiazine or cotrimoxazole. Other cases receiveditraconazole or ketoconazole at higher doses than usual. The mortality was 23.1%, higher than the 5% observed in immunocompetent patients. Long term chemoprophylaxis with oral sulfonamides or azoles is recommended (52).

Alternative Therapy

Sulfonamide Derivatives

Sulfonamides have a fungistatic activity against P. brasiliensis, are inexpensive, and well tolerated orally. The success rate is over 60% (10). Two types of sulfonamides are given: rapidly eliminated sulfa drugs (e. g. sulfadiazine) and slowly eliminated sulfa drugs (e. g. sulfamethoxypyridazine). Sulfadiazine is given every 6 h orally and sulfamethoxypyridazine every 12 h orally. The daily dose of rapidly eliminated compounds is 3-5 g in adults or 0.15 g/kg in children while the slowly eliminated sulfonamides are given at a dose of 1 or 2 g/day (57, 89). Serum sulfonamide levels should be maintained above 50 mg/L. The treatment duration should be 3 years. Early discontinuation of therapy often results in resistance of P. brasiliensis to sulfonamides. Side effects are rarely observed. A few patients develop crystalluria with hematuria, which can be controlled by increasing the water intake or drinking bicarbonated water. Leukopenia, rash, fever, photosensitization and digestive disturbances are rarely reported (67, 73, 89).

Combination of sulfamethoxazole with trimethoprim (cotrimoxazole) has proved effective in the initial treatment of paracoccidioidomycosis (5, 45). This drug can be administered orally or intravenously. The preparation most often used contains 800 mg of sulfamethoxazole and 160 mg of trimethoprim. The usual dose is 1 tablet every 12 hours for 1 year. Cotrimazole does not appear to be superior to sulfonamides, but some patients who are resistant to sulfonamides have improved on cotrimoxazole. A combination of sulfadiazine and trimethoprim (cotrimazine) is used in Brazil and has proven effective in the treatment of neuroparacoccidioidomycosis. Dosages are similar to those of cotrimoxazole (6).

Sulfonamides may be used (a) for adult patients suffering chronic unifocal disease, with no previous therapy and in good overall condition; (b) when paracoccidioidomycosis is associated with tuberculosis and (c) as maintenance therapy after course of amphotericin B.

Amphotericin B

Amphotericin is effective, although side effects are frequent (23). This drug causes rapid regression of all lesions of paracoccidioidomycosis, but it does not prevent relapses. Therefore, it is usually indicated for use in severe cases and discontinuation when clinical remission is achieved.Sulfonamides are then given as maintenance therapy.

Amphotericin B is administered intravenously at an initial dose of 0.2 mg/kg/day, increasing up to 0.8 mg/kg/day until a total of 1.5 g is given. Sulfonamides should then be given at the usual dosage for 2 to 3 years. Combination therapy gives better results than administration of amphotericin B alone; relapses were reduced from 60 to 18 % of cases (23, 89).

Amphotericin B combined with rifampin was used in four patients who had not shown clinical improvement with amphotericin B alone. Rifampin was given at a daily dose of 600 mg and amphotericin B was administered at a dose of 25 or 50 mg, three times a week. Clinical remission of the disease was achieved after several months (108).

Amphotericin B should be used with caution and treatment should be monitored. The following laboratory tests should be done once a week: electrolytes, serum creatinine, blood urea nitrogen and urinalysis. Hemogram and electrocardiography may be performed every two weeks (57, 73).

Liposomal formulations of amphotericin B have been rarely used in paracoccidioidomycosis. Four patients suffering the juvenile form of paracoccidioidomycosis were treated with 3 mg/ kg/ day of amphotericin B colloidal dispersion for at least 28 days. Although, all the patients showed an initial clinical improvement, all of them relapsed within six months (21).

Ketoconazole

Ketoconazole has a good digestive absorption at acid pH, but its bioavailability decreases in patients who receive medication to reduce gastric acidity. Excellent results have been reported with over 90% clinical cure after 1 year of therapy. The drug is administered orally at a daily dose of 200 or 400 mg in adults or 5 to 8 mg/kg in children. Peak serum concentrations of 2 to 4 µg/ml are usually obtained with these doses (75). Relapses were observed in less than 10% of cases; patients who relapse can still be treated successfully with ketoconazole. Treatment failures have been documented in patients with low gastric acidity, those whose digestive absorption is impaired by lymphatic blockage in the acute juvenile type of the disease and patients who receivedrifampin (68, 75).

Treatment may be discontinued when the patient achieves complete clinical remission, serologic tests become negative or decrease to low titers, and paracoccidioidin skin tests become positive in those who displayed negative reactions before treatment.

Ketoconazole is well tolerated; side effects have been registered in less than 10% of patients, and they were usually minor. An asymptomatic increase of hepatic enzymes levels was reported in 8% of cases. Symptomatic hepatitis was unusual in paracoccidioidomycosis patients. Endocrine side effects (e. g., decreased libido and gynecomastia) were rarely observed, and although this drug interferes with corticosteroids synthesis, symptoms of hypoadrenalism were extremely rare. Endocrine effects have been reversible and dose dependent (69). Ketoconazole is an excellent alternative drug (but it is somewhat less effective than itraconazole). It is the cheapest treatment for paracoccidioidomycosis (85).

Fluconazole

This triazole has been used in few cases of paracoccidioidomycosis. It has excellent and rapid digestive absorption, independent of meals and gastric acidity. Peak serum concentrations of 6 to 10 µg/ml are frequently obtained 2 hours after administration of 200 mg. It may also be given intravenously. Its bioavailability is over 80%. Binding to plasma proteins is low, about 12%, which allows free circulation of the drug and easy passage through the blood-brain barrier. Levels in cerebrospinal fluid reach 80% of those in blood, but its tissue affinity is lower than that of itraconazole. Over 62% of this triazole is eliminated in its active form in urine and less than 20% is metabolized in the liver where it is transformed into inactive compounds. Steady state is achieved after 3 to 4 days and is influenced by renal function. With normal renal function, half-life is approximately 31 h (57).

Fluconazole has been used to treat 37 patients at a daily dose of 200-400 mg for at least 6 months. Significant improvement was seen in 34 cases, but one sudden death was observed. There was one failure and one patient could not be evaluated because of noncompliance. Eight patients received cotrimoxazole as a suppressive treatment after achieving clinical remission with fluconazole. Only 25 patients were followed for more than 6 months after treatment and only one relapse was observed. No side effects were registered in this group of patients (74).

Both in vitro studies and treatment of experimental paracoccidioidomycosis in animal models have shown fluconazole to be less active than itraconazole. The clinical use of fluconazole is less than that of itraconazole, nevertheless it has some potential advantages: it can be administered parenterally, reaches a higher concentration in cerebrospinal fluid and urine, has more predictable digestive absorption, has fewer drug interaction and fewer side effects. Fluconazole should be given in special situations such as renal or brain involvement.

Terbinafine

Terbinafine has been successfully used in the treatment of one case suffering chronic paracoccidioidomycosis (77).

ADJUNCTIVE THERAPY

Treatment should include general measures such as rest, appropriate diet rich in proteins and calories, vitamin supplementation and discontinuation of alcohol and tobacco. Intestinal parasitosis and tuberculosis are also common in these patients and require therapy.

Surgery may be necessary to remove cerebral granulomas, and to treat fibrous stenosis of the mouth, larynx and trachea (73, 74).

As mentioned above, cell-mediated immunity is often depressed in severe cases of paracoccidioidomycosis. The use of immunomodulating drugs has been studied in animal models: dialysable leukocyte extracts, cyclophosphamide and glucan directly affect the outcome of experimental disease by reducing the number of lesions. Interferon-( improves the azoles activity against paracoccidioidomycosis (9, 37, 57, 79).

A glucan (ß-1.3 plyglucose), extracted from Saccharomyces cerevisiae, has been administered to a limited number of patients. It was given intravenously or intramuscularly, once a week at a dose of 10 mg, and after a month, it was administered once a month for one year. In one study, one group of 10 patients received intravenous glucan once a week for 1 month, followed by monthly doses over an 11-month period, together with antifungal agent as a immunostimulant. A second group of 8 patients that were less severely ill were given only antifungal agents. In the group treated with glucan there was only one relapse compared with 5 relapses in the group that received antifungal agents only. Patients treated with glucan exhibited a better clinical outcome, a faster recovery of cell mediated immunity, a marked decrease in erythrocyte sedimentation rate, and a decrease in serum specific antibodies (55, 57).

ENDPOINTS FOR MONITORING THERAPY

Untreated cases are always fatal after several years (66). Death may occur from respiratory failure, malnutrition, or intercurrent infections.

Post therapeutic latency exists. After successful treatment, some lesions can remain latent with occasional relapses. The paracoccidioidin skin test is usually positive, and serologic reactions show low titers or negative results. In severe cases, intense fibrosis observed as a consequence of healing lesions may cause tracheal and laryngeal stenosis, buccal atresia, or cardiorespiratory impairment (paradoxical cure) (30, 45, 66).

Criteria for cure of paracoccidioidomycosis include (a) clinical remission of all active lesions of at least 6 months duration; (b) eradication of P. brasiliensis from secretions; (c) stabilization of the radiologic pattern of the lungs and (d) immunologic cure, with positive paracoccidioidin skin test and reversion of serologic test from positive to negative or stable serology that remains at low values. Clinical remission is usually observed after 3 months of treatment; mycologic cure occurs even earlier. Radiologic stabilization often takes more than 6 months, and serologic conversion occurs about 4 years after the beginning of treatment (57, 64).

Patient follow-up should be continued for at least two years after treatment has been completed. Evaluation should be carried out at three-month intervals and should include clinical and immunological evaluations. Patients who exhibit negative paracoccidioidin skin test and positive serology one year after ending the initial treatment are at high risk of relapse.

VACCINES

Although some immunogenic preparations have been successfully applied in experimental paracoccidioidomycosis in mice, no commercially available vaccine is available (13, 33, 82).

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