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Background: Intravenous amphotericin B, with or without flucytosine, is usually standard therapy for cryptococcal meningitis in patients with the acquired immunodeficiency syndrome (AIDS). Fluconazole, an oral triazole agent, represents a promising new approach to the treatment of cryptococcal disease., Methods: In a randomized multicenter trial, we compared intravenous amphotericin B with oral fluconazole as primary therapy for AIDS-associated acute cryptococcal meningitis. Eligible patients, in all of whom the diagnosis had been confirmed by culture, were randomly assigned in a 2:1 ratio to receive either fluconazole (200 mg per day) or amphotericin B. Treatment was considered successful if the patient had had two consecutive negative cerebrospinal fluid cultures by the end of the 10-week treatment period., Results: Of the 194 eligible patients, 131 received fluconazole and 63 received amphotericin B (mean daily dose, 0.4 mg per kilogram of body weight in patients with successful treatment and 0.5 mg per kilogram in patients with treatment failure; P = 0.34). Treatment was successful in 25 of the 63 amphotericin B recipients (40 percent; 95 percent confidence interval, 26 percent to 53 percent) and in 44 of the 131 fluconazole recipients (34 percent; 95 percent confidence interval, 25 percent to 42 percent) (P = 0.40). There was no significant difference between the groups in overall mortality due to cryptococcosis (amphotericin vs. fluconazole, 9 of 63 [14 percent] vs. 24 of 131 [18 percent]; P = 0.48); however, mortality during the first two weeks of therapy was higher in the fluconazole group (15 percent vs. 8 percent; P = 0.25). The median length of time to the first negative cerebrospinal fluid culture was 42 days (95 percent confidence interval, 28 to 71) in the amphotericin B group and 64 days (95 percent confidence interval, 53 to 67) in the fluconazole group (P = 0.25). Multivariate analyses identified abnormal mental status (lethargy, somnolence, or obtundation) as the most important predictive factor of a high risk of death during therapy (P less than 0.0001)., Conclusions: Fluconazole is an effective alternative to amphotericin B as primary treatment of cryptococcal meningitis in patients with AIDS. Single-drug therapy with either drug is most effective in patients who are at low risk for treatment failure. The optimal therapy for patients at high risk remains to be determined.

The in vivo interactions of cilofungin, an echinocandin antifungal agent, and amphotericin B, a polyene derivative, in a murine model of disseminated candidiasis have been investigated. While single therapy with either drug alone prolonged survival of infected mice, kidney colony counts were not appreciably reduced. In contrast, combination therapy, especially at higher doses of both drugs, resulted in significant prolongation of survival and suppression of growth of yeast cells in the kidneys. Combination therapy of experimental candidiasis with cilofungin and amphotericin B did not result in antagonism; rather, additive or synergistic effects were seen. Future preclinical work with other echinocandin and polyene derivatives should include studies evaluating the in vivo interactions of both classes of compounds.

Mice infected intravenously with Candida albicans were treated with SCH 39304, a new triazole antifungal compound, amphotericin B, or both. Two dose levels of each drug were evaluated in an attempt to identify potential helpful or harmful effects on survival and kidney colony counts. Contrary to theoretical predictions, combination therapy was not antagonistic and some additive or synergistic effects were observed. The results obtained in this study suggest that antagonism between polyene and azole antifungal drugs is not inevitable and that additive or synergistic effects may be possible.

Cryptococcosis is currently the most common life threatening mycoses found in patients with the acquired immunodeficiency syndrome (AIDS). Extrapulmonary involvement is most frequently seen, especially in the central nervous system and skin. Clinical findings are non-specific, even in patients with meningitis. Threshold for diagnosis of this infection should be low, with serum cryptococcal antigens, blood, urine and sputum cultures for Cryptococcus neoformans performed in febrile AIDS patients. Lumbar puncture should also be performed if unexplained headaches are included in a patient's complaints. There is currently no consensus for the most appropriate treatment strategy and the role of oral azoles versus amphotericin B or amphotericin B with flucytosine remains a serious question in need of further controlled studies. Patients eligible for multicentered trials should be encouraged to participate. Therapy for others should be individualized. This review will address some of these issues.

Described is the case of a 73-yr-old woman with metastatic pancreatic islet carcinoma that manifested initially as Zollinger-Ellison syndrome followed by onset of endogenous Cushing's syndrome, who developed Pneumocystis carinii pneumonia while on therapy with a long-acting somatostatin analog. Although P. carinii pneumonia has been observed in patients with Cushing's syndrome associated with other conditions, this is the first reported case in a patient with Zollinger-Ellison syndrome. Heightened awareness of the possibility of opportunistic infections in patients receiving somatostatin therapy for Cushing's syndrome of any cause, particularly Zollinger-Ellison syndrome, may be warranted.

Conidia, produced by the mycelial phase of dimorphic fungi, are thought to represent the infectious form of the organism but must complete a transition to the tissue-invasive, yeast-like phase for infection to ensue. Preventing such transition should effectively eliminate pathogenicity. Using Blastomyces dermatitidis as a target, murine bronchoalveolar macrophages preferentially blocked phase transition after 4 h of incubation with conidia, relatively sparing the ability of conidia to produce hyphae. H2O2, in relatively high concentrations, demonstrated the same activity. The effects of H2O2 seem irreversible, since H2O2-treated conidia that germinated at 48 h at 25 degrees C were still unable to produce yeasts over the next 5 days when incubated at 37 degrees C. Catalase could not reverse the macrophage-induced inhibition of phase transition, suggesting that nonoxidative defense mechanisms may be operative in vivo. Since conidia do not form mycelia at temperatures found in mammalian hosts, these effects may represent a novel host defense mechanism against dimorphic fungal pathogens.

Fungal meningitis tends to be a subacute or chronic process; however, it may be just as lethal as bacterial meningitis if untreated. There are many similarities between the pathogenic fungi. Most of the fungi are aerosolized and inhaled, and initiate a primary pulmonary infection which is usually self-limited. Hematogenous dissemination may follow the initial infection, with subsequent involvement of the CNS. Rarely, trauma or local extension provides the route to CNS infection. The host is frequently, although not always, immunosuppressed. The hyphae of molds generally cause focal disease with hemorrhagic necrosis secondary to vascular thrombosis. The yeasts tend to cause a more diffuse process with the base of the brain being primarily affected, such that hydrocephalus is seen as a frequent complication of chronic disease. Diagnosis may be difficult, as the CSF may be normal, with negative smears and sterile cultures, although more often there is at least one abnormality indicating disease. Serologies (if available, depending on the fungus) may point towards the proper diagnosis, as may a careful travel history. Currently, amphotericin B is still the drug of choice in most situations; however, the newer azole antifungal agents offer great promise, especially in the treatment of cryptococcal meningitis. The precise role of such agents will remain unclear until appropriate large-scale studies of their effectiveness have been completed. The treatment of the unusual CNS mycoses will continue to be based on clinical experience, and reports of the use of new azoles in these diseases need to be critically evaluated.

We describe two patients with osteomyelitis due to Candida spp. treated with fluconazole, a new triazole antifungal. One patient had extensive involvement of ribs and costochondral regions of the anterior chest, and the other had vertebral infection. Both were cured with courses of 10 and 14 months, with greater than or equal to 1 year of follow-up after fluconazole was discontinued. Fluconazole is an attractive agent for the treatment of Candida osteomyelitis and deserves to be studied more extensively for this indication.

Persistent fever that is refractory to broad-spectrum antibacterials is common in neutropenic patients undergoing induction chemotherapy of acute leukemia. Clinical experience suggests that many of these patients are infected with fungi. Until recently, data supporting the role of empiric antifungal therapy in this setting were limited to small groups of patients or postmortem reports. Evolving evidence in larger patient populations supports data from smaller series: febrile neutropenic patients who have failed to respond to a 4- to 7-day course of broad-spectrum antibacterials may benefit from the early initiation of antifungal therapy. Patients with fungal colonization or pulmonary infiltrates and adult patients who have not received previous fungal prophylaxis may especially benefit from the early use of antifungal drugs. Amphotericin B has been the "gold standard" for empiric antifungal therapy, although the newer azoles may be useful in certain situations.

SCH 39304, a broad-spectrum azole derivative, was evaluated in an experimental mouse model of blastomycosis pneumonia. Five days after being inoculated with Blastomyces dermatitidis, infected mice were treated with either oral SCH 39304, fluconazole, or intraperitoneal amphotericin B. A dose response protective effect was observed with SCH 39304 at 5 to 100 mg/kg of body weight per day, with 5 mg of SCH 39304 per kg per day providing activity similar to that of 100 mg of fluconazole per kg per day. Colony counts of yeasts in the lungs of mice sacrificed while on therapy with SCH 39304 were consistently below those of controls, and several lungs were sterile. We conclude that SCH 39304 is effective in murine blastomycosis treatment and deserves to be evaluated in the treatment of human blastomycosis.

Fluconazole is a new antifungal agent available in both oral and parenteral formulations. According to the experts in this roundtable discussion, fluconazole represents a major clinical advance in the treatment of candidiasis and cryptococcosis in cancer patients, patients with AIDS, organ transplant recipients, and other patients at risk for opportunistic mycoses. The pharmacokinetic profile for fluconazole permits infrequent dosing and also makes it ideal for tissue site infections. Fluconazole's low toxicity gives it an advantage over currently available antifungal therapy and will permit prompt presumptive treatment of selected infections.

Infections caused by Cryptococcus neoformans cause significant morbidity and high mortality, particularly among immunocompromised patients. Cryptococcal meningitis is an important cause of central nervous system disease and death in patients with AIDS. Although the introduction of amphotericin B has greatly improved the prognosis of patients with cryptococcal meningitis, 30 years of experience have revealed important clinical limitations, including modest efficacy, nephrotoxicity, other clinically significant toxicities, and the inconvenience of intravenous dosing. The discovery of the additive effects of amphotericin B and flucytosine in cryptococcosis resulted in some improvement in efficacy and reduction in amphotericin B-related toxicity. However, approximately 30% of patients with cryptococcal meningitis still fail to respond to therapy. Ketoconazole has not proved useful in treating cryptococcal meningitis. Accumulating evidence suggests that the antifungal triazoles fluconazole, itraconazole, and SCH 39304 represent an advance in the treatment of cryptococcal meningitis, particularly in AIDS patients. Preliminary clinical trials in patients with and without AIDS have indicated that fluconazole and intraconazole are effective and well tolerated as either initial or maintenance therapy. Two large comparative trials of fluconazole and amphotericin B in patients with cryptococcal meningitis (mostly those with AIDS) are under way.

We studied the mechanisms of adherence of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages and the ability of the conidia to elicit an increase in macrophage O2- production, using an avirulent fungal strain. The number of cell associated conidia was counted by visual inspection of 2 hour macrophage monolayers incubated with conidia and O2- was measured by reduction of ferricytochrome c. Adherence of conidia to bronchoalveolar macrophages was time dependent and reached a plateau after 30 min (36 +/- 5%, 51 +/- 22%, and 36 +/- 17% macrophages with adherent conidia after 15, 30, and 60 min, respectively). Both Ca+2 and Mg+2 were required. The carbohydrates mannose, mannan, fucose, alphamethylmannoside, beta-glucan, galactose, N-acetylglucosamine and chitotriose (100-1000 micrograms/ml) did not inhibit adherence of conidia to macrophages. Trypsin treatment of macrophages or conidia did not affect binding. Conidia did not stimulate bronchoalveolar macrophage production of O2- above baseline concentrations (2.0 +/- 0.9 vs 0.8 +/- 0.5 nmol O2-, p greater than 0.05). We conclude that murine bronchoalveolar macrophage--B. dermatitidis conidia interactions occur primarily by a non-lectin-like attachment and do not result in the production of macrophage derived O2-.

Mycoses due to a variety of organisms have been implicated as opportunistic pathogens in patients infected with HIV. These infections are a cause of significant morbidity and mortality, and most often require life-long antifungal medication to suppress reactivation of the fungal process. With the advent of newer antifungal drugs, such as fluconazole and itraconazole, options are available so that appropriate nontoxic, long-term therapy can be administered. As the number of HIV-infected people increases and their longevity is extended, fungi will continue to pose challenging problems in diagnosis and therapy.

Optimal yields of pulmonary macrophages (PuM) from lavage of normal murine lungs have been obtained by the study of several different lung lavage fluids. Approximately 1 X 10(6) PuM can be consistently recovered from mice weighing 20 to 30 g, using 15 ml of phosphate buffered saline with 0.1% EDTA, with or without 10% fetal calf serum at 37 degrees C. Use of Terasaki tray wells for monolayer formation has allowed a relatively small number of cells (75,000) to be used to prepare multiple replicate wells from each mouse. Simultaneous functional comparisons with other cell types, e.g., peritoneal macrophages (PeM) were performed using this practical and economical system. Data on the phagocytosis and killing of Candida albicans by PuM and PeM are presented as evidence of the utility of this miniaturized system.

Ketoconazole, an antifungal drug, causes gynecomastia in some patients. It also inhibits androgen and glucocorticoid synthesis. In four volunteer male subjects, 600-mg doses of ketoconazole depressed serum testosterone concentrations markedly, but serum estradiol to a much lesser degree. The bound and free percentages of both hormones were not significantly altered. The net result was a significant elevation of the estradiol-testosterone ratio, expressed as either total circulating hormone or free hormone. In five male patients receiving long-term high-dose ketoconazole therapy, the testosterone concentrations fell, but the effect on estradiol was variable. In these patients the estradiol-testosterone ratio was persistently increased. Since gynecomastia appears to be the result of an elevated estradiol-testosterone ratio, the selective hormonal effect demonstrated may explain the side effect of gynecomastia after ketoconazole therapy.

Forty-seven courses of oral ketoconazole therapy administered to 46 patients with progressive coccidioidomycosis were evaluated. Responses, of varying degrees evaluated by a point system, occurred in 81, 94, and 91 percent of courses evaluated for skeletal, chronic pulmonary, and cutaneous disease, respectively. Presumed side effects occurred in 26 percent of courses and were generally minor and reversible. Relapse occurred in 33, 33, and 11 percent of evaluable responding patients with skeletal, chronic pulmonary and cutaneous disease, respectively. From this experience, ketoconazole appears to be an important advance in the therapy of chronic coccidioidomycosis. Whether cure is possible will require further study and follow-up.

We describe a murine model of acute blastomycosis pneumonia induced by Blastomyces dermatitidis conidia. Anesthetized mice were infected by placing a droplet containing conidia suspended in sterile saline on the nares and allowing the suspension to be aspirated into the lungs. Conidia were obtained from mycelia derived from the yeast forms of two well characterized strains of B. dermatitidis, ATCC 26199 and 26197. ATCC 26199 yeast and conidia were highly virulent in BALB/cByJ mice whereas both forms of ATCC 26197 were incapable of causing disease in 60 days. Conidia and yeasts of the virulent strain caused pathologic changes primarily in the lungs, whereas no abnormalities were seen in the lungs, or elsewhere, in mice infected with the a virulent strain. Small numbers of ATCC 26199 yeast forms were found in scattered foci in liver and kidneys, but no inflammatory reaction surrounded the fungus. This murine model of acute blastomycosis pneumonia mimics the acute disease in humans and is initiated with the naturally infectious particle.

Cefotaxime, a cephalosporin drug, has been shown to be active in vitro against nocardiae, a finding confirmed in this study. Pharmacokinetic studies were performed in mice to define regimens which provided peak serum levels comparable to that achieved in man with currently used doses. These regimens were shown to be effective with only short courses of therapy of rapidly progressive and highly lethal N. asteroides infection, produced by pulmonary challenge of mice. This suggests the possible utility of this drug in human nocardiosis.

One hundred sixty patients were entered in two multicenter protocols to receive 400 to 2,000 mg of ketoconazole once daily for nonmeningeal or meningeal coccidiodomycosis. For 24 h after administration of all doses, mean concentrations in serum exceeded MICs for Coccidioides immitis (trough concentrations, greater than 1 microgram/ml). Mean peak concentrations occurred 4 to 6 h after administration, ranging from 7 to 17 micrograms/ml for doses of 400 to 2,000 mg. Incremental increases in peak concentrations in serum were greatest at doses of less than or equal to 1,200 mg. To investigate whether long-term therapy altered concentrations in serum, serial data were studied by several methods. The results suggested a trend to increased levels in serum with prolonged therapy, but were not statistically significant. All 168 cerebrospinal fluid (CSF) samples from meningitis patients contained less than or equal to 2.9 micrograms/ml, and only 6 contained greater than 1 microgram/ml. There was no apparent relation between dose, time after dose, site of CSF sampling, or concurrent inflammation and CSF ketoconazole concentration. Neither concentration in serum, toxicity, nor outcome correlated with dose, calculated in milligrams per kilogram at the fixed doses (400-mg increments) under study. Likewise, at the various doses, concentration in serum did not correlate with outcome or toxicity, suggesting that individual drug disposition was not an important factor in outcome or toxicity. Toxicity was reversible, and principal side effects were nausea and vomiting (50%), gynecomastia (21%), decreased libido (13%), alopecia (8%), elevated liver function tests (5%), pruritus (5%), and rash (4%). Gastrointestinal and endocrinologic toxicity were dose related and increased at doses greater than 800 mg. The cumulative percent toxicity requiring discontinuation of drug was 6, 17, 23, and 56% at 400-, 800-, 1,200-, and 1,600-mg doses. Doses of >400 mg are thus markedly more toxic, and efficacy data for nonmeningeal disease have not demonstrated that they are more efficacious.

The effectiveness of toxic oxygen metabolites in killing the yeast form of Paracoccidioides brasiliensis (the form that occurs in host tissues) was studied with a fluorescence method in vitro. The two isolates studied were similar in susceptibility and H2O2 alone was lethal with an LD50 of 15-25 mM. The addition of halide (5 X 10(-4) M) augmented the lethality of H2O2 and in that setting H2O2 was c.90% lethal at 5 X 10(-5) M. Killing was most effective in the presence of peroxidase, when only 5 X 10(-6) M H2O2 (a concentration attainable in vivo by phagocytes) was required for a 95% kill. Kinetic studies revealed that toxic concentrations of H2O2 alone or of the H2O2-halide-peroxidase (PPH) system produced significant killing in 1 min; killing was maximal in 15 min. The PPH system was the more rapid in action. The dependence of the PPH killing system on H2O2 was demonstrated by an absence of killing in the presence of catalase. The susceptibility of P. brasiliensis to H2O2 and the PPH system appeared different in some respects from that noted for other dimorphic fungal pathogens. These studies suggest that toxic oxygen metabolites are important in host defence against P. brasiliensis.

UK-49,858, a new antifungal triazole derivative, was compared with amphotericin B in the treatment of pulmonary infections by Blastomyces dermatitidis in male BALB/cByJ mice. Administration of UK-49,858 in daily doses of 25 or 50 mg/kg for 21 days gave 30 and 100% survival rates, respectively. These results compared with 100% mortality in infected controls and 100% survival among mice treated with amphotericin B. UK-49,858 did not eradicate the fungus from the lungs of surviving animals, while amphotericin B effected sterilization of the lungs in 66% of the survivors.

The interaction of elicited murine polymorphonuclear neutrophils (PMN) and the thermally dimorphic fungal pathogen Blastomyces dermatitidis in vitro was studied. The PMN elicited intraperitoneally with thioglycollate, in normal mice or mice immune to B dermatitidis, failed to reduce colony forming units (CFU) of B dermatitidis in the inoculum in a 4-hr in vitro assay, even in the presence of 10% fresh immune serum. In contrast, PMN elicited intraperitoneally in immune mice by injection of nonviable B dermatitidis cells significantly reduced inoculum CFU (60 +/- 5%) under the same conditions. Furthermore, nonviable B dermatitidis intraperitoneally (i.p.) in normal mice or nonviable Candida albicans i.p. in immune mice failed to elicit peritoneal exudate cells that reduced inoculum CFU in this system. These results support the concept that PMN, elicited in a site by means of an immunological reaction, acquired enhanced microbicidal activity. The fungicidal activity of immunologically elicited PMN was shown to be most effective at high effector to target cell ratios (1,000:1), maximal within 2 hr of coculture, and significantly enhanced in the presence of fresh immune serum compared to heat-inactivated immune serum, normal mouse serum, or fetal bovine serum. Such PMN also had significantly enhanced fungicidal activity against C albicans compared to normal PMN. Fungicidal activity was abrogated in the presence of catalase, implicating hydrogen peroxide generation as the killing mechanism in the activated cells.

Diagnosis of nocardiosis is often delayed because of the frequently nonspecific nature of the illness and because of the long incubation period required to isolate the organism in the microbiology laboratory. Past experience with serological methods for diagnosing nocardiosis have met with little success, primarily because there was no suitable antigen. Using a previously identified Nocardia asteroides-specific protein, we developed an enzyme immunoassay for the rapid diagnosis of nocardiosis. We evaluated sera from 22 patients with nocardiosis, 83 hospitalized controls, and 21 patients with tuberculosis. Twenty (91%) of the sera from patients with nocardiosis had titers of antibody greater than or equal to 1:256, whereas all of the sera from controls and from patients with tuberculosis had titers less than 1:256. This assay represents a highly sensitive and specific serodiagnostic tool for evaluating patients with possible nocardiosis.

The fungicidal activity of murine broncho-alveolar macrophages (BAM) against the yeast form of a virulent strain of Blastomyces dermatitidis was studied in the small-volume wells of a Terasaki plate. In a 4-h fungicidal assay, significant killing (16-33%) of the fungus by unstimulated BAM was demonstrated with BAM from normal mice and from mice rendered immune to lethal infection with B. dermatitidis. No significant differences between the activity of BAM from these two sources could be identified. Addition of 10% autologous normal or immune serum did not augment the macrophage fungicidal activity. Simultaneous experiments with peritoneal macrophages (PM) also gave reproducible killing of the yeast form in the wells of the Terasaki plate, but in the larger wells of microtitration plates, PM showed no significant fungicidal activity. On the other hand, BAM had similar fungicidal activity against B. dermatitidis in Terasaki and microtitration-plate wells. The modest fungicidal activity of BAM from immune mice against B. dermatitidis suggests that the resistance of immune mice to respiratory challenge is likely to be based on some augmentation of this first line of defence.

A rapid spectrofluorometric method for determining the levels of both trimethoprim and sulfamethoxazole from the same specimen of serum is described. The method involves stepwise extraction of the specimen first with chloroform at an alkaline pH (pH 9.0) for trimethoprim followed by n-butyl chloride at an acidic pH (pH 2.0) for sulfamethoxazole. To quantitate trimethoprim, the chloroform layer was subjected to fluorometry by exciting the specimen at 295 nm and measuring the relative intensity at 330 nm. To determine sulfamethoxazole levels, the n-butyl chloride layer was subjected to fluorometry by exciting the specimen at 285 nm and measuring the relative intensity at 330 nm. Relative intensities were linear (r greater than 0.99) over the concentration ranges of 0.5 to 40 microgram/ml for trimethoprim and 1 to 400 microgram/ml for sulfamethoxazole. Values obtained by this spectrofluorometric procedure were in excellent agreement with those obtained by a conventional fluorometric assay for trimethoprim and a colorimetric assay for sulfamethoxazole. Elevated levels of endogenous metabolic products and numerous other drugs, including a number of antimicrobial agents, did not interfere with the method. Although salicylates interfere with the determination of sulfamethoxazole, an appropriate correction can be made. This method can also be used to determine the drug levels in cerebrospinal fluid.

With a new short-term assay, where the reduction of CFUs in the inoculum could be measured, we investigated the killing of the dimorphic fungal pathogen Blastomyces dermatitidis in its yeast phase by murine peritoneal macrophages. Peritoneal macrophages from concanavalin A- or Mycobacterium bovis BCG-treated mice, but not resident or thioglycolate-elicited macrophages, significantly reduced the CFUs of B. dermatitidis in the inoculum. The activation of peritoneal macrophages for fungicidal activity by concanavalin A treatment was shown to be dose dependent and transient, i.e., absent after 72 h. These results indicate that it is possible for murine peritoneal macrophages to kill B. dermatitidis in vitro. The addition of specific antibody or complement or both did not enhance the killing of B. dermatitidis by these nonspecifically activated macrophages.

The effects of cilofungin, a lipopeptide antifungal agent active against several species of Candida, on human neutrophil candidacidal activity were investigated. While no increase or decrease in neutrophil killing of blastospores was observed when cilofungin was simultaneously incubated with neutrophils and yeasts, preincubation of yeasts with serum containing cilofungin resulted in approximately 90% decrease in killing of five of six strains. Attachment/phagocytosis of these strains was unchanged from controls. We conclude that cilofungin interferes with opsonization of most strains of C. albicans, resulting in a marked decrease in neutrophil candidacidal activity, a phenomenon which may be of significance in vivo in certain tissues where access to antibodies and/or complement is limited.

Diagnosis of systemic nocardiosis has been hampered by difficulty in the rapid isolation and identification of the organism and by the frequent need for invasive biopsies. We show that a 55,000-Mr protein previously found in Nocardia asteroides is also present in N. brasiliensis and N. caviae culture filtrates. We also used the antigen in a dot blot assay to identify patients infected with N. brasiliensis. Sera from patients with mycetoma secondary to N. brasiliensis (n = 6) or Actinomadura madurae (n = 2) and 10 hospitalized controls were tested. Six (100%) of the sera from the patients with nocardial mycetoma had positive dot blots to purified antigen, whereas all 10 of the control sera were negative. Serum from one of two patients infected with A. madurae also reacted with the antigen. Thus, the 55-kilodalton protein appears to be produced by three medically important species of Nocardia and is useful when used in an immunoblot assay for identifying patients infected with these organisms.

Polymorphonuclear neutrophils (PMN) induced locally in immune mice by intraperitoneal injection of antigen exhibit enhanced fungicidal activity compared with PMN elicited with thioglycolate. The mechanism of the differences in these PMN populations was studied. Sublethal infection was used to produce immunity to Blastomyces dermatitidis. A correlation was sought between the ability of PMN to kill, or not kill, B. dermatitidis and the production of the oxidative burst, as measured by luminol-enhanced chemiluminescence (CL). Although elicited PMN cocultured with Candida albicans produced a burst of CL and were candidacidal, killing did not occur when PMN were cocultured with B. dermatitidis. Lack of killing of B. dermatitidis by elicited PMN correlated with lack of stimulation of a brisk oxidative burst. In contrast to elicited PMN, PMN induced by B. dermatitidis antigen responded to this fungus with a burst of CL and a significant reduction of inoculum CFU (80%). Furthermore, these PMN when cocultured with C. albicans produced an enhanced burst of CL, and killing was enhanced compared with that by elicited PMN, e.g., 86 versus 58%. The CL burst and killing of B. dermatitidis by antigen-induced PMN was abrogated in the presence of catalase, implying a critical role for hydrogen peroxide. Partial but significant depression of CL and killing in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, identified hydroxyl radical, or its metabolites, as a toxic product(s) responsible for a significant fraction of fungicidal activity. These results indicate that the metabolic activity and microbicidal activity of PMN can be altered (enhanced) at the site of an immunological reaction and thus could constitute an important factor in resistance.

With the increased number of immunocompromised patients there has been a concomitant increase in patient morbidity and mortality due to fungi. The etiologic microorganisms vary depending upon the type of immune dysfunction. Patients with malignancies and chemotherapy-induced neutropenia commonly are infected with Candida and Aspergillus. Other ubiquitous fungi such as Rhizopus, Fusarium, and Trichosporon are more frequently implicated as agents of disease in these patients. Patients with cell-mediated immune dysfunction such as acquired immune deficiency syndrome (AIDS) are susceptible to mucocutaneous candidiasis and pulmonary and disseminated cryptococcosis. Histoplasmosis and coccidioidomycosis have been particularly lethal infections in AIDS patients. Contributing factors such as broad-spectrum antibiotic use, intravenous catheterization, malnutrition, hyperalimentation, multiple surgical procedures and/or trauma, and steroids used either singly or in combination may also predispose patients to invasive fungal disease. Definitive diagnosis is often difficult to establish and usually requires invasive biopsy. Delay of culture results due to the time required to process specimens and to allow the fungus to grow also contributes to the poor results of therapy. Biopsy of skin lesions represents a useful technique for making a diagnosis. Recent advances in antifungal therapeutics promise to change the current approach to treatment for several of the mycoses. The availability of new oral azoles with spectra of activity that include aspergillosis and cryptococcosis, which currently require treatment with parenteral amphotericin B, may prove practical for prolonged oral therapy of otherwise lethal mycoses.

Fifteen patients with coccidioidal meningitis were treated with high doses of ketoconazole for up to 4 years. Five patients were treated with ketoconazole alone. One clinically failed, one developed hepatotoxicity, and three achieved remission of meningitis. One patient received intrathecal AMB in addition to ketoconazole for only 2 weeks before continuing on ketoconazole alone. He improved, but discontinued ketoconazole because of nausea and vomiting, and suffered a lethal relapse. Nine patients received ketoconazole in combination with prolonged courses of intrathecal AMB. Two patients were failures from nausea and vomiting, and the remaining seven either improved or experienced remission. The clinical responses appeared to be similar in patients receiving high-dose ketoconazole, either alone or combined with AMB, suggesting that there is no clinically significant antagonism of the drugs. Nausea and vomiting are significant limitations of high-dose ketoconazole. Ketoconazole alone is effective in some patients with coccidioidomycotic meningitis.

Candida rugosa was isolated from two patients. One patient had acute leukemia and developed invasive disease due to this yeast on two occasions while granulocytopenic. Her infection was eventually cured after treatment with amphotericin B. In another immunocompromised patient, the yeast was isolated from the sputum in the presence of a pulmonary infiltrate, but there was no other evidence for a pathogenic role. Antifungal susceptibility testing of the first patient's isolate and three environmental isolates showed all four to be susceptible to amphotericin B, miconazole, and flucytosine, and only the patient isolate was resistant to ketoconazole. These results suggest possibilities for therapy in future encounters. It appears that C. rugosa, a common pathogen in cattle, can be pathogenic in humans under the appropriate circumstances.

Paracoccidioidomycosis is the designation for the disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is a disease of great importance in Latin America and constitutes an important diagnosis for consideration in patients from endemic areas who have pulmonary or systemic disease compatible with fungal infection. In the laboratory, basic work has uncovered important interactions between the fungus and the human sex steroid system. These studies may have important implications in our approach to this and other infectious diseases.

Three strains of Blastomyces dermatitidis which differ in their virulence for mice were exposed in their yeast form to various components of the peroxidase-hydrogen peroxide-halide system. Susceptibility to H2O2 alone correlated with virulence, with the most virulent strain (ATCC 26199) least susceptible (50% lethal dose, greater than 50 mM) and an avirulent strain (ATCC 26197) most susceptible (50% lethal dose less than 3.3 mM). A strain of intermediate virulence (ATCC 26198) was of intermediate susceptibility (50% lethal dose, 11.5 mM). The addition of a nontoxic concentration of KI (5 X 10(-4) M) did not increase H2O2 toxicity. However, the addition of either myeloperoxidase or horseradish peroxidase and KI markedly decreased the amount of H2O2 required to kill the organisms, with 100 +/- 0% of all strains killed at 5 X 10(-5) M H2O2 and 97 +/- 4, 100 +/- 0, and 94 +/- 8% of ATCC 26199, ATCC 26198, and ATCC 26197 killed, respectively, at 5 X 10(-6) M H2O2. Kinetic studies with H2O2 alone revealed a delayed onset of killing, but virtually 100% of organisms were killed by 120 min of exposure in all strains. By comparison, the peroxidase-hydrogen peroxide-halide system was 100% lethal for all strains at 1 min. The relatively high concentrations of H2O2 required to kill the yeast phase of B. dermatitidis suggest that H2O2 alone does not account for host resistance to the organism. However, the rapidly lethal effect of the peroxidase-hydrogen peroxide-halide system at physiologically relevant concentrations suggests that this may be one mechanism of host defense to B. dermatitidis.

In order to assess the cellular responses in the lung in murine pulmonary blastomycosis, serial lung lavages were performed in normal BALB/cByJ mice that had received an intranasal inoculation with 1 of 2 strains of Blastomyces dermatitidis of opposite virulence. The virulent strain, ATCC 26199, induced an increasing number of neutrophils recovered from the lung lavages. An early peak of incoming neutrophils was seen at 1 to 2 days, followed by a more rapid accumulation of intraalveolar neutrophils, until by the ninth day after infection, 80% of the cells recovered from the mice were neutrophils. In contrast, the avirulent strain, ATCC 26197, induced the same degree (20%) of neutrophil influx on Days 1 and 2. Thereafter, the percentage of neutrophils consistently declined, so that by Day 6 after inoculation, the differential counts were normal. The total number of cells recovered from the mice challenged with the virulent strain did not increase significantly until Day 4; then the total number of cells consistently increased until the experiment was terminated on Day 14. The total number of cells obtained from the mice challenged with the avirulent organism never exceeded the normal range. To assess the effects of nonviable inert particles, mice were given Sephadex G-25 beads by the intranasal route, and the total number of cells recovered from the lung lavage fluid and the differential counts were determined. The peak neutrophil influx occurred 6 h after aspiration of the beads, compared with 24 to 48 h with the fungi.(ABSTRACT TRUNCATED AT 250 WORDS)

The first case of gonococcal endocarditis on a prolapsing mitral valve is reported. The organism was found to be highly sensitive to penicillin G, Arg- Hyp- Ura-, and sensitive to the bactericidal action of normal serum. This combination of characteristics in a strain of Neisseria gonorrheae causing systemic disease is distinctly unusual. With high dose penicillin therapy the immunologic parameters returned to normal and the patient made an uneventful recovery. High levels of circulating immune complexes were detected in the patient's serum.

An ammonium sulfate precipitate derived from a culture filtrate of Nocardia asteroides contained 20-30 protein subunits, as identified by gel electrophoresis. Western blot analysis of these proteins (obtained from serum samples from patients with systemic nocardiosis) revealed that all of the 17 samples reacted with a subunit of a protein with an apparent molecular weight of 55,000 and that 11 of the 17 samples reacted with a subunit with a molecular weight of 31,000. In contrast, only two of 25 serum samples obtained from healthy controls or patients hospitalized without nocardial or mycobacterial disease reacted with these subunits. Sera from 21 patients infected with Mycobacterium tuberculosis, a historically troublesome cross-reactive group, failed to react with either the 55,000 or the 31,000 molecular weight subunit. The presence of only two protein subunits that reacted with sera from humans with nocardiosis suggests that these molecules may have use as diagnostic reagents or probes of the immunologic reaction to Nocardia.

A new, rapid method for measuring serum levels of nafcillin by spectrofluorometry is described. The method involves extraction of 2 ml of acidified serum with n-butyl chloride, subjecting the organic solvent layer to excitation at 340 nm, and measuring the relative intensity of emission fluorescence at 380 nm. An excellent linear correlation exists between serum levels of nafcillin and the relative intensity in a drug concentration range of 0.25 to 150 mug/ml. The results obtained by this spectrofluorometric technique are in complete accord with those obtained by the conventional microbiological assay using Staphylococcus aureus ATCC 6538P. The method is not interfered with by elevated levels of endogenous metabolic products or the presence of other drugs, including a number of antimicrobial agents. The assay is interfered with, however, by the presence of salicylates, for which appropriate correction can easily be made. A salicylate assay employing a spectrofluorometric technique is also described.

A case of reactivated paracoccidioidomycosis in an immunosuppressed host is described, the first association of this disease with renal transplantation. His presentation and clinical course was similar to the general experience with this mycosis. his response to ketoconazole was delayed when compared with that in non-immunosuppressed patients. Ketoconazole produced a dramatic resolution of all symptoms within 7 months. Serial determinations of paracoccidioides complement fixing antibodies showed falling titers, accurately reflecting his clinical status. Comparisons are made with other endemic mycoses in immunosuppressed patients. More cases of paracoccidioidomycosis in immunosuppressed patients can be expected to occur as immunosuppressive therapy becomes more common in areas endemic for Paracoccidioides brasiliensis, and with current trends in migration and travel.

Strongyloidiasis in immunosuppressed patients often requires prolonged administration of the antihelminthic, thiabendazole. The case described herein represents the first report of theophylline toxicity induced by concurrent administration of thiabendazole. Preliminary studies suggest that interference with theophylline clearance by thiabendazole may be the mechanism involved in this drug interaction.

The syndrome of granulomatous angiitis related to varicella zoster virus infection often manifests as herpes zoster ophthalmicus followed by contralateral hemiplegia. Forty-five cases have been reported to date, and the authors' experience with two additional cases seen in a one-year period is described. Given the frequency of both stroke and herpes zoster ophthalmicus in an aging population, the authors postulate that granulomatous angiitis is underdiagnosed. There is need for increased awareness of this disease by the non-neurologist. Diagnostic and therapeutic considerations are reviewed.

A series of experiments show the potency of a newly described microbicidal system, involving iron, H2O2, and halide, in killing a fungus (Blastomyces dermatitidis). B dermatitidis has previously been shown susceptible to the myeloperoxidase-H2O2-halide system. The present studies show killing of either of two strains in 1 hour if Fe++ at 5 X 10(-5)M, H2O2 at 5 X 10(-5)M and Kl at 5 X 10(-4)M are all present (P less than 0.001). EDTA, a Fe++ chelator, abrogates killing. The mechanism presumably utilizes hydroxyl radical, since an inhibitor, ethanol, also neutralizes the system. The bactericidal and fungicidal system is of great potential importance in vivo.