Limitations of antifungal prophylaxis in preventing invasive Candida surgical site infections after liver transplant surgery.

Invasive primary Candida surgical site infections (IP-SSIs) are a common complication of liver transplantation, and targeted antifungal prophylaxis is an efficient strategy to limit their occurrence. We performed a retrospective single-center cohort study among adult single liver transplant recipients at Duke University Hospital in the period between 1 January 2015 and 31 December 2020. The study aimed to determine the rate of Candida IP-SSI according to the peri-transplant antifungal prophylaxis received. Of 470 adult single liver transplant recipients, 53 (11.3%) received micafungin prophylaxis, 100 (21.3%) received fluconazole prophylaxis, and 317 (67.4%) did not receive systemic antifungal prophylaxis in the peri-transplant period. Ten Candida IP-SSIs occurred among 5 of 53 (9.4%) micafungin recipients, 1 of 100 (1.0%) fluconazole recipients, and 4 of 317 (1.3%) recipients who did not receive antifungal prophylaxis. Our study highlights the limitations of antifungal prophylaxis in preventing invasive Candida IP-SSI after liver transplant surgery. We hypothesize that pathogen, host, and pharmacokinetic-related factors contributed to the occurrence of Candida IP-SSI despite antifungal prophylaxis. Our study reinforces the need for a risk-based, multi-pronged approach to fungal prevention, including targeted antifungal administration in patients with risks for invasive candidiasis and close monitoring, especially among patients with surgically complex procedures, with timely control of surgical leaks.

Management of an Outbreak of Exophiala dermatitidis Bloodstream Infections at an Outpatient Oncology Clinic.

We report the presentation and management of 17 cases of Exophiala dermatitidis and Rhodotorula mucilaginosa bloodstream infections caused by a compounded parenteral medication at an oncology clinic. Twelve patients were asymptomatic. All central venous catheters were removed and antifungal therapy, primarily voriconazole, was administered to patients. Three patients died.

Autophagy is redundant for the host defense against systemic Candida albicans infections.

Autophagy has been demonstrated to play an important role in the immunity against intracellular pathogens, but very little is known about its role in the host defense against fungal pathogens such as Candida albicans. Therefore, the role of autophagy for the host defense against C. albicans was assessed by complementary approaches using mice defective in autophagy, as well as immunological and genetic studies in humans. Although C. albicans induced LC3-II formation in macrophages, myeloid cell-specific ATG7(-/-) mice with defects in autophagy did not display an increased susceptibility to disseminated candidiasis. In in vitro experiments in human blood mononuclear cells, blocking autophagy modulated cytokine production induced by lipopolysaccharide, but not by C. albicans. Furthermore, autophagy modulation in human monocytes did not influence the phagocytosis and killing of C. albicans. Finally, 18 single-nucleotide polymorphisms in 13 autophagy genes were not associated with susceptibility to candidemia or clinical outcome of disease in a large cohort of patients, and there was no correlation between these genetic variants and cytokine production in either candidemia patients or healthy controls. Based on these complementary in vitro and in vivo studies, it can be concluded that autophagy is redundant for the host response against systemic infections with C. albicans.

Pericarditis mediated by respiratory syncytial virus in a hematopoietic stem cell transplant patient.

We describe a case of pericarditis and large pericardial effusion in a 63-year-old African-American man undergoing autologous hematopoietic stem cell transplant for multiple myeloma. Pericardial tissue biopsy demonstrated fibrinous pericarditis, and immunohistochemistry stains were positive for respiratory syncytial virus. The patient improved with oral ribavirin and intravenous immune globulin infusions.

The impact of caspase-12 on susceptibility to candidemia.

Candida is one of the leading causes of sepsis, and an effective host immune response to Candida critically depends on the cytokines IL-1ß and IL-18, which need caspase-1 cleavage to become bioactive. Caspase-12 has been suggested to inhibit caspase-1 activation and has been implicated as a susceptibility factor for bacterial sepsis. In populations of African descent, CASPASE-12 is either functional or non-functional. Here, we have assessed the frequencies of both CASPASE-12 alleles in an African-American Candida sepsis patients cohort compared to uninfected patients with similar predisposing factors. African-American Candida sepsis patients (n = 93) and non-infected African-American patients (n = 88) were genotyped for the CASPASE-12 genotype. Serum cytokine concentrations of IL-6, IL-8, and IFNγ were measured in the serum of infected patients. Statistical comparisons were performed in order to assess the effect of the CASPASE-12 genotype on susceptibility to candidemia and on serum cytokine concentrations. Our findings demonstrate that CASPASE-12 does not influence the susceptibility to Candida sepsis, nor has any effect on the serum cytokine concentrations in Candida sepsis patients during the course of infection. Although the functional CASPASE-12 allele has been suggested to increase susceptibility to bacterial sepsis, this could not be confirmed in our larger cohort of fungal sepsis patients.

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood.

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.

To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper-binding site. When this base change was introduced into CNLAC1 by site-directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.

Posaconazole as salvage treatment of invasive fungal infections in patients with underlying renal impairment.

OBJECTIVES: The aim of this study is to determine the efficacy and safety of posaconazole in patients with underlying renal impairment. Patients and methods We analysed the efficacy and safety of posaconazole in patients with renal impairment in a post hoc subanalysis of a Phase 3, multicentre, open-label trial in patients with invasive fungal infections (IFIs). In the Phase 3 study, 330 patients intolerant of or with IFIs refractory to standard antifungal therapy received posaconazole 800 mg daily in divided doses. In our subanalysis, 238 patients with proven/probable IFIs, including 65 patients with renal impairment (creatinine clearance < 50 mL/min or serum creatinine (sCR) level >2 mg/dL at baseline) and 173 patients with greater renal function [creatinine clearance >/= 50 mL/min (acceptable renal function)], formed the modified intent-to-treat population. Success was defined as complete or partial response, and non-success was defined as stable disease or treatment failure. RESULTS: Overall response rates were similar in the renal impairment group (49%) and in the acceptable renal function (50%) group. Seventeen of the 41 patients with renal impairment and aspergillosis responded. Adverse events occurred in 32/65 (49%) patients with renal impairment and in 72/173 (42%) patients with acceptable renal function. The most common adverse events in both groups were nausea (14% patients with renal impairment versus 8% with acceptable renal function), altered/elevated levels of other medications (8% versus 2%), increased sCR levels (6% versus 0%), vomiting (6% versus 4%), abdominal pain (5% versus 5%) and dizziness (5% versus 1%). CONCLUSIONS: These results suggest that posaconazole is effective and well tolerated in patients with refractory IFIs regardless of renal impairment.

Pulmonary cryptococcosis in patients without HIV infection: factors associated with disseminated disease.

Cryptococcus neoformans is an uncommonly recognized cause of pneumonia in HIV-negative patients. Because of its propensity to disseminate to the meninges and other sites, a lumbar puncture is recommended for patients with pulmonary cryptococcosis, regardless of other risk factors. This study explored clinical and laboratory features to help predict which patients had pulmonary disease alone versus those who had pulmonary plus extrapulmonary disease. A retrospective chart review at 15 medical centers was performed from 1990 to 2000 of all HIV-negative patients who had pulmonary cryptococcosis. Demographic, clinical, radiographic, and laboratory features were evaluated to determine factors that differentiated those patients who had extrapulmonary disease. Among 166 patients who had pulmonary cryptococcosis, 122 had pulmonary infection only and 44 had pulmonary plus extrapulmonary (disseminated) disease. A negative serum cryptococcal antigen titer was more common in patients with pulmonary disease alone (p < 0.01). Multivariate analysis demonstrated that patients who had disseminated disease were more likely than those who only had pulmonary disease to have cirrhosis (p = 0.049), headache (p < 0.001), weight loss (p = 0.003), fever (p = 0.035), altered mental status (p < 0.001), and to be receiving high-dose corticosteroids (p = 0.008). In this large cohort of HIV-negative patients with pulmonary cryptococcosis, there were easily distinguished clinical and laboratory features among patients with pulmonary disease alone versus those with pulmonary plus extrapulmonary disease. These findings may be helpful in the evaluation of HIV-negative patients with pulmonary cryptococcosis with regard to the need for lumbar puncture or to search for disseminated disease.

Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide.

Cryptococcus neoformans is variably encapsulated in vitro, whereas in tissues it develops a large capsule. We observed that cells of a strain with thin capsules, when growing in a standard fungal culture medium, became heavily encapsulated when incubated in serum-free cell culture medium (Dulbecco's modified Eagle's medium [DME]). Capsule size was quantitated physically by measuring cell volume, and chemically by determining the content of a capsular monosaccharide, glucuronate. The CO2/HCO-3 couple stimulated capsule development, resulting in visible enlargement by 3 h after exposure to high CO2/HCO-3. The amount of capsule per cell was directly proportional to the total millimolar CO2/HCO-3 concentration between 24 and 2.4 mM at pH 7.35, but at constant PCO2 (40 torr) and varying [HCO-3], the cells were heavily encapsulated down to pH 6.8. Concentration of CO2/HCO-3 in the physiologic range increased elaboration of polysaccharide into the medium and slowed the cell generation time from 2 to 6 h. Four other first-passage clinical isolates were all heavily encapsulated in DME with CO2/HCO-3, but variably encapsulated in DME without CO2/HCO-3. Exposure of yeast to increased CO2/HCO-3 caused a marked reduction in complement-mediated phagocytosis by mouse macrophages. A stable clone was isolated which contained capsular polysaccharide, but lacked the CO2-inducible phenotype. This clone was avirulent for steroid-treated rabbits. Thus, the prevailing CO2 concentration in mammalian tissues may be one stimulus for capsular polysaccharide synthesis. This could serve as an adaptive mechanism favoring parasite survival in the host.

Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages.

The microbiostatic action of macrophages was studied in vitro employing peritoneal cytotoxic macrophages (CM) from mice acting against Cryptococcus neoformans cultured in Dulbecco's medium with 10% dialyzed fetal bovine serum. Fungistasis was measured using electronic particle counting after lysis of macrophages with detergent. Macrophage fungistasis failed in medium lacking only L-arginine. Complete fungistasis was restored by L-arginine; restoration was concentration dependent, maximal at 200 microM. Deletion of all other essential amino acids did not abrogate fungistasis provided that L-arginine was present. Of twenty guanido compounds, including D-arginine, only three (L-arginine, L-homoarginine, and L-arginine methylester) supported fungistasis. Known activators or mediators of macrophage cytotoxicity (endotoxin, interferon gamma, tumor necrosis factor) did not replace L-arginine for CM-mediated fungistasis. The guanido analogue NG-monomethyl-L-arginine was a potent competitive inhibitor of CM-mediated fungistasis giving 50% inhibition at an inhibitor/L-arginine ratio of 1:27. Although CM completely blocked fungal reproduction via an L-arginine-dependent mechanism, the majority of the dormant fungi remained viable. Thus, this mechanism is viewed as a microbiostatic process similar or identical to the tumoristatic effect of macrophages. This suggests the production of a broad spectrum biostatic metabolite(s) upon consumption of L-arginine by cytotoxic macrophages.

Metabolic fate of L-arginine in relation to microbiostatic capability of murine macrophages.

L-arginine is required for the fungistatic action of murine macrophages in vitro. To further investigate this requirement, L-arginine metabolism by macrophages was measured under conditions where fungistasis either succeeded or failed. Macrophage fungistasis correlated with metabolism of L-arginine to citrulline, nitrite, and nitrate. The metabolic rate was dependent on extracellular L-arginine concentration, reaching a maximum of 67 nmol nitrite/h per mg protein. It accounted for one-third of arginine consumed by fungistatic macrophages. Equimolar amounts of citrulline and total nitrite plus nitrate accumulated in medium. This was consistent with the hypothesis that one of the equivalent guanidino nitrogens of L-arginine was oxidized to both nitrite and nitrate leaving L-citrulline as the amino acid reaction product. The analogue, NG-mono-methyl-L-arginine, selectively inhibited nitrogen oxidation and it was shown previously that it inhibited fungistatic capability. Resident macrophages were not fungistatic and their nitrogen oxidation was low. Once macrophages began producing nitrite/nitrate, protein synthesis was not required during the next 8 h for either fungistasis or nitrogen oxidation. Two-thirds of L-arginine consumption was due to macrophage arginase yielding L-ornithine and urea, which accumulated in medium. This activity was dissociated from macrophage fungistasis. Nitrogen oxidation metabolism by macrophages is linked to a mechanism that inhibits proliferation of fungi. This may involve synthesis of an intermediate compound(s) that has antimicrobial properties.

Combination antifungal therapy: what can and should we expect?

Invasive fungal infections are associated with significant morbidity and mortality among immunocompromised patients. Recent advances in antifungal development have afforded us more pharmacologic compounds to choose from when managing these fungal infections. The role of combination antifungal therapy has been well established for fungal infections such as cryptococcal meningitis. The availability of new antifungals, increased incidence of mould infections and high mortality among certain affected populations, such as hematopoietic stem cell transplant recipients, has stimulated interest in the clinical use of combination antifungal therapy. In this paper, we review supporting evidence for the use of combination antifungals in the treatment of cryptococcal meningitis, invasive candidiasis, invasive aspergillosis and zygomycosis. Several controlled clinical trials have demonstrated benefits of combination antifungal approaches for patients with cryptococcal meningitis and invasive candidiasis, but variable effects when using different agents in combination have been reported. Randomized prospective studies of combination antifungal therapy in mould infections are lacking but some series provide supportive evidence for this approach. We also describe limitations of the data and these study designs, including the fact that we still need randomized controlled multicenter studies of combination antifungal therapy for mould infections. Trials in this area should be performed with efficiency and economics in mind, and could potentially use surrogate markers as end points. Therefore, we suggest future investigations of combination antifungal therapy should include a randomized, comparative trial of primary therapy for invasive aspergillosis.

The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle. The gene encoding a heterotrimeric G-protein beta subunit, GPB1, was cloned and disrupted. gpb1 mutant strains are sterile, indicating a role for this gene in mating. GPB1 plays an active role in mediating responses to pheromones in early mating steps (conjugation tube formation and cell fusion) and signals via a mitogen-activated protein (MAP) kinase cascade in both MATalpha and MATa cells. The functions of GPB1 are distinct from those of the Galpha protein GPA1, which functions in a nutrient-sensing cyclic AMP (cAMP) pathway required for mating, virulence factor induction, and virulence. gpb1 mutant strains are also defective in monokaryotic fruiting in response to nitrogen starvation. We show that MATa cells stimulate monokaryotic fruiting of MATalpha cells, possibly in response to mating pheromone, which may serve to disperse cells and spores to locate mating partners. In summary, the Gbeta subunit GPB1 and the Galpha subunit GPA1 function in distinct signaling pathways: one (GPB1) senses pheromones and regulates mating and haploid fruiting via a MAP kinase cascade, and the other (GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.

Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents.

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

Caspofungin for the treatment of azole resistant candidemia in a premature infant.

Candidemia is common in extremely low birth weight infants and is associated with substantial mortality and morbidity. Treatment options have traditionally been limited to amphotericin B deoxycholate or fluconazole. We present a case of a premature infant with persistent candidemia despite antifungal treatment that responded to therapy with caspofungin, an echinocandin antifungal. The infant's Candida isolate developed resistance to azoles during fluconazole administration and also suffered from severe hypercalcemia during the initiation of caspofungin therapy.

Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target.

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.

The STE12alpha homolog is required for haploid filamentation but largely dispensable for mating and virulence in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised hosts. The organism has a known sexual cycle, and strains of the MATalpha mating type are more virulent than isogenic MATa strains in mice, and they are more common in the environment and infected hosts. A C. neoformans homolog of the STE12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae and Candida albicans was identified previously, found to be encoded by a novel region of the MATalpha mating type locus, and shown to enhance filamentous growth when overexpressed. We have disrupted the C. neoformans STE12 gene in a pathogenic serotype A isolate. ste12 mutant strains exhibit a severe defect in filamentation and sporulation (haploid fruiting) in response to nitrogen starvation. In contrast, ste12 mutant strains have only modest mating defects and are fully virulent in two animal models compared to the STE12 wild-type strain. In genetic epistasis experiments, STE12 functions in a MAP kinase cascade to regulate fruiting, but not mating. Thus, the C. neoformans STE12alpha transcription factor homolog plays a specialized function in haploid fruiting, but it is dispensable or redundant for mating and virulence. The association of the MATalpha locus with virulence may involve additional genes, and other transcription factors that regulate mating and virulence remain to be identified.

Oropharyngeal yeast flora and fluconazole resistance in HIV-infected patients receiving long-term continuous versus intermittent fluconazole therapy.

OBJECTIVE: To examine the impact of continuous versus intermittent fluconazole therapy on fungal colonization and fluconazole resistance in the oropharynx of HIV-infected patients. DESIGN: Case-control study. SETTING: Duke University Adult Infectious Diseases Clinic, a tertiary referral center in North Carolina which provides care for 700 HIV-infected persons. PATIENTS: Nineteen HIV-infected patients on daily continuous fluconazole for a minimum of 6 months and eleven HIV-infected patients on intermittent fluconazole for a minimum of 6 months were matched by sex and CD4 cell count to HIV-infected patients who had not received fluconazole in the preceding 6 months. MAIN OUTCOME MEASURES: Fungal isolation and fluconazole susceptibility testing were performed on oral saline rinses from each patient. RESULTS: The patients taking continuous fluconazole were more likely than matched controls to have had sterile mouth rinses (14 out of 19 versus five out of 19; P < 0.001), and the yeasts that were isolated were more likely than matched controls to be non-Candida albicans species and to have minimum inhibitory concentrations (MIC) to fluconazole > or = 16 micrograms/ml. None of these isolates were associated with symptoms. In contrast, none of the patients in the intermittent fluconazole group had sterile cultures. When this group was compared to controls, they were more likely to have had non-C. albicans species, and the C. albicans isolates obtained had higher MIC to fluconazole. CONCLUSIONS: Long-term continuous therapy with fluconazole may prevent the appearance of Candida in the oral cavity. This finding may reduce recurrence rates and might favorably impact on the clinical appearance of mucosal candidiasis with resistant C. albicans.