Biomedical subjects
Publications and source records attributed to W R Kirkpatrick.
Combinations of caspofungin and posaconazole were evaluated by fractional inhibitory concentration index against 119 Candida glabrata isolates. Synergy was seen in 18% of all isolates and in 4% of fluconazole-resistant isolates at 48 h without evidence of antagonism. This antifungal combination may have utility against this organism.
Candida glabrata has emerged as a common cause of fungal sepsis in bone marrow transplant patients, particularly those receiving fluconazole prophylaxis. Colonization of the lower GI tract and indwelling catheters have been thought to be the primary sources of systemic infection with Candida. We report on a bone marrow transplant patient who developed Candida glabrata sepsis from pre-existing oral colonization.
The antifungal susceptibilities of 79 oral Candida glabrata isolates to fluconazole and voriconazole were compared. The MICs at which 90% of the isolates tested were inhibited were 1 microg of voriconazole/ml and 32 microg of fluconazole/ml. Oral C. glabrata isolates for which the fluconazole MICs are elevated are commonly those for which the voriconazole MICs are elevated, but these increases may be transient for voriconazole, as they are for fluconazole.
Molecular mechanisms of azole resistance in Candida albicans include alterations in the target enzyme and increased efflux of drug, but the impact of specific treatment regimens on resistance has not been established. A patient with advanced AIDS was enrolled in a longitudinal study to receive continuous oral fluconazole (FLU) 200 mg/day for the treatment of oropharyngeal candidosis (OPC). Oral cultures were obtained at time of enrollment, during episodes of OPC and quarterly for surveillance. The patient had five symptomatic relapses on continuous FLU during 43 months. All OPC episodes were successfully treated with increasing doses of FLU although increased FLU MICs were detected for C. albicans isolates with progression of time. DNA-typing techniques demonstrated that resistance developed in a persistent strain of C. albicans. Both FLU-resistant and isogenic isolates with reduced susceptibility were detected in the same clinical samples through multiple episodes. Analysis of molecular mechanisms of resistance revealed overexpression of MDR and CDR genes encoding efflux pumps (but not ERG11) in isolates with decreased FLU susceptibility. In addition, the presence of the G464S amino acid substitution in their lanosterol demethylase, affecting its affinity for FLU, was also detected. However, other isogenic, but FLU-susceptible isolates recovered from the same samples did not harbour the mutation, indicating microevolution of yeast populations within the oral cavity. In this patient, the continuous antifungal pressure exerted by FLU resulted in development of resistance of multifactorial nature. Despite their clonal origin, different subpopulations of C. albicans demonstrated distinct resistance mechanisms, including concomitant presence and absence of functional point mutations in ERG11 genes.
Candida dubliniensis is a recently described species that has been shown to cause oropharyngeal candidiasis in patients with HIV. We present a detailed evaluation of a patient undergoing head and neck radiation for oral cancer who developed oropharyngeal candidiasis from a mixed infection of C dubliniensis and Candida albicans. To our knowledge, this is the first described case of C dubliniensis contributing to oropharyngeal candidiasis in this patient population.
Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in C. albicans strains displaying high-level fluconazole resistance (MICs, > or =64 microg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14alpha-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.
A review of representative cases of invasive aspergillosis was conducted to describe current treatment practices and outcomes. Eighty-nine physicians experienced with aspergillosis completed case forms on 595 patients with proven or probable invasive aspergillosis diagnosed using modifications of the Mycoses Study Group criteria. Pulmonary disease was present in 56%, with disseminated infection in 19%. The major risk factors for aspergillosis were bone marrow transplantation (32%) and hematologic malignancy (29%), but patients had a variety of underlying conditions including solid organ transplants (9%), AIDS (8%), and pulmonary diseases (9%). Overall, high antifungal failure rates occurred (36%), and complete antifungal responses were noted in only 27%. Treatment practices revealed that amphotericin B alone (187 patients) was used in most severely immunosuppressed patients while itraconazole alone (58 patients) or sequential amphotericin B followed by itraconazole (93 patients) was used in patients who were less immunosuppressed than patients receiving amphotericin B alone. Response rate for patients receiving amphotericin B alone was poor, with complete responses noted in only 25% and death due to or with aspergillosis in 65%. In contrast, patients receiving itraconazole alone or following amphotericin B had death due to or with Aspergillus in 26% and 36%, respectively. These results confirm that mortality from invasive aspergillosis in severely immunosuppressed patients remains high even with standard amphotericin B. Improved responses were seen in the less immunosuppressed patients receiving sequential amphotericin B followed by itraconazole and those receiving itraconazole alone. New approaches and new therapies are needed to improve the outcome of invasive aspergillosis in high-risk patients.
Voriconazole (VRC) was evaluated in an immunosuppressed-guinea pig model of invasive aspergillosis. VRC was more effective than amphotericin B or similar doses of itraconazole in the clearance of Aspergillus from tissues. VRC treatment regimens improved survival and significantly reduced tissue colony counts compared with those of controls.
SCH56592 (SCH) was evaluated in an immunosuppressed rabbit model of invasive aspergillosis. SCH was more effective than similar doses of itraconazole and as effective as amphotericin B in the clearance of Aspergillus spp. from tissues. Compared with controls, SCH regimens reduced mortality, improved survival, and significantly reduced tissue colony counts.
Seven isolates each of Candida albicans and Candida dubliniensis were paired (11 pairs) and examined for competitive interaction. Equal numbers of CFU of each competitor were inoculated into Sabouraud dextrose broth and incubated at 37 degrees C with vigorous shaking under conditions favorable to either broth or biofilm growth. Surviving proportions of each competitor were calculated from the broth culture at 24 and 96 h and the biofilm culture at 96 h, with species differentiation done on CHROMagar Candida medium. C. albicans had a competitive advantage over C. dubliniensis in broth culture and under biofilm growing conditions; however, with the presence of a supporting structure for biofilm formation, C. dubliniensis was able to better withstand the competitive pressures from C. albicans.
Three serial isolates of Candida albicans were obtained from each of five HIV infected patients with recurrent oropharyngeal candidiasis from the same geographical area. Isolates from one patient remained susceptible to fluconazole whereas serial isolates from the other four patients showed decreasing susceptibilities to the drug. Strain identity was investigated by pulse-field gel electrophoretic (PFGE) separation of chromosomes, restriction fragment length polymorphism (RFLP) of chromosomal DNA, Southern blot analysis with the moderately repetitive probe Ca3 of the materials present in the RFLP gels after transfer to nylon membranes, and random amplification of polymorphic DNA (RAPD). All techniques were able to group isolates obtained from the same patient. Techniques resulting in more complex banding profiles exhibited increased discriminatory power allowing detection of strain variants. Methods resulting in less complex banding patterns, especially Southern hybridization of SfiI digested chromosomal DNA with the moderately repetitive probe Ca3, were more helpful to determine isogenicity among isolates obtained from the same patient. The combination of results from methods with high discriminatory power (to maximize detection of strain variants) and methods resulting in less complex banding patterns (to allow determination of isogenic isolates) should facilitate the delineation of the epidemiology of C. albicans infection.
Mechanisms of resistance to azoles in Candida albicans, the main etiologic agent of oropharyngeal candidiasis (OPC), include alterations in the target enzyme (lanosterol demethylase) and increased efflux of drug. Previous studies on mechanisms of resistance have been limited by the fact that only a single isolate from each OPC episode was available for study. Multiple isolates from each OPC episode were evaluated with oral samples plated in CHROMagar Candida with and without fluconazole to maximize detection of resistant yeasts. A total of 101 isolates from each of three serial episodes of OPC from four different patients were evaluated. Decreasing geometric means of fluconazole MICs with serial episodes of infection were detected in the four patients. However, 8-fold or larger (up to 32-fold) differences in fluconazole MICs were detected within isolates recovered at the same time point in 7 of 12 episodes. Strain identity was analyzed by DNA typing techniques and indicated that isolates from each patient represented mainly isogenic strains, but differed among patients. A Northern blot technique was used to monitor expression of ERG11 (encoding lanosterol demethylase) and genes coding for efflux pumps. This analysis revealed that clinical isolates obtained from the same patient and episode were phenotypically heterogeneous in their patterns of expression of these genes involved in fluconazole resistance. These results demonstrate the complexity of the distribution of the molecular mechanisms of antifungal drug resistance and indicate that different subpopulations of yeasts may coexist at a given time in the same patient and may develop resistance through different mechanisms.
Three serial isolates of Candida albicans were obtained by direct swab or by oral saline rinses from each of five human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis. Genotyping techniques confirmed the presence of a persistent strain in multiple episodes from the same patient, which was different from the strains isolated from other patients. Fluconazole susceptibility was determined by both an agar dilution method and the National Committee for Clinical Laboratory Standards macrobroth procedure. In four of these patients the strains developed fluconazole resistance, and in one patient the strain remained susceptible. The different isolates were propagated as yeast cells on a synthetic medium, and their cell wall proteinaceous components were extracted by treatment with beta-mercaptoethanol. Protein and mannoprotein components present in the extracts were analyzed by electrophoresis, immunoblotting, and lectin-blotting techniques. The analysis showed a similar composition, with only minor qualitative and quantitative differences in the polypeptidic and antigenic patterns associated with the cell wall extracts from serial isolates from the same patient, as well as those from different strains isolated from different patients. Use of monospecific antibodies generated against two immunodominant antigens during candidiasis (enolase and the 58-kDa fibrinogen-binding mannoprotein) demonstrated their expression in all isolates tested. Overall, the antigenic makeup of C. albicans strains remained constant during the course of infection and was not affected by development of fluconazole resistance. In contrast to previous reports, the low degree of antigenic variability observed in this study may be due to the fact that the isolates were obtained from a highly homogeneous population of patients and to the uniformity in techniques used for the isolation, storage, and culture of the different strains, as well as extraction methodologies.
Oral mucosal colonization and infection with Candida are common in patients receiving radiation therapy for head and neck cancer. Infection is marked by oral pain and/or burning and can lead to significant patient morbidity. The purpose of this study was to identify Candida strain diversity in this population by using a chromogenic medium, subculturing, molecular typing, and antifungal susceptibility testing of clinical isolates. These results were then correlated with clinical outcome in patients treated with fluconazole for infection. Specimens from 30 patients receiving radiation therapy for head and neck cancer were cultured weekly for Candida. Patients exhibiting clinical infection were treated with oral fluconazole. All isolates were plated on CHROMagar Candida and RPMI medium, subcultured, and submitted for antifungal susceptibility testing and molecular typing. Infections occurred in 27% of the patients and were predominantly due to Candida albicans (78%). Candida carriage occurred in 73% of patients and at 51% of patient visits. Yeasts other than C. albicans predominated in carriage, as they were isolated from 59% of patients and at 52% of patient visits. All infections responded clinically, and all isolates were susceptible to fluconazole. Molecular typing showed that most patients had similar strains throughout their radiation treatment. One patient, however, did show the acquisition of a new strain. With this high rate of infection (27%), prophylaxis to prevent infection should be evaluated for these patients.
PURPOSE: The effects of continuous or intermittent therapy with fluconazole on the recurrence of and the development of fluconazole resistance are not known. PATIENTS AND METHODS: We studied human immunodeficiency virus (HIV)-positive patients with CD4 cell count <350 x 10(6)/L and oropharyngeal candidiasis in a prospective, randomized study. After initial treatment, 20 patients (16 of whom completed 3 months of follow-up) received continuous fluconazole at 200 mg/day, and 48 patients (28 of whom completed follow-up) received intermittent therapy at the time of symptomatic relapses. Oral samples were obtained weekly during episodes of infection and quarterly as surveillance cultures. Development of resistance was defined as a fourfold rise in minimum inhibitory concentration (MIC) to at least 16 microg/mL from the initial culture in the same species, the emergence of new, resistant (MIC > or =16 microg/mL) species, or a significant increase in the proportion of resistant isolates. RESULTS: During a mean follow-up of 11 months, median annual relapse rates were lower in patients on continuous therapy (0 episodes/year) than in patients on intermittent therapy (4.1 episodes/year; P <0.001). Sterile cultures were seen in 6 of 16 (38%) patients on continuous therapy compared with 3 of 28 (11%) on intermittent therapy (P = 0.04). Microbiological resistance developed in 9 of 16 (56%) patients on continuous treatment, compared with 13 of 28 (46%) on intermittent treatment (P = 0.75). However, despite isolates with increased MICs, 42 of 44 patients responded to fluconazole in doses up to 800 mg/day. CONCLUSIONS: In patients with frequent recurrences, continuous suppressive therapy significantly reduced relapses and colonization. Resistance occurred with both continuous and intermittent therapy; however, therapeutic responses were excellent.
Signs and symptoms of oropharyngeal candidiasis (OPC) were correlated with microbiology and clinical response to fluconazole in a cohort of patients with advanced human immunodeficiency virus (HIV) infection and recurrent OPC. Sixty-four HIV-infected patients with a median CD4 cell count of < 50/mm3 (range, 3-318/mm3) who presented with OPC were enrolled in a longitudinal study. Specimens for cultures were taken weekly until clinical resolution. Therapy with fluconazole was increased weekly as required to a maximum daily dose of 800 mg until resolution of symptoms and oral lesions. Resistant or dose-dependent susceptible yeasts, defined as a minimum inhibitory concentration of > or = 16 micrograms/mL, were detected in 48 (31%) of 155 episodes. Clinical resolution with fluconazole therapy occurred in 107 (100%) of 107 episodes with susceptible yeasts vs. 44 (92%) of 48 episodes with resistant or dose-dependent susceptible strains (P = .008). Patients from whom fluconazole-resistant yeasts were isolated required longer courses of therapy and higher doses of fluconazole for response, but overall, excellent responses to fluconazole were seen in patients with advanced HIV infection.
Resistance to fluconazole is becoming an increasing problem in the management of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Strains obtained from five patients developed decreased fluconazole susceptibility over time. DNA strain typing confirmed the high degree of relatedness among isolates from one patient and the variability among isolates from different patients. Expression of genes involved in development of fluconazole resistance was monitored in each isolate using probes specific for ERG11 (lanosterol 14alpha-demethylase), MDR1 (a major facilitator), and CDR (ATP-binding cassette or ABC transporter) genes. Increased expression of CDR genes was detected in the series of isolates from two patients. Isolates from one of the two patients also demonstrated increased ERG11 expression, whereas isolates from the other patient did not. Increased levels of MDR1 mRNA correlated with increased resistance in sequential isolates from another patient. Initial overexpression of MDR1 with subsequent overexpression of CDR genes and a final isolate again overexpressing MDR1 were detected in serial isolates from another patient. In another patient, overexpression of these genes was not detected despite an eightfold increase in fluconazole MIC. In this patient, sequence data of the ERG11 gene revealed no point mutations associated with decreased susceptibility. Five different patterns of gene expression were observed in isolates recovered from five patients who developed resistance. Therefore, these experiments demonstrate that a variety of mechanisms or combinations of mechanisms are associated with the development of fluconazole drug resistance. Additional studies are needed to estimate the frequency and clinical impact of these mechanisms of resistance.
A murine model of systemic candidiasis was used to assess the virulence of serial Candida albicans strains for which fluconazole MICs were increasing. Serial isolates from five patients with 17 episodes of oropharyngeal candidiasis were evaluated. The MICs for these isolates exhibited at least an eightfold progressive increase from susceptible (MIC < 8 microg/ml; range, 0.25 to 4 microg/ml) to resistant (MIC >/= 16 microg/ml; range, 16 to >/=128 microg/ml). Virulence of the serial isolates from three of five patients showed a more than fivefold progressive decrease in the dose accounting for 50% mortality and was associated with development of fluconazole resistance. Low doses of fluconazole prolonged survival of mice infected with susceptible yeasts but failed to prolong survival following challenge with a resistant strain. In addition, a decreased burden of renal infection was noted in mice challenged with two of the three resistant strains. This was consistent with reduced virulence. Fluconazole did not further decrease the level of infection. In the isolates with a decrease in virulence, two exhibited overexpression of CDR, which encodes an ABC drug efflux pump. In contrast, serial isolates from the remaining two patients with the development of resistance did not demonstrate a change in virulence and fluconazole remained effective in prolonging survival, although significantly higher doses of fluconazole were required for efficacy. Resistant isolates from both of these patients exhibited overexpression of MDR. This study demonstrates that decreased virulence of serial C. albicans isolates is associated with increasing fluconazole MICs in some cases but not in others and shows that these low-virulence strains may not consistently cause infection.