Quantitation of Candida albicans ergosterol content improves the correlation between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive candidiasis.

MIC end point determination for the most commonly prescribed azole antifungal drug, fluconazole, can be complicated by "trailing" growth of the organism during susceptibility testing by the National Committee for Clinical Laboratory Standards approved M27-A broth macrodilution method and its modified broth microdilution format. To address this problem, we previously developed the sterol quantitation method (SQM) for in vitro determination of fluconazole susceptibility, which measures cellular ergosterol content rather than growth inhibition after exposure to fluconazole. To determine if SQM MICs of fluconazole correlated better with in vivo outcome than M27-A MICs, we used a murine model of invasive candidiasis and analyzed the capacity of fluconazole to treat infections caused by C. albicans isolates which were trailers (M27-A MICs at 24 and 48 h, /=64 microg/ml, respectively; SQM MIC, /=64 microg/ml; SQM MIC, 54 microg/ml). Compared with the untreated controls, fluconazole therapy increased the survival of mice infected with a sensitive isolate and both trailing isolates but did not increase the survival of mice infected with a resistant isolate. These results indicate that the SQM is more predictive of in vivo outcome than the M27-A method for isolates that give unclear MIC end points due to trailing growth in fluconazole.

Itraconazole resistance in Aspergillus fumigatus.

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

Fungal infections: a survey of laboratory services for diagnosis and treatment.

A questionnaire on the services provided and the methods used for the diagnosis of fungal infections and for the support of antifungal chemotherapy was sent to members of the British Society for Medical Mycology (BSMM) and the British Society for Antimicrobial Chemotherapy (BSAC). Ninety-five responses from general microbiology laboratories in the United Kingdom were analysed, and we compared services provided by laboratories that serve a transplant unit with those offered by other laboratories. We estimate that about 150 cases of cryptococcosis, 500 to 600 of candidaemia, and 300 to 400 of invasive aspergillosis are identified by laboratories in the United Kingdom (UK) each year. The clinical laboratories are aware of the importance of fungal infection, but rely heavily on reference services. In some laboratories, however, the degree of investigation of specimens and the procedures in use are inadequate for diagnosing systemic mycoses and determining the susceptibility of isolates to antifungal agents. The balance between reference and local services requires attention and external quality assurance needs to be applied effectively. In addition, effective methods for the diagnosis of systemic mycoses, and reliable and practicable methods for determining the susceptibility of isolates to antifungal agents, are needed urgently.

Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis.

We examined the effect of different fluconazole treatment regimens on the emergence of azole drug resistance among Candida species recovered from the mouths of 54 HIV-infected individuals. Patients were assigned to one of three treatment groups depending on their history of oral candidosis and fluconazole use. Mouthwashes obtained at regular intervals were cultured and isolates identified using standard methods. Antifungal broth micro-dilution tests were performed to determine IC30s of fluconazole and ketoconazole. Sixty-four Candida albicans isolates from 20 patients with no evidence of oral candidosis who had not received fluconazole all had IC30s of < or = 4 mg/L. Thirty-four (83%) of 41 C. albicans isolates from ten patients receiving intermittent, short-term fluconazole treatment for oral candidosis had IC30s of < or = 4 mg/L, but only two isolates (5%) had IC30s > or = 64 mg/L. In contrast, 26 (40%) of 65 C. albicans isolates from 15 patients given long-term fluconazole (50-200 mg/day or 150 mg/week) were classified as resistant having IC30s of fluconazole of > or = 64 mg/L. Ten of these 26 fluconazole-resistant isolates were susceptible to ketoconazole with IC30s of < or = 4 mg/L suggesting azole drug cross-resistance is not inevitable. Tests on multiple colonies from individual isolation plates showed that it was not unusual to obtain differing IC30 values, indicating that a sweep inoculum is essential if resistance is to be detected. Nine (60%) of the 15 patients given long-term fluconazole harboured isolates of C. albicans that were resistant to fluconazole at some time during the study period. All had low CD4 counts and were approaching the final stage of their illness. Three patients on long-term treatment had resistant organisms at the outset of the study; in the remainder, resistant strains emerged during the study period. In six of the nine cases, emergence of resistance in vitro correlated with persistent clinical signs of oral infection. Thirty-six isolates of Candida species other than C. albicans were also recovered from patients receiving long-term fluconazole and 29 (81%) of these had IC30s of > or = 64 mg/L. Our experience with C. albicans in patients with HIV infection, suggests that the long-term azole drug use may be an important factor in the development of fluconazole resistance as such resistance was rare and transient in patients on intermittent short-term treatment.

Management of persistent vulvo vaginal candidosis due to azole-resistant Candida glabrata.

CASE REPORT--SUBJECTS--Three cases are described of long-standing vaginal candidosis due to Candida glabrata. These had failed to respond to local and systemic antifungals. In each case the infecting strain appeared resistant to a range of azole drugs in vitro. CLINICAL COURSE--Case one--This patient recovered following prolonged treatment with oral itraconazole in combination with oral and vaginal nystatin. Case two. Yeasts were eradicated from this patient following cyclical treatment with oral dydrogesterone; prolonged vaginal treatment with nystatin may have helped. Case three. This patient did not respond to a prolonged course of oral itraconazole in combination with vaginal and oral nystatin, oral medroxyprogesterone or intravaginal boric acid. Eradication of C glabrata was finally achieved by local application of 1% gentian violet. Shortly after eradication of the C glabrata infection, both Case two and Case three developed infections with other Candida species responsive to azole antifungals.

Effect of simultaneous oral and vaginal treatment on the rate of cure and relapse in vaginal candidosis.

One hundred patients with vulvovaginal candidosis were entered in a double-blind trial to compare the effect of six days' local treatment with clotrimazole with that of the same treatment plus 10 days' oral treatment with nystatin. No significant differences were detected in the rate of cure or relapse between the treatment groups. The cure rate was lowest and the relapse rate highest in patients in whom vaginal candidosis had last been diagnosed during the preceding 12 months.

Grass pollen content of the air in the Bristol Channel region in 1976.

The grass pollen content of the air at four sites bordering the Bristol Channel (at Bristol, Cardiff, Penarth and Penmaen) was studied. The trap at Penmaen was situated at an official climatological station which provided the meteorological data used in the surveys. Although at certain times large differences could be seen between the concentrations of grass pollen at the different sites, overall the findings for the four sites were similar. Changes in the atmospheric pollen concentration related to variations in meteorological conditions. At the peak of the season high concentrations of grass pollen persisted throughout the night.