No abstract

We investigated the possible presence of DNA specific for Aspergillus species in serum samples of patients with invasive aspergillosis (IA) by the nested PCR method. Fourteen strains of fungi including 5 strains of Aspergillus species and 10 strains of common bacteria were used for examination of specificity and sensitivity of the nested PCR. Two sets of oligonucleotide primers were derived from the sequence of the variable regions V7 to V9 of the 18S rRNA genes of Aspergillus fumigatus. The specific fragment was amplified from five strains of Aspergillus species in the single and nested PCR but not from other microorganisms. Target DNA was detected by the nested PCR with as little as 50 fg of the extracted DNA of A. fumigatus. We investigated the detection of DNA specific for Aspergillus species in serum samples of a murine model of aspergillosis and 20 patients with IA. The specific fragment was detected by the nested PCR in 71% of serum samples of infected mice and 70% of serum samples of patients with IA, while galactomannan antigen was detected in 43 and 60% of samples, respectively. The high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for immunocompromised patients with IA.

The G test containing factor G, fractioned from the Limulus lysate, was used to detect (1-3)-beta-D-glucan in a rat model of aspergillosis. Aspergillus fumigatus strain MF-13, 1 x 10(4) conidia, were inoculated transtracheally into rats treated with cortisone acetate (100 mg/kg) and fed a low-protein (8%) diet. Increased serum (1-3)-beta-D-glucan was found on the sixth day after inoculation in concentrations of 370 +/- 178 pg/ml (mean +/- SD) in untreated controls, and 154 +/- 43 pg/ml in rats treated with 0.5 mg/kg of amphotericin B. On day 11 (1-3)-beta-D-glucan concentrations were 2,590 +/- 2,940 pg/ml and 448 +/- 442 pg/ml, respectively. The elevation in levels of (1-3)-beta-D-glucan increased in correlation with the elevation of galactomannan antigen titers; (1-3)-beta-D-glucan is thus measurable during experimental aspergillosis in rats.

The activities of amphotericin B, miconazole, fluconazole, and itraconazole against Trichosporon beigelii were assessed in a mouse model of disseminated infection. Cyclophosphamide plus prednisolone-immunosuppressed ICR mice, intravenously challenged with a lethal inoculum of (6 × 10⁶ CFU/mouse), were assigned to receive 7 days of therapy with amphotericin B (0.5 or 2 mg/kg/day), miconazole (10 or 40 mg/kg/day), fluconazole (10 or 40 mg/kg/day), or itraconazole (10 and 40 mg/kg/day). The efficacy of a combination of amphotericin B (1 mg/kg/day) with fluconazole (10 mg/kg/day) or itraconazole (20 mg/kg/day) with that of each agent alone was also compared. Both amphotericin B and azoles improved survival and reduced the fungal counts in kidneys of infected mice in a dose-dependent pattern. In general, fluconazole was superior to amphotericin B and the other azoles, whereas the latter two drugs were as effective as amphotericin B. The activity of amphotericin B combined with fluconazole appeared to be superior to that of each agent alone, especially in reducing the organ fungal burden. The other combination (amphotericin B plus itraconazole) had a weaker effect, but no antagonism was observed. In conclusion, azoles may be an alternative to amphotericin B for the treatment of T. beigelii infection. Furthermore, their combination with amphotericin B may improve the poor outcome seen in profoundly neutropenic patients with disseminated trichosporonosis.

The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B, flucytosine, miconazole, fluconazole and itraconazole against 21 isolates of Trichosporon beigelii in RPMI-1640 medium were determined using National Committee for Clinical Laboratory Standards (NCCLS) methodology in microdilution method. Most isolates were sensitive to miconazole (MIC90 0.78 microgram/ml), fluconazole (MIC90 6.25 micrograms/ml), and itraconazole (MIC90 0.19 microgram/ml), with the former being the most active agent tested (MFC90 3.12 mu/ml). Although amphotericin B inhibited most strains (MIC range, 0.78-3.12 micrograms/ml), poor fungicidal activity was observed (MFC range, 1.56-12.5 micrograms/ml) showing a pattern of relative resistance in vitro. Flucytosine showed generally poor activity against most isolates tested. These in vitro findings confirm the resistance of T.beigelii to amphotericin B and suggest that azoles may be an alternative to the former for the treatment of disseminated trichosporonosis. However, in vivo studies would better validate these in vitro findings.