A novel high-throughput cellular screening assay for the discovery of HIV-1 integrase inhibitors.

The discovery of HIV-1 integrase inhibitors has been enabled by high-throughput screening and rational design of novel chemotypes. Traditionally, biochemical assays focusing on the strand transfer activity of integrase have been used to screen compound libraries for identification of novel inhibitors. In contrast, cellular screening assays enable a phenotypic or multi-target approach, and may result in identification of compounds inhibiting integrase in its natural context, the pre-integration complex. Furthermore, a cellular assay encompassing 3' processing, strand transfer and nuclear import may lead to the identification of compounds with novel mechanisms of action targeting cellular and viral factors. Therefore, a cellular screening assay was developed, which focused on integrase activity, where infection of MT4 cells with an HIV-1 based lentiviral vector was synchronized by temporary arrest at the reverse transcriptase step and subsequent release to enable integration. The assay was validated using a panel of antivirals and proved to be a robust cellular screening assay for the identification of novel integrase inhibitors.

Prevalence of Candida dubliniensis isolates in a yeast stock collection.

To establish the historical prevalence of the novel yeast species Candida dubliniensis, a survey of 2,589 yeasts originally identified as Candida albicans and maintained in a stock collection dating back to the early 1970s was undertaken. A total of 590 yeasts, including 93 (18.5%) beta-glucosidase-negative isolates among 502 isolates that showed abnormal colony colors on a differential chromogenic agar and 497 other isolates, were subjected to DNA fingerprinting with the moderately repetitive sequence Ca3. On this basis, 53 yeasts were reidentified as C. dubliniensis (including the C. dubliniensis type strain, included as a blind control in the panel of yeasts). The 52 newly found isolates came from 36 different persons, and a further 3 C. dubliniensis isolates were detected by DNA fingerprinting of previously untested isolates from one of these individuals. The prevalence of C. dubliniensis among yeasts in oral and fecal samples was significantly higher than that among yeasts from other anatomical sites and was significantly higher among human immunodeficiency virus (HIV)-infected individuals than among known or presumed HIV-negative individuals. However, a single vaginal isolate and two oral isolates from healthy volunteers confirmed that the species is restricted neither to gastrointestinal sites nor to patients with overt disease. The oldest examples of C. dubliniensis were from oral samples of three patients in the United Kingdom in 1973 and 1975. In comparison with age-matched control isolates of C. albicans, the C. dubliniensis isolates showed slightly higher levels of susceptibility in vitro to amphotericin B and flucytosine and slightly lower levels of susceptibility to three azole antifungal agents.

Susceptibilities of Candida spp. to antifungal agents visualized by two-dimensional scatterplots of relative growth.

The growth of 811 clinical yeast isolates in the presence of single concentrations of antifungal agents was measured spectrophotometrically and expressed as a percentage of growth in inhibitor-free control cultures. Two-dimensional scatterplots of the relative growth data allowed for the simple visual determination of some susceptibility trends, including correlations in relative growth between different agents and in relative susceptibilities between different yeast species. A positive susceptibility correlation was found for relative growth results with the azole antifungal agents fluconazole, itraconazole, and ketoconazole for 504 Candida albicans isolates. The relative growth scatterplots for fluconazole versus itraconazole showed that 50 (9.9%) of 504 C. albicans isolates were outliers with respect to the 95% confidence limits for a line of correlated relative growth established with an initial test panel of 59 isolates of this species. The outlying isolates were relatively less susceptible to fluconazole than to itraconazole under the conditions of the test. Most of the outliers were received in 1993 and 1994; only 3.9% of the isolates received in 1991 and 1992 and 1.7% of the isolates received before 1991 showed this differential susceptibility. In addition, most of the outliers came from patients with human immunodeficiency virus infections. The relative growth scatterplots confirmed the known high susceptibility of most Candida parapsilosis isolates to both fluconazole and itraconazole and the specifically low susceptibility of Candida krusei isolates to fluconazole. The scatterplots also illustrated a tendency towards lower (and correlative) relative growth among oral isolates obtained from AIDS patients who responded to azole antifungal treatment than among isolates from clinical nonresponders.

Oxygen as limiting nutrient for growth of Cryptococcus neoformans.

In microbroth cultures with RPMI 1640 medium, the growth yield of seven Cryptococcus neoformans isolates was unaffected by augmentation of the normal (0.2%) glucose concentration in the medium to 2%, and the addition of other potential carbon, nitrogen, and vitamin sources to the medium also failed to produce large changes in growth yield. However, macrobroth cultures of C. neoformans in RPMI 1640 that were agitated by rotation in air gave turbidities 6 to 37 times greater than those in identical cultures incubated statically, and similar levels of increase were seen whether the medium contained 0.2 or 2% glucose. Incubation of microplates under an oxygen atmosphere or with agitation by rotation led to an increase of up to twofold in growth turbidity of the yeast. The maximum increase was achieved by incubation with rotation and was dependent on the brand of microplate used. The findings implicate oxygen as a growth-limiting nutrient for C. neoformans. Incubation of microbroth cultures under conditions that enhance oxygen availability for antifungal susceptibility testing purposes may increase the speed of such tests and enhance the determination of MIC endpoints.