RANDOM AMPLIFICATION OF POLYMORPHIC DNA AND MICROSATELLITE GENOTYPINGOF PRETREATMENT AND POSTTREATMENT ISOLATES OF CANDIDA SPP. FROM HUMANIMMUNODEFICIENCY VIRUS-INFECTED PATIENTS ON DIFFERENT FLUCONAZOLE REGIMENS

Twelve patients infected with the human immunodeficiency virus (HIV) a nd with CD4 cell counts below 100 cells/mu l received fluconazole dail y (200 mg; five patients) or weekly (400 mg; seven patients) for funga l prophylaxis during a 6-month period. Oropharyngeal swabs were taken at regular intervals in order to detect colonization with Candida spp. All yeast isolates were examined with respect to the development over time of fluconazole resistance. Genetic diversity among the strains w as assessed in order to discriminate between selection of a resistant subclone and patient recolonization. Genotyping was performed through random amplification of polymorphic DNA (RAPD) analysis. Specific site polymorphisms were assayed by tracking length variability in several microsatellite loci. Finally, to maximize resolution, one of these loc i (ERK1) was analyzed by nucleotide sequencing. Although the number of strains analyzed was too small to allow statistical verification, it appeared that when fluconazole was given weekly, a smaller fraction of the strains showed diminished sensitivity than when it was given dail y. Genetic analyses allowed three different scenarios to be discerned. Resistance development in an otherwise apparently unchanged strain wa s seen for 1 of the 12 patients. Clear strain replacement was observed for 3 of the remaining 11 patients. For all other patients minor diff erences were seen in either the RAPD genotype or the microsatellite al lele composition during the course of treatment. In general, microsate llite sequence data is in agreement with data obtained by other method s, but occasionally within-patient heterogeneity is indicated. The pre sent results show that during fluconazole treatment colonizing strains can remain identical, be replaced by clearly different strains, or un dergo small changes. Within a patient there may be different levels of intrastrain variation.