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Journal of Clinical Microbiology, August 1998, p. 2308-2313, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Random Amplification of Polymorphic DNA and Microsatellite Genotyping of Pre- and Posttreatment Isolates of Candida spp. from Human Immunodeficiency Virus-Infected Patients on Different Fluconazole Regimens

David Metzgar,1 Alex van Belkum,2,* Dawn Field,1 Richard Haubrich,3 and Christopher Wills4

Department of Biology1 and Center for Molecular Genetics,4 University of California at San Diego, La Jolla, California 92093-0116; Department of Medicine, University of California at San Diego Treatment Center, San Diego, California 921033; and Department of Medical Microbiology & Infectious Diseases, Erasmus Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands2

Received 31 December 1997/Returned for modification 12 March 1998/Accepted 26 May 1998

Twelve patients infected with the human immunodeficiency virus (HIV) and with CD4 cell counts below 100 cells/µl received fluconazole daily (200 mg; five patients) or weekly (400 mg; seven patients) for fungal 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 was 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 loci (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 daily. Genetic analyses allowed three different scenarios to be discerned. Resistance development in an otherwise apparently unchanged strain was seen for 1 of the 12 patients. Clear strain replacement was observed for 3 of the remaining 11 patients. For all other patients minor differences were seen in either the RAPD genotype or the microsatellite allele composition during the course of treatment. In general, microsatellite sequence data is in agreement with data obtained by other methods, but occasionally within-patient heterogeneity is indicated. The present results show that during fluconazole treatment colonizing strains can remain identical, be replaced by clearly different strains, or undergo small changes. Within a patient there may be different levels of intrastrain variation.

* Corresponding author. Mailing address: Erasmus Medical Center Rotterdam, Department of Medical Microbiology & Infectious Diseases, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 31-10-4635813. Fax: 31-10-4633875. E-mail: vanbelkum{at}bacl.azr.nl.

Journal of Clinical Microbiology, August 1998, p. 2308-2313, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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