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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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