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Antimicrobial Agents and Chemotherapy, March 2001, p. 776-780, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.776-780.2001

Rapid Detection of Mutations in the Human-Derived Pneumocystis carinii Dihydropteroate Synthase Gene Associated with Sulfa Resistance

Liang Ma and Joseph A. Kovacs^*

Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland

Received 27 September 2000/Returned for modification 27 November 2000/Accepted 11 December 2000

Recent studies have shown that point mutations in the dihydropteroate synthase (DHPS) gene of human-derived Pneumocystis carinii are related to exposure to sulfa drugs and possibly represent the emergence of sulfa resistance. We developed a simple single-strand conformation polymorphism (SSCP) method to permit rapid detection of these mutations. With plasmid constructs, SSCP was able to detect as little as 10% of a minority population. The SSCP assay was compared to direct sequencing for typing the DHPS gene by examining 37 clinical isolates with known DHPS sequences and 41 clinical isolates with unknown DHPS sequences. The typing results were consistent between these two methods for all isolates except 11 in which mutations were detected by SSCP but not by direct sequencing. Sequencing of individual clones after subcloning confirmed the presence of mutations in a minority population as determined by SSCP. SSCP is a very simple and sensitive method for rapid identification of P. camii DHPS mutations.

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^* Corresponding author. Mailing address: Building 10, Room 7D43, National Institutes of Health, 10 Center Dr. MSC 1662, Bethesda, MD 20892-1662. Phone: (301) 496-9907. Fax: (301) 402-1213. E-mail: jkovacs{at}nih.gov.

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Antimicrobial Agents and Chemotherapy, March 2001, p. 776-780, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.776-780.2001

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• Zingale, A., Carrera, P., Lazzarin, A., Scarpellini, P. (2003). Detection of Pneumocystis carinii and Characterization of Mutations Associated with Sulfa Resistance in Bronchoalveolar Lavage Samples from Human Immunodeficiency Virus-Infected Subjects. J. Clin. Microbiol. 41: 2709-2712 [Abstract] [Full Text]  
• Ma, L., Kovacs, J. A. (2001). Genetic Analysis of Multiple Loci Suggests that Mutations in the Pneumocystis carinii f. sp. hominis Dihydropteroate Synthase Gene Arose Independently in Multiple Strains. Antimicrob. Agents Chemother. 45: 3213-3215 [Abstract] [Full Text]