Rapid detection of dihydropteroate polymorphism in AIDS-related pneumocystis carinii pneumonia by restriction fragment length polymorphism

Sulpha agents, which act by inhibiting the enzyme dihydropteroate synthase (DHPS), are used widely for the treatment and prophylaxis of Pneumocystis c arinii pneumonia (PCP). Recently, we have shown that mutations in the dihyd ropteroate synthase (DHPS) gene of Pneumocystis carinii f.sp hominis are as sociated with failure of sulpha prophylaxis and increased mortality in HIV- 1 positive patients with PCP, suggesting that DHPS mutations may cause sulp ha resistance. To facilitate detection of DHPS mutations we developed a res triction fragment length polymorphism (RFLP) assay, detecting mutations at codon 55 and 57 of the P. carinii DHPS gene. The RFLP-assay was compared wi th direct DNA sequencing on 27 PCP isolates from HIV-1 positive patients wi th a mixture of wildtype and mutant DHPS types. In all samples the RFLP-ass ay correctly identified wildtype or DHPS mutation at codon 55 or 57. Combin ed with DNA extraction by a Chelex-based method, this method can be perform ed within 1 d and allows a fast, cost-efficient and reliable method of dete ction of DHPS mutations in P. carinii.