Characterisation of Pneumocystis jirovecii DHPS genotypes using a simple, inexpensive restriction fragment length polymorphism analysis

Abstract

Pneumocystis jirovecii is an opportunistic fungal pathogen in immunocompromised patients, causing a life-threatening disease, Pneumocystis pneumonia (PCP). The introduction of highly active antiretroviral therapy (HAART) has significantly reduced the incidence of the disease; however, PCP remains one of the leading causes of pneumonia in immunosuppressed patients, especially in developing countries. The drug of choice for PCP treatment is trimethoprim-sulfamethoxazole. However, the widespread use of this drug has raised concerns regarding possible resistance due to dihydropteroate synthase (DHPS) mutations in P. jirovecii infected patients. Molecular epidemiological studies are needed to estimate the prevalence of mutant DHPS genotypes in PCP patients. We characterised genetic polymorphisms in the DHPS region of P. jirovecii using a rapid, cost-effective restriction fragment length polymorphism (RFLP) method. We used a nested PCR to amplify a DHPS fas gene segment, followed by digestion of the product with restriction endonucleases AccI and HaeIII that identified the presence of DHPS mutations at codons 55 and 57 respectively. A total of 671 samples from 585 patients was characterised by RFLP, with an overall 87% prevalence of DHPS mutations. Most samples had single mutations (50%), 28% had double mutations and the remaining (22%) showed mixed genotypes that could not be resolved by RFLP. These significant results highlight the need for ongoing molecular epidemiological studies on the relationship between P. jirovecii DHPS mutants and sulfa drug resistance.Copyright ©SAJID.