Investigation of genotypic variations and sulfonamide drug resistance in isolats of pneumocystis jirovecii

Pneumocystis jirovecii (P. jirovecii) is a human-specific species known as Pnemocystis jirovecii pneumonia (PCP) that can cause severe interstitial pneumonia, which can be fatal, in immunocompromised individuals. Direct microscopy is the gold standard in the diagnosis of the organism. In cases where low organism load is observed, molecular methods such as PCR are also needed. In our study, mtLSU-rRNA and DHPS gene polymorphism in P. jirovecii isolates were investigated from Turkey's Izmir province and its surroundings. For this purpose, a total of 157 P. jirovecii isolates obtained from BAL samples of patients of hospital origin and lung tissue samples of autopsy cases were examined. After nested PCR, mtLSU-rRNA genotyping was performed by Sanger sequencing and DHPS polymorphism was evaluated by RFLP method. Among the 138 P. jirovecii isolates that can be genotyped, mtLSU-rRNA genotype was found as the most common genotype with 2, 58%. The most common genotypes found after genotype 2 were genotype 3 (13%), genotype 1 (11.6%) and genotype 4 (5.1%), while genotype 5 (0.7%) was detected in only one autopsy sample. In addition, 16 (11.6%) cases had double or triple coinfections. It was observed that features such as age, gender, immune status and PCP/colonization difference did not affect genotype distribution. But, genotype 2 dominance in solid organ tumors and mixed infection height in patients with lung disease were statistically significant. DHPS gene was reproduced in 137 (87.3%) of 157 samples included in the study. As a result of fragment lenght polymorphism (RFLP), no mutation was found in 135 samples (wild type). In two hospital-based samples (1.5%), mixed infection (wild type + mut 57) with wild type and 57th codon mutations were detected. In this study, it was shown time that DHPS gene mutations associated with drug resistance can be found in our society. In addition, important epidemiological data on the distribution of mtLSU-rRNA genotypes were obtained. On the other hand, our study demonstrated that real-time PCR is the effective method in the diagnosis of P. jirovecii laboratory. Conventional n-PCR method was found to be is less successful than real-time PCR in the presence of very low density organism.