Prevalence and genotyping of Pneumocystis jirovecii in renal transplant recipients-preliminary report Author Szydłowicz, Magdalena Author Jakuszko, Katarzyna Author Szymczak, Anna Author Piesiak, Paweł Author Kowal, Aneta Author Kopacz, Żaneta Author Wesołowska, Maria Author Lobo, Maria Luísa Author Matos, Olga Author Hendrich, Andrzej B. Author Kicia, Marta text Parasitology Research 2019 181 2018-11-03 118 1 181 189 http://dx.doi.org/10.1007/s00436-018-6131-0 journal article 298235 10.1007/s00436-018-6131-0 2ef6c7d0-8965-433b-8b05-a9afefaf4f42 1432-1955 PMC6329730 30392033 11376753 P. jirovecii prevalence and patients ’ characteristics Among all renal transplant recipients tested ( n = 72, including 36 males , 36 females ), the mean age was 52.5 ± 13.9 years, range 21–76 years. The mean time after kidney transplantation was 78.7 months, ranging from 5 days to 19 years. Nine patients had undergone the second kidney transplantation, and two had undergone the third transplantation. One patient had undergone both kidney and heart transplantation. Immunosuppressive treatment included prednisone, calcineurin inhibitors (tacrolimus or cyclosporine), proliferation signal inhibitors (sirolimus or everolimus), mycophenolate mofetil, or azathioprine. Forty-four (61.1%) patients were receiving treatment combining three of the above drugs, 25 (34.7%) were receiving a dual combination, and 3 (4.2%) were treated with one type of immunosuppressant only (patients who required dialysis after kidney transplant failure). The routine anti- Pneumocystis prophylaxis regimen contained co-trimoxazole at a dose of 480 mg a day for 6 months after transplantation. Among all patients examined, 13 were receiving co-trimoxazole prophylaxis during sputum collection, and one patient was receiving only trimethoprim due to a previous allergic reaction to sulfamethoxazole. Nested PCR amplifying the mtLSU rRNA gene of P. jirovecii was positive in eight of the 72 (11.11%) patients’ samples. Three of these eight patients showed symptoms compatible of pneumonia (including low-grade fever, dyspnea, and radiological presentation). In samples from only three patients (37.5%), P. jirovecii presence was confirmed by IF staining, and only in one of these cases the number of cysts observed on the whole microscope slide was above five. That was the only case with both a positive result of IF staining and the presence of respiratory symptoms typical for PcP. Anti- Pneumocystis therapy was introduced in this patient, in view of PcP suspicion. Due to the patient’ s allergy to sulfamethoxazole, it consisted of intravenous clindamycin ( 900 mg every 8 h on the first day and 600 mg every 6 h afterwards) and primaquine for 29 days. The treatment was completed after the negative result of control PCR analysis and pentamidine was introduced for prevention. The most important characteristics of P. jirovecii -positive and P. jirovecii -negative patients are listed in Table 1. A statistically significant correlation with colonization was observed for the employment of a dual immunosuppressive regimen consisting of calcineurin inhibitors and prednisone ( P = 0.041, Fisher’ s exact test). Moreover, the mean eosinophil level was lower in P. jirovecii- positive patients, as compared to negative ones ( P = 0.040, Student’ s t test). There were no significant differences in results of basic laboratory tests for other parameters, CMV infection, or other co-morbidities. Multilocus typing Only mtLSU rRNA was fully genotyped in all analyzed specimens. The ratios of efficient amplification of the other genetic fragments were 50% for SOD , 62.5% for DHPS , and 87.5% for CYB ( Table 2 ). Since multilocus genotype is a combination of at least two loci, such complex analysis was not possible due to incomplete data required. Therefore, single genetic fragments were analyzed individually in order to verify the presence of any statistically significant correlations between SNP distribution and patients’ data. Three of the five previously described mtLSU rRNA genotypes were identified: genotype 1 (wild type ) was found in four patients’ samples (50%), while genotypes 2 and 3 were detected in one (12.5%) and three (37.5%) other patients, respectively. The most common CYB genotypes were CYB 1 and 2, both occurring in two (28.6%) cases. The remaining identified genotypes were CYB 5, 7, and 8, occurring in one case each (14.3%). SOD polymorphisms were identified in four samples only, half of which referred to wild-type genotype ( SOD 1), and the other half to SOD 2. Finally, DHPS typing revealed the presence of genotype 1 (wild-type only) in all five successfully amplified samples. There were no significant differences in SNP distribution and gender, immunosuppressive regimen, or PcP symptoms. However, it was observed that detection of CYB 2 genotype was significantly correlated with the ongoing prophylaxis regimen ( P = 0.047, Fisher’ s exact test). Moreover, both mean age ( P = 0.033, Student’ s t test) and time after kidney transplantation ( P = 0.028, Student’ s t test) were significantly lower in patients with detected wild-type mtLSU rRNA genotype (44.5 years, 58.1 months, respectively) than in those with mutant genotypes (65.8 years, 78.3 months, respectively). DHPS typing in patients with various pulmonary diseases was possible in 53% of available samples. Similarly, only genotype 1 was detected. Comparison of RTRs and patients with various pulmonary diseases (whose data on demographic, prevalence, and genotype distribution were described previously; Sokulska et al. 2017 ) did not reveal any significant differences in SNP distribution or P. jirovecii prevalence.