Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases

Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases Abstract Pneumocystis jirovecii is an opportunistic fungus causing Pneumocystis pneumonia primarily in immunosuppressed patients. However, immunocompetent individuals may become colonized and, as asymptomatic carriers, serve as reservoirs of the pathogen. Moreover, these asymptomatic carriers are at higher risk of developing pneumonia if favorable conditions occur. This study aimed to determine the prevalence of P. jirovecii in patients with various pulmonary diseases and to characterize the genetic diversity of organisms circulating in the studied population. Bronchial washing specimens from 105 patients were tested for presence of P. jirovecii using nested polymerase chain reaction (PCR) targeting the mtLSU rRNA gene, as well as immunofluorescence microscopy. Multilocus sequence typing involving analysis of three loci—mtLSU rRNA, CYB, and SOD—was used for genotyping analysis. P. jirovecii DNA was detected in 17 (16.2%) patients. Amplification of the SOD locus was successful only in five cases (29.4% of the positive patients), while mtLSU rRNA and CYB were genotyped in all positive samples. Therefore, combined genotypes were identified based only on mtLSU rRNA and CYB loci. Eight different genotypes were identified, with Pj 1 and Pj 2 being the most prevalent (29.4% of patients each). There was no statistical correlation between these genotypes and demographic or clinical data; however, we found that infection with mutant CYB strains occurred only in patients diagnosed with lung cancer. Of the potential predictors examined, only immunosuppressive treatment was significantly associated with colonization. In conclusion, patients with various respiratory diseases, especially when immunosuppressed, are at risk of Pneumocystis colonization. Pneumocystis jirovecii, colonization, prevalence, genotyping, respiratory diseases Introduction Pneumocystis jirovecii, formerly Pneumocystis carinii f. sp. hominis, is a unicellular fungal species occurring in human lungs.1 Since Pneumocystis is an opportunistic pathogen, its infection may lead to the development of Pneumocystis pneumonia (PcP) primarily in immunocompromised individuals, who represent the main group at risk.2 In turn, its presence in immunocompetent people usually does not trigger any clinical signs or symptoms. It may, however, persist as an asymptomatic carriage, defined as colonization.3 This phenomenon, in contrast to active infection, is characterized by the low fungal burden and does not cause the disease in the carrier. Pneumocystis presence in a biological specimen from a person without clinical symptoms of pneumonia or related radiological signs may be confirmed by molecular methods but not necessarily by microscopic examination, due to the low fungal burden.4 It is assumed that the concept of colonization is used whenever P. jirovecii DNA is detected in specimens taken from asymptomatic patients at risk of infection.5 It has been shown that colonization occurs among individuals with pulmonary diseases, with a frequency depending on the geographical location and type of respiratory diagnosis.6,7 The causal link, however, has not been elucidated and requires further investigation. On the one hand, even asymptomatic presence of the pathogen in the lungs may induce a host inflammatory response and lung damage, thereby potentially contributing to the infection of other pathogens or development of chronic lung diseases.7 On the other hand, pulmonary lesions and lung tissue damage induced by preexisting respiratory ailments might favor Pneumocystis colonization by facilitating its pulmonary involvement.8 The research on Pneumocystis has been hindered due to the lack of appropriate systems for in vitro culture of this organism.9 Therefore, molecular methods have been applied for the investigation of Pneumocystis transmission and diversity.10 Multilocus typing is considered to be the gold standard for such evaluation, as it involves analysis of two or more loci, thereby providing higher sensitivity and accuracy than single locus typing.11 Maitte et al.12 proposed a simplified approach involving analysis of three loci: mitochondrial large subunit (mtLSU) rRNA, cytochrome b (CYB), and superoxide dismutase (SOD). Such a scheme provides a sufficiently high discriminatory power for genetic diversity analysis, required for preliminary epidemiological investigation of Pneumocystis occurrence. The incidence of particular genotypes in various populations may indeed be influenced by the underlying disease, immunosuppressive treatment, climatic and geographical factors as well as divergent application of PcP prophylaxis in different health care systems.13 Owing to the arguments presented above, exploration of P. jirovecii epidemiology and transmission in specific populations is an important issue. However, to date they have not been sufficiently elucidated in Poland, so there are few data available on the prevalence of Pneumocystis and epidemiological profiles occurring in the country (and particularly in Lower Silesia).14 Therefore, the main objectives of the present study were to: (i) determine the prevalence of P. jirovecii among patients with a variety of respiratory diseases; and (ii) characterize potential associations between demographic and clinical data and distribution of the multilocus genotypes (MLGs), combining three independent polymorphic loci: mtLSU rRNA, CYB, and SOD. Methods Patients and specimens The study involved 105 human immunodeficiency virus (HIV)-negative patients who had been receiving care at the Department of Pulmonology and Lung Cancer of Wroclaw Medical University (Wroclaw, Poland) between July 2015 and September 2016. All of these patients required flexible bronchoscopy as part of their diagnostic management for possible respiratory disease. During the procedure, the bronchoscope was introduced into the main bronchi and 30–60 ml of the warmed saline (room temperature) was instilled through the working channel into the airways. The bronchial washing (BW) was recovered via a suction channel into a suitable receptacle. Fresh BW samples were centrifuged, and the sediment was resuspended in the remnant of supernatant. In sum, 20 μl of cell pellets were used to smear slides. High quality acetone was used to fix the slides to be stained. The remaining amount was frozen at −20°C for maximum 2 weeks without preservatives before molecular analysis. Since BW collection is an invasive method, samples were taken only once from each patient. Informed consent, approved by the Human Research Ethics Committee of Wroclaw Medical University according to agreement no. KB648/2014, was obtained from all individual participants included in the study. Detection of P. jirovecii After homogenization by bead disruption using a Precellys24 Instrument (Bertin Technologies, France) and digestion with proteinase K at 56°C for 1 hour, total DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The detection of P. jirovecii organisms was performed using a nested polymerase chain reaction (PCR) protocol amplifying the mtLSU rRNA gene using Taq polymerase, as described previously,15,16 with a sample of human-derived P. jirovecii DNA used as a positive control in each experiment. Molecular examination was repeated two or three times for each sample. Additionally, indirect immunofluorescence (IF) assay (MonoFluo kit P. jirovecii; Bio-Rad) was applied. Two to three slides were prepared for each patient sample. They were examined with a fluorescence microscope, using 20× and 40× dry objectives. The entire stained region was screened if no P. jirovecii was seen. It enabled assessment of fungal burden by scoring the total number of cysts observed.17 Case definition The diagnosis of PcP was made on the basis of specific clinical symptoms of pneumonia (low-grade fever, dyspnea, unproductive cough) and/or typical radiologic findings (bilateral ground glass opacity on chest radiography or chest computed tomography), confirmed by microscopic and molecular examination of BW specimens. A P. jirovecii-colonized patient was defined as an individual without symptoms or thoracic radiography signs of PcP, from whom a BW specimen contained P. jirovecii DNA detectable by nested PCR and was or was not positive by IF assay. An IF sample was assumed to be positive if five or more fluorescing cysts or clusters of cysts were identified; one to five fluorescing cysts recorded on a whole slide were interpreted as an equivocal result, suggesting a colonization case.17,18 When no cysts were identified on a whole slide, an IF sample was considered negative. Multilocus typing All positive products of nested PCR amplifying the mtLSU rRNA locus were sequenced to detect polymorphisms at two informative positions (85 and 248). Bands of the expected size (260 bp) were visualized by agarose gel electrophoresis and purified with the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). Products were sequenced bi-directionally by a company offering this service commercially. All samples with detectable DNA of Pneumocystis were also subjected to nested PCRs with Phusion HotStart II DNA polymerase (Thermo Scientific, Waltham, MA, USA) amplifying the CYB and SOD loci, as described previously.19,20 Bands of expected sizes (578 bp for SOD and 620 bp for CYB) were visualized, purified and sequenced as described above, in order to detect polymorphisms at informative positions (110 and 215 for SOD; 279, 348, 516, 547, 566 and 838 for CYB).19,20 Sequence alignment was performed manually using ChromasPro (version 2.1.1, Technelysium Pty Ltd., Australia) and BLAST software accessible online (https://blast.ncbi.nlm.nih.gov). Results were compared with P. jirovecii reference gene sequences (GenBank accession no. M58605.1 for mtLSU rRNA, KT592347.1 for SOD, and AF321304.1 for CYB). Genotypes were determined according to the available nomenclature.12,19,21 Statistical analysis The χ2 or Fisher's exact test was used to compare categorical variables (sex, final respiratory diagnosis, immunosuppressive treatment) between Pneumocystis-positive and -negative patients, while the continuous variable (age) was compared using Student's t test. A value of P < .05 was considered significant. Results P. jirovecii prevalence and patients’ characteristics The mean age of all patients (n = 105, including 70 males, 35 females) was 62 ± 12.7 years, range 27–87 years. None of the examined patients was subjected to anti-Pneumocystis prophylaxis. DNA of P. jirovecii was detected in 17 of the 105 (16.2%) subjects studied. Moreover, eight out of these 17 patients (47%) were confirmed as Pneumocystis-positive by IF staining as well. However, the low number of cysts (less than five) observed in all these cases and the lack of clinical signs and symptoms compatible with PcP (except for one case of dyspnea) suggested colonization. Since none of the patients included in this study was diagnosed with PcP, no specific treatment was prescribed and their further outcome was not followed after specimens’ collection. Basic characteristics of P. jirovecii-positive and -negative patients, underlying diseases potentially associated with Pneumocystis colonization, as well as immunosuppressive regimens are listed in Table 1. There were no significant differences in sex, age, or the frequency of certain underlying respiratory diseases. The only one statistically significant correlation was observed between the application of immunosuppressive agents (cytotoxic drugs or steroids) and Pneumocystis colonization (P = .016, Fisher's exact test). Table 1. Comparison of Pneumocystis jirovecii-positive and -negative patients’ basic characteristics. Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Data represent number (%) unless otherwise indicated. COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease. aSteroids or cytotoxic drugs. bOnly conditions potentially associated with P. jirovecii colonization are selected. cIncluding: sarcoidosis, pneumoconiosis, allergic alveolitis and cryptogenic organizing pneumonia. *P value < .05. View Large Table 1. Comparison of Pneumocystis jirovecii-positive and -negative patients’ basic characteristics. Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Data represent number (%) unless otherwise indicated. COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease. aSteroids or cytotoxic drugs. bOnly conditions potentially associated with P. jirovecii colonization are selected. cIncluding: sarcoidosis, pneumoconiosis, allergic alveolitis and cryptogenic organizing pneumonia. *P value < .05. View Large Multilocus typing Both mtLSU rRNA and CYB genes were amplified successfully in all analyzed specimens, whereas the SOD gene was amplified only in five of the 17 (29.4%) P. jirovecii-positive patients (Table 2). Therefore, genotyping was performed combining mtLSU rRNA and CYB types. As a result, eight genotypes were determined (Table 3). The two most common were Pj 1 (comprising wild-type versions of both genotypes) and Pj 2, found in five specimens each (29.4%). Table 2. Polymorphisms identified at the three studied loci. Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Nonsynonymous mutations are underlined. View Large Table 2. Polymorphisms identified at the three studied loci. Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Nonsynonymous mutations are underlined. View Large Table 3. Genotyping of Pneumocystis in the studied population. Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) aFor further details, see Table 2. View Large Table 3. Genotyping of Pneumocystis in the studied population. Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) aFor further details, see Table 2. View Large For mtLSU rRNA, three of the five previously described genotypes were identified: genotype 2 occurred in eight patients’ samples (47%), while genotypes 1 and 3 were detected in five (29.5%) and four (23.5%) patients’ samples, respectively. Mutant CYB genotypes were identified in five samples (29.4%). These included isolates with genotypes CYB 2, 5, 6, 7, and 8, observed in one case each. There was no statistically significant correlation between MLG distribution and patients’ underlying conditions or immune status, or the season in which infection occurred. However, taking into account single genotypes, we observed that infection with mutant CYB strains occurred in patients diagnosed only with lung cancer (P = .029, Fisher's exact test). Discussion Pneumocystis infection is considered mainly in terms of HIV diagnosis. Nevertheless, other groups of patients with impaired immunity, such as organ transplant recipients and oncologically treated individuals, are also at high risk of being infected with Pneumocystis and developing PcP.22 Moreover, growing evidence proves that patients with various lung diseases constitute a group susceptible to Pneumocystis infection as well.6 Its prevalence among this group of patients varies between different areas, depending on epidemiological factors, such as climatic characteristics of a specific geographical location.23 In Europe, the level of Pneumocystis colonization among non–HIV-infected patients with lung diseases varies from 2.5% in Italy,24 4.4% in Denmark,25 12.5% in France,26 24.4% in Portugal,27 up to 27.1% in Spain.28 Our findings (16.2%) correlate with published data from other European countries located in a similar climate zone: Germany (19%)29 and the United Kingdom (18%).30 Despite being asymptomatic, Pneumocystis colonization remains an epidemiological problem. First of all, not only may colonized individuals transmit the pathogen to other people,31 but also, if the immune status of the carrier deteriorates, colonization may develop into pneumonia. Second, application of anti-Pneumocystis prophylaxis in colonized individuals is difficult to implement and could lead to the selection of drug-resistant strains.32 Finally, presence of the pathogen in the lungs may induce a host inflammatory response and tissue damage, which in turn can lead to the development of pulmonary diseases.6 In fact, it has been proposed that high rates of colonization may be associated with specific chronic lung diseases (interstitial lung diseases, COPD or lung cancer).7,8,33,34 However, there was no significant difference in the frequency of certain disease entities between Pneumocystis-positive and -negative individuals in our study. These data are not consistent with previous reports,6–8 which may be explained by the influence of other factors, such as those associated with geographical location, on Pneumocystis prevalence in patients at risk.35 Also, we observed a statistically significant correlation between the application of immunosuppressive agents and presence of P. jirovecii in BW specimens. Since generally the immunosuppressive treatment increases susceptibility to colonization of this opportunistic pathogen regardless of the type of basic disease entity, such a correlation seems to be predictable.4 Our results confirm that, similarly as in organ transplant recipients and other groups at risk, the inclusion of immunosuppressive treatment may further support the colonization of Pneumocystis in the lungs and PcP should be considered as a possible complication in these individuals.36,37 Besides their effect on the prevalence of Pneumocystis, the above factors may also be associated with the occurrence of genetic variations among circulating Pneumocystis strains.13 The most robust information can be achieved by genotyping more than one locus simultaneously. The mtLSU rRNA gene, involved in basic mechanisms of translation by providing activity of peptidyl transferase to the mitochondrial ribosome,38 has been widely used for genetic characterization of P. jirovecii isolates from different geographic areas.13,20,21,28 Sequencing of this locus in our studies revealed the presence of three different genotypes, with genotype 2 observed most frequently. This genotype was also the most common among patients suspected of pulmonary infection residing in Rome, Italy.39 In contrast, genotype 1 is the most frequent among HIV-positive and -negative patients from other European countries, such as Portugal, Spain, or France.13,26 These differences in genotype distribution support the assumption of the epidemiological impact on circulation of particular Pneumocystis strains in the designated areas.13 The SOD gene encodes an enzyme responsible for protection against free oxygen radicals, capable of causing harmful oxidative stress, to which Pneumocystis is particularly exposed due to its pulmonary tropism.40 In the present study, successful amplification of this locus occurred in only 29.4% of cases. This is probably associated with the fact that the SOD gene occurs in a single copy in the Pneumocystis genome, so the efficiency of its amplification is relatively low.12,20 This is particularly problematic in the case of a low fungal burden, characteristic for colonized patients included in this study. Nevertheless, out of five previously described genotypes, we detected only SOD 1 and SOD 2, which are also the ones most often found in France, Portugal, and Cuba.12,19,20 Cytochrome b, encoded by CYB gene, is a target for atovaquone, a drug used in prophylaxis for Pneumocystis infection. It has been shown that the occurrence of polymorphisms within the CYB locus may be associated with previous exposure to atovaquone41 or even lead to drug resistance.32 However, nearly one-third of patients enrolled in this study were infected with organisms carrying polymorphisms at the CYB locus, even though none of them was subjected to any Pneumocystis prophylaxis regimen, suggesting that alterations in this gene might be induced by other factors as well. This is in agreement with the fact that the non-synonymous mutations detected in our samples (genotypes CYB 2 and CYB 7, Table 2) do not concern the atovaquone-binding site of cytochrome b.19 Interestingly, all samples with identified CYB versions differing from the wild type were collected solely from patients diagnosed with lung cancer. A possible explanation is that these individuals are characterized by conditions promoting colonization of P. jirovecii organisms with certain genetic variations. Similar findings concerning the occurrence of specific genotypes in a particular group of patients have been described for mtLSU rRNA genotypes when comparing non-HIV-infected and HIV-infected individuals.28,42 Another reason for such variability may be the influence of some agents used in treatment of patients with lung cancer on promoting the development of such polymorphisms. Taken together, these reports suggest that different underlying conditions might determine the specific pattern of genotype distribution. This assumption, however, should be taken with caution. The group of patients included in this study is very heterogeneous and patients with lung cancer constitute the majority. Therefore, even though statistically significant, the associations of mutations in a single genotype may be distorted by the unbalanced ratio of patients. Further research with the respective demographic and clinical information should be conducted in order to clarify these associations, preferably in a study including a larger sample. As a result of this study eight genotypes based on mtLSU rRNA and CYB loci were identified using multilocus genotyping methodology. Among them the most prevalent were Pj 1 and Pj 2, occurring in 29.4% of patients each. Other genotypes were identified in individual patients only (except Pj 3, found in two cases), which may suggest that inter-human transmission is the main source of Pneumocystis infection, involving strains with genetic alterations, and appearance of mutant genotypes may result from specific patient conditions and/or selective pressure of treatment. This is of particular importance for immunosuppressed patients, the main group at risk of PcP, since certain mutations may be associated with drug resistance or specific clinical parameters of infection.43 In conclusion, the prevalence of 16.2% observed in this study testifies to the fact that patients with various pulmonary diseases are at risk of Pneumocystis colonization. Apart from its potential role in the development or maintenance of chronic lung diseases, investigation on Pneumocystis prevalence is also important due to the fact that carriers may constitute a reservoir of this pathogen, including resistant forms. Circulating strains may in fact comprise genetic variations, detected even among individuals not exposed to known selective pressure, providing evidence for inter-human transmission as the main source of infection. Finally, our results suggest that if asymptomatic Pneumocystis infection occurs among non–HIV-infected individuals, it is likely that HIV-infected patients remain at risk of colonization, the former being a possible source of infection. In this respect, the significance of colonization in an epidemiological context requires further thorough analysis. To our knowledge, this is the first study in Poland concerning P. jirovecii genetic diversity and the prevalence of this organism among non–HIV-infected patients with a variety of pulmonary diseases. Funding This work was supported by the National Science Centre, Poland [DEC-2012/05/D/NZ6/00615] and Wroclaw Medical University Grant for Young Scientists [Pbmn 191]. The founders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. 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Emerg Infect Dis. 2004 ; 10 : 1729 – 1735 . Google Scholar Crossref Search ADS PubMed 11. Matos O , Esteves F . Pneumocystis jirovecii multilocus gene sequencing: findings and implications . Future Microbiol . 2010 ; 5 : 1257 – 1268 . Google Scholar Crossref Search ADS PubMed 12. Maitte C , Leterrier M , Le Pape P , Miegeville M , Morio F . Multilocus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used? J Clin Microbiol . 2013 ; 51 : 2843 – 2849 . Google Scholar Crossref Search ADS PubMed 13. Esteves F , Montes-Cano MA , de la Horra C et al. Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain . Clin Microbiol Infect . 2008 ; 14 : 356 – 362 . Google Scholar Crossref Search ADS PubMed 14. Zajac-Spychała O , Gowin E , Fichna P et al. Pneumocystis pneumonia in children – the relevance of chemoprophylaxis in different groups of immunocompromised and immunocompetent paediatric patients . Cent Eur J Immunol . 2015 ; 40 : 91 – 95 . Google Scholar Crossref Search ADS PubMed 15. Wakefield AE , Pixley FJ , Banerji S et al. Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin . Mol Biochem Parasitol . 1990 ; 43 : 69 – 76 . Google Scholar Crossref Search ADS PubMed 16. Wakefield AE. DNA Sequences Identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in Samples of Air Spora . J Clin Microbiol . 1996 ; 34 : 1754 – 1759 . Google Scholar PubMed 17. Gill V , Evans G , Stock F et al. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies . J Clin Microbiol . 1987 ; 25 : 1837 – 1840 . Google Scholar PubMed 18. Midgley J , Parsons PA , Shanson DC , Husain OAN , Francis N . Monoclonal immunofluorescence compared with silver stain for investigating Pneumocystis carinii pneumonia . J Clin Pathol . 1991 ; 44 : 75 – 76 . Google Scholar Crossref Search ADS PubMed 19. Esteves F , Gaspar J , Tavares A et al. Population structure of Pneumocystis jirovecii isolated from immunodeficiency virus-positive patients . Infect Genet Evol. 2010 ; 10 : 192 – 199 . Google Scholar Crossref Search ADS PubMed 20. Monroy-Vaca EX , de Armas Y , Illnait-Zaragozí MT et al. Genetic diversity of Pneumocystis jirovecii in colonized Cuban infants and toddlers . Infect Genet Evol. 2014 ; 22 : 60 – 66 . Google Scholar Crossref Search ADS PubMed 21. Beard CB , Carter JL , Keely SP et al. Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission . Emerg Infect Dis. 2000 ; 6 : 265 – 272 . Google Scholar Crossref Search ADS PubMed 22. Roblot F , Godet C , Le Moal G et al. Analysis of underlying diseases and prognosis factors associated with Pneumocystis carinii pneumonia in immunocompromised HIV-negative patients . Eur J Clin Microbiol Infect Dis . 2002 ; 21 : 523 – 531 . Google Scholar Crossref Search ADS PubMed 23. Varela JM , Regordán C , Medrano FJ et al. Climatic factors and Pneumocystis jiroveci infection in southern Spain. Clin Microbiol Infect . 2004 ; 10 : 770 – 772 . Google Scholar Crossref Search ADS PubMed 24. Visconti E , Marinaci S , Zolfo M et al. Very low frequency of Pneumocystis carinii DNA detection by PCR in specimens from patients with lung damage . J Clin Microbiol. 2000 ; 38 : 1307 – 1308 . Google Scholar PubMed 25. Helweg-Larsen J , Jensen JS , Dohn B , Benfield TL , Lundgren B . Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia-a case-control study . BMC Infect Dis. 2002 ; 28 : 1 – 6 . 26. Hernández-Hernández F , Fréalle E , Caneiro P et al. Prospective multicenter study of Pneumocystis jirovecii colonization among cystic fibrosis patients in France. J Clin Microbiol . 2012 ; 50 : 4107 – 4110 . Google Scholar Crossref Search ADS PubMed 27. Matos O , Costa MC , Correia I et al. Pneumocystis jirovecii infection in immunocompetent patients with pulmonary disorders, in Portugal . Acta Med Port . 2006 ; 19 : 121 – 126 . Google Scholar PubMed 28. Montes-Cano MA , de la Horra C , Martin-Juan J et al. Pneumocystis jiroveci genotypes in the Spanish population . Clin Infect Dis. 2004 ; 39 : 123 – 128 . Google Scholar Crossref Search ADS PubMed 29. Sing A , Roggenkamp A , Autenrieth IB , Heesemann J . Pneumocystis carinii carriage in immunocompetent patients with primary pulmonary disorders as detected by single or nested PCR . J Clin Microbiol . 1999 ; 37 : 3409 – 3410 . Google Scholar PubMed 30. Maskell NA , Waine DJ , Lindley A et al. Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study . Thorax . 2003 ; 58 : 594 – 597 . Google Scholar Crossref Search ADS PubMed 31. Rivero L , de la Horra C , Montes-Cano MA et al. Pneumocystis jirovecii transmission from immunocompetent carriers to infant . Emerg Infect Dis. 2008 ; 14 : 1116 – 1118 . Google Scholar Crossref Search ADS PubMed 32. Walker DJ , Wakefield AE , Dohn MN et al. Sequence polymorphisms in the Pneumocystis carinii cytochrome b gene and their association with atovaquone prophylaxis failure . J Infect Dis. 1998 ; 178 : 1767 – 1775 . Google Scholar Crossref Search ADS PubMed 33. Vidal S , de la Horra C , Martín J et al. Pneumocystis jirovecii colonisation in patients with interstitial lung disease . Clin Microbiol Infect . 2006 ; 12 : 231 – 235 . Google Scholar Crossref Search ADS PubMed 34. Mori H , Ohno Y , Ito F et al. Polymerase chain reaction positivity of Pneumocystis jirovecii during primary lung cancer treatment . Jpn J Clin Oncol. 2010 ; 40 : 658 – 662 . Google Scholar Crossref Search ADS PubMed 35. Togashi Y , Masago K , Ito Y et al. Pneumocystis jiroveci pneumonia and colonization in patients with advanced lung cancer . Oncol Lett . 2013 ; 5 : 601 – 604 . Google Scholar Crossref Search ADS PubMed 36. Msaad S , Yangui I , Bahloul N et al. Do inhaled corticosteroids increase the risk of Pneumocystis pneumonia in people with lung cancer? World J Clin Cases . 2015 ; 3 : 843 – 847 . Google Scholar Crossref Search ADS PubMed 37. Krakówka P , Miksiewicz-Wasilewska H , Kotecki NR . Pneumocystis carinii pneumonia in patients with lung neoplasms. Pulmonol Pol . 1981 ; 49 : 145 – 150 . 38. Noller HF . Structure of Ribosomal RNA. Annu Rev Biochem . 1984 ; 53 : 119 – 162 . Google Scholar Crossref Search ADS PubMed 39. Dimonte S , Berrilli F , D’Orazi C et al. Molecular analysis based on mtLSU-rRNA and DHPS sequences of Pneumocystis jirovecii from immunocompromised and immunocompetent patients in Italy. Infect Genet Evol . 2013 ; 14 : 68 – 72 . Google Scholar Crossref Search ADS PubMed 40. Denis C , Guyot K , Wakefield AE et al. Molecular cloning and characterization of a superoxide dismutase (sod) gene in Pneumocystis carinii . J Euk Microbiol . 1998 ; 45 : 475 – 483 . Google Scholar Crossref Search ADS PubMed 41. Kazanjian P , Armstrong W , Hossler PA et al. Pneumocystis carinii cytochrome b mutations are associated with atovaquone exposure in patients with AIDS . J Infect Dis . 2001 ; 183 : 819 – 822 . Google Scholar Crossref Search ADS PubMed 42. Gupta R , Mirdha BR , Guleria R et al. Genotypic variation of Pneumocystis jirovecii isolates in India based on sequence diversity at mitochondrial large subunit rRNA. Int J Med Microbiol . 2011 ; 301 : 267 – 272 . Google Scholar Crossref Search ADS PubMed 43. Esteves F , Gaspar J , Marques T et al. Identification of relevant single-nucleotide polymorphisms in Pneumocystis jirovecii: relationship with clinical data. Clin Microbiol Infect . 2010 ; 16 : 878 – 884 . © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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1369-3786
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1460-2709
D.O.I.
10.1093/mmy/myx121
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Abstract

Abstract Pneumocystis jirovecii is an opportunistic fungus causing Pneumocystis pneumonia primarily in immunosuppressed patients. However, immunocompetent individuals may become colonized and, as asymptomatic carriers, serve as reservoirs of the pathogen. Moreover, these asymptomatic carriers are at higher risk of developing pneumonia if favorable conditions occur. This study aimed to determine the prevalence of P. jirovecii in patients with various pulmonary diseases and to characterize the genetic diversity of organisms circulating in the studied population. Bronchial washing specimens from 105 patients were tested for presence of P. jirovecii using nested polymerase chain reaction (PCR) targeting the mtLSU rRNA gene, as well as immunofluorescence microscopy. Multilocus sequence typing involving analysis of three loci—mtLSU rRNA, CYB, and SOD—was used for genotyping analysis. P. jirovecii DNA was detected in 17 (16.2%) patients. Amplification of the SOD locus was successful only in five cases (29.4% of the positive patients), while mtLSU rRNA and CYB were genotyped in all positive samples. Therefore, combined genotypes were identified based only on mtLSU rRNA and CYB loci. Eight different genotypes were identified, with Pj 1 and Pj 2 being the most prevalent (29.4% of patients each). There was no statistical correlation between these genotypes and demographic or clinical data; however, we found that infection with mutant CYB strains occurred only in patients diagnosed with lung cancer. Of the potential predictors examined, only immunosuppressive treatment was significantly associated with colonization. In conclusion, patients with various respiratory diseases, especially when immunosuppressed, are at risk of Pneumocystis colonization. Pneumocystis jirovecii, colonization, prevalence, genotyping, respiratory diseases Introduction Pneumocystis jirovecii, formerly Pneumocystis carinii f. sp. hominis, is a unicellular fungal species occurring in human lungs.1 Since Pneumocystis is an opportunistic pathogen, its infection may lead to the development of Pneumocystis pneumonia (PcP) primarily in immunocompromised individuals, who represent the main group at risk.2 In turn, its presence in immunocompetent people usually does not trigger any clinical signs or symptoms. It may, however, persist as an asymptomatic carriage, defined as colonization.3 This phenomenon, in contrast to active infection, is characterized by the low fungal burden and does not cause the disease in the carrier. Pneumocystis presence in a biological specimen from a person without clinical symptoms of pneumonia or related radiological signs may be confirmed by molecular methods but not necessarily by microscopic examination, due to the low fungal burden.4 It is assumed that the concept of colonization is used whenever P. jirovecii DNA is detected in specimens taken from asymptomatic patients at risk of infection.5 It has been shown that colonization occurs among individuals with pulmonary diseases, with a frequency depending on the geographical location and type of respiratory diagnosis.6,7 The causal link, however, has not been elucidated and requires further investigation. On the one hand, even asymptomatic presence of the pathogen in the lungs may induce a host inflammatory response and lung damage, thereby potentially contributing to the infection of other pathogens or development of chronic lung diseases.7 On the other hand, pulmonary lesions and lung tissue damage induced by preexisting respiratory ailments might favor Pneumocystis colonization by facilitating its pulmonary involvement.8 The research on Pneumocystis has been hindered due to the lack of appropriate systems for in vitro culture of this organism.9 Therefore, molecular methods have been applied for the investigation of Pneumocystis transmission and diversity.10 Multilocus typing is considered to be the gold standard for such evaluation, as it involves analysis of two or more loci, thereby providing higher sensitivity and accuracy than single locus typing.11 Maitte et al.12 proposed a simplified approach involving analysis of three loci: mitochondrial large subunit (mtLSU) rRNA, cytochrome b (CYB), and superoxide dismutase (SOD). Such a scheme provides a sufficiently high discriminatory power for genetic diversity analysis, required for preliminary epidemiological investigation of Pneumocystis occurrence. The incidence of particular genotypes in various populations may indeed be influenced by the underlying disease, immunosuppressive treatment, climatic and geographical factors as well as divergent application of PcP prophylaxis in different health care systems.13 Owing to the arguments presented above, exploration of P. jirovecii epidemiology and transmission in specific populations is an important issue. However, to date they have not been sufficiently elucidated in Poland, so there are few data available on the prevalence of Pneumocystis and epidemiological profiles occurring in the country (and particularly in Lower Silesia).14 Therefore, the main objectives of the present study were to: (i) determine the prevalence of P. jirovecii among patients with a variety of respiratory diseases; and (ii) characterize potential associations between demographic and clinical data and distribution of the multilocus genotypes (MLGs), combining three independent polymorphic loci: mtLSU rRNA, CYB, and SOD. Methods Patients and specimens The study involved 105 human immunodeficiency virus (HIV)-negative patients who had been receiving care at the Department of Pulmonology and Lung Cancer of Wroclaw Medical University (Wroclaw, Poland) between July 2015 and September 2016. All of these patients required flexible bronchoscopy as part of their diagnostic management for possible respiratory disease. During the procedure, the bronchoscope was introduced into the main bronchi and 30–60 ml of the warmed saline (room temperature) was instilled through the working channel into the airways. The bronchial washing (BW) was recovered via a suction channel into a suitable receptacle. Fresh BW samples were centrifuged, and the sediment was resuspended in the remnant of supernatant. In sum, 20 μl of cell pellets were used to smear slides. High quality acetone was used to fix the slides to be stained. The remaining amount was frozen at −20°C for maximum 2 weeks without preservatives before molecular analysis. Since BW collection is an invasive method, samples were taken only once from each patient. Informed consent, approved by the Human Research Ethics Committee of Wroclaw Medical University according to agreement no. KB648/2014, was obtained from all individual participants included in the study. Detection of P. jirovecii After homogenization by bead disruption using a Precellys24 Instrument (Bertin Technologies, France) and digestion with proteinase K at 56°C for 1 hour, total DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The detection of P. jirovecii organisms was performed using a nested polymerase chain reaction (PCR) protocol amplifying the mtLSU rRNA gene using Taq polymerase, as described previously,15,16 with a sample of human-derived P. jirovecii DNA used as a positive control in each experiment. Molecular examination was repeated two or three times for each sample. Additionally, indirect immunofluorescence (IF) assay (MonoFluo kit P. jirovecii; Bio-Rad) was applied. Two to three slides were prepared for each patient sample. They were examined with a fluorescence microscope, using 20× and 40× dry objectives. The entire stained region was screened if no P. jirovecii was seen. It enabled assessment of fungal burden by scoring the total number of cysts observed.17 Case definition The diagnosis of PcP was made on the basis of specific clinical symptoms of pneumonia (low-grade fever, dyspnea, unproductive cough) and/or typical radiologic findings (bilateral ground glass opacity on chest radiography or chest computed tomography), confirmed by microscopic and molecular examination of BW specimens. A P. jirovecii-colonized patient was defined as an individual without symptoms or thoracic radiography signs of PcP, from whom a BW specimen contained P. jirovecii DNA detectable by nested PCR and was or was not positive by IF assay. An IF sample was assumed to be positive if five or more fluorescing cysts or clusters of cysts were identified; one to five fluorescing cysts recorded on a whole slide were interpreted as an equivocal result, suggesting a colonization case.17,18 When no cysts were identified on a whole slide, an IF sample was considered negative. Multilocus typing All positive products of nested PCR amplifying the mtLSU rRNA locus were sequenced to detect polymorphisms at two informative positions (85 and 248). Bands of the expected size (260 bp) were visualized by agarose gel electrophoresis and purified with the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). Products were sequenced bi-directionally by a company offering this service commercially. All samples with detectable DNA of Pneumocystis were also subjected to nested PCRs with Phusion HotStart II DNA polymerase (Thermo Scientific, Waltham, MA, USA) amplifying the CYB and SOD loci, as described previously.19,20 Bands of expected sizes (578 bp for SOD and 620 bp for CYB) were visualized, purified and sequenced as described above, in order to detect polymorphisms at informative positions (110 and 215 for SOD; 279, 348, 516, 547, 566 and 838 for CYB).19,20 Sequence alignment was performed manually using ChromasPro (version 2.1.1, Technelysium Pty Ltd., Australia) and BLAST software accessible online (https://blast.ncbi.nlm.nih.gov). Results were compared with P. jirovecii reference gene sequences (GenBank accession no. M58605.1 for mtLSU rRNA, KT592347.1 for SOD, and AF321304.1 for CYB). Genotypes were determined according to the available nomenclature.12,19,21 Statistical analysis The χ2 or Fisher's exact test was used to compare categorical variables (sex, final respiratory diagnosis, immunosuppressive treatment) between Pneumocystis-positive and -negative patients, while the continuous variable (age) was compared using Student's t test. A value of P < .05 was considered significant. Results P. jirovecii prevalence and patients’ characteristics The mean age of all patients (n = 105, including 70 males, 35 females) was 62 ± 12.7 years, range 27–87 years. None of the examined patients was subjected to anti-Pneumocystis prophylaxis. DNA of P. jirovecii was detected in 17 of the 105 (16.2%) subjects studied. Moreover, eight out of these 17 patients (47%) were confirmed as Pneumocystis-positive by IF staining as well. However, the low number of cysts (less than five) observed in all these cases and the lack of clinical signs and symptoms compatible with PcP (except for one case of dyspnea) suggested colonization. Since none of the patients included in this study was diagnosed with PcP, no specific treatment was prescribed and their further outcome was not followed after specimens’ collection. Basic characteristics of P. jirovecii-positive and -negative patients, underlying diseases potentially associated with Pneumocystis colonization, as well as immunosuppressive regimens are listed in Table 1. There were no significant differences in sex, age, or the frequency of certain underlying respiratory diseases. The only one statistically significant correlation was observed between the application of immunosuppressive agents (cytotoxic drugs or steroids) and Pneumocystis colonization (P = .016, Fisher's exact test). Table 1. Comparison of Pneumocystis jirovecii-positive and -negative patients’ basic characteristics. Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Data represent number (%) unless otherwise indicated. COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease. aSteroids or cytotoxic drugs. bOnly conditions potentially associated with P. jirovecii colonization are selected. cIncluding: sarcoidosis, pneumoconiosis, allergic alveolitis and cryptogenic organizing pneumonia. *P value < .05. View Large Table 1. Comparison of Pneumocystis jirovecii-positive and -negative patients’ basic characteristics. Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Pneumocystis jirovecii Characteristic Positive (n = 17) Negative (n = 88) P value Age in years, mean (range) 62 (27–75) 62 (28–87) .954 Sex  Male 12 (71) 58 (66) .708  Female 5 (29) 30 (34) .708 Immunosuppressive treatmenta 5 (29) 6 (8) .016* Final respiratory diagnosisb  Lung cancer 9 (53) 34 (38) .272  ILDsc 2 (12) 9 (10) 1  COPD 1 (6) 11 (12.5) .685 Data represent number (%) unless otherwise indicated. COPD, chronic obstructive pulmonary disease; ILD, interstitial lung disease. aSteroids or cytotoxic drugs. bOnly conditions potentially associated with P. jirovecii colonization are selected. cIncluding: sarcoidosis, pneumoconiosis, allergic alveolitis and cryptogenic organizing pneumonia. *P value < .05. View Large Multilocus typing Both mtLSU rRNA and CYB genes were amplified successfully in all analyzed specimens, whereas the SOD gene was amplified only in five of the 17 (29.4%) P. jirovecii-positive patients (Table 2). Therefore, genotyping was performed combining mtLSU rRNA and CYB types. As a result, eight genotypes were determined (Table 3). The two most common were Pj 1 (comprising wild-type versions of both genotypes) and Pj 2, found in five specimens each (29.4%). Table 2. Polymorphisms identified at the three studied loci. Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Nonsynonymous mutations are underlined. View Large Table 2. Polymorphisms identified at the three studied loci. Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Number of Locus Genotype specimens (%) Single nucleotide polymorphism (amino acid) position and identity mtLSU rRNA Genotype 1 5 (29.5) 85C; 248C Genotype 2 8 (47) 85A; 248C Genotype 3 4 (23.5) 85T; 248C CYB CYB 1 12 (70.5) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 2 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838T(280Phe) CYB 5 1 (5.9) 279T(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 6 1 (5.9) 279C(93Ile); 348A(116Gly); 516T(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) CYB 7 1 (5.9) 279C(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566T(189Leu); 838C(280Leu) CYB 8 1 (5.9) 279T(93Ile); 348A(116Gly); 516C(172Ile); 547C(183Leu); 566C(189Ser); 838C(280Leu) SOD SOD 1 3 (60) 110C; 215T(41Asp) SOD 2 2 (40) 110T; 215C(41Asp) Nonsynonymous mutations are underlined. View Large Table 3. Genotyping of Pneumocystis in the studied population. Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) aFor further details, see Table 2. View Large Table 3. Genotyping of Pneumocystis in the studied population. Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) Genotypes at each locusa Combined No. of specimens genotype mtLSU rRNA CYB (%) Pj 1 Genotype 1 CYB 1 5 (29.4) Pj 2 Genotype 2 CYB 1 5 (29.4) Pj 3 Genotype 3 CYB 1 2 (11.7) Pj 4 Genotype 2 CYB 2 1 (5.9) Pj 5 Genotype 3 CYB 5 1 (5.9) Pj 6 Genotype 2 CYB 6 1 (5.9) Pj 7 Genotype 3 CYB 7 1 (5.9) Pj 8 Genotype 2 CYB 8 1 (5.9) aFor further details, see Table 2. View Large For mtLSU rRNA, three of the five previously described genotypes were identified: genotype 2 occurred in eight patients’ samples (47%), while genotypes 1 and 3 were detected in five (29.5%) and four (23.5%) patients’ samples, respectively. Mutant CYB genotypes were identified in five samples (29.4%). These included isolates with genotypes CYB 2, 5, 6, 7, and 8, observed in one case each. There was no statistically significant correlation between MLG distribution and patients’ underlying conditions or immune status, or the season in which infection occurred. However, taking into account single genotypes, we observed that infection with mutant CYB strains occurred in patients diagnosed only with lung cancer (P = .029, Fisher's exact test). Discussion Pneumocystis infection is considered mainly in terms of HIV diagnosis. Nevertheless, other groups of patients with impaired immunity, such as organ transplant recipients and oncologically treated individuals, are also at high risk of being infected with Pneumocystis and developing PcP.22 Moreover, growing evidence proves that patients with various lung diseases constitute a group susceptible to Pneumocystis infection as well.6 Its prevalence among this group of patients varies between different areas, depending on epidemiological factors, such as climatic characteristics of a specific geographical location.23 In Europe, the level of Pneumocystis colonization among non–HIV-infected patients with lung diseases varies from 2.5% in Italy,24 4.4% in Denmark,25 12.5% in France,26 24.4% in Portugal,27 up to 27.1% in Spain.28 Our findings (16.2%) correlate with published data from other European countries located in a similar climate zone: Germany (19%)29 and the United Kingdom (18%).30 Despite being asymptomatic, Pneumocystis colonization remains an epidemiological problem. First of all, not only may colonized individuals transmit the pathogen to other people,31 but also, if the immune status of the carrier deteriorates, colonization may develop into pneumonia. Second, application of anti-Pneumocystis prophylaxis in colonized individuals is difficult to implement and could lead to the selection of drug-resistant strains.32 Finally, presence of the pathogen in the lungs may induce a host inflammatory response and tissue damage, which in turn can lead to the development of pulmonary diseases.6 In fact, it has been proposed that high rates of colonization may be associated with specific chronic lung diseases (interstitial lung diseases, COPD or lung cancer).7,8,33,34 However, there was no significant difference in the frequency of certain disease entities between Pneumocystis-positive and -negative individuals in our study. These data are not consistent with previous reports,6–8 which may be explained by the influence of other factors, such as those associated with geographical location, on Pneumocystis prevalence in patients at risk.35 Also, we observed a statistically significant correlation between the application of immunosuppressive agents and presence of P. jirovecii in BW specimens. Since generally the immunosuppressive treatment increases susceptibility to colonization of this opportunistic pathogen regardless of the type of basic disease entity, such a correlation seems to be predictable.4 Our results confirm that, similarly as in organ transplant recipients and other groups at risk, the inclusion of immunosuppressive treatment may further support the colonization of Pneumocystis in the lungs and PcP should be considered as a possible complication in these individuals.36,37 Besides their effect on the prevalence of Pneumocystis, the above factors may also be associated with the occurrence of genetic variations among circulating Pneumocystis strains.13 The most robust information can be achieved by genotyping more than one locus simultaneously. The mtLSU rRNA gene, involved in basic mechanisms of translation by providing activity of peptidyl transferase to the mitochondrial ribosome,38 has been widely used for genetic characterization of P. jirovecii isolates from different geographic areas.13,20,21,28 Sequencing of this locus in our studies revealed the presence of three different genotypes, with genotype 2 observed most frequently. This genotype was also the most common among patients suspected of pulmonary infection residing in Rome, Italy.39 In contrast, genotype 1 is the most frequent among HIV-positive and -negative patients from other European countries, such as Portugal, Spain, or France.13,26 These differences in genotype distribution support the assumption of the epidemiological impact on circulation of particular Pneumocystis strains in the designated areas.13 The SOD gene encodes an enzyme responsible for protection against free oxygen radicals, capable of causing harmful oxidative stress, to which Pneumocystis is particularly exposed due to its pulmonary tropism.40 In the present study, successful amplification of this locus occurred in only 29.4% of cases. This is probably associated with the fact that the SOD gene occurs in a single copy in the Pneumocystis genome, so the efficiency of its amplification is relatively low.12,20 This is particularly problematic in the case of a low fungal burden, characteristic for colonized patients included in this study. Nevertheless, out of five previously described genotypes, we detected only SOD 1 and SOD 2, which are also the ones most often found in France, Portugal, and Cuba.12,19,20 Cytochrome b, encoded by CYB gene, is a target for atovaquone, a drug used in prophylaxis for Pneumocystis infection. It has been shown that the occurrence of polymorphisms within the CYB locus may be associated with previous exposure to atovaquone41 or even lead to drug resistance.32 However, nearly one-third of patients enrolled in this study were infected with organisms carrying polymorphisms at the CYB locus, even though none of them was subjected to any Pneumocystis prophylaxis regimen, suggesting that alterations in this gene might be induced by other factors as well. This is in agreement with the fact that the non-synonymous mutations detected in our samples (genotypes CYB 2 and CYB 7, Table 2) do not concern the atovaquone-binding site of cytochrome b.19 Interestingly, all samples with identified CYB versions differing from the wild type were collected solely from patients diagnosed with lung cancer. A possible explanation is that these individuals are characterized by conditions promoting colonization of P. jirovecii organisms with certain genetic variations. Similar findings concerning the occurrence of specific genotypes in a particular group of patients have been described for mtLSU rRNA genotypes when comparing non-HIV-infected and HIV-infected individuals.28,42 Another reason for such variability may be the influence of some agents used in treatment of patients with lung cancer on promoting the development of such polymorphisms. Taken together, these reports suggest that different underlying conditions might determine the specific pattern of genotype distribution. This assumption, however, should be taken with caution. The group of patients included in this study is very heterogeneous and patients with lung cancer constitute the majority. Therefore, even though statistically significant, the associations of mutations in a single genotype may be distorted by the unbalanced ratio of patients. Further research with the respective demographic and clinical information should be conducted in order to clarify these associations, preferably in a study including a larger sample. As a result of this study eight genotypes based on mtLSU rRNA and CYB loci were identified using multilocus genotyping methodology. Among them the most prevalent were Pj 1 and Pj 2, occurring in 29.4% of patients each. Other genotypes were identified in individual patients only (except Pj 3, found in two cases), which may suggest that inter-human transmission is the main source of Pneumocystis infection, involving strains with genetic alterations, and appearance of mutant genotypes may result from specific patient conditions and/or selective pressure of treatment. This is of particular importance for immunosuppressed patients, the main group at risk of PcP, since certain mutations may be associated with drug resistance or specific clinical parameters of infection.43 In conclusion, the prevalence of 16.2% observed in this study testifies to the fact that patients with various pulmonary diseases are at risk of Pneumocystis colonization. Apart from its potential role in the development or maintenance of chronic lung diseases, investigation on Pneumocystis prevalence is also important due to the fact that carriers may constitute a reservoir of this pathogen, including resistant forms. Circulating strains may in fact comprise genetic variations, detected even among individuals not exposed to known selective pressure, providing evidence for inter-human transmission as the main source of infection. Finally, our results suggest that if asymptomatic Pneumocystis infection occurs among non–HIV-infected individuals, it is likely that HIV-infected patients remain at risk of colonization, the former being a possible source of infection. In this respect, the significance of colonization in an epidemiological context requires further thorough analysis. To our knowledge, this is the first study in Poland concerning P. jirovecii genetic diversity and the prevalence of this organism among non–HIV-infected patients with a variety of pulmonary diseases. Funding This work was supported by the National Science Centre, Poland [DEC-2012/05/D/NZ6/00615] and Wroclaw Medical University Grant for Young Scientists [Pbmn 191]. The founders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Medical MycologyOxford University Press

Published: Oct 1, 2018

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