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Risk factors for Pneumocystis jirovecii pneumonia (PJP) in kidney transplantation recipients

Risk factors for Pneumocystis jirovecii pneumonia (PJP) in kidney transplantation recipients www.nature.com/scientificreports OPEN Risk factors for Pneumocystis jirovecii pneumonia (PJP) in kidney transplantation recipients Received: 17 January 2017 1,3 2 2 2 2 2 Su Hwan Lee , Kyu Ha Huh , Dong Jin Joo , Myoung Soo Kim , Soon Il Kim , Juhan Lee , 1 1 1 1 2 1 Accepted: 4 April 2017 Moo Suk Park , Young Sam Kim , Se Kyu Kim , Joon Chang , Yu Seun Kim & Song Yee Kim Published: xx xx xxxx Pneumocystis jirovecii pneumonia (PJP) is a potentially life-threatening infection that occurs in immunocompromised patients. The aim of this study was to evaluate risk factors for PJP in kidney transplantation recipients. We conducted a retrospective analysis of patient data from 500 consecutive kidney transplants performed at Severance Hospital between April 2011 and April 2014. Eighteen kidney transplantation recipients (3.6%) were diagnosed with PJP. In the univariate analysis, acute graft rejection, CMV infection, use of medication for diabetes mellitus, and lowest lymphocyte count were associated with PJP. Recipients who experienced acute graft rejection (odds ratio [OR] 11.81, 95% confidence interval [CI] 3.06–45.57, P < 0.001) or developed CMV infection (OR 5.42, 95% CI 1.69– 17.39, P = 0.005) had high odds of PJP in multivariate analysis. In the acute graft rejection subgroup, patients treated with anti-thymocyte globulin (ATG) had significantly higher odds of PJP (OR 5.25, 95% CI 1.01–27.36, P = 0.006) than those who were not. Our data suggest that acute graft rejection and CMV infection may be risk factors for PJP in kidney transplant patients. The use of ATG for acute graft rejection may increase the risk of PJP. Pneumocystis jirovecii is an opportunistic fungal pathogen . Pneumocystis jirovecii pneumonia (PJP) previously known as Pneumocystis carinii pneumonia is a potentially life-threatening infection that occurs in immunocom- 1, 2 promised patients . In the absence of prophylaxis, PJP occurs in approximately 5–15% of transplant patients, 2, 3 depending on the transplanted organ or transplant center . 4, 5 In kidney transplantation, PJP is a very serious risk factor for graft loss and patient mortality . In the absence of appropriate treatment, the mortality rate of PJP is 90–100%, and can be as high as 50% despite adequate ther- 6, 7 apy . er Th efore, several guidelines—such as the Kidney Disease Improving Global Outcomes (KDIGO) guide- line, the European Renal Best Practice guideline, and other reports— usually recommend PJP prophylaxis by 8–10 using TMP/SMX for 3–6 months after renal transplantation . The incidence rate of PJP has decreased with the use of prophylaxis, however, an increasing number of PJP outbreaks in kidney transplant centers has been 4, 5, 11 reported worldwide in recent years . The causes of those outbreaks have not fully been evaluated. Risk factors for the development of PJP in kidney transplant patients are still not confirmed. The overall load of immunosuppressive therapy, higher donor age, higher recipient age, lymphopenia, previous cytomegalovirus (CMV) infection, or treatment used for episodes of graft rejection have been reported as risk factors for PJP in 12–17 kidney transplant patients . However, factors identified in some studies are not always confirmed in other studies. Thus, further research is needed to evaluate the risk factors for PJP in kidney transplant patients in the era of routine PJP prophylaxis. The aim of this study was to evaluate the risk factors for PJP in kidney transplantation recipients. Patients and Methods Study design and population. This single center, retrospective clinical study included all kidney transplant patients aged ≥18 years who underwent kidney transplantation at the Severance Hospital, a 2000-bed university Division of Pulmonology, Department of Internal Medicine, Severance Hospital, Institute of Chest Diseases, Yonsei University College of Medicine, Seoul, Republic of Korea. Department of Surgery, Research Institute for Transplantation, Yonsei University College of Medicine, Seoul, Republic of Korea. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Ewha Medical Research Institute, Ewha Womans University School of Medicine, Seoul, Korea. Correspondence and requests for materials should be addressed to S.Y.K. (email: dobie@ yuhs.ac) Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 1 www.nature.com/scientificreports/ tertiary referral hospital in South Korea, from April 2011 to April 2014. During this period, 500 patients under- went kidney transplantation; they were followed up until October 2015. We divided the patients into two groups according to the occurrence of PJP—the case group developed PJP whereas the control group did not—and then compared the groups. In our center, all kidney transplant recipients receive PJP prophylaxis using trimethoprim/ sulfamethoxazole (TMP/SMX) 160/800 mg per day for 12 months post-transplantation. After transplantation, patients received a tacrolimus-based combination regimen or a cyclosporine-based combination regimen for maintaining immunosuppression. Data collection. Data from all kidney transplant recipients were collected from the hospital’s electronic med- ical records. Clinical data on mortality, development of PJP, demographic characteristics, graft origin (deceased vs. living), immunosuppressive regimen, data about acute graft rejection such as frequency of such episodes or treatment received, smoking status, history of infections including CMV, BK virus, hepatitis, tuberculosis (TB), and comorbidities were evaluated. Definition. We defined PJP when the following two conditions were satisfied; first, a positive result on Pneumocystis jirovecii real-time polymerase chain reaction (PCR) testing or direct immunou fl orescence testing of microbiological samples (sputum, tracheal aspirate, bronchial washing fluid or bronchoalveolar lavage fluid) 18–20 and second, identification of lung infiltration on chest computed tomography (CT) . CMV infection was defined as a fourfold elevation of the CMV PCR titer and the use of CMV medication (ganciclovir or valganciclovir). Smoking status was categorized as ever smoker or never smoked; the latter cate- gory included those who smoked fewer than 100 cigarettes in their lifetime. Development of acute graft rejection was established by biopsy of the transplanted kidney. Steroid pulse therapy (methylprednisolone [500 mg/day × 4 for 5 days]) was considered the first line therapy for acute graft rejection. When there was an inadequate response to steroid pulse therapy, an anti-thyomcyte globulin (ATG) was used. Lowest lymphocyte count was reviewed during follow-up in the PJP negative group aer ft first hospital discharge and within 1 month before diagnosis of PJP in the PJP group. Clinical characteristics and events were reviewed until the last follow-up date in the PJP negative group and were reviewed until the date of PJP development in the PJP group. Ethical approval. The study protocol was approved by the Institutional Review Board (IRB) of Severance Hospital (IRB number: 4-2015-1051). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was waived by the IRB because of the study’s retrospective nature. Statistical analysis. Statistical analysis was performed using SPSS version 20 (IBM, Armonk, New York, USA). Data are described as medians (interquartile range [IQR]) or numbers (percentages). The Chi-squared and Fisher’s exact test or the Mann-Whitney test was used to assess differences between two groups. The independent risk for PJP was identified using logistic regression modeling. The fundamental variable (such as age, gender, BMI or kidney transplantation type) and variable which were meaningful risk factors in univariate analysis were used in multivariate analysis. A two-tailed P-value < 0.05 was considered statistically significant. Results Characteristics of the overall study population and patients diagnosed with PJP. Demographic and clinical characteristics of the total study population are presented in Table 1. Overall, 500 patients were enrolled over the 3-year study period, with a similar number enrolled each year. The median age of all recipients was 47 years (range, 18–71) and men accounted for 61.4%. The major cause of kidney transplantation was hyper - tension (39.6%); the other cause was diabetes mellitus (17.0%). Transplanted kidneys were sourced more oen ft from living than deceased donors (64.4% living vs. 35.6% deceased). In terms of immunosuppressive medication, 440 (88%) patients received a tacrolimus-based combination regimen and 60 (12%) received a cyclosporine-based combination regimen. The median duration of follow up was 36.2 month (range, 18.4–54.6). Eighteen patients were diagnosed with PJP during the study period, and most cases developed 12–24 months aer k ft idney transplantation. All patients diagnosed with PJP were started on TMP/SMX. Five patients required second-line medication because of disease progression despite TMP-SMX use; four were treated with primaquine and clindamycin, while one was treated with pentamidine. Twelve (66.7%) patients with PJP improved aer t ft reat- ment, with no sequelae. However, three (16.7%) patients had residual sequelae and three (16.7%) died. Table 2 describes clinical characteristics and prognosis of PJP patients. Risk factors for PJP in the total population. Table 3 shows the risk factors for PJP. In the univariate analysis, more men than women developed PJP, although this was not statistically significant (60.6% vs. 83.3%, respectively, P = 0.052). Median age, duration of follow up, gra ft source (deceased vs. living donor), proportion of re-transplant patients, and smoking status did not differ significantly between those who had PJP and those who did not. The underlying cause of kidney disease, necessitating transplantation, varied. However, hypertension, diabetes mellitus (DM), and IgA nephropathy were the major causes in both groups (P = 0.102). More acute gra ft rejection aer t ft ransplantation wwere observed in the PJP group compared with the PJP negative group (83.3% vs. 20.1%, respectively, P < 0.001). And CMV infection was more common in the PJP group than the PJP nega- tive group (61.1% vs. 12.9%, respectively, P < 0.001). Acute rejection and CMV infection were checked until PJP pneumonia in PJP group. Among those patients in the PJP group, 50% used medication for DM, compared to 26.1% in the PJP negative group (P = 0.032). Lowest lymphocyte counts were lower in the PJP group than in the 3 3 control group (0.3 × 10 /µL vs. 0.7 × 10 /µL, P = 0.007). In terms of immunosuppressive agents, patients received either tacrolimus- or cyclosporine-based regimens; the regimen used did not differ between the PJP group and the control group (P = 0.710). In the multivariate analysis, acute graft rejection [odds ratio (OR), 11.81; 95% Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 2 www.nature.com/scientificreports/ Characteristics N = 500 Sex, N (%)  Men 307 (61.4)  Women 193 (38.6) Age, years (median, range) 47 (18–71) Transplantation era, N (%)  2011.4–2012.3 167 (33.4)  2012.4–2013.3 158 (31.6)  2013.4–2014.4 175 (35.0) BMI, kg/m2 (median, IQR) 22 (20.1–24.2) KT type, N (%)  Deceased 178 (35.6)  Living 322 (64.4) Re-transplantation, N (%) 41 (8.2) Primary underlying disease, N (%)  Polycystic kidney 17 (3.4)  HTN 198 (39.6)  DM 85 (17.0)  IgA nephropathy 63 (12.6)  Autoimmune disease 7 (1.4)  Chronic glomerulonephritis 36 (7.2)  Nephrotic syndrome 41 (8.2)  Recurrent pyelonephritis/other 21 (4.2)  Unknown 32 (6.4) Immunosuppressive agent, N (%)  Cyclosporine based regimen 60 (12.0)  Tacrolimus based regimen 440 (88.0) Follow up duration, month (median, range) 36.2 (18.4–54.5) Development of PJP, N (%) 18 (3.6) Interval between PJP and graft, month (median 17.4 (11.2–27.9) IQR)  −12 month, N (%) 4 (22.2)  12–24 month, N (%) 7 (38.9)  24–36 month, N (%) 6 (33.3)  36- month, N (%) 1 (5.6) Treatment medication of PJP, N (%)  TMP-SMX alone 13 (72.2)  TMP-SMX prior to primaqiune + clinadamycin 4 (22.2)  TMP-SMX prior to pentamidine 1 (5.6) Table 1. Demographic and clinical characteristics of total study population and PJP patients. Abbreviations: PJP, Pneumocystis jirovecii pneumonia; IQR, interquartile range; BMI, body mass index; KT, kidney transplantation; HTN, hypertension; DM, diabetes mellitus; TMP-SMX, trimethoprim/sulfamethoxazole. confidence interval (CI), 3.06–45.57] and CMV infection (OR, 5.42; 95% CI, 1.69–17.93) were associated with PJP, whereas sex, age, body mass index (BMI), and graft source were not. Acute graft rejection and CMV infection were important risk factors for PJP, as presented in Table  3. To under- stand the impact of acute graft rejection and CMV pneumonia on PJP, we investigated the timing of the onset of PJP aer ac ft ute graft rejection or CMV infection. The median interval between acute graft rejection and PJP was 6.1 (IQR, 3.3–12.9) months, while the median interval between CMV infection and PJP was 1.5 (IQR, 0.1–7.1) months (Fig. 1a and b). Risk factors for PJP in patients experiencing acute graft rejection. As shown in Table 3, most cases of PJP (83.3%) occurred aer ac ft ute graft rejection. Hence, we performed a subgroup analysis of the risk factors for PJP in patients who developed acute graft rejection (Table  4). Of the 500 kidney transplant patients, 112 experienced acute graft rejection. Of those, 15 were diagnosed with PJP. Sex, median age, BMI, graft source, smoking status, BK virus infection, use of DM medication, development of acute graft rejection within 1-year post-transplant, number of acute graft rejection episodes, and lowest lymphocyte count did not differ between patients who developed PJP and those who did not. All patients who developed acute graft rejection were man - aged with steroid pulse therapy. However, additional use of ATG for acute gra r ft ejection treatment was more used in the PJP group (73.3% vs. 39.2%, P = 0.013). In addition, the proportion of patients with CMV infection was Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 3 www.nature.com/scientificreports/ Interval between Rejection CMV rejection and Sex Age Donor type number infection Immunosuppression Treatment PCP Outcome Case1 Male 42 Living 1 Yes Tac, Cor, MMF TMP-SMX → Pentamidine 3.3 month Die Case2 Male 35 Living 4 Yes Tac, Cor, Miz TMP-SMX → Primaquine + Clindamycin 3.4 month Recover Case3 Male 54 Living 1 No Tac, Cor, MMF TMP-SMX 6.9 month Die Case4 Male 51 Deceased 0 No Tac, Cor, MMF TMP-SMX — Recover Case5 Male 58 Deceased 1 No Cys, Cor, MMF TMP-SMX 11 month Graft fail Case6 Male 50 Deceased 1 Yes Tac, Cor, MMF TMP-SMX 6.2 month Recover Case7 Male 53 Deceased 1 No Tac, Cor, MMF TMP-SMX 29 month Graft fail Case8 Female 55 Living 1 Yes Tac, Cor, MMF TMP-SMX → Primaquine + Clindamycin 13 month Recover Case9 Male 54 Deceased 2 Yes Tac, Cor, MMF TMP-SMX → Primaquine + Clindamycin 1.5 month Die Case10 Male 55 Deceased 1 No Tac, Cor, MMF TMP-SMX 29 month Recover Case11 Male 61 Living 1 Yes Tac, Cor, MMF TMP-SMX 5.7 month Recover Case12 Male 43 Living 1 Yes Tac, Cor TMP-SMX 6.1 month Graft fail Case 13 Female 25 Living 0 Yes Tac, Cor, MMF TMP-SMX — Recover Case 14 Male 53 Deceased 2 No Tac, Cor, MMF TMP-SMX 2.8 month Recover Case 15 Male 52 Living 2 Yes Tac, Cor, MMF TMP-SMX 4.8 month Recover Case 16 Female 56 Deceased 2 Yes Tac, Cor, MMF TMP-SMX → Primaquine + Clidamycin 17 month Recover Case 17 Male 65 Deceased 0 No Tac, Cor, MMF TMP-SMX — Recover Case 18 Male 40 Deceased 2 Yes Tac, Cor, MMF TMP-SMX 1.2 month Recover Table 2. Clinical characteristic and prognosis of PJP patients in kidney transplantation. Abbreviations: PJP, Pneumocystis jirovecii pneumonia; CMV, cytomegalovirus; KT, kidney transplantation; MMF, mycophenolate mofetil; Cor, corticosteroid; Tac, tacrolimus; TMP-SMX, trimethoprim/sulfamethoxazole; Miz, Mizoribine; Cys, cyclosporine. higher in the PJP group than in the PJP negative group (66.7% vs. 34%, respectively, P = 0.016). In the multivari- ate analysis, the independent risk factors for PJP in the acute graft rejection subgroup were identified as male sex (OR, 6.45; 95% CI, 1.24–33.73) and the use of ATG (OR, 5.25; 95% CI, 1.01–27.36). Discussion In our study, of 500 kidney transplant patients, 18 developed PJP. Acute graft rejection and CMV infection were identified as risk factors for PJP. Within the subgroup of patients who experienced acute graft rejection, male sex and the use of ATG were risk factors for PJP. Our data showed that more patients in the PJP group than in the PJP negative group had CMV infection. This 17, 21 result is in agreement with that of previous studies . CMV is known to modify host immune responses by var- ious mechanisms, suppressing helper T-cell and antigen presenting cell functions . Therefore, CMV infection is both a marker of an immunocompromised state and has, itself, an immunosuppressive effect . er Th efore, when one opportunistic infection is diagnosed in transplant patients, physicians should consider that patients may at the same time or in the following weeks/months present with another opportunistic infection. 10, 13, 15, 17 Several articles have reported that the development of acute graft rejection is a risk factor for PJP . Our data were similar. When acute graft rejection occurs, patients are treated with steroid pulse therapy or other immunosuppressive agents. These results support the hypothesis that severe immunosuppression may increase the occurrence of PJP. Similarly, low lymphocyte counts have been reported to be associated with the develop- ment of PJP . Our data showed that the median lowest lymphocyte count was relatively low in the PJP group. Although this result was not evident in multivariate analysis, the univariate analysis still suggests the importance of lymphocytes in the development of PJP infection. Previous studies have reported other risk factors for PJP in patients who have undergone kidney transplan- 3, 15, 24 tation . Lufft et al . first reported that different immunosuppressive regimens could ae ff ct the occurrence of PJP in renal transplant recipients . In their report, tacrolimus-based regimens seemed more likely to trigger PJP, but this was not confirmed. MMF was suggested for use as an immunosuppressive agent having anti-PJP 25, 26 effects . In our study, there was no difference in the occurrence of PJP between the groups of patients receiving tacrolimus- or cyclosporine-based regimens. We could not analyze the effect of MMF on PJP because kidney transplant recipients at our center almost used MMF. Further research on the ee ff ct of immunosuppressive regi - mens on development of PJP is needed. We performed further evaluation of patients (n = 112) who experienced acute graft rejection. Arend et al . reported that the incidence of PJP among patients treated for 0, 1, 2, or ≥3 rejection episodes increased with the increasing number of rejection episodes. The proportion of patients requiring additional ATG for an episode of rejection was higher in the PJP positive group than in the control group . In our study, there was no associ- ation between the number of acute graft rejection episodes and PJP. Only additional use of ATG for treatment of acute graft rejection was associated with the development of PJP in the acute graft rejection subgroup. PJP occurred in four patients whilst taking PJP prophylaxis, three of whom had used additional ATG treatment prior Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 4 www.nature.com/scientificreports/ Univariate Multivariate PJP PJP negative positive Variable (n = 482) (n = 18) P OR (95% CI) P 3.93 (1.00– Men, sex, N (%) 292(60.6) 15 (83.3) 0.052 0.050 15.47) 47.0 53.0 Age, years (median, range) 0.076 1.02 (0.96–1.08) 0.536 (18.0–71.0) (25.0–65.0) Follow up duration, month 36.2 36.3 0.688 (median, range) (18.7–54.6) (18.4–54.5) 22.2 21.1 BMI, kg/m2 (median, IQR) 0.200 0.94 (0.78–1.14) 0.525 (20.1–24.2) (18.5–23.7) KT type, living, N (%) 314 (65.1) 8 (44.4) 0.072 0.50(0.17–1.44) 0.198 Re-transplantation, N (%) 40 (8.3) 1 (5.6) 1.000 Ever smoker, N (%) 133 (27.6) 6 (33.3) 0.594 Primary underlying disease, N (%) 0.102  Polycystic kidney 15 (3.1) 2 (11.1)  HTN 194 (40.2) 4 (22.2)  DM 81 (16.8) 4 (22.2)  IgA nephropathy 60 (12.4) 3 (16.7)  Autoimmune disease 6 (1.2) 1 (5.6)  Chronic Glomerulonephritis 35 (7.3) 1 (5.6)  Nephrotic syndrome 40 (8.3) 1 (5.6)  Recurrent pyelonephritis/other 19 (3.9) 2 (11.1)  Unknown 32 (6.6) 0 (0) 11.81 (3.06– Acute graft rejection, N (%) 97 (20.1) 15 (83.3) <0.001 <0.001 45.57) 5.42 (1.69– CMV infection, N (%) 62 (12.9) 11 (61.1) <0.001 0.005 17.39) BK virus infection, N (%) 88 (18.3) 7 (38.9) 0.058 Using DM medication, N (%) 126 (26.1) 9 (50.0) 0.032 1.59 (0.51–5.02) 0.427 History of TB, N (%) 29 (6.0) 1 (5.6) 1.000 Lowest lymphocyte, 10 /μ 0.7 0.3 0.007 1.21 (0.83–1.70) 0.290 (0.4–1.2) (0.1–0.6) (median, IQR) Immunosuppressive agent, N (%) 0.710  Cyclosporine based regimen 59 (12.2) 1 (5.6)  Tacrolimus based regimen 423 (87.8) 17 (94.4) Table 3. Risk factors for PJP pneumonia in total population (univariate and multivariate analysis). Abbreviations: PJP, Pneumocystis jirovecii pneumonia; OR, odds ratio; CI, confidence interval; IQR, interquartile range; BMI, body mass index; KT, kidney transplantation; TB, tuberculosis; HTN, hypertension; DM, diabetes mellitus. Figure 1. Interval time of PJP aer ac ft ute graft rejection or CMV infection in PJP patients. (a) Acute rejection and PJP: Median month (IQR): 6.1 (3.3–12.9), (b) CMV infection and PJP: Median month (IQR): 1.5 (0.1–7.1). Note: PJP, Pneumocystis jirovecii pneumonia. Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 5 www.nature.com/scientificreports/ Univariate Multivariate PJP negative PJP positive OR (95% Variable (n = 97) (n = 15) P CI) P 6.45 (1.24– Men, sex, N (%) 62 (63.9) 13 (86.7) 0.138 0.027 33.73) 1.03 Age, years (median, IQR) 48.0 (38.5–55.0) 53.0 (43.0–55.0) 0.128 0.331 (0.97–1.10) 0.96 BMI, kg/m (median, IQR) 22.1 (20.2–24.3) 21.5 (18.6–23.7) 0.492 0.677 (0.77–1.18) 0.59 KT type, living, N (%) 60 (61.9) 7 (53.3) 0.264 0.394 (0.18–1.97) Ever smoker, N (%) 27 (27.8) 5 (13.4) 0.760 CMV infection, N (%) 33 (34.0) 10 (66.7) 0.016 BK virus infection, N (%) 27 (27.8) 7 (46.7) 0.225 Steroid pulse, N (%) 97 (100.0) 15 (100.0) 1.000 Using DM medication, N (%) 34 (35.1) 7 (46.7) 0.385 5.25 (1.01– Using of ATG, N (%) 38 (39.2) 11 (73.3) 0.013 0.006 27.36) Acute graft rejection within 1 82 (84.5) 12 (80.0) 0.706 year aer KT ft , N (%) Lowest lymphocyte, 10 /μ 0.26 (0.1–0.6) 0.28 (0.1–0.4) 0.617 (median, IQR) Rejection number ≥2, N (%) 28 (28.9) 6 (40.0) 0.382 Re-transplantation, N (%) 7 (7.2) 1 (6.7) 1.000 History of TB, N (%) 29 (6.0) 1 (6.7) 1.000 Immunosuppressive agent, N (%) 1.000  Cyclosporine based regimen 7 (7.2) 1 (6.7)  Tacrolimus based regimen 90 (92.8) 14 (93.3) Table 4. Risk factors for PJP in acute graft rejection patients (univariate and multivariate analysis). Abbreviations: PJP, Pneumocystis jirovecii pneumonia; OR, Odds ratio; CI, confidence interval; IQR, interquartile range; BMI, body mass index; KT, kidney transplantation; DM, diabetes mellitus; ATG, anti- thymocyte globulin; TB, tuberculosis. to developing PJP. These results suggest that immunosuppressive status is more important than other factors in the development of PJP. All guidelines recommend PJP prophylaxis for a certain period aer k ft idney transplantation, although no uni- 8–10 versal consensus exists on the optimal duration of prophylaxis . Our data indicated that acute graft rejection, CMV infection, and ATG use in the acute graft rejection subgroup could be the risk factors for PJP. Thus, further PJP prophylaxis is suggested in patients who have these risk factors, based on our data. In terms of the duration of prophylaxis, most cases of PJP occurring aer ac ft ute graft rejection occurred within 12 months, and most cases of PJP occurring aer a ft n episode of CMV infection occurred within 6 months. Hence, we suggest a 6–12 month course of prophylaxis following either an episode of acute graft rejection or CMV infection. Although some com- plications have been reported with the use of TMP/SMX for PJP prophylaxis, major complications are relatively rare ; therefore, the benefits of PJP prophylaxis outweigh the risks. There are several limitations to our study. First, we used the results of PCR as well as direct immunouo fl res- cence to diagnose PJP. Thus, our data might include patients with false positive results. In general, confirmation of PJP requires special microbiological stains of sputum or bronchoalveolar lavage (BAL) specimens . However, the proportion of PJP cases in HIV-uninfected patients in which is confirmed as organism in respiratory spec- 30–32 imens is relatively low (compared with HIV-infected patients) because of the low burden of organism . Pneumocystis jirovecii PCR assays are sensitive and increase the diagnostic yield in HIV-uninfected immunocom- 33–35 promised patients . We used chest CT findings at initial diagnosis of PJP to exclude patients with false positive Pneumocystis jirovecii PCR results. Second, the number of PJP patients in our study was relatively small. However, unlike other studies, our study collected data on a large number of consecutive kidney transplant patients, instead of selecting particular patients; this point can be its strength. Third, patients with CMV infection and patients who used ATG overlapped in the acute graft rejection subgroup. Thus, we could not analyze the independent effect of CMV infection or ATG use on PJP. Fourth, this study was retrospective, thus, we could not systemically analyze multiple laboratory test results. There is a need for further, prospective studies to assess the risk factors for PJP. Conclusion Our data suggest that acute graft rejection and CMV infection may be risk factors for PJP in kidney transplant patients. In patients who develop acute graft rejection, the use of ATG may increase the risk of PJP. We suggest providing further PJP prophylaxis for kidney transplant patients who develop CMV infection or acute graft rejec- tion, especially for those treated with ATG. Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 6 www.nature.com/scientificreports/ References 1. o Th mas, C. F. Jr. & Limper, A. H. Pneumocystis pneumonia. N Engl J Med 350, 2487–2498, doi:10.1056/NEJMra032588 (2004). 2. Sepkowitz, K. A., Brown, A. E. & Armstrong, D. Pneumocystis carinii pneumonia without acquired immunodeficiency syndrome. More patients, same risk. 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Screening Pneumocystis carinii pneumonia in non-HIV-infected immunocompromised patients using polymerase chain reaction. Diagn Microbiol Infect Dis 64, 396–401, doi:10.1016/j.diagmicrobio.2009.04.009 (2009). Author Contributions S.H. Lee: participated in data collection and analysis, wrote the manuscript. M.S. Park, Y.S. Kim, S.K. Kim, J. Chang: assisted in patient data assessment and supervision of the project. K.H. Huh, D.J. Joo, M.S. Kim, S.I. Kim, J.H. Lee, Y.S. Kim: provided patient data, participated in data and follow-up of patients. S.Y. Kim: performed study conception and design, final approval of the version to be published. Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 7 www.nature.com/scientificreports/ Additional Information Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Risk factors for Pneumocystis jirovecii pneumonia (PJP) in kidney transplantation recipients

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www.nature.com/scientificreports OPEN Risk factors for Pneumocystis jirovecii pneumonia (PJP) in kidney transplantation recipients Received: 17 January 2017 1,3 2 2 2 2 2 Su Hwan Lee , Kyu Ha Huh , Dong Jin Joo , Myoung Soo Kim , Soon Il Kim , Juhan Lee , 1 1 1 1 2 1 Accepted: 4 April 2017 Moo Suk Park , Young Sam Kim , Se Kyu Kim , Joon Chang , Yu Seun Kim & Song Yee Kim Published: xx xx xxxx Pneumocystis jirovecii pneumonia (PJP) is a potentially life-threatening infection that occurs in immunocompromised patients. The aim of this study was to evaluate risk factors for PJP in kidney transplantation recipients. We conducted a retrospective analysis of patient data from 500 consecutive kidney transplants performed at Severance Hospital between April 2011 and April 2014. Eighteen kidney transplantation recipients (3.6%) were diagnosed with PJP. In the univariate analysis, acute graft rejection, CMV infection, use of medication for diabetes mellitus, and lowest lymphocyte count were associated with PJP. Recipients who experienced acute graft rejection (odds ratio [OR] 11.81, 95% confidence interval [CI] 3.06–45.57, P < 0.001) or developed CMV infection (OR 5.42, 95% CI 1.69– 17.39, P = 0.005) had high odds of PJP in multivariate analysis. In the acute graft rejection subgroup, patients treated with anti-thymocyte globulin (ATG) had significantly higher odds of PJP (OR 5.25, 95% CI 1.01–27.36, P = 0.006) than those who were not. Our data suggest that acute graft rejection and CMV infection may be risk factors for PJP in kidney transplant patients. The use of ATG for acute graft rejection may increase the risk of PJP. Pneumocystis jirovecii is an opportunistic fungal pathogen . Pneumocystis jirovecii pneumonia (PJP) previously known as Pneumocystis carinii pneumonia is a potentially life-threatening infection that occurs in immunocom- 1, 2 promised patients . In the absence of prophylaxis, PJP occurs in approximately 5–15% of transplant patients, 2, 3 depending on the transplanted organ or transplant center . 4, 5 In kidney transplantation, PJP is a very serious risk factor for graft loss and patient mortality . In the absence of appropriate treatment, the mortality rate of PJP is 90–100%, and can be as high as 50% despite adequate ther- 6, 7 apy . er Th efore, several guidelines—such as the Kidney Disease Improving Global Outcomes (KDIGO) guide- line, the European Renal Best Practice guideline, and other reports— usually recommend PJP prophylaxis by 8–10 using TMP/SMX for 3–6 months after renal transplantation . The incidence rate of PJP has decreased with the use of prophylaxis, however, an increasing number of PJP outbreaks in kidney transplant centers has been 4, 5, 11 reported worldwide in recent years . The causes of those outbreaks have not fully been evaluated. Risk factors for the development of PJP in kidney transplant patients are still not confirmed. The overall load of immunosuppressive therapy, higher donor age, higher recipient age, lymphopenia, previous cytomegalovirus (CMV) infection, or treatment used for episodes of graft rejection have been reported as risk factors for PJP in 12–17 kidney transplant patients . However, factors identified in some studies are not always confirmed in other studies. Thus, further research is needed to evaluate the risk factors for PJP in kidney transplant patients in the era of routine PJP prophylaxis. The aim of this study was to evaluate the risk factors for PJP in kidney transplantation recipients. Patients and Methods Study design and population. This single center, retrospective clinical study included all kidney transplant patients aged ≥18 years who underwent kidney transplantation at the Severance Hospital, a 2000-bed university Division of Pulmonology, Department of Internal Medicine, Severance Hospital, Institute of Chest Diseases, Yonsei University College of Medicine, Seoul, Republic of Korea. Department of Surgery, Research Institute for Transplantation, Yonsei University College of Medicine, Seoul, Republic of Korea. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Ewha Medical Research Institute, Ewha Womans University School of Medicine, Seoul, Korea. Correspondence and requests for materials should be addressed to S.Y.K. (email: dobie@ yuhs.ac) Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 1 www.nature.com/scientificreports/ tertiary referral hospital in South Korea, from April 2011 to April 2014. During this period, 500 patients under- went kidney transplantation; they were followed up until October 2015. We divided the patients into two groups according to the occurrence of PJP—the case group developed PJP whereas the control group did not—and then compared the groups. In our center, all kidney transplant recipients receive PJP prophylaxis using trimethoprim/ sulfamethoxazole (TMP/SMX) 160/800 mg per day for 12 months post-transplantation. After transplantation, patients received a tacrolimus-based combination regimen or a cyclosporine-based combination regimen for maintaining immunosuppression. Data collection. Data from all kidney transplant recipients were collected from the hospital’s electronic med- ical records. Clinical data on mortality, development of PJP, demographic characteristics, graft origin (deceased vs. living), immunosuppressive regimen, data about acute graft rejection such as frequency of such episodes or treatment received, smoking status, history of infections including CMV, BK virus, hepatitis, tuberculosis (TB), and comorbidities were evaluated. Definition. We defined PJP when the following two conditions were satisfied; first, a positive result on Pneumocystis jirovecii real-time polymerase chain reaction (PCR) testing or direct immunou fl orescence testing of microbiological samples (sputum, tracheal aspirate, bronchial washing fluid or bronchoalveolar lavage fluid) 18–20 and second, identification of lung infiltration on chest computed tomography (CT) . CMV infection was defined as a fourfold elevation of the CMV PCR titer and the use of CMV medication (ganciclovir or valganciclovir). Smoking status was categorized as ever smoker or never smoked; the latter cate- gory included those who smoked fewer than 100 cigarettes in their lifetime. Development of acute graft rejection was established by biopsy of the transplanted kidney. Steroid pulse therapy (methylprednisolone [500 mg/day × 4 for 5 days]) was considered the first line therapy for acute graft rejection. When there was an inadequate response to steroid pulse therapy, an anti-thyomcyte globulin (ATG) was used. Lowest lymphocyte count was reviewed during follow-up in the PJP negative group aer ft first hospital discharge and within 1 month before diagnosis of PJP in the PJP group. Clinical characteristics and events were reviewed until the last follow-up date in the PJP negative group and were reviewed until the date of PJP development in the PJP group. Ethical approval. The study protocol was approved by the Institutional Review Board (IRB) of Severance Hospital (IRB number: 4-2015-1051). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was waived by the IRB because of the study’s retrospective nature. Statistical analysis. Statistical analysis was performed using SPSS version 20 (IBM, Armonk, New York, USA). Data are described as medians (interquartile range [IQR]) or numbers (percentages). The Chi-squared and Fisher’s exact test or the Mann-Whitney test was used to assess differences between two groups. The independent risk for PJP was identified using logistic regression modeling. The fundamental variable (such as age, gender, BMI or kidney transplantation type) and variable which were meaningful risk factors in univariate analysis were used in multivariate analysis. A two-tailed P-value < 0.05 was considered statistically significant. Results Characteristics of the overall study population and patients diagnosed with PJP. Demographic and clinical characteristics of the total study population are presented in Table 1. Overall, 500 patients were enrolled over the 3-year study period, with a similar number enrolled each year. The median age of all recipients was 47 years (range, 18–71) and men accounted for 61.4%. The major cause of kidney transplantation was hyper - tension (39.6%); the other cause was diabetes mellitus (17.0%). Transplanted kidneys were sourced more oen ft from living than deceased donors (64.4% living vs. 35.6% deceased). In terms of immunosuppressive medication, 440 (88%) patients received a tacrolimus-based combination regimen and 60 (12%) received a cyclosporine-based combination regimen. The median duration of follow up was 36.2 month (range, 18.4–54.6). Eighteen patients were diagnosed with PJP during the study period, and most cases developed 12–24 months aer k ft idney transplantation. All patients diagnosed with PJP were started on TMP/SMX. Five patients required second-line medication because of disease progression despite TMP-SMX use; four were treated with primaquine and clindamycin, while one was treated with pentamidine. Twelve (66.7%) patients with PJP improved aer t ft reat- ment, with no sequelae. However, three (16.7%) patients had residual sequelae and three (16.7%) died. Table 2 describes clinical characteristics and prognosis of PJP patients. Risk factors for PJP in the total population. Table 3 shows the risk factors for PJP. In the univariate analysis, more men than women developed PJP, although this was not statistically significant (60.6% vs. 83.3%, respectively, P = 0.052). Median age, duration of follow up, gra ft source (deceased vs. living donor), proportion of re-transplant patients, and smoking status did not differ significantly between those who had PJP and those who did not. The underlying cause of kidney disease, necessitating transplantation, varied. However, hypertension, diabetes mellitus (DM), and IgA nephropathy were the major causes in both groups (P = 0.102). More acute gra ft rejection aer t ft ransplantation wwere observed in the PJP group compared with the PJP negative group (83.3% vs. 20.1%, respectively, P < 0.001). And CMV infection was more common in the PJP group than the PJP nega- tive group (61.1% vs. 12.9%, respectively, P < 0.001). Acute rejection and CMV infection were checked until PJP pneumonia in PJP group. Among those patients in the PJP group, 50% used medication for DM, compared to 26.1% in the PJP negative group (P = 0.032). Lowest lymphocyte counts were lower in the PJP group than in the 3 3 control group (0.3 × 10 /µL vs. 0.7 × 10 /µL, P = 0.007). In terms of immunosuppressive agents, patients received either tacrolimus- or cyclosporine-based regimens; the regimen used did not differ between the PJP group and the control group (P = 0.710). In the multivariate analysis, acute graft rejection [odds ratio (OR), 11.81; 95% Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 2 www.nature.com/scientificreports/ Characteristics N = 500 Sex, N (%)  Men 307 (61.4)  Women 193 (38.6) Age, years (median, range) 47 (18–71) Transplantation era, N (%)  2011.4–2012.3 167 (33.4)  2012.4–2013.3 158 (31.6)  2013.4–2014.4 175 (35.0) BMI, kg/m2 (median, IQR) 22 (20.1–24.2) KT type, N (%)  Deceased 178 (35.6)  Living 322 (64.4) Re-transplantation, N (%) 41 (8.2) Primary underlying disease, N (%)  Polycystic kidney 17 (3.4)  HTN 198 (39.6)  DM 85 (17.0)  IgA nephropathy 63 (12.6)  Autoimmune disease 7 (1.4)  Chronic glomerulonephritis 36 (7.2)  Nephrotic syndrome 41 (8.2)  Recurrent pyelonephritis/other 21 (4.2)  Unknown 32 (6.4) Immunosuppressive agent, N (%)  Cyclosporine based regimen 60 (12.0)  Tacrolimus based regimen 440 (88.0) Follow up duration, month (median, range) 36.2 (18.4–54.5) Development of PJP, N (%) 18 (3.6) Interval between PJP and graft, month (median 17.4 (11.2–27.9) IQR)  −12 month, N (%) 4 (22.2)  12–24 month, N (%) 7 (38.9)  24–36 month, N (%) 6 (33.3)  36- month, N (%) 1 (5.6) Treatment medication of PJP, N (%)  TMP-SMX alone 13 (72.2)  TMP-SMX prior to primaqiune + clinadamycin 4 (22.2)  TMP-SMX prior to pentamidine 1 (5.6) Table 1. Demographic and clinical characteristics of total study population and PJP patients. Abbreviations: PJP, Pneumocystis jirovecii pneumonia; IQR, interquartile range; BMI, body mass index; KT, kidney transplantation; HTN, hypertension; DM, diabetes mellitus; TMP-SMX, trimethoprim/sulfamethoxazole. confidence interval (CI), 3.06–45.57] and CMV infection (OR, 5.42; 95% CI, 1.69–17.93) were associated with PJP, whereas sex, age, body mass index (BMI), and graft source were not. Acute graft rejection and CMV infection were important risk factors for PJP, as presented in Table  3. To under- stand the impact of acute graft rejection and CMV pneumonia on PJP, we investigated the timing of the onset of PJP aer ac ft ute graft rejection or CMV infection. The median interval between acute graft rejection and PJP was 6.1 (IQR, 3.3–12.9) months, while the median interval between CMV infection and PJP was 1.5 (IQR, 0.1–7.1) months (Fig. 1a and b). Risk factors for PJP in patients experiencing acute graft rejection. As shown in Table 3, most cases of PJP (83.3%) occurred aer ac ft ute graft rejection. Hence, we performed a subgroup analysis of the risk factors for PJP in patients who developed acute graft rejection (Table  4). Of the 500 kidney transplant patients, 112 experienced acute graft rejection. Of those, 15 were diagnosed with PJP. Sex, median age, BMI, graft source, smoking status, BK virus infection, use of DM medication, development of acute graft rejection within 1-year post-transplant, number of acute graft rejection episodes, and lowest lymphocyte count did not differ between patients who developed PJP and those who did not. All patients who developed acute graft rejection were man - aged with steroid pulse therapy. However, additional use of ATG for acute gra r ft ejection treatment was more used in the PJP group (73.3% vs. 39.2%, P = 0.013). In addition, the proportion of patients with CMV infection was Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 3 www.nature.com/scientificreports/ Interval between Rejection CMV rejection and Sex Age Donor type number infection Immunosuppression Treatment PCP Outcome Case1 Male 42 Living 1 Yes Tac, Cor, MMF TMP-SMX → Pentamidine 3.3 month Die Case2 Male 35 Living 4 Yes Tac, Cor, Miz TMP-SMX → Primaquine + Clindamycin 3.4 month Recover Case3 Male 54 Living 1 No Tac, Cor, MMF TMP-SMX 6.9 month Die Case4 Male 51 Deceased 0 No Tac, Cor, MMF TMP-SMX — Recover Case5 Male 58 Deceased 1 No Cys, Cor, MMF TMP-SMX 11 month Graft fail Case6 Male 50 Deceased 1 Yes Tac, Cor, MMF TMP-SMX 6.2 month Recover Case7 Male 53 Deceased 1 No Tac, Cor, MMF TMP-SMX 29 month Graft fail Case8 Female 55 Living 1 Yes Tac, Cor, MMF TMP-SMX → Primaquine + Clindamycin 13 month Recover Case9 Male 54 Deceased 2 Yes Tac, Cor, MMF TMP-SMX → Primaquine + Clindamycin 1.5 month Die Case10 Male 55 Deceased 1 No Tac, Cor, MMF TMP-SMX 29 month Recover Case11 Male 61 Living 1 Yes Tac, Cor, MMF TMP-SMX 5.7 month Recover Case12 Male 43 Living 1 Yes Tac, Cor TMP-SMX 6.1 month Graft fail Case 13 Female 25 Living 0 Yes Tac, Cor, MMF TMP-SMX — Recover Case 14 Male 53 Deceased 2 No Tac, Cor, MMF TMP-SMX 2.8 month Recover Case 15 Male 52 Living 2 Yes Tac, Cor, MMF TMP-SMX 4.8 month Recover Case 16 Female 56 Deceased 2 Yes Tac, Cor, MMF TMP-SMX → Primaquine + Clidamycin 17 month Recover Case 17 Male 65 Deceased 0 No Tac, Cor, MMF TMP-SMX — Recover Case 18 Male 40 Deceased 2 Yes Tac, Cor, MMF TMP-SMX 1.2 month Recover Table 2. Clinical characteristic and prognosis of PJP patients in kidney transplantation. Abbreviations: PJP, Pneumocystis jirovecii pneumonia; CMV, cytomegalovirus; KT, kidney transplantation; MMF, mycophenolate mofetil; Cor, corticosteroid; Tac, tacrolimus; TMP-SMX, trimethoprim/sulfamethoxazole; Miz, Mizoribine; Cys, cyclosporine. higher in the PJP group than in the PJP negative group (66.7% vs. 34%, respectively, P = 0.016). In the multivari- ate analysis, the independent risk factors for PJP in the acute graft rejection subgroup were identified as male sex (OR, 6.45; 95% CI, 1.24–33.73) and the use of ATG (OR, 5.25; 95% CI, 1.01–27.36). Discussion In our study, of 500 kidney transplant patients, 18 developed PJP. Acute graft rejection and CMV infection were identified as risk factors for PJP. Within the subgroup of patients who experienced acute graft rejection, male sex and the use of ATG were risk factors for PJP. Our data showed that more patients in the PJP group than in the PJP negative group had CMV infection. This 17, 21 result is in agreement with that of previous studies . CMV is known to modify host immune responses by var- ious mechanisms, suppressing helper T-cell and antigen presenting cell functions . Therefore, CMV infection is both a marker of an immunocompromised state and has, itself, an immunosuppressive effect . er Th efore, when one opportunistic infection is diagnosed in transplant patients, physicians should consider that patients may at the same time or in the following weeks/months present with another opportunistic infection. 10, 13, 15, 17 Several articles have reported that the development of acute graft rejection is a risk factor for PJP . Our data were similar. When acute graft rejection occurs, patients are treated with steroid pulse therapy or other immunosuppressive agents. These results support the hypothesis that severe immunosuppression may increase the occurrence of PJP. Similarly, low lymphocyte counts have been reported to be associated with the develop- ment of PJP . Our data showed that the median lowest lymphocyte count was relatively low in the PJP group. Although this result was not evident in multivariate analysis, the univariate analysis still suggests the importance of lymphocytes in the development of PJP infection. Previous studies have reported other risk factors for PJP in patients who have undergone kidney transplan- 3, 15, 24 tation . Lufft et al . first reported that different immunosuppressive regimens could ae ff ct the occurrence of PJP in renal transplant recipients . In their report, tacrolimus-based regimens seemed more likely to trigger PJP, but this was not confirmed. MMF was suggested for use as an immunosuppressive agent having anti-PJP 25, 26 effects . In our study, there was no difference in the occurrence of PJP between the groups of patients receiving tacrolimus- or cyclosporine-based regimens. We could not analyze the effect of MMF on PJP because kidney transplant recipients at our center almost used MMF. Further research on the ee ff ct of immunosuppressive regi - mens on development of PJP is needed. We performed further evaluation of patients (n = 112) who experienced acute graft rejection. Arend et al . reported that the incidence of PJP among patients treated for 0, 1, 2, or ≥3 rejection episodes increased with the increasing number of rejection episodes. The proportion of patients requiring additional ATG for an episode of rejection was higher in the PJP positive group than in the control group . In our study, there was no associ- ation between the number of acute graft rejection episodes and PJP. Only additional use of ATG for treatment of acute graft rejection was associated with the development of PJP in the acute graft rejection subgroup. PJP occurred in four patients whilst taking PJP prophylaxis, three of whom had used additional ATG treatment prior Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 4 www.nature.com/scientificreports/ Univariate Multivariate PJP PJP negative positive Variable (n = 482) (n = 18) P OR (95% CI) P 3.93 (1.00– Men, sex, N (%) 292(60.6) 15 (83.3) 0.052 0.050 15.47) 47.0 53.0 Age, years (median, range) 0.076 1.02 (0.96–1.08) 0.536 (18.0–71.0) (25.0–65.0) Follow up duration, month 36.2 36.3 0.688 (median, range) (18.7–54.6) (18.4–54.5) 22.2 21.1 BMI, kg/m2 (median, IQR) 0.200 0.94 (0.78–1.14) 0.525 (20.1–24.2) (18.5–23.7) KT type, living, N (%) 314 (65.1) 8 (44.4) 0.072 0.50(0.17–1.44) 0.198 Re-transplantation, N (%) 40 (8.3) 1 (5.6) 1.000 Ever smoker, N (%) 133 (27.6) 6 (33.3) 0.594 Primary underlying disease, N (%) 0.102  Polycystic kidney 15 (3.1) 2 (11.1)  HTN 194 (40.2) 4 (22.2)  DM 81 (16.8) 4 (22.2)  IgA nephropathy 60 (12.4) 3 (16.7)  Autoimmune disease 6 (1.2) 1 (5.6)  Chronic Glomerulonephritis 35 (7.3) 1 (5.6)  Nephrotic syndrome 40 (8.3) 1 (5.6)  Recurrent pyelonephritis/other 19 (3.9) 2 (11.1)  Unknown 32 (6.6) 0 (0) 11.81 (3.06– Acute graft rejection, N (%) 97 (20.1) 15 (83.3) <0.001 <0.001 45.57) 5.42 (1.69– CMV infection, N (%) 62 (12.9) 11 (61.1) <0.001 0.005 17.39) BK virus infection, N (%) 88 (18.3) 7 (38.9) 0.058 Using DM medication, N (%) 126 (26.1) 9 (50.0) 0.032 1.59 (0.51–5.02) 0.427 History of TB, N (%) 29 (6.0) 1 (5.6) 1.000 Lowest lymphocyte, 10 /μ 0.7 0.3 0.007 1.21 (0.83–1.70) 0.290 (0.4–1.2) (0.1–0.6) (median, IQR) Immunosuppressive agent, N (%) 0.710  Cyclosporine based regimen 59 (12.2) 1 (5.6)  Tacrolimus based regimen 423 (87.8) 17 (94.4) Table 3. Risk factors for PJP pneumonia in total population (univariate and multivariate analysis). Abbreviations: PJP, Pneumocystis jirovecii pneumonia; OR, odds ratio; CI, confidence interval; IQR, interquartile range; BMI, body mass index; KT, kidney transplantation; TB, tuberculosis; HTN, hypertension; DM, diabetes mellitus. Figure 1. Interval time of PJP aer ac ft ute graft rejection or CMV infection in PJP patients. (a) Acute rejection and PJP: Median month (IQR): 6.1 (3.3–12.9), (b) CMV infection and PJP: Median month (IQR): 1.5 (0.1–7.1). Note: PJP, Pneumocystis jirovecii pneumonia. Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 5 www.nature.com/scientificreports/ Univariate Multivariate PJP negative PJP positive OR (95% Variable (n = 97) (n = 15) P CI) P 6.45 (1.24– Men, sex, N (%) 62 (63.9) 13 (86.7) 0.138 0.027 33.73) 1.03 Age, years (median, IQR) 48.0 (38.5–55.0) 53.0 (43.0–55.0) 0.128 0.331 (0.97–1.10) 0.96 BMI, kg/m (median, IQR) 22.1 (20.2–24.3) 21.5 (18.6–23.7) 0.492 0.677 (0.77–1.18) 0.59 KT type, living, N (%) 60 (61.9) 7 (53.3) 0.264 0.394 (0.18–1.97) Ever smoker, N (%) 27 (27.8) 5 (13.4) 0.760 CMV infection, N (%) 33 (34.0) 10 (66.7) 0.016 BK virus infection, N (%) 27 (27.8) 7 (46.7) 0.225 Steroid pulse, N (%) 97 (100.0) 15 (100.0) 1.000 Using DM medication, N (%) 34 (35.1) 7 (46.7) 0.385 5.25 (1.01– Using of ATG, N (%) 38 (39.2) 11 (73.3) 0.013 0.006 27.36) Acute graft rejection within 1 82 (84.5) 12 (80.0) 0.706 year aer KT ft , N (%) Lowest lymphocyte, 10 /μ 0.26 (0.1–0.6) 0.28 (0.1–0.4) 0.617 (median, IQR) Rejection number ≥2, N (%) 28 (28.9) 6 (40.0) 0.382 Re-transplantation, N (%) 7 (7.2) 1 (6.7) 1.000 History of TB, N (%) 29 (6.0) 1 (6.7) 1.000 Immunosuppressive agent, N (%) 1.000  Cyclosporine based regimen 7 (7.2) 1 (6.7)  Tacrolimus based regimen 90 (92.8) 14 (93.3) Table 4. Risk factors for PJP in acute graft rejection patients (univariate and multivariate analysis). Abbreviations: PJP, Pneumocystis jirovecii pneumonia; OR, Odds ratio; CI, confidence interval; IQR, interquartile range; BMI, body mass index; KT, kidney transplantation; DM, diabetes mellitus; ATG, anti- thymocyte globulin; TB, tuberculosis. to developing PJP. These results suggest that immunosuppressive status is more important than other factors in the development of PJP. All guidelines recommend PJP prophylaxis for a certain period aer k ft idney transplantation, although no uni- 8–10 versal consensus exists on the optimal duration of prophylaxis . Our data indicated that acute graft rejection, CMV infection, and ATG use in the acute graft rejection subgroup could be the risk factors for PJP. Thus, further PJP prophylaxis is suggested in patients who have these risk factors, based on our data. In terms of the duration of prophylaxis, most cases of PJP occurring aer ac ft ute graft rejection occurred within 12 months, and most cases of PJP occurring aer a ft n episode of CMV infection occurred within 6 months. Hence, we suggest a 6–12 month course of prophylaxis following either an episode of acute graft rejection or CMV infection. Although some com- plications have been reported with the use of TMP/SMX for PJP prophylaxis, major complications are relatively rare ; therefore, the benefits of PJP prophylaxis outweigh the risks. There are several limitations to our study. First, we used the results of PCR as well as direct immunouo fl res- cence to diagnose PJP. Thus, our data might include patients with false positive results. In general, confirmation of PJP requires special microbiological stains of sputum or bronchoalveolar lavage (BAL) specimens . However, the proportion of PJP cases in HIV-uninfected patients in which is confirmed as organism in respiratory spec- 30–32 imens is relatively low (compared with HIV-infected patients) because of the low burden of organism . Pneumocystis jirovecii PCR assays are sensitive and increase the diagnostic yield in HIV-uninfected immunocom- 33–35 promised patients . We used chest CT findings at initial diagnosis of PJP to exclude patients with false positive Pneumocystis jirovecii PCR results. Second, the number of PJP patients in our study was relatively small. However, unlike other studies, our study collected data on a large number of consecutive kidney transplant patients, instead of selecting particular patients; this point can be its strength. Third, patients with CMV infection and patients who used ATG overlapped in the acute graft rejection subgroup. Thus, we could not analyze the independent effect of CMV infection or ATG use on PJP. 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Screening Pneumocystis carinii pneumonia in non-HIV-infected immunocompromised patients using polymerase chain reaction. Diagn Microbiol Infect Dis 64, 396–401, doi:10.1016/j.diagmicrobio.2009.04.009 (2009). Author Contributions S.H. Lee: participated in data collection and analysis, wrote the manuscript. M.S. Park, Y.S. Kim, S.K. Kim, J. Chang: assisted in patient data assessment and supervision of the project. K.H. Huh, D.J. Joo, M.S. Kim, S.I. Kim, J.H. Lee, Y.S. Kim: provided patient data, participated in data and follow-up of patients. S.Y. Kim: performed study conception and design, final approval of the version to be published. Scientific Repo R ts | 7: 1571 | DOI:10.1038/s41598-017-01818-w 7 www.nature.com/scientificreports/ Additional Information Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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