Male Renal Transplant Recipients' Experiences From Their Wives' Pregnancy and Childbirth.

This study aimed to analyze male renal transplant recipients' experience with their partners' pregnancy and childbirth and to investigate their methods of nursing their condition. We performed semistructured interviews and collected data from 6 Japanese males who underwent a kidney transplant after their partner had given birth. The data were analyzed using the Qualitative Synthesis Method (KJ Method). The mean age of the participants at data collection was 40.3 ± 4.7 years, whereas it was 34.7 ± 5.8 years when the transplant was performed. The Qualitative Synthesis Method revealed 7 symbols related to the pregnancy and childbirth experience of the partners of male kidney transplant recipients. Males who received a kidney transplant struggled with severe renal disease before the transplant. They also experienced indecisiveness about whether they should go through with the transplant. However, their lives changed because of the transplant and having children. This situation resulted in a sense of responsibility and a reason to live robustly for the male kidney transplant recipients. Nevertheless, they faced distress as kidney transplant patients. Their wives supported them through this experience. They communicated to their children what they learned from the experience while effectively dealing with their condition. The improvement in their sexual function resulting from the transplant influenced their determination to get married. It is necessary to offer information about the recovery of fertility and the possibility of having a child when choosing renal replacement therapy, give explanations based on evidence, and construct a counseling system.

A Case of Bariatric Surgery for a Japanese Kidney Transplant Recipient With Diabetes Mellitus: A Case Report.

The patient, a 54-year-old woman, underwent a living donor kidney transplant at Osaka City University Hospital 7 years before the bariatric surgery. Her comorbidities were diabetes, sleep apnea, and severe obesity (weight 103 kg, body mass index [BMI] 36 kg/m2), and her diabetes was poorly controlled with an HbA1c of 8.5%. On admission, she weighed 99 kg, BMI was 34 kg/m2, Serum creatinine (S-Cre) was 1.54 mg/dL, and HbA1c was 7.1%. A laparoscopic sleeve gastrectomy was performed, and her weight decreased without complications during the perioperative period. She was discharged on postoperative day 28. Two months after surgery, her weight was 87 kg, BMI 30 kg/m2, S-Cre 1.34 mg/dL, HbA1c 6.7 %, renal function improved, urine protein decreased, and insulin dosage decreased dramatically. We report this valuable case because there are no reports of bariatric surgery in Japanese renal transplant recipients.

Development and nationwide validation of kidney graft injury markers using urinary exosomes and microvesicles (complete English translation of the Japanese version).

BACKGROUND: Non-invasive, prompt, and proper detection tools for kidney graft injuries (KGIs) are awaited to ensure graft longevity. We screened diagnostic biomarkers for KGIs following kidney transplantation using extracellular vesicles (EVs; exosomes and microvesicles) from the urine samples of patients. METHODS: One hundred and twenty-seven kidney recipients at 11 Japanese institutions were enrolled in this study; urine samples were obtained prior to protocol/episode biopsies. EVs were isolated from urine samples, and EV RNA markers were assayed using quantitative reverse transcription polymerase chain reaction. Diagnostic performance of EV RNA markers and diagnostic formulas comprising these markers were evaluated by comparison with the corresponding pathological diagnoses. RESULTS: EV CXCL9, CXCL10, and UMOD were elevated in T-cell-mediated rejection samples compared with other KGI samples, while SPNS2 was elevated in chronic antibody-mediated rejection (cABMR) samples. A diagnostic formula developed through Sparse Logistic Regression analysis using EV RNA markers allowed us to accurately (with an area under the receiver operator characteristic curve [AUC] of 0.875) distinguish cABMR from other KGI samples. EV B4GALT1 and SPNS2 were also elevated in cABMR, and a diagnostic formula using these markers was able to distinguish between cABMR and chronic calcineurin toxicity accurately (AUC 0.886). In interstitial fibrosis and tubular atrophy (IFTA) urine samples and those with high Banff chronicity score sums (BChS), POTEM levels may reflect disease severity, and diagnostic formulas using POTEM detected IFTA (AUC 0.830) and high BChS (AUC 0.850). CONCLUSIONS: KGIs could be diagnosed with urinary EV mRNA analysis with relatively high accuracy.

Frailty and sarcopenia in older kidney transplant recipients: a cross-sectional study.

PURPOSE: The aging of the kidney transplant population is accelerating, and measures against geriatric syndromes including frailty and sarcopenia, which elevate the risk of needing long-term care and even death, are being considered important. Recently, both the frailty and sarcopenia criteria for Asians were revised based on various research reports and clinical experiences. The purpose of this study is twofold: firstly, to investigate the prevalence of frailty based on the revised Japanese version of the Cardiovascular Health Study (J-CHS) criteria and the Kihon Checklist (KCL) and that of sarcopenia based on the Asian Working Group for Sarcopenia (AWGS) 2019 as well as the relationship between frailty and sarcopenia, and secondly, to determine the concurrent validity of the KCL with the revised J-CHScriteria in older kidney transplant recipients. METHODS: This study was a single-center cross-sectional investigation carried out on older kidney transplant recipients who visited our hospital from August 2017 to February 2019. The diagnosis of frailty was assessed using the revised J-CHS criteria and the KCL. The diagnosis of sarcopenia was made by low skeletal muscle mass and either low physical performance or low muscle strength based on the AWGS 2019. To examine the relationship between frailty and sarcopenia, categorical variables were compared using chi-squared test and continuous variables Mann-Whitney U test. Spearman's correlation analysis was used to investigate the correlation between the KCL score and the revised J-CHS score. The concurrent validity of the KCL for estimating frailty based on the revised J-CHS criteria was evaluated using the receiver operating characteristics (ROC) curve analysis. RESULTS: A total of 100 older kidney transplant recipients were enrolled in this study. The median age was 67, 63 (63%) were males, and the median time after transplant was 95 months. The prevalence of frailty based on the revised J-CHS criteria and the KCL, and sarcopenia based on the AWGS 2019 was 15%, and 19%, and 16% respectively. Sarcopenia was significantly associated with frailty based on the KCL (p = 0.016), while not with frailty based on the revised J-CHS criteria (p = 0.11). The KCL score significantly correlated with the revised J-CHS score (p < 0.001). The area under the ROC curve was 0.91. CONCLUSION: Frailty and sarcopenia are interrelated complex geriatric syndromes that are risk factors for adverse health outcomes. In older kidney transplant recipients, frailty and sarcopenia were highly prevalent and frequently co-existed. Furthermore, the KCL was verified as a useful tool for frailty screening in these patient. Easy identification of patients with frailty, which is reversible, can help clinicians institute appropriate corrective measures for kidney transplant recipients to improve transplant outcomes.

Kidney Transplantation After Renal Transcatheter Arterial Embolization for Cyst Infection in a Hemodialysis Patient With Autosomal Dominant Polycystic Kidney Disease: A Case Report.

A 31-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) required antibiotic therapy for repeated renal cyst infections. The patient was scheduled for a living donor renal transplant with her mother as the donor. Two months before surgery, the patient was admitted to the hospital due to a severe renal cyst infection that improved with antibiotic treatment and percutaneous drainage, but the scheduled surgery was postponed. Transcatheter arterial embolization (TAE) was performed to control repeated renal cyst infections. Seven months after TAE, the patient underwent living donor renal transplantation. The postoperative course was uneventful, and the patient was discharged from the hospital on immunosuppressive medication 26 days after surgery with no evidence of recurrent infection or deterioration of renal function. Thirty months after transplantation, there has been no recurrence of infection.

Reliability and Validity of the Japanese Version of the Basel Assessment of Adherence to Immunosuppressive Medications Scale in Kidney Transplant Recipients.

A valid and reliable instrument that can measure adherence is needed to identify nonadherent patients and to improve adherence. However, there is no validated Japanese self-report instrument to evaluate adherence to immunosuppressive medications for transplant patients. The purpose of this study was to determine the reliability and validity of the Japanese version of the Basel Assessment of Adherence to Immunosuppressive Medications Scale (BAASIS). Methods: We translated the BAASIS into Japanese and developed the Japanese version of the BAASIS (J-BAASIS) according to the International Society of Pharmacoeconomics and Outcomes Research task force guidelines. We analyzed the reliability (test-retest reliability and measurement error) and validity of the J-BAASIS (concurrent validity with the medication event monitoring system and the 12-item Medication Adherence Scale) referring to the COSMIN Risk of Bias checklist. Results: A total of 106 kidney transplant recipients were included in this study. In the analysis of test-retest reliability, Cohen's kappa coefficient was found to be 0.62. In the analysis of measurement error, the positive and negative agreement were 0.78 and 0.84, respectively. In the analysis of concurrent validity with the medication event monitoring system, sensitivity and specificity were 0.84 and 0.90, respectively. In the analysis of concurrent validity with the 12-item Medication Adherence Scale, the point-biserial correlation coefficient for the "medication compliance" subscale was 0.38 (P < 0.001). Conclusions: The J-BAASIS was determined to have good reliability and validity. Using the J-BAASIS to evaluate adherence can help clinicians to identify medication nonadherence and institute appropriate corrective measures to improve transplant outcomes.

Kidney transplantation for a transgender male.

The proportion of transgender people has increased over time, but few cases of transgender people undergoing kidney transplantation have been described. A 41-year-old transgender man (female-to-male) had chronic kidney disease caused by IgA nephropathy. He had received testosterone therapy and sex reassignment surgeries (chest masculinization surgery, metoidioplasty, scrotoplasty, and hysterectomy-ovariectomy) since he was 19 years due to gender incongruence. He underwent a preemptive living-donor kidney transplantation from his wife. His skeletal muscle mass was closer to that of a female than that of a male and suggested that eGFR should be calculated with the equation based on the gender assigned at birth (female) rather than the gender identity (male). Moreover, the recovery of kidney function due to successful kidney transplantation decreased serum gonadotropin levels, but normalization of his sex hormone profile was not achieved. Further accumulation of experience with kidney transplantation for transgender people is needed.

Peripelvic Extravasation After Living Donor Kidney Transplant: A Case Report.

The patient was a 33-year-old man. A living donor kidney transplant from his father was performed, and a double-J ureteric stent was placed in the ureter of the transplanted kidney during surgery. Postoperatively, after the urethral catheter was removed, he presented with lower right abdominal pain when excessively strained during defecation. A computed tomography scan showed fluid retention in the retroperitoneal space around the transplanted kidney, and a drainage tube was placed. Urinary components were detected in the drainage, and the patient was diagnosed with peripelvic extravasation. Because the surgical wound opened during the course of treatment, debridement and wound treatment were performed. The patient underwent hyperbaric oxygen therapy, and peripelvic extravasation and wound dehiscence both improved.

Influence of protein intake on the changes in skeletal muscle mass after kidney transplantation.

BACKGROUND & AIMS: Skeletal muscle mass decreases in patients with chronic kidney disease, especially those on dialysis with end-stage kidney disease. On the other hand, the recovery of renal function due to successful kidney transplantation (KT) improves skeletal muscle mass loss. However, low protein intake may influence the changes in skeletal muscle mass after KT. The aim of the present study is to examine the association of the changes in skeletal muscle mass with protein intake in kidney transplant recipients (KTRs). METHODS: A cohort study was conducted in KTRs and living-kidney donors (LKDs). Skeletal muscle mass index (SMI) was measured using bioelectrical impedance analysis before KT and at 1 month and 12 months after KT. Protein intake was calculated with 24-h urine urea nitrogen from the Maroni formula at 12 months after KT. To evaluate the association between protein intake and the changes in SMI during the first year after KT, we performed a multivariable regression analysis adjusted for covariates including age, sex, cumulative glucocorticoids, cumulative hospitalization, diabetes mellitus, and SMI before KT. RESULTS: In KTRs (n = 64), the median SMI was 7.26 kg/m2 before KT, which decreased to 7.01 kg/m2 at 1 month after KT and increased to 7.55 kg/m2 at 12 months after KT. In LKDs (n = 17), the median SMI was 6.24 kg/m2 before KT which increased to 6.40 kg/m2 at 1 month after KT and further increased to 6.95 kg/m2 at 12 months after KT. The changes in SMI during the 1-year period after KT exhibited a positive correlation with protein intake (p = 0.015) after adjustment. The predicted value of protein intake in KTRs, whose values of SMI before KT and at 12 months after KT were the same, was 0.72 g/kg ideal body weight (IBW)/day using the multivariable non-linear regression model. CONCLUSIONS: In KTRs, insufficient protein intake adversely affected the recovery from skeletal muscle mass loss after KT. Therefore, a protein intake of at least more than 0.72 g/kg IBW/day, the predicted value obtained in the present study, might be recommended for KTRs suffering from skeletal muscle mass loss.

Hyperpolypharmacy and Frailty in Kidney Transplant Recipients.

BACKGROUND: Kidney transplant recipients (KTRs) take multiple medications including immunosuppressants every day. Although polypharmacy is associated with frailty, the situation remains unknown in KTRs. The aim of the present study is to investigate the association between hyperpolypharmacy and frailty in KTRs. METHODS: This study was a single-center, cross-sectional investigation carried out on KTRs between August 2018 and February 2019 at Osaka City University Hospital. Frailty was evaluated using the Kihon Checklist (KCL). The number of medications was determined from the regular medicines the participants took by mouth every day. Hyperpolypharmacy was defined as 10 or more medications. Statistical analyses were performed using multivariable logistic regression analyses and multivariable linear regression analyses. RESULTS: Of 211 KTRs enrolled in this study, the mean (SD) number of medicines taken orally regularly was 9.4 (3.4), and hyperpolypharmacy participants accounted for 41%. Hyperpolypharmacy was associated with both the total KCL score (odds ratio, 1.13; P = .016) and being frail compared with being robust (odds ratio, 5.70; P = .007) after adjustments for age, sex, and body mass index. The number of medications was associated with both the total KCL score (ß = 0.20; P < .001) and being frail compared with being robust (ß = 2.51; P < .001) after adjustments for age, sex, body mass index, dialysis vintage, time after transplant, serum albumin, and estimated glomerular filtration rate. The optimal cutoff value for the number of medications to detect frailty was 12 (area under the curve, 0.81). CONCLUSIONS: In KTRs, hyperpolypharmacy was prevalent and was associated with frailty.

Clinical Outcomes of Everolimus With Reduced-Dose Tacrolimus vs Mycophenolate Mofetil With Standard-Dose Tacrolimus in De Novo ABO-Incompatible Kidney Transplant Recipients: 1-Year Follow-up.

BACKGROUND: One of the major barriers for long-term renal graft survival is considered to be calcineurin inhibitor nephrotoxicity, contributing to chronic graft dysfunction. Thus, recent immunosuppressive strategies are focused on regimens that can reduce or avoid exposure to calcineurin inhibitors. Herein, we carried out a small-scale pilot study to assess whether everolimus (EVR) with reduced-dose tacrolimus (Tac) is an acceptable immunosuppressive regimen for patients with de novo ABO-incompatible kidney transplant compared with mycophenolate mofetil (MMF) with standard-dose Tac. METHODS: This retrospective single-center cohort study included patients who underwent ABO-incompatible kidney transplant at our institution between January 2016 and December 2019. Those whose immunosuppressive regimen was changed by reasons other than rejection during the 1-year follow-up period were excluded. RESULTS: A total of 24 patients were enrolled in this study: 10 patients who received an EVR with reduced-dose Tac regimen and 14 patients who received an MMF with standard-dose Tac regimen. Tac trough levels in the EVR group were significantly lower than those in the MMF group (P < .001). No patients died or lost their grafts during the follow-up period. There were no significant differences in renal function, proteinuria, and prevalence of hyperlipidemia between the 2 groups at 1 year after transplant. There were no significant differences in the incidence of rejection (acute cellular rejection, steroid-resistant acute cellular rejection, acute antibody-mediated rejection) and infection (cytomegalovirus viremia, cytomegalovirus disease, BK viremia, BK virus nephropathy) between the 2 groups. CONCLUSIONS: Comparable with MMF with standard-dose Tac, EVR with reduced-dose Tac might be an acceptable immunosuppressive regimen for patients with de novo ABO-incompatible kidney transplant.

The Change in Muscle Mass Among Kidney Transplant Recipients: A Prospective Cohort Study.

BACKGROUND: Recovery of renal function after transplantation leads to improved uremic conditions, increased physical activity, and liberation from severe dietary restrictions. Consequently, the muscle mass of kidney transplant recipients increases for several years after their transplant. However, the change in muscle mass and its associated factors among these patients remain largely unknown. Herein, we carried out a prospective cohort study with 1-year follow-up to investigate how muscle mass changes and to identify its risk factors among kidney transplant recipients. PATIENTS AND METHODS: We performed a single-center, 1-year, prospective, observational cohort study from August 2017 to February 2019 at Osaka City University Hospital in Japan. The skeletal muscle mass index (SMI) was measured by bioelectrical impedance analysis. The risk factors related to the change in muscle mass were analyzed using multivariate linear regression models of age, sex, body mass index (BMI), dialysis vintage, transplant vintage, diabetes mellitus, hemoglobin, C-reactive protein, estimated glomerular filtration rate, and SMI at baseline. RESULTS: A total of 180 kidney transplant recipients were enrolled in the present study. The median age was 55 years, and the median transplant vintage was 78 months. The median rate of change in SMI was +2.07%, and SMI increased in 118 (66%) patients during the 1-year follow-up. By multivariate analysis, the change in SMI at 1-year follow-up was independently associated with age (P = 0.017) and BMI (P = .023). CONCLUSIONS: SMI increased in most of the kidney transplant recipients, and age and BMI might be the risk factors for this change in muscle mass among these patients.

ABO-Incompatible Kidney Transplantation After Bone Marrow Transplantation: A Case Report.

Many studies have been made on ABO-compatible kidney transplants following hematopoietic stem cell transplantation. However, there have been few reports on ABO-incompatible kidney transplantation following hematopoietic stem cell transplantation (HSCT). We report on the case of a successful ABO-incompatible kidney transplantation with high titers after bone marrow transplantation experienced no infectious episodes. The patient was a 38-year-old man with end-stage kidney disease resulting from interstitial nephritis induced by drug toxicity or graft-vs-host disease (GVHD). He had received allogeneic bone marrow transplantation from a human leukocyte antigen-identical unrelated donor to treat chronic myelogenous leukemia. The patient with high anti-B antibody titers (IgM 1:1024 IgG 1:256) received a desensitization protocol consisting of 2 doses of rituximab and 5 courses of plasmapheresis. The patient had prolonged depletion of circulating B cells 2 years after the transplant and was infected with cytomegalovirus viremia, pneumocystis jiroveci pneumonia, and adenovirus urinary tract infection at 2, 3, and 17 months post-transplant, respectively. Currently, at 6 years after his transplant, the patient has had no rejection and is in good clinical condition with only mild renal insufficiency. Our results suggest that ABO-incompatible kidney transplantation may be an effective renal replacement therapy for patients with end-stage kidney disease after HSCT, but desensitization in combination with immunosuppressants could lead to a state of over-immunosuppression, causing various infections.

Splenectomy for ABO-Incompatible Kidney Transplantation and Very Late-Onset Cytomegalovirus Disease.

INTRODUCTION: Splenectomy had been previously performed in ABO-incompatible kidney transplantation to reduce the B cell pool. However, studies have shown that patients undergoing splenectomy may have a lifelong susceptibility to infection and mortality. Splenectomy may affect the incidence of cytomegalovirus (CMV) disease even at a very late stage after transplantation in ABO-incompatible recipients. PATIENTS AND METHODS: Seven patients received their graft from an ABO-incompatible living donor at our institution and underwent splenectomy for B cell reduction. Among them, 3 recipients experienced very late-onset CMV disease approximately 10 years after their transplant and were enrolled in this study. RESULTS: Very late-onset CMV disease occurred at 9 years and 9 months, 15 years, and 13 years and 5 months after transplantation, respectively. Two recipients suffered from CMV retinitis, while one experienced colitis. The age of the patients at onset of CMV disease was 69 years, 42 years, and 71 years, respectively. CONCLUSION: This may be the first report on very late-onset CMV disease after splenectomy in ABO-incompatible kidney transplantation. We should be aware that these recipients can experience very late-onset CMV disease even approximately 10 years after their transplant.

Frailty is associated with dialysis duration before transplantation in kidney transplant recipients: A Japanese single-center cross-sectional study.

OBJECTIVES: To investigate the prevalence of frailty, and the relationship of frailty based on the Kihon Checklist criteria with dialysis duration before transplantation and time after transplantation in kidney transplant recipients. METHODS: This study was a single-center, cross-sectional investigation carried out on kidney transplant recipients. To examine the association between the total Kihon Checklist score with time after transplant and dialysis duration before transplant, the multivariable proportional odds logistic regression model was used with adjustment for age, sex, body mass index, estimated glomerular filtration rate and serum albumin levels. RESULTS: Out of 205 kidney transplant recipients enrolled in this study, frail, prefrail and robust recipients accounted for 11.2%, 26.8% and 62.0%, respectively. Dialysis duration before transplantation was associated with frailty, but time after transplant was not associated with frailty. CONCLUSIONS: The prevalence of frailty in kidney transplant recipients is approximately 11%, and it is associated with the duration of pretransplant dialysis. These findings suggest that a shorter dialysis duration might be beneficial for preventing frailty in kidney transplant recipients.

Association of sarcopenia with phase angle and body mass index in kidney transplant recipients.

Malnutrition is an important risk factor for the development of sarcopenia. Recently, phase angle (PhA) obtained from the bioelectrical impedance analysis is increasingly becoming known as a nutritional status marker and may be considered a good indicator to identify elderly patients at risk of sarcopenia. In this study, we investigated the prevalence of sarcopenia and the relationship between sarcopenia and PhA or body mass index (BMI) as nutritional factors, and evaluated the discrimination performance of these nutritional factors for sarcopenia in 210 kidney transplant recipients. The median age was 55 years and 11.1% had sarcopenia. This prevalence of sarcopenia was lower than previous reports in kidney transplant recipients, maybe because of the differences in sarcopenia definitions and population demographics such as age, sex, race, and comorbidities. Both PhA and BMI were negatively correlated with sarcopenia after adjusting for age, sex, dialysis vintage, time after transplant, presence of diabetes mellitus, hemoglobin, estimated glomerular filtration rate, and the other nutritional factor. The discrimination performance for PhA and BMI had enough power to detect sarcopenia. These results suggest that PhA and BMI can be used in clinical practice to predict sarcopenia in kidney transplant patients.

Latest insights on ABO-incompatible living-donor renal transplantation.

This review summarizes the latest insights on ABO-incompatible living-donor renal transplantation. Desensitization protocols and clinical outcomes were investigated, and a comparison was made with kidney-paired donation, which is not permitted in Japan for ethical reasons. Although renal transplantation is greatly beneficial for most patients with end-stage kidney disease, many of these patients must remain on dialysis therapy for extended periods due to the scarcity of organs from deceased donors. ABO blood type incompatibility was once believed to be a contraindication to renal transplantation due to the increased risk for antibody-mediated rejection and early graft loss attributable to isoagglutinins. Recently, pretransplant desensitization strategies, such as removal of isoagglutinins and antibody-producing cells, have achieved successful outcomes, although it remains unclear whether graft survival and patient morbidity are equivalent to those for ABO-compatible renal transplantation. The present review suggested that ABO-incompatible living-donor renal transplantation might be a favorable radical renal replacement therapy for patients with end-stage kidney disease.

Conversion From Cyclosporine to Once-Daily Tacrolimus on 50:1 mg Basis: A Short-Term Pilot Study.

OBJECTIVES: In addition to graft dysfunction, renal transplant recipients on cyclosporine may be switched to tacrolimus to reduce its drug-related secondary clinical effects and undesirable cosmetic side effects. However, the dose level of once-daily tacrolimus for these patients has yet to be established. The objective of this prospective study was to confirm the safety of converting stable renal transplant recipients on cyclosporine to once-daily tacrolimus at a 50:1 mg ratio. MATERIALS AND METHODS: Our study enrolled 17 patients receiving cyclosporine who were observed for 3 months. Graft biopsies did not reveal any acute rejection, and the conversion ratio to once-daily tacrolimus was 50:1 mg. Dose adjustments were made to achieve a target tacrolimus trough concentration of 3 to 5 ng/mL at 2 weeks, and graft biopsies were taken after the 3-month observation period. RESULTS: Dose adjustment was required in 7 recipients (41.2%) within 3 months of conversion. None of the recipients had acute cellular rejection or C4d deposition, and the mean estimated glomerular filtration rate of 38.7 ± 11.0 mL/min/1.73 m2 at baseline was significantly improved to 42.0 ± 10.0 mL/min/1.73 m2 at month 3. CONCLUSIONS: Although recipients of renal transplant can be forced to discontinue cyclosporine administration due to undesirable adverse effects, our study showed that a once-daily dose of tacrolimus may be safe when administered at a conversion ratio of 50:1.