Years of life lost and long-term outcomes due to glomerular disease in a Southeast Asian Cohort.

Death and end-stage kidney disease (ESKD) are major outcomes of glomerular disease. (GD) The years of potential life lost (YLL) may provide additional insight into the disease burden beyond death rates. There is limited data on premature mortality in GD. In this retrospective observational cohort study, we evaluated the mortality, ESKD rates, and YLL in Thais with biopsy-proven GD. The mortality and combined outcome rates were determined by log-rank test and ESKD by using a competing risk model. YLL and premature life lost before age 60 were calculated for different GD based on the life expectancy of the Thai population. Patients with GD (n = 949) were followed for 5237 patient years. The death rate and ESKD rates (95%CI) were 4.2 (3.7-4.9) and 3.3 (2.9-3.9) per 100 patient-years, respectively. Paraprotein-related kidney disease had the highest death rate, and diabetic nephropathy had the highest ESKD rate. Despite not having the highest death rate, lupus nephritis (LN) had the highest YLL (41% of all GD) and premature loss of life before age 60. In conclusion, YLL provided a different disease burden assessment compared to mortality rates and identified LN as the major cause of premature death due to GD in a Southeast Asian cohort.

Economic evaluation of peritoneal dialysis and hemodialysis in Thai population with End-stage Kidney Disease.

BACKGROUND: This study aimed to conduct a cost-utility analysis of the "Peritoneal Dialysis (PD)-First" policy in 2008 under a universal health coverage scheme and hemodialysis (HD) in Thai patients with End-stage Kidney Disease (ESKD) using updated real-practice data. METHODS: Markov model was used to evaluate the cost-utility of two modalities, stratified into five age groups based on the first modality taken at 20, 30, 40, 50, and 60 years old from government and societal perspectives. Input parameters related to clinical aspects and cost were obtained from 15 hospitals throughout Thailand and Thai Renal Replacement Therapy databases. Both costs and outcomes were discounted at 3%, adjusted to 2021, and converted to USD (1 USD = 33.57 Thai Baht). One-way analysis and probabilistic sensitivity analysis were performed to assess the uncertainty surrounding model parameters. RESULTS: From the government perspective, compared to PD-first policy, the incremental cost-effectiveness ratio (ICER) was between 19,434 and 23,796 USD per QALY. Conversely, from a societal perspective, the ICER was between 31,913 and 39,912 USD per QALY. Both are higher than the willingness to pay threshold of 4,766 USD per QALY. CONCLUSION: By applying the updated real-practice data, PD-first policy still remains more cost-effective than HD-first policy at the current willingness to pay. However, HD gained more quality-adjusted life years than PD. This information will assist clinicians and policymakers in determining the future direction of dialysis modality selection and kidney replacement therapy reimbursement policies for ESKD patients.

Durability of Humoral and Cellular Immunity after an Extended Primary Series with Heterologous Inactivated SARS-CoV-2 Prime-Boost and ChAdOx1 nCoV-19 in Dialysis Patients (ICON3).

The durability of a three-dose extended primary series of COVID-19 vaccine in dialysis patients remains unknown. Here, we assessed dynamic changes in SARS-CoV-2-specific humoral and cell-mediated immunity at baseline, 3 months, and 6 months after the extended primary series in 29 hemodialyzed (HD), 28 peritoneal dialyzed (PD) patients, and 14 healthy controls. Participants received two doses of inactivated SARS-CoV-2 vaccine followed by a dose of ChAdOx1 nCoV-19 vaccine. At 6 months, median anti-RBD IgG titers (IQR) significantly declined from baseline in the HD (1741 (1136−3083) BAU/mL vs. 373 (188−607) BAU/mL) and PD (1093 (617−1911) BAU/mL vs. 180 (126−320) BAU/mL) groups, as did the mean percent inhibition of neutralizing antibodies (HD: 96% vs. 81%; PD: 95% vs. 73%) (all p < 0.01). Age and post-vaccination serological response intensity were predictors of early humoral seroprotection loss. In contrast, cell-mediated immunity remained unchanged. In conclusion, humoral immunity declined substantially in dialysis patients, while cell-mediated immunity remained stable 6 months after the extended heterologous primary series of two inactivated SARS-CoV-2/ChAdOx1 nCoV-19 vaccine. A booster dose could be considered in dialysis patients 3 months after this unique regimen, particularly in the elderly or those with a modest initial humoral response.

An additional dose of viral vector COVID-19 vaccine and mRNA COVID-19 vaccine in kidney transplant recipients: A randomized controlled trial (CVIM 4 study).

Immunogenicity following an additional dose of Coronavirus disease 2019 (COVID-19) vaccine was investigated in an extended primary series among kidney transplant (KT) recipients. Eighty-five KT participants were randomized to receive either an mRNA (M group; n = 43) or viral vector (V group; n = 42) vaccine. Among them, 62% were male, with a median (IQR) age of 50 (43-59) years and post-transplantation duration of 46 (26-82) months. At 2 weeks post-additional dose, there was no difference in the seroconversion rate between the M and V groups (70% vs. 65%, p = .63). A median (IQR) of anti-RBD antibody level was not statistically different between the M group compared with the V group (51.8 [5.1-591] vs. 28.5 [2.9-119.3] BAU/ml, p = .18). Furthermore, the percentage of participants with positive SARS-CoV-2 surrogate virus neutralization test results was not statistically different between groups (20% vs. 15%, p = .40). S1-specific T cell and RBD-specific B cell responses were also comparable between the M and V groups (230 [41-420] vs. 268 [118-510], p = .65 and 2 [0-10] vs. 2 [0-13] spot-forming units/106 peripheral blood mononuclear cells, p = .60). In conclusion, compared with an additional dose of viral vector COVID-19 vaccine, a dose of mRNA COVID-19 vaccine did not elicit significantly different responses in KT recipients, regarding either humoral or cell-mediated immunity. (TCTR20211102003).

Immunogenicity of ChAdOx1 nCoV-19 vaccine after a two-dose inactivated SARS-CoV-2 vaccination of dialysis patients and kidney transplant recipients.

Vaccination with inactivated SARS-CoV-2 virus produces suboptimal immune responses among kidney transplant (KT), peritoneal dialyzed (PD), and hemodialyzed (HD) patients. Participants were vaccinated with two-dose inactivated SARS-CoV-2 vaccine (V2) and a third dose of ChAdOx1 nCoV-19 vaccine (V3) at 1-2 months after V2. We enrolled 106 participants: 31 KT, 28 PD, and 31 HD patients and 16 controls. Among KT, PD, and HD groups, median (IQR) of anti-receptor binding domain antibody levels were 1.0 (0.4-26.8), 1092.5 (606.9-1927.2), and 1740.9 (1106-3762.3) BAU/mL, and percent neutralization was 0.9 (0-9.9), 98.8 (95.9-99.5), and 99.4 (98.8-99.7), respectively, at two weeks after V3. Both parameters were significantly increased from V2 across all groups (p < 0.05). Seroconversion and neutralization positivity rates in PD, HD, and control groups were 100% but were impaired in KT patients (39% and 16%, respectively). S1-specific T-cell counts were increased in PD and HD groups (p < 0.05) but not in KT patients. The positive S1-specific T-cell responder rate was > 90% in PD, HD, and control groups, which was higher than that in KT recipients (74%, p < 0.05). The heterologous inactivated virus/ChAdOx1 nCoV-19 vaccination strategy elicited greater immunogenicity among dialysis patients; however, inadequate responses remained among KT recipients (TCTR20210226002).

Predicting treatment response and clinicopathological findings in lupus nephritis with urine epidermal growth factor, monocyte chemoattractant protein-1 or their ratios.

INTRODUCTION: There is a need for sensitive and specific biomarkers to predict kidney damage and therapeutic response in lupus nephritis (LN). Monocyte chemoattractant protein-1 (MCP-1) and epidermal growth factor (EGF) are cytokines with divergent roles. EGF or EGF/MCP1 ratio have been shown to correlate with prognosis in primary glomerulonephritis, but there is limited information in lupus nephritis (LN). This study evaluated the roles of MCP-1, EGF or their ratio as biomarkers of histopathology and response to treatment in LN. METHODS: This was a cross-sectional and observational study. Baseline urine MCP-1 and EGF levels in systemic lupus erythematosus (SLE) patients and controls (total n = 101) were compared, and levels were correlated with clinicopathological findings and subsequent response to treatment. RESULTS: MCP-1 was higher in active LN (n = 69) compared to other SLE groups and controls, whereas EGF was not different. MCP-1 correlated with disease activity (proteinuria, renal SLEDAI, classes III/IV/V, and high activity index.) By contrast, EGF correlated with eGFR, but not with proteinuria, activity index, or class III/IV/V. MCP-1 was higher, and EGF was lower in high chronicity index. EGF/MCP-1 decreased with greater clinicopathological severity. In a subgroup with proliferative LN who completed six months of induction therapy (n = 41), EGF at baseline was lower in non-responders compared to responders, whereas MCP-1 was similar. By multivariable analysis, baseline EGF was independently associated with subsequent treatment response. Area under the curve for EGF to predict response was 0.80 (0.66-0.95). EGF ≥ 65.6 ng/ mgCr demonstrated 85% sensitivity and 71% specificity for response. EGF/MCP-1 did not improve the prediction for response compared to EGF alone. CONCLUSION: MCP-1 increased with disease activity, whereas EGF decreased with low GFR and chronic damage. Urine EGF may be a promising biomarker to predict therapeutic response in LN. EGF/MCP-1 did not improve the prediction of response.

SARS-CoV-2-specific humoral and cell-mediated immune responses after immunization with inactivated COVID-19 vaccine in kidney transplant recipients (CVIM 1 study).

Immunogenicity following inactivated SARS-CoV-2 vaccination among solid organ transplant recipients has not been assessed. Seventy-five patients (37 kidney transplant [KT] recipients and 38 healthy controls) received two doses, at 4-week intervals, of an inactivated whole-virus SARS-CoV-2 vaccine. SARS-CoV-2-specific humoral (HMI) and cell-mediated immunity (CMI) were measured before, 4 weeks post-first dose, and 2 weeks post-second dose. The median (IQR) age of KT recipients was 50 (42-54) years and 89% were receiving calcineurin inhibitors/mycophenolate/corticosteroid regimens. The median (IQR) time since transplant was 4.5 (2-9.5) years. Among 35 KT patients, the median (IQR) of anti-RBD IgG level measured by CLIA after vaccination was not different from baseline, but was significantly lower than in controls (2.4 [1.1-3.7] vs. 1742.0 [747.7-3783.0] AU/ml, p < .01) as well as percentages of neutralizing antibody inhibition measured by surrogate viral neutralization test (0 [0-0] vs. 71.2 [56.8-92.2]%, p < .01). However, the median (IQR) of SARS-CoV-2 mixed peptides-specific T cell responses measured by ELISpot was significantly increased compared with baseline (30 [4-120] vs. 12 [0-56] T cells/106  PBMCs, p = .02) and not different from the controls. Our findings revealed weak HMI but comparable CMI responses in fully vaccinated KT recipients receiving inactivated SARS-CoV-2 vaccination compared to immunocompetent individuals (Thai Clinical Trials Registry, TCTR20210226002).

Short-Term Immunogenicity Profiles and Predictors for Suboptimal Immune Responses in Patients with End-Stage Kidney Disease Immunized with Inactivated SARS-CoV-2 Vaccine.

INTRODUCTION: Patients with end-stage kidney disease (ESKD) are at risk of severe coronavirus disease and mortality. Immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated whole-virus vaccine in patients with ESKD has never been explored. METHODS: We conducted a prospective cohort study of 60 patients with ESKD and 30 healthy controls. All participants received two doses of an inactivated whole-virus SARS-CoV-2 vaccine (Sinovac Biotech Ltd) 4 weeks apart. SARS-CoV-2-specific humoral and cell-mediated immune responses were investigated and referenced with healthy controls. RESULTS: After two doses, an anti-receptor-binding domain immunoglobulin G of 50 AU/ml or greater was present in 53 of 60 patients (88%) in the ESKD group and all participants (100%) in the control group (P = 0.05). The percentage of patients with ESKD and controls with neutralizing antibodies of 35% threshold or greater was 58% and 88%, respectively (P = 0.01). Furthermore, the proportion of patients with ESKD and S1-specific T cell response was comparable with controls (82% vs. 77%, P = 0.45). Old age, high ferritin level, and low absolute lymphocyte count were independently associated with poor humoral immune responses. CONCLUSIONS: Patients with ESKD could develop similar SARS-CoV-2-specific cell-mediated immune responses compared to healthy controls, although suboptimal humoral immune responses were observed following two doses of SARS-CoV-2 vaccination. Therefore, patients with ESKD and the abovementioned factors are at risk of generating inadequate humoral immune responses, and a vaccine strategy to elicit greater immunogenicity among these relatively immunocompromised patients is warranted. (Thai Clinical Trials Registry, TCTR20210226002).

Bioimpedance Analysis-Guided Volume Expansion for the Prevention of Contrast-Induced Acute Kidney Injury (the BELIEVE Pilot Randomized Controlled Trial).

INTRODUCTION: Peri-procedural i.v. fluid administration is important for the prevention of contrast-induced acute kidney injury (CI-AKI). However, standardized fluid management protocols may not be suitable for all patients. We therefore wished to determine whether an individualized fluid administration protocol guided by measuring extracellular water (ECW) using bioimpedance analysis (BIA) would be safe and would reduce the incidence CI-AKI compared to a standardized fluid administration prescription. METHODS: In this pilot, randomized, parallel-group, single-blind, controlled trial, we compared the effect of BIA-guided isotonic bicarbonate administration according to the ratio of ECW to total body water (ECW/TBW) to our standard isotonic bicarbonate protocol in regard to the safety and efficacy of preventing CI-AKI in chronic kidney disease patients undergoing elective cardiac angiography. Our primary outcome was the incidence of CI-AKI, which was defined as a ≥0.3 mg/dl or 150% increase in serum creatinine concentration within 48 to 72 hours after cardiac angiography. RESULTS: We studied 61 patients, 30 in the bioimpedance group and 31 in the control group. Age was similar (72.5 ± 7 vs. 71.4 ± 7.9 years), as were body mass index (25.5 vs. 25.8 kg/m2) and baseline serum creatinine (1.3 ± 0.3 vs. 1.4 ± 0.4 mg/dl). The peri-procedural fluid volume administered was significantly greater in the BIA-guided hydration group (899.0 ± 252.7 ml vs. 594.4 ± 125.9 ml, P < .01). The incidence of CI-AKI was 3.3% in BIA-guided hydration group and 6.5% in the control group (relative risk = 0.52, 95% confidence interval = 0.05-5.40, P = 1.00). Adverse events reported were comparable between groups (6.7% vs. 6.5%, P = 1.00). CONCLUSIONS: The overall incidence of CI-AKI after cardiac angiography in our patients with mild-to-moderate renal insufficiency was lower than anticipated. Isotonic bicarbonate administration guided by bioimpedance measurements was safe, and probably led to a lower incidence of CI-AKI, although this not reach statistical significance.

Urine epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors of complete remission in primary glomerulonephritis.

BACKGROUND: The balance of several cytokines likely influences the resolution of glomerulonephritis. Monocyte chemoattractant protein-1(MCP-1) is a chemokine that promotes renal inflammation whereas epidermal growth factor (EGF) stimulates protective responses. Previously, high urine MCP-1(MCP-1) and low urine EGF (EGF) levels were found to be associated with tubulointerstitial fibrosis, but there is limited information on the value of these mediators as predictors of therapeutic responses or long term outcome in primary glomerulonephritis. OBJECTIVES: To determine the performance of urine EGF, MCP-1 or their ratio at baseline as biomarkers to predict complete remission, and the relationship of these mediators with subsequent renal function 24 months later in primary glomerulonephritis. METHODS: This is a prospective study of patients with biopsy-proven primary glomerulonephritis. Baseline urine samples were collected at biopsy before therapy. MCP-1 and EGF were analyzed by enzyme-linked immunosorbent assays and expressed as a ratio to urine creatinine (ng/mgCr) or as EGF/MCP-1 ratio (ng/ng). Proteinuria and estimated glomerular filtration rate (eGRF) were monitored after therapy. Complete remission (CR) was defined as proteinuria ≤ 0.3 g/gCr. RESULTS: Median follow-up was 20 months. Of all patients (n = 74), 38 patients (51.4%) subsequently achieved CR. Baseline urine EGF and EGF/MCP-1 levels were significantly higher in CR compared to Not CR. By contrast, MCP-1 was not different. High EGF (EGF > 75 ng/mgCr) was a significant predictor (OR 2.28) for CR by multivariate analysis after adjusting for proteinuria, blood pressure, baseline eGFR. In patients who completed 24 months follow-up (n = 43), baseline EGF correlated inversely with proteinuria and positively with eGFR at 24 months. CONCLUSION: High urine EGF level is a promising biomarker of CR. Baseline EGF levels correlated with kidney function at 2 years. EGF/MCP-1 was not superior to EGF alone. Further studies are necessary to determine the role of urine EGF as a guide to therapy in primary GN.

Urine Epidermal Growth Factor, Monocyte Chemoattractant Protein-1 or Their Ratio as Biomarkers for Interstitial Fibrosis and Tubular Atrophy in Primary Glomerulonephritis.

BACKGROUND/AIMS: The degree of tubular atrophy and interstitial fibrosis (IFTA) is an important prognostic factor in glomerulonephritis. Imbalance between pro-inflammatory cytokines such as monocyte chemoattractant protein- 1 (MCP-1) and protective cytokines such as epidermal growth factor (EGF) likely determine IFTA severity. In separate studies, elevated MCP-1 and decreased EGF have been shown to be associated with IFTA severity. In this study, we aim to evaluate the predictive value of urinary EGF/MCP-1 ratio compared to each biomarker individually for moderate to severe IFTA in primary glomerulonephritis (GN). METHODS: Urine samples were collected at biopsy from primary GN (IgA nephropathy, focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy). MCP-1 and EGF were analyzed by enzyme-linked immunosorbent assay. RESULTS: EGF, MCP-1 and EGF/MCP-1 ratio from primary GN, all correlated with IFTA (n=58). By univariate analysis, glomerular filtration rate, EGF, and EGF/MCP-1 ratio were associated with IFTA. By multivariate analysis, only EGF/MCP-1 ratio was independently associated with IFTA. EGF/MCP-1 ratio had a sensitivity of 88% and specificity of 74 % for IFTA. EGF/MCP-1 had good discrimination for IFTA (AUC=0.85), but the improvement over EGF alone was not significant. CONCLUSION: EGF/MCP-1 ratio is independently associated IFTA severity in primary glomerulonephritis, but the ability of EGF/MCP-1 ratio to discriminate moderate to severe IFTA may not be much better than EGF alone.

Hypokalemic Hypertension Leading to a Diagnosis of Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease. Hypertension is common and occurs before decline in renal function. However, the coexistence of hypertension and hypokalemia is rare in ADPKD patients. We report on a 32-year-old woman with secondary aldosteronism. Magnetic resonance imaging of the renal arteries revealed multiple cysts of varying sizes in both the kidneys and the liver, compatible with ADPKD. Increased reninangiotensin-aldosterone system activity was secondary to cyst expansion. After initiation of angiotensin II receptor blocker, her blood pressure was controlled without additional requirement of potassium.

Strategies for preserving residual renal function in peritoneal dialysis patients.

Although there have been many advancements in the treatment of patients with chronic kidney disease (CKD) over the last 50 years, in terms of reducing cardiovascular risk, mortality remains unacceptably high, particularly for those patients who progress to stage 5 CKD and initiate dialysis (CKD5d). As mortality risk increases exponentially with progressive CKD stage, the question arises as to whether preservation of residual renal function once dialysis has been initiated can reduce mortality risk. Observational studies to date have reported an association between even small amounts of residual renal function and improved patient survival and quality of life. Dialysis therapies predominantly provide clearance for small water-soluble solutes, volume and acid-base control, but cannot reproduce the metabolic functions of the kidney. As such, protein-bound solutes, advanced glycosylation end-products, middle molecules and other azotaemic toxins accumulate over time in the anuric CKD5d patient. Apart from avoiding potential nephrotoxic insults, observational and interventional trials have suggested that a number of interventions and treatments may potentially reduce the progression of earlier stages of CKD, including targeted blood pressure control, reducing proteinuria and dietary intervention using combinations of protein restriction with keto acid supplementation. However, many interventions which have been proven to be effective in the general population have not been equally effective in the CKD5d patient, and so the question arises as to whether these treatment options are equally applicable to CKD5d patients. As strategies to help preserve residual renal function in CKD5d patients are not well established, we have reviewed the evidence for preserving or losing residual renal function in peritoneal dialysis patients, as urine collections are routinely collected, whereas few centres regularly collect urine from haemodialysis patients, and haemodialysis dialysis patients are at risk of sudden intravascular volume shifts associated with dialysis treatments. On the other hand, peritoneal dialysis patients are exposed to a variety of hypertonic dialysates and episodes of peritonitis. Whereas blood pressure control, using an angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB), and low-protein diets along with keto acid supplementation have been shown to reduce the rate of progression in patients with earlier stages of CKD, the strategies to preserve residual renal function (RRF) in dialysis patients are not well established. For peritoneal dialysis patients, there are additional technical factors that might aggravate the rate of loss of residual renal function including peritoneal dialysis prescriptions and modality, bio-incompatible dialysis fluid and over ultrafiltration of fluid causing dehydration. In this review, we aim to evaluate the evidence of interventions and treatments, which may sustain residual renal function in peritoneal dialysis patients.

Chromosome 20p inverted duplication deletion identified in a Thai female adult with mental retardation, obesity, chronic kidney disease and characteristic facial features.

We report on a 21-year-old Thai woman presenting with mental retardation, developmental delays, selective mutism, distinctive facial features, sensorineural hearing loss, single right kidney, uterine didelphys and obesity. A longitudinal clinical course beginning in childhood revealed excessive weight gain, poor language skills and poor school performance. Chronic kidney disease stage 4, with elevated blood pressure, was first noted in adulthood. Array comparative genomic hybridization detected a copy loss at 20p13 co-existing with a copy gain at 20p13-20p11.22. A conventional cytogenetic study revealed the complex structural rearrangement of chromosome 20 [der (20) dup (20) (p11.2p13) del (20) (p13.pter)]. A FISH analysis, using probes against duplication and deletion regions, confirmed that there was an inverted duplication of p11.2-p13 and a deletion in the subtelomere region. Previous reports have identified this cytogenetic characterization in a Caucasian boy. Therefore, this is the first reported case of chromosome 20p inverted duplication deletion syndrome in an adult from the Southeast Asian population group.

Mineral metabolism and outcomes in chronic kidney disease stage 2-4 patients.

BACKGROUND: Marked hyperphosphatemia, hyperparathyroidism and 25-hydroxyvitamin D deficiency are associated with mortality in dialysis patients. Such data in chronic kidney disease stage 2-4 population are limited. It has been suggested that high-normal serum phosphate predicts worse renal and patient outcomes. The data regarding parathyroid hormone and outcomes in this population is limited. The present study examined mineral metabolism and its association with the development of end-stage renal disease and mortality in stage 2-4 chronic kidney disease patients. METHODS: This is a prospective cohort study that included 466 non-dialysis chronic kidney disease stage 2-4 patients. Mineral parameters were obtained at the time of enrollment and the patients were followed prospectively for 25 (1-44) months or until they reached the endpoints of end-stage renal disease or mortality. RESULTS: Hyperparathyroidism and 25-hydroxyvitamin D deficiency began to occur in the early stages of chronic kidney disease, whereas significant hyperphosphatemia only developed in the later stages. High-normal and mildly elevated serum phosphate (>4.2 mg/dL) predicted the composite outcome of end-stage renal disease or mortality after adjustments for cardiovascular risk factors, chronic kidney disease stage and other mineral parameters. Parathyroid hormone levels above the upper limit of normal (>65 pg/mL) predicted the future development of end-stage renal disease and the composite outcome of end-stage renal disease or mortality after adjustments. 25-hydroxyvitamin D deficiency (<15 ng/mL) was also associated with worse outcomes. CONCLUSIONS: In chronic kidney disease, hyperparathyroidism developed prior to significant hyperphosphatemia confirming the presence phosphate retention early in the course of chronic kidney disease. High-normal serum phosphate and mildly elevated parathyroid hormone levels predicted worse renal and patient outcomes. This data emphasizes the need for early intervention in the care of chronic kidney disease stage 2-4 patients.