Post-acute sequelae of SARS-CoV-2 infection: relationship of central nervous system manifestations with physical disability and systemic inflammation.

BACKGROUND: Despite the multitude of clinical manifestations of post-acute sequelae of SARS-CoV-2 infection (PASC), studies applying statistical methods to directly investigate patterns of symptom co-occurrence and their biological correlates are scarce. METHODS: We assessed 30 symptoms pertaining to different organ systems in 749 adults (age = 55 ± 14 years; 47% female) during in-person visits conducted at 6-11 months after hospitalization due to coronavirus disease 2019 (COVID-19), including six psychiatric and cognitive manifestations. Symptom co-occurrence was initially investigated using exploratory factor analysis (EFA), and latent variable modeling was then conducted using Item Response Theory (IRT). We investigated associations of latent variable severity with objective indices of persistent physical disability, pulmonary and kidney dysfunction, and C-reactive protein and D-dimer blood levels, measured at the same follow-up assessment. RESULTS: The EFA extracted one factor, explaining 64.8% of variance; loadings were positive for all symptoms, and above 0.35 for 16 of them. The latent trait generated using IRT placed fatigue, psychiatric, and cognitive manifestations as the most discriminative symptoms (coefficients > 1.5, p < 0.001). Latent trait severity was associated with decreased body weight and poorer physical performance (coefficients > 0.240; p ⩽ 0.003), and elevated blood levels of C-reactive protein (coefficient = 0.378; 95% CI 0.215-0.541; p < 0.001) and D-dimer (coefficient = 0.412; 95% CI 0.123-0.702; p = 0.005). Results were similar after excluding subjects with pro-inflammatory comorbidities. CONCLUSIONS: Different symptoms that persist for several months after moderate or severe COVID-19 may unite within one latent trait of PASC. This trait is dominated by fatigue and psychiatric symptoms, and is associated with objective signs of physical disability and persistent systemic inflammation.

A continuous fall of PSA use for prostate cancer screening among Brazilian doctors since 2001. Good or bad notice?

PURPOSE: To evaluate the trend of use of Prostate Specifi c Antigen (PSA) for screening of prostate cancer (PC) among Brazilian doctors, from the beginning of its regular availability in clinical laboratories. MATERIAL AND METHODS: A serial cross-sectional study was performed using data obtained from a large database between 1997 and 2016. The general PSA screening trend during this period, adjusted for the total number of exams performed in men, was analyzed. Time-series analysis was performed through observation of the general regression curve using the generalized least squares method, and the impact of the recommendations was assessed with autoregressive integrated moving average (ARIMA) models. RESULTS: During the period studied 2,521,383 PSA determinations were done. The age of the participants ranged from 21 to 111 years, with an average of 56.7 ± 22.7 years. The relative number of PSA tests/100.000 exams in males showed a constant reduction since 2001, and this trend was more evident in the group aged 55-69 years. Although statistically signifi cant, the impact of reduced PSA screening after the 2012 USPSTF publication was clinically irrelevant. CONCLUSIONS: Our results indicated a continuous reduction in the use of PSA screening over time, regardless of the publication of recommendations or clinical guidelines. The fact that this trend was more pronounced among those with a greater benefi t potential (55-69 years), relative to groups with a greater damage potential due to overdiagnosis and overtreatment (aged >74 years and < 40 years), is a matter of concern. Follow-up studies of these trends are advisable.

Clinical Applications of Point-of-Care Testing in Different Conditions.

BACKGROUND: The use of point-of-care testing (POCT) in different clinical applications is justified by the fact that the time to release the result is shortened, allowing the physician to define the diagnosis and most appropriate therapy in a shorter time. However, the negative aspects must also be highlighted and studied so that we can move forward with the use of these devices. These negative aspects include greater analytical imprecision compared to laboratory automation, the variability between different equipment from different manufacturers, the risk of inappropriate use, a low level of global regulation, higher costs compared with laboratory testing and cost ineffectiveness in terms of health care. Methods and. RESULTS: This review presents some clinical applications of POCT in different scenarios, such as for diabetes mellitus, infectious diseases, pediatrics, and chronic kidney disease, among others. CONCLUSIONS: We hope to see a global consensus on an acceptable quality standard for performing POCT that is adaptable, practical, and cost effective in primary care settings, ensuring patient safety, and minimizing the risk of harm.

Point-of-Care Testing: General Aspects.

Point-of-Care Testing (POCT) has been highlighted in the health care sector in recent decades. On the other hand, due to its low demand, POCT is at a disadvantage compared to conventional equipment, since its cost is inversely proportional to the volume of use. In addition, for the implementation of POCT to succeed, it is essential to rely on the work of a multidisciplinary team. The awareness of health professionals of the importance of each step is perhaps the critical success factor. The trend towards the continuous advancement of the use of POCT and the great potential of its contributions reinforce the need to implement quality management tools, including performance indicators, to ensure their results. This review presents some advantages and disadvantages concerning POCT and the real need to use it. A worldwide call for the availability of easy-to-use health technologies that are increasingly closer to the final user is one of the main reasons for this focus.

PSA kinetics before 40 years of age.

PURPOSE: The baseline PSA has been proposed as a possible marker for prostate cancer. The PSA determination before 40 years seems interesting because it not suffers yet the drawbacks related to more advanced ages. Considering the scarcity of data on this topic, an analysis of PSA kinetics in this period seems interesting. MATERIALS AND METHODS: A retrospective assay in a database of a private diagnostic center was performed from 2003 to 2016. All subjects with a PSA before 40 years were included. RESULTS: 92995 patients performed PSA between the ages of 21 - 39. The mean value ranged from 0.66 ng / mL (at age 22) to 0.76 ng / mL (at age 39) and the overall mean was 0.73 ng / mL. As for outliers, 3783 individuals presented a baseline PSA > 1.6 ng / mL (p95). A linear regression model showed that each year there is a PSA increase of 0.0055 ng / mL (ß = 0.0055; r² = 0.0020; p < 0.001). A plateau in PSA between 23 and 32 years was found and there were only minimal variations among the ages regardless of the evaluated percentile. CONCLUSION: It was demonstrated that PSA kinetics before 40 years is a very slow and progressive phenomenon regardless of the assessed percentile. Considering our results, it could be suggested that any PSA performed in this period could represent the baseline value without significant distortions.

N-acetyl-cysteine is associated to renal function improvement in patients with nephropathic cystinosis.

BACKGROUND: Nephropathic cystinosis is an autosomal recessive systemic severe disease characterized by intralysosomal cystine storage. Cysteamine is an essential component of treatment. There is solid evidence that cystine accumulation itself is not responsible for all abnormalities in cystinosis; there is also a deficiency of glutathione in the cytosol. Patients with cystinosis can be more susceptible to oxidative stress. CASE-DIAGNOSIS/TREATMENT: The patient cohort comprised 23 cystinosis patients (16 males) aged <18 years (mean age 8.0 ± 3.6 years) with chronic kidney disease class I-IV with good adherence to treatment, including cysteamine. Oxidative stress was evaluated based on the levels of serum thiobarbituric acid-reactive substances (TBARS), and renal function was evaluated based on serum creatinine and cystatin C levels and creatinine clearance (Schwartz formula). N-Acetylcysteine (NAC), an antioxidant drug was given to all patients for 3 months (T1) at 25 mg/kg/day divided in three doses per day. The measured values at just before the initiation of NAC treatment (T0) served as the control for each patient. RESULTS: Median serum TBARS levels at T0 and T1 were 6.92 (range 3.3-29.0) and 1.7 (0.6-7.2)  nmol/mL, respectively (p < 0.0001). In terms of renal function at T0 and T1, serum creatinine levels (1.1 ± 0.5 vs. 0.9 ± 0.5 mg/dL, respectively; p < 0.0001), creatinine clearance (69.7 ± 32.2 vs. T1 = 78.5 ± 33.9 mL/min/1.73 m(2), respectively; p = 0.006), and cystatin c level (1.33 ± 0.53 vs. 1.15 ± 0.54 mg/l, respectively; p = 0.0057) were all significantly different at these two time points. Serum creatinine measurements at 6 (T -6) and 3 months (T -3) before NAC initiation and at 3 (T +3) and 6 months (T +6) after NAC had been withdrawn were also evaluated. CONCLUSION: During the 3-month period that our 23 cystinosis patients were treated with NAC, oxidative stress was reduced and renal function significantly improved. No side-effects were detected. Larger and controlled studies are needed to confirm these findings.

Immunothrombosis and COVID-19 ‒ a nested post-hoc analysis from a 3186 patient cohort in a Latin American public reference hospital.

OBJECTIVE: COVID-19 is associated with an elevated risk of thromboembolism and excess mortality. Difficulties with best anticoagulation practices and their implementation motivated the current analysis of COVID-19 patients who developed Venous Thromboembolism (VTE). METHOD: This is a post-hoc analysis of a COVID-19 cohort, described in an economic study already published. The authors analyzed a subset of patients with confirmed VTE. We described the characteristics of the cohort, such as demographics, clinical status, and laboratory results. We tested differences amid two subgroups of patients, those with VTE or not, with the competitive risk Fine and Gray model. RESULTS: Out of 3186 adult patients with COVID-19, 245 (7.7%) were diagnosed with VTE, 174 (5.4%) of them during admission to the hospital. Four (2.3% of these 174) did not receive prophylactic anticoagulation and 19 (11%) discontinued anticoagulation for at least 3 days, resulting in 170 analyzed. During the first week of hospitalization, the laboratory most altered results were C-reactive protein and D-dimer. Patients with VTE were more critical, had a higher mortality rate, worse SOFA score, and, on average, 50% longer hospital stay. CONCLUSION: Proven VTE incidence in this severe COVID-19 cohort was 7.7%, despite 87% of them complying completely with VTE prophylaxis. The clinician must be aware of the diagnosis of VTE in COVID-19, even in patients receiving proper prophylaxis.

Diminished mycophenolic acid exposure caused by omeprazole may be clinically relevant in the first week posttransplantation.

BACKGROUND: Some studies have reported a decreased absorption of mycophenolic acid (MPA) from mycophenolate mofetil (MMF) in renal transplanted (RTx) patients under proton-pump inhibitors (PPIs). There is still a lack of information regarding (1) whether this effect occurs when MMF is administered with either tacrolimus or cyclosporine A [calcineurin inhibitors (CNIs)], (2) whether the effect has the same amplitude during the first year after RTx, and finally (3) whether this decrease in exposure is clinically relevant. METHODS: We retrospectively analyzed the omeprazole effect in 348 12-hour pharmacokinetic samplings [area under the curve (AUC)(0-12h)] performed on days 7, 14, 30, 60, 180, and 360 after RTx in 77 patients who participated in previous trials. RESULTS: For all periods, the groups with and without PPI did not differ in all variables. By mixed-model analysis of variance, PPI reduced the MPA AUC(0-12h) (P < 0.0008) in the patients under both CNIs mainly due to decreased absorption (P = 0.049). In the tacrolimus group, a lower exposure seemed also due to a decreased MPA reabsorption at 10-12 hours. The PPI effect remains throughout the first year but was clinically more important on day 7. By Cox analysis, the use of PPI was associated with a 25% less chance of being adequately exposed to MPA (95% confidence interval 0.58-0.99, P = 0.04). Similarly, the number of patients underexposed to MPA (AUC < 30 ng·h/mL) was higher at most periods in the PPI group but again not statistically significant. CONCLUSIONS: These data indicate that PPI decreases the MPA exposure when associated with both CNIs but particularly in the first week after RTx. In this period, the MMF dose should be increased. This effect lasts throughout the first year but does not seem to be clinically relevant after the first week.

Analytical Performance of COVID-19 Detection Methods (RT-PCR): Scientific and Societal Concerns.

Background. Health and social management of the SARS-CoV-2 epidemic, responsible for the COVID-19 disease, requires both screening tools and diagnostic procedures. Reliable screening tests aim at identifying (truely) infectious individuals that can spread the viral infection and therefore are essential for tracing and harnessing the epidemic diffusion. Instead, diagnostic tests should supplement clinical and radiological findings, thus helping in establishing the diagnosis. Several analytical assays, mostly using RT-PCR-based technologies, have become commercially available for healthcare workers and clinical laboratories. However, such tests showed some critical limitations, given that a relevant number of both false-positive and false-negative cases have been so far reported. Moreover, those analytical techniques demonstrated to be significantly influenced by pre-analytical biases, while the sensitivity showed a dramatic time dependency. Aim. Herein, we critically investigate limits and perspectives of currently available RT-PCR techniques, especially when referring to the required performances in providing reliable epidemiological and clinical information. Key Concepts. Current data cast doubt on the use of RT-PCR swabs as a screening procedure for tracing the evolution of the current SARS-COV-2 pandemic. Indeed, the huge number of both false-positive and false-negative results deprives the trustworthiness of decision making based on those data. Therefore, we should refine current available analytical tests to quickly identify individuals able to really transmit the virus, with the aim to control and prevent large outbreaks.

Perception of usefulness of laboratory tests ordering by internal medicine residents in ambulatory setting: A single-center prospective cohort study.

The demand for high value health care uncovered a steady trend in laboratory tests ordering and inappropriate testing practices. Residents' training in laboratory ordering practice provides an opportunity for quality improvement. We collected information on demographics, the main reason for the appointment, preexisting medical conditions and presence of co-morbidities from first-visit patients to the internal medicine outpatient service of our university general hospital. We also collected information on all laboratory tests ordered by the attending medical residents. At a follow-up visit, we recorded residents' subjective perception on the usefulness of each ordered laboratory test for the purposes of diagnosis, prognosis, treatment or screening. We observed that 17.3% of all ordered tests had no perceived utility by the attending resident. Tests were usually ordered to exclude differential diagnoses (26.7%) and to help prognosis estimation (19.1%). Age and co-morbidity influenced the chosen category to legitimate usefulness of tests ordering. This study suggests that clinical objectives (diagnosis, prognosis, treatment or prevention) as well as personalization to age and previous health conditions should be considered before test ordering to allow a more appropriate laboratory tests ordering, but further studies are necessary to examine this framework beyond this medical training scenario.

Use and misuse of biomarkers and the role of D-dimer and C-reactive protein in the management of COVID-19: A post-hoc analysis of a prospective cohort study.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is associated with high mortality among hospitalized patients and incurs high costs. Severe acute respiratory syndrome coronavirus 2 infection can trigger both inflammatory and thrombotic processes, and these complications can lead to a poorer prognosis. This study aimed to evaluate the association and temporal trends of D-dimer and C-reactive protein (CRP) levels with the incidence of venous thromboembolism (VTE), hospital mortality, and costs among inpatients with COVID-19. METHODS: Data were extracted from electronic patient records and laboratory databases. Crude and adjusted associations for age, sex, number of comorbidities, Sequential Organ Failure Assessment score at admission, and D-dimer or CRP logistic regression models were used to evaluate associations. RESULTS: Between March and June 2020, COVID-19 was documented in 3,254 inpatients. The D-dimer level ≥4,000 ng/mL fibrinogen equivalent unit (FEU) mortality odds ratio (OR) was 4.48 (adjusted OR: 1.97). The CRP level ≥220 mg/dL OR for death was 7.73 (adjusted OR: 3.93). The D-dimer level ≥4,000 ng/mL FEU VTE OR was 3.96 (adjusted OR: 3.26). The CRP level ≥220 mg/dL OR for VTE was 2.71 (adjusted OR: 1.92). All these analyses were statistically significant (p<0.001). Stratified hospital costs demonstrated a dose-response pattern. Adjusted D-dimer and CRP levels were associated with higher mortality and doubled hospital costs. In the first week, elevated D-dimer levels predicted VTE occurrence and systemic inflammatory harm, while CRP was a hospital mortality predictor. CONCLUSION: D-dimer and CRP levels were associated with higher hospital mortality and a higher incidence of VTE. D-dimer was more strongly associated with VTE, although its discriminative ability was poor, while CRP was a stronger predictor of hospital mortality. Their use outside the usual indications should not be modified and should be discouraged.

Improving serum calcium test ordering according to a decision algorithm.

AIM: To detect differences in the pattern of serum calcium tests ordering before and after the implementation of a decision algorithm. METHODS: We studied patients admitted to an internal medicine ward of a university hospital on April 2013 and April 2016. Patients were classified as critical or non-critical on the day when each test was performed. Adequacy of ordering was defined according to adherence to a decision algorithm implemented in 2014. RESULTS: Total and ionised calcium tests per patient-day of hospitalisation significantly decreased after the algorithm implementation; and duplication of tests (total and ionised calcium measured in the same blood sample) was reduced by 49%. Overall adequacy of ionised calcium determinations increased by 23% (P=0.0001) due to the increase in the adequacy of ionised calcium ordering in non-critical conditions. CONCLUSIONS: A decision algorithm can be a useful educational tool to improve adequacy of the process of ordering serum calcium tests.

Longitudinal Pharmacokinetics of Mycophenolic Acid in Elderly Renal Transplant Recipients Compared to a Younger Control Group: Data from the nEverOld Trial.

BACKGROUND AND OBJECTIVES: Elderly patients are increasingly likely to be recipients of transplants. However, the pharmacokinetics of mycophenolic acid (MPA) in this population are yet to be studied in detail. The objective of this study was to assess whether there were differences in MPA pharmacokinetic parameter values between elderly recipients and younger-adult recipients during the 6 months immediately following renal transplantation. METHODS: In this analysis, the longitudinal 12-h pharmacokinetics of MPA, administered as enteric-coated mycophenolate sodium (EC-MPS), were evaluated in 44 elderly renal transplant recipients and compared with the corresponding pharmacokinetics of MPA in 31 younger adult recipients. Measurements were performed at 7, 30, 60, 90, and 180 days post-transplantation. All patients received tacrolimus and prednisone. RESULTS: The elderly patients were 30 years older than the younger controls, with a predominance of males and Caucasians. Elderly patients had lower serum albumin than the younger controls during the first 6 months after transplantation. The mean estimated total body MPA clearance of the elderly recipients was not significantly different from that of the controls at any analyzed time point (the mean clearance across all time points was 0.31 ± 0.17 vs 0.30 ± 0.25 L/h/kg). MPA exposure, as evaluated from the area under the 12-h time versus measured MPA concentration (adjusted for dose/body weight) curve, did not differ between the groups at any time point (mean exposure across all time points was 4.68 ± 3.61 vs 5.95 ± 4.29 µg·h/mL per mg/kg for the elderly recipients and the controls). CONCLUSIONS: These data show that the pharmacokinetics of MPA in elderly renal transplant recipients were no different to those of younger-adult recipients in this study population. CLINICALTRIALS.GOV: NCT 01631058.

UPLC-MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+ and B CD19+ lymphocytes.

Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC-MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid-liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters® Acquity™ UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters® MassTrak TDM C18 (2.1 × 10 mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4 mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0-5.2 ng/mL in PBMC, 0-5.0 ng/mL in T CD4+ Lymphocytes and 0-5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90 days either at room temperature or at -30 °C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/106 cells in PBMC, from 0.081 to 1.432 ng/106 cells in T CD4+, and from 0.197 to 1.564 ng/106 cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients.

Immunothrombosis and COVID-19 ‒ a nested post-hoc analysis from a 3186 patient cohort in a Latin American public reference hospital

Abstract Objective COVID-19 is associated with an elevated risk of thromboembolism and excess mortality. Difficulties with best anticoagulation practices and their implementation motivated the current analysis of COVID-19 patients who developed Venous Thromboembolism (VTE). Method This is a post-hoc analysis of a COVID-19 cohort, described in an economic study already published. The authors analyzed a subset of patients with confirmed VTE. We described the characteristics of the cohort, such as demographics, clinical status, and laboratory results. We tested differences amid two subgroups of patients, those with VTE or not, with the competitive risk Fine and Gray model. Results Out of 3186 adult patients with COVID-19, 245 (7.7%) were diagnosed with VTE, 174 (5.4%) of them during admission to the hospital. Four (2.3% of these 174) did not receive prophylactic anticoagulation and 19 (11%) discontinued anticoagulation for at least 3 days, resulting in 170 analyzed. During the first week of hospitalization, the laboratory most altered results were C-reactive protein and D-dimer. Patients with VTE were more critical, had a higher mortality rate, worse SOFA score, and, on average, 50% longer hospital stay. Conclusion Proven VTE incidence in this severe COVID-19 cohort was 7.7%, despite 87% of them complying completely with VTE prophylaxis. The clinician must be aware of the diagnosis of VTE in COVID-19, even in patients receiving proper prophylaxis.

Trace element contents in serum of healthy elderly population of metropolitan São Paulo area in Brazil.

In this study, the elements Br, Ca, Cl, Fe, Na, Rb, Se and Zn were determined in serum of a healthy elderly population residing in the São Paulo Metropolitan area, using instrumental neutron activation analysis. Comparison studies indicated that only Se concentration was significantly lower in the elderly group aged 75-91 years than those obtained for the group aged 60-74 years. Between genders, males presented lower Br concentrations and, the females lower Fe. Mean concentrations obtained for most elements were within the values reported in the literature.

Laboratory Tests Ordering Pattern by Medical Residents From a Brazilian University Hospital.

OBJECTIVES: The adequacy of laboratory test orders by medical residents is a longstanding issue. The aim of this study is to analyze the number, types, and pattern of repetition of tests ordered by medical residents. METHODS: We studied all tests ordered over a 1-year period for inpatients of an internal medicine ward in a university hospital. Types, results, and repetition pattern of tests were analyzed in relation to patients' diagnoses. RESULTS: We evaluated 117,666 tests, requested for 1,024 inpatients. The mean number of tests was 9.5 per day. The test repetition pattern was similar, regardless of patients' diagnoses, previous test results, or duration of stay. The probability of an abnormal result after a sequence of three normal tests was lower than 25%, regardless of the diagnosis. CONCLUSIONS: Number of tests and repetition were both high, imposing costs, discomfort, and risks to patients, thus warranting further investigation.

Interferon lambda and hepatitis C virus core protein polymorphisms associated with liver cancer.

BACKGROUND: Hepatitis C virus (HCV) infection is often persistent and gradually advances from chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (HCC). Worldwide, hepatocellular carcinoma is the fifth most common neoplasm. METHOD OF STUDY: the Interferon lambda (IFNL) polymorphisms genotypes (rs8099917, rs12979860 and rs12980275) and the presence of mutations in HCV core protein were analyzed in 59 patients with HCC, and also in 50 cirrhotic patients (without HCC). RESULTS: the rs12980275-AG genotype was associated with HCC on age-adjusted analysis (OR 2.42, 95% CI 1.03-5.69, P=0.043). Core substitutions R70Q and L91M were mainly found in genotype 1b isolates. Furthermore, a borderline level of statistical significance association was found among the presence of amino acid Glutamine (Q) in the position 70 and IFNL3 genotype AG (P=0.054). CONCLUSIONS: the screening of these polymorphisms and functional studies would be useful in clinical practice for identifying groups at high risk of HCC development.