Meals and Room Temperature Storage do not Significantly Affect Feasibility of Direct RT-PCR Tests for SARS-CoV-2 Using Saliva.

BACKGROUND: Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using saliva samples has emerged as a preferred technique since sample collection is easy and noninvasive. In addition, several commercial high-throughput PCR kits that do not require RNA extraction/purification have been developed and are now available for testing saliva samples. However, an optimal protocol for SARS-CoV-2 RT-PCR testing of saliva samples using the RNA extraction/purification-free kits has not yet been established. The aim of this study was to establish optimal preanalytical conditions, including saliva sample collection, storage, and dilution for RNA extraction/purification-free RT-PCR (direct RT-PCR). METHODS: Patients suspected with COVID-19 from March 02 to August 31, 2020, were enrolled in this study. A total of 248 samples, including 43 nasopharyngeal swabs and 205 saliva samples, were collected from 66 patients (37 outpatients and 29 inpatients) and tested using the 2019 Novel Coronavirus Detection Kit (nCoV-DK, Shimadzu Corporation, Kyoto, Japan). RESULTS: The detection results obtained using nasopharyngeal swabs and saliva samples matched 100%. The sampling time, i.e., either awakening time or post-breakfast, had no significant effect on the viral load of the saliva samples. Although saliva samples are routinely diluted to reduce viscosity, we observed that dilution negatively affected PCR sensitivity. Saliva samples could be stored at room temperature (25°C) for 24 hours or at 4°C for up to 48 hours. CONCLUSIONS: This study demonstrated the appropriate conditions of saliva sample collection, processing, and storage, and indicated that the nCoV-DK is applicable to saliva samples, making the diagnosis method simple and safe.

Achievements and challenges of the Sakigake designation system in Japan.

The Sakigake designation system (Sakigake) has been launched to encourage the pioneered development of innovative new medical products for the effective treatment of severe illness in Japan, which allows leveraging the several advantages in prioritized consultation, rapid review, premium drug pricing and extended data-protection period. We retrospectively analysed the Sakigake products including drugs and regenerative medical products to clarify the achievements and the future issues in this system. From April 2015 to August 2020 (the first 5-year trial period of Sakigake), 37 products were designated, and 10 of those were approved in Japan in which 7 new active substances achieved the first-in-world approvals. Oncology, neurology and cardiovascular disease were the major therapeutic areas, and those 3 accounted for 75.7% of all products. Sakigake achieved some first-in-world approvals by the Pharmaceuticals and Medical Devices Agency/the Ministry of Health, Labor and Welfare of innovative new medical products, although in some therapeutic areas, there remains room in stimulating drug development.

Rationales of delay and difference in regulatory review by Japan, the USA and Europe among new drugs first approved in Japan.

AIMS: To clarify the rationales of delay or difference in the review of new drug applications among regulatory authorities for new drugs, those first approved in the world being in Japan. METHODS: Among 80 new drugs first approved in Japan from 2008 to 2019, we identified those subsequently approved in the USA or Europe. Significant delays in approval time (boxplot outliers) and the rationales for the delays were assessed among the Pharmaceuticals and Medical Devices Agency (PMDA), the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). RESULTS: Of the 80 Japan-first approvals, 25 and 24 were approved in the USA and Europe, respectively, and their median approval times in Japan, the USA and Europe were 285, 334 and 477 days, respectively. Significant delays were identified for pirfenidone (1806 days, FDA), alogliptin benzoate (1856 days, FDA), insulin degludec (1457 days, FDA) and romosozumab (750 days, PMDA; 994 days, FDA; 748 days, EMA). Due to concerns about cardiovascular risk, alogliptin benzoate and insulin degludec were requested for additional clinical trials by the FDA, and romosozumab required a much longer review period than the standard approval time in all three regions. CONCLUSIONS: Among the new drugs significantly delayed in approval time in Japan, the USA or Europe, there were some differences in the requirements, the participating regions and the assessment of clinical trials. The regulatory views on the cardiovascular risk also differed among the three regions. These divergences may be associated with the differences in approval histories.

[How to Interpret Data from Routine Laboratory Tests].

The reversed clinicopathological conference (RCPC) is one of the most effective training methods to inter- pret routine laboratory data. In this RCPC, three specialists in laboratory medicine discussed laboratory data of a patient with upper gastrointestinal bleeding. Even though they interpreted the data with different methods, they arrived at the same conclusion. This discussion shows that it is possible to grasp the disease condition by RCPC training. In addition, combining laboratory data, the medical history, and physical find- ings helps improve the diagnostic ability. [Review].

Performance evaluation of the digital cell imaging analyzer DI-60 integrated into the fully automated Sysmex XN hematology analyzer system.

BACKGROUND: The XN-Series (Sysmex, Kobe, Japan) have been equipped with the automated digital cell imaging analyzer DI-60, which provides complete automation of the sample processing with automated complete blood counts (CBC), slide making/staining, and digital scanning with cell pre-classification. The aim of this study was to evaluate the efficacy of the XN-Series as an integrated blood cell analysis system. METHODS: White blood cell (WBC) morphological analysis by the DI-60 was evaluated using 232 blood samples from patients. Routine analysis of a total of 2000 blood samples has been performed to evaluate the processing ability of the XN-Series connected to the DI-60. RESULTS: The overall analysis accuracy of pre-classification of WBC by the DI-60 was 88.4%. Good correlation was observed between final results of the DI-60 analysis and manual differentiation with high sensitivity and specificity for blasts and immature granulocytes. The sample processing time of the XN-Series, from automated CBC to cell pre-classification, was 38±1 min/single run and 165±12 min/500 CBC samples run (slide preparation rate 15.6%) with no sample hold-up at the DI-60. CONCLUSIONS: The automated morphological analysis capability of the DI-60 has potential usefulness in the integrated automated hematology analysis system of XN-Series.

Performance evaluation of the automated morphological analysis of erythrocytes by CellaVision DM96.

BACKGROUND: Automated digital morphology systems are utilized for blood cell morphological examination. The aim of this study is to evaluate the accuracy and efficacy of RBC morphological anomaly screening using the CellaVision DM96 (DM96) automated image analysis system. METHODS: The automated analysis of RBC shape, size, and chromasia abnormalities was conducted on the DM96 using 478 blood samples. A manual microscopic review was independently performed. RESULTS: The DM96 preclassified samples as poikilocytosis-positive for 98% of cases with schistocytosis or echinocytosis, 97% of elliptocytosis, and 92% or 65% of cases that were positive for teardrop cells or for target cells, respectively. The accuracy of the DM96 in the detection of RBC size and chromasia abnormalities of iron deficiency anemia cases was higher than direct microscopic observation. CONCLUSIONS: Automated morphological analysis with the DM96 has potential utility in the morphological screening of RBC anomalies that are associated with disease.

A cross-sectional survey on principal investigators' clinical research knowledge in Japan.

Principal investigators (PIs) play a key role in clinical research, and must thus understand the role of clinical research support staff to conduct successful and appropriate clinical research. This study evaluates clinical research capabilities by examining the clinical research knowledge of PIs and clinical research support staff. The participants of this cross-sectional study were academic researchers and clinical research support staff from Japanese universities and research institutions. The participants comprised of 54 respondents, among whom 36 were PIs (physicians) and 18 were clinical research support staff. A self-administered electronic survey was created and evaluated by experts, with 50 knowledge items. Mann-Whitney U tests were used to determine the significance of the differences in knowledge between clinical research support staff and PIs. We compared the correct answer rate of clinical research support staff and PIs for each knowledge category and observed that the clinical research support staff scored significantly higher than the PIs in all aspects of clinical research knowledge sections, including total score. Our findings showed that PIs did not have the same amount of clinical research knowledge as the clinical research support staff. Clinical research knowledge is essential for investigators, especially PIs, to protect patients and promote medical breakthroughs. Thus, more accessible clinical research education and mandatory knowledge testing will allow PIs to lead successful clinical research and further the level of medical research in Japan.

Effluent N-terminal expressed in renal cell carcinoma/mesothelin predicts increased peritoneal permeability in patients undergoing peritoneal dialysis.

INTRODUCTION: We investigated whether N-terminal and C-terminal products of expressed in renal cell carcinoma/mesothelin (N-ERC and C-ERC) in peritoneal effluent can predict peritoneal permeability in patients undergoing peritoneal dialysis (PD). METHODS: Thirty-seven peritoneal effluent samples were obtained from 26 PD patients. High transport status was determined by the peritoneal equilibration test as a dialysate/plasma ratio of creatinine (D/P Cr) ≥ 0.81. Effluent cancer antigen 125 (CA125) was used as a reference. RESULTS: Effluent N-ERC concentration was better correlated with D/P Cr than effluent C-ERC or CA125 concentration. In multivariate analyses, effluent N-ERC and C-ERC, but not CA125, were significant predictors of high transport status after adjusting for age, PD duration, and residual renal Kt/V. ROC analysis showed that effluent N-ERC was the best predictor of high transport status among those three biomarkers. CONCLUSION: Effluent N-ERC predicts increased peritoneal permeability in patients undergoing PD.

Ultrafiltration volume by once-weekly hemodialysis is a predictor of technique survival of combination therapy with peritoneal dialysis and hemodialysis.

Overhydration is a major cause of technique failure of peritoneal dialysis (PD). Hence, we investigated the impact of ultrafiltration (UF) volume by once-weekly hemodialysis (HD), excess volume beyond their dry weight, on technique survival of PD and HD combination therapy (PD+HD). Forty-six anuric PD+HD patients were divided into three groups according to baseline UF volume by HD: low-UF (

Evolving Landscape of New Drug Approval in Japan and Lags from International Birth Dates: Retrospective Regulatory Analysis.

The Pharmaceuticals and Medical Devices Agency (PMDA) has approved hundreds of new drugs in recent years. We retrospectively analyzed the new drugs approved in Japan from 2008 to 2019, and identify the first-in-world approvals and clarify the current drug lag. The new drug and the drug lag were defined as a drug with a new active substance and a difference between the approval date in Japan and the international birth date, respectively. Among 400 new drugs approved in Japan during the last 12 years, 80 (20.0%) were first approved in Japan, and 320 were outside Japan (the United States: 202, 50.5%; Europe: 82, 20.5%; other regions: 36, 9.0%). Of these, 45 new drugs have not yet been approved outside Japan, and the remaining 355 have been globally approved in Japan and overseas. The number of new drug approvals were the largest in oncology followed by metabolic/endocrine and infectious diseases. The median drug lags (year) among all 400 new drugs and 355 new drugs with global approvals were 4.3 and 4.7 in the first tertile (2008-2011), 1.5 and 2.6 in the second tertile (2012-2015), and reduced to 1.3 and 2.2 in the third tertile (2016-2019), respectively. Substantial drug lag remains in neurology, psychiatry, and therapeutic areas where the number of new drug approvals was relatively small. Collectively, one-fifth of the new drugs approved in Japan are first-in-world approvals. Drug lag has been greatly decreased, although it still exists.

Seroprevalence of anti-SARS-CoV-2 antibodies in Japanese COVID-19 patients.

OBJECTIVES: To determine the seroprevalence of anti-SARS-CoV-2 IgG and IgM antibodies in symptomatic Japanese COVID-19 patients. METHODS: Serum samples (n = 114) from 34 COVID-19 patients with mild to critical clinical manifestations were examined. The presence and titers of IgG antibody for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were determined by a chemiluminescent microparticle immunoassay (CMIA) using Alinity i SARS-CoV-2 IgG and by an immunochromatographic (IC) IgM/IgG antibody assay using the Anti-SARS-CoV-2 Rapid Test. RESULTS: IgG was detected by the CMIA in 40%, 88%, and 100% of samples collected within 1 week, 1-2 weeks, and 2 weeks after symptom onset in severe and critical cases, and 0%, 38%, and 100% in mild/moderate cases, respectively. In severe and critical cases, the positive IgG detection rate with the IC assay was 60% within one week and 63% between one and two weeks. In mild/moderate cases, the positive IgG rate was 17% within one week and 63% between one and two weeks; IgM was positive in 80% and 75% of severe and critical cases, and 42% and 88% of mild/moderate cases, respectively. On the CMIA, no anti-SARS-CoV-2 IgG antibodies were detected in COVID-19 outpatients with mild symptoms within 10 days from onset, whereas 50% of samples from severe inpatients were IgG-positive in the same period. The IC assay detected higher IgM positivity earlier from symptom onset in severe and critical cases than in mild/moderate cases. CONCLUSIONS: A serologic anti-SARS-CoV-2 antibody analysis can complement PCR for diagnosing COVID-19 14 days after symptom onset.

Comparison of the clinical performance and usefulness of five SARS-CoV-2 antibody tests.

We examined the usefulness of five COVID-19 antibody detection tests using 114 serum samples at various time points from 34 Japanese COVID-19 patients. We examined Elecsys Anti-SARS-CoV-2 from Roche, and four immunochromatography tests from Hangzhou Laihe Biotech, Artron Laboratories, Chil, and Nadal. In the first week after onset, Elecsys had 40% positivity in Group S (severe cases) but was negative in Group M (mild-moderate cases). The immunochromatography kits showed 40-60% and 0-8% positivity in Groups S and M, respectively. In the second week, Elecsys showed 75% and 50% positivity, and the immunochromatography tests showed 5-80% and 50-75% positivity in Groups S and M, respectively. After the third week, Elecsys showed 100% positivity in both groups. The immunochromatography kits showed 100% positivity in Group S. In Group M, positivity decreased to 50% for Chil and 75-89% for Artron and Lyher. Elecsys and immunochromatography kits had 91-100% specificity. Elecsys had comparable chronological change of cut-off index values in the two groups from the second week to the sixth week. The current SARS-CoV-2 antibody detection tests do not provide meaningful interpretation of severity and infection status. Its use might be limited to short-term epidemiological studies.

SARS-CoV-2 seroprevalence in healthcare workers at a frontline hospital in Tokyo.

Healthcare workers (HCWs) are highly exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The actual coronavirus disease (COVID-19) situation, especially in regions that are less affected, has not yet been determined. This study aimed to assess the seroprevalence of SARS-CoV-2 in HCWs working in a frontline hospital in Tokyo, Japan. In this cross-sectional observational study, screening was performed on consented HCWs, including medical, nursing, and other workers, as part of a mandatory health checkup. The screening test results and clinical characteristics of the participants were recorded. The antibody seroprevalence rate among the 4147 participants screened between July 6 and August 21, 2020, was 0.34% (14/4147). There was no significant difference in the seroprevalence rate between frontline HCWs with a high exposure risk and HCWs working in other settings with a low exposure risk. Of those seropositive for SARS-CoV-2, 64% (9/14) were not aware of any symptoms and had not previously been diagnosed with COVID-19. In conclusion, this study provides insights into the extent of infection and immune status in HCWs in Japan, which has a relatively low prevalence of COVID-19. Our findings aid in formulating public health policies to control virus spread in regions with low-intensity COVID-19.

[Serum zinc concentration decreases with age and is associated with anemia in middle-aged and elderly people].

Zinc (Zn) is an essential trace element for humans and its deficiency can lead to several clinical problems. This study examined the relationship between the serum zinc concentration and anemia in middle-aged and elderly people. Samples were obtained from 150 men and 303 women who received health checkups over the course of 40 years. The serum concentration of Zn was measured as well as the complete blood count (CBC), alanine aminotransferase (AST), aspartate aminotransferase (ALT), gamma-glutamyl transferase (gamma GT), total cholesterol (TC), high density lipoprotein (HDL-C), triglyceride (TG), creatinine (Cr), uric acid (UA) and fasting plasma glucose (FPG). The serum Zn concentration was 77.4 +/- 9.7 microg/dL in men and 79.1 +/- 10.4 microg/dL in women (p= 0.09). The serum Zn concentration correlated inversely with age (r=-0.11, p=0.018). Anemia diagnosed by the World Health Organization criteria, was present in 17.3% of men and in 13.5% of women. However, more than 80% of the anemia was normocytic (men 86%, women 81%). The serum Zn concentration was significantly lower in those with anemia than in those without anemia. The Hb level correlated with the serum Zn concentration (men r=0.25, p=0.002, women r=0.23, p<0.001). A multiple regression analysis confirmed a low serum Zn concentration to be associated with a low Hb level. In conclusion, this study indicates that the serum Zn concentration decreases with age and that a low Zn concentration is associated with normocytic anemia, thus suggesting that a Zn deficiency may therefore be one of the causes of anemia in elderly people.

Preß1-high-density lipoprotein metabolism is delayed in patients with chronic kidney disease not on hemodialysis.

BACKGROUND: Preß1-high-density lipoprotein (HDL) is a lipid-poor cholesterol acceptor that is converted to lipid-rich HDL by lecithin-cholesterol acyltransferase (LCAT). In patients receiving hemodialysis, preß1-HDL metabolism is hampered even if HDL cholesterol is normal. Hemodialysis may affect preß1-HDL metabolism by releasing lipases from the vascular wall due to heparin. OBJECTIVES: We investigated whether preß1-HDL metabolism is delayed in patients with chronic kidney disease (CKD) who are not receiving hemodialysis. METHODS: We examined 44 patients with Stage 3 or higher CKD and 22 healthy volunteers (Control group). The patients with CKD were divided into those without renal replacement therapy (CKD group, n = 22) and those undergoing continuous ambulatory peritoneal dialysis (CAPD group, n = 22). Plasma preß1-HDL concentrations were determined by immunoassay. During incubation at 37°C, we used 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) to inhibit LCAT activity and defined the conversion halftime of preß1-HDL (CHTpreß1) as the time required for the difference in preß1-HDL concentration in the presence and absence of 5,5-DTNB to reach half the baseline concentration. RESULTS: The absolute and relative preß1-HDL concentrations were higher, and CHTpreß1 was longer in the CKD and CAPD groups than in the Control group. Preß1-HDL concentration was significantly correlated with CHTpreß1 but not with LCAT activity in patients with CKD and CAPD. CONCLUSION: Preß1-HDL metabolism is delayed in patients with CKD who are not on hemodialysis. This preß1-HDL metabolic delay may progress as renal function declines.

Bioimpedance Spectroscopy-Based Fluid Status in Combined Dialysis Compared With Hemodialysis and Peritoneal Dialysis: A Cross-Sectional Study.

Combination therapy with peritoneal dialysis and hemodialysis (PD+HD) is widely used in Japan for PD patients with decreased residual renal function. However, fluid status in PD+HD patients has not been well studied. In this cross-sectional study, we compared fluid status in 41 PD+HD patients with that in 103 HD and 92 PD patients using the bioimpedance spectroscopy. Extracellular water normalized to patient height (NECW, kg/m) was the highest in pre-HD (8.3 ± 1.6) followed by PD (7.9 ± 2.7), PD+HD (7.5 ± 2.5), and post-HD patients (6.9 ± 1.5) (P < 0.01). By multiple linear regression analysis, PD+HD was associated with a significantly lower NECW than pre-HD (ß = -0.8, P = 0.03) and similar to PD (ß = -0.5, P = 0.24) and post-HD (ß = 0.6, P = 0.08) after adjustment for age, sex, diabetic nephropathy, ischemic heart disease, dialysis period, and daily urine volume. There was no correlation between NECW and daily urine volume in all dialysis groups. Average daily fluid removal (a sum of urine volume and ultrafiltration volume by dialysis) was positively correlated with NECW in PD+HD and pre-HD, but not in PD and post-HD patients. Our results suggest that fluid status in PD+HD patients with decreased residual renal function is acceptable as compared with that in HD and PD patients.

Health-related quality of life on combination therapy with peritoneal dialysis and hemodialysis in comparison with hemodialysis and peritoneal dialysis: A cross-sectional study.

BACKGROUND: The health-related quality of life (HRQOL) of dialysis patients has not been well examined, especially in combination therapy with peritoneal dialysis and hemodialysis (PD+HD) patients. We compared the HRQOL of PD+HD patients with that of HD and PD patients. METHODS: A multicenter, cross-sectional study was conducted on 36 PD+HD, 103 HD, and 90 PD patients in Japan who completed the Kidney Disease Quality of Life Short Form 36, version 1.3. HRQOL scores were summarized into physical- (PCS), mental- (MCS), role/social- (RCS), and kidney disease component summaries (KDCS). RESULTS: Of the PD+HD patients, 31 (86%) transferred from PD and 5 (14%) transferred from HD. They had the longest dialysis vintage and the smallest urine volume. PCS, MCS, and KDCS HRQOL scores of PD+HD patients were comparable with those of HD and PD patients. However, the RCS score for PD+HD was significantly higher than that for HD (p = 0.020) and comparable with that for PD. PD+HD and PD were associated with significantly higher RCS scores than HD after adjusting for age, gender, diabetic nephropathy, dialysis vintage, ischemic heart disease, and peripheral arterial disease. CONCLUSIONS: For RCS, HRQOL in PD+HD patients was better than that in HD and comparable with that in PD patients, whereas the PCS, MCS, and KDCS HRQOL scores of PD+HD patients were comparable with those of HD and PD patients.

Hospitalization for Patients on Combination Therapy With Peritoneal Dialysis and Hemodialysis Compared With Hemodialysis.

INTRODUCTION: Combination therapy with peritoneal dialysis and hemodialysis (PD+HD) is widely used for PD patients with decreased residual kidney function in Japan; however, hospitalization for this combined dialysis has not been investigated so far. We compared the risk of hospitalization for PD+HD with that for HD. METHODS: A multicenter, prospective observational study was conducted on 42 PD+HD and 42 HD patients matched for age and diabetic nephropathy. The main outcome measure was the cumulative incidence of hospitalization for any cause assessed with the Kaplan-Meier method. Hospitalization rates (the number of admissions per 100 patient-years) associated with dialysis modality were also calculated. The impact of dialysis modality on time to hospitalization was analyzed using the Cox proportional hazard model. RESULTS: There was no significant difference between groups in terms of age, sex, dialysis vintage, diabetic nephropathy, and comorbidities. The cumulative incidence of hospitalization did not significantly differ between the groups (log-rank test, P = 0.36). Although total hospitalization rates were 66.0 in PD+HD and 59.2 in HD, hospitalization rates for the sum of PD-related infections (a composite of catheter-related infection and peritonitis) and vascular access troubles were 21.7 in PD+HD and 7.2 in HD. On univariate Cox proportional hazard analysis, dialysis modality had no significant impact on time to hospitalization. CONCLUSION: The risk of hospitalization was not significantly different between PD+HD and HD, although PD+HD patients had a higher risk of dialysis access-related complications than HD patients.

Apolipoproteins C-II and C-III as nutritional markers unaffected by inflammation.

BACKGROUND: Rapid turnover proteins (RTPs), such as transthyretin (TTR), retinol binding protein (RBP), and transferrin (Tf), provide an accurate assessment of nutritional status but are susceptible to inflammation. Lipid-related markers, which have short half-lives in serum, may be better suited for nutritional assessment. We sought to identify sensitive nutritional markers unaffected by inflammation. METHODS: Fasting serum samples were collected from 30 malnourished inpatients and 25 healthy volunteers. Malnourished inpatients were divided into 2 groups: a low-C-reactive protein (CRP) group (CRP < 20 mg/l, n = 15) and a high-CRP group (CRP ≥ 20 mg/l, n = 15). Lipid-related markers, traditional nutritional markers, RTPs, micronutrients, and ketone bodies were measured and compared among the groups. RESULTS: Apolipoprotein (Apo)C-II and ApoC-III concentrations were lower in malnourished inpatients than in the control group. There was no significant difference in ApoC-II and ApoC-III between the low- and high-CRP groups. Carnitine transporters and ketone bodies did not show a significant difference among the three groups. Albumin, TTR, RBP, and Tf concentrations were lowest in the high-CRP group, intermediate in the low-CRP group, and highest in the control group. CONCLUSIONS: These results indicate that ApoC-II and ApoC-III are appropriate nutritional biomarkers unaffected by inflammation.