Factors associated with prolonged duration of viral clearance in non-severe SARS-CoV-2 patients in Osaka, Japan.

BACKGROUND: We investigated factors associated with prolonged viral clearance of SARS-CoV-2 among non-severe adult patients in Osaka, Japan. A total of 706 laboratory-confirmed COVID-19 patients were enrolled in this longitudinal observational study between 29 January 2020 and 31 May 2020, across 62 hospitals and three non-hospital recuperation facilities. METHODS: Logistic regression analysis was performed to investigate the factors associated with prolonged (29 days: upper 25% in duration) viral clearance of SARS-CoV-2. Linear regression analysis was conducted to assess these factors 14 days after symptom onset. RESULTS: The median duration of viral clearance was 22 days from symptom onset. After adjustment for sex, age, symptoms, comorbidity, and location of recuperation, comorbidities were associated with prolonged duration: (OR, 1.77 [95% CI, 1.11-2.82]) for one, (OR, 2.47 [95% CI, 1.32-4.61]) for two or more comorbidities. Viral clearance 14 days after symptom onset was 3 days longer for one comorbidity and 4 days longer for two or more comorbidities compared to clearance when there was no comorbidity. CONCLUSION: The presence of comorbidity was a robust factor associated with a longer duration of viral clearance, extending by 3 to 4 days compared to patients with no comorbidity.

Risk factors for fecal carriage of IMP-6-producing Enterobacteriaceae at a long-term care hospital in Japan: A follow-up report from the northern Osaka multicentre study group.

The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing at medical institutions in Japan without even noticing. Recently, we performed a point prevalence survey for CRE carriage at a medical facility in northern Osaka that demonstrated an unexpectedly high prevalence of blaIMP-6-positive CRE, particularly at long-term care hospitals (LTCH). To identify the risk factors for CRE carriage, we collected clinical data of patients at a representative LTCH. Of 140 patients who were included in this study, 27 (19.3%) were colonized with metallo-beta-lactamase (IMP-6) producers. Pulsed-field gel electrophoresis of the IMP-6 producing Enterobacteriaceae suggested a non-clonal transmission of Escherichia coli, while a clonal spread was shown for Klebsiella pneumoniae. Risk factors for CRE colonization were a longer stay at the hospital stay and a lower independence state, as measured by Norton scales. We propose that a paradigm shift in infection control, inciting a coordinated regional effort to involve LTCHs, should be discussed in the aging society of Japan.

Development of selective medium for IMP-type carbapenemase-producing Enterobacteriaceae in stool specimens.

BACKGROUND: Identification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE. METHODS: Fifteen reference CPE strains producing different types of ß-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium. RESULTS: All tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%). CONCLUSIONS: M-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.

Coagulation factors in whole blood collected from pregnant women and stored at 4°C.

The present study aimed to measure the levels of coagulation factors in stored whole blood of pregnant women and to determine their usefulness in treating pregnant women who developed coagulopathy. A prospective study to measure coagulation factors in stored donated whole blood from pregnant and non-pregnant women was conducted. Fibrinogen, FV, FVII, FVIII, FXIII, and von Willebrand factor were measured in blood stored at 4°C for 0, 1, 3, and 5 weeks. All coagulation factors except for factor XIII decreased during storage. Fibrinogen and factor VII in the blood collected from pregnant women gradually decreased over time and their levels were significantly higher after 5 weeks of storage than those of non-pregnant women at week 0. Whole blood donated by pregnant women for autologous blood transfusion and stored at 4°C may be expected being effectively for the prevention of coagulopathy and the treatment of circulatory blood volume loss.

[Emergency incompatible red cell transfusion to a patient whose blood type was suspended from identification].

We report a case of a 59-year-old woman who presented with hypovolemic shock and compensated acidosis (preoperative arterial blood gases: pH 7.3, P(CO2) 31.9 mmHg, Pa(O2) 112.3 mmHg, base excess -9.8, Hb 6.4 g x dl(-1)) due to perforated descending colon, necessitating emergency surgery. Tracheal intubation had been performed preoperatively. Prior to induction of anesthesia, blood pressure was 106/74 mmHg, heart rate 119 beats x min(-1), and Sp(O2) 100% breathing room air. Anesthesia was induced with remifentanil influsion at a rate of 0.05 mg x kg(-1) x min(-1), sevoflurane 1% and rocuronium bromide 30mg, and was maintained with oxygen, air, remifentanil and sevoflurane. For a critical hypovolemia, in accordance to the guidelines for intraoperative critical hemorrhage and the Japanese practical guidelines for blood components therapy, we started to transfuse incompatible red cell (O+) since the identification of blood typing was suspended. The duration of surgery was 104 min, with an intraoperative total bleeding of 125 ml. Four units of total blood transfusion and 3,050 ml of infusion of Ringer's acetate solution were administered. The patient was transferred to ICU with tracheal intubation. No adverse reactions associated with blood type incompatibility were recognized.

Linguistic Features of Clients and Counselors for Early Detection of Mental Health Issues in Online Text-based Counseling.

Online counseling is essential for overcoming mobility restrictions, schedule limitations, and mental health stigma. However, government counseling offices are being inun-dated with consultations for which non-mental health supports are targeted. Therefore, we aim to create a classification model that classifies whether the clients have mental health issues or other issues. We expect to support counselors by presenting the classification results. We conducted the first automatic detection of clients who might be suffering from mental health issues and used almost 1000 actual counseling sessions for our machine learning framework. We achieved an F1-score of 0.646 by classifying dialogue sessions using features such as frequency-inverse, document frequency, document embedding of a large-scale language model, linguistic inquiry and word count, topic modeling, and statistics of dialogue sentences. In addition, we performed dimensionality reduction with principal component analysis. We also conducted evaluation experiments using dialogue sentences from the beginning to the middle of sessions as input and clarified the relationship between the number of messages in the dialogues and the transition in the classification performance. We also identified the words that contribute to detecting mental health issues for each client and counselor. Clinical relevance-This study makes it possible to detect the trends identified in a client's anxieties during counseling. Our findings are critical for designing systems that assist counselors.

The characteristics and clinical course of patients with COVID-19 who received invasive mechanical ventilation in Osaka, Japan.

OBJECTIVE: To describe the detailed clinical course of patients with coronavirus disease 2019 (COVID-19) who received invasive mechanical ventilation. METHODS: We conducted a case series of patients with COVID-19 who received invasive mechanical ventilation in Osaka, Japan, between January 29 and May 28, 2020. We describe the patient characteristics and clinical course from onset. Additionally, we fitted logistic regression models to investigate the associations between patient characteristics and the 30-day mortality rate. RESULTS: A total of 125 patients who received invasive mechanical ventilation (median age [interquartile range], 68 [57-73] years; male, 77.6%) were enrolled. Overall, the 30-day mortality was 24.0%, and the median (interquartile range) length of ICU stay and length of invasive mechanical ventilation use were 16 (12-29) days and 13 (9-26) days, respectively. From clinical onset, 121 patients (96.8%) were intubated within 14 days. In multivariable logistic regression analysis, age of 65 years or older (odds ratio, 3.56; 95% confidence interval, 1.21-10.49; P = 0.02) and male sex (odds ratio, 3.75; 95% confidence interval, 1.00-11.24, P = 0.04) were significantly associated with a higher 30-day mortality rate. CONCLUSIONS: In this case series of patients with COVID-19 who received invasive mechanical ventilation in Japan, the 30-day mortality rate was 24.0%, and age 65 years or older and male sex were associated with higher 30-day mortality rate.

DNA cytosine methylation profile in various cancer-related genes is altered in cultured rat hepatocyte cell lines as compared with primary hepatocytes.

We determined the DNA cytosine methylation status in the promoter CpG islands of eight cancer-related genes (p16, Socs-1, Rassf1A, Hic-1, Dlc-1, Timp-1, Timp-2, and Timp-3) in five rat hepatocyte cell lines, including normal cell lines (Clone 9 and CWSV-1) and tumor cell lines (H4-II-E-C3, MH1C1, and McA-RH7777). The experimental methods used to assess the methylation profile were methylation-specific PCR (MSP) and methylation-sensitive digestion combined with PCR. The results were compared with the methylation status of rat primary hepatocytes. To evaluate methylation-mediated gene induction/silencing, the expression of gene transcripts was semi-quantitatively assessed using RT-PCR. In primary cells, the CpG islands of all genes tested were unmethylated. In contrast, there was at least one hypermethylated gene in the cultured cell lines. Three genes (p16, Socs-1 and Rassf1A) were hypermethylated in Clone 9 cells; among the other five genes, three genes (Hic-1, Timp-1 and Timp-3) were hypermethylated in the CWSV-1 cell lines and two genes (Dlc-1 and Timp-2) were hypermethylated only in the tumor cell lines. The methylation status in some of the tested genes was altered at an early stage of cell culture as compared to primary cells. It is also noteworthy that hypermethylation in Socs-1, Rassf1, Hic-1, and Timp-3 was widespread among the cell lines tested, but not in the primary cells and Clone 9 cells. This study suggests that a cautious approach is required when cell lines are utilized to study methylation-related carcinogenic, metastatic or tumoricidal mechanisms.

Identification of long-term repopulating potential of human cord blood-derived CD34-flt3- severe combined immunodeficiency-repopulating cells by intra-bone marrow injection.

Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003;101:2924). In contrast to murine CD34(-) Kit(+)Sca-1(+)Lineage(-) (KSL) cells, human CB-derived Lin(-)CD34(-) cells did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34(-) SRCs. Both CD34(+)flt3(+/-) cells showed SRC activity. In the CD34(-) cell fraction, only CD34(-)flt3(-) cells showed distinct SRC activity by IBMI. Although CD34(+)flt3(+) cells showed a rather weak secondary repopulating activity, CD34(+)flt3(-) cells repopulated many more secondary recipient mice. However, CD34(-)flt3(-) cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34(-)flt3(-) cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34(+)flt3(+/-) cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34(-)flt3(-) cells produced CD34(+)flt3(-) as well as CD34(+)flt3(+) SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34(-) SRCs, like murine CD34(-) KSL cells, do not express flt3. On the basis of these data, we propose that the immunophenotype of very primitive long-term repopulating human hematopoietic stem cells is Lin(-)CD34(-)c-kit(-)flt3(-). Disclosure of potential conflicts of interest is found at the end of this article.

LINE-1 hypomethylation in a choline-deficiency-induced liver cancer in rats: dependence on feeding period.

Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.

SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection.

Precise analysis of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34(-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin(-)) CD34(-) cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4- mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34(+) progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34(+) cells in vivo. These in vivo-generated CD34(+) cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34(-) SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel class of primitive human CD34(-) SRCs by IBMI will provide a new concept of the hierarchy in the human HSC compartment and has important implications for clinical HSC transplantation as well as for basic research of HSC.