A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020.

INTRODUCTION: The Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo. METHODS: We diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method. RESULTS: As a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative. CONCLUSION: The false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.

Molecular Characteristics of Novel Mono-Reassortant G9P[8] Rotavirus A Strains Possessing the NSP4 Gene of the E2 Genotype Detected in Tokyo, Japan.

Rotavirus A (RVA) has been detected in patients with gastroenteritis even after vaccine introduction in Japan. To investigate circulating RVA strains, RVA-positive stool specimens obtained in Tokyo in 2017 and 2018 were analyzed using next-generation sequencing. A total of 50 and 21 RVA samples were obtained in 2017 and 2018, respectively. In 2017, G2P[4] (40.0%) was the most prevalent strain, followed by G3P[8] (DS-1-like) (28.0%), G8P[8] (10.0%), G3P[8] (Wa-like) (8.0%), G9P[8]-E1 (8.0%), and mixed infection (6.0%). In 2018, G3P[8] (DS-1-like) (28.6%) and G9P[8]-E2 (28.6%) were the most prevalent strains, followed by G9P[8]-E1 (19.0%), G2P[4] (9.5%), G8P[8] (9.5%), and mixed infection (4.8%). Six G9P[8]-E2 strains detected in 2018 showed an atypical genotype constellation (G9P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), which had not been reported previously. Phylogenetic analyses suggested that the RVA virus was generated by inter-genogroup reassortment between commonly circulating G9P[8] and G2P[4] strains in Japan. The G9P[8] strain seemed to be reassorted with only the NSP4 gene of the E2 genotype of the G2P[4] strain. Since this newly-emerged G9P[8]-E2 virus was detected in different locations in Tokyo, the virus appears to have already begun to spread to a wider area.

Characteristics of acute hepatitis A virus infection before and after 2001: A hospital-based study in Tokyo, Japan.

BACKGROUND AND AIM: The incidence of acute hepatitis A [AH (A)] is decreasing because of improvements in hygiene; however, cases of sporadic severe hepatitis are still being reported. We assessed the epidemiology of AH (A) in Japan. METHODS: This was a hospital-based retrospective study, in which 126 AH (A) patients (96 men [76%], median age 39 [range, 19-66] years) were enrolled. Patients diagnosed with AH (A) before 2001 (n = 79) and after 2001 (n = 47) were compared. RESULTS: The incidence of AH (A) showed peaks in 1990, 1999, and 2018. After 2001, one patient had hepatitis B virus, four had human immunodeficiency virus, and three had syphilis coinfections. Before and after 2001, HAV was transmitted, respectively, by raw oysters (28% and 26%), overseas travel (19% and 28%), and sexual contact (0% and 19%) (P < 0.01). The frequencies of symptoms were appetite loss (51% and 32%), fever (63% and 81%), and diarrhea (3% and 13%) (all P < 0.05), respectively. On admission, the median levels of alanine aminotransferase (1455 and 3069 U/L) and γ-glutamyl transpeptidase (221 and 345 U/L) were significantly higher (P < 0.01), and the prothrombin time (77.5% and 65.9%) and platelet count (22.7 and 16.4 × 10/µL) were significantly lower after 2001 (P < 0.05). A time to normalization of the bilirubin level ≥ 30 days was associated with older age and a diagnosis of AH (A) after 2001. CONCLUSIONS: Outbreaks and severe AH (A) cases due to sexual transmission have been reported recently. It is necessary to examine their sexual behavior and other sexual infection.

Whole genomic analysis of human G8P[14] group A rotavirus detected from community gastroenteritis outbreak.

Several suspected cases of zoonotic transmission of group A rotavirus (RVA)-related gastroenteritis were reported previously. In August 2012, G8P[14] RVA was detected in fecal specimens from a community gastroenteritis outbreak occurring during a school trip. In this study, additional analyses were performed and it was found that this strain had the G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 sequence, similar to bovine-like RVA strains. Some contamination by emesis and diarrheic feces was observed near a rest room in the lodging area. Contact history with animals was unknown in members of this school trip, and this case implied that the strain may have acquired the ability for person-to-person transmission.

Comparison between patients with norovirus-related gastroenteritis and asymptomatic carriers with respect to distribution of antibody-complexed viral particles and intestinal flora.

Asymptomatic carriers have a major influence on the spreading of norovirus infections. The objective of this study was to examine the characteristics of patients and asymptomatic carriers affected by norovirus-related community gastroenteritis outbreaks. No significant difference between the two groups was observed in terms of the number of norovirus-antibody complexes with respect to total numbers. Principal coordinates analysis of the intestinal flora based on ß-diversity analysis, revealed a different bacterial composition between patients and asymptomatic carriers, particularly regarding the genera Pseudomonas, Bacteroides, and Erwinia, as well as the Ruminococcaceae family. Although the proportional changes between these intestinal microorganisms were not sufficient to explain gastroenteritis symptoms, they represent possible markers shared by asymptomatic norovirus carriers.

[Molecular Epidemiological Analysis of Hepatitis A Viruses Circulating in Tokyo, 2016-2017].

Hepatitis A virus (HAV) is a common infectious agent that causes acute hepatitis worldwide. Since the incubation period of HAV infection is about one month, it is difficult to identify the source of infection based only on medical interviews. Molecular epidemiological analysis of HAV isolated from patients can help to reveal the infection route and to identify diffuse outbreaks caused by common food vehicles. In the present study, samples were collected from 108 cases notified to the active epidemiological investigation system in Tokyo between 2016 and 2017. Samples found to be HAV-positive by semi-nested RT-PCR were subjected to sequencing and phylogenetic analysis; the results were analyzed together with the epidemiological data. HAV was detected in 99 out of 108 cases. Phylogenetic analysis showed that the 99 HAV strains were divided into 91 of genotype IA, two of IB, and six of IIIA. The 91 HAV strains typed as IA were further divided into four main line-ages, IA-1, IA-2, IA-3, and IA-4, each with a unique epidemiological background. Our nucleotide sequence database of HAV and epidemiological background data will be helpful to investigate sources of infection and the epidemiology of hepatitis A cases in the future.

Detection of Norovirus in Swab Specimens of Restrooms and Kitchens Collected for Investigation of Suspected Food Poisoning Outbreaks in Tokyo.

During 2015-2016, we examined norovirus (NoV) RNA in swab specimens collected for investigation of suspected food poisoning outbreaks in Tokyo by real-time RT-PCR. Of 1,726 swab samples, 65 (3.8%) were NoV-positive and all positive swab samples were derived from NoV-positive outbreaks. Swab specimens were positive in 41 of 181 (22.7%) NoV outbreaks, while no positive swabs were detected in NoV-negative outbreaks. PCR fragments amplified from 32 swabs were sequenced, and all of them displayed complete homology with sequences from clinical and food samples. Though the results of swabs may be useful for determining the causative agent and infection route in some outbreaks, there was no case in which the results of swabs alone could elucidate the cause of food poisoning. Swabs may be useful in food poisoning investigations, if the results are interpreted in conjunction with epidemiological findings and clinical data. Swab samples are often collected several days after an outbreak, and the influence of disinfection should be taken into consideration. In NoV outbreaks, 55 out of 640 (8.6%) restroom swab specimens were NoV-positive whereas six of 618 (1.0%) were positive among kitchen swab specimens. In the restroom, the toilet bowl (43.6%) showed the highest positive rate and next was the toilet seat (14.5%). Additionally, NoV was detected at various sites in the restroom, including doorknob and floor. Since NoV-positive swab specimens may suggest that sanitation management is not performed properly in the facility, swab results may be utilized as a basis for hygiene guidance.

[Serial Food Poisoning Outbreaks Caused by Norovirus-Contaminated Shredded Dried Laver Seaweed Provided at School Lunch, Tokyo, 2017].

In February 2017, four food poisoning outbreaks occurred in Tokyo, involving ten schools. Shredded dried laver seaweed processed by a single food manufacturer in December 2016 was provided in common for the school meals that caused all four outbreaks. Of 4,209 persons exposed, 1,193 (28.3%) had symptoms of gastroenteritis. Norovirus (NoV) GII was detected in 207 (78.1%) of 265 cases by real-time RT-PCR. Thirty-one shredded dried laver seaweed samples were examined and seven (22.6%) of them were positive for NoV GII. PCR fragments of NoV ORF1/2 junction region (302 bp) from seven shredded dried laver seaweed samples and 20 clinical samples derived from the four outbreaks were sequenced. All of them displayed complete homology, and the genotype was classified as GII.17. A nearly full-length sequence (7,420 bp) of NoV RNA derived from a case was obtained by next-generation sequencer analysis and phylogenetic analysis indicated that this strain belongs to the same cluster as Hu/GII/JP/2015/GII.P17_GII.17/Kawasaki308. Thus, our investigation elucidated that the causative agent of these four serial food poisoning outbreaks was NoV GII.17 and the infectious source was a single batch of shredded dried laver seaweed. The water activity of the shredded dried laver seaweed was found to be 0.119 to 0.129. It was epidemiologically clarified that NoV does not lose infectivity for about two months even in the dry state. We conclude that a large diffuse outbreak of food poisoning caused by NoV GII.17 contamination of shredded dried laver seaweed had occurred in Tokyo. Our elucidation of the causative agent indicated that the food poisoning outbreaks in multiple areas of Japan, including Tokyo, during January to February 2017 were caused by the same contaminated food.

Chemerin promotes the proliferation and migration of vascular smooth muscle and increases mouse blood pressure.

Blood chemerin concentration shows positive correlation not only with body mass index and serum triglyceride level but also with systolic blood pressure. While it seems likely that chemerin influences vascular smooth muscle cell (SMC) proliferation and migration, which are crucial to the development of hypertension, this remains to be clarified. In the present study, we investigated whether chemerin controls SMC proliferation and migration in vitro and also affects blood pressure in vivo. In vitro, chemerin significantly stimulated rat mesenteric arterial SMC proliferation and migration, as determined by a cell counting assay and Boyden chamber assay, respectively. The migratory effect of chemerin was confirmed in human aortic SMCs. Chemerin significantly increased ROS production in SMCs and phosphorylation of Akt (Ser(473)) and ERK, as measured by fluorescent staining and Western blot analysis, respectively. Various inhibitors (ROS inhibitor: N-acetyl-l-cysteine, phosphatidylinositol 3-kinase inhibitor: LY-294002, MAPKK inhibitor: PD-98059, NADPH oxidase inhibitor: gp91 ds-tat, and xanthine oxidase inhibitor: allopurinol) as well as chemokine-like receptor 1 small interfering RNA significantly inhibited chemerin-induced SMC proliferation and migration. Furthermore, chemerin-neutralizing antibody prevented carotid neointimal hyperplasia in the mouse ligation model. In vivo, chronic chemerin treatment (6 µg/kg, 6 wk) increased systolic blood pressure as well as phosphorylation of Akt and ERK in the mouse isolated aorta. In summary, we, for the first time, demonstrate that chemerin/chemokine-like receptor 1 stimulates SMC proliferation and migration via ROS-dependent phosphorylation of Akt/ERK, which may lead to vascular structural remodeling and an increase in systolic blood pressure.

HDAC4 mediates development of hypertension via vascular inflammation in spontaneous hypertensive rats.

Histone deacetylases (HDACs) are transcriptional corepressors. Our recent study demonstrated that HDAC4 protein specifically increases in mesenteric artery from spontaneous hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Vascular inflammation is important for pathogenesis of hypertension. We examined whether HDAC4 affects vascular inflammatory responses and promotes hypertension. In vivo, blood pressure, reactive oxygen species (ROS) production, and VCAM-1 expression in isolated mesenteric artery were elevated in young SHR (7 wk old) compared with age-matched WKY, which were prevented by long-term treatment of SHR with an HDACs inhibitor, trichostatin A (TSA; 500 µg·kg(-1)·day(-1) for 3 wk). In isolated mesenteric artery, the increased angiotensin II-induced contraction in SHR was reversed by TSA. The endothelium-dependent relaxation induced by ACh in SHR was augmented by TSA. In cultured rat mesenteric arterial smooth muscle cells (SMCs), expression of HDAC4 mRNA and protein was increased by TNF-α (10 ng/ml). TSA (10 µM, pretreatment for 30 min) inhibited VCAM-1 expression and NF-κB phosphorylation induced by TNF (10 ng/ml, 24 h or 20 min) in SMCs. HDAC4 small interfering RNA inhibited TNF-induced monocyte adhesion, VCAM-1 expression, transcriptional activity of NF-κB, and ROS production in SMCs. The present results demonstrated that proinflammatory effects of HDACs may mediate the further development of hypertension in SHR. It is also suggested in cultured vascular SMCs that TNF-induced HDAC4 mediates vascular inflammation likely via VCAM-1 induction through ROS-dependent NF-κB activation.

A proposed mechanism for amitriptyline neurotoxicity based on its detergent nature.

Although amitriptyline has gained attention as a potent local anesthetic, recent animal studies showed that it can cause irreversible neural impairment. We hypothesized that nerve membrane disruption caused by solubilization, a common detergent property, accounted for amitriptyline neurotoxicity. We used a two-phase approach to test our hypothesis. Firstly, we determined (1) the molecular aggregation concentration of amitriptyline, (2) the concentration of amitriptyline that disrupts artificial lipid membranes and (3) the concentration of amitriptyline that causes hemolysis. Secondly, we compared these levels with neurotoxic concentrations determined from assessment in a rat model of spinal anesthesia using changes in cutaneous stimulus threshold (CST). Amitriptyline concentrations that caused molecular aggregation, model membrane disruption and hemolysis were 0.46%, 0.35% and 0.3%, respectively. Animal study showed a significant increase in CST at >or=0.3% of amitriptyline, indicating neurological impairment. Since amitriptyline caused model membrane disruption and hemolysis at the molecular aggregation concentration, solubilization plays a role in the destruction of artificial membranes and erythrocytes. Furthermore, these concentrations are also in good agreement with the minimum concentration causing neurological injury. Therefore, while additional studies, including histopathology, are necessary to clarify this observation, amitriptyline neurotoxicity appears to be associated with its detergent nature.

Quantitative and fiber-selective evaluation of dose-dependent nerve blockade by intrathecal lidocaine in rats.

We investigated whether cutaneous stimulus threshold (CST), as determined using a Neurometer, could be used for quantitative and differential nerve evaluation of reversible and irreversible nerve block following intrathecal lidocaine administration in rats. Rats with intrathecal catheters were randomly assigned to one of five groups (saline or 2, 5, 10, or 20% lidocaine). Prior to and 4 days after drug administration, CST was determined at 5, 250, and 2000 Hz. In the 2% lidocaine group, CST from end of lidocaine infusion to recovery from anesthesia was also monitored. Skin-clamp testing and gait observation were performed for comparison with CST findings. Behavioral examinations revealed persistent sensory or motor impairment lasting 4 days in groups receiving >/=5% lidocaine but not in the saline and 2% lidocaine groups. With 2% lidocaine, return to baseline CSTs at 5 and 250 Hz was delayed compared with thresholds at 2000 Hz. Although CSTs in the 5% group at 5 and 250 Hz increased significantly, thresholds at 2000 Hz did not differ from those in rats administered saline. CSTs with >/=10% lidocaine displayed no differences between frequencies. At each frequency, CSTs for rats with >/=5% lidocaine increased in a clearly concentration-dependent manner. These results suggest that CST testing enables evaluation of the different nerve functions for Abeta, Adelta, and C fibers in rats for lidocaine concentrations

Proper shoulder position for subclavian venipuncture: a prospective randomized clinical trial and anatomical perspectives using multislice computed tomography.

BACKGROUND: Although the Trendelenburg position and shoulder bracing are recommended for safe subclavian venipuncture, the optimal shoulder position remains unclear. The current study observed spatial relations between the subclavian vein and surrounding structures using multislice computed tomography to determine optimal shoulder position for safe subclavian venipuncture and then conducted a small follow-up clinical trial to confirm these findings. METHODS: Thoracic multislice computed tomography was performed for seven adult volunteers at three shoulder positions: elevated (up); neutral; and lowered caudally (down). Overlap and distance between the clavicle and the subclavian vein and the diameter of the subclavian vein were measured. Anatomical relations between the subclavian artery and vein were also observed. The success rate for subclavian venipuncture was then compared between the up and down shoulder positions in 30 patients. RESULTS: In the multislice computed tomography study, the mean overlap ratios between clavicle and subclavian vein in the up, neutral, and down positions were 33.5, 36.9, and 40.0%, respectively. Overlap increased with lower shoulder position (up < neutral < down; P < 0.05). The mean distances between the clavicle and the subclavian vein in the up, neutral, and down positions were 6.8, 5.0, and 3.6 mm, respectively. Again, distance decreased with lower shoulder position (up < neutral < down; P < 0.05). The diameter of the subclavian vein did not differ among the three shoulder positions. The success rate for subclavian venipuncture was significantly higher in the down position compared with the up position (P = 0.003). CONCLUSIONS: Lowered shoulder position increases both overlap and proximity between the clavicle and the subclavian vein, producing a more constant relation between the clavicle and the subclavian vein, without affecting vein diameter. Proper use of a lowered shoulder position should thus increase the safety and reliability of subclavian venipuncture compared with other shoulder positions.

Neurological evaluation by intraoperative wake-up during carotid endarterectomy under general anesthesia.

Although general anesthesia allows relief from stressors such as pain, discomfort, or anxiety for patients undergoing carotid endarterectomy, neurologic assessment is less reliable than under local anesthesia. We describe a unique anesthetic management strategy for carotid endarterectomy patients incorporating the advantages of both general and local anesthesia. The technique allows thorough assessment of neurologic function during carotid cross-clamping by intraoperative wake-up, and guarantees airway management by tracheal intubation.

Possible mechanism of irreversible nerve injury caused by local anesthetics: detergent properties of local anesthetics and membrane disruption.

BACKGROUND: Irreversible nerve injury may result from neural membrane lysis due to the detergent properties of local anesthetics. This study aimed to investigate whether local anesthetics display the same properties as detergents and whether they disrupt the model membrane at high concentrations. METHODS: Concentrations at which dodecyltrimethylammonium chloride and four local anesthetic (dibucaine, tetracaine, lidocaine, and procaine) molecules exhibit self-aggregation in aqueous solutions were measured using an anesthetic cation-sensitive electrode. Light-scattering measurements in a model membrane solution were also performed at increasing drug concentrations. The concentration at which drugs caused membrane disruption was determined as the point at which scattering intensity decreased. Osmotic pressures of anesthetic agents at these concentrations were also determined. RESULTS: Concentrations of dodecyltrimethylammonium chloride, dibucaine, tetracaine, lidocaine, and procaine at which aggregation occurred were 0.15, 0.6, 1.1, 5.3, and 7.6%, respectively. Drug concentrations causing membrane disruption were 0.09% (dodecyltrimethylammonium chloride), 0.5% (dibucaine), 1.0% (tetracaine), 5.0% (lidocaine), 10.2% (procaine), and 20% (glucose), and osmotic pressures at these concentrations were 278, 293, 329, 581, 728, and 1,868 mOsm/kg H2O, respectively. CONCLUSIONS: These results show that all four local anesthetics form molecular aggregations in the same manner as dodecyltrimethylammonium chloride, a common surfactant. At osmotic pressures insufficient to affect the membrane, local anesthetics caused membrane disruption at the same concentrations at which molecular aggregation occurred. This shows that disruption of the model membrane results from the detergent nature of local anesthetics. Nerve membrane solubilization by highly concentrated local anesthetics may cause irreversible neural injury.

Lidocaine spray used to capture a live Clinostomum parasite causing human laryngitis.

This case report describes a patient who developed Clinostomum laryngitis after eating raw fresh-water fish. Parasite removal was performed under general anesthesia using a laryngomicroscopic method. Because it was difficult to capture the worm intact using forceps, it was sprayed with 8% lidocaine solution. This immediately inhibited peristaltic movement of the parasite allowing easy retrieval without tearing any part of the organism, thus facilitating parasite identification.