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.

Biomechanical Study of the Digital Flexor Tendon Sliding Lengthening Technique.

PURPOSE: To compare the mechanical properties of sliding lengthening (SL) and Z-lengthening (ZL) for flexor tendon elongation used for conditions such as Volkmann contracture, cerebral palsy, and poststroke spasticity. METHODS: We harvested 56 flexor tendons, including flexor pollicis longus tendons, flexor digitorum superficialis tendons (zones II to IV), and flexor digitorum profundus tendons (zones II to V) from 24 upper limbs of 12 fresh cadavers. Each tendon was harvested together with its homonymous tendon from the opposite side of the cadaver and paired. We used 28 pairs of tendons and divided them randomly into 4 groups depending on the lengthening distance (20 or 30 mm) and type of stitching (single or double mattress sutures). Then we divided each pair into either the SL or ZL group. Each group was composed of 7 specimens. The same surgeon lengthened all tendons and stitched them with 2-0 polyester sutures. We tested biomechanical tensile strength immediately after completing lengthening and suturing in each group. RESULTS: Ultimate tensile strengths were: 23 N for the SL 20-mm lengthening and single mattress suture and 7 N for the ZL; 25 N for the SL 20-mm lengthening and double mattress suture and 10 N for the ZL; 15 N for the SL 30-mm lengthening and single mattress suture and 8 N for the ZL; and 18 N for the SL 30-mm lengthening and double mattress suture and 10 N for the ZL. CONCLUSIONS: The SL technique may be a good alternative to the ZL technique because it provides higher ultimate tensile strength. CLINICAL RELEVANCE: Because of its higher ultimate tensile strength, the SL technique may allow for earlier rehabilitation and reduced risk of postoperative complications.

A case of middle-aged central sleep apnea due to Joubert syndrome with different treatment effects of oxygen and acetazolamide.

We report a case of severe central sleep apnea incidentally diagnosed during polysomnography for suspected obstructive sleep apnea. Characteristic clinical features included episodic hyperventilation followed by apnea from hypocapnia, which did not follow a Cheyne-Stokes pattern. Combined with the identification of cerebellar and brainstem malformations known as the "molar tooth sign" on a brain MRI, developmental delay, and motor coordination problems, Joubert syndrome (a congenital disease) was first diagnosed at the age of 50 years. Central apneas were also observed during wakefulness, although not continuously. During sleep, continuous positive airway pressure and adaptive servo-ventilation were ineffective at the referring clinic and at our hospital. Supplemental oxygen decreased the frequency of central apneas and significantly shortened the duration of each central sleep apnea compared with room air. In contrast, the opposite response was observed with acetazolamide administration.

[Case of Heerfordt's syndrome with prolonged peripheral nerve involvement].

A 28-year-old man complaining of myiodesopsia was given a diagnosis of uveitis. Subsequently he complained facial nerve palsy and enlargement of parotid gland. Heerfordt's syndrome was diagnosed based on the results of several examinations. Facial nerve palsy, enlargement of the parotid gland and uveitis were improved by systemic corticosteroid therapy. At present he is receiving systemic corticosteroid therapy, but numbness in the mouth, thought to be the involvement of the trigeminal nerve, remains. Systemic corticosteroid therapy is usually effective for most cases with Heerford's syndrome. On the other hand, there are some cases with the prolonged peripheral nerve involvement despite systemic corticosteroid therapy, as seen in this case. If peripheral nerve involvement is prolonged, it is necessary to consider small-fibre neuropathy as one possible cause.

Longitudinal evaluation of local muscle conditions in a rat model of gastrocnemius muscle injury using an in vivo imaging system.

This study aimed to evaluate the time course of local changes during the acute phase of gastrocnemius muscle strain, in a rat model, using an in vivo imaging system. Thirty-eight, 8-week-old Sprague-Dawley male rats were used in our study. Experimental injury of the right gastrocnemius muscle was achieved using the drop-mass method. After inducing muscle injury, a liposomally formulated indocyanine green derivative (LP-iDOPE, 7 mg/kg) was injected intraperitoneally. We evaluated the muscle injuries using in vivo imaging, histological examinations, and enzyme-linked immunosorbent assays. The fluorescence peaked approximately 18 h after the injury, and decreased thereafter. Histological examinations revealed that repair of the injured tissue occurred between 18 and 24 h after injury. Quantitative analyses for various cytokines demonstrated significant elevations of interleukin-6 and tumor necrosis factor-α at 3 and 18 h post-injury, respectively. The time course of fluorescence intensity, measured using in vivo imaging, demonstrated that the changes in cytokine levels and histopathologic characteristics were consistent. Specifically, these changes reached peaked 18 h post-injury, followed by trends toward recovery.

Anomalous vertebral arteries in the extra- and intraosseous regions of the craniovertebral junction visualized by 3-dimensional computed tomographic angiography: analysis of 100 consecutive surgical cases and review of the literature.

STUDY DESIGN: Consecutive case series and literature review. OBJECTIVE: To describe the utility of 3-dimensional computed tomographic angiography (3D CTA) for evaluating vertebral artery (VA) anomalies before surgery. SUMMARY OF BACKGROUND DATA: Recent advances in instrumentation surgery at the craniovertebral junction (CVJ) enable us to perform rigid internal fixation. However, the risk of VA injury as a complication of the surgery has become a major problem. Thus, the importance of preoperative evaluation of the VA course has been emphasized. METHODS: Cases of 100 consecutive patients who underwent CVJ instrumentation surgery since July 1998 were analyzed. Occipitocervical/thoracic or C1-C2 posterior fusion was performed for atlantoaxial subluxation (AAS) in 59 patients and cervical fixation including C2 was required for middle-to-lower cervical lesions in 41 patients. Twenty-seven patients with AAS had a congenital skeletal anomaly (CSA) at the CVJ including os odontoideum and occipitalization of C1 (AAS-CSA[+] group). Anomalous VAs at the extra- and intraosseous regions were evaluated by 3D CTA. RESULTS: No neurovascular injury occurred during surgery. Abnormal courses of the VA at the extraosseous region were detected in 10 cases: 2 had fenestration and 8 had a persistent first intersegmental artery. All 10 cases were in the AAS-CSA(+) group. A high-riding VA was detected in 31 cases. Fourteen out of the 31 cases were in the AAS-CSA(+) group, indicating 51.9% of the AAS-CSA(+) group had high-riding VA. In the AAS-CSA(+) group, a C1-C2 transarticular screw and C2 pedicle screw were actually inserted in 58% and 31% of the planned insertions, respectively. CONCLUSION: The present findings suggest that the frequency of an abnormal VA at the extra- and intraosseous regions is increased when patients have AAS and CSA at the CVJ. Using preoperative 3D CTA, we can precisely identify anomalous VAs and thereby reduce the risk of their intraoperative injury.

Platelet-rich plasma combined with hydroxyapatite for lumbar interbody fusion promoted bone formation and decreased an inflammatory pain neuropeptide in rats.

STUDY DESIGN: A prospective interventional trial, using a rat model of lumbar interbody fusion. OBJECTIVE: To examine the potential efficacy of platelet-rich plasma (PRP) for lumbar interbody fusion, using hydroxyapatite (HA). SUMMARY OF BACKGROUND DATA: PRP is an autologous product containing a high concentration of platelets in a small volume of plasma and has osteoinductive effects. HA has osteoconductive ability and has been used in combination with autogenous bone for spine fusion. However, reports using PRP with HA for spine fusion are very few. The purpose of this study was to examine the efficacy of PRP with HA for spinal interbody fusion and at the same time to estimate the change in immunoreactivity of the inflammatory neuropeptide, calcitonin gene-related peptide (CGRP), in dorsal root ganglion (DRG) neurons innervating spinal discs. METHODS: A total of 35 Sprague-Dawley rats were used in this study. Twenty-one rats were used for conducting interbody fusion experiments, 7 rats were used as immunostaining controls, and 7 other rats were used as blood donors for making PRP. L5-L6 interbody fusion was performed on 21 rats using HA + PRP (n = 7), HA + platelet-poor plasma (n = 7), or HA + saline (n = 7). Simultaneously, Fluoro-Gold neurotracer was applied to the intervertebral space to detect DRG neurons innervating the discs. L5-L6 lumbar radiographs were obtained and lumbar DRGs were immunostained for CGRP. The rate of bone union and the change in CGRP immunoreactive DRG neurons innervating the discs were evaluated and compared among groups. RESULTS: All L5-L6 lumbar discs were fused in the PRP + HA group (fused 7/total 7), whereas only 1 case was fused in the platelet-poor plasma group (1 of 7) and no cases in the HA-only group (0 of 7), which was a significant difference. Upon immunohistochemical analysis, CGRP-positive neurons innervated L5-L6 intervertebral discs in nonunion cases, and these were significantly increased compared with those in union cases. CONCLUSION: Our study suggests that using PRP with HA was beneficial for spine fusion. This combination may promote bone union and also decrease inflammatory neuropeptide in sensory neurons innervating the discs.

Assessment of gait in a rat model of myofascial inflammation using the CatWalk system.

STUDY DESIGN: Gait analysis and immunohistological analysis in a rat model of myofascial inflammation in low back. OBJECTIVE: To investigate gait in a rat model of myofascial inflammation using the CatWalk gait analysis system. SUMMARY OF BACKGROUND DATA: There are few reports examining low back pain behavior in animal models. The CatWalk is a computer-assisted gait analysis system that provides an automated way to assess gait function and this behavior during pain. METHODS: In a myofascial inflammation group, 0.5 mL of 4% paraformaldehyde buffer and 0.5 mL of 5% Fluoro-Gold (FG) buffer were injected into bilateral multifidus muscles of rats. In a control group, FG buffer alone was injected. Five days after surgery, the gait of rats in both groups was investigated using the CatWalk system. In the present study a total of 36 gait parameters were quantified and used to judge pain-related behavior. Bilateral dorsal root ganglia (DRGs) from L1 to L6 levels were resected, and immunostained for calcitonin gene-related peptide (CGRP). RESULTS: In the myofascial inflammation group, the mean duty cycle (duration of paw contact divided by time between consecutive paw contacts) of each paws (front and hind) were significantly higher and mean stride length (the distance between successive placements of the same paw) of each paws were significantly shorter compared with the control group. Furthermore, mean minimum contact intensity of the complete paw and mean contact intensity of each paws in the myofascial inflammation group were significantly higher compared with the control group. The proportion of CGRP-immunoreactive FG-labeled neurons among all FG-labeled DRG neurons in the myofascial inflammation group was significantly higher than the proportion in the control group. CONCLUSION: These results suggest that myofascial inflammation in low back caused the changes to the rat's gait, including long stands, short stride, and strong paw contact.

Biomechanical study of human cadaveric lumbar spine reinforced by newly developed hydroxyapatite bone cement.

The compression strength of the lumbar spine reinforced by newly developed hydroxyapatite (HA) bone cement was evaluated using a mechanical testing machine. Sixteen cadaveric lumbar vertebrae obtained from nine subjects (five men, four women) were used. The specimens were randomly divided into two groups. In group A ( n = 8), HA bone cement was injected into the vertebral body through curetted pedicles using specially designed needles and then pushed into the vertebral body by the surgeon's finger, simulating open surgery. In group B ( n = 8) the cement was injected using 16-gauge Ostycut biopsy needles via the pedicles through both sides, simulating percutaneous injection. The initial ultimate compression strength of the specimens was 28.6 +/- 13.4 MPa in group A and 25.2 +/- 12.6 MPa in group B. The value after reinforcement was 35.6 +/- 12.9 MPa in group A and 30.4 +/- 14.8 MPa in group B. There was no significant difference between the ultimate strength of the intact specimen and that after reinforcement. The present study demonstrated biomechanical characteristics of vertebral body fractures reinforced with newly developed HA bone cement.