Effective inactivation of Nipah virus in serum samples for safe processing in low-containment laboratories.

BACKGROUND: Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe encephalitis and respiratory disease with a high mortality rate in humans. During large outbreaks of the viral disease, serological testing of serum samples could be a useful diagnostic tool, which could provide information on not only the diagnosis of NiV disease but also the history of an individual with previous exposure to the virus, thereby supporting disease control. Therefore, an efficient method for the inactivation of NiV in serum samples is required for serological diagnosis. METHODS: We determined the optimal conditions for the inactivation of NiV infectivity in human serum using heating and UV treatment. The inactivation method comprised UV irradiation with a cover of aluminum foil for 30 min and heating at 56 °C for 30 min. RESULTS: With an optimized protocol for virus inactivation, NiV infectivity in serum samples (containing 6.0 × 105 TCID50) was completely inactivated. CONCLUSIONS: We developed a recommended protocol for the effective inactivation of NiV. This protocol would enable a regional or local laboratory to safely transport or process samples, including NiV, for serological testing in its biosafety level-2 facility.

[Clinical Features and Treatment Strategy of Vertebral Artery Injury Associated with Cervical Spine Trauma].

OBJECTIVE: Vertebral artery injury(VAI)associated with cervical spine trauma has the potential to cause catastrophic vertebrobasilar stroke. However, there are no well-defined treatment recommendations for VAI. The purpose of this study was to identify an effective treatment strategy for VAI following cervical spine trauma. METHODS: Ninety-seven patients with blunt cervical spine trauma were treated at Hyogo Prefectural Kakogawa Medical Center between January 2013 and September 2017. Of these patients, 49 underwent computed tomographic angiography or magnetic resonance angiography for evaluation of the vertebral artery. Eighteen patients(36.7%)had a diagnosis of VAI. We retrospectively analyzed the clinical features, treatment, and outcomes in these 18 patients. RESULTS: Seven patients(38.9%)had bilateral VAI, 16(88.9%)had cervical dislocation, and 2(11.1%)had transverse process fractures extending into the transverse foramen. Surgical reduction was performed in 14 patients. Five patients with either bilateral or unilateral occlusion underwent parent artery occlusion before reduction. There were no complications after this procedure. Two patients with bilateral VAI had a stroke before treatment. There were no infarctions in the distribution of the vertebrobasilar artery after intervention. The perioperative stroke rate was relatively good, and almost all Glasgow Outcome Scale scores were related to the degree of spinal cord injury. CONCLUSIONS: Aggressive screening for VAI is important in patients with cervical spine trauma in order to ensure adequate treatment. Although the treatment strategy described here could yield good results, it may require modification according to the needs of the individual patient.

Inactivation of SARS-CoV-2 in serum using physical methods.

Since 2019, many studies on COVID-19, which has caused extensive damage as a pandemic, have been ongoing worldwide. These include serological and biochemical studies using sera from patients and animal models. Testing with these sera must be performed after the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Heat treatment, UV irradiation, and/or gamma-ray irradiation are used to inactivate viruses in serum. Determining the inactivation conditions that ensure the inactivation of viruses and minimize the effect on test results after inactivation is important to ensure worker safety and accuracy of test results. In this study, serum samples containing SARS-CoV-2 were subjected to heat, UV irradiation, and gamma irradiation to determine their inactivation conditions. The viral titers were below the detection limit after heating at 56°C for 1 h or 60°C for 15 min, UV-B irradiation with a transilluminator for 30 min, or gamma ray irradiation with 60Co at 10 kGy. These results provide useful information for safe serological and biochemical experiments.

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice.

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.

Phagocytic entry of Legionella pneumophila into macrophages through phosphatidylinositol 3,4,5-trisphosphate-independent pathway.

Legionella pneumophila, a causative agent of Legionnaire's disease, is an intracellular pathogen. It intervenes in the signal transduction of macrophages by secreting effector molecules through the Icm/Dot type IV secretion system (T4SS). There is a connection between signaling cascades that regulate phagocytosis and the production of reactive oxygen species (ROS). Class I phosphatidylinositol 3-kinase (PI3-K) and its product phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) play key roles in the reorganization of cytoskeleton (phagocytosis) and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (ROS production). We investigated the production of PI(3,4,5)P3 and recruitment of class I PI3-K and Rac1 during phagocytosis of L. pneumophila by macrophages. Transient recruitment of class I PI3-K as well as PI(3,4,5)P3 production was observed around a phagocytosed T4SS mutant LELA3118 or avirulent mutant 25D in an early stage of infection. In contrast, class I PI3-K was recruited while accumulation of PI(3,4,5)P3 was not observed around wild type JR32. Immunoglobulin G (IgG)-opsonized live JR32, which would activate class I PI3-K through the Fcγ receptor pathway, did not induce PI(3,4,5)P3 production. Regardless of whether wild type or mutants were used, transient Rac1 accumulation was observed around bacteria. These results indicate that the phagocytosis of wild type L. pneumophila occurs via a special mechanism in which PI(3,4,5)P3 production is absent. This suggests that L. pneumophila may inhibit the production of PI(3,4,5)P3, but not the recruitment of class I PI3-K and Rac1, in a T4SS-dependent manner. L. pneumophila may start the modulation of host signaling cascade immediately after contact with host cells to evade the ROS-dependent bactericidal system while completing entry into macrophages.

Practical removal of radioactivity from sediment mud in a swimming pool in Fukushima, Japan by immobilized photosynthetic bacteria.

About 90% of the radioactive Cs in the sediment mud of a school's swimming pool in Fukushima, Japan was removed by treatment for 3 d using the alginate immobilized photosynthetic bacterium Rhodobcater sphaeroides SSI. Even though batch treatment was carried out 3 times repeatedly, the activity of immobilized cells in removing Cs was maintained at levels of about 84% (second batch) and 78% (third batch). Cs was strongly attached to the sediment mud because, even with HNO(3) treatment at pH of 2.00-1.60 for 24 h, it was not eluted into the water. Furthermore, more than 75% of the Cs could be removed without solubilization with HNO(3). This suggests that the Cs attached to the sediment mud was transformed into immobilized cells via the Cs(+) ion by the negative charge of the immobilized cell surface and/or the potassium transport system of the photosynthetic bacterium.

Practical removal of radioactivity from soil in Fukushima using immobilized photosynthetic bacteria combined with anaerobic digestion and lactic acid fermentation as pre-treatment.

Practical removal of radioactivity from polluted soil in Fukushima, Japan was done using a photosynthetic bacterium, Rhodobacter sphaeroides SSI, immobilized in alginate beads. The beads were put in a mesh bag and soaked in which soil was suspended (5 kg of soil/10 L of tap water). The radioactivity of the broth decreased by 31% after 15 d of aerobic treatment. When lactic acid bacterial culture broth was added to the suspend broth, about 50% of the radioactivity was transferred to a suspend broth fraction consisting of small particles from the soil after 3 d of fermentation and 20 s of sedimentation. The results suggest that organic matter in the soil was decomposed by anaerobic digestion and lactic acid fermentation simultaneously, and was then transferred into the liquid as small particles. With combined treatment by anaerobic digestion and lactic acid fermentation for 5 d and immobilized bead aerobic treatment for an additional 19 d, the radioactivity of suspend broth decreased by 66%. The radioactivity of the original soil (10.56 µSv/h) ultimately decreased by 67% (3.52 µSv/h) after the combined treatment.

Continuous Local Antibiotics Perfusion Therapy for Acute Deep Infections after Open Fractures.

Unresolved bone and soft tissue infections remain a great hindrance to fracture management worldwide, both economically and functionally for the patient. For this purpose, the benefits of local antibiotic administration besides systemic therapy have been elucidated. We present a retrospective descriptive analysis of six patients (4 males and 2 females) with acute deep infections after open fractures managed using the continuous local antibiotic perfusion (CLAP) therapy. After sufficient debridement, gentamicin solution concentrated at 1,200 µg/mL was continuously infused (2 mL/h) for 7-12 days by syringe pump through an inlet tube placed on the infected area. The antibiotics injected into the infected area were both collected and perfused by negative pressure using a negative-pressure wound therapy system. After an average of 9.5 days of CLAP therapy, symptoms of infection disappeared, and the bacterial culture was negative. There were no cases of recurrence during the follow-up period, and no complications, such as acute renal failure, ototoxicity, allergic or hypersensitivity reactions, and impaired fracture healing, were observed. All six cases were successfully managed with the CLAP therapy without any serious side effects. CLAP therapy may be a potential treatment option for acute deep infections after open fractures.

Ampicillin/sulbactam versus cefazolin plus aminoglycosides for antimicrobial prophylaxis in management of Gustilo type IIIA open fractures: A retrospective cohort study.

BACKGROUND: The antibiotic regimens for prophylaxis in the management of open fractures remain controversial. Although the use of aminoglycosides is widely accepted for treatment of Gustilo type III open fractures, aminoglycosides are often avoided in patients with risk factors. This study aimed to compare efficacy and safety of two regimens, cephazolin plus aminoglycoside (amikacin or gentamicin) and ampicillin/sulbactam (ABPC/SBT), in patients with Gustilo type IIIA open fractures. METHODS: A total of 95 Gustilo type IIIA fractures in 90 patients were retrospectively reviewed in this study. The cohort was categorized into two groups that were treated in accordance with the institutional prescribed regimen in different periods: (1) cefazolin plus aminoglycoside (January 1, 2014-September 30, 2017) and (2) ABPC/SBT monotherapy (October 1, 2017-September 30, 2020). Cefazolin was used at 1-2 g every 8 h, aminoglycoside (amikacin or gentamicin) was used daily depending on body weight, and ABPC/SBT was used at 3 g every 8 h The antibiotic administration was continued within 3 days or until successful soft tissue coverage was achieved. The infection rate and the incidence of acute kidney injury (AKI) in both groups were assessed. RESULTS: ABPC/SBT was used in 34 patients (36 fractures), and 56 patients (59 fractures) received cefazolin plus aminoglycoside for antibiotic prophylaxis. Infection developed in 2 of 36 fractures in ABPC/SBT group and 4 of 59 fractures in the cefazolin plus aminoglycoside group (p > 0.99). The average serum creatinine levels on admission, baseline, and peak during the hospital stay were not significantly different between the two groups. One case of AKI was identified in each group, indicating that incidence rate of AKI was not significantly different between the two groups. CONCLUSION: We demonstrated the non-inferiority of ABPC/SBT therapy over cefazolin plus aminoglycoside regimen for type IIIA open fractures. The ABPC/SBT regimen may be an alternative option for managing Gustilo type IIIA open fractures. Further prospective studies with larger samples are needed to verify these results.

Potential risk of SARS-CoV-2 infection among people handling linens used by COVID-19 patients before and after washing.

The risk of SARS-CoV-2 infection when people handle linens is uncertain. We examined the presence of SARS-CoV-2 on linens, in the air, and on personal protective equipment (PPE) to assess potential infection risk among individuals who handle linens used by SARS-CoV-2-infected people. Patients in a hospital and an accommodation facility who tested positive for SARS-CoV-2 participated in this study in 2020. Linen samples before washing or disinfection, rinse water after washing or disinfection, air in the workplace at the hospital and an accommodation facility, and the PPE worn by linen-handling people were tested for SARS-CoV-2 RNA and viable viruses. Among 700 samples from 13 SARS-CoV-2-infected participants and their surrounding environment, SARS-CoV-2 RNA was detected from 14% (52/362) of the linens used by COVID-19 patients (cycle threshold [Ct] value: 33-40). SARS-CoV-2 RNA was detected from 8% (2/26) of rinse water after washing or disinfection, from 15% (16/104) of air samples in the workspace, and from 10% (5/52) of gowns worn by linen-handling people, all with high Ct values (> 36). No SARS-CoV-2 was isolated from any samples. The potential risk of SARS-CoV-2 infection from handling linens used by SARS-CoV-2-infected people exists but appears to below.

Avulsion fracture of the posterior oblique ligament associated with acute tear of the medial collateral ligament.

The term posterior oblique ligament was proposed in 1973 but the specific anatomy of this region has been controversial. It has been concluded that the posterior oblique ligament is separated from the medial collateral ligament, each with distinctly different bony attachment points. In this article, we report a posterior oblique ligament avulsion fracture associated with a medial collateral ligament rupture.

Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat.

Campylobacter jejuni is the major cause of human gastroenteritis worldwide. Under stress conditions, C. jejuni can enter a viable but non-culturable (VBNC) state. We found that the C. jejuni was able to enter a VBNC state by prolonged incubation at 4°C. The standard isolation methods using pre-enrichment steps in Bolton broth or Preston broth could not detect the VBNC cells in spiked chicken meat. The transcription levels of virulence-associated genes (flaA, flaB, cadF, ciaB, cdtA, cdtB and cdtC) were expressed in VBNC cells but in low levels. The VBNC cells retained the ability to invade Caco-2 human intestinal epithelial cells in vitro. In most cases, VBNC cells failed to resuscitate in Caco-2 cells, but in some experiments, they formed colonies after co-incubation with host cells. Collectively, C. jejuni enters into a VBNC state at 4°C and the VBNC C. jejuni remains virulent which may possibly lead to disease in humans. C. jejuni in VBNC state is a potential concern for food safety.

Elevated production of Legionella-specific immunoglobulin A in A/J mice is accompanied by T-helper 1-type polarization.

Legionella pneumophila (Lpn) is a Gram-negative bacterium and an intracellular parasite that causes Legionnaires' disease. Secretion of immunoglobulin A (IgA) against Lpn on the mucosal surface of the upper respiratory tract may be important as a self-defense mechanism. A/J mice have been demonstrated to be permissive as to Lpn replication in macrophages due to a natural mutation in neuronal apoptosis inhibitory protein 5. We compared A/J and BALB/c mice as to IgA production after repeated intranasal immunization using a heat-killed Lpn in the presence of cholera toxin as a mucosal adjuvant. A/J mice secreted more Lpn-specific IgA in nasal washes than BALB/c mice. The Lpn-specific serum IgA level was also higher in A/J than BALB/c mice. Because both BALB/c and A/J mice are known to exhibit T-helper 2 (Th2)-biased immune responses, we examined whether the Lpn-specific IgA production is related to the stronger Th2 bias. There was no difference in IgG1 (Th2-controlled) while A/J mice produced more IgG2a (Th1-controlled), suggesting that the elevated IgA response was rather correlated with Th1-controlled isotype switching. Our results also suggest that A/J mice will be useful hosts for Lpn-specific IgA production such as for the preparation of IgA monoclonal antibodies.

Evasion of Legionella pneumophila from the bactericidal system by reactive oxygen species (ROS) in macrophages.

We demonstrated that the production of reactive oxygen species (ROS) by U937 macrophage-like cells was suppressed upon infection with a wild type Legionella pneumophila strain, whereas such suppression was not observed in the case of infection with intracellular growth-deficient mutants. This was supported not only by measuring ROS released into the supernatants of cell cultures by chemiluminescence assaying but also by detecting intracellular ROS with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), under a confocal laser scanning microscope. Furthermore, more than 60% of the phagosomes containing intracellular growth-deficient mutants were colocalized with p47(phox), which is the cytosolic subunit of NADPH oxidase, consistently throughout the observation period in an early stage of bacterial infection. In contrast, the colocalization of p47(phox) was suppressed after infection with the wild type strain. These results suggest that the interference with ROS production by U937 cells infected with wild type L. pneumophila is due to a failure of NADPH oxidase activation through inhibition of p47(phox) recruitment to phagosomes harboring bacteria. The results also highlighted the difference in the nature of phagosomes between ones harboring the wild type and ones the intracellular growth-deficient strains.

Evaluation of impairment of hand function in patients with cervical myelopathy.

Clumsiness of the hands is one of the most common complaints in patients with cervical myelopathy. However, few reports have been published that evaluate hand dysfunction. One hundred and twenty-nine patients (36 women and 93 men) with cervical myelopathy were examined and results compared with those of age- and sex-matched controls. Hand function was evaluated as scores of 10 subtests designed on the basis of various hand activities. Patients with cervical myelopathy obtained significantly lower scores on the hand function test in each age-group than controls. There was good correlation between hand function scores and the extent of cervical myelopathy. Therefore, the hand function test is a useful battery to objectively and quantitatively evaluate impairment of cervical myelopathy, although longitudinal studies are needed to determine its practical usability.

The influence of proprioceptive impairment on hand function in patients with cervical myelopathy.

STUDY DESIGN: A prospective clinical study. OBJECTIVES: To quantitatively evaluate hand function in patients with cervical myelopathy and to assess the influence of proprioceptive impairment on hand function in patients with cervical myelopathy. SUMMARY OF BACKGROUND DATA: Clumsiness of the hands is one of the most common complaints in patients with cervical myelopathy. However, few reports have been published that evaluate hand dysfunction, and no reports have assessed the association between hand function and proprioceptive impairment. PATIENTS AND METHODS: A total of 129 patients (36 women and 93 men) with cervical myelopathy were examined. Hand function was evaluated as scores of ten subtests designed on the basis of various hand activities. The degree of proprioceptive impairment of the hand as well as that of light touch sensation loss is classified into four grades. Scores of the hand function test are compared according to the grade of proprioceptive impairment of the hands. RESULTS: Significant relation between the impairment of hand function and the extent of cervical myelopathy was observed. There was good correlation between the hand function scores and the grade of impairment of proprioception and light touch sensation. Furthermore, patients with impairment of proprioception obtained significantly lower scores in the hand function test than those with loss of light touch sensation. CONCLUSIONS: The hand function test is a useful battery to objectively and quantitatively evaluate impairment of cervical myelopathy, although longitudinal studies are needed to determine its practical usability. There was some relation between scores of hand function test and the extent of proprioceptive impairment. Thus, hand function test may be partly influenced by proprioceptive loss.

Lumbar facet joint infection associated with epidural and paraspinal abscess: a case report with review of the literature.

A case of lumbar facet joint infection associated with epidural and paraspinal abscess is presented. A 57-year-old man was hospitalized with severe low back pain that radiated into the bilateral buttocks and legs. The patient had a temperature of 37.3 degrees C, and the biochemical inflammatory parameters were elevated. Initially on admission, we administered antibiotics intravenously. However, pain increased, fever elevated gradually, and bladder-bowel disturbance appeared. Magnetic resonance imaging (MRI) showed effusion in the widened facet joint, paraspinal muscles, and epidural space, which compressed the theca severely. We performed an immediate surgical debridement combined with thorough antibiotic therapy and achieved excellent results. MRI was useful for precise diagnosis and operative planning.