ST-246 Inhibits In Vivo Poxvirus Dissemination,Virus Shedding,and Systemic Disease Manifestation

Orthopoxvirus infections, such as smallpox, can lead to severe systemic disease and result in considerable morbidity and mortality in immunologically naïve individuals. Treatment with ST-246, a small-molecule inhibitor of virus egress, has been shown to provide protection against severe disease and death induced by several members of the poxvirus family, including vaccinia, variola, and monkeypox viruses. Here, we show that ST-246 treatment not only results in the significant inhibition of vaccinia virus dissemination from the site of inoculation to distal organs, such as the spleen and liver, but also reduces the viral load in organs targeted by the dissemination. In mice intranasally infected with vaccinia virus, virus shedding from the nasal and lung mucosa was significantly lower (∼22- and 528-fold, respectively) upon ST-246 treatment. Consequently, virus dissemination from the nasal site of replication to the lung also was dramatically reduced, as evidenced by a 179-fold difference in virus levels in nasal versus bronchoalveolar lavage. Furthermore, in ACAM2000-immunized mice, vaccination site swabs showed that ST-246 treatment results in a major (∼3,900-fold by day 21) reduction in virus detected at the outside surfaces of lesions. Taken together, these data suggest that ST-246 would play a dual protective role if used during a smallpox bioterrorist attack. First, ST-246 would provide therapeutic benefit by reducing the disease burden and lethality in infected individuals. Second, by reducing virus shedding from those prophylactically immunized with a smallpox vaccine or harboring variola virus infection, ST-246 could reduce the risk of virus transmission to susceptible contacts.Smallpox disease is marked by the dissemination of variola virus within the infected host and the appearance of skin lesions or pocks (reviewed in reference 3 and at http://whqlibdoc.who.int/smallpox/9241561106.pdf). The virus is transmitted between humans mainly by aerosol droplets; however, contact with variola virus-contaminated clothing or bedding may contribute to transmission. Once inhaled, variola virus appears to first infect the upper- or lower-respiratory-tract mucosa and spread to and replicate within the local lymph nodes. The virus then disseminates to the spleen and liver via a transient viremia and replicates in reticuloendothelial cells through an average incubation period of 10 to 12 days. During the high-fever prodrome period that follows, a second wave of viremia occurs and results in the dissemination of the virus to mucous membranes of the mouth and pharynx and to the dermal epithelium of the skin. This dissemination leads to the eruption of lesions over the tongue, mouth, and oropharynx and of rashes that start at the face and extremities and may eventually envelope the whole body. Studies performed for 2 to 3 weeks after the onset of fever indicated that infectious virus is excreted in the throat, urine, and conjunctiva of smallpox patients (20). Additionally, the level and duration of secretion was higher in clinically severe (those with hemorrhagic and confluent lesions) cases than less severe (those with discrete lesions) cases (20). Evidence from epidemiological studies further suggests that transmission from smallpox patients to their contacts occurs only from the time of the earliest appearance of rash, and that excretions from the mouth and nose are the most important source of infectious virus for transmission (8). The transmission rate of smallpox to unvaccinated contacts is estimated to be in the range of 37 to 88% (3). Smallpox was declared eradicated from the natural environment in 1980 as a result of a worldwide vaccination campaign conducted by the World Health Organization. Despite the potential threat of the intentional release of variola virus as a biological weapon in the future, routine mass vaccinations are not implemented due to adverse events associated with live vaccinia virus (VV) immunization (6). The occurrence of vaccine-associated adverse events, such as progressive vaccinia, eczema vaccinatum, generalized vaccinia, and postvaccinial encephalitis, suggests that uncontrolled VV dissemination in the vicinity of the vaccination site or to distal sites elsewhere on the body may have serious consequences in vaccinees, especially those that are immunocompromised. In addition, virus shed from the vaccination site and inadvertently transferred to a household contact with a history of atopic dermatitis may lead to a life-threatening case of eczema vaccinatum (26).VV, the prototypic member of the Orthopoxvirus genus, is used to study virus infection, replication, morphogenesis, and dissemination (reviewed in reference 19). After DNA replication in the cytoplasm, two infectious but structurally and functionally different forms of the virus are formed: intracellular mature virus (IMV or MV) and extracellular enveloped virus (EEV or EV). MVs remain confined to the cytoplasm and do not disseminate from the cell until cell lysis occurs. However, MVs that acquire a double membrane from early endosomes or the trans-Golgi network form intracellular enveloped virus (IEV) and get transported to the cell surface. After the fusion of its outer membrane with the plasma membrane, IEV may be either retained as cell-associated enveloped virus (CEV) or released from the cell surface as EV. In vitro, CEV is involved in the cell-to-cell spreading of VV, whereas EV is involved in virus dissemination to nonadjoining nearby or distal cells (2, 15). The 37-kDa protein p37, encoded by the VV F13L gene, plays a critical role in this process, since the deletion of the gene results in the blockade of MV envelopment and the inhibition of plaque formation (2). In vivo, EV is implicated in virus dissemination within the host and has been shown to be released from squamous epithelial cells of the nasal cavity in mice intranasally (i.n.) infected with the IHD-J strain of VV (15, 16). Despite the abundance of MVs in the cytoplasm of nasal squamous epithelial cells, no MVs are detected extracellularly and almost all free extracellular virions are EV, suggesting that the mechanism of virus release in vivo is by the exocytosis of EV, not by cell disruption (16). Although the original form of infectious VV released from infected hosts appears to be EV (not MV), due to the fragility of the EV membrane to withstand environmental factors, it is thought that the more physically stable MV released by the disruption of the EV outer membrane is responsible for the infection of susceptible hosts (22). As such, reducing the amount of EV released from epithelial cells of the respiratory tract might lower the risk of person-to-person transmission of VV by limiting the amount of shed EV available for conversion to MV, thus resulting in the infection of contacts.ST-246 is an orally bioavailable low-molecular-size (376 g/mol) compound that is selectively active against several orthopoxviruses, including VV and ectromelia, cowpox, camelpox, monkeypox, and variola viruses (4, 23, 28). In vitro, ST-246 treatment resulted in a 10-fold reduction of EV formation (without affecting MV production) and in the inhibition of plaque formation and virus-induced cytopathic effect (28). The analysis of ST-246-resistant cowpox virus variants in vitro has revealed that the antiviral activity of ST-246 is targeted against a product of the V061 gene, which is homologous to the VV F13L gene required for MV envelopment (28). In vivo, ST-246 treatment has been shown to protect mice (ectromelia, vaccinia, or cowpox viruses), rabbits (rabbitpox), and ground squirrels (monkeypox) from lethal orthopoxvirus infections (14, 17, 21, 28). ST-246 treatment additionally reduced virus replication in organs such as spleen and liver (17, 28). In nonhuman primates, ST-246 fully protected cynomolgus monkeys from lethal intravenous (i.v.) challenges with monkeypox and variola virus and significantly reduced viral load and lesion formation (9, 10). In this study, we extended on these previous reports and examined the impact of ST-246 treatment on the in vivo dissemination of virus from the site of inoculation to various distal organs in BALB/c mice infected with the Western Reserve strain of VV (VV-WR) by i.n., percutaneous, i.v., subcutaneous (s.c.), and intraperitoneal (i.p.) routes. We also investigated the effect of ST-246 treatment on the level of virus shedding from the nasal and lung cavities of mice challenged i.n. and from vaccine-induced lesions of ACAM2000-vaccinated mice.     

Characterization of Neisseria meningitidis isolates from recent outbreaks in Ethiopia and comparison with those recovered during the epidemic of 1988 to 1989

The objectives of this study were to collect and characterize epidemic meningococcal isolates from Ethiopia from 2002 to 2003 and to compare them to 21 strains recovered during the previous large epidemic of 1988 to 1989. Ninety-five patients in all age groups with clinical signs of meningitis and a turbid cerebrospinal fluid (CSF) sample were included in the study of isolates from 2002 to 2003. Seventy-one patients (74.7%) were confirmed as having Neisseria meningitidis either by culture (n = 40) or by porA PCR (n = 31) of their CSF. The overall case fatality rate (CFR) was 11.6%; the N. meningitidis-specific CFR was 4.2%. All 40 strains were fully susceptible to all antibiotics tested except sulfonamide, were serotyped as A:4/21:P1.20,9, and belonged to sequence type 7 (ST-7). The strains from 1988 to 1989 were also equally susceptible and were characterized as A:4/21:P1.20,9, but they belonged to ST-5. Antigenic characterization of the strains revealed differences in the repertoire of lipooligosaccharides and Opa proteins between the old and the recent strains. PCR analysis of the nine lgt genes revealed the presence of the lgtAHFG genes in both old and recent strains; lgtB was present in only some of the strains, but no correlation with sequence type was observed. Further analysis showed that in addition to their pgm alleles, the Ethiopian ST-5 and ST-7 strains also differed in their tbpB, opa, fetA, and lgtA genes. The occurrence of new antigenic structures in strains sharing the same serogroup, PorA, and PorB may help explain the replacement of ST-5 by ST-7 in the African meningitis belt.     

Prevalence and Associated Factors of Psychological Distress among Nurses in Public Hospitals,Southwest, Ethiopia: A cross-sectional Study

BackgroundPsychological distress is a state of emotional suffering and also characterized by somatic symptoms. Health care workers more prone psychological distress than general population. However, little attention was paid on psychological distress among nurses particularly in Ethiopia. Therefore, this study aimed at assessing the prevalence of psychological distress and its'' associated factors among nurses in public hospitals, Southwest Ethiopia.MethodAn institutional-based cross-sectional study was conducted from February 1^st, 2018 to April 1^st, 2018. All 282 eligible nurses in the selected public hospitals were enrolled. Data was collected using the predesigned tool like Self-Reporting Questionnaire version 20. Data were entered using EPI INFO version 7 and was exported to statistical packages for social science (SPSS) version 21.0 for analysis. Logistic regression analysis was employed and variables with a P-value of < 0.05 were considered as statistically significant.ResultA total of 282 eligible nurses were enrolled in the study with mean age of 28.71 [SD ±7.047]. The prevalence of psychological distress among nurses was 78(27.7%). Predictor variables like; nurses with job title of staff nurse, less working experience, poor interaction with staffs, fatigue, poor social support, perfectionism, and insomnia were more prone to develop the psychological distress.ConclusionThe study revealed that a considerable proportion of nurses had psychological distress. Therefore, it needs to develop psychological support strategies to improve the mental health resilience of nurses.     

Establishing a Self-sustaining Emergency Medicine Point-of-Care Ultrasound Curriculum in an Academic Teaching Hospital in Ethiopia

BackgroundPoint-of-care ultrasound (POCUS) training has become a standard component of Canadian emergency medicine (EM) residency programs. In resource-limited contexts, including Ethiopia, there is a critical shortage of local clinicians who can perform and teach POCUS. Our aim was to establish an introductory POCUS rotation within the EM residency program at Addis Ababa University (AAU) through The Toronto Addis Ababa Academic Collaboration in Emergency Medicine (TAAAC-EM).MethodsThrough stakeholder engagement, the authors completed a quality improvement initiative and conducted a survey of AAU EM faculty and residents to understand which POCUS scans should be included in a core residency POCUS curriculum, “POCUS1”.Results17 residents completed the POCUS1 program and 16 residents completed the written survey. Focused assessment with sonography for trauma, inferior vena cava, and lung (pneumothorax, pleural effusions, and interstitial syndrome) were identified as core introductory topics. Seventeen residents completed the initial POCUS1 program. Three program graduates were supported to become “POCUS1 Master Instructors” to continue the program during the SARS-CoV-2 global pandemic.ConclusionThe authors identified the highest yield POCUS scans through a written survey, successfully introduced a sustainable core POCUS curriculum at AAU for EM residents, and graduated three master instructors for curriculum continuation. We outline the structure and materials for implementation of POCUS programs for EM trainees and staff in similar low- and middle-income countries.     

Coagulation parameters in lung cancer patients: A systematic review and meta‐analysis

BackgroundHypercoagulability in lung cancer patients is associated with a high incidence of mortality and morbidity in the world. Therefore, this meta‐analysis aimed to explore the correlation of the basic coagulation abnormalities in lung cancer patients compared with the control.MethodPubMed, Scopus, and other sources were employed to identify eligible studies. The outcome variable was expressed using mean ± standard deviation (SD). Heterogeneity among studies and publication bias were evaluated. The quality of included studies was also assessed based on Newcastle–Ottawa Scale checklist.ResultFinally, through a total of eight studies, prolonged prothrombin time (PT; standard mean difference [SMD]: 1.29; 95% CI: 0.47–2.11), plasma D‐dimer value (SMD 3.10; 95% CI 2.08–4.12), fibrinogen (SMD 2.18; 95% CI:1.30–3.06), and platelet (PLT) count (SMD 1.00; 95% CI 0.84–1.16) were significantly higher in lung cancer patients when compared with the control group. The single‐arm meta‐analysis also showed that compared with control, lung cancer patients had high pooled PT 13.7 (95% CI:12.2–15.58) versus 11.79 (95% CI = 10.56–13.02), high D‐dimer 275.99 (95% CI:172.9–11735.9) versus 0.2 (95% CI:0.20–0.37), high plasma fibrinogen 5.50 (95% CI:4.21–6.79) versus 2.5 (95% CI:2.04–2.91), and high PLT count 342.3 (95% CI:236.1–448.5) versus 206.6 (95% CI:176.4–236.7).ConclusionIn conclusion, almost all the coagulation abnormalities were closely associated with lung cancer, and hence coagulation indexes provide an urgent clue for early diagnosis and timely management.     

Superior mesenteric artery syndrome: a rare cause of duodenal obstruction. Cases reports and review of literature

The Superior Mesenteric Artery Syndrome is a rare cause of duodenal obstruction. We present two young Ethiopian female patients who were diagnosed with this rare condition. After a duodeno-jejunostomy, the patients showed significant improvement and were discharged in a very good condition. This is the first report of this rare case from Ethiopia and a brief literature review is also presented.     

Real‐world evidence concerning clinical and economic outcomes of switching to insulin glargine 300 units/mL vs other basal insulins in patients with type 2 diabetes using basal insulin

This retrospective cohort study compared real‐world clinical and healthcare‐resource utilization (HCRU) data in patients with type 2 diabetes using basal insulin (BI) who switched to insulin glargine 300 units/mL (Gla‐300) or another BI. Data from the Predictive Health Intelligence Environment database 12 months before (baseline) and 6 months after (follow‐up) the switch date (index date, March 1, 2015 to May 31, 2016) included glycated haemoglobin A1c (HbA1c), hypoglycaemia, HCRU and associated costs. Baseline characteristics were balanced using propensity score matching. Change in HbA1c from baseline was similar in both matched cohorts (n = 1819 in each). Hypoglycaemia incidence and adjusted event rate were significantly lower with Gla‐300. Patients switching to Gla‐300 had a significantly lower incidence of HCRU related to hypoglycaemia. All‐cause and diabetes‐related hospitalization and emergency‐department HCRU were also favourable for Gla‐300. Lower HCRU translated to lower costs in patients using Gla‐300. In this real‐world study, switching to Gla‐300 reduced the risk of hypoglycaemia in patients with type 2 diabetes when compared with those switching to another BI, resulting in less HCRU and potential savings of associated costs.     

Insulin internalization and degradation in adipocytes from normal and type II diabetic subjects

We studied the ability of isolated adipocytes from normal and type II diabetic subjects to internalize and process [125I]insulin. Adipocytes were incubated with [125I]insulin at 16 or 37 C, and at various times total cell-associated, surface-bound, and intracellular insulin were quantitated using an acid-barbital extraction technique which quickly removes cell surface insulin, leaving behind the intracellular insulin. Insulin internalization was slow in normal adipocytes at 16 C, such that only 13% of total cell-associated insulin was intracellular after 2 h of incubation. In contrast, internalization was rapid at 37 C, such that the intracellular pool of insulin was near maximal by 30 min and accounted for approximately 40% of the total cell-associated insulin. Sephadex G-50 column chromatography of the intracellular insulin demonstrated that more than 95% of this pool coeluted with native insulin. In adipocytes from the diabetic subjects, approximately 45% of total cell-associated insulin was intracellular after 30 min of incubation at 37 C. After 60 min of incubation at 37 C, the percentages of total cell-associated and surface-bound insulin were significantly lower in adipocytes from diabetic compared to normal subjects [1.81 +/- 0.31% (+/- SEM) vs. 2.92 +/- 0.24% (P less than 0.05) and 0.97 +/- 0.14% vs. 1.72 +/- 0.15% (P less than 0.01), respectively]. The percentage of insulin in the intracellular compartment was also slightly lower in adipocytes from diabetic compared to normal subjects (0.84 +/- 0.19% vs. 1.20 +/- 0.16%; P greater than 0.05). The lysosomotropic agent chloroquine increased total cell-associated insulin, and this was due entirely to an increase in intracellular insulin. In adipocytes from normal subjects, chloroquine increased intracellular insulin by 32% at 30 min, by 89% at 60 min, by 140% at 90 min, and by 178% at 120 min. In comparison to the normal adipocytes, the chloroquine-mediated increase in intracellular insulin was lower in adipocytes from the diabetic subjects (-8.1% at 30 min, 37% at 60 min, 58% at 90 min, and 63% at 120 min; P less than 0.05 at all time points). These results indicate that insulin is rapidly internalized in human adipocytes at 37 C such that approximately half of total cell-associated insulin is intracellular the intracellular insulin is largely intact; and intracellular processing of insulin by a chloroquine-sensitive pathway(s) is impaired in adipocytes from type II diabetic subjects.