title: Potent and specific inhibition of SARS-CoV antigen expression by RNA interference.

text: BACKGROUND Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. METHODS The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot. RESULTS The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription. CONCLUSIONS Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.


title: Severe acute respiratory syndrome.

text: Severe acute respiratory syndrome (SARS) is a new respiratory illness due to a novel coronavirus called SARS-CoV. Cases of SARS were first identified from Guangdong Province in southern China in November 2002. Over the next several months, international travel allowed for the rapid spread of >8,000 cases over three continents. The economic and psychological impact of this mysterious illness was profound. In order to better educate the public about this emerging infectious disease, we describe the characteristics of SARS-CoV and review the epidemiology, clinical presentation, laboratory, radiographic and histopathological features, diagnosis, treatment, prevention and infection control issues pertaining to SARS. While a significant amount of information about this disease has been gained in a brief period of time, details regarding the origin of SARS-CoV, its stability in the environment and in human body fluids, and whether it has become established in humans remain unknown.


title: The Presentation and Diagnosis of the First Known Community-Transmitted Case of SARS-CoV-2 in the Republic of Ireland.

text: Introduction This case series describes the diagnosis of the first case of community transmission of SARS-CoV-2 in the Republic of Ireland. Cases Case 1: A 25 year old male presented with dyspnoea, cough and high fevers for 4 days. He was commenced on broad-spectrum antimicrobials and oxygen therapy. His respiratory function deteriorated in spite of these measures and he required mechanical ventilation. CT showed left upper lobe consolidation as well as multifocal ground-glass opacification. Case 2: A 43 year-old male presented with headache and was found incidentally to have pneumonia. He was recently diagnosed with pituitary apoplexy secondary to an adenoma with resultant pituitary insufficiency but MRI brain was stable. His respiratory function deteriorated in spite of antibiotics and he required mechanical ventilation. CT showed likely atypical infection with resultant ARDS. Outcome Both underwent nasopharyngeal RT-PCR testing for SARS-CoV-2. Patient 2 was positive. Patient 1 was extubated and made a good recovery. Patient 2 was transferred to another centre for ECMO therapy. He died 27 days after transfer. Conclusion Given the atypical presentations in generally otherwise young and healthy individuals, the decision was made outside of national guidance to perform testing for SARS-CoV-2. This diagnosis had far-reaching implications for the SARS-CoV-2 pandemic within Ireland.


title: A complex zinc finger controls the enzymatic activities of nidovirus helicases.

text: Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.


title: A novel "small round virus" inducing poult enteritis and mortality syndrome and associated immune alterations.

text: The role of a novel "small round virus" (SRV) isolated from poult enteritis and mortality syndrome (PEMS) cases in inducing PEMS and associated immune alterations was examined in this study. Specific-pathogen-free and conventional poults were orally challenged with SRV and/or turkey coronavirus and monitored for clinical signs. Intestines, thymus, bursa, and spleens were examined for SRV antigen at various days postinoculation (DPI). Peripheral blood lymphocytes (PBLs), thymocytes, and splenic lymphocytes from inoculated poults or lymphocytes isolated from healthy poults after incubation with SRV in vitro were examined for lymphoproliferative potential against concanavalin A (Con A). The incidence of lymphocyte subpopulations in the peripheral blood and thymic lymphocytes of SRV-challenged poults was examined by flow cytometry. The results of these studies showed that the SRV challenge induced diarrhea, growth suppression, and atrophy of thymus and bursa resembling those of PEMS in field and/or experimental infections. The SRV antigen was detected in intestinal tissues soon after infection (i.e., at 2 and 4 DPI), whereas lymphoid tissues such as thymus, bursa, and spleen were positive for SRV antigen starting at 4 DPI until 8 DPI, suggesting virus translocation to lymphoid organs. The responsiveness of PBLs to Con A at 2 DPI was significantly reduced in all virus challenge groups (e.g., 28% and 22% in the SRV-alone group in studies 1 and 2, respectively) below the uninfected group. However, this suppressed response was no longer evident in the SRV group by 7 DPI. The SRV incubation with normal thymocytes and splenocytes in vitro resulted in significantly reduced lymphoproliferative response against Con A (41.2% and 10.49% reductions at 1:50 SRV dilution vs. controls in thymocytes and splenocytes, respectively). Flow cytometry analysis revealed a sudden decline at 2 DPI in the numbers of CD4- CD8+ lymphocyte subset in PBLs of SRV-infected poults. However, by 8 DPI, SRV-challenged poults had relatively higher CD4- CD8+ lymphocytes in PBLs. On the contrary, thymocytes had higher percentages of CD4- CD8+ lymphocytes at 2 and 4 DPI and reached comparable levels at 8 DPI in controls and SRV-infected poults. No differences were observed in CD4+ CD8- lymphocyte numbers in controls vs. SRV-infected poults. The findings of these studies imply that SRV may be a promising primary etiologic agent of PEMS. Furthermore, the SRV infection may compromise the lymphocyte-mediated immune defenses by reducing lymphoproliferation and the CD4- CD8+ (presumably T-cytotoxic cells) lymphocytes during the acute stage of SRV infection.


title: [Avian influenza and the Severe Acute Respiratory Syndrome (SARS) - experiences and perspectives].

text: New respiratory viruses associated with pneumonia have in the past few years been detected in humans. The sudden appearance of the severe acute respiratory syndrome (SARS) in 2003 demonstrated that an emerging and highly infectious disease caused by a hitherto unknown virus was able to spread rapidly, but could finally be contained by stringent measures. The avian influenza A-H5N1-virus of high pathogenicity has crossed in multiple instances the species barriers between humans, mammals, and birds posing a serious pandemic threat. The application of the so far learnt and the continued development of preventive strategies, efficient vaccines, and antiviral substances are besides worldwide surveillance decisive to rapidly detect the repeated, enforced, or new appearance of viruses like the SARS-CoV, influenza A-H5N1 virus, or of new viruses, to contain their spread, and to defeat them.


title: Molecular evolution meets the genomics revolution.

text: Changes in technology in the past decade have had such an impact on the way that molecular evolution research is done that it is difficult now to imagine working in a world without genomics or the Internet. In 1992, GenBank was less than a hundredth of its current size and was updated every three months on a huge spool of tape. Homology searches took 30 minutes and rarely found a hit. Now it is difficult to find sequences with only a few homologs to use as examples for teaching bioinformatics. For molecular evolution researchers, the genomics revolution has showered us with raw data and the information revolution has given us the wherewithal to analyze it. In broad terms, the most significant outcome from these changes has been our newfound ability to examine the evolution of genomes as a whole, enabling us to infer genome-wide evolutionary patterns and to identify subsets of genes whose evolution has been in some way atypical.


title: CD8(+) T cell-mediated immune responses in West Nile virus (Sarafend strain) encephalitis are independent of gamma interferon.

text: The flavivirus West Nile virus (WNV) can cause fatal encephalitis in humans and mice. It has recently been demonstrated, in an experimental model using WNV strain Sarafend and C57BL/6 mice, that both virus- and immune-mediated pathology is involved in WNV encephalitis, with CD8(+) T cells being the dominant subpopulation of lymphocyte infiltrates in the brain. Here, the role of activated WNV-immune CD8(+) T cells in mouse WNV encephalitis was investigated further. Passive transfer of WNV-immune CD8(+) T cells reduced mortality significantly and prolonged survival times of mice infected with WNV. Early infiltration of WNV-immune CD8(+) T cells into infected brains is shown, suggesting a beneficial contribution of these lymphocytes to recovery from encephalitis. This antiviral function was not markedly mediated by gamma interferon (IFN-gamma), as a deficiency in IFN-gamma did not affect mortality to two strains of WNV (Sarafend and Kunjin) or brain virus titres significantly. The cytolytic potential, as well as precursor frequency, of WNV-immune CD8(+) T cells were not altered by the absence of IFN-gamma. This was reflected in transfer experiments of WNV-immune CD8(+) T cells from IFN-gamma(-/-) mice into WNV-infected wild-type mice, which showed that IFN-gamma-deficient T cells were as effective as those from WNV-immune wild-type mice in ameliorating disease outcome. It is speculated here that one of the pleiotropic functions of IFN-gamma is mimicked by WNV-Sarafend-mediated upregulation of cell-surface expression of major histocompatibility complex antigens, which may explain the lack of phenotype of IFN-gamma(-/-) mice in response to WNV.


title: Pentaglobin in steroid-resistant severe acute respiratory syndrome.

text: SETTING The treatment of severe acute respiratory syndrome (SARS) is at best controversial, although there is considerable anecdotal experience to show the benefits of corticosteroid therapy for selected patients. Some patients deteriorate relentlessly despite treatment with antibiotic, corticosteroid and mechanical ventilation. OBJECTIVE To attempt to determine the clinical efficacy of pentaglobin, an IgM-enriched immunoglobulin preparation, on 12 severe SARS patients who continued to deteriorate despite corticosteroid and ribavirin therapy. DESIGN Retrospective analysis of daily quantitative and radiographic data on the cohort in a regional teaching hospital. RESULTS AND CONCLUSION There was significant improvement in radiographic scores, when compared with day 1, on days 5, 6 and 7 (P < 0.05) after commencement of pentaglobin treatment. Similarly, there was significant improvement in oxygen requirement, when compared with day 1, on days 6 and 7 (P < 0.05) after commencement of pentaglobin treatment. There were no reported adverse events attributable to pentaglobin administration. Ten patients made an uneventful recovery after treatment. One elderly man died from cardiorespiratory arrest despite clinical and radiological improvement, and another patient is making good progress. Pentaglobin is safe and probably effective in the treatment of steroid-resistant SARS. A double-blind placebo-controlled study should therefore be considered.


title: Modes to facilitate ventilator weaning.

text: Weaning comprises 40 percent of the duration of mechanical ventilation. Protocols to reduce weaning time and to identify candidates at the earliest possible moment have been introduced to reduce complications and costs. Increased demand for mechanical ventilation, an increase in the number of patients requiring prolonged ventilation, and resource/staffing issues have created an environment where automated weaning may play a role. A number of closed loop techniques have been introduced since the early 1990s, with increasing sophistication. Preliminary research has demonstrated mixed results. Current systems continue to be evaluated in different patient populations and environments. Automated weaning is part of the ICU armamentarium, and identification of the patient populations most likely to benefit needs to be further defined.


title: Epidemiological study on risk factors for anxiety disorder among Chinese doctors.

text: OBJECTIVES Anxiety disorders are the most prevalent mental health problem. However, few studies are available pertaining to this problem among Chinese doctors, a special occupational population exposed to high stress, especially during the implementation of health system reform. The aim of this study was to assess anxiety disorder among Chinese doctors and clarify its risk factors. METHODS A cross-sectional study was conducted in 7 teaching hospitals of China Medical University. Questionnaires pertaining to anxiety disorder indicated by the Zung Self-Rating Anxiety Scale (SAS) and demographic characteristics, health status, lifestyle factors, work conditions and work-related psychological status were distributed to all registered doctors during April-June 2009. There were 1,134 effective respondents (effective response rate 71.2%), and these respondents became our subjects (539 male doctors and 595 female doctors). RESULTS The average standard scores of SAS for the male and female doctors were 46.8 and 46.7. General linear model analysis indicated that SAS was associated with, in standardized estimate (β) sequence, cynicism, emotional exhaustion, professional efficacy, sleeping problem, chronic diseases and physical exercise in men and with professional efficacy, emotional exhaustion, cynicism, sleeping problem, chronic diseases, job satisfaction, alcohol consumption and meals in women. Work-related psychological status contributed the most to the model R-square. CONCLUSION Chinese doctors were at considerably high risk of anxiety disorder and job burnout and sleeping problem had prominent roles. Interventions such as preventing exhaustion at work and improving sleep quality should be given attention during health care system reform with the aim of lessening anxiety disorder in doctors in China.


title: Laparoscopic-assisted Ventral Hernia Repair: Primary Fascial Repair with Polyester Mesh versus Polyester Mesh Alone.

text: Laparoscopic-assisted ventral hernia repair (LAVHR) with mesh is well established as the preferred technique for hernia repair. We sought to determine whether primary fascial closure and/or overlap of the mesh reduced recurrence and/or complications. We conducted a retrospective review on 57 LAVHR patients using polyester composite mesh between August 2010 and July 2013. They were divided into mesh-only (nonclosure) and primary fascial closure with mesh (closure) groups. Patient demographics, prior surgical history, mesh overlap, complications, and recurrence rates were compared. Thirty-nine (68%) of 57 patients were in the closure group and 18 (32%) in the nonclosure group. Mean defect sizes were 15.5 and 22.5 cm(2), respectively. Participants were followed for a mean of 1.3 years [standard deviation (SD) = 0.7]. Recurrence rates were 2/39 (5.1%) in the closure group and 1/18 (5.6%) in the nonclosure group (P = 0.947). There were no major postoperative complications in the nonclosure group. The closure group experienced four (10.3%) complications. This was not a statistically significant difference (P = 0.159). The median mesh-to-hernia ratio for all repairs was 15.2 (surface area) and 3.9 (diameter). Median length of stay was 14.5 hours (1.7-99.3) for patients with nonclosure and 11.9 hours (6.9-90.3 hours) for patients with closure (P = 0.625). In conclusion, this is one of the largest series of LAVHR exclusively using polyester dual-sided mesh. Our recurrence rate was about 5 per cent. Significant mesh overlap is needed to achieve such low recurrence rates. Primary closure of hernias seems less important than adequate mesh overlap in preventing recurrence after LAVHR.


Free 24-hour specialist support during Covid-19.


title: Feline infectious peritonitis presenting as a tumour in the abdominal cavity.

text: This paper describes six cases of feline infectious peritonitis (FIP) in which an abdominal tumour had been suspected clinically. Pathological changes were mainly restricted to the massive enlargement of a mesenteric lymph node due to necrogranulomatous lymphadenitis. FIP was diagnosed on the basis of the immunohistological demonstration of coronavirus antigen in intact macrophages within the necrogranulomatous lesions. In the affected lymph node lymphoid tissue was either almost completely effaced or restricted to follicles composed mainly of variable numbers of blasts. From one to many plasma cells positive for coronavirus-specific antibodies were present in the marginal sinuses or capsules. In addition, necrogranulomas were present in the gut-associated lymphoid tissue of the caecum of one cat, and adjacent to the affected lymph node of another.


title: Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

text: We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.


title: The community's attitude towards swine flu and pandemic influenza.

text: OBJECTIVE To ascertain the beliefs, perceived risks and initial attitudes of the Australian community towards the influenza pandemic declared by the World Health Organization in response to the emergence of an A(H1N1) influenza subtype. DESIGN, SETTING AND PARTICIPANTS Cross-sectional survey of Sydney residents during WHO Phase 5 of pandemic (H1N1) 2009. Members of the public were approached in shopping and pedestrian malls in seven areas of Sydney between 2 May and 29 May 2009 to undertake the survey. The survey was also made available by email. MAIN OUTCOME MEASURES Perceived personal risk and seriousness of the disease, opinion on the government and health authorities' response, feelings about quarantine and infection control methods, and potential compliance with antiviral prophylaxis. RESULTS Of 620 respondents, 596 (96%) were aware of pandemic (H1N1) 2009, but 44% (273/620) felt they did not have enough information about the situation. More than a third (38%; 235/620) ranked their risk of catching influenza during a pandemic as low. When asked how they felt pandemic influenza would affect their health if they were infected, only a third (33%; 206/620) said "very seriously". Just over half of the respondents (58%; 360/620) believed the pandemic would be over within a year. Respondents rated quarantine and vaccination with a pandemic vaccine as more effective than hand hygiene for the prevention of pandemic influenza. CONCLUSIONS Emphasising the efficacy of recommended actions (such as hand hygiene), risks from the disease and the possible duration of the outbreak may help to promote compliance with official advice.


The chickens come home to roost.


title: [Changes of blood cells in patients with severe acute respiratory syndrome].

text: OBJECTIVE To investigate the changes of blood corpuscles of patients with severe acute respiratory syndrome (SARS) in Beijing. METHODS Totally 43 patients (21 male and 22 female, 19-74 years old age range) diagnosed as of probable SARS were included in this study. Their corpuscles in the peripheral blood were tested every two days, and the results were analyzed. RESULTS Patients with SARS were more likely to develop leukocytopenia, lymphopenia and thrombocytopenia in the early period of disease than those in control group. The situation, especially lymphopenia and thrombocytopenia, could not be reversed in patients who died. Persistent low counts of lymphocytes and platelets at presentation might be associated with adverse outcomes. CONCLUSION Low counts of leukocytes, lymphocytes and platelets were common among patients in the early stage of SARS. Persistent Lymphopenia and thrombocytopenia may be associated with the prognosis.


title: [Laparoscopic partial pericystectomy of hepatic hydatid cysts --Papadimitriou operation: a case report].

text: Operative treatment of hepatic hydatid cyst is technically demanding procedure. The method of choice is conservative treatment with Albendazolum followed by surgery. Open laparotomy or laparoscopic operation can be performed. We prefere laparoscopic exploration followed by laparoscopic total pericystectomy or laparoscopic partial pericystectomy. If laparoscopic operation is not possible due to technical reasons and patient safety, conversion to an open operation should be done, followed by total or partial pericystectomy. The case and our detailed technique of laparoscopic partial pericystectomy with biliostasis and omentoplasty is described. Laparoscopic operation is equally safe for the patient, yet with minor trauma and better aesthetic effect.


title: Inactivation of SARS coronavirus by means of povidone-iodine, physical conditions, and chemical reagents.

text: The efficacy of several povidone-iodine (PVP-I) products, a number of other chemical agents, and various physical conditions were evaluated for their ability to inactivate the severe acute respiratory syndrome coronavirus (SARS-CoV). Treatment of SARS-CoV with PVP-I products for 2 min reduced the virus infectivity from 1.17 x 10(6) TCID50/ml to below the detectable level. The efficacy of 70% ethanol was equivalent to that of PVP-I products. Fixation of SARS-CoV-infected Vero E6 cells with a fixative including formalin, glutaraldehyde, methanol, and acetone for 5 min or longer eliminated all infectivity. Heating the virus at 56 degrees C for 5 min dramatically reduced the infectivity of the virus from 2.6 x 10(7) to 40 TCID50/ml, whereas heating the virus for 60 min or longer eliminated all infectivity. Irradiation with ultraviolet light at 134 microW/cm2 for 15 min reduced the infectivity from 3.8 x 10(7) to 180 TCID50/ml; however, prolonged irradiation (60 min) failed to eliminate the remaining virus, leaving 18.8 TCID50/ml. We believe that these findings will be useful for the implementation of infection control measures against SARS, and for the establishment of effective guidelines for the prevention of SARS outbreaks.


title: Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization.

text: Five strains of infectious bronchitis virus isolated from commercial chickens from the state of Pennsylvania, USA during the years 1998 and 1999 were studied. The strains were selected for cross-challenge in specific pathogen free chickens and virus neutralization in chick embryos on the basis of partial S1 sequence amino acid identity values. The partial sequences analysed spanned the hypervariable amino terminus region of S1 from amino acid residues 48 to 219, based on the Beaudette strain. Using their S1 identity values, the strains represented a continuum of genetic, and thus antigenic, relationships. When compared with strain PA/5083/99, strain PA/Wolgemuth/98 had high sequence identity (96%) followed by PA/171/99 (85%), PA/5344/98 (70%) and PA/1220/98 (34%). The method of Archetti and Horsfall was used for calculating antigenic relatedness values of virus neutralization tests. The same formula was also applied to the percentage protection values of cross-challenge tests to derive protective relatedness values among the strains. The antigenic relatedness values, protective relatedness values, and the partial S1 sequence identity values were then analysed. The findings indicated partial S1 sequence identity values were more strongly correlated with protective relatedness values and than antigenic relatedness values.


title: [Comparison between the number and nature of fecal clostridia and other risk factors implicated in the intestinal pathology of newborn infants].

text: One-hundred and fifteen infants aged 1 to 31 days from two intensive-care units were grouped into 6 classes according to clinical criteria (enterocolitis with or without anatomopathological examination and pneumatosis intestinalis, "haemorrhagic colitis", acute diarrhoea or absence of intestinal disorders). The total number of viable bacteria, the number of Clostridium and, in some cases, the presence of rota- and/or coronavirus were determined in their stools. The incidence of Clostridium in the stools of infants with enterocolitis (with or without pneumatosis intestinalis) or haemorrhagic colitis was not significantly different from that of infants without intestinal disorders, whereas stools of infants with acute diarrhoea less often contained Clostridium than those of other infants. C. butyricum, C. difficile, C. perfringens, C. tertium, and C. sordellii were identified. Correspondence analysis comparing the variable, "clinical profile", with 23 other variables, suggested that the variables of gemellity, a birthweight below 1900 g, a gestational age of less than 35 weeks, respiratory distress, umbilical catheterization and a Clostridium count above 10(7)/g at the onset of clinical signs, i.e. between 8 to 12 days of age, were linked to the clinical profile of necrotizing enterocolitis with pneumatosis intestinalis. Conversely, the absence of gemellity, a high birthweight and gestational age, the absence of respiratory distress or umbilical catheterization, the onset of diarrhoea within 8 days, and the presence of rota- and/or coronavirus in the stools were linked with a clinical profile of acute diarrhoea.


title: [Differential diagnosis of viral diarrheas in calves].

text: There is a description of the enzymoimmunologic method (ELISA), which was used for demonstration of rotaviruses and coronaviruses in the samples of excrements of the calves suffering from diarrheas. It is shown by a comparison with the results obtained by electron microscopy that the ELISA method provides by up to 50% higher capture rate and the reaction is highly specific. The method can also be applied to a detection of human rotavirus in the children's stool.


title: Self-adhesive mesh for Lichtenstein inguinal hernia repair. Experience of a single center.

text: AIM Lichtenstein tension-free mesh repair is the most frequently performed procedure for inguinal hernioplasty. In the past surgery aimed to control recurrences. Nowadays it is important to avoid postoperative chronic pain and thus several studies have examined the potential role of meshes in causing postoperative pain. The purpose of this study was to retrospectively assess the early and long-term results after Lichtenstein tension-free repair using a self-adhesive mesh (Parietex ProgripTM - Covidien, Germany) in a single center. METHODS The study enrolled 211 patients, 199 males (94.3%) and 12 females (5.7%), mean age 62 years (28-90 years), between January 2008 and December 2011. Of these, 206 had primary inguinal hernias while 5 were recurrences following previous tension repair. Ten different general surgeons, including residents, performed Lichtenstein hernia repair using a 12 x 8-cm Parietex ProgripTM mesh. In 88.1% of patients no additional fixation was used, while in 11.9% a single 2-0 polypropylene stitch was placed on the pubic bone. A 1-10 visual analog scale (VAS) was used to assess postoperative pain, evaluating it at 1 week, 1 month and 12, 24 and 36 months. Local paresthesia was assessed at same intervals. Any pain sensation lasting longer than 3 months postoperatively, or requiring injection of analgesics was defined as chronic pain. RESULTS Mean operating time was 64.1 minutes (SD ± 21.14). There were no intraoperative complications. Early postoperative complications included hematoma-seroma (5.7% cases), superficial wound infection (1%), urinary retention (0.5%), and scrotal swelling (1%). The main follow-up period was 3 years, although patients operated between 2009 and 2011 underwent a shorter follow-up. At one-year follow-up, 17 patients reported groin discomfort, but did not require analgesics. Three patients reported moderate pain, requiring occasional use of oral analgesics, and 2 of these described a discontinuous pain mainly during movement. One patient reported severe pain requiring local injection of analgesics. At 2-year follow-up, 3 patients reported groin discomfort. Five of the 17 patients who reported discomfort at 1 year were lost to the 2-year follow-up. One patient kept reporting a high VAS score (6), though slightly reduced from the previously reported at 1-year follow-up. Recurrence was observed in 0.5% at 1 year and in 2.4% at 2 years. At 3 years only half of the patients (102) were still on follow-up. Of these, 1 reported mild discomfort and 3 developed hernia recurrence. Globally a decrease in pain and local discomfort was observed. No cases of seroma, testicular complications or mesh infection were reported at 1-, 2- and 3-year follow-up. CONCLUSION Self-gripping mesh for inguinal hernia repair is a good and safe option, easy to handle and with a low incidence of chronic pain (<3%). A sutureless fixation seems to prevent the development of postoperative chronic pain, without increasing recurrence rates. Using a self-adhesive mesh also slightly reduce operating times, and costs are lower when compared to biological glue used to fix the mesh. In conclusion, our experience with the self-gripping mesh is limited but positive, randomized clinical trials are warranted to confirm our results.


Spinal epidural abscess.


title: CHRONOBIOLOGY OF HIGH BLOOD PRESSURE.

text: BIOCOS, the project aimed at studying BIOlogical systems in their COSmos, has obtained a great deal of expertise in the fields of blood pressure (BP) and heart rate (HR) monitoring and of marker rhythmometry for the purposes of screening, diagnosis, treatment, and prognosis. Prolonging the monitoring reduces the uncertainty in the estimation of circadian parameters; the current recommendation of BIOCOS requires monitoring for at least 7 days. The BIOCOS approach consists of a parametric and a non-parametric analysis of the data, in which the results from the individual subject are being compared with gender- and age-specified reference values in health.Chronobiological designs can offer important new information regarding the optimization of treatment by timing its administration as a function of circadian and other rhythms.New technological developments are needed to close the loop between the monitoring of blood pressure and the administration of antihypertensive drugs.


HBO2 for COVID-19: Clinical trials at clinicaltrials.gov.


title: Effects of hyperventilation on cerebral oxygen saturation estimated using near-infrared spectroscopy: A randomised comparison between propofol and sevoflurane anaesthesia.

text: BACKGROUND Near-infrared spectroscopy estimates cerebral regional tissue oxygen saturation (rSO2), which may decrease under hyperventilation. Propofol and sevoflurane act differently on cerebral blood vessels. Consequently, cerebral blood flow during hyperventilation with propofol and sevoflurane anaesthesia may differ. OBJECTIVES The first aim of this study was to compare the changes in rSO2 between propofol and sevoflurane anaesthesia during hyperventilation. The second aim was to assess changes in rSO2 with ventilation changes. DESIGN A randomised, open-label study. SETTING University of Yamanashi Hospital, Yamanashi, Japan from January 2014 to September 2014. PARTICIPANTS Fifty American Society of Anesthesiologists physical status 1 or 2 adult patients who were scheduled for elective abdominal surgery were assigned randomly to receive either propofol or sevoflurane anaesthesia. Exclusion criterion was a known history of cerebral disease such as cerebral infarction, cerebral haemorrhage, transient ischaemic attack and subarachnoid haemorrhage. INTERVENTIONS After induction of anaesthesia but before the start of surgery, rSO2, arterial carbon dioxide partial pressure (PaCO2) and arterial oxygen saturation were measured. Measurements were repeated at 5-min intervals during 15 min of hyperventilation with a PaCO2 around 30 mmHg (4 kPa), and again after ventilation was normalised. MAIN OUTCOME MEASURES The primary outcome was the difference of changes in rSO2 between propofol anaesthesia and sevoflurane anaesthesia during and after hyperventilation. The second outcome was change in rSO2 after the initiation of hyperventilation and after the normalisation of ventilation. RESULTS Changes of rSO2 during hyperventilation were -10 ± 7% (left) and -11 ± 8% (right) in the propofol group, and -10 ± 8% (left) and -9 ± 7% (right) in the sevoflurane group. After normalisation of PaCO2, rSO2 returned to baseline values. Arterial oxygen saturation remained stable throughout the measurement period. The rSO2 values were similar in the propofol and the sevoflurane groups at each time point. CONCLUSION The effects of hyperventilation on estimated rSO2 were similar with propofol and sevoflurane anaesthesia. Changes in rSO2 correlated well with ventilation changes. TRIAL REGISTRATION Japan Primary Registries Network (JPRN); UMIN-CTR ID; UMIN000010640.


title: Epidemiology of a primary pneumonic plague in Kantoshu, Manchuria, from 1910 to 1911: statistical analysis of individual records collected by the Japanese Empire.

text: BACKGROUND Among the potential uses of Yersinia pestis, intentional release of its aerosolized form, causing person-to-person transmission, is thought to be the most threatening. With the current rarity of pneumonic plague epidemics, our epidemiological knowledge remains insufficient for detailed characterization of effective control measures. METHODS Temporal patterns and key biological parameters of a pneumonic plague epidemic in Manchuria from 1910 to 1911 were analysed based on historical records collected by Kanto Totokufu, the administration of the Japanese Empire in Manchuria at that time. The serial intervals were fitted to gamma distribution using the maximum likelihood method, and time-delay distributions from onset-to-admission, admission-to-death, and onset-to-death were investigated. RESULTS Whereas a total of 228 cases were diagnosed with pneumonic plague in areas under direct control of the Japanese Empire, 4,781 cases were also recorded in surrounding areas. Although the epidemic grew exponentially in the early phase, the average doubling time steadily increased reflecting successful control efforts. The estimated mean serial interval (and standard deviation) was 5.7 (3.6) days. All cases with known dates of onset were admitted to hospital within 4 days after onset, and the mean time from onset to admission was 1.1 (0.4) days. CONCLUSIONS The increase in doubling time demonstrates the efficient and rapid countermeasures employed. Since the short interval from onset to death implies the importance of rapid responses, the challenge in confronting a future bioterrorist attack is to implement rapid and appropriate integration of control measures both at the individual and community level to prevent further transmissions as well as lower case fatality.


title: Patterns of anti-TNF use and associated treatment outcomes in inflammatory bowel disease patients: results from an analysis of Dutch health insurance claims data.

text: INTRODUCTION Real-life patterns of anti-tumour necrosis factor (anti-TNF) use remain largely unknown. We aimed to investigate survival rates, clinical outcomes and costs of anti-TNF agents in a large population of patients with inflammatory bowel disease (IBD). METHODS Health insurance data from 22,082 IBD patients were provided by Achmea Healthcare. Time to anti-TNF discontinuation, treatment intensification, corticosteroid initiation and hospitalisation were analysed in patients starting on anti-TNF treatment from January 2008 until December 2014. Treatment regimens were analysed at different time points. RESULTS In this cohort, 855 and 1199 subjects started infliximab and adalimumab treatment, respectively. The median time to anti-TNF discontinuation was 600 days (IQR 156-1693). The proportion of subjects receiving intensified treatment increased over time (infliximab at 3 vs. 24 months: 22.2% vs. 33.6%, p = 0.01; adalimumab at 3 vs. 24 months: 10.5% vs. 19.3%, p < 0.001). Cessation of anti-TNF treatment was less common in Crohn's disease patients (HR 0.79, p = 0.001) and in patients receiving intensified treatment (HR 0.62, p = 0.001). Immunomodulator use was associated with a longer time to corticosteroid initiation (HR 0.80, p = 0.048), but not with longer drug survival (HR 0.99, p = 0.617). Hospitalisation was more common in Crohn's patients (HR 1.49, p = 0.011). Corticosteroid initiation was lower in Crohn's patients (HR 0.57, p < 0.001) and in patients using infliximab (HR 0.55, p < 0.001). CONCLUSIONS Discontinuation of anti-TNF therapy occurred earlier than previously reported and was associated with a diagnosis of ulcerative colitis and non-intensified anti-TNF treatment. Immunomodulator use at the start of anti-TNF treatment was associated with a longer time to corticosteroid initiation, but not with longer drug survival.


title: [Roles of multidisciplinary team (MDT) in diagnosis and treatment of suspected cases of corona virus disease 2019 (COVID-19)].

text: OBJECTIVE To analyze the roles of multidisciplinary team (MDT) in the diagnosis and treatment of suspected cases of corona virus disease 2019 (COVID-19). METHODS The clinical data of 48 patients with suspected COVID-19 admitted in Jinhua Central Hospital from January 21, 2020 to March 20, 2020 were retrospectively analyzed. RESULTS Of the 48 suspected cases, 18 were diagnosed with COVID-19, and 30 were excluded. Each of the confirmed cases were discussed among MDT for 2 to 12 times with an average of (4.7±3.2) times; while for non-COVID-19 patients were discussed for 2 to 4 times with an average of (2.3±0.6) times per case. With the guidance of MDT, one COVID-19 patient was transferred to designated provincial hospital after effective treatment; one patient complicated with acute cholecystitis underwent gallbladder puncture and drainage; and COVID-19 was excluded in a highly suspected patient after alveolar lavage fluid examination. Except one transferred patient, all 17 confirmed COVID-19 patients were cured and discharged; there was no cross-infection occurred in suspected patients during the hospitalization; there were no deaths and no medical staff infections. CONCLUSIONS The efficiency of diagnosis and treatment for suspected COVID-19 patients can be improved under MDT mode, particularly for complicated and refractory cases.


title: Open access: a giant leap towards bridging health inequities.

text: AbstractAccess to health research publications is an essential requirement in securing the chain of communication from the researcher to the front-line health worker. As the diagram of the knowledge cycle from the Canadian Institutes of Health Research shows, health knowledge generated in the world's laboratories is passed down the information chain through publications, through its impact and application, its subsequent "translation" into appropriate contexts for different user communities, arriving finally with health workers and the general public. This article focuses on the first link in the chain, from research author to reader, and the free online access to peer-reviewed published articles that are the building blocks for future health innovation developments.


title: The rational clinician in a pandemic setting.

text: Pandemic (H1N1) 2009 influenza has generated many controversies in Australia around case definitions, laboratory diagnosis, case management, medical logistics and travel restrictions. Our experience as clinical advisers in the Victorian Department of Human Services Emergency Operations Centre suggests the following: Case definitions may change frequently, and will tend to become more clinically specific over time. Early in a pandemic, laboratory diagnosis plays a critical role in case finding and pathogen identification. Later in the pandemic, standardised case management applied to well crafted case definitions should reduce reliance on the diagnostic laboratory in clinical management. The diagnostic laboratory will remain critical to monitoring disease surveillance, pathogen virulence, and drug susceptibility. Medical logistics will continue to challenge pandemic managers as the health sector struggles to do the most good for the greatest number of people. Travel restrictions remain scientifically controversial public health recommendations. Issues of scalability (escalation and de-escalation of the response) relating to virus lethality need to be resolved in current pandemic planning.


title: Mobilizing Primary Health Care:Cuba's Powerful Weapon against COVID-19.

text: A strong foundation of primary care is critical to the health system and is particularly important during pandemics like COVID-19. Primary care practices should be a natural fi t for triaging, testing, treating, and educating patients.-Corinne Lewis, Shanoor Seervai, Tanya Shah, Melinda K. Abrams, and Laurie Zephyrin MD The Commonwealth Fund, April 22, 2020[1.


title: Structural and biological basis of CTL escape in coronavirus-infected mice.

text: Cytotoxic T lymphocyte escape occurs in many human infections, as well as mice infected with the JHM strain of mouse hepatitis virus, which exhibit CTL escape variants with mutations in a single epitope from the spike glycoprotein (S510). In all CTL epitopes prone to escape, only a subset of all potential variants is generally detected, even though many of the changes that are not selected would result in evasion of the T cell response. It is postulated that these unselected mutations significantly impair virus fitness. To define more precisely the basis for this preferential selection, we combine x-ray crystallographic studies of the MHC class I (D(b))/S510 complexes with viral reverse genetics to identify a prominent TCR contact residue (tryptophan at position 4) prone to escape mutations. The data show that a mutation that is commonly detected in chronically infected mice (tryptophan to arginine) potently disrupts the topology of the complex, explaining its selection. However, other mutations at this residue, which also abrogate the CTL response, are never selected in vivo even though they do not compromise virus fitness in acutely infected animals or induce a significant de novo CTL response. Thus, while structural analyses of the S510/D(b) complex provide a strong basis for why some CTL escape variants are selected, our results also show that factors other than effects on virus fitness limit the diversification of CD8 T cell epitopes.


title: Has allergenic pollen an impact on non-allergic diseases?

text: Recent observations suggest that pollen do not only interacts with the human immune system to elicit an allergic response in susceptible individuals. It would have a much broader impact on human health. This applies more especially, yet not exclusively, to three groups of diseases: non-allergic respiratory conditions, cardio- and cerebrovascular accidents, and psychiatric disorders including suicide and suicide attempt. At present, the reasons for these unexpected connections are only hypothetical, and require further exploration in larger samples, but there is perhaps a multitude of them. One must therefore favour a holistic approach of pollen and its impact on human health.


title: Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication.

text: The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.


title: Current challenges in providing bariatric surgery in France: A nationwide study.

text: Bariatric surgery is a well-accepted procedure for severe and massive obesity management. We aimed to determine trends, geographical variations, and factors influencing bariatric surgery and the choice of procedure in France in a large observational study.The Health Insurance Fund for Salaried Workers (Caisse National Assurance Maladie Travailleurs Salariés) covers about 86% of the French population. The Système National d'Information Inter-régimes de l'Assurance Maladie database contains individualized and anonymized patient data on all reimbursements for healthcare expenditure. All types of primary bariatric procedures (Roux-en-Y gastric bypass [RYGB] or omega loop, adjustable gastric banding [AGB], or longitudinal sleeve gastrectomy [LSG]) performed during 2011 to 2013 were systematically recorded. Surgical techniques performed by region of residence and age-range relative risks with 95% confidence intervals of undergoing LSG or RYGB versus AGB were computed.In 2013, LSG was performed more frequently than RYGB and AGB (57% vs 31% and 13%, respectively). A total of 41,648 patients underwent a bariatric procedure; they were predominantly female (82%) with a mean (±standard deviation) age of 40 (±12) years and a body mass index ≥40 kg/m for 68% of them. A total of 114 procedures were performed in patients younger than 18 years and 2381 procedures were performed in patients aged 60 years and older. Beneficiaries of the French universal health insurance coverage for low-income patients were more likely to undergo surgery than the general population. Large nationwide variations were observed in the type choice of bariatric surgical procedures. Significant positive predictors for undergoing RYGB compared to those for undergoing AGB were as follows: referral to a center performing a large number of surgeries or to a public hospital, older age, female gender, body mass index ≥50 kg/m, and treatment for obstructive sleep apnea syndrome, diabetes, or depression. Universal health insurance coverage for low-income patients was inversely correlated with the probability of RYGB.Differences in access to surgery have been observed in terms of the patient's profile, geographical variations, and predictors of types of procedures. Several challenges must be met when organizing the medical care of this growing number of patients, when delivering surgery through qualified centers while assuring the quality of long-term follow-up for all patients.


title: Evidence and effectiveness in decisionmaking for quarantine.

text: When public health decisionmakers turned to quarantine during the recent severe acute respiratory syndrome (SARS) epidemic, difficult questions were raised about the legitimacy and acceptability of restrictive measures to attain public health goals. SARS also brought to light how scientific uncertainty can permeate public health decisionmaking, leading us to think about the relationship between the adequacy of evidence of the effectiveness of an intervention and its role in the justification of public health action. In this article, we critically examine the role of evidence and effectiveness in decision-making for quarantine. It is our contention that the effectiveness of a public health intervention should not be defined exclusively in (absolute and objective) scientific terms but rather conceptualized relationally and normatively in public health decisionmaking.


Who should manage the airway?


title: Mass spectrometric characterization of proteins from the SARS virus: a preliminary report.

text: A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a approximately 46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted approximately 139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.


Frequency of diarrhea in novel coronavirus 2019 infection.


Severe acute respiratory syndrome: guidelines were drawn up collaboratively to protect healthcare workers in British Columbia.


title: The SARS outbreak in a general hospital in Tianjin, China -- the case of super-spreader.

text: Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease with a high case-fatality rate and devastating socio-economic impact. In this report we summarized the results from an epidemiological investigation of a SARS outbreak in a hospital in Tianjin, between April and May 2003. We collected epidemiological and clinical data on 111 suspect and probable cases of SARS associated with the outbreak. Transmission chain and outbreak clusters were investigated. The outbreak was single sourced and had eight clusters. All SARS cases in the hospital were traced to a single patient who directly infected 33 people. The patients ranged from 16 to 82 years of age (mean age 38.5 years); 38.7% were men. The overall case fatality in the SARS outbreak was 11.7% (13/111). The outbreak lasted around 4 weeks after the index case was identified. SARS is a highly contagious condition associated with substantial case fatality; an outbreak can result from one patient in a relatively short period. However, stringent public health measures seemed to be effective in breaking the disease transmission chain.


title: Instent restenosis after carotid stenting: treatment using an off-label cardiac scoring balloon.

text: BACKGROUND Treatment of instent restenosis after carotid artery stenting because of circumferential or calcified lesions can be difficult and refractory to conventional balloon angioplasty. We describe the off-label use of a cardiac scoring balloon that was used for lesions refractory to angioplasty with other balloons. CASE DESCRIPTIONS Two patients with a history of carotid artery stenting 6 and 8 years ago, presented with symptomatic carotid instent restenosis caused by circumferential and calcified lesions, respectively. Angioplasty with conventional compliant and noncompliant balloons was unsuccessful. An AngioSculpt percutaneous transluminal coronary angioplasty (PTCA) scoring balloon catheter (AngioScore, Fremont CA, USA) was successful in achieving vessel recanalization despite the refractory nature of these lesions. No further conventional balloons or use of cutting balloons was required. CONCLUSION The AngioSculpt PTCA scoring balloon catheter can be a useful option for treatment of refractory calcified or circumferential carotid instent restenosis.


title: Inhaling to mitigate exhaled bioaerosols.

text: Humans commonly exhale aerosols comprised of small droplets of airway-lining fluid during normal breathing. These "exhaled bioaerosols" may carry airborne pathogens and thereby magnify the spread of certain infectious diseases, such as influenza, tuberculosis, and severe acute respiratory syndrome. We hypothesize that, by altering lung airway surface properties through an inhaled nontoxic aerosol, we might substantially diminish the number of exhaled bioaerosol droplets and thereby provide a simple means to potentially mitigate the spread of airborne infectious disease independently of the identity of the airborne pathogen or the nature of any specific therapy. We find that some normal human subjects expire many more bioaerosol particles than other individuals during quiet breathing and therefore bear the burden of production of exhaled bioaerosols. Administering nebulized isotonic saline to these "high-producer" individuals diminishes the number of exhaled bioaerosol particles expired by 72.10 +/- 8.19% for up to 6 h. In vitro and in vivo experiments with saline and surfactants suggest that the mechanism of action of the nebulized saline relates to modification of the physical properties of the airway-lining fluid, notably surface tension.


title: The complexities of multi-level governance in public health.

text: This article reviews some of the challenges to developing national public health programs, focussing on the distribution of constitutional authority for public health and governance challenges that arise from this. Constitutional authority for public health resides primarily with the provinces. The federal government has obtained the authority to legislate in this area primarily through its power over criminal law. Challenges facing the establishment of national public health programs include the ambiguity over constitutional responsibility, challenges in managing externalities and spillovers, and issues related to funding and data ownership. Policy-making is also complicated by the importance of municipal and supranational governments in public health. National programs need to be structured in a way that balances the advantages of regional approaches to public health challenges with the benefits of a coordinated central response. To do so, policy-makers need to address unique challenges to public health governance.


Serological Assays for SARS-CoV-2 Infectious Disease: Benefits, Limitations and Perspectives.


title: Further characterisation of the coronavirus IBV ORF 1a products encoded by the 3C-like proteinase domain and the flanking regions.

text: Coronavirus IBV encodes a piconarvirus 3C-like proteinase. In a previous report, this proteinase was shown to undergo rapid degradation in vitro in reticulocyte lysate due to a posttranslational event involving ubiquitination of the protein. Several lines of evidence presented here indicate that the proteinase itself is stable. Translation of the IBV sequence from nucleotide 8864 to 9787 resulted in the synthesis of a 33 kDa protein, representing the full-length 3C-like proteinase. Pulse-chase and time-course experiments showed that this protein was stable in reticulocyte lysate for up to 2 hours. However, a 45 kDa protein encoded by the IBV sequence from nucleotide 8693 to 9911 underwent rapid degradation in reticulocyte lysate, but was stable in wheat germ extract, suggesting that an ATP-dependent protein degradation pathway may be involved in the turnover of the 45 kDa protein. To identify the IBV sequence responsible for the instability of this 45 kDa protein species, the region from nucleotide 8693 to 9787 was translated both in vitro and in vivo, leading to the synthesis of a stable 43 kDa protein. These results suggest that a destabilising signal may be located in the IBV sequences between the nucleotides 9787 and 9911. Meanwhile, protein aggregation was observed when the product encoded by the IBV sequence from nucleotide 9911 to 10,510 was boiled for 5 minutes before being analysed in SDS-PAGE; when the same product was treated at 37 degrees C for 15 minutes, however, protein aggregation was not detected. Deletion studies indicate that the presence of a hydrophobic domain downstream of the 3C-like proteinase-encoding region may be the cause for the aggregation of the product encoded by this region of ORF 1a.


title: Mixed bacterial-viral infections in septic children with leukemia.

text: BACKGROUND Febrile infections in children with leukemia are common. The occurrence of possible mixed bacterial-viral infections is unknown. METHODS We searched for viruses in leukemic children with blood culture-positive bacterial infections. The prospective multicenter survey included 156 febrile episodes in 51 children with acute leukemia. The mean follow-up time was 1.5 years per patient (27,743 patient-days at risk). Sixteen viruses were searched for from nasal swab and stool samples using virus culture, virus antigen detection, and polymerase chain reaction tests. RESULTS Bacterial blood cultures were positive in 19 (11%) febrile episodes among 17 children. In half of the septic episodes (11 of 19), a virus was also found. Rhinovirus and respiratory syncytial virus were the most common viruses detected. CONCLUSIONS Our findings suggest that invasive bacterial infections are commonly associated with viral infections in children with leukemia.


title: Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways.

text: We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.


title: Comparison of two region of interest definition methods for metabolic response evaluation with [18F]FDG-PET.

text: AIM: In therapy response monitoring by [18F]2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET), different tumor delineations are used, resulting in different values for change in glucose metabolic rate (DMRglu). We propose a technique to compare metabolic rates in a region of interest (ROI) based on fixed volumes rather than on fixed thresholds. This method involves change in lesion size. METHODS: In 49 patients with colorectal carcinoma (CRC) and 50 patients with non-small cell lung carcinoma (NSCLC) scheduled for chemotherapy, FDG-PET was performed at baseline and during chemotherapy. A ROIfixed thresholds was determined by using a 50% threshold on both baseline and follow-up FDG-PET. A ROIfixed volumes was determined by using a 50% threshold, determined on the series with the largest tumor volume. This ROIfixed volumes is used on consecutive scans. Predictive effects of both methods were investigated by survival analysis for overall and progression free survival. RESULTS: In CRC, only ROIfixed volumes based DMRglu showed significant predictive ability. In NSCLC, both techniques showed significant predictive ability. During multivariate analysis, ROIfixed volumes determined DMRglu was an independent predictor for both overall and progression free survival in NSCLC whereas ROIfixed thresholds determined MRglu was not. After dichotomization at the median DMRglu, median survival ratio was higher in ROIfixed volumes than ROIfixed thresholds for CRC (overall survival: 1.78 vs 1.25, progression free survival: 1.57 vs 1.21) and NSCLC (overall survival: 2.01 vs 2.01, progression free survival: 2.93 vs 2.13). CONCLUSION: ROIfixed volumes based DMRglu shows better correlation with survival than DMRglu determined from a ROIfixed thresholds.


title: Intrahepatic alpha beta-TcRintermediate LFA-1high T cells are stimulated during mouse hepatitis viral infection.

text: Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of thymic and extrathymic T cell subpopulation disorders induced during the viral hepatitis. To understand local hepatic immune responses, the phenotypes of resident hepatic lymphocytes were determined and compared that of splenic and thymic T cell subpopulations during the acute viral hepatitis induced by MHV3 in susceptible C57BL/6 mice. Single positive (SP) CD4+ or CD8+ cells strongly increased in the liver. A specific cell population, the double positive (CD4+ C8+) cells, normally present in liver and thymic cell preparations, decreased in C57BL/6 mice following the viral infection. alpha beta-TcRintermediate T cells shifted toward alpha beta-TcRhigh T cells in the liver and thymus of infected mice, but not in their spleen. The specific alpha beta-TcRint or high lymphocytes occurring in the liver of MHV3-infected mice expressed higher levels of leukocyte function antigen-1 (LFA-1) and Pgp-1 (CD44) activation markers, suggesting that they were either activated or antigen-experienced during the viral infection. No significant changes in T cell subpopulations were detected in the spleen. These observations suggest that MHV3 infection could induce an early in situ stimulation of resident hepatic T cells, despite a peripheral immunodeficiency in the thymus and spleen.


[The minimal surgical access for appendectomy and complication of the acute appendicitis].


title: Gene therapy prospects--intranasal delivery of therapeutic genes.

text: Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.


title: Assessment of risk conferred by coding and regulatory variations of TMPRSS2 and CD26 in susceptibility to SARS-CoV-2 infection in human.

text: At present, more than 200 countries and territories are directly affected by the coronavirus disease-19 (COVID-19) pandemic. Incidence and case fatality rate are significantly higher among elderly individuals (age>60 years), type 2 diabetes and hypertension patients. Cellular receptor ACE2, serine protease TMPRSS2 and exopeptidase CD26 (also known as DPP4) are the three membrane bound proteins potentially implicated in SARS-CoV-2 infection. We hypothesised that common variants from TMPRSS2 and CD26 may play critical role in infection susceptibility of predisposed population or group of individuals. Coding (missense) and regulatory variants from TMPRSS2 and CD26 were studied across 26 global populations. Two missense and five regulatory SNPs were identified to have differential allelic frequency. Significant linkage disequilibrium (LD) signature was observed in different populations. Modelled protein-protein interaction (PPI) predicted strong molecular interaction between these two receptors and SARS-CoV-2 spike protein (S1 domain). However, two missense SNPs, rs12329760 (TMPRSS2) and rs1129599 (CD26), were not found to be involved physically in the said interaction. Four regulatory variants (rs112657409, rs11910678, rs77675406 and rs713400) from TMPRSS2 were found to influence the expression of TMPRSS2 and pathologically relevant MX1. rs13015258 a 50 UTR variant from CD26 have significant role in regulation of expression of key regulatory genes that could be involved in SARS-CoV-2 internalization. Overexpression of CD26 through epigenetic modification at rs13015258-C allele was found critical and could explain the higher SARS-CoV-2 infected fatality rate among type 2 diabetes.


title: Cutting edge: inhibition of TLR and FcR responses in macrophages by triggering receptor expressed on myeloid cells (TREM)-2 and DAP12.

text: DAP12 is an ITAM-containing adapter that associates with receptors in myeloid and NK cells. DAP12-associated receptors can give activation signals leading to cytokine production; however, in some situations, DAP12 inhibits cytokine production stimulated through TLRs and FcRs. Here we show that Triggering Receptor Expressed on Myeloid cells (TREM)-2 is responsible for the DAP12-mediated inhibition in mouse macrophages. A chimeric receptor composed of the extracellular domain of TREM-2 and the cytoplasmic domain of DAP12 inhibited the TLR- and FcR-induced TNF production of DAP12-deficient macrophages, whereas a TREM-1 chimera did not. In wild-type macrophages, TREM-2 knockdown increased TLR-induced TNF production. A TREM-2 Fc fusion protein bound to macrophages, indicating that macrophages express a TREM-2 ligand. Thus, the interaction of TREM-2 and its ligand results in an inhibitory signal that can reduce the inflammatory response.


title: EUS-guided choledochoduodenostomy for malignant distal biliary obstruction using a lumen-apposing fully covered metal stent after failed ERCP.

text: BACKGROUND A novel lumen-apposing, self-expanding metal stent to perform EUS-guided drainage procedures has been recently developed. The aim of this study was to analyze the safety, technical and clinical effectiveness of this device for EUS-guided choledochoduodenostomy (EUS-CD) with palliative intent. METHODS Retrospective analysis of all consecutive patients with unresectable malignant distal bile duct obstruction who, between March 2012 and September 2014, underwent EUS-CD using the study devices (AXIOS™ and Hot AXIOS™, Xlumena Inc., Mountain View, CA, USA) after unsuccessful ERCP in seven European centers was carried out. RESULTS Fifty-seven patients (M/F 31/26; median age 73) underwent EUS-CD using the AXIOS™ stent or the Hot AXIOS™ delivery system. ERCP failure was due to duodenal obstruction in 41 patients (71.9 %) and to inability to cannulate the papilla in the remaining 16 patients (28.1 %). The procedure was technically successful in 56/57 patients (98.2 %), with a mean procedural time of 22.4 min (range 11-65). Clinical success was achieved in 54 of these 56 patients (96.4 %; 94.7 % of the entire cohort). Overall major procedural complication rate was 7 % (two duodenal perforations, one bleeding and one transient cholangitis). During follow-up, 5 out of 54 (9.3 %) patients with clinica success required re-intervention for stent migration in one case and a sump syndrome with transient increase in serum bilirubin concentrations with sludge in the distal duct reservoir in the remaining four patients. CONCLUSIONS Our study shows that EUS-CD using the AXIOS™ and the Hot AXIOS™ devices is a safe procedure, with high technical and clinical success rates.


title: Application of polymerase chain reaction for the correlation of Salmonella serovars recovered from greyhound feces with their diet.

text: The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With the onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five "normal" fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.


title: The effectiveness of surgical face masks: what the literature shows.

text: The use and withdrawal of surgical face masks in recent years has occurred in an ad hoc manner that is incompatible with evidence-based practice. Much of the literature on masks consists of anecdotal evidence or summaries of previous studies. The rationale for wearing masks has shifted from protection of the patient to protection of the health care professional wearing the mask. Currently there is little evidence that wearing a surgical mask provides sufficient protection from all the hazards likely to be encountered in an acute health care setting: the use of a respirator and face shield should be considered depending on the circumstances.


title: Consumer Health Informatics: Promoting Patient Self-care Management of Illnesses and Health.

text: Consumer health informatics (CHI) is propelling important changes for medical providers and the lives of patients through information and communications technology. Independently, medical consumers seek, collect, and use health information for decision making. However, when constructing a CHI-based medical platform, high technology must be applied in a fully understandable and usable format for both health care providers and consumers. This study examines the present status of CHI and its effect on medical consumers. For the development of CHI, we discuss the need for tailored health communications and capacity building with chronic patients at the medical center. First, empowerment is a key characteristic needed for medical consumer health care management. However, promoting patient self-care management of illnesses and health is necessary to create conjugation where cooperation with medical service providers is possible. Also, establishing a health care delivery system that will support cooperation is necessary. Second, tailored health communications can uniquely construct the health information of patients, which prevents unnecessary or excessive information from leading patients to confused and inappropriate decisions. Ultimately, through the present environment of health communication, the innovation of a consumer health care information system has become the tide of the times and the positive effect of improved health can be expected.


Hearing the Call of Duty: What We Must Do to Allow Medical Students to Respond to the COVID-19 Pandemic.


title: Robotic assisted versus pure laparoscopic surgery of the adrenal glands: a case-control study comparing surgical techniques.

text: PURPOSE The role of the da Vinci Robotic System ® in adrenal gland surgery is not yet well defined. The goal of this study was to compare robotic-assisted surgery with pure laparoscopic surgery in a single center. METHODS One hundred and 16 patients underwent minimally invasive adrenalectomies in our department between June 1994 and December 2014, 41 of whom were treated with a robotic-assisted approach (robotic adrenalectomy, RA). Patients who underwent RA were matched according to BMI, age, gender, and nodule dimensions, and compared with 41 patients who had undergone laparoscopic adrenalectomies (LA). Statistical analysis was performed using the Student's t test for independent samples, and the relationship between the operative time and other covariates were evaluated with a multivariable linear regression model. P < 0.05 was considered significant. RESULTS Mean operative time was significantly shorter in the RA group compared to the LA group. The subgroup analysis showed a shorter mean operative time in the RA group in patients with nodules ≥6 cm, BMI ≥ 30 kg/m2 and in those who had previous abdominal surgery (p < 0.05). Results from the multiple regression model confirmed a shorter mean operative time with RA with nodules ≥6 cm (p = 0.010). Conversion rate and postoperative complications were 2.4 and 4.8 % in the LA group and 0 and 4.8 % in the RA group. CONCLUSIONS In our experience, RA shows potential benefits compared to classic LA, in particular on patients with nodules ≥6 cm, BMI ≥ 30 kg/m2, and with previous abdominal surgery.


title: Immunome-derived vaccines.

text: Immune response to a subset of antigens and epitopes derived from an infectious pathogen may be sufficient for competent protection; immune recognition of every potential epitope derived from the pathogen's genome does not appear to be required. The pneumococcal and hepatitis vaccines, both of which are subunit vaccines, illustrate this premise. Similarly, 'immunome-derived vaccines' are based on the concept that response to the subset of antigens and epitopes that interface with the host immune system (the immunome) and not the whole organism (represented by the proteome or genome) can be sufficient for protection. Competent immune responses to cancer are also probably restricted to the neoplasm's 'immunome', although the set of antigens that drive successful immune response to cancer cells has proven more difficult to uncover. Researchers are now using bioinformatics sequence analysis tools, epitope mapping tools, microarrays and high-throughput immunology assays to discover the components of the immunome, which are then used to compose these new vaccines. At least one immunome-derived vaccine is in clinical trials and many others are in the vaccine pipeline. Due to the rapid improvement of immunoinformatics tools and immunological assays, the era of immunome-derived vaccines has begun.


title: Human-specific regulation of alpha 2-6-linked sialic acids.

text: Many microbial pathogens and toxins recognize animal cells via cell surface sialic acids (Sias) that are alpha 2-3- or alpha 2-8-linked to the underlying glycan chain. Human influenza A/B viruses are unusual in preferring alpha 2-6-linked Sias, undergoing a switch from alpha 2-3 linkage preference during adaptation from animals to humans. This correlates with the expression of alpha 2-6-linked Sias on ciliated human airway epithelial target cells and of alpha 2-3-linked Sias on secreted soluble airway mucins, which are unable to inhibit virus binding. Given several known differences in Sia biology between humans and apes, we asked whether this pattern of airway epithelial Sia linkages is also human-specific. Indeed, we show that since the last common ancestor with apes, humans underwent a concerted bidirectional switch in alpha 2-6-linked Sia expression between airway epithelial cell surfaces and secreted mucins. This can explain why the chimpanzee appears relatively resistant to experimental infection with human Influenza viruses. Other tissues showed additional examples of human-specific increases or decreases in alpha 2-6-linked Sia expression and only one example of a change specific to certain great apes. Furthermore, while human and great ape leukocytes both express alpha 2-6-linked Sias, only human erythrocytes have markedly up-regulated expression. These cell type-specific changes in alpha 2-6-Sia expression during human evolution represent another example of a human-specific change in Sia biology. Because the data set involves multiple great apes, we can also conclude that Sia linkage expression patterns can be conserved during millions of years of evolution within some vertebrate taxa while undergoing sudden major changes in other closely related ones.


The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring.


title: Severe acute respiratory syndrome in surgical patients: a diagnostic dilemma.

text: BACKGROUND The diagnosis of severe acute respiratory syndrome (SARS) in surgical patients can potentially be missed based on current World Health Organization (WHO) case definitions. METHOD We report a retrospective case series of 10 surgical inpatients diagnosed with SARS following an outbreak in the surgical wards. Patients were included if they fulfilled the WHO case definition of probable SARS, had an active surgical problem and were admitted to the surgical wards during the outbreak period. Clinical histories, laboratory investigations and radiological findings were reviewed and analyzed. RESULTS The mean age of the cohort was 57.6 years (range: 38-78 years). Nine patients had concomitant medical conditions. Three patients were in the early postoperative period, while the remaining seven were admitted for surgical related infections. All patients presented with fever, but only eight had accompanying respiratory symptoms. Lymphopenia and raised lactate dehydrogenase (LDH) was seen in seven patients. Eight patients had positive bacterial cultures. The primary abnormality on chest radiograph was air-space opacification. Rapid progression of radiological changes was seen in seven patients. Mortality rate for our cohort is 20%. CONCLUSION The diagnosis of SARS in surgical patients differs from that previously described in normal patients. An apparent cause of fever and positive blood cultures cannot exclude a diagnosis of SARS. The current WHO case definition could result in delayed or even missed diagnosis. Early isolation of febrile patients with a positive contact history must be undertaken, even in the face of another identifiable cause.


Regulation of kinase activity.


title: Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation.

text: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. Since its associated morbidity and mortality have been postulated to be due to immune dysregulation, we investigated which of the viral proteins is responsible for chemokine overexpression. To delineate the viral and cellular factor interactions, the role of four SARS coronavirus proteins, including nonstructural protein 1 (nsp-1), nsp-5, envelope, and membrane, were examined in terms of cytokine induction. Our results showed that the SARS coronavirus nsp-1 plays an important role in CCL5, CXCL10, and CCL3 expression in human lung epithelial cells via the activation of NF-kappaB.


title: Assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the E1 glycoprotein of coronavirus mouse hepatitis virus A59.

text: The E1 glycoprotein of coronavirus mouse hepatitis virus A59 was synthesized in vitro by translation of viral mRNA in the presence of dog pancreatic microsomes. Its disposition in the membrane was investigated by digestion with proteases and by selective NH2-terminal labeling. The protein spans the membrane, but only small portions from the NH2 and COOH terminus are exposed respectively in the lumenal and cytoplasmic domains; the bulk of the molecule is apparently buried in the membrane. The protein lacks a cleavable leader sequence and does not acquire its characteristic O-linked oligosaccharides in rough microsomes. It may enter the membrane at any stage during synthesis of the first 150 amino acid residues. These unusual features of the protein might help to explain why it is not transported to the cell surface in vivo but remains in intracellular membranes, causing the virus to bud there.


title: Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease.

text: Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)), a protein required for the maturation of SARS-CoV, is vital for its life cycle, making it an attractive target for structure-based drug design of anti-SARS drugs. The structure-based virtual screening of a chemical database containing 58,855 compounds followed by the testing of potential compounds for SARS-CoV M(pro) inhibition leads to two hit compounds. The core structures of these two hits, defined by the docking study, are used for further analogue search. Twenty-one analogues derived from these two hits exhibited IC50 values below 50 microM, with the most potent one showing 0.3 microM. Furthermore, the complex structures of two potent inhibitors with SARS-CoV M(pro) were solved by X-ray crystallography. They bind to the protein in a distinct manner compared to all published SARS-CoV M(pro) complex structures. They inhibit SARS-CoV M(pro) activity via intensive H-bond network and hydrophobic interactions, without the formation of a covalent bond. Interestingly, the most potent inhibitor induces protein conformational changes, and the inhibition mechanisms, particularly the disruption of catalytic dyad (His41 and Cys145), are elaborated.


[Viral pneumonia].


title: Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model.

text: BACKGROUND The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. METHODS The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein. RESULTS The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. CONCLUSIONS The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).


title: Phenotypic and functional characterization of CD8+ T lymphocytes from the central nervous system of rats with coronavirus JHM induced demyelinating encephalomyelitis.

text: Intracerebral infection of Lewis (LEW) inbred rats with the neurotropic strain of the murine coronavirus JHM (JHMV) frequently results in a monophasic paralytic disease. In contrast, infection of Brown Norway (BN) inbred rats does not lead to clinical disease. Previous findings indicated that in both rat strains brain-infiltrating leukocytes consisted mainly of CD8+ T lymphocytes. Here, we phenotypically as well as functionally characterised this T cell subset after isolation from the central nervous system (CNS). Using JHMV-infected target cells, MHC class I restricted, cytotoxic T lymphocytes were demonstrated to be present in the leukocyte fraction from the CNS of both, susceptible LEW and disease-resistant BN rats. However, compared to infected, but healthy BN rats, diseased LEW rats generated an enhanced cytotoxic immune response which became most prominent at the maximum of neurological disease. Recently published observations from our laboratory demonstrated a strong virus-specific antibody response in the CNS of BN rats. In LEW rats, however, the response was delayed and of low magnitude. This suggests, that consequences of cytotoxic T lymphocyte action in JHMV-infected CNS tissue largely depend on the efficacy of an accompanying virus-specific humoral immune response.


title: ASSOCIATION BETWEEN MANNOSE-BINDING LECTIN GENE POLYMORPHISMS AND SUSCEPTIBILITY TO DENGUE VIRUS INFECTION: A PRELIMINARY REPORT.

text: Mannose-binding lectin (MBL) can bind with a wide range of pathogens and can activate through lectin pathway or enhances opsonophagocytosis. MBL is encoded by the MBL2 gene and single-nucleotide polymorphisms (SNPs) in the promoter and exon have functional effects on serum levels of MBL. MBL deficiency has been shown to predispose to infectious diseases. We assess whether or not, the variant MBL alleles are associated with susceptibility to dengue infection. Patients with confirmed dengue infection who were admitted to King Chulalongkorn Memorial Hospital during a calendar year were studied. Controls were patients without dengue infection. Deoxyribonucleic acid (DNA) was extracted from 50 μl of peripheral blood mononuclear cell (PBMC) using the DNA Blood Mini Kit. The SNPs in the promoter (-221 X/Y) and exon 1 (codon 54 A/B) of MBL2 gene were genotyped by using 2 separate cycling reactions of the TaqMan allele discrimination system. Serum levels of MBL were determined by double-antibody sandwich ELISA. Chi-square was used for statistical analysis. Serum MBL levels and genotypes were determined in 110 dengue patients (mean age 18.1 years; 62 males and 48 females) and 42 controls (mean age 25.8 years; males: females = 1:1). Our study showed that YB haplotype is associated with low serum levels of MBL. There was no association between MBL2 gene polymorphisms and susceptibility to dengue infection. The higher frequency of YB in dengue patients than in controls suggesting the likelihood of an association. Further studies are warranted.


title: Metered-dose inhaler with spacer instead of nebulizer during the outbreak of severe acute respiratory syndrome in Singapore.

text: BACKGROUND The use of metered-dose inhaler (MDI) with spacer instead of nebulizer may be important during an outbreak of an airborne infection. However, there is a paucity of data on patients' and nurses' abilities and perspectives on MDI with spacer for the treatment of acute airway obstruction during such an outbreak. METHODS We evaluated 50 consecutive MDI-with-spacer treatments administered in the respiratory wards of the National University Hospital of Singapore, and interviewed the patients after each treatment during the outbreak of severe acute respiratory syndrome (SARS). We also conducted interviews with 50 nurses who had experience in administering bronchodilators via both nebulizer and MDI with spacer. RESULTS Forty-six patients (92%) were able to use MDI with spacer effectively. Sixteen percent of the patients preferred nebulizer over MDI with spacer. Fifty-eight percent of the patients thought MDI with spacer was easier to use than nebulizer, and 34% thought MDI was as easy to use as nebulizer. Sixteen percent of the patients thought that nebulizer was more effective than MDI with spacer in relieving their symptoms. Ninety-six percent of the nurses preferred nebulizer over MDI with spacer. Forty-two nurses (84%) thought that nebulizer was more effective for treating acute airflow obstruction in the hospital. CONCLUSIONS In the in-patient setting during an outbreak of an airborne infection, for treatment of acute airflow obstruction, MDI with spacer was acceptable and preferred by a high percentage of patients. However, a high percentage of nurses had misconceptions regarding the efficacy of and patients' ability to use MDI with spacer.


title: Laparoscopic nephrectomy in a hemophilia B patient.

text: Surgery in patients with hemophilia is a serious challenge. It requires a comprehensive approach, as well as careful postoperative monitoring. We present here the first case of a transperitoneal laparoscopic radical nephrectomy (TLRN) for renal cell carcinoma, of the clear-cell type, performed in a hemophilia B patient. The level of factor IX clotting activity before surgery and on postoperative days 1-6 was maintained at 65-130% and at 30-40% on subsequent days until healing of the post-operative wound was achieved. The intraoperative and postoperative courses were uneventful. TLRN can therefore be considered safe and effective for renal cell carcinoma. In hemophilia patients, the TLRN procedure requires proper preparation, as well as adequate substitution therapy for the deficient coagulation factor provided by a multidisciplinary team in a comprehensive center.


title: Development of an empirical model to aid in designing airborne infection isolation rooms.

text: Airborne infection isolation rooms (AIIRs) house patients with tuberculosis, severe acute respiratory syndrome (SARS), and many other airborne infectious diseases. Currently, facility engineers and designers of heating, ventilation, and air-conditioning (HVAC) systems have few analytical tools to estimate a room's leakage area and establish an appropriate flow differential (DeltaQ) in hospitals, shelters, and other facilities where communicable diseases are present. An accurate estimate of leakage area and selection of DeltaQ is essential for ensuring that there is negative pressure (i.e., pressure differential [DeltaP]) between an AIIR and adjoining areas. National Institute for Occupational Safety and Health (NIOSH) researchers evaluated the relationship between DeltaQ and DeltaP in 67 AIIRs across the United States and in simulated AIIR. Data gathered in the simulated AIIR was used to develop an empirical model describing the relationship between DeltaQ, DeltaP, and leakage area. Data collected in health care facilities showed that the model accurately predicted the leakage area 44 of 48 times. Statistical analysis of the model and experimental validation showed that the model effectively estimated the actual leakage area from -39% to +22% with 90% confidence. The NIOSH model is an effective, cost-cutting tool that can be used by HVAC engineers and designers to estimate leakage area and select an appropriate DeltaQ in AIIRs to reduce the airborne transmission of disease.