First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.

BACKGROUND: Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. METHODS: For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. RESULTS AND CONCLUSION: A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.

Successful management of the first reported case in Austria of COVID-19 with ARDS.

We report the successful management of a patient with severe respiratory failure due to COVID-19 admitted to an intensive care unit complicated by secondary catheter-related infection of Candida glabrata. We are discussing some of the clinical challenges and the pitfalls in molecular diagnosis of SARS-CoV-2, including the fact that a positive PCR result may not always reflect infectiousness.

West Nile virus lineage 2 infection in a blood donor from Vienna, Austria, August 2014.

Eastern Austria is neighbouring regions with ongoing West Nile virus (WNV) transmissions. Three human WNV infections had been diagnosed during the past decade in Austria. The Austrian Red Cross Blood Service (ARC-BS) started a first voluntary screening for WNV in blood donors from Eastern Austria by Nucleic Acid Testing (NAT) in June 2014. This is also the most extensive WNV surveillance programme in humans in Austria so far. In August 2014, one autochthonous WNV infection was detected in a blood donor from Vienna. By now, one in 67,800 whole blood donations was found to be positive for WNV RNA.

Detection of HHV-6-specific mRNA and antigens in PBMCs of individuals with chromosomally integrated HHV-6 (ciHHV-6).

After inheritance of chromosomally integrated HHV-6 (ciHHV-6), viral DNA is found in every nucleated cell. The prevalence of ciHHV-6 is estimated to be 0.2-5% of humans. There are conflicting data on the potential for replication, possibly leading to clinical implications. We analysed peripheral blood mononuclear cells (PBMCs) from individuals with ciHHV-6 proven by fluorescence in situ hybridization (FISH) for HHV-6-specific mRNA (U94, U42, U22) and antigens by means of reverse transcription PCR and an indirect immunoperoxidase staining. U94 transcripts indicative of latent infection were detected in six (54.5%) out of 11 individuals at least once. Transcripts indicative of lytic infection (i.e. U42 and U22) were detected in four (36.4%) out of 11 individuals at least once. HHV-6 antigen was detected in seven (70%) out of 10 individuals at least once. The presence of viral mRNA and proteins supports virus gene expression from ciHHV-6, which may lead to virus replication. Considering the properties of active HHV-6 infection together with obvious replicative activity in individuals with ciHHV-6, pathophysiological effects leading to clinical consequences of chromosomally integrated viral DNA might be considered.

Retrospective identification of human cases of West Nile virus infection in Austria (2009 to 2010) by serological differentiation from Usutu and other flavivirus infections.

There is increasing evidence for the spread of West Nile virus (WNV) in southern, eastern and central Europe. In parallel, another flavivirus, the antigenically closely related Usutu virus, was introduced from Africa and first detected in Austria (2001), followed by Spain (2003), Hungary (2005), Italy (2006), Switzerland (2006) and Germany (2007). In Austria, human WNV infections have not previously been documented, although the virus was isolated from birds and detected in mosquitoes in 2008 and 2009. We therefore conducted a retrospective search for human cases of WNV infection using serum and cerebrospinal fluid samples collected from patients with central nervous system (CNS) disease in the summers of 2009, 2010 and 2011. Although all samples were negative for WNV by polymerase chain reaction, quantitative evaluation of standardised antibody assays with purified flavivirus antigens (including Usutu virus, which cross-reacts with WNV even in neutralisation assays) provided serological evidence for three autochthonous WNV infections in Austria: two in 2009 and one in 2010. Our data highlight the importance of raising awareness of WNV infections in Austria and neighbouring countries and suggest including testing for this infection in routine diagnostic practice of CNS diseases.

Rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus A(H1N1), A(H1N1)2009, and A(H3N2) subtypes by melting point analysis.

The high mutation rate of influenza virus, combined with the increasing worldwide use of influenza virus-specific drugs, allows the selection of viruses that are resistant to the currently available antiviral medications. Therefore, reliable tests for the rapid detection of drug-resistant influenza virus strains are required. We evaluated the use of a procedure involving real-time polymerase chain reaction (PCR) followed by melting point analysis (MPA) of hybrids formed between the PCR product and a specific oligonucleotide probe for the identification of point mutations in the influenza A virus neuraminidase gene (NA) that are associated with oseltamivir resistance [resulting in the amino acid change H275Y for seasonal and pandemic influenza A(H1N1) viruses and E119V for A(H3N2) viruses]. Therefore, 54 seasonal A(H1N1) (12 oseltamivir-resistant and 42 sensitive strains), 222 A(H1N1)2009 (5 resistant, 217 sensitive), and 51 A(H3N2) viruses (2 resistant, 49 sensitive) were tested by MPA, and the results were compared to those obtained by sequencing the NA gene. The results clearly indicate that the identification of drug resistance mutations by MPA is as accurate as sequencing, irrespective of whether MPA is performed using clinical material or the corresponding isolate. MPA enables a clear identification of mutations associated with antiviral resistance.

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

Measles outbreak linked to a minority group in Austria, 2008.

We report on a measles outbreak originating in an anthroposophic community in Austria, 2008. A total of 394 (94.9%) cases fulfilled the outbreak case definition including 168 cases affiliated to the anthroposophic community. The source case was a school pupil from Switzerland. The Austrian outbreak strain was genotype D5, indistinguishable from the Swiss outbreak strain. A school-based retrospective cohort study in the anthroposophic school demonstrated a vaccine effectiveness of 97.3% in pupils who had received a single dose of measles-containing vaccine and 100% in those who had received two doses. The vaccination coverage of the cases in the anthroposophic community was 0.6%. Of the 226 outbreak cases not belonging to the anthroposophic community, the 10-24 years age group was the most affected. Our findings underline the epidemiological significance of suboptimal vaccination coverage in anthroposophic communities and in older age groups of the general population in facilitating measles virus circulation. The findings of this outbreak investigation suggest that the WHO European Region is unlikely to achieve its 2010 target for measles and rubella elimination.

Measles outbreak in Styria, Austria, March-May 2009.

In the last week of March 2009, five measles cases among students of an anthroposophic school were reported to the public health authorities in the Austrian province of Styria where only five cases had been reported in the whole of 2008. A descriptive epidemiological investigation of the measles outbreak was performed. Between 2 March and 10 May 2009, 37 cases of measles were identified in Styria: 33 confirmed outbreak cases and four probable outbreak cases. The measles outbreak spread from the general population (12 cases) to an anthroposophic community (25 cases). Cases outside of the anthroposophic community were mostly over 10 years of age (10/12). Thirty-five cases were unvaccinated, and two of the 37 had received one dose of measles, mumps, rubella vaccine. Following a measles outbreak in Salzburg in 2008 with 394 cases, this outbreak reemphasises the continued need for additional vaccination campaigns in population groups over the age of 10 years.

Ongoing rubella outbreak in Austria, 2008-2009.

Since October 2008, a total of 143 cases of rubella have affected the two Austrian provinces Styria and Burgenland. The index case occurred in mid-October 2008, but was not notified to the public health authorities until February 2009, when the Austrian Agency for Health and Food Safety was asked to investigate a cluster of 32 rubella cases (24 laboratory-confirmed and eight clinically suspected cases). No case of rubella had been reported in the two affected provinces between February 2007 - when statutory notification for rubella was implemented - and mid-October 2008. 113 of the 143 cases (79%) were confirmed: 101 (89.3% of the 113 cases) clinical-laboratory confirmed and 12 clinical-epidemiological confirmed. Thirty cases fulfilled the criteria of a probable outbreak case only (laboratory results or data on epidemiological link are pending). For 140 outbreak cases data on age was known; the median age was 19 years (range: 2-60 years). 20 cases occurred in soldiers in seven military camps in the area. 55 cases (38.5 %) were female. One case of a laboratory-confirmed rubella infection, affecting an unvaccinated pregnant 18-years old native Austrian in the early first trimenon of pregnancy, led to voluntary abortion

Human parvovirus B19: a new emerging pathogen of inflammatory cardiomyopathy.

The human parvovirus B19 (PVB19), an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes such as hydrops fetalis, is the only known human pathogenic parvovirus so far. Although enteroviruses have long been considered the most common cause of inflammatory cardiomyopathy, PVB19 is emerging as a important candidate. Recent studies have indicated an association of PVB19 with paediatric and adult inflammatory cardiac disease. However, whether or not PVB19 has an impact on inflammatory cardiomyopathy in adult patients is still unclear. The first hints for a possible aetiopathogenetic role of the PVB19-infection and the development of cardiac dysfunction were demonstrated by molecular biology utilizing in situ hybridization (ISH) and polymerase chain reaction (PCR). According to available evidence, PVB19-associated inflammatory cardiomyopathy is characterized by infection of endothelial cells of small intracardiac arterioles and venules, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, and penetration of inflammatory cells into the myocardium.

Decreased interferon-gamma response in respiratory syncytial virus compared to other respiratory viral infections in infants.

An inappropriate interferon-gamma response has been implicated in the pathogenesis of severe respiratory syncytial virus (RSV) lower respiratory tract illness (LRTI). To assess whether this is unique for RSV primary LRTI compared to a first non-RSV LRTI, intracellular interferon-gamma was determined by flow cytometry in peripheral blood mononuclear cells from 32 infants with a primary RSV infection, 28 with a first non-RSV LRTI due to adenoviral, parainfluenzaviral and rhinoviral infection and 13 healthy infants. Interferon-gamma responses were increased significantly during adenoviral, parainfluenzaviral and the majority of the rhinoviral infections, but remained low during RSV and severe rhinoviral infection. Low interferon-gamma responses were associated with a more severe clinical course of LRTI. This indicates that depending on the nature of the viral pathogen, respiratory virus infections in infants differ significantly with regard to the quantity of the interferon-gamma production and that this may contribute to the clinical course of the disease.

Pre-emptive treatment of CMV DNAemia in paediatric stem cell transplantation: the impact of recipient and donor CMV serostatus on the incidence of CMV disease and CMV-related mortality.

Cytomegalovirus (CMV) DNAemia was detected by PCR in 30/125 (24%) consecutive paediatric patients undergoing allogeneic stem cell transplantation. All patients with CMV DNAemia received pre-emptive ganciclovir until two consecutive negative results were obtained. CMV-IgG-positive patients (R+) had a significantly increased risk of DNAemia as compared to CMV-IgG-negative (R-) patients (62% vs 8%) P<0.0001. The incidence of DNAemia was 71% (10/14) in R+ transplanted from seronegative donors (D-) compared to 54% (13/32) in those transplanted from seropositive donors (D+). Of 30 (40%) children with DNAemia, 12 developed CMV disease despite pre-emptive treatment. The overall incidence of disease was 0% (0/59) for R-/D-, 9% (3/23) for R+/D+, 7% (2/29) for R-/D+ and 57% (8/14) for R+/D-. In patients with DNAemia, 4/20 (20%) patients with D+ and 8/10 (80%) with D- became symptomatic. In the multivariate analysis of both groups, patients at risk (R+ and/or D+) and patients with DNAemia, a negative donor serostatus was the only factor associated with a significantly increased incidence of disease. Seven of 9 patients with lethal CMV disease had received CMV-IgG-negative grafts. The data suggest that in CMV seropositive recipients donor CMV seropositivity is associated with a reduced incidence of CMV disease and a favourable outcome following pre-emptive treatment.

Screening for possible failure of herpes simplex virus PCR in cerebrospinal fluid for the diagnosis of herpes simplex encephalitis.

The objectives of this study were to evaluate the reliability of herpes simplex virus (HSV) PCR testing in cerebrospinal fluid (CSF) for the detection of herpes simplex encephalitis. This was done by examining retrospectively the clinical follow-up of a large group of patients tested routinely by HSV-PCR. In addition, an attempt was made to assess the incidence of herpes simplex encephalitis in a central European population. CSF samples from 1,427 patients from all Vienna hospitals were submitted for HSV-PCR testing during a period of 4 years and 8 months. Herpes simplex encephalitis was detected by PCR in 12 cases and by serological methods in one additional patient. Retrospective analysis of the course of disease, which was possible in 799 PCR-negative patients, led to the identification of three additional cases in which herpes simplex encephalitis appears to have occurred despite negative PCR results. Failure of the PCR in these patients is most likely due to the time of obtaining CSF during the course of disease. A high specificity of the assay was demonstrated by the lack of false positive results in any of the 708 cases in which other causes for the neurological symptoms had been identified in the follow-up. The incidence of herpes simplex encephalitis in the population of Vienna was between 1 case/469,000-577,000 individuals/year. The highest annual incidence was detected in the age group between 3 months and 3 years, which, however, could not be confirmed statistically.

Comparison of sequence analysis and the INNO-LiPA HBV DR line probe assay for detection of lamivudine-resistant hepatitis B virus strains in patients under various clinical conditions.

A line probe assay (INNO-LiPA HBV DR) detecting drug-resistant hepatitis B virus (HBV) strains was evaluated. Results concordant with sequence analysis were obtained with 48 of 56 serum samples from HBV-infected patients undergoing lamivudine therapy. In eight cases, additional minor subpopulations could be identified by the line probe assay.

Early detection of acute rhinovirus infections by a rapid reverse transcription-PCR assay.

The development of a rhinovirus (RV)-RNA-specific reverse transcription (RT)-PCR assay is complicated by the close homology between the RV and enterovirus (EV) genomes in the highly conserved 5'-noncoding region, which is chosen for primer design in most RT-PCR assays. We have developed a sensitive, rapid, and RV-specific nested RT-PCR assay and have used it to test nasopharyngeal aspirates from 556 patients presenting with acute respiratory tract infections. RV RNA was detected by nested RT-PCR not only in all of 52 samples that were RV positive by virus isolation methods but also in 124 of 367 samples that were negative by virus isolation methods and enzyme-linked immunosorbent assay (ELISA). In addition, in 23 of 137 samples that were positive for a different respiratory virus by virus isolation and/or ELISA, RV RNA was detected by RT-PCR. EVs, adenoviruses, respiratory syncytial viruses, coronaviruses, and influenza and parainfluenza viruses, including clinical isolates as well as stock viruses, were not amplified in our RV-specific RT-PCR assay, indicating that this assay was highly specific. The processing time was less than 2 days for the RT-PCR, as opposed to up to 2 weeks for virus isolation. These results indicate that nested RT-PCR is more sensitive than conventional methods for the detection of RV in patients experiencing acute respiratory tract infections and represents the only reliable tool for the early laboratory diagnosis of RV infections. This is especially important in light of new opportunities for therapy currently being developed.

Changes of endothelial nitric oxide synthase level and activity during endothelial cell proliferation.

The goal of this study was to investigate the effect of endothelial cell proliferation on the expression and activity of endothelial nitric oxide synthase (eNOS). Bovine atrial endothelial cells (BAtEC) were studied between day 1 and 6 after seeding. During this period the number of cells in S-phase decreased progressively, while cell number and protein content increased, reaching a maximum at confluence (day 4). Expression of eNOS (determined by ELISA) and eNOS activity (determined by L-arginine to L-citrulline conversion) increased with culture duration with a maximum at confluence. Nitric oxide (*NO) release from BAtEC was determined after stimulation with Ca2+ ionophore A23187 (10 microM, 30 min) by .NO chemiluminescence in the absence of a chemical reduction system. Total *NO release (measured in the presence of 100 U/ml superoxide dismutase) did not change with state of cell proliferation/growth, whereas "bioavailable" *NO (measured in the absence of superoxide dismutase) was low in highly proliferating BAtEC. Relative eNOS activity (.NO and L-citrulline production per eNOS protein) was highest in proliferating BAtEC. The novel finding of this study is that the specific eNOS activity is upregulated in proliferating BAtEC and downregulated in quiescent BAtEC. The amount of "bioavailable" *NO is determined by eNOS activity and *NO inactivation (probably by superoxide), both high in proliferating BAtEC.

Monitoring the virus load can predict the emergence of drug-resistant hepatitis B virus strains in renal transplantation patients during lamivudine therapy.

The development of resistant hepatitis B virus (HBV) strains during lamivudine treatment has been described repeatedly. To investigate whether the development of such resistant HBV strains can be predicted in an early phase of therapy, the HBV loads of 11 renal transplantation patients were screened at 3-month intervals by a quantitative HBV polymerase chain reaction (PCR) assay. Lamivudine resistance was detected by sequence analysis. Five patients developed resistance to lamivudine in the 12-15-month follow-up period. In all of them, a virus load of 1x103 HBV DNA copies still was detectable after 3 months of therapy. This was statistically significantly different from those patients who did not develop lamivudine resistance within the observation period, all of whom had no HBV DNA detectable after 3 months of treatment (P=.0022). Thus, virus load testing by use of a sensitive PCR assay allows the early prediction of the emergence of lamivudine-resistant HBV strains.

Identification of selective estrogen receptor modulators by their gene expression fingerprints.

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.