A novel PCR-based point-of-care method facilitates rapid, efficient, and sensitive diagnosis of influenza virus infection.

OBJECTIVE: The aim of this single-centre study was the comparative analysis of the GeneXpert (Cepheid Inc.) and the LIAT (Roche) system for the rapid polymerase chain reaction (PCR)-based detection of influenza A (IA) and influenza B (IB) viruses. PATIENTS AND METHODS: During the 2017-2018 flu season, 651 prospectively collected samples (throat and nasal swabs) of patients with symptoms of influenza-like illness or acute respiratory infection were tested for the presence of IA and IB viruses using the GeneXpert and LIAT systems. To evaluate the usefulness for near-patient testing, a LIAT system was installed at the Department of Emergency Medicine, and sample testing was performed on site. Reference testing of all samples was performed with the Xpert Flu assay and for 313 samples in addition with the Xpert Xpress Flu/RSV (respiratory syncytial virus) assay at the central laboratory. Analysis of all samples was carried out within 24 hr after collection. RESULTS: Overall, 267 of the 651 samples analysed were positive for influenza viruses in at least one of the three assays investigated (IA, 88; IB, 179). The overall rates of agreement between the LIAT assay and the Xpert Flu assay was 96.0% for the detection of IA and IB viruses. The sensitivity and specificity of the LIAT assay compared to the Xpert Flu assay for the detection of IA was 98.80% (95% confidence interval (CI) 93.47-99.97%) and 99.12% (95% CI, 97.96% to 99.71%) and for the detection of IB 98.76% (95% CI 95.58-99.85%), and 96.33% (95% CI 94.26-97.81%), respectively. The LIAT assay showed a statistically significant higher detection rate of IB virus than the Xpert Flu assay (p <0.01). No significant difference was found between the detection rate of the LIAT assay and the Xpert Xpress Flu/RSV assay. The mean time to the availability of a definite test result was significantly shorter with the on-site LIAT system than the GeneXpert system (mean 59 min saving time; p <0.01). CONCLUSION: The LIAT system represents a robust and highly sensitive point-of-care device for the rapid PCR-based detection of influenza A and influenza B viruses.

Viral determinants predicting hepatitis B surface antigen (HBsAg) seroclearance in HIV-/HBV-coinfected patients.

The aim of this retrospective study was the identification of clinically useful viral determinants for the prediction of hepatitis B surface antigen (HBsAg) seroclearance and sustained virological response in hepatitis B virus/human immunodeficiency virus (HBV-/HIV)-coinfected patients receiving HBV-active combined antiretroviral therapy (cART). Quantification of HBsAg, HBeAg and HBV DNA before and after initiation of HBV-active cART in a cohort of 59 HIV-/HBV-coinfected patients was performed. Calculations of receiver operating characteristics (ROC) and Kaplan-Meier analysis were used for the identification of predictors of HBsAg seroclearance for HBeAg-positive [HBeAg(+); n = 36] and HBeAg-negative [HBeAg(-);n = 23] patients. HBeAg(+) patients with an HBsAg on-treatment decline ≥ 1 log IU/mL per year achieved higher HBsAg loss rates (P = 0.0294), whereas the quantification of HBeAg had no predictive value for HBsAg seroclearance. Among HBeAg(-) patients, a pretreatment baseline cut-off level of HBsAg ≤ 100 IU/mL was highly predictive for HBsAg seroclearance. No significant influence of the HBV genotype on HBsAg seroclearance was observed among the entire cohort. Quantitative determination of HBsAg provides a clinically useful viral parameter for the prediction of HBsAg seroclearance both in HBeAg(+) and HBeAg(-) HIV-/HBV-coinfected patients receiving HBV-active cART.

Validation of the modified hemagglutination inhibition assay (mHAI), a robust and sensitive serological test for analysis of influenza virus-specific immune response.

BACKGROUND: The hemagglutination inhibition assay (HAI) is universally regarded as the gold standard in influenza virus serology. Nevertheless, difficulties in titre readouts are common and interlaboratory variations are frequently reported. OBJECTIVE: We developed and validated the modified HAI to facilitate reliable, accurate and reproducible analysis of sera derived from influenza vaccination studies. STUDY DESIGN: Clinical and preclinical serum samples, NIBSC reference sera and seasonal influenza virus type A (H1N1 and H3N2) and type B antigens were employed to validate the mHAI. Moreover, pandemic virus strains (H5N1 and H1N1pdm09) were used to prove assay robustness. RESULTS: Utilisation of a 0.08% solution of stabilised human erythrocytes, assay buffer containing bovine serum albumin and microscopical plate readout are the major differences between the modified and standard HAI assay protocols. Validation experiments revealed that the mHAI is linear, specific and up to eightfold more sensitive than the standard HAI. In 95.6% of all measurements mHAI titres were precisely measured irrespective of the assay day, run or operator. Moreover, 96.4% (H1N1) or 95.2% (H3N2 and B), respectively, of all serum samples were determined within one dilution step of the nominal values for spiked samples. Finally, the mHAI results remained unaffected by variations in virus antigens, erythrocytes, reagents, laboratory location, sample storage conditions or matrix components. CONCLUSION: The modified HAI is easy to analyse, requires only a single source of erythrocytes and allows utilisation of numerous influenza virus antigens, also including virus strains which are difficult to handle by the standard HAI (e.g. H3N2, H5N1 and H1N1pdm09).

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.

Antibodies induced with recombinant VP1 from human rhinovirus exhibit cross-neutralisation.

Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations.

Relative seroprevalence of human herpes viruses in patients with chronic lymphocytic leukaemia.

BACKGROUND: Herpes virus infections may have a significant role in chronic lymphocytic leukaemia (CLL) due to their ability to modulate the host's immune system. MATERIALS AND METHODS: We examined the seroprevalence of four herpes viruses [Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), human herpes virus (HHV)-6 and -7] in a cohort of European CLL patients (cohort 1, n = 100) in relation to the immunoglobulin variable heavy (IGHV) chain gene use and compared serological results with those obtained from age- and gender-matched healthy adults (n = 100). RESULTS: CMV-seroprevalence was significantly higher in CLL cohort 1 (79%) than in the control cohort (57%, P = 0.001); the seroprevalence of EBV (89% vs. 94%), HHV-6 (73% vs. 60%), or HHV-7 (35% vs. 35%) was not. In CLL cohort 1, use of IGHV3-30 was more prevalent among CMV-seropositive and of IGHV3-21 among HHV-7-seronegative cases. To investigate the generalizability of these findings, we investigated the herpes virus seroprevalence in a second cohort of age-matched CLL patients from a different geographical area (USA, n = 100, cohort 2). In cohort 2, CMV-seroprevalence was comparable with that of the control cohort (53%). Seroprevalence of EBV, HHV-6 and HHV-7 were 85%, 88% and 73% respectively. In CLL cohort 2, use of IGHV3-30 or IGHV3-21 was not associated with any of the herpes viruses investigated. CONCLUSIONS: CMV-seropositivity is associated with CLL in selected patient cohorts. However, the considerable variation in herpes virus-specific seropositivity between geographically distinct CLL cohorts indicates that seropositivity for any of the four human herpes viruses investigated is not generally associated with CLL.

Near-patient assays for diagnosis of influenza virus infection in adult patients.

Rapid and reliable diagnosis of influenza is essential for identification of contagious patients and effective patient management. Near-patient assays allow establishment of the diagnosis within minutes in young children, and this study aimed to evaluate near-patient assays in relation to the patient's age. A total of 194 patients with laboratory-confirmed influenza A/H3N2 virus infection, diagnosed within a prospective cohort study, were included. Cryopreserved nasopharyngeal swabs collected from these patients were tested by four near-patient assays (Binax Now Influenza A&B, Quick S-Influ A/B, Influ-A&B Respi-Strip, and Actim Influenza A&B). The main outcome measure was sensitivity of the near-patient assays in relation to the age of patients. The Binax Now, Quick S-Influ, Influ-A&B Respi-Strip and Actim assays had overall sensitivities of 19%, 18%, 26%, and 40%, respectively. The estimated sensitivity for influenza A/H3N2 virus detection in nasopharyngeal swabs was 17-56% in children 1 year of age and decreased to 8-22% in patients 80 years of age (logistic regression). The sensitivity of the Influ-A&B Respi-Strip and Actim assays decreased significantly with increasing age (p 0.014 and p 0.033, respectively (logistic regression)), a trend for decrease was observed for the Binax Now assay (p 0.074 (logistic regression)), and the low sensitivity of the Quick S-Influ assay was similar in children and adults. Less than one-fourth of diagnosed influenza A/H3N2 virus infections can be identified in elderly patients using a near-patient assay. Consequently, near-patient assays are of limited value for confirming the diagnosis when influenza is clinically suspected in adults. Antiviral therapy and additional diagnostic procedures cannot be withheld on the basis of a negative near-patient assay result, particularly in adult patients.

Mumps outbreak in young adults following a festival in Austria, 2006.

Mumps is not a mandatorily notifiable disease in Austria. However, in the first week of May 2006, a sudden increase in serologically confirmed cases of mumps, confined to three public health districts of the southern Austrian province of Carinthia, was identified by the Austrian Reference laboratory for MMR. An epidemiological investigation of this cluster of mumps cases was performed. A total of 214 cases fulfilled the outbreak case definition; 143 cases were laboratory confirmed and 71 cases were epidemiologically linked and fulfilled the clinical picture of the case definition. The vaccination status was known for 169 patients. Nearly half of the cases for whom the vaccination status was known occurred in non-vaccinated persons, another 40% were vaccinated with one dose of the vaccine and 11% had received two doses. Only four mumps cases occurred in children aged 14 years or younger, indicating that the vaccination coverage and the acceptance of the recommended childhood vaccinations have strongly improved within the past 15 years. Vaccination scheme failure but not vaccine failure is primarily to blame for this mumps outbreak.

A human case of travel-related rabies in Austria, September 2004.

A young male Austrian tourist, aged 23 years and unvaccinated against rabies, was bitten by a dog in Morocco in July 2004. One month later he was hospitalised in Ceuta with symptoms compatible with rabies. He died on 23 September in an Austrian hospital after a diagnosis of rabies was confirmed by FAT, IHC and RT-PCR (including sequencing) of the neck skin and the RT-PCR (including sequencing) of the pharyngeal swab. This Austrian case of laboratory confirmed rabies highlights the urgent need for reinforcement of the international recommendations for travel vaccinations.

A human case of travel-related rabies in Austria, September 2004.

A young male Austrian tourist, aged 23 years and unvaccinated against rabies, was bitten by a dog in Morocco in July 2004. One month later he was hospitalised in Ceuta with symptoms compatible with rabies. He died on 23 September in an Austrian hospital after a diagnosis of rabies was confirmed by FAT, IHC and RT-PCR (including sequencing) of the neck skin and the RT-PCR (including sequencing) of the pharyngeal swab. This Austrian case of laboratory confirmed rabies highlights the urgent need for reinforcement of the international recommendations for travel vaccinations.

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.

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.

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.

Reduced interferon-gamma expression in peripheral blood mononuclear cells of infants with severe respiratory syncytial virus disease.

We examined the in vivo cell-mediated immune response in infants with respiratory syncytial virus (RSV) infection in order to gain information about the pathogenesis of severe RSV disease in infancy. Semiquantitative reverse transcription-polymerase chain reaction and three-color flow cytometry were used to determine the levels of messenger RNA (mRNA) for interferon (IFN)-gamma in peripheral blood mononuclear cells, and the distribution of lymphocyte subsets in infants with acute RSV infection. The findings were correlated with the severity of the patients' illness and the production of RSV-specific IgE antibodies (RSV-IgE). Significantly lower IFN-gamma levels and T-lymphocyte counts in the acute phase of illness were observed in infants with severe RSV disease than in those with a milder clinical course of illness. The induction of RSV-IgE was not related to IFN-gamma levels in the acute phase of illness, but rather correlated with IFN-gamma expression during convalescence. The data indicate that reduced IFN-gamma expression may be an important factor in the pathogenesis of severe RSV disease in infancy.

Nephropathia epidemica and Puumala virus in Austria.

To study the epidemiology of hantavirus infections in Austria, 1215 humans and 596 rodents of different species were tested for the presence of antibodies against Puumala and Hantaan virus. Direct virus identification by polymerase chain reaction in lung tissue of serologically positive rodents was performed to verify antibody results and to determine the genetic identity of viral RNA by phylogenetic analysis of a part of the hantavirus M segment. For 32 of the 37 cases of nephropathia epidemica diagnosed in Austria, the location where transmission took place could be traced to specific areas in the Austrian federal states of Carinthia and Styria. The overall seroprevalence in humans was 1.2% and ranged from 0.02% in Villach, Carinthia, to 0.8% in Korneuburg, Lower Austria, and 1.8% in Wolfsberg, Carinthia. Virus RNA could be amplified from three Clethrionomys glareolus voles collected in Klippitztörl, Carinthia, and from one collected in Ernstbrunn, Lower Austria. The sequences were all identified as Puumala virus by phylogenetic analysis and were found to be most closely related to the western European Puumala viruses from Germany and France. No evidence of the existence of Hantaan-like infections and viruses in Austria was found.

Antigenic and genomic diversity of central European respiratory syncytial virus strains.

Thirty-two RSV strains recovered during the winter months of 1987/88 to 1993/94 from hospitalized children in Vienna, Austria and Zagreb, Croatia were analysed for antigenic and genetic variations. Twenty-nine of the 32 isolates investigated belonged to group A and 3 to group B, with the majority of infections caused by subgroup A1 (21 of 29). Restriction endonuclease mapping of PCR products derived from parts of the N and G gene of 18 group A strains identified 3 distinct lineages, very similar to those defined by analysis of recurrent epidemics in Birmingham, United Kingdom during the same period. Results of this study provide further information on the global pattern of RSV and show that very similar viruses are present simultaneously in widely separated areas.

CMV-viraemia during allogenic bone marrow transplantation in paediatric patients: association with survival and graft-versus-host disease.

Sixty-one consecutive paediatric patients undergoing allogenic bone marrow transplantation (BMT) were screened prospectively for cytomegalovirus (CMV)-viraemia by PCR. Sixteen patients (26%) presented with single or recurrent CMV-viraemia between day -7 and + 100. Although only four of them had evidence of CMV-disease, there was a significant difference in the incidence of acute Graft versus Host Disease (GVHD) grade III-IV (75% vs 15.5%), liver-involvement (68% vs 13%) and the incidence of chronic GVHD (83% vs 13.8%) between CMV-PCR-positive and CMV-PCR-negative patients. Transplant-related mortality (TRM) was 43.7% in the CMV-PCR-positive group versus 13% in patients which had no evidence for CMV-viraemia. In all but one cases mortality in CMV-PCR positive patients was GVHD-associated. Pre-emptive therapy with gancyclovir in case of CMV-viraemia seemed to have no impact on incidence and severity of GVHD.

Increase in CD23+ B cells in infants with bronchiolitis is accompanied by appearance of IgE and IgG4 antibodies specific for respiratory syncytial virus.

Infection with respiratory syncytial virus (RSV) may induce asthma-like symptoms and RSV-specific IgE in infected infants as a result of Th2-like response to RSV. The effect of RSV infection on the expression of B cell antigens CD21 and CD23, putative participants in Th2 responses, was investigated. Samples from bronchiolitic infants (n = 19) were tested by three-color immunofluorescence flow cytometry during the acute phase of infection and 4-6 weeks later. In 6 of 10 RSV-positive infants, the percentage of CD23+ B cells was higher than in 9 RSV-negative children and in controls. Both CD21+ and CD21- B cells exhibited a higher percentage of CD23. The group with increased expression of CD23 antigen had RSV-specific IgE and IgG4 antibodies. These findings corroborate the hypothesis that RSV could provoke a Th2-type response, but the relationship between CD23 antigen and RSV infection must be determined.