H.G.M. Niesters

Clinical virology in real time

n Abstractn n The ability to detect nucleic acids has had and still has a major impact on diagnostics in clinical virology. Both quantitative and qualitative techniques, whether signal or target amplification based systems, are currently used routinely in most if not all virology laboratories. Technological improvements, from automated sample isolation to real time amplification technology, have given the ability to develop and introduce systems for most viruses of clinical interest, and to obtain clinical relevant information needed for optimal antiviral treatment options. Both polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) can currently be used together with real time detection to generate results in a short turn-around time and to determine whether variants relevant for antiviral resistance are present. These new technologies enable the introduction of an individual patient disease management concept. Within our clinical setting, we have introduced this e.g. for quantitative detection of Epstein–Barr Virus (EBV) in T-dell depleted allogeneic stem cell transplant patients. This enabled us to develop models for pre-emptive anti B-cell immunotherapy for EBV reactivation, thereby effectively reducing not the incidence of EBV-lymphoproliferative disease but the virus related mortality. Furthermore, additional clinically relevant viruses can now easily be detected simultaneously. It also becomes more feasible to introduce molecular testing for those viruses that can easily be detected using classical virological methods, like culture techniques or antigen detection. Prospective studies are needed to evaluate the clinical importance of the additional positive samples detected. It should however be made clear that a complete exchange of technologies is unlikely to occur, and that some complementary technologies should stay operational enabling the discovery of new viruses. The implementation of these molecular diagnostic technologies furthermore warrants the use and introduction of standardized materials as well as participation in international quality control programs. Finally, the use of an internal control throughout the whole procedure not only ensures the accuracy of the results generated, but also is necessary to enable precise quantification of these results and to determine detection thresholds more accurately. Since so many targets do have clinical implications, laboratories might prefer to use universal internal controls before the in-house developed assays should be introduced in clinical virology.n n

Prevalence of hepatitis E virus infection in liver transplant recipients

Hepatitis E virus (HEV) infection is known to run a self‐limited course. Recently, chronic hepatitis E has been described in several immunosuppressed patients after solid organ transplantation. The prevalence of HEV infection after transplantation, however, is unknown. We studied HEV parameters [HEV RNA, HEV immunoglobulin M (IgM), and HEV immunoglobulin G (IgG) by enzyme‐linked immunosorbent assay and confirmatory immunoblotting] in a cohort of 285 adult liver transplant recipients. The most recent freeze‐stored sera were investigated, and if they were positive, a retrospective analysis was performed. Samples from 274 patients (96.1%) tested negative for all HEV parameters. This included a patient described earlier as having experienced an episode of chronic HEV hepatitis in the past. One patient was found positive for HEV RNA without HEV antibodies. She presently suffers from chronic HEV hepatitis and has also been described before. Sera from 9 patients tested positive for HEV IgG without HEV IgM or HEV RNA. Six of these 9 patients (2.1% of the total) were found to have HEV IgG antibodies in retrospect related to an HEV infection at some time pre‐transplant as they also tested positive in a pretransplant serum sample. One of these 9 patients suffered in retrospect from a chronic HEV infection with mild hepatitis between 2 and 5 years after liver transplantation on the basis of the course of HEV RNA, IgM, and IgG, aminotransferases, and liver histology. Overall, the prevalence of acquired HEV hepatitis after liver transplantation was 1% in this cohort. We conclude that liver transplant recipients have a risk for chronic HEV infection, but the prevalence is low. Liver Transpl 15:1225–1228, 2009.

Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2b.

Hepatitis E virus (HEV) infections are known to run a self‐limiting course. Recently, chronic hepatitis E has been described in immunosuppressed patients after solid‐organ transplantation. Besides the general recommendation to lower the immunosuppressive medication in these patients, there is currently no specific treatment. We here describe the successful use of pegylated interferon alpha‐2b in the treatment of 2 liver transplant recipients who suffered a chronic HEV infection for 9 years (case A) or 9 months (case B). After 4 weeks of therapy, a 2‐log decrease (case A) and a 3‐log decrease (case B) in the viral load were observed. In case A, who received treatment for 1 year, serum viral RNA became undetectable from week 20 onward, and serum liver enzymes normalized completely. In case B, interferon was discontinued at week 16 because of a lack of a further decline in the viral load. However, 4 weeks after the cessation of therapy, viral RNA was no longer detectable in the serum, and this was probably related to a further decline in the immunosuppressive medication. Liver tests normalized completely. In both cases, no relapse has been noted so far. We conclude that pegylated interferon alpha‐2b may be useful in the treatment of chronic HEV infections in patients in whom the reduction of the immunosuppressive medication alone is not sufficient. Liver Transpl , 2010.

Upsurge of human enterovirus 68 infections in patients with severe respiratory tract infections

BACKGROUNDnEnterovirus 68 (EV68) belongs to species Human enterovirus D. It is unique among enteroviruses because it shares properties with human rhinoviruses. After the first isolation in 1962 from four children with respiratory illness, reports of (clusters of) EV68 infections have been rare. During the autumn of 2010, we noticed an upsurge of EV68 infections in the Northern part of the Netherlands in patients with severe respiratory illness.nnnOBJECTIVESnTo give a detailed description of the clinical and virological data of patients with EV68 infection identified in 2010, and compare these with data collected in 2009.nnnSTUDY DESIGNnWe systematically collected clinical data from patients with an EV68 infection detected in 2010. We added four patients with an EV68 infection from 2009. Further characterization of EV68 was performed by partial sequence analysis of the VP1 genomic region.nnnRESULTSnIn 2010, EV68 was identified as the only cause of respiratory illness in 24 patients, of which 5 had to be admitted to the intensive care unit. Sequence analysis revealed different lineages in the majority of EV68 detected in 2010 as compared to the 2009 isolates.nnnCONCLUSIONSnWe noticed an increase of EV68 infections and present clinical as well as sequence data, in which two distinct phylogenetic clusters could be identified.

An international external quality assessment of nucleic acid amplification of herpes simplex virus

BACKGROUNDnThere is an increasing awareness of the need for external quality control of diagnostic virology.nnnOBJECTIVESnTo assess the quality of nucleic acid amplification tests (NAT) of herpes simplex within Europe.nnnSTUDY DESIGNnHerpes simplex virus (HSV) proficiency panels were produced at the Swedish Institute for Infectious Disease Control on behalf of the European Union Concerted Action for Quality Control of Nucleic Acid Amplification in 1999 and 2000. Nine reference laboratories evaluated the production process. Each panel consisted of 12 coded samples with various concentrations of inactivated, freeze-dried HSV type 1 (HSV-1), and HSV type 2 (HSV-2), or negative controls. Positive samples included HSV-1 and HSV-2 in a range of concentrations (2 x 10(2) to 2 x 10(7) genome copies per ml) similar to those found in cerebrospinal fluids from patients with HSV encephalitis.nnnRESULTSnSixty-six participants reported a total of 76 data sets for panel 1, and 71 reported 78 data sets for panel 2. The majority of the participants employed qualitative in-house polymerase chain reaction (PCR) methods, either in a single, nested or semi-nested format. For panel 2, 9 laboratories reported use of real-time PCR in contrast to 3 for panel 1. Three laboratories submitted quantitative results on both panels. Thirty percent of the data sets had correct results for the entire panel 1. In 6 data sets (8%) a total of 11 false positive results were reported. For panel 2, 28% of the data sets had correct result. Nineteen false positive results were reported in 14 data sets (18%), but most of the incorrect results reflected a lack of test sensitivity.nnnCONCLUSIONSnThe relatively high frequency of false positive results and the large number of false-negative results, albeit at low copy number, stress the need for improvement in the quality of HSV NAT and for external quality control programmes.

The emergence of enterovirus D68 in a Dutch University Medical Center and the necessity for routinely screening for respiratory viruses

n Abstractn n Backgroundn Since August 2014, an increase in infections caused by enterovirus D68 (EV-D68) was reported in the USA and Canada, for the most part in children presenting with severe respiratory symptoms.n n n Objectivesn To determine whether an increase in severe EV-D68 respiratory infections was observed in our region.n n n Study designn Samples from patients with respiratory symptoms were screened for viral pathogens, including rhinovirus and enterovirus. Subsequently, samples positive for rhinovirus and enterovirus were routinely sequenced for phylogenetic analysis. Furthermore, an additional method was used to detect EV-D68 specifically.n n n Resultsn During the first three quarters of the year 2014, 1896 respiratory samples were analyzed; 39 (2%) of them tested positive for enterovirus. Eighteen samples tested positive for EV-D68, obtained from 16 different patients admitted to our hospital. Eleven were children below the age of 18, of whom five children needed intensive care treatment. The remaining five samples were from adults, who all had an underlying disease; three were transplant patients (heart, lung and renal transplantation), the other two had an underlying lung condition (COPD, asthma). Phylogenetic analysis showed a close relationship with the strains circulating currently in the USA, all belonging to the known EV-D68 genetic subtypes.n n n Conclusionsn We observed an increase of EV-D68 infections in our population, both in children as well as in adult. In 2014 there have been 16 cases so far, compared to none in 2011 and 2013 and a single case in 2012. Phylogenetic analysis identified two similar clusters as shown in the USA and Canada.n n

Development of hepatitis B virus resistance for lamivudine in chronic hepatitis B patients co-infected with the human immunodeficiency virus in a Dutch cohort

INTRODUCTIONnWith the introduction of HAART, the HIV-1 has turned from a lethal into a chronic infection in the majority of patients. In homosexual populations, 20% of HIV-1 infected patients suffer from a chronic HBV infection, which may eventually lead to complications of the liver disease because of prolonged survival. Lamivudine is effective in reducing both HIV-1 and HBV viral replication. However, resistance for lamivudine may complicate the course of the HBV disease in HIV-1-infected patients. We, therefore, conducted a retrospective study in HIV-1-HBV co-infected patients on lamivudine therapy.nnnPATIENTS AND METHODSnAll HIV-1-HBV co-infected patients who were treated with lamivudine for over 6 months in five major referral clinics in The Netherlands with HBV DNA above 2.0 x 10(5) geq ml(-1) at baseline, were evaluated. Retrospectively, the course of HBV DNA in available serum samples was established. If HBV DNA was detectable with the sensitive PCR-assay, YMDD-analyses of the polymerase gene of the hepatitis B virus was executed with the INNO-LiPA-DR-strip.nnnRESULTSnForty-six patients were evaluated. The median level of HBV DNA at start of lamivudine therapy was 1.31 x 10(9) geq ml(-1) (range 3.5 x 10(5) - 2.0 x 10(10), n=43). Of three patients no baseline sample was available, but since HBV DNA was still above 2.0 x 10(5) geq ml(-1) at week 3, 7 and 11, these patients were included. Median duration of lamivudine therapy was 97 weeks (range 27-263). The percentage of detected mutations was 25 and 52% at 1 and 2 years, respectively. Twenty-two patients ultimately developed a mutation. Both baseline Body Mass Index (BMI) and the decrease in CD4 cell count as a time dependent factor were significantly related to the emergence of mutations. In 10 out of 12 evaluated patients, HBV DNA levels returned to baseline level or even above baseline level after the development of mutant virus. One patient (5%) developed a flare of serum transaminases (ALT>10 x ULN) 24 weeks after first detection of variant virus.nnnCONCLUSIONnThere is a linear time-dependent appearance of HBV mutations for lamivudine in our population. In a minority of patients (5%), development of a mutation was followed by a significant elevation of serum transaminases. A decline in CD4 cell count, which may indicate less response to HAART, induces a faster emergence of mutations and close surveillance of HBV co-infected patients on therapy may be indicated due to the prolonged survival of HIV-1 patients.

Pediatric parechovirus infections

Human parechoviruses (HPeVs) are members of the large and growing family of Picornaviridae. Although 16 types have been described on the basis of the phylogenetic analyses of the VP1 encoding region, the majority of published reports relate to the HPeV types 1-8. In pediatrics, HPeV1, HPeV2 and HPeV4-8 mainly cause mild gastrointestinal or respiratory illness; only occasionally more serious diseases have been reported, including myocarditis, encephalitis, pneumonia, meningitis, flaccid paralysis, Reye syndrome and fatal neonatal infection. In contrast, HPeV3 causes severe illness in young infants, including sepsis and conditions involving the central nervous system. Currently, the most sensitive method for detecting HPeV is real-time polymerase chain reaction assays on stools, respiratory swabs, blood and cerebrospinal fluid. However, although it is known that HPeVs play a significant role in various severe pediatric infectious diseases, diagnostic assays are not routinely available in clinical practice and the involvement of HPeV is therefore substantially underestimated. Despite long-term efforts, the development of antiviral therapy against HPeVs is limited; no antiviral medication is available and the use of monoclonal antibodies is still being evaluated. More research is therefore needed to clarify the specific characteristics of this relevant group of viruses and to develop appropriate treatment strategies.

Paired measurements of quantitative hepatitis B virus DNA in saliva and serum of chronic hepatitis B patients: implications for saliva as infectious agent

BACKGROUNDnAfter intensive source and contact tracing 20 % of acute Hepatitis B virus (HBV) infections remain unexplained. Saliva may be an unexpected vehicle of HBV DNA transmission.nnnOBJECTIVEnTo further explore this hypothesis we evaluated the quantitative levels of HBV DNA in saliva and compared these with the HBV DNA levels measured in serum.nnnSTUDY DESIGNnSerum and saliva were collected from 27 chronic HBV patients attending our outpatient clinic.nnnRESULTSnThere were 16 men and 11 women; 15 patients were HBeAg positive, anti-HBe negative and 11 patients were HBeAg negative, anti-HBe positive. One patient was HBeAg and anti-HBe negative. Samples of serum and saliva were collected on the same day. All saliva specimens were clear on inspection. HBV DNA in serum was measured by the Digene Hybrid Capture II microplate assay (Digene Diagnostics), the HBV Monitor assay (Roche Diagnostics) as well as an in-house developed HBV DNA TaqMan assay. The HBV DNA TaqMan assay was used for the quantitative measurement of HBV DNA in saliva. Median HBV DNA levels in serum were 2.10 x 10(5) geq/ml and ranged from 373 genome equivalents per ml (geq/ml) to 4.13 x 10(9) geq/ml; median HBV DNA levels in saliva were 2.27 x 10(4) geq/ml and ranged from 373 geq/ml to 9.25 x 10(6) geq/ml. A clear correlation was shown between HBV DNA in serum and saliva; log HBV DNA in saliva=1.01 + 0.56 x (log HBV DNA in serum).nnnCONCLUSIONSnthis is the first report of precise quantitative measurements of HBV DNA levels in saliva and the relationship with HBV DNA levels in serum. Our findings show that saliva is a source of HBV DNA. This finding may have implications in selected patients for the infectivity of saliva and offer further insight in the routes of transmission of HBV infection.

European surveillance for enterovirus D68 during the emerging North-American outbreak in 2014

BACKGROUNDnIn August and September 2014, unexpected clusters of enterovirus-D68 (EV-D68) infections associated with severe respiratory disease emerged from North-America. In September, the European Centre for Disease Prevention and Control (ECDC) asked European countries to strengthen respiratory sample screening for enterovirus detection and typing in cases with severe respiratory presentations.nnnOBJECTIVESnTo provide a detailed picture of EV-D68 epidemiology in Europe by conducting a retrospective and prospective laboratory analysis of clinical specimens.nnnSTUDY DESIGNnAn initiative supported by the European Society for Clinical Virology (ESCV) and ECDC was launched to screen for EV-D68 in respiratory specimens between July 1st and December 1st 2014 in Europe and to sequence the VP1 region of detected viruses for phylogenetic analytic purposes.nnnRESULTSnForty-two institutes, representing 51 laboratories from 17 European countries, analyzed 17,248 specimens yielding 389 EV-D68 positive samples (2.26%) in 14 countries. The proportion of positive samples ranged between 0 and 25% per country. These infections resulted primarily in mild respiratory disease, mainly detected in young children presenting with wheezing and in immuno-compromised adults. The viruses detected in Europe are genetically very similar to those of the North-American epidemic and the majority (83%) could be assigned to clade B. Except for 3 acute flaccid paralysis (AFP) cases, one death and limited ICU admissions, no severe cases were reported.nnnCONCLUSIONSnThe European study showed that EV-D68 circulated in Europe during summer and fall of 2014 with a moderate disease burden and different pathogenic profile compared to the North-American epidemic.