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Clinical Virology

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The section Clinical Virology has a number of specific research projects.

Hepatitis E virus genotype 3 infection in immuno-compromised patients
Dr. A. Riezebos-Brilman, R. van der Heide, BSc, Prof. dr. H.G.M. Niesters
Collaborations:UMCG Department of Hepatology (Dr. E. Haagsma, Dr. A. van den Berg)
UMCG Department of Lungtransplantation (Dr. E. Verschuuren)
UMCG Department of Nephrology (Prof. dr. W. van Son).
Municipal Health Service Groningen (Dr. B. Wolters)
Medical University of Vienna, Department of Virology (Prof. E. Puchhammer-Stockl)
QCMD Glasgow, Dr. P. Wallace


Hepatitis E virus (HEV) has long been known as a major cause of acute hepatitis in developing countries, with occasional travel-related cases in developed countries. Until recently, HEV was considered as an agent responsible for an acute hepatitis, incapable of progressing to a chronic state. Travel-related HEV is mainly caused by HEV genotype 1 and 2, strictly human viruses. Recently, HEV genotype 3 infection is being recognized as a public health issue in developed countries and is believed to be a porcine zoonosis. There are now increasing numbers of reports showing that HEV genotype 3 infection may lead to a chronic hepatitis in immuno-compromised patients.

Besides the general recommendation to lower the immuno-suppressive medication in these immuno-compromised patients, there is currently no specific treatment option. However, both pegylated interferon alpha-2b and oral ribavirin have been described as potentially effective treatment of HEV infection. Early detection of HEV infection will be essential to minimize liver damage and maximize the effect of antiviral therapy.

Since second half of 2007, HEV diagnostics is performed in patients treated in the UMCG with unexplained hepatitis. HEV RNA is being detected in plasma, serum or feces using a real-time PCR method. Subsequently, the HEV is characterized by sequencing of the ORF1 region. Over 20 patients have now been diagnosed with hepatitis E virus infection in the UMCG. As described in the literature, viral genotyping in these infected HEV patients revealed only genotype 3 strains. The majority of the patients is immuno-compromised.

Little is known about the route of transmission and the relation with the zoonotic origin. Factors predicting the development of chronic infection in immuno-suppressed individuals exposed to HEV are not yet fully unraveled. Also the effect of antiviral treatment in different groups of immunocompromised patients is thus far indefinite. These issues are part of our investigations.

Publication and abstract:
Haagsma EB, Riezebos-Brilman A, van den Berg AP, Porte RJ, Niesters HG. Liver Transpl. 2010 Apr;16(4):474-7.
Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2b.

Abstract: 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).

The clinical significance of respiratory and enteric viruses in children.
Drs. J. Rahamat-Langendoen, Dr. A. Riezebos-Brilman, R. Poelman, R. Borger BSc, R. van der Heide BSc, Dr. T. Schuurman, Prof. dr. H.G.M. Niesters
Collaborations:UMCG Department of Infectious Disease, Beatrix Childrens Hospital (Dr. E. Scholvinck)
University of Groningen RuG, Department of Epidemiology (Prof.dr. E. Hak)

 
A prospective study is performed since 2009 to collect both diagnostic  and clinical data from patients admitted to the Beatrix Childrens Hospital. This allows us to predict more carefully the clinical parameters related to specific viral targets (currently focusing on respiratory viruses), to understand transmission routes within our hospitalized population (and nosocomial infections), as well as to determine what the value is of a relative viral load in clinical specimens.
Part of this work is related to the epidemiological characterization of viruses and to TYPENED information. Within several projects, sequence information is collected for enteroviruses, parechoviruses, noroviruses, influenza viruses, rhinoviruses.

Publication and abstract:
- Rahamat-Langendoen J, Riezebos-Brilman A, Borger R, van der Heide R, Brandenburg A,
Schölvinck E, Niesters HG.
J Clin Virol. 2011 Oct;52(2):103-6. Epub 2011 Jul 29.

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

BK virus in kidney transplant patients
Dr. Annelies Riezebos-Brilman, Lilli Gard, BSc, Prof. dr. H.G.M. Niesters
Collaborations:UMCG Department of Nephrology (Prof. dr. W. van Son).


BK virus belongs to the family Polyomaviridae. Polyomaviruses are small non-enveloped, double-stranded DNA viruses. Thus far, 6 human polyomaviruses are described; BK and JC virus, WU and KI virus, Merkel cell carcinoma virus and Trichodysplasia spinolosa virus. These viruses are ubiquitous human viruses and known to cause persistent or latent, but primarily asymptomatic, infections. In immunocompromised patients, however, these viruses may cause severe disease.

BK virus is urotheliotropic and its reactivation is linked to two major complications in transplant recipients. Polyomavirus-associated nephropathie (PyVAN) and polyomavirus-associated hemorrhagic cystitis (PyVHC). Most cases of PyVAN occur in kidney transplant patients. BK virus reactivation most likely results from reactivating BK virus in tubular epithelial cell of the donor kidney. Viruria can be detected in up to 80% of renal transplant patients and 5-10% will progress to PyVAN. Patients with PyVAN present with increasing renal dysfunction. Graft-failure is reported in 50-70% of the patients diagnosed with PyVAN.

Within the UMCG about 120 kidney transplantations are being performed every year. Routinely, urine and plasma samples of these patients will be screened for BK virus by quantitative real-time PCR assay. A BK-virus specific serology assay is being implemented to investigate the serostatus pre-transplantation. Four different BK-virus genotypes have been identified. For the differentiation of these variants, a multiplex RT-PCR is under development.

The significance of BK virus subtypes and serostatus in relation to particular clinical symptoms or to different groups of immunocompromised patients is part of our study.

The Development of FlowG, a MiddleWare solution for molecular diagnostics
Prof. dr. H.G.M. Niesters, F. Klijn BSc
Collaborations: NL: Labhelp, J. Poodt MSc.


A standard routine molecular diagnostic laboratory has currently implemented several real time amplification equipment, automated isolation devices and amplification set-up pipetting robots. Mostly devices from different vendors are present and all have a short technical life span. All these individual devices must be linked using an open MiddleWare software program, called Flow Groningen or FlowG.

The automation of molecular diagnostic hardware can be linked to a Laboratory Information System (ISMED) using Microsoft Access and Visual Basic for Applications. This includes text file based interaction between a database and different devices, the automated analysis of internal and run controls, as well as the validation of individual diagnostic parameters. The software is implemented around our laboratory routine diagnostics set-up.

PCR Job-files from our LIS enables the selection of both qualitative and quantitative assays. These are linked with internal controls, positive and negative controls for various isolation devices (EasyMag, MP32, MP96). Simultaneously, information for CAS1200/Qiagility pipetting robots is generated, which can be used for the PCR set-up on ABI7500 amplification machines. Primer and probe batches and control reagents (internal and positive controls) with specifications (95% confidence intervals) are used for the validation of the results. Out of range data are located separately, while the accepted values are interpreted electronically with visual green/red colors results. For quantitative  laboratory developed assays, values are automatically assigned based on validated and pre-calculated standard curves. Finally, using a PCR-RES file, the data are uploaded into our LIS. QC data are available in the database to enable time independent evaluation of reagents and controls

The implementation of this open software solution to connect equipment from different vendors has enabled us to reduce turnaround time, reduce mistakes, limits the use of copying results by using pen or pencil, enables the automated analysis and return of results, enabled the QC of laboratory developed tests (primer and probe batches, internal and positive controls). Further development of automation of laboratory processes into LEAN concepts are tested.