Laboratory Products
Clinical Evaluation of a Multiplex Real-time PCR Assay for the Detection and Quantifi cation of Hepatitis E Virus
Heiner Wedemeyer1 , Birgit Bremer1 , William F. Carman2 , Camilla Gruhn2 , Madeleine Heckmann2 , Miriam Steimer2
1. Medizinische Hochschule Hannover, Klinik für Gastroenterologie, Hepatologie und Endokrinologie, Carl-Neuberg-Str. 1, 30625 Hannover 2. Fast Track Diagnostics Luxembourg S.a.r.l, 29, rue Henri Koch, Esch-sur-Alzette L-4354, Luxembourg
Every year an estimated 20 million individuals are infected by the hepatitis E virus. Over 3.3 million symptomatic cases of hepatitis E and 56,600 hepatitis E related deaths occur as a result [1]. Hepatitis E is a liver disease caused by the hepatitis E virus: a non-enveloped, positive-sense, single-stranded ribonucleic acid (RNA) virus transmitted mainly through the faecal-oral route, via contaminated water and some foods such as pork.
Following exposure to the hepatitis E virus the incubation period ranges from 3 to 8 weeks, and the virus can cause both acute sporadic and epidemic viral hepatitis. Symptomatic infection is most common in young adults aged 15-40 years, with typical signs and symptoms including jaundice, anorexia, enlarged liver, abdominal pain and tenderness, nausea, vomiting and fever.
Outbreaks and sporadic cases of hepatitis E occur around the world. Disease prevalence is highest in developing countries where accesses to essential water, sanitation, hygiene and health services are limited [1]. In developed countries hepatitis E carries a different threat, as disease transmission can occur through the transfusion of blood products. Hepatitis E is usually self-limiting and is not considered to be fatal. However, it is imperative to avoid transmission and disease progression in immunosuppressed, pregnant and transplant patients, and in cases where HBV or HCV infection is negative but symptoms persist [2]. These groups of patients are more susceptible to acute liver failure which can cause death. Therefore it is essential that diagnostics methods utilised are sensitive and specifi c, to allow accurate patient diagnosis, and prompt treatment options to be employed.
Diagnosing hepatitis E
The clinical signs, symptoms and laboratory fi ndings for the diagnosis of hepatitis E often overlap with other etiologies, which can make confi rming a diagnosis problematic. Diagnostic methods fall into two categories: direct and indirect. Direct methods of detecting the virus include real-time polymerase chain reaction (RT-PCR), whilst indirect methods include detection of the anti-HEV IgM and IgG antibodies. Anti-HEV IgM and IgG are reliable methods of diagnosis in immune-competent hosts, however within immunocompromised hosts false negatives are frequently presented, posing a diagnostic challenge [3].
To diagnose HEV infection effi ciently in immunocompromised patients, RT-PCR is becoming the gold standard test due to its accurate, sensitive and fast diagnosis [4]. Choosing the right assay that offers specifi c and accurate results in a timely fashion is an important factor within RT-PCR, and for precise diagnosis, as any degree of performance variability can ultimately lead to inaccurate diagnoses with disastrous consequences.
A range of assays from various manufacturers are currently on the market to detect the hepatitis E virus, one of which is an assay produced by Fast-track diagnostics. An internal evaluation performed by Fast-track diagnostics showed that 100% of samples were correctly detected by the assay, exhibiting specifi city, sensitivity and precision of analysis. Following internal evaluation, the same variables were measured in an external quality control evaluation. The Quality Control for Molecular Diagnostics (QCMD) confi rmed the results observed in the internal evaluation, as shown in Table 1.
Table 1. Number of samples correctly detected by FTD HEV assay.
Table 2. Overview of the clinical samples tested by FTD HEV assay, MHH in-house assay 1 and competitor kit.
A Comparison Study to Clinically Evaluate a Novel Fast-track Diagnostics Multiplex Real-Time PCR Assay
Objective
The MHH, relies on prompt and effi cient disease diagnosis, to provide effective patient care. To ensure the most effi cient assay was being utilised for accurate patient diagnosis, an external comparison study to evaluate the Fast-track diagnostics Hepatitis E RNA (FTD HEV) assay, against their HEV in-house singleplex assay (MHH) and a competitor assay (detecting HEV and internal control) as reference [3].
Methods
One hundred and four clinical specimens as represented in Table 2 including EDTA blood, serum, stool, ascites and CPDA were extracted, and were tested with two competitor assays against the in-house assay currently used by MHH.
200µl input volume was extracted and eluted in 75µl and tested on a medium to high- throughput PCR platform. Fast-track diagnostics hepatitis E assay was tested with Fast- track mastermix (Fast-track diagnostics), and the PCR setups were performed in accordance with the instructions of the kit or assay.
The following comparison study highlights the results of a comparison between the Fast-track diagnostics assay, a competitor assay and in-house assay already used at MHH, to detect hepatitis E virus [5].
INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2016
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