Post on 31-May-2015
transcript
Rapid Virological Diagnosis of
Central Nervous System Infections
by Use of a Multiplex Reverse Transcription-PCR
DNA Microarray
Viktoriya Morozova
Preliminary evaluation of the analytical and clinical performances multiplex RT-PCR DNA microarray, the Clart Enthepex kit.
Aim of the study
This kit has been developed to determine in a fast way the agent(s) responsible for the illness (Herpesvirus or Enterovirus), thus allowing the establishment of the most effective clinical treatment in each case.
CLART® ENTHERPEX kit
This kit is based on viral genome-specific fragment amplification by multiplex PCR and its subsequent detection via hybridization with a microorganism-specific binding probe on low-density microarrays.
Clart Entherpex kit
allows simultaneous detection and identification of the eight human herpesviruses and enteroviruses.
Coxsackivirus
HHV-8
Clart Entherpex kit
HSV-Herpes Simplex virus
VZV-Varicella Zoster virus
CMV- Cytomegalovirus
EBV-Epstein-Barr virus
HHV-6, HHV-7, HHV-8 -human herpesvirus
HEVs – human enteroviruses
Viruses
Evaluation
Since neither QCMD panels nor known clinical samples containing HHV-7 or HHV-8 were available, the performances of the DNA microarray regarding the detection of both these viruses have not been evaluated.
…
4 proficiency samples for each of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6 and HEVs with concentrations ranging from 10² to 10⁵ copies/ml were selected and independently tested 4 times each in order to assess the specificity, the sensitivity, and reproducibility of the DNA microarray.
Phase 1
Limit of detection of less than 500 copies/ml for all of the 6 herpesviruses tested.
HEVs- the low viral loads corresponding to
280 and 390 genome copies/ml were not detected
HEVs – the viral load corresponding to 1480 genome copies/ml was detected in 3 out 4 analyses (75%)
Results
The analytical sensitivities of the test corresponding to the copy number of each one of the viruses that can be detected in 100% of the analyses performed were :
HHV-6 between 500 to 1,000 copies/ml
HSV-1, HSV-2, VZV, and EBV >2,000 copies/ml
Results
Results
The manufacturer's claims regarding the analytical sensitivities of the assay are:
10 copies for VZV, HHV-7, and HSV-1
100 copies for HSV-2, CMV, EBV, HHV-6, HHV-8, and the coxsackieviruses
1,000 copies for the echoviruses and the polioviruses
per PCR mixture containing 5 μl of DNA/RNA extract.
…
Retrospective analysis of 78 CSF samples obtained from patients hospitalized for suspected neurological virus infections from 2002 to 2009
Phase 2
28 samples previously tested positive for herpesviruses (7 HSV-1, 2 HSV-2, 8 VZV, 1 CMV, 4 EBV, 6 HHV-6) and negative for HEV
30 samples previously positively tested for HEV and negative for herpesviruses.
20 samples previously tested negative for both HEVs and herpesviruses.
Phase 2 continued….
27 of the 28 herpesvirus-positive CSF samples were detected (96%)
30 of the 30 HEV-positive CSF samples were detected (100%)
Were detected 11 (37%) HHV-7 and HEV mixed infections among the 30 pediatric aseptic meningitis cases initially related to HEVs
Phase 2. Results
Results
Results
Conclusion
High sensitivity allowing the detection of minimum quantities of DNA.
High specificity, when using a sequence corresponding to a highly preserved region within the viral genome and binding probes specific to each human herpes and enterovirus type.
Fast. Allowing the laboratory to provide the answer to the clinician in a single work day
Simultaneous detection of multiple DNA & RNA viruses present in a single sample
Pros
Limited automation (requires numerous handlings)
Kit cannot be used without a microarray reader piloted by specific software provided by the manufactures, thus limiting the implementation of the kit
Risk of contamination during handling amplicons
Cons
Due to the small number of CSF specimens tested, further studies are needed to better characterize the performances of this test before its use in routine patient care.
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The end
http://www.genomica.es/en/in_vitro_diagnostics_products_clart_entherpex.cfm
http://www.qcmd.org/index.php?pageId=34&pageVersion=EN
http://www.journalofclinicalvirology.com/article/S1386-6532(02)00028-8
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522074
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209095/?log$=activity
References