rapid virological diagnosis of central nervous system infections 22
TRANSCRIPT
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Rapid Virological Diagnosis of
Central Nervous System Infections
by Use of a Multiplex Reverse Transcription-PCR
DNA Microarray
Viktoriya Morozova
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Preliminary evaluation of the analytical and clinical performances multiplex RT-PCR DNA microarray, the Clart Enthepex kit.
Aim of the study
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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
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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
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allows simultaneous detection and identification of the eight human herpesviruses and enteroviruses.
Coxsackivirus
HHV-8
Clart Entherpex kit
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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
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Evaluation
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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.
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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
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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
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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
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Results
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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.
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Retrospective analysis of 78 CSF samples obtained from patients hospitalized for suspected neurological virus infections from 2002 to 2009
Phase 2
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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….
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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
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Results
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Results
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Conclusion
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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
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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
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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
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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