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Real-time PCR detection of polyomaviruses BK and JC in urine
Abstracts: Posters
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HHV6 quantification by real-time-PCR in different body compartments
I. Garrigue 1 , L. Roncin 1 , S. Boucher 1 , R. Tabrizi 2, N. Milpied 2, H. Fleury 1 , M.E. Lafon 1 . 1Laboratoire de Virologie EA296& 2
Service d'H6matologie, Univers#6 Victor Segalen Bordeaux 2 and Centre Hospitalier Universitaire de Bordeaux, 146 rue L~o Saignat, 33 076 BORDEAUX C6dex, France Background and Aims: HHV6 infection is currently diagnosed in our setting by a qualitative Taqman-based real-time PCR [1]. However, positivity is sometimes difficult to interpret, depending on the nature of the biological sample. The aim of the present study was to transform our routine assay into a quantitative one and investigate whether viral load measurement could improve diagnostic procedures. Methods: A plasmidic standard was constructed from HHV6positive samples. The external standard curve added to the routine diagnostic assay included 5 successive dilutions. The 118 samples tested comprised 61 whole blood samples, 6 respiratory secretions, 14 broncho-alveolar lavages, 4 bone marrow samples and 33 biopsies, for 69 symptomatic immunocompromised patients treated in the Haematology unit. Results were expressed as DNA copies/mL in liquid samples and DNA copies/Fg extracted DNA in biopsies. Results: The quantitative assay threshold of detection was 500 DNA copies/mL whole blood with a linearity range from 103 to 108 copies/mE Inter- and intra- experiment reproducibility was ascertained. The highest load values were observed in whole blood (median: 24.7x 103 copies/mL, extreme: 11 x 106). The median value in biopsies was 1.4 copies/Fg DNA, with a single exceptional pulmonary sample (415 copies/Fg DNA). In a few patients, viremia remained limited or undetectable in spite of significant biopsy HHV6 load. Co-infections with other Herpesviridae were occasionally observed. Conclusions and Discussion: Real-time PCR quantification could prove useful in determining whether HHV6 is the causative agent of the observed symptoms. References [1] Aberle SW, Puchhammer-Stockl E. J Clin Virol. 2002; 25(Suppl 1): $79-85. I ~
Significance of cut-off ratios with high-risk HPV hybrid capture 2
M. Raymaekers, A. Broekmans, R Declercq, B. Maes, K. Magerman, A. Mewis, V. Peeters, J.-L. Rummens, R. Cartuyvels.
Laboratory of Molecular Biology, Virga Jesse Hospital, Hasselt, Belgium Aim: This study investigated the significance of ratios (relative light units (RLU) sample/RLU calibrator) near the kit cut-off (~>1.0) for high-risk HPV (HRHPV), obtained by the Hybrid Capture 2 assay (HC2, Digene). Methods: Forty-seven cervical cytological samples, showing cytological abnormalities (ASCUS and LSIL), with ratios for HRHPV (HC2) between 0.8 and 2.5, were retested with HC2 and analyzed simultaneously with Inno-LiPA HPV Genotyping kit v2 (Innogenetics). Results: Only 16 (34%) of the 36 (77%) samples with an initial ratio between 1 and 2.5 showed a ratio ~>1. An additional positive result was found in one sample. In 26/47 (55%) samples, Inno-LiPA revealed the presence of at least one HRHPV type. Only 20/47 (43%) of these samples had an initial ratio ~>1.0. In 7/47 (15%) samples, Inno-LiPA revealed the presence of only low-risk HPV types. Of these samples 5/47 (11%) had an initial ratio ~>1.0. In 7/47 (15%) samples, Inno-LiPA revealed the presence of HPV type 53 and 66, of which no consensus exists concerning clinical significance. Six (13%) of these samples had a ratio ~>1.0. A negative result was obtained with Inno-LiPA in 6 (13%) samples. Five (11%) of these samples had a ratio ~>1.0. Conclusion: In conclusion, HC2 HRHPV ratios between 0.8 and 2.5 should be interpreted with caution because of the risk of crosshybridization and limited sensitivity. Therefore we advise to retest these samples with a more sensitive and specific technique. Retesting with HC2 has no additional benefit.
$31
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Real-time PCR detection of polyomaviruses BK and JC in urine
J. Entwistle, M. Feola, M. Adelson, E. Mordechai. Medical
Diagnostic Laboratories, L.L.C., 2439 Kuser Road, Hamilton, NJ 08690, USA Background and Aims: To develop a rapid and accurate method for identifying Polyomaviruses BK and JC in urine specimens. Polyomaviruses are shed in immunosuppressed patients, including those receiving renal transplants. Both viruses are very widespread as approximately 80% of the American adult population has Polyomavirusspecific antibodies. Methods: Two distinct real-time PCR assays specific for Polyomaviruses BK and JC were developed and optimized for use on a Rotor-Gene RG-3000 platform. DNA was extracted from the urine using an X-tractor Gene (Corbett Robotics). Virus stability was assessed by incubating a biologically equivalent concentration of each virus on UroSwab (Copan Italia, Brescia) for up to five days prior to extraction. Urine specimens from 120 patients were analyzed by real-time PCR and compared with a Pyrosequencing assay to calculate sensitivity and specificity. Results: Sensitivity and specificity were calculated for each reaction: Polyomavirus BK, 100% and 100% (5 positives/115 negatives) and Polyomavirus JC, 100% and 100% (9 positives/111 negatives). The quantitative capabilities of each assay extended over seven orders of magnitude; no cross-reactivity was observed. Pyrosequencing was used to verify the presence of Polyomavirus BK (sequencing of the real-time PCR amplicon) and JC (sequencing of an alternate gene) in the specimens. Stability assays verified detection of the virus for up to five days post-incubation on UroSwabs stored at room temperature. Conclusions and Discussion: This study, along with the accompanying Pyrosequencing assays, validates the use of real-time PCR for the rapid quantitative detection of two urological pathogens associated with systemic infections. Both procedures were highly sensitive and specific.
1•
High level HHV-6 DNA and viral chromosomal integration
K.N. Ward 1, H.N. Leong 2, E.R Nacheva 3, J. Howard 3, C.E. Atkinson 2, N.W.S. Davies 1 , RD. Griffiths 2, D.A. Clark 1 . 1Centre
for Virology, Bloomsbury Campus, 2Centre for Virology, Hampstead Campus, 3Department of Haematology, Hampstead Campus, Royal Free and University College Medical School, London, UK Background and Aims: The level of HHV-6 DNA was measured in the whole blood, serum and hair follicles of 6 immunocompetent patients with human herpesvirus-6 (HHV-6) chromosomal integration. Methods: HHV-6 and I-~-globin DNA levels were quantified in whole blood, serum and hair follicles by real time PCR. The number of HHV-6 DNA copies/cell or lysed cell was determined by comparison with the number of I-~-globin DNA copies. Results: The mean HHV-6 DNA concentration (IOgl0copies/ml) in blood was 7.0 (~>1 HHV-6 DNA copies/leucocyte) and in serum 5.3 (~>1 HHV-6 DNA copies/lysed cell). The mean HHV-6 DNA load (log10 copies)/hair follicle was 4.2 (~>1 copies/hair follicle cell). Conclusion and Discussion: Viral integration is not confined to blood cells. The characteristically high levels of HHV-6 DNA whole blood and serum in chromosomal integration may confound diagnosis since the finding of HHV-6 DNA in serum is usually interpreted as indicating virus replication due to primary infection, reactivation or reinfection. However, viral chromosomal integration can be differentiated from active infection by comparing the HHV-6 DNA load in blood with that in serum.
I•
HHV6 quantification by real-time-PCR in different body compartments
I. Garrigue 1 , L. Roncin 1 , S. Boucher 1 , R. Tabrizi 2, N. Milpied 2, H. Fleury 1 , M.E. Lafon 1 . 1Laboratoire de Virologie EA296& 2
Service d'H6matologie, Univers#6 Victor Segalen Bordeaux 2 and Centre Hospitalier Universitaire de Bordeaux, 146 rue L~o Saignat, 33 076 BORDEAUX C6dex, France Background and Aims: HHV6 infection is currently diagnosed in our setting by a qualitative Taqman-based real-time PCR [1]. However, positivity is sometimes difficult to interpret, depending on the nature of the biological sample. The aim of the present study was to transform our routine assay into a quantitative one and investigate whether viral load measurement could improve diagnostic procedures. Methods: A plasmidic standard was constructed from HHV6positive samples. The external standard curve added to the routine diagnostic assay included 5 successive dilutions. The 118 samples tested comprised 61 whole blood samples, 6 respiratory secretions, 14 broncho-alveolar lavages, 4 bone marrow samples and 33 biopsies, for 69 symptomatic immunocompromised patients treated in the Haematology unit. Results were expressed as DNA copies/mL in liquid samples and DNA copies/Fg extracted DNA in biopsies. Results: The quantitative assay threshold of detection was 500 DNA copies/mL whole blood with a linearity range from 103 to 108 copies/mE Inter- and intra- experiment reproducibility was ascertained. The highest load values were observed in whole blood (median: 24.7x 103 copies/mL, extreme: 11 x 106). The median value in biopsies was 1.4 copies/Fg DNA, with a single exceptional pulmonary sample (415 copies/Fg DNA). In a few patients, viremia remained limited or undetectable in spite of significant biopsy HHV6 load. Co-infections with other Herpesviridae were occasionally observed. Conclusions and Discussion: Real-time PCR quantification could prove useful in determining whether HHV6 is the causative agent of the observed symptoms. References [1] Aberle SW, Puchhammer-Stockl E. J Clin Virol. 2002; 25(Suppl 1): $79-85. I ~
Significance of cut-off ratios with high-risk HPV hybrid capture 2
M. Raymaekers, A. Broekmans, R Declercq, B. Maes, K. Magerman, A. Mewis, V. Peeters, J.-L. Rummens, R. Cartuyvels.
Laboratory of Molecular Biology, Virga Jesse Hospital, Hasselt, Belgium Aim: This study investigated the significance of ratios (relative light units (RLU) sample/RLU calibrator) near the kit cut-off (~>1.0) for high-risk HPV (HRHPV), obtained by the Hybrid Capture 2 assay (HC2, Digene). Methods: Forty-seven cervical cytological samples, showing cytological abnormalities (ASCUS and LSIL), with ratios for HRHPV (HC2) between 0.8 and 2.5, were retested with HC2 and analyzed simultaneously with Inno-LiPA HPV Genotyping kit v2 (Innogenetics). Results: Only 16 (34%) of the 36 (77%) samples with an initial ratio between 1 and 2.5 showed a ratio ~>1. An additional positive result was found in one sample. In 26/47 (55%) samples, Inno-LiPA revealed the presence of at least one HRHPV type. Only 20/47 (43%) of these samples had an initial ratio ~>1.0. In 7/47 (15%) samples, Inno-LiPA revealed the presence of only low-risk HPV types. Of these samples 5/47 (11%) had an initial ratio ~>1.0. In 7/47 (15%) samples, Inno-LiPA revealed the presence of HPV type 53 and 66, of which no consensus exists concerning clinical significance. Six (13%) of these samples had a ratio ~>1.0. A negative result was obtained with Inno-LiPA in 6 (13%) samples. Five (11%) of these samples had a ratio ~>1.0. Conclusion: In conclusion, HC2 HRHPV ratios between 0.8 and 2.5 should be interpreted with caution because of the risk of crosshybridization and limited sensitivity. Therefore we advise to retest these samples with a more sensitive and specific technique. Retesting with HC2 has no additional benefit.
$31
1•
Real-time PCR detection of polyomaviruses BK and JC in urine
J. Entwistle, M. Feola, M. Adelson, E. Mordechai. Medical
Diagnostic Laboratories, L.L.C., 2439 Kuser Road, Hamilton, NJ 08690, USA Background and Aims: To develop a rapid and accurate method for identifying Polyomaviruses BK and JC in urine specimens. Polyomaviruses are shed in immunosuppressed patients, including those receiving renal transplants. Both viruses are very widespread as approximately 80% of the American adult population has Polyomavirusspecific antibodies. Methods: Two distinct real-time PCR assays specific for Polyomaviruses BK and JC were developed and optimized for use on a Rotor-Gene RG-3000 platform. DNA was extracted from the urine using an X-tractor Gene (Corbett Robotics). Virus stability was assessed by incubating a biologically equivalent concentration of each virus on UroSwab (Copan Italia, Brescia) for up to five days prior to extraction. Urine specimens from 120 patients were analyzed by real-time PCR and compared with a Pyrosequencing assay to calculate sensitivity and specificity. Results: Sensitivity and specificity were calculated for each reaction: Polyomavirus BK, 100% and 100% (5 positives/115 negatives) and Polyomavirus JC, 100% and 100% (9 positives/111 negatives). The quantitative capabilities of each assay extended over seven orders of magnitude; no cross-reactivity was observed. Pyrosequencing was used to verify the presence of Polyomavirus BK (sequencing of the real-time PCR amplicon) and JC (sequencing of an alternate gene) in the specimens. Stability assays verified detection of the virus for up to five days post-incubation on UroSwabs stored at room temperature. Conclusions and Discussion: This study, along with the accompanying Pyrosequencing assays, validates the use of real-time PCR for the rapid quantitative detection of two urological pathogens associated with systemic infections. Both procedures were highly sensitive and specific.
1•
High level HHV-6 DNA and viral chromosomal integration
K.N. Ward 1, H.N. Leong 2, E.R Nacheva 3, J. Howard 3, C.E. Atkinson 2, N.W.S. Davies 1 , RD. Griffiths 2, D.A. Clark 1 . 1Centre
for Virology, Bloomsbury Campus, 2Centre for Virology, Hampstead Campus, 3Department of Haematology, Hampstead Campus, Royal Free and University College Medical School, London, UK Background and Aims: The level of HHV-6 DNA was measured in the whole blood, serum and hair follicles of 6 immunocompetent patients with human herpesvirus-6 (HHV-6) chromosomal integration. Methods: HHV-6 and I-~-globin DNA levels were quantified in whole blood, serum and hair follicles by real time PCR. The number of HHV-6 DNA copies/cell or lysed cell was determined by comparison with the number of I-~-globin DNA copies. Results: The mean HHV-6 DNA concentration (IOgl0copies/ml) in blood was 7.0 (~>1 HHV-6 DNA copies/leucocyte) and in serum 5.3 (~>1 HHV-6 DNA copies/lysed cell). The mean HHV-6 DNA load (log10 copies)/hair follicle was 4.2 (~>1 copies/hair follicle cell). Conclusion and Discussion: Viral integration is not confined to blood cells. The characteristically high levels of HHV-6 DNA whole blood and serum in chromosomal integration may confound diagnosis since the finding of HHV-6 DNA in serum is usually interpreted as indicating virus replication due to primary infection, reactivation or reinfection. However, viral chromosomal integration can be differentiated from active infection by comparing the HHV-6 DNA load in blood with that in serum.