- Email: [email protected]
HIV and HCV RNA quantification in semen by m2000rt technology
Abstracts: Posters
I•
Cytomegalovirus monitoring using two different commercial PCR assays
$27
l
•
C
a
n
rubella be detected in amniotic fluid stored on filter-paper?
U. Sester 1, M. Sester 1, N. Finnstr6m 2, B. G~rtner 1.
C. Vauloup-Fellous, L. Grangeot-Keros. Microbiology and
l Universit~tsklinikum, Homburg, Germany, 2Sangtec Molecular Diagnostics, Bromma, Sweden
Immunology department, Antoine B6cl6re Hospital, Clamart, Faculty of Medicine Paris-Sud, France
It is of great importance to monitor Cytomegalovirus (CMV) in immuno-suppressed patients especially following transplantation. These patients are usually treated on a pre-emptive basis, i.e. when the viral load is rising a preventive treatment is started. Therefore it is important to detect the viral load as early as possible to prevent infection. Objectives: To compare affigene | CMV trender and Cobas Amplicor CMV Monitor in the monitoring of CMV infection in transplant recipient patients. Method: Eight patients after solid-organ transplantation (7 kidney and 1 lung transplantation) were monitored for CMV utilising affigene | CMV trender or Cobas Amplicor CMV Monitor. Whole blood was collected from each patient one time per week over at least four weeks. DNA was prepared from 50 ul whole blood using affigene | DNA extraction kit for affigene | CMV trender assay and from 500 ul whole blood using MagNAPure kit for Cobas Amplicor CMV Monitor assay. Results: In two of the patients CMV was monitored 24 and 26 days earlier using affigene | CMV trender compared to Cobas Amplicor CMV Monitor. These were the two patients with highest numbers of consecutive samples. There were no such clear indications for the other six patients. The opposite fact, earlier detection with Cobas Amplicor CMV Monitor was not seen. Conclusion: In this study it was shown that the affigene | CMV trender assay early detects CMV during monitoring of organ transplanted patients. An early detection is of importance for the preemptive treatment in this patient category.
Background and Aims: Rubella is now rare in developed countries that have introduced rubella vaccination programmes in the early 1980s. However, in many developing countries, rubella still remains a major cause of congenital abnormalities such as blindness, deafness and mental retardation. Following maternal infection, prenatal diagnosis of congenital rubella infection (CRI), relies on sensitive RT-PCR tests on amniotic fluid (AF), less invasive that fetal blood sampling. This test may be very difficult to carry out, when storage and shipment of AF samples at controlled temperature (-20~ is difficult. The purpose of our work was to investigate whether it was possible to detect rubella RNA in AF stored on filter paper. Methods: 100 ml of infected AF were spotted onto filter paper, dried, and subjected to 7 days storage at fluctuating room temperatures (14 to 26 ~ simulating specimen transportation conditions. Rubella RNA was then recovered, and amplified by our commonly used RT- PCR. Results: rubella RNA was detected in both liquid AF stored at -80~ and AF stored on filter paper, whereas, as previously described, rubella RNA may be no longer detected when liquid AF was stored 7 days at room temperature. Conclusion and Discussion: For CRI diagnosis, storage of AF on filter paper may be a reliable alternative to sending frozen AF, especially in areas where immediate and prolonged refrigeration during transportation are unavailable.
I•
Humoral immune response after rubella primary infection and after vaccination
1~
Withdrawn
C. Vauloup-Fellous, L. Grangeot-Keros. Microbiology and
Immunology department, Antoine B6cl6re Hospital, Clamart, Faculty of Medicine Paris-Sud, France Background: It is well known that after rubella primary infection, IgG level is usually higher than after vaccination. It has also been published that rubella specific IgM is detected for a longer time after vaccination. Less is known about the IgG avidity index which is an important part of the quality of the humoral immune response. Methods: We measured Rubella IgG and IgM levels as well as the IgG avidity index in serum samples taken less than 6 months after infection (N=10) or after vaccination (N=15). These parameters were also measured in serum samples taken more than 6 months after the onset of infection (N = 15) or after vaccination (N = 15). Results: We confirmed that the level of rubella-specific IgG was lower after rubella vaccination than after primary infection. By contrast with primary infection, we also confirmed that IgM antibody can persist at a steady level a very long time after vaccination. As for the IgG avidity index, we showed that it increases much more slowly after vaccination and that it usually stabilizes at a level lower than after primary infection. Conclusions: These observations may be very useful in systemic screening to discriminate primary infection from vaccination when IgM antibody is detected. Furthermore, the results confirmed that the quality of the humoral immune response seems lower after vaccination than after rubella infection. However, as it is well known that rubella vaccine is very efficient, we may suppose that the induced cellular immune response plays an important role in the protection observed.
I
•
H
I
V
and HCVRNA quantification in semen by m2000rt technology
T. Bourlet 1 , R. Levy2, H. Saoundin 1 , S. Pillet 1 , B. Pozzetto 1.
1Laboratoire de Bact6riologie-Virologie and 2Laboratoire de Biologie de la Reproduction, GIMAR Faculty of Medicine and University Hospital of Saint-Etienne, 42055 Saint-Etienne, France Background and Aims: In France, guidelines have been edicted for the management of serodiscordant couples with viral risk, who are candidate to Assisted Reproductive Techniques. The aim of this study was to evaluate the performance of the automated-extraction apparatus m2000rt combined to the Real-Time HIV-1 and HCVTM (Abbott Diagnostics) techniques for the quantification of HIV and HCV RNA in semen. Methods: Pools of seminal plasmas collected from men attending medically-assisted procreation and negative for HCV and HIV were contaminated with dilutions of HIV RNA - f r o m 0 to 4 Iogcopies/ml or HCV RNA - f r o m 0 to 3.7 IoglU/ml. The same dilutions were also performed in human blood plasma. Ten different seminal samples were artificially contaminated with either 3.7 or 21oglU/ml of HCV RNA and tested. Extraction volumes were 0.5 and 0.2 ml for HCV, and 0.5ml for HIV. Results: The sensitivity threshold was about 501U/ml and 100 copies/ml, for HCV and HIV RNA, respectively. No false positive result occurred for both viruses. The variability was less than 5% between samples tested in duplicate for both viruses. The intersemen variability was less than 5%, compared to the theoretical value of HCV RNA (not tested for HIV). No significant change in viral load was observed between blood and seminal plasma for both viruses. No difference in quantification of HCV RNA was observed between the 2 extraction volumes (not tested for HIV). Conclusion: HIV-1 and HCV RNA can be quantified adequately in semen samples by real-time PCR using the m2000rt technology.
I•
Cytomegalovirus monitoring using two different commercial PCR assays
$27
l
•
C
a
n
rubella be detected in amniotic fluid stored on filter-paper?
U. Sester 1, M. Sester 1, N. Finnstr6m 2, B. G~rtner 1.
C. Vauloup-Fellous, L. Grangeot-Keros. Microbiology and
l Universit~tsklinikum, Homburg, Germany, 2Sangtec Molecular Diagnostics, Bromma, Sweden
Immunology department, Antoine B6cl6re Hospital, Clamart, Faculty of Medicine Paris-Sud, France
It is of great importance to monitor Cytomegalovirus (CMV) in immuno-suppressed patients especially following transplantation. These patients are usually treated on a pre-emptive basis, i.e. when the viral load is rising a preventive treatment is started. Therefore it is important to detect the viral load as early as possible to prevent infection. Objectives: To compare affigene | CMV trender and Cobas Amplicor CMV Monitor in the monitoring of CMV infection in transplant recipient patients. Method: Eight patients after solid-organ transplantation (7 kidney and 1 lung transplantation) were monitored for CMV utilising affigene | CMV trender or Cobas Amplicor CMV Monitor. Whole blood was collected from each patient one time per week over at least four weeks. DNA was prepared from 50 ul whole blood using affigene | DNA extraction kit for affigene | CMV trender assay and from 500 ul whole blood using MagNAPure kit for Cobas Amplicor CMV Monitor assay. Results: In two of the patients CMV was monitored 24 and 26 days earlier using affigene | CMV trender compared to Cobas Amplicor CMV Monitor. These were the two patients with highest numbers of consecutive samples. There were no such clear indications for the other six patients. The opposite fact, earlier detection with Cobas Amplicor CMV Monitor was not seen. Conclusion: In this study it was shown that the affigene | CMV trender assay early detects CMV during monitoring of organ transplanted patients. An early detection is of importance for the preemptive treatment in this patient category.
Background and Aims: Rubella is now rare in developed countries that have introduced rubella vaccination programmes in the early 1980s. However, in many developing countries, rubella still remains a major cause of congenital abnormalities such as blindness, deafness and mental retardation. Following maternal infection, prenatal diagnosis of congenital rubella infection (CRI), relies on sensitive RT-PCR tests on amniotic fluid (AF), less invasive that fetal blood sampling. This test may be very difficult to carry out, when storage and shipment of AF samples at controlled temperature (-20~ is difficult. The purpose of our work was to investigate whether it was possible to detect rubella RNA in AF stored on filter paper. Methods: 100 ml of infected AF were spotted onto filter paper, dried, and subjected to 7 days storage at fluctuating room temperatures (14 to 26 ~ simulating specimen transportation conditions. Rubella RNA was then recovered, and amplified by our commonly used RT- PCR. Results: rubella RNA was detected in both liquid AF stored at -80~ and AF stored on filter paper, whereas, as previously described, rubella RNA may be no longer detected when liquid AF was stored 7 days at room temperature. Conclusion and Discussion: For CRI diagnosis, storage of AF on filter paper may be a reliable alternative to sending frozen AF, especially in areas where immediate and prolonged refrigeration during transportation are unavailable.
I•
Humoral immune response after rubella primary infection and after vaccination
1~
Withdrawn
C. Vauloup-Fellous, L. Grangeot-Keros. Microbiology and
Immunology department, Antoine B6cl6re Hospital, Clamart, Faculty of Medicine Paris-Sud, France Background: It is well known that after rubella primary infection, IgG level is usually higher than after vaccination. It has also been published that rubella specific IgM is detected for a longer time after vaccination. Less is known about the IgG avidity index which is an important part of the quality of the humoral immune response. Methods: We measured Rubella IgG and IgM levels as well as the IgG avidity index in serum samples taken less than 6 months after infection (N=10) or after vaccination (N=15). These parameters were also measured in serum samples taken more than 6 months after the onset of infection (N = 15) or after vaccination (N = 15). Results: We confirmed that the level of rubella-specific IgG was lower after rubella vaccination than after primary infection. By contrast with primary infection, we also confirmed that IgM antibody can persist at a steady level a very long time after vaccination. As for the IgG avidity index, we showed that it increases much more slowly after vaccination and that it usually stabilizes at a level lower than after primary infection. Conclusions: These observations may be very useful in systemic screening to discriminate primary infection from vaccination when IgM antibody is detected. Furthermore, the results confirmed that the quality of the humoral immune response seems lower after vaccination than after rubella infection. However, as it is well known that rubella vaccine is very efficient, we may suppose that the induced cellular immune response plays an important role in the protection observed.
I
•
H
I
V
and HCVRNA quantification in semen by m2000rt technology
T. Bourlet 1 , R. Levy2, H. Saoundin 1 , S. Pillet 1 , B. Pozzetto 1.
1Laboratoire de Bact6riologie-Virologie and 2Laboratoire de Biologie de la Reproduction, GIMAR Faculty of Medicine and University Hospital of Saint-Etienne, 42055 Saint-Etienne, France Background and Aims: In France, guidelines have been edicted for the management of serodiscordant couples with viral risk, who are candidate to Assisted Reproductive Techniques. The aim of this study was to evaluate the performance of the automated-extraction apparatus m2000rt combined to the Real-Time HIV-1 and HCVTM (Abbott Diagnostics) techniques for the quantification of HIV and HCV RNA in semen. Methods: Pools of seminal plasmas collected from men attending medically-assisted procreation and negative for HCV and HIV were contaminated with dilutions of HIV RNA - f r o m 0 to 4 Iogcopies/ml or HCV RNA - f r o m 0 to 3.7 IoglU/ml. The same dilutions were also performed in human blood plasma. Ten different seminal samples were artificially contaminated with either 3.7 or 21oglU/ml of HCV RNA and tested. Extraction volumes were 0.5 and 0.2 ml for HCV, and 0.5ml for HIV. Results: The sensitivity threshold was about 501U/ml and 100 copies/ml, for HCV and HIV RNA, respectively. No false positive result occurred for both viruses. The variability was less than 5% between samples tested in duplicate for both viruses. The intersemen variability was less than 5%, compared to the theoretical value of HCV RNA (not tested for HIV). No significant change in viral load was observed between blood and seminal plasma for both viruses. No difference in quantification of HCV RNA was observed between the 2 extraction volumes (not tested for HIV). Conclusion: HIV-1 and HCV RNA can be quantified adequately in semen samples by real-time PCR using the m2000rt technology.