| 1. |
- Engstrand, Mats, et al.
(author)
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Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants
- 2000
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In: Transplantation. - : Ovid Technologies (Wolters Kluwer Health). - 0041-1337 .- 1534-6080. ; 69:11, s. 2243-2250
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Journal article (peer-reviewed)abstract
- BACKGROUND: Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 50-90% of individuals in different populations. After primary infection, the virus persists latently in myeloid cells under the control of specific T-cells. Reactivation of CMV infection may cause lethal organ dysfunction and is frequently seen in immunosuppressed individuals. CD8+ cytotoxic T-cells (CTL) have a primary role in suppressing CMV reactivation, and the dominating CTL response is directed against pp65. METHODS: MHC tetramers, that is, complexes between HLA class I (or class II) molecules and antigenic peptides conjugated to fluorochromes allow the direct visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodominant NLVPMVATV peptide. RESULTS: The A2/pp65 tetramer specifically stained CMV-directed T-cell lines, and sorted cells showed CMV-specific cytotoxicity. High proportions (0.1-9%) of the CD8+ T-cells were A2/pp65 tetramer+ in healthy HLA-A2+ CMV carriers and in immunosuppressed kidney transplant patients with latent infection. Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tetramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 tetramer+ cells expressed T-cell activation markers. CONCLUSIONS: The construction of a novel A2/pp65 MHC tetramer enabled the design of a rapid and precise flow cytometric method allowing quantitative and qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetramer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.
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| 2. |
- Eriksson, Britt-Marie, et al.
(author)
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A prospective study of rapid methods of detecting cytomegalovirus in the blood of renal transplant recipients in relation to patient and graft survival
- 1996
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In: Clinical Transplantation. - 0902-0063 .- 1399-0012. ; 10:6 Pt 1, s. 494-502
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Journal article (peer-reviewed)abstract
- Eighty-five renal transplant recipients were prospectively monitored for CMV infection up to 4 months post-transplantation by virus isolation from leukocytes, CMV antigen detection (pp65) in peripheral blood leukocytes (PBL), polymerase chain reaction (PCR) of alkaline treated plasma (P-PCR), PCR of extracted DNA from PBL (L-PCR) and serology. Additionally univariate and multivariate analyses of risk factors for patient and graft survival up to 4 yr post-transplantation were performed. The incidence of CMV infection was 78% and of CMV disease 33%. Antigen detection in PBL was positive before or at onset of symptoms in 23/24 (96%) evaluable patients with CMV disease. The corresponding figures for virus isolation were 22/24 (92%), P-PCR 21/24 (88%) and for L-PCR 18/24 (75%). The percentage of negative samples in patients without CMV disease was 89% for the antigen test, 92% for L-PCR and 83% for virus isolation and P-PCR. One rapid test (antigen test, P-PCR or L-PCR) was positive at a median of 16 d before the onset of symptoms. The antigen test was generally the first rapid test to become positive. CMV disease did not affect graft survival in the multivariate analysis but was associated with decreased patient survival.
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| 3. |
- Eriksson, Britt-Marie, et al.
(author)
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Circulating soluble vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in immunocompetent and renal transplant patients:correlation with cytomegalovirus disease and renal function
- 2001
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In: Scandinavian Journal of Infectious Diseases. - : Informa UK Limited. - 0036-5548 .- 1651-1980. ; 33:5, s. 350-354
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Journal article (peer-reviewed)abstract
- The plasma levels of the soluble adhesion molecules, soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1), were measured before and after transplantation in 26 renal transplant recipients, and in 173 longitudinally collected samples in 17 of the patients. The patients were carefully monitored for the presence of cytomegalovirus (CMV) infection and rejection. Forty healthy blood donors and 12 otherwise healthy subjects with symptomatic primary CMV infections served as controls. During CMV disease, plasma levels of sVCAM-1 and sICAM-1 were elevated in both renal transplant patients and otherwise healthy subjects with CMV disease. The sVCAM-1 levels were strongly elevated before transplantation in renal transplant recipients and correlated with creatinine levels. Increased sVCAM-1 levels were also registered during rejection episodes. CMV disease, per se, is associated with markedly increased levels of sVCAM-1 and sICAM-1. There is also a correlation of sVCAM-1 levels with serum creatinine levels. Thus, the presence of CMV infection and renal function are factors that must be considered in further studies of soluble adhesion molecules.
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| 4. |
- Johansen, Kari, et al.
(author)
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Norovirus strains belonging to the GII.4 genotype dominate as a cause of nosocomial outbreaks of viral gastroenteritis in Sweden 1997-2005 - Arrival of new variants is associated with large nation-wide epidemics
- 2008
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In: Journal of Clinical Virology. - : Elsevier BV. - 1386-6532 .- 1873-5967. ; 42:2, s. 129-134
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Journal article (peer-reviewed)abstract
- Background: In recent years an increase of the incidence of nosocomial outbreaks caused by noroviruses has been observed throughout Sweden, with high peaks noted in the winter seasons 2002/2003 and 2004/2005, respectively. Objectives: To phylogenetically characterize norovirus strains causing nosocomial outbreaks from 1997 to 2005 and estimate the impact of norovirus-like disease on the Swedish health care system during the peak season 2002/2003 when a new variant of norovirus occurred. Study design: Stool samples from 115 randomly selected nosocomial outbreaks occurring during 1997-2005 throughout Sweden were studied by RT-PCR and sequencing. In addition, to investigate the impact on the health-care system, a questionnaire was distributed to infection control units (n = 90) serving all Swedish hospitals, nursing homes and other health-care institutions during the largest epidemic of nosocomial outbreaks. Results: Sequencing of 279 nucleotides of the norovirus RNA polymerase gene in stools containing norovirus RNA showed that strains belonging to the GII.4 genotype dominated. Each of the two large epidemics was due to a new variant within this cluster. The questionnaire revealed that 30,000-35,000 episodes of nosocomial norovirus-like infections occurred in 80 of 82 major Swedish hospitals affected in 2002/2003. Conclusion: New norovirus variants within the cluster GGII.4 may have a major impact on the health-care system. (c) 2008 Elsevier B.V. All rights reserved.
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| 5. |
- Mousavi-Jazi, Mehrdad, et al.
(author)
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Sequence analysis of UL54 and UL97 genes and evaluation of antiviral susceptibility of human cytomegalovirus isolates obtained from kidney allograft recipients before and after treatment
- 2001
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In: Transplant Infectious Disease. - : Wiley. - 1398-2273 .- 1399-3062. ; 3:4, s. 195-202
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Journal article (peer-reviewed)abstract
- The frequency of infections caused by drug-resistant cytomegalovirus (CMV) in solid-organ transplant recipients is not known. Only a few resistant strains have been described in transplant recipients. Antiviral susceptibility to ganciclovir (GCV) and foscarnet (PFA) of CMV isolates from 24 renal transplant patients with CMV viremia and CMV disease before and after therapy were investigated by a solid phase ELISA. The CMV DNA polymerase (UL54) and viral phosphotransferase (UL97) genes were also sequenced. Ten patients did not receive antiviral treatment; five and nine patients were treated with PFA and GCV, respectively. No appearance of drug-resistant viruses was observed in the present study, but one isolate showed a reduced sensitivity to PFA after treatment with GCV. This finding could not be explained by the presence or development of mutations that have been associated with drug resistance in UL54. We found no evidence that short-term treatment of CMV with PFA- or GCV-induced resistance.
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| 6. |
- Tiveljung Lindell, Annika, et al.
(author)
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Molecular epidemiology of norovirus infections in Stockholm, Sweden, during the years 2000 to 2003 : Association of the GGIIb genetic cluster with infection in children
- 2005
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In: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 43:3, s. 1086-1092
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Journal article (peer-reviewed)abstract
- The incidence of norovirus-associated gastroenteritis and the molecular epidemiology of norovirus strains were studied during three seasons (2000-2001, 2001-2002, and 2002-2003) among patients of all ages, mainly from the Stockholm region in Sweden. A total of 3,252 fecal samples were analyzed by reverse transcription-PCR. The incidences of norovirus infection among adults were 23, 26, and 30% during the three seasons studied and 18,11, and 15% among children 0 to 15 years of age. During the first season, all norovirus strains detected by PCR were typed either by reverse line blot hybridization or nucleotide sequence analysis. During the two successive seasons, a total of 60 norovirus-positive strains from the beginning, peak, and end of the seasons were selected for nucleotide sequence analysis. We identified two dominant noroviras variants over the seasons: a new norovirus variant, recently described as the GGIIb genetic cluster, dominated among children during the first season, and during the following two seasons, a GGII-4 variant dominated. Our data suggest that norovirus infections are common, not only among adults, but also among children, and that some strains may predominantly affect children. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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| 7. |
- Wirgart, Benita Zweygberg, et al.
(author)
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Cytomegalovirus (CMV) DNA amplification from plasma compared with CMV pp65 antigen (ppUL83) detection in leukocytes for early diagnosis of symptomatic CMV infection in kidney transplant patients
- 1996
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In: Clinical and Diagnostic Virology. - 0928-0197 .- 1873-4901. ; 7:2, s. 99-110
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Journal article (peer-reviewed)abstract
- BACKGROUND: Rapid laboratory methods for the early detection of cytomegalovirus (CMV) are needed for the prevention of CMV disease in transplant recipients. These methods should not only be able to detect the virus but also be highly predictive for CMV disease. OBJECTIVE: The clinical value of a simple and rapid nested plasma polymerase chain reaction (PCR) was evaluated by comparing the results with CMV pp65 antigen detection in leukocytes (CMV antigenemia assay), virus isolation from leukocytes, CMV IgG and IgM antibody response and clinical data. STUDY DESIGN: A total of 471 EDTA blood samples were collected from 85 kidney transplant patients during a 3-4 month period after transplantation. CMV DNA was amplified directly from 10 microliters of plasma while 150000 separated leukocytes were stained for CMV pp65 antigen by each of two monoclonal antibodies. A total of one million leukocytes were used for virus isolation. The PCR protocol used in the present study involves a simple alkaline lysis technique for isolating DNA directly from plasma which is easy and rapid to perform. RESULTS: Twenty-eight patients developed symptomatic CMV infection while asymptomatic infection occurred in 29 patients. CMV pp65 antigen detection had a 75% sensitivity and a 57% positive predictive value for CMV disease development, compared with 64% and 79% sensitivity and 49% and 46% positive predictive value for CMV DNA and viremia, respectively. The median time until detection of CMV in patients with symptomatic CMV infection was 26 days after transplantation, compared with 49 days in asymptomatic patients by any of the methods used. Early appearance (within 8 weeks) of CMV pp65 antigen and CMV DNA had high predictive values for symptomatic infection; repeated detection of pp65 antigen and CMV DNA were more common in symptomatic patients. CONCLUSIONS: CMV antigenemia assay and plasma PCR can be used for pre-symptomatic diagnosis of CMV infection. Virus isolation and CMV serology in most cases provide a post-symptomatic diagnosis. The best marker for monitoring kidney transplant patients might be the quantitative CMV antigenemia assay.
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| 8. |
- Zweygberg Wirgart, Benita
(author)
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Early diagnosis of cytomegalovirus infection using monoclonal antibodies and DNA amplification
- 1997
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Doctoral thesis (other academic/artistic)abstract
- Human cytomegalovirus (HCMV) is ubiquitous and frequently infects normal individuals with few serious clinical consequences. However, patients with impaired cellular immunity frequently develop life-threatening opportunistic HCMV infections due to either primary infection or reactivation of latent virus. Pneumonia is the most common manifestation of HCMV visceral organ disease in solid organ and bonemarrow transplant recipients while retinitis is the most common manifestation in AlDS patients. Exposure of the fetus to HCMV in utero may lead to multi-organ system damage and permanent neurological sequelae. Early intervention with antiviral drugs depends on early sensitive and specific virological diagnosis. Detection of anti-CMV antibodies and virus isolation do not allow an early and presymptomatic diagnosis of HCMV infection. The work described has focused on the development of methods to enable the rapid and simple detection of CMV immediate early (IE), early (E) and structural CMV proteins or the detection of amplified CMV DNA in urine, bronchoalveolar lavage (BAL) or blood samples obtained from immunocompetent or immunosuppressed patients. CMV mouse monoclonal antibodies were produced and three of them, (DDG9, CCH2 and AAC 10) were further characterized and used in the different assays. Urine samples from children attending day care centres and from kidney transplant recipients were used for the development of a new method for the detection of early CMV antigen in infected cell cultures. IF staining with the CCH2 monoclonal antibody at 24 hours post-inoculation using urine from kidney transplant recipients revealed a sensitivity of 70% compared to virus isolation. The sensitivity was increased to 85% by the use of biotin-streptavidin and a new immunoenzymatic staining (IENZ) procedure. However, viremia is a better marker for disease development and further studies concentrated on the detection of CMV in blood. The CMV antigenemia assay, which can quantitatively detect CMV pp65 antigen in leukocytes during CMV infection, and a nested polymerase chain reaction (PCR) for amplification of CMV DNA from plasma were established and evaluated. We found plasma-PCR and the CMV antigenemia assay to be almost equally sensitive. However, CMV antigenemia gave the highest positive predictor value (ppv) (57%), for the development of symptomatic CMV infection in kidney transplant recipients, compared to 49% ppv for CMV DNA in plasma. In immunosuppressed patients with symptoms of pulmonary infection, amplification of CMV DNA by PCR from bronchoalveolar lavage (BAL) fluids was the most sensitive method for detection of CMV. The sensitivity was lower but the specificity was higher for the demonstration CMV E antigen and E antigen in infected cell cultures and for the direct detection of CMV antigens in BAL cells. For rapid diagnosis, a combination of these three methods are recommended. A negative PCR result had a 100% negative predictive value for the development of CMV pneumonia for the following two months. Three CMV gene regions, (major immediate early (ME)), DNA polymerase, and glycoprotein B (gB)) were sequenced for 46 isolates obtained from four patient populations in order to assess the nucleotide stability and to evaluate the suitability of the three gene regions as targets for CMV DNA amplification. In diagnostic PCR assays, most laboratories use primers from the MIE gene region. However, we found the MIE to be the most variable gene region, indicating that this gene region is less suitable for diagnostic PCR primer selection compared to the more conserved DNA polymerase and gB gene regions. Our studies have contributed to the development of new, more optimal diagnostic methods for early diagnosis of CMV infection. The monoclonal antibodies have proved to be powerful tools for the detection of CMV in infected cells. With the new sequence data, new more optimal primer sequences can be selected and used in diagnostic PCR assays in order to detect the majority of CMV strains circulating in society.
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