Benita Zweygberg Wirgart

Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants

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.

Molecular Epidemiology of Norovirus Infections in Stockholm, Sweden, during the Years 2000 to 2003: Association of the GGIIb Genetic Cluster with Infection in Children

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 norovirus 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.

Identification of viral agents associated with diarrhea in young children during a winter season in Beijing, China

Abstract Background Viral diarrhea remains a major cause of childhood morbidity and mortality worldwide. Although rotavirus was extensively studied in China, few comprehensive studies of all viral agents related to diarrhea in children have been conducted. Objectives Our study was performed to investigate the role of enteric viruses in acute diarrhea in our country and to evaluate methods that could be used in routine diagnostics. Study design One hundred stool samples were collected from children under 5 years of age seeking medical care for acute diarrhea during the winter season 2000/2001 in Beijing Childrens Hospital. All specimens were initially screened microscopically for leucocytes/red blood cells. Samples with negative results were analyzed for virus presence using commercial EIAs and/or in-house RT-PCRs. Results At least one viral agent was found in 67% of the specimens. The frequency of rotavirus, astrovirus, norovirus and enteric adenovirus was 59%, 8%, 6% and 2%, respectively. Dual infections were found in 9.0% (6/67) of the positive samples. The results from rotavirus and astrovirus EIAs were concordant with those of rotavirus and astrovirus RT-PCRs. Conclusions Enteric viruses play an important role in pediatric diarrhea during the winter season in China. A combination of microscopic examination of stool samples with specific EIA assays to detect virus antigen in stool specimens may be suitable for routine diagnostics.

The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.

Transmission of cytomegalovirus to extremely preterm infants through breast milk

Aim: To evaluate the rate and clinical expression of postnatal cytomegalovirus (CMV) infection transmitted through breast milk in extremely preterm infants.

Early detection of cytomegalovirus in cell culture by a new monoclonal antibody, CCH2

A CMV monoclonal antibody, CCH2, produced in this laboratory was evaluated for rapid detection of CMV. Two staining procedures, immunofluorescence and an immunoenzymatic technique using biotin-streptavidin peroxidase, were compared. The CCH2 monoclonal antibody was used to demonstrate early CMV antigen in cell culture 24 h after inoculation of 598 urine samples from kidney transplanted patients by indirect immunofluorescence in comparison with virus isolation. One hundred and sixty of the specimens were stained additionally by an immunoenzymatic technique and the results were compared. CMV was isolated from 170 out of 598 specimens within 6 weeks. Early CMV antigen was demonstrated in 114 of these specimens by immunofluorescence giving a sensitivity of 67% and a specificity of 95%. In the comparison with the immunoenzymatic staining procedure the results for all three tests agreed for 81% (130/160) of the specimens. After resolving discordant results into true positives and true negatives, the sensitivity was 87, 85 and 70%, respectively for virus isolation, immunoenzymatic staining and immunofluorescence and the specificity 100, 96 and 99%. The CCH2 monoclonal antibody proved to be useful for rapid detection of CMV in urine specimens and using immunoenzymatic staining with biotin-streptavidin a sensitivity comparable to that of virus isolation was found.

Intrauterine fetal death due to echovirus 11.

In a case of intrauterine fetal death in the 29th week of gestation, echovirus 11 could be isolated from the umbilical cord of the fetus. The mother had no apparent signs of infection but serological evidence of current echovirus 11 infection. Enterovirus PCR performed on paraffin-embedded specimens of various tissues (myocardium, lung, liver and placenta) from the fetus yielded positive results in all cases. These findings, together with supporting serological and epidemiological findings--e.g. proven echovirus 11 infection 3 weeks before in the 18-month-old son of the woman--constituted strong evidence that echovirus 11 infection was responsible for the fetal death.

Respiratory Virus Infections in Stockholm During Seven Seasons: A Retrospective Study of Laboratory Diagnosis

A retrospective analysis of the virological findings in all respiratory samples (7303) analysed at the laboratory of Karolinska Hospital between 1993 and 2000 was performed. The findings were studied according to age and seasonal variation, and the methods were evaluated. Most samples were from children. RSV was the dominant agent, found in 34% of all samples from children 0–1 y of age. Influenza A was found in 13% of samples from the age group 2–5 y. Influenza A dominated among adults and the elderly. RSV was found only in 2% of samples from patients 81 y or older. Adenovirus was found among children and adults, but not at all among the elderly. Both antigen detection and virus isolation were performed on 79% (5776) of the samples. For diagnosis of influenza A, virus isolation was more sensitive than immunofluorescence, but for diagnosis of RSV immunofluorescence was more sensitive than virus isolation. Thus, the analysis verified that influenza A is common not only among adults and the elderly, but also among small children. RSV was an uncommon finding among the elderly. Immunofluorescence is sensitive and rapid for the diagnosis of particularly RSV among small children and influenza in all age groups.

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

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.

Early detection of cytomegalovirus in cell culture by a monoclonal antibody

A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytopathogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70 degrees C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods.