Reconstitution of cytomegalovirus specific T cells after pediatric allogeneic stem cell transplantation: results from a pilot study using a multi-allele CMV tetramer group.

BACKGROUND: Recovery of cytomegalovirus (CMV)-specific T cell mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. Tetramer-based technologies have been shown to be a sensitive tool in the enumeration of specific T cells, but have the disadvantage of HLA-restriction of the peptides. PATIENTS AND METHODS: In this pilot study, we tested the feasibility of a panel of 6 CMV-specific tetrameric HLA/CMV-peptide complexes to enumerate CMV-specific CD8 +T cells (CTLs). The reconstitution of CMV-specific CTLs was assessed in 16 children in the first year after allogeneic SCT (median age, 8 years). RESULTS: The presented assay covered more than 85% of our patients transplanted in the last 3 years. During CMV-reactivation, all 4 of the 16 analyzed patients with a high virus-load showed less than 10 CMV-specific CTLs/microl; out of these, three had not any detectable CMV-CTLs. On the other hand, five of the children with less than 10 CMV-specific CTLs/microl did not develop CMV reactivation. When enumeration of T cells was performed by means of different tetrameric HLA/CMV-peptide complexes simultaneously, the numbers of CMV-specific CTLs cells widely differed according to the HLA-type. CONCLUSIONS: Our pilot study suggests that enumeration of CMV-specific T cells by means of a panel of 6 tetramers might be a useful tool in the risk assessment for CMV reactivation in the majority of patients undergoing allogeneic SCT, but future trials have to evaluate whether this method is appropriate in tailoring antiviral therapy in the individual patient.

Infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus.

To identify a model for the study of intestinal pathogenesis of severe acute respiratory syndrome (SARS) we tested the sensitivity of six human intestinal epithelial cell lines to infection with SARS coronavirus (SARS-CoV). In permissive cell lines, effects of SARS-CoV on cellular gene expression were analysed using high-density oligonucleotide arrays. Caco-2 and CL-14 cell lines were found to be highly permissive to SARS-CoV, due to the presence of angiotensin-converting enzyme 2 as a functional receptor. In both cell lines, SARS-CoV infection deregulated expression of cellular genes which may be important for the intestinal pathogenesis of SARS.

Acute retinal necrosis six years after herpes simplex encephalitis: an elusive immune deficit suggested by insufficient test sensitivity.

A patient presented with acute retinal necrosis of the left eye. Demonstration of herpes simplex virus (HSV) DNA in the aqueous humour confirmed the diagnosis. Negative results of HSV type-specific antibody tests based on gG antigens suggested a primary HSV infection. However, the patient had a past history of laboratory-confirmed herpes simplex encephalitis 6 years ago. Using antibody tests based on whole viral lysate antigens, he was seropositive from the onset, and immunoblot testing confirmed a lack of anti-gG reactivity. To be able to assess whether this might be related to the apparent inability of his immune system to suppress clinically symptomatic HSV infection, serial samples were tested by an HSV neutralisation test and a whole-blood flow cytometric assay to determine the frequency of HSV-specific CD4 lymphocytes. However, this did not yield evidence of obvious immunodeficiency; the patient reacted similarly to known positive controls by both assays. Although type-specific HSV serological tests based on gG are generally more specific than those based on whole viral lysate antigens, they have a somewhat lower sensitivity, as a certain percentage of HSV-infected individuals do not develop antibodies against gG, and others may suffer a secondary loss of anti-gG reactivity. Thus there is a risk of missing individual infected patients. Unless this potential problem is recognised, serious consequences might possibly result. We therefore urge virologists and clinicians to exercise great care if highly specific antibody assays based on recombinant proteins are employed.

Primary cytomegalovirus infection in an outpatient setting--laboratory markers and clinical aspects.

BACKGROUND: Occasionally, primary cytomegalovirus (CMV) infection may give rise to more or less severe clinical illness in immunocompetent adults. We retrospectively analyzed cases of acute CMV infection in medical outpatients. PATIENTS AND METHODS: Over a 6-year period, we identified 22 patients with a febrile illness and hepatitis suffering from primary CMV infection. This was diagnosed on the basis of a strongly positive CMV IgM antibody test result and/or CMV IgG seroconversion. Clinical features as well as relevant laboratory results were analyzed. We also tested available samples for CMV glycoprotein B-specific antibodies and CMV IgG avidity and analyzed results of Epstein-Barr virus (EBV)-specific antibody assays. In addition, current age-specific CMV IgG seroprevalence rates were determined using 9,870 routine patient samples. RESULTS: At presentation, all patients complained of malaise and fever higher than 38 degrees C, and many also complained of cephalgia. Most patients who underwent abdominal ultrasonography had an enlargement of the spleen. Most patients had a relative lymphocytosis but only three had a mild leukocytosis. C-reactive protein was only slightly elevated in 13 patients; all 22 patients had elevated levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). Half the patients reported travel to areas outside western Europe, mostly to tropical and subtropical areas, within 3 weeks before onset of illness. Primary CMV infection was confirmed by negative anti-gB antibody test results and the absence of high-avidity CMV antibodies. In contrast, despite past EBV infection demonstrated by positive anti-EBNA-1 results, 15 out of 21 patients tested for EBV markers had positive or nonspecific IgM test results. The overall CMV IgG seroprevalence rate in the routine samples was 64.4%, with marked age-dependent increases. CONCLUSION: CMV is a relevant differential diagnosis in feverish illnesses accompanied by hepatitis in otherwise healthy adults, about 40% of whom are CMV-naïve. Half our patients seem to have acquired their CMV infection abroad, so that a diagnosis of CMV infection needs to be taken into account in travelers, in addition to infectious illnesses more commonly considered in this context, such as dengue or hepatitis A. For diagnosis, both CMV and EBV antibody studies should be performed and the inclusion of assays able to demonstrate past infection is helpful for achieving a definite diagnosis.

Viral zoonoses - a threat under control?

Despite intensive research and considerable effort to eradicate infectious diseases, modern medicine has failed to control many infectious diseases which have been thought to be easy to overcome with advances in medical science and technology. In fact, infectious diseases remain a dominant feature in public health considerations for the 21st century. Some infectious agents already known to be pathogenic have gained increasing importance in recent decades due to changes in disease patterns. Furthermore, many new, previously unknown infectious agents with a high pathogenic potential have been identified. Nearly all of these emergent disease episodes have involved zoonotic or species-jumping infectious agents. The complex interaction of factors like environmental and ecological changes, social factors, decline of health care, human demographics and behaviour influences the emergence of re-emergence of such diseases. Viruses, especially RNA viruses with their ability to adapt quickly to changing environmental conditions, are among the most prominent examples of emerging pathogens. In this review, we present the important examples of zoonotic viruses and discuss the factors playing a key role in the emergence and resurgence of these diseases.

Polyomavirus viruria in bone marrow transplant recipients: lack of correlation with clinical symptoms.

BACKGROUND: Human polyomavirus (HPV) infection is controversially discussed as a factor influencing the outcome of bone marrow transplantation (BMT). PATIENTS AND METHODS: Here we report on 62 patients undergoing BMT with clinical signs of urocystitis, such as micro- or macrohematuria with more or less severe dysuria. These patients were tested for the presence of HPV in urine specimens (n = 80) by transmission electron microscopy (TEM). RESULTS: HPV viruria was found in 35 patients (56%); 33 (94%) of them had hemorrhagic cystitis, whereas two (6%) were suffering from dysuria only. Among the patients with hemorrhagic cystitis, ten (30%) presented with macrohematuria, four (12%) with microhematuria and 19 (58%) with micro- or macrohematuria and severe dysuria. 26 of 27 HPV-negative patients (96%) showed hemorrhagic cystitis with either macrohematuria (n = 7) (26%) or microhematuria (n = 15) (56%). Four patients (15%) suffered from hematuria and dysuria, one patient (4%) from dysuria only. CONCLUSION: Although HPV-negative patients tended to present with less severe clinical symptoms, overall no statistically significant influence on the outcome of BMT was seen.

Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients of allogeneic stem cell transplants.

BACKGROUND: Although several diagnostic methods are available for the surveillance of patients at risk of human cytomegalovirus (CMV) infection and disease, little data is available on their comparative performances in the diagnostic setting. OBJECTIVES: To compare different assays for CMV detection, especially assays based on (quantitative) DNA and mRNA detection. STUDY DESIGN: Eight allogeneic bone marrow and stem cell transplant recipients at high risk for developing CMV disease (donor CMV-negative, recipient positive) were regularly tested for 7-20 weeks post-transplant by spin-amplification rapid culture from urine (viruria), antigenemia (pp65 assay), pp67 mRNA in whole blood (NASBA), and CMV DNA both qualitatively (in-house PCR, whole blood) and quantitatively (in-house PCR, plasma; Cobas Amplicor CMV Monitor Test, plasma and whole blood; Hybrid Capture, whole blood). RESULTS: Four patients (50%) suffered CMV reactivation during follow-up. Out of 104 sample dates, 41 (39.4%) yielded a positive CMV result in at least one assay. Out of the 28 samples tested by all assays, the highest percentage of positive results was obtained with the in-house quantitative PCR (60.7%), followed by the Hybrid Capture system (39.3%), the Cobas Amplicor CMV Monitor Test, plasma version (35.7%), the Cobas Amplicor CMV Monitor Test, whole blood version (32.1%), in-house qualitative PCR (28.6%), and the mRNA assay (21.4%). Viruria was positive in one sample and pp65 antigenemia was found in two samples. CONCLUSIONS: Despite a considerable incidence of CMV reactivations, pre-emptive anti-CMV chemotherapy prevented the development of CMV disease with the exception of one case. The molecular assays had superior sensitivity to conventional ones. The antigenemia assay proved unsuitable for the surveillance of hematological transplant patients. However, none of the tests recognized all timepoints with CMV reactivation. Further comparative studies are needed to determine their respective diagnostic values.

Kaposi's sarcoma-associated herpesvirus seroprevalence in selected german patients: evaluation by different test systems.

A total of 603 serum samples obtained from 12 different patient and control groups, including potentially cross-reactive sera, were tested for the presence of antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8). The assays used were an inhouse immunofluorescence test (IFT) employing latent KSHV antigens and a prototype enzyme-linked immunosorbent assay (ELISA) coated with recombinant latency-associated KSHV nuclear antigen (LANA, open reading frame 73) and the K8.1 protein. Sera giving discrepant results were additionally tested with two commercial IFTs employing KSHV latent and lytic antigens, respectively. The low KSHV seroprevalence rate found in blood donors (3.0% by in-house IFT, 2.0% by recombinant ELISA) was comparable to that found previously in Western European countries. The highest KSHV seroprevalence rates were found in patients with Kaposi's sarcoma (100% by both assays), followed by HIV-infected men without Kaposi's sarcoma (23.3% by in-house IFT and 17.8% by ELISA) and women (15.7% by in-house IFT and 13.7% by ELISA). Overall correlation between both assays was 91.2%, with the highest rate of discordant results occurring in HIV-infected male subjects. Retesting of the 53 discrepant samples by the commercial IFTs revealed the best, albeit low, correlation between the in-house IFT and the commercial latent antigen IFT and poor correlations between the other assays. Apart from patients with autoimmune antibodies, there was no significant degree of non-specific reactivity in either of the KSHV tests due to antibodies against Epstein-Barr virus and human herpesvirus 6. Despite the lack of a "gold standard" for KSHV antibody detection, the fact that the results obtained overall agreed rather well indicates their suitability for conducting seroepidemiological studies.

Extended routine polymerase chain reaction surveillance and pre-emptive antiviral therapy for cytomegalovirus after allogeneic transplantation.

Pre-emptive treatment strategies based on sensitive screening for cytomegalovirus (CMV) infection up to day +100 after allogeneic transplantation have been shown to reduce the incidence of CMV disease during the period of surveillance. However, the use of ganciclovir has been associated with delays in immune reconstitution and an increased incidence of late CMV disease after day +100. In the present study, 81 patients undergoing allogeneic transplantation received polymerase chain reaction (PCR)-guided pre-emptive therapy based on detection of CMV DNA by PCR on 2 consecutive weeks up to day +180. Thirty-three of the 52 high-risk patients (CMV-seropositive donor or recipient) received a total of 45 treatment episodes up to day +100. Three of these patients (5.7%) developed CMV disease, with one fatality. Twelve of the surviving 44 high-risk patients (27%) required pre-emptive treatment between days +101 and +192, but none of these patients developed late CMV disease with a median follow-up of 402 d (range 117-952 d). Antiviral therapy was stopped after a single negative PCR result with no subsequent episodes of CMV disease while patients remained off antiviral treatment. As all initial episodes of CMV DNA detection occurred within 60 d of transplantation, it may be possible to discontinue monitoring beyond day +100 in patients who have remained CMV PCR negative before this. Thus, we have confirmed that PCR-guided pre-emptive therapy results in a low incidence of CMV disease before day +100 and that discontinuing treatment on the basis of viral clearance as determined by CMV PCR appears to be safe practice. In addition, we have observed no episodes of late CMV disease with an extension of surveillance to 26 weeks.

Cytomegalovirus infection decreases expression of thrombospondin-1 and -2 in cultured human retinal glial cells: effects of antiviral agents.

In fibroblasts, infection with human cytomegalovirus (HCMV) inhibits expression of the extracellular matrix proteins thrombospondin-1 and -2 (TSP-1 and TSP-2). These effects may depend on expression of HCMV immediate-early (IE) genes, which are activated by cellular transcription factor NF-kappaB. The influence of HCMV infection on TSP-1 and TSP-2 expression and the ability of different antiviral drugs to prevent these cellular changes in permissive cultures of human retinal glial cells were observed. Ganciclovir inhibited only HCMV late antigen (LA) expression, whereas antisense oligonucleotide ISIS 2922 and peptide SN50, inhibitors of HCMV IE expression and NF-kappaB activity, respectively, inhibited both IE and LA expression. ISIS 2922 and SN50, but not ganciclovir, prevented down-modulation of TSP-1 and TSP-2. The results showed that HCMV-induced down-modulation of TSP-1 and TSP-2 in retinal glial cells is prevented by inhibition of HCMV IE expression. These findings may be relevant to pathogenesis and treatment of HCMV retinitis.

No apparent effect of cidofovir in epidermodysplasia verruciformis.

This paper reports the failure of a patient suffering from Epidermodysplasia verruciformis, characterised by widespread infection of the skin with human papillomaviruses, to respond to topical and systemic treatment with the antiviral agent, Cidofovir, despite its previously demonstrated effectiveness against a range of different papillomavirus-associated conditions.

Quantitative detection of human immunodeficiency virus (HIV) antigen by the Enzymun-Test: comparison with alternative assays and nucleic acid sequence-based amplification of HIV type 1 RNA.

A new modular automated enzyme immunoassay (EIA) (Enzymun-Test HIV Ag: Boehringer Mannheim) for quantitative human immunodeficiency virus (HIV) antigen detection was evaluated by testing a panel of 1,506 serum samples, including seroconversions, dilution series, follow-up samples from patients under antiretroviral therapy, single serum specimens from HIV-seropositive individuals in different stages of infection, potentially cross-reactive samples, and sera from HIV-negative hospitalized patients. The Abbott HIV type 1 (HIV-1) antigen monoclonal antibody assay served as the reference assay, and nucleic acid sequence-based amplification (Organon Teknika) for quantitative amplification of HIV-1 RNA was used for follow-up of patients under antiretroviral chemotherapy. The Boehringer Mannheim and Abbott EIAs showed concordant results for the early detection of HIV antigen in all the seroconversion panels. The follow-up samples from 29 HIV-infected individuals under antiretroviral therapy gave divergent results between both antigen tests. For the detection of HIV antigen in single serum samples from HIV-infected patients in different stages of HIV infection, a higher number of positive samples was detected with the Abbott HIV-1 antigen monoclonal antibody assay in samples from patients in stages II and III of HIV infection. The Enzymun-Test detected three or more positive samples than did the Abbott assay among the samples of patients with AIDS. The concordance on a sample-to-sample basis between the Boehringer Mannheim and Abbott EIAs was 98.6%. The sensitivity of the Enzymun-Test in comparison to the reference assay was 97.2%; the specificity was 98.8%. Although no close correlation could be found between the amount of viral RNA in serum detected by nucleic acid sequence-based amplification and the concentration of HIV antigen, a high HIV-1 RNA copy number was mostly associated with high levels of HIV antigen. In conclusion, the Enzymun-Test permits accurate HIV antigen detection and offers, in contrast to previous assays, the possibility of completely automated detection.

Lack of correlation between different hepatitis C virus screening and confirmatory assays.

Numerous 2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was to compare the performance of five different commercially available screening assays and four 'confirmatory' assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative 'core' region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NS1 antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively. A clear association was observed between HCV-RNAemia in serum samples and index values (O.D. sample/O.D. cut-off) of the screening EIAs as well as with the number of reactive proteins in the confirmatory assays. In conclusion, the results of current screening and confirmatory assays are highly divergent. The additional diagnostic significance of the relatively expensive and labour-intensive immunoblots appears to be very limited. For the serological diagnosis of HCV infection and for blood donor screening, confirmatory assays should only be used if there is a borderline result by HCV EIA. The determination of infectivity by qualitative PCR and the follow-up of patients undergoing IFN therapy by HCV-RNA quantification appears to be much more useful.

Evaluation of 11 enzyme immunoassays for the detection of immunoglobulin M antibodies to Epstein-Barr virus.

Laboratory diagnosis of Epstein-Barr virus (EBV) infection is based mainly on serological tests. The analysis of the pattern of class-specific immunoglobulin response against defined viral antigens, i.e. virus capsid antigen (VCA), early antigen (EA) and Epstein-Barr nuclear antigen (EBNA), permits the differentiation between primary, latent and secondary (reactivated) EBV infection. In recent months, numerous test kits for the detection of VCA specific IgM antibody have been introduced on the international market. With a panel of well defined sera, the sensitivity and specificity of eleven different commercially available IgM ELISAs was evaluated. A well established, commercially available, indirect immunofluorescence assay (IFA) served as the reference test. Compared to the IFA, the Biotest and Sigma Diagnostic assays had the highest sensitivity for the detection of IgM antibody to VCA or EA. A variable number of false positive results (n = 0-9) was obtained with the EBV-IgM assays by testing potentially cross-reactive serum samples. Up to 19 serum samples from immunocompromised organ transplant recipients were found positive with the Sigma Diagnostics assay. The results of this study show that there are great differences in quality of current EBV-IgM-ELISA test kits. Depending on the clinical setting, it may be important to use a test kit which detects immunoglobulin M reactivity to EA in order to warrant an optimal sensitivity for the serological diagnosis of EBV reactivation in immunocompromised patients. However, since most immunosuppressed patients have serological reactivations, and in most cases these are asymptomatic, the clinical relevance of the detection of EA-IgM is very low.