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SUMMARYMeasles, mumps, rubella (MMR) and varicella zoster virus (VZV) infection can cause serious diseases and complications in the HIV-positive population. Due to successful vaccination programmes measles, mumps and congenital rubella syndrome has become neglected in Germany. However, recent outbreaks of measles have occurred from import-associated cases. In this cross-sectional study the serostatus for MMR and VZV in 2013 HIV-positive adults from three different university outpatient clinics in Bonn (n = 544), Cologne (n = 995) and Munich (n = 474) was analysed. Sera were tested for MMR- and VZV-specific immunglobulin G antibodies using commercial immunoassays. Seronegativity was found in 3% for measles, 26% for mumps, 11% for rubella and 2% for VZV. Regarding MMR, 35% of patients lacked seropositivity against at least one infectious agent. In multivariable analysis younger age was strongly associated with seronegativity against all four viruses, measles, mumps, rubella (P < 0·001, P < 0·001 and P = 0·001, respectively) and VZV (P = 0·001). In conclusion, there is high need for MMR and VZV vaccination in people living with HIV in Germany born in 1970 or later. Thus, systematic MMR and VZV antibody screening and vaccination should be implemented in the HIV-positive population to prevent serious disease and complications of vaccine-preventable diseases.

Background Adenovirus (ADV) infections can have a high mortality in immunocompromised patients and are difficult to treat. Objectives and study design We retrospectively analyzed occurrence and risk factors of ADV infection in 399 adults with hematological disorders undergoing hematopoietic stem cell transplantation (allo-HSCT), focusing on alternative donor transplantation (ADT) and disseminated disease. Results ADV infection occurred in 42 patients (10.5%). Disease was localized in 18 and disseminated in 6 patients. ADV infection was observed in 15% after ADT, performed in 29% of all recipients, and was less frequent (6%) in T-cell-replete (TCR) haploidentical transplantation using post-transplantation cyclophosphamide (PTCY) than in other ADT protocols. Lower age, the use of alternative donor grafts and acute graft-versus-host disease (GvHD) ≥ grade II were risk factors for ADV infection. After failure of standard antiviral treatment, three patients with disseminated ADV disease received one dose of ADV-specific T cells, resulting in virological response in 2/3 patients, clearance of ADV viremia in 2/2 patients, and survival of 1/3 patients; both patients with pneumonia died. Conclusions ADV infection was of moderate occurrence in our adult recipients of allo-HSCT despite a high proportion of potential high-risk patients receiving ADT. TCR strategies using PTCY might limit ADV complications in haploidentical transplantation. Despite feasible adoptive therapy strategies, outcome of disseminated disease remains dismal.

The determination of pathogen-specific antibody indices (AIs) of CSF and serum is an essential cornerstone in assessing neurological diseases and demands reliable high precision. Various companies provide ELISA kits for the detection of respective antibody concentrations and base AI calculation on a single CSF/serum pair of optical densities (ODs), combined with selection rules. The remainder of OD measurements is not used. There is no averaging of measurement errors and result stabilization. OD data from Siemens Enzygnost ELISA measurements of 2012-2016 proficiency survey samples for measles/rubella/varicella zoster/herpes simplex virus (MRZH) reaction (INSTAND e.V.) were reanalyzed. Several reference methods for calculating Q values from ODs using multiple-point evaluation are described. The methods are based on the α method and the four-parameter logistic (4PL) equation. Statistical analysis shows standard deviations of relative AI differences from AI target values to be significantly lower if derived from multiple-point evaluation instead of single-pair evaluation. Thus, the virus-averaged hit rate of a 10% target AI environment can be improved from 49% up to 69%. Waiving the usage of a standard curve in favor of parameter fitting significantly improves calculational precision for Siemens Enzygnost assays. Patient safety, diagnostic assay costs, and laboratory effectiveness might be improved for other test distributors as well.

We retrospectively compared the incidence of virus infections and outcome in the context of immune reconstitution in two different HLA-haploidentical transplantation (haplo-HSCT) settings. The first was a combined T-cell-replete and T-cell-deplete approach using antithymocyte globulin (ATG) prior to transplantation in patients with hematological diseases (cTCR/TCD group, 28 patients; median age 31 years). The second was a T-cell-replete (TCR) approach using high-dose posttransplantation cyclophosphamide (TCR/PTCY group, 27 patients; median age 43 years). The incidence of herpesvirus infection was markedly lower in the TCR/PTCY (22 %) than in the cTCR/TCD group (93 %). Recovery of CD4+ T cells on day +100 was faster in the TCR/PTCY group. CMV reactivation was 30 % in the TCR/PTCY compared to 57 % in the cTCR/TCD group, and control with antiviral treatment was superior after TCR/PTCY transplantation (100 vs 50 % cTCR/TCD). Twenty-five percent of the patients in the cTCR/TCD group but no patient in the TCR/PTCY group developed PTLD. While 1-year OS was not different (TCR/PTCY 59 % vs cTCR/TCD 39 %; p = 0.28), virus infection-related mortality (VIRM) was significantly lower after TCR/PTCY transplantation (1-year VIRM, 0 % TCR/PTCY vs 29 % cTCR/TCD; p = 0.009). On day +100, predictors of better OS were lymphocytes >300/μl, CD3+ T cells >200/μl, and CD4+ T cells >150/μl, whereas the application of steroids >1 mg/kg was correlated with worse outcome. Our results suggest that by presumably preserving antiviral immunity and allowing fast immune recovery of CD4+ T cells, the TCR approach using posttransplantation cyclophosphamide is well suited to handle the important issue of herpesvirus infection after haplo-HSCT.

Summary We investigated whether children after heart- (HTx) or heart–lung transplantation (HLTx) show protective antibody levels against recommended vaccinations, whether vaccination schedules are completed and which factors influence serologic immunity. We performed a cross sectional ELISA – quantification of specific antibodies in 46 patients after pediatric thoracic Tx. Findings were correlated to vaccination history, age at Tx, clinical course and immunosuppressive regimen. We found protective antibody levels against diphtheria in 74% of patients, against tetanus in 22%, against Haemophilus influenzae type b in 30% and against Streptococcus pneumoniae in 59%. Antibody concentrations against live attenuated vaccines were significantly lower in children transplanted in the first 2 years of life. Antibodies were absent for measles in 55% of late – and 81% of early transplanted children, for mumps in 66%/94%, for rubella in 30%/56% and for Varicella in 34%/63%. We found significant correlation of low antibody concentrations and age at Tx. Patients without protective antibody concentrations had significantly longer use of steroids. Vaccination schedules were incomplete or delayed in the majority of patients associated with more days in hospital pre-Tx. Our study shows that closer adherence to pretransplantation vaccination schedules and also post-transplantation monitoring of antibody levels are required in transplant patients.

Cells of the Reh line, originally derived from an ALL of the ‘Non-T, Non-B’ type were cultured in diffusion chambers implanted intraperitoneally into preirradiated CBA mice. At different intervals over a period of 20 d changes in surface characteristics were examined by labelling the cells with AcALLG, ATCG as well as with polyvalent AIg. The evaluation was performed by using direct immunofluorescence. In addition, the ability to form rosettes with SRBC, AET-treated sheep erythrocytes, mouse red blood cells and EoxAC was tested. On day 0 of the diffusion chamber culture the cells only carried cALLA, and no rosette formation was observed. In the course of the diffusion chamber culture the cells unequivocally developed T-cell antigen, and in 1 of 2 experiments they further acquired a receptor for forming AET-and E-rosettes. Conversely, in the other experiment a receptor for mouse red blood cells was detected in a considerable portion of the cells. Our data show that the rather undifferentiated Reh line cells in vitro are able to develop features of mature T-cells and attributes of early B-cells during the diffusion chamber culture. In vitro they apparently retain a bivalent potentiality of lymphatic maturation. The diffusion chamber system proves to be a suitable tool for promoting differentiation in these cells.

Environmental factors have long been known to influence immune responses. In particular, clinical studies about the association between migration and increased risk of atopy/asthma have provided important information on the role of migration associated large sets of environmental exposures in the development of allergic diseases. However, investigations about environmental effects on immune responses are mostly limited in candidate environmental exposures, such as air pollution. The influences of large sets of environmental exposures on immune responses are still largely unknown. A simulated 520-d Mars mission provided an opportunity to investigate this topic. Six healthy males lived in a closed habitat simulating a spacecraft for 520 days. When they exited their “spacecraft” after the mission, the scenario was similar to that of migration, involving exposure to a new set of environmental pollutants and allergens. We measured multiple immune parameters with blood samples at chosen time points after the mission. At the early adaptation stage, highly enhanced cytokine responses were observed upon ex vivo antigen stimulations. For cell population frequencies, we found the subjects displayed increased neutrophils. These results may presumably represent the immune changes occurred in healthy humans when migrating, indicating that large sets of environmental exposures may trigger aberrant immune activity.

A voluntary recall of VAQTA in prefilled syringes was implemented in Europe in late 2001 after the manufacturer noted a slight decrease in the antigen content of a small percentage of the syringes manufactured over a particular time frame. In Germany, a large-scale serologic testing program was implemented. The assay results were conveyed to the subject's physician, and free vaccine was provided for anyone requesting revaccination. An analysis was performed on a subset of 58,546 vaccine recipients with hepatitis A antibody results. Of the 28,681 persons who received either two 25 units (25 U) or two 50 units (50 U) doses of VAQTA, the seropositivity rate (SPR) was 99.5% after receipt of 2 doses, similar to the results in prelicensure clinical trials. The SPR was similar among recipients of lots that had been manufactured over the time frame associated with the recalled lots versus those receiving lots not associated with the recalled lots (25U: 99.7% versus 99,7%; 50U: 98.6% versus 99.6%, respectively). There were less recipients of 25U doses of the affected lots, who had high hepatitis A antibody titers (or =100 mIU/mL), compared to recipients of unaffected lots.

We studied 228 consecutive stem cell transplant recipients, screened for reactivation of human herpesvirus-6 (HHV-6) in peripheral blood and other specimens as clinically indicated by means of qualitative polymerase chain reaction. Among them, 197 received an allograft and 31 autograft. Ninety-six of 228 patients (42.1%) showed HHV-6 reactivation in peripheral blood and 129 of 228 (56.6%) demonstrated HHV-6 in at least one of the specimens tested. 41.9% of patients were asymptomatic when HHV-6 was identified. Clinical features, noted when HHV-6 was detected, included interstitial or alveolar pneumonia, gastroduodenal and colorectal disease, bone marrow suppression and liver disease. However, based on clinical and histopathological criteria, HHV-6 was considered a causal agent in only a minority of patients, in particular, those suffering from bone marrow suppression (n = 11), gastroduodenitis (five), colitis (three), interstitial/alveolar pneumonia (five), skin rash (one), pericarditis (two) and encephalitis (one). HHV-6 reactivation was significantly associated with the occurrence of graft-versus-host disease [odds ratio (OR) 5.31], Epstein-Barr virus coinfection (OR 8.89) and unrelated donor transplantation (OR 5.67) indicating an increased stage of immunosuppression.

Background. Transplant vasculopathy (TVP) is the most common cause of death and retransplantation after heart transplantation. Human cytomegalovirus (HCMV) infection has been linked to atherosclerosis and to the development of TVP. A prospective study evaluating the relation between CMV infection and progression of TVP is lacking thus far. The purpose of the present study was to investigate the influence of CMV infection status on the progression of TVP within 1 year. Methods. We enrolled 103 consecutive heart-transplant recipients who underwent routine cardiac catheterization and intracoronary ultrasound examination at study entry and after 1 year. Plaque progression determined by quantitative intracoronary ultrasound was used to define the severity of disease at baseline and at 1-year follow-up. At study entry, HCMV infection status was evaluated by immunological assays and reverse-transcriptase polymerase chain reaction (RT-PCR). Results. HCMV immunoglobulin (Ig)G/IgM seropositivity was found in 34 (33%) of transplant recipients, 11 of whom were HCMV PCR positive. The HCMV-positive group showed more advanced, calcified lesions (64.7% vs. 27.5%, P=0.002), and the maximal plaque thickness was significantly different from the HCMV IgG/IgM-negative group (median [quartile] 1.36 [0.85, 1.88] vs. 1.05 [0.58, 1.34], P=0.02). In a logistic regression model, we demonstrate that HCMV IgG/IgM positivity is a predictor for the progression of TVP independent of cardiovascular risk factors, inflammatory markers, and platelet activation (P=0.038). In addition, HCMV PCR positivity even increases the risk for accelerated TVP (P=0.017) and, consecutively, transplant failure. Conclusions. HCMV infection status in transplant patients detects patients with increased risk for transplant failure caused by TVP.

Measles virus induces an acute disease with rash and fever. Despite ongoing vaccination and elimination campaigns, the measles virus still sustains long-lasting transmission chains in Europe. Here we report the complete genome sequence of a wild-type measles virus isolated from a patient in Munich (MVi/Muenchen.DEU/19.13[D8]) during a German measles outbreak in 2013.

Human herpes virus type 7 (HHV-7) has been associated with unspecific febrile syndrome, exanthem subitum (ES), viral rashes and Epstein-Barr virus (EBV) like syndrome. Neurological complications such as hemiplegia or seizures have been described in a few children with ES. Whether HHV-7 may also affect the CNS in the absence of ES is unknown. In this study, we investigated CSF samples from children with different neurological diseases for the presence of HHV-7 specific DNA. A HHV-7 specific nested polymerase chain reaction (PCR) was established amplifying a 478 bp DNA sequence of the glycoprotein U23 of HHV-7 strain SB. 68 children with CNS diseases with inflammatory CSF findings (n = 24), CNS diseases without inflammatory CSF findings (n = 18) and febrile seizures (n = 26) were examined. A total of 26 children with infectious diseases in the absence of neurological disease and 11 children without signs of a peripheral infection and without neurological disease served as controls. The CSF samples of six children from the study groups were HHV-7 PCR positive, but none from the controls. These children were diagnosed with aseptic meningitis (n = 1), viral encephalitis/meningoencephalitis (n = 2), facial palsy (n = 1), vestibular neuritis (n = 1) and febrile seizure (n = 1).These results indicate that human herpes virus type 7 infection is associated with central nervous system disease in children and should be considered in children whether inflammation in the cerebrospinal fluid is present or not.

Human parvovirus B19 can persist in immunocompromised patients and may produce severe clinical illness. In this retrospective study the incidence of B19-associated infections in bone marrow transplant patients was investigated. During 1 year 60 patients received bone marrow grafts (eight autografts and 52 allogeneic transplantations). In case of early onset, atypical or steroid-resistant erythrodermia the patients' blood and/or tissue specimens were screened for B19 infection by polymerase chain reaction (PCR). Additionally, specimens of patients with severe organ failure were tested. A total of 64 PCRs was performed in 27 patients. Seven patients with erythrodermia and one with vulvovaginitis proved to be PCR positive. In patients with organ failure B19 DNA was detected in the myocardium and liver. The incidence of B19 infections in this cohort was 15% and the B19-associated mortality rate 7%. In conclusion, parvovirus B19-associated infections may be more common in immunocompromised patients than previously anticipated.

A previously healthy 10–year–old Greek boy born to nonconsanguineous healthy parents developed progressive liver disease after acute infectious mononucleosis. EBV–induced autoimmune hepatitis was suspected and treatment was started with high–dose prednisolone, acyclovir and intravenous immunoglobulins. Despite therapy, his liver function continuously deteriorated and the child died 9 months later in profound immune deficiency from candida septicemia. Flow cytometric analysis of his lymphocytes revealed a major subpopulation of atypical cells (20.3%) which were CD3+, fitted into the lymphocyte gate but showed a very low level of CD4 expression, comparable to that of monocytes. After short–time cell culture, the cells became adherent and developed granules and dendrites. We conclude that these cells may represent strongly activated CD4+ T lymphocytes with downregulated CD4 expression or a subtype of dendritic cells.

Several enzyme immunoassays (EIA) for the detection of parvovirus B19 IgM (anti-B19 IgM) are now commercially available. In this study, seven commercial EIAs (Biotrin, DAKO, Viramed, Viratech, R-Biopharm, Mast) were compared with an in-house EIA (MvP-EIA) using native viral B19 particles and the reference IgM radioimmunoassay (MACRIA). A total of 88 sera were tested. Results agreed in 39/88 (44.3%) sera, whereas 47/88 (53.4%) were discrepant and 2/88 (2.3%) gave an equivocal result. Assay sensitivity ranged from 70.3 to 100% and specificity, from 75.9 to 100%. The best results were obtained with two EIAs (Biotrin, DAKO) using baculovirus-expressed B19 proteins as antigen. This study has shown that baculovirus-expressed B19 antibody tests are suitable tools for detecting anti-B19 IgM.

Viral hemorrhagic fever has re-emerged in the United Arab Emirates (UAE) since November 1993. Genomic RNA of Crimean-Congo hemorrhagic virus (C-CHFV) was detected by a newly developed, nested reverse transcriptase polymerase chain reaction (RT-PCR) in the sera of four (25.0%) of 16 suspected cases of viral hemorrhagic fever. The RT-PCR was based on oligonucleotide primers deducted from the small RNA segment encoding the nucleoprotein of the virus. By comparison with a nucleotide sequence of a C-CHFV isolate from a Chinese sheep, a divergence of 10.0-11.8% was detected in the C-CHFV variants causing the UAE outbreak. In the four positive sera, three phylogenetically distinct C-CHFV variants were amplified and confirmed by direct sequencing of the PCR fragments. These C-CHFV sequences were obtained directly from sera of infected humans without prior propagation in cell culture. The RT-PCR allows rapid detection of genomic C-CHFV RNA in clinical specimens and study of the molecular epidemiology of this infection.

Sera from patients with sandfly fever caused by Toscana virus (TOSV) infection were tested by immunoblot for specific antibody response to TOSV derived from infected Vero-E6 cells. The 28 kDa TOSV nucleoprotein (N) was identified as the major immunodominant protein recognized by immunoblot. In sera of patients with acute TOSV infection, specific antibodies of the IgM, IgA, and IgG class were detected. Using sandfly fever virus, serotypes Sicilian (SFSV) and Naples (SFNV), as antigens for immunoblot, TOSV antibody-positive sera cross-reacted with the corresponding N proteins. These sera reacted for IgM and IgG by SFSV immunoblot, and for IgM by SFNV immunoblot. The diagnosis of sandfly fever may be confirmed by TOSV immunoblot.

Introduction BK nephropathy has emerged as an important cause for allograft failure in renal transplant patients. The kidney tubules are the main target of BK virus infiltration. Neutrophil gelatinase-associated lipocalin (NGAL) has been proven to be a powerful biomarker for tubular damage. Therefore, we investigated the suitability of plasma NGAL as new diagnostic tool in patients with BK infection. Material and methods We retrospectively analyzed 240 renal transplant recipients. Systematic BKV screening by plasma PCR was performed one month after transplantation and every three month thereafter for two yr. Plasma NGAL concentration was investigated using a commercial ELISA. Medical records and electronic databases were reviewed for clinical parameters. Results BK viremia (BKV+) was diagnosed in 5.0% (12/240) and BK nephropathy in 3.3% (8/240) of our patients. BKV+ patients received more induction therapy (p = 0.03) and experienced a higher rate of biopsy-proven rejections compared to 13 control patients with similar graft function but negative BKV PCR. Contrary to our hypothesis, there was no difference in plasma NGAL expression between both groups (128.6 vs. 172.2 ng/mL; p = 0.68). Conclusions Intensified immunosuppressive therapy is associated with an increased risk for BK nephropathy. Plasma NGAL is neither suitable for diagnosing BK nephropathy nor helpful in predicting the individual course of patients with BKV infection.

Human parvovirus B19 causes erythema infectiosum (fifth disease) and aplastic crisis. In pregnancy, B19 may be transmitted on to the fetus resulting in hydrops fetalis. B19 is sometimes associated with thrombocytopenia and vasculitis. In immunocompromised patients, B19 may persist causing chronic infection. Several methods for detecting B19 virus or specific antibodies are now available. B19 infection does not usually require therapy. Efficacy of immunoglobulins in chronic B19 infection has been demonstrated.

Summary A single tube, reverse transcriptase/polymerase chain reaction (RT-PCR) was developed and evaluated for detecting a 400-bp product of the small RIMA of sandfly fever virus, serotype Toscana (TOS). For more sensitive detection of genomic TOS RNA, a nested PCR amplifying a 243-bp cDNA within the RT-PCR product was established. Nucleotide sequence analysis of first- and second-round PCR products using the dideoxy cycle sequencing technique confirmed a previously published sequence of the TOS reference strain (ISS. Phi. 3). By nested PCR, genomic TOS RNA was amplified from two consecutive sera taken 3 and 7 weeks after the onset of illness in one patient, and from CSF of a second patient obtained at the onset of meningitis. Authenticity of amplified PCR products was confirmed by nucleotide sequence analysis, revealing a sequence identical to the TOS reference strain. RT-PCR and nested PCR are useful for laboratory diagnosis and studies of the molecular epidemiology of TOS infection.

Objective Monitoring the cellular immunity for Cytomegalovirus (CMV) in organ transplant recipients is a promising tool to support prevention strategies for posttransplant CMV infection or reactivation. Commercially available in vitro test systems differ substantially in their capacity to stimulate subpopulations of T-lymphocytes. We compared two assays for CMV immune monitoring in respect of their clinical practicability and significance. Methods Blood samples of 30 lung transplant recipients (LuTRs) were examined before transplantation and over a period of six months afterwards with T-Track® CMV (Lophius Biosciences GmbH, Regensburg) and QuantiFERON®-CMV (Cellestis GmbH, Darmstadt). The T-Track® CMV is based on ELISpot-technology, allowing quantification of interferon-gamma (IFNγ) secreting CD4+ and CD8+ T-cells after specific stimulation. Contrarily, the QuantiFERON®-CMV assay is restricted to detection of IFNγ secreted by CD8+ T-lymphocytes with ELISA. The data are evaluated in the context of transplant outcome and determination of viral load in plasma by qPCR. Results and Conclusion Both approaches provide similar results while exhibiting certain advantages and limitations. Early during immune suppressive therapy, QuantiFERON®-CMV generates often indeterminate results as depletion of T-cells is not taken into account. Although a comparatively large volume of blood is required, T-Track® CMV circumvents this drawback. In addition, T-Track® CMV reaches a higher sensitivity prior to LuTx and after onset of immunosuppression.

A serological survey for antibodies to hepatitis C virus (HCV), dengue viruses (DEN), West Nile virus (WN), and sindbis virus (SIN) was carried out in sera of selected groups of the population of the Islands of Mauritius (n = 449) and Rodrigues (n = 115), Indian Ocean. 8.3% of 564 sera were positive for anti-HCV. In Mauritius, 2.1% of sera of healthy individuals were found with anti-HCV. The highest prevalence was found in sexually transmitted disease (STD) patients and prison inmates with 46.2% and 43.8%, respectively. None of the sera from blood donors sampled from Rodrigues Island had anti-HCV. Antibodies to arbo-viruses were detected in sera of individuals from both islands. Anti-DEN IgG was detected in 3.8% of sera from Mauritius and 0.9% from Rodrigues. Anti-WN IgG was detected in 2.2% of sera from Mauritius and 0.9% from Rodrigues. All sera from Rodrigues were without anti-SIN IgG, 1.1% of those from Mauritius were positive. This suggests that arboviruses occur on these islands. © 1994 Wiley-Liss, Inc.

Desensitization strategies for ABO-incompatible renal transplants with plasma exchange (PE) or specific immunoadsorption (IA) decrease immunoglobulin levels. After recent measles outbreak and decreasing vaccination rates, we studied the impact of apheresis on anti-measles antibodies. Anti-measles antibodies were measured before desensitization, before transplantation and during followup in 12 patients with ABO incompatibility (2x PE only, 8x IA only, and 2x IA and PE) and 3 patients with donor-specific HLA antibodies (all PE). Patients received rituximab, IVIG, and standard immunosuppressive therapy. All patients had detectable anti-measles antibodies before desensitization (mean 3238 mU/l, range 560–8100). After 3–6 PE sessions, titers decreased significantly to 1710 mU/l ( 𝑃 0 . 0 5 ), in one patient to nondetectable values, while IA only maintained protective titers. After a median followup of 64 days, anti-measles antibodies returned to baseline in all patients. Immunity against measles was temporarily reduced by apheresis but remained detectable in most patients at time of transplantation. Desensitization maintains long-term protective immunity against measles.

We investigated whether children after heart- (HTx) or heart-lung transplantation (HLTx) show protective antibody levels against recommended vaccinations, whether vaccination schedules are completed and which factors influence serologic immunity. We performed a cross sectional ELISA - quantification of specific antibodies in 46 patients after pediatric thoracic Tx. Findings were correlated to vaccination history, age at Tx, clinical course and immunosuppressive regimen. We found protective antibody levels against diphtheria in 74% of patients, against tetanus in 22%, against Haemophilus influenzae type b in 30% and against Streptococcus pneumoniae in 59%. Antibody concentrations against live attenuated vaccines were significantly lower in children transplanted in the first 2 years of life. Antibodies were absent for measles in 55% of late - and 81% of early transplanted children, for mumps in 66%/94%, for rubella in 30%/56% and for Varicella in 34%/63%. We found significant correlation of low antibody concentrations and age at Tx. Patients without protective antibody concentrations had significantly longer use of steroids. Vaccination schedules were incomplete or delayed in the majority of patients associated with more days in hospital pre-Tx. Our study shows that closer adherence to pretransplantation vaccination schedules and also post-transplantation monitoring of antibody levels are required in transplant patients.

Objectives: To describe a previously healthy patient now suffering from monophasic encephalitis caused by a primary infection with BK virus and to discuss possible risk factors for developing BK virus encephalitis. Design: Case report. Setting: Referral hospital. Patient: The patient was examined on referral. Main Outcome Measures: The main diagnostic tests performed were serology, polymerase chain reaction on cerebrospinal fluid samples, and cranial magnetic resonance imaging. Results: During the course of the patient's encephalitis, an IgM titer developed against polyomavirus, followed by anti—polyomavirus IgG. Wild-type BK virus was demonstrated in cerebrospinal fluid samples. Cranial magnetic resonance imaging showed diffuse reversible white matter changes most prominent on T2-weighted images. Conclusion: We conclude that diagnostic tests for BK, a human polyomavirus, should be included in the screening program for encephalitogenic pathogens.

In a retrospective analysis, we identified 38 evaluable patients who received intravenous ribavirin after adenovirus or RSV detection in the respiratory and/or gastrointestinal tract throughout the years 1998 and 2001. A total of 43 treatment cycles are analyzed. Intravenous ribavirin was combined with cidofovir in about half of the patients. In six out of eight patients treated because of RSV isolates from the respiratory tract, the virus was no longer detectable after treatment. In case of adenovirus isolates, the treatment was efficacious in eradicating the virus from the respiratory tract in more than 60% and from the gastrointestinal tract in 75% of treatment cycles. The addition of cidofovir was not beneficial in eradicating RSV isolates, but somewhat improved the virus clearance of adenovirus. Virus clearance was associated with a trend to a better median survival after virus detection. There were some episodes of suspected hemolysis and a trend towards lower leukocyte counts, reaching grade 3 toxicity in about 15% of treatment cycles. However, in general, intravenous ribavirin was well tolerated. In conclusion, the possible efficacy of intravenous Ribavirin in controlling RSV or adenovirus infections after allogeneic stem cell transplantations should be evaluated in prospective clinical trials. The Hematology Journal (2004) 5, 135–144. doi:10.1038/sj.thj.6200358

The proteomic approach is a valuable tool to detect and identify proteins that are associated with cancer. In previous investigations on experimentally induced rat hepatomas, we detected aldose reductase-like protein (ARLP) as a highly significant marker protein. Our present study was intended to look for the presence of similar tumor-associated marker proteins on human hepatocellular carcinomas (HCC). We found several novel tumor-associated protein variants that represent members of the aldo-keto reductase (AKR) superfamily. Human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumor-associated AKR member detected in HCC by 2-dimensional electrophoresis (2-DE) and identified by mass spectrometric fingerprinting. The enzyme was found in 4 distinct forms (hARLP-1, 36/7.4 (kd/pI); hARLP-2, 36/7.2; hARLP-3, 36/6.4; and hARLP-4, 33/7.35). In addition, a human aldose reductase-like protein (hARLP-5, 36/7.6) was identified that differed from hARLP-1 by 1 amino acid (D313N), indicating 2 allelic forms of the human aldose reductase-like gene. A novel antibody directed against common parts of the hARLPs revealed hARLP reactivity in human HCC by immunohistochemistry. Furthermore, aldose reductase (AR) was identified and characterized as a tumor-associated variant. In conclusion, in all investigated human HCCs at least one of the various types of the described tumor-associated proteins of the AKR superfamily was clearly present. Of these HCC samples, 95% were positive for hARLPs as proven by 2-DE analysis and/or by use of the antibody directed against hARLP. Thus, hARLP is a strong candidate for use as an immunohistochemical diagnostic marker of human HCC.

Sandfly fever virus, serotype Toscana (TOS), is endemic in some Mediterranean countries and causes sandfly fever (pappataci fever). In some patients, TOS may cause meningitis and meningoencephalitis. We report on two German adults returning from Italy with TOS-related meningitis, complicated in one case by abducens nerve palsy. TOS infection should be considered as a cause of acute central nervous system disorders in patients returning from areas of endemicity.

2, not significant), respectively. sACE (U/L) tracked, codominantly, with the D allele in both groups (sarcoidosis group sACE against genotype p=0·01, control group p

Laboratory diagnosis of imported, vector-borne virus diseases during a 22-month-period in Munich, Germany, is summarized. In 13/317 Germans returning from the Mediterranean with suspected sandfly fever, acute sandfly fever, serotype Toscana, was confirmed serologically: 84.6% of the infections were acquired in Italy. Of 249 German tourists with febrile disease returning from the tropics, acute infection with dengue virus was diagnosed serologically in 26 (10.4%): most infections were acquired in Thailand (57.7%). In a seroepidemiological study of 670 German aid workers who had spent two years in the tropics, 49 (7.3%) were positive for antibodies to dengue, 9 (1.3%) to Chikungunya, and 1 (0.1%) to Sindbis virus. Of 17 Middle Eastern patients with suspected viral haemorrhagic fever, genomic Crimean-Congo haemorrhagic fever virus RNA was amplified in 4 (23.5%) by semi-nested reverse transcriptase polymerase chain reaction, and confirmed by molecular characterization of nucleic acid. With the increase in travel to and from endemic areas, imported vector-borne virus infections are increasingly important in Germany.

Summary Dengue viruses (DEN) are increasingly becoming endemic and epidemic in tropical and subtropical countries. In this study, 17 serologically confirmed clinical cases of DEN infection diagnosed in German tourists over the period from May 1993 until May 1994 are presented. Thirteen out of 17 (76.S%) infections occurred in southeast Asia (Thailand S8.9%), and 4/17 (23.S%) in Central and South America. All sera screened by means of the indirect immunofluorescence assay (IIFA) were positive for anti-DEN IgM. Acute infection was confirmed by demonstrating anti-DEN IgM using a “μ-capture assay”. Sixteen out of 17 (94.1%) of patients presenting with fever upon admission were positive for anti-DEN IgG, with titres of ≥ 128 in the IIFA. This report indicates the rising significance of DEN as a travel-associated infection in German tourists.

SUMMARYOf eight acute infections in German tourists caused by sandfly fever virus, serotype Toscana (TOS). and diagnosed clinically and serologically, seven were acquired during visits to Tuscany, Italy, and one to Coimbra, Portugal. An indirect immunofluorescence assay (IFA) using infected cells, and a newly developed enzyme-immunoassay (EIA) using crude virus antigen prepared from infected Vero-E6 cells was used to detect anti-TOS IgM and IgG. In a seroepidemiological survey of 859 health care workers and medical students, anti-TOS IgG was detected in 1·0% by IFA, and in 0·7% by EIA. In 2034 German patients hospitalized for various diseases, 1·6% were positive for anti-TOS IgG by IFA, and 0·8% by EIA. Anti-TOS IgG was detected in 43 samples of commercial immunoglobulins at titres of 10–1000 by EIA. Although the seroprevalence of antibodies to TOS is low in Germany, TOS infection should be considered in patients returning from endemic areas who complain of fever, and headaches, and have symptoms of meningitis.

Summary Laboratory diagnosis of parvovirus B19 (B19) infection has been hampered by the limited availability of B19 virus. Recombinant viral proteins are now available for use as antigen in serological assays. We compared detection of anti-B19 IgM by “μ-capture assay” using viral B19 particles to a recombinant (rec.) immunoblot and a rec. enzyme-immunoassay (ELISA) using viral structural proteins as antigens expressed in E. coli . The rec. immunoblot was 94.3% sensitive and 96.4% specific for anti-B19 IgM, and the sensitivity of the rec. ELISA was 94.3% and the specificity, only 72.7%. There was an agreement between the “μ-capture assay” and the rec. immunoblot in 87.8% and the rec. ELISA in only 74.4%. For detection of anti-B19 IgG in patients with acute B19 infection, the rec. immunoblot was 94.3% and the rec. ELISA 85.7% sensitive. The rec. immunoblot is more reliable for detection of acute B19 infection than the rec. ELISA.

Fetal tissues from 16 spontaneous abortions, two terminations, and one perinatal death, 18 of which were associated with maternal human parvovirus B19 infection, were examined for B19 infection by histology and in situ hybridization using a digoxigenin-labeled B19-DNA probe. In 15 spontaneous abortions and one termination, erythroblasts with intranuclear inclusions (lantern cells) reacted with B19-DNA by in situ hybridization. No internal or external fetal malformations were observed. Because 13 (86.7%) spontaneous abortions with lantern cells occurred between the 20th and 28th weeks of gestation, it is postulated that B19 infection may be a particular threat to the fetus during this stage of gestation.

Fetal tissues from 16 spontaneous abortions, two terminations, and one perinatal death, 18 of which were associated with maternal human parvovirus B19 infection, were examined for B19 infection by histology and in situ hybridization using a digoxigenin-labeled B19-DNA probe. In 15 spontaneous abortions and one termination, erythroblasts with intranuclear inclusions (lantern cells) reacted with B19-DNA by in situ hybridization. No internal or external fetal malformations were observed. Because 13 (86.7%) spontaneous abortions with lantern cells occurred between the 20th and 28th weeks of gestation, it is postulated that B19 infection may be a particular threat to the fetus during this stage of gestation.

Detection of Human Herpesvirus Type 7 (Hhv7) in the Cerebrospinal Fluid(Csf) of Children With Aseptic Meningitis/Encephalitis and Other Central Nervous System Diseases By Polymerase Chain Reaction (Pcr)

Necrotizing enterocolitis can occur in epidemics. Various infectious agents have been associated with these clusters, but so far there has been no report of Parvovirus B19 infection and NEC in previously healthy premature infants. We report here an epidemic of NEC linked to Parvo B19 infection Within 10 days 8 premature infants developed the clinical picture of NEC, 6 of them within 24 hours. Their GA ranged from 27 - 32 weeks, their postnatal age from 4 days to 6 weeks. Prior to the onset of severe abdominal pain wiih bloody stools all infants had tolerated their enteral feedings well. Three infants required exploratory laparatomy. The entire small and large intestines were intraoperatively found to be cyanotic with no focal lesion or focal necrosis. Stool and blood cultures were negative for enteric pathogens and Rotavirus Parvo B19 DNA was found in stool and serum samples of 3 infants by the Polymcrase-Chain-Reaction (PCR), 3 infants were negative and 2 infants were not studied. Primers were selected from the VP1 region, so they bracketed a 240 base pair fragment of the Parvovirus B19 genome. Sequence analysis of the PCR-products revealed characteristics typical of Parvo B19 in all investigated positive cases. There were 2 isolated cases of Parvo B19 pos. NEC 4 month later. Summary: Parvovirus B 19 could be detected with PCR in 5 of 10 infants with NEC, but only in 2 of 19 healthy control infants. Since 2 sick infants were not examined no definite statement can be made, but we believe that there is now some evidence that Parvovirus B19 infection can cause necrotizing enterocolitis in neonates. Further prospective studies are needed to elucidate this association.

In order to explore their differentiation capacity peripheral blasts from 4 children with acute lymphoblastic leukemia of the common type (cALL) as well as cells of the Reh line were cultured in diffusion chambers implanted intraperitoneally into preirradiated CBA mice (8 gy). At various times after initiation of the culture, changes in the membrane marker profile were studied by investigation of surface antigens and rosetting phenomena. In the course of the culture, the cells of two patients, T. H. and I.G., developed surface immunoglobulin (sIg) in combination with the CALL antigen (cALLA). Simultaneously, the cells of I.G. containing cytoplasmic Ig prior to cultivation acquired a receptor for mouse erythrocytes (EM). Besides quite a few cells developing the T-cell antigen, the majority of the cell yield in patient V.M. expressed sIg alone or together with the cALLA. Comparable results together with an increasing portion of mouse rosette forming cells were obtained in the fourth patient, C.M. In case of the Reh-line the cells developed the T-cell antigen, and in one of two experiments they further acquired a receptor for sheep erythrocytes. conversely, in a second experiment and EM-receptor was detected. Our data suggest that leukemic blast cells carrying the cALLA are representative of an early developmental stage of lymphatic ontogeny.

cALL positive blast cells from peripheral blood of four children as well as cells from the Reh line were cultivated in DC in order to promote cell differentiation. DC were implanted into the peritoneal cavity of pre-irradiated CBA mice. At different intervals during the culture period changes in the membrane marker profile were determined by direct immunofluorescence after labeling the cells with AcALLG, ATCG and polyvalent AIg. In addition, the ability of cells to form rosettes with SRBC, AET-treated sheep erythrocytes and mouse red blood cells was tested. In the course of the culture the majority of cALL positive cells from three patients (T.H., V.M., I.G.) simultaneously developed a positive reaction with polyvalent AIg. Furthermore, a remarkable portion of cells in V.M. turned out to become positive with only AIg. Comparable results together with an increasing percentage of M-RFC were obtained in the fourth patient, C.M., using AIg(Fab) 2 . Besides this development, quite a few cells expressing the T-cell antigen occurred in the latter two patients. In the case of the Reh line, the cells developed T-cell antigen, and in one of two experiments they further acquired an E-receptor. Conversely, in the second experiment a receptor for mouse red blood cells was detected. Our data suggest that malignant cells carrying the cALLA are representative of an early developmental stage of lymphatic ontogeny.

Blast cells derived from blood and marrow samples of a patient with acute lymphoblastic leukemia (ALL), as well as from the Reh line originally established from an ALL patient, were cultured in diffusion chambers implanted i.p. into preirradiated CBA mice. At different intervals over a period of up to 20 days, surface immunoglobulins, ALL antigen, and T-cell antigen were investigated by using direct immunofluorescence. Rosette formation was tested with sheep and mouse erythrocytes. On day 0, the cells expressed only ALL antigen at the surface, and no rosette formation was observed. During culture, the patient's lymphoblasts, which originally had cytoplasmic IgM in addition to ALL antigen, expressed surface immunoglobulins as well as mouse erythrocyte receptors. The Reh line cells were ambivalent in two experiments developing T-cell antigens and sheep erythrocyte receptors as well as mouse erythrocyte receptors. Our data suggest that the differentiation arrest in leukemic lymphoblasts can be overcome, thus entailing a surface pattern similar to mature T- or B-lymphocytes.

9 erwachsene Patienten mit vom T-Zellsystem ausgehenden lymphatischen Systemerkrankungen wurden untersucht: 4 Patienten mit T-ALL; 1 T-ALL-CLL-Mischform; 2 T-CLL; 2 Sezary-Syndrom. Die klinischen Besondernheiten der verschiedenen Leukamieformen wurden verglichen, morphologische und zytochemische Befunde beschrieben. Die T-Zelleigenschaft der jeweiligen malignen Zellpopulation sowie die B- und T-Zellzusammensetzung der morphologisch normalen Rest-Lymphozytenpopulation wurden durch folgende Zellmarker festgestellt: Nachweis von T-Zellantigen bzw. Membranimmunglobulin durch spezifische heterologe Antiseren, konjugiert mit Peroxidase,125Jod oder Fluoreszein; KBR oder Zytotoxizitat durch derartige Antiseren; spontane Rosettenbildung mit Schaferythrozyten oder mit Acrylsaure-Perlen. Die Stimulierbarkeit der malignen Zellen, bzw. der restlichen, morphologisch normalen Zellen durch pflanzliche Mitogene oder allogene Zellen wurde gepruft. Unter Berucksichtigung aller Befunde stellen die T-Zell-Leukamien des Erwachsenen eine besondere Krankheitsgruppe dar, deren einzelne Unterformen Gemeinsamkeiten und fliesende Ubergange aufweisen.

Auer-rod positive acute myelocytic leukemia (AML) blasts from a 33-year-old patient were cultured in diffusion chambers (DC) in order to test their differentiation potential. The cells were labeled with anti-human granulocyte anti-serum known to be negative for eosinophils, and evaluated using the unlabeled peroxidase-antiperoxidase (PAP) method. Parallel to a decline in the number of leukemic blasts there was an increase of up to 86% in the number of granulocytic cells belonging to the eosinophilic series. Auer-rod bodies were found in the eosinophilic cells even after 20 days in culture. Staining with anti-granulocyte antiserum failed to demonstrate positive cells at any time during DC culture. Based on the negative reaction with the anti-granulocytic antibodies already at an early stage of development evidence is provided for the existence of a progenitor cell exclusively committed to the eosinophilic pathway.

SummaryWe report the case of a young female lung transplant recipient with difficult-to-treat cytomegalovirus (CMV) disease. While treatment with intravenous (IV) ganciclovir failed due to antiviral drug resistance, a trial with foscarnet resulted in severe side effects. In addition, the patient received IV CMV-specific immune globulins as adjunctive therapy and leflunomide as experimental therapy. In this context, CMV-specific immune monitoring was performed and was successfully implemented in management decisions. The patient was screened for acquisition of an adaptive immune response, and antiviral prophylaxis and therapy was tailored according to results. This report highlights the impact of CMV-specific immune monitoring on individualized therapy for appropriate prophylaxis and management of CMV infection and diseases.

Peripheral mononuclear blood cells of 2 healthy individuals were cultured over a 13 d period in diffusion chambers. A high percentage of cALL antigen positive cells and later on TdT containing cells appeared during culture. The cALL+ cells could be clearly distinguished into 2 groups: a population with weak and diffuse fluorescence and a small number of ring-like brightly dyed cells. The first group develops into TdT+ cells and therefore resembles immature T-cells. The interpretation of the ring-like stained cells is still preliminary, but it cannot be excluded, that these cells represent a cell population with more than lymphatic differentiation capacity.