Shedding of norovirus in symptomatic and asymptomatic infections.

Norovirus is the most frequent cause of acute infectious gastroenteritis and it is difficult to control in crowded environments like hospitals and nursing homes. Transmission depends on oral intake of virus deposited in the environment by infectious subjects. Data from volunteer studies indicate that virus concentrations in stool are highly variable, but systematic studies of the time-course of shedding and its individual variation are lacking. This paper quantifies norovirus shedding in a large population of 102 subjects, including asymptomatic shedders, and uses a longitudinal model to generalize shedding patterns. Enhanced surveillance for studies of transmission of norovirus in hospital outbreaks has yielded a considerable number of faecal samples from symptomatic and asymptomatic shedders, both from patients and staff. Norovirus concentrations were determined by real-time PCR. A quantitative dynamic model was fitted to the shedding data, in a multilevel Bayesian framework, to study the time-course of shedding and its variation. The results indicate that shedding in asymptomatic subjects is similar to that in symptomatic infections, both showing considerable variation in peak levels (average 105-109 /g faeces) as well as duration of virus shedding (average 8-60 days). Patients appear to shed higher numbers of virus than staff, for slightly longer durations, but the differences are too small to be significant. Given equal shedding, the greater contribution of symptomatic cases to transmission must be caused by their higher efficiency in spreading these viruses. The results of this study will be helpful for risk studies that need to quantify the deposition of virus in the environment.

Leflunomide as part of the treatment for multidrug-resistant cytomegalovirus disease after lung transplantation: case report and review of the literature.

Treatment of cytomegalovirus (CMV) disease in transplant patients is challenging and, with antiviral resistance to first-line drugs, it remains uncertain which treatment algorithm to follow. Some data suggest that leflunomide, a pyrimidine synthesis inhibitor, can be used to treat resistant CMV infections. We report a 57-year-old CMV immunoglobulin-G (IgG)-seronegative woman, who received a bilateral lung transplant (LuTx) from a CMV IgG-positive donor with CMV primary disease. The CMV strain was genotypically resistant to ganciclovir, foscarnet, and cidofovir. After starting leflunomide as add-on therapy to a multidrug anti-CMV regimen, viral load declined substantially in 2 months without adverse events. This experience is discussed against the background of existing literature on the use of leflunomide as an anti-CMV agent in LuTx recipients.

An outbreak of severe respiratory tract infection caused by human metapneumovirus in a residential care facility for elderly in Utrecht, the Netherlands, January to March 2010.

Recognition of infections with human metapneumovirus (HMPV) among institutionalised elderly is rising. When HMPV was found to be the causative agent of an outbreak of pneumonia in a residential care facility for elderly in the Netherlands, an elaborate outbreak investigation was set up, including active surveillance for new cases. From clinical cases, defined by fever (> 38°C) and symptoms of respiratory tract infections, respiratory samples for analyses of viral pathogens by real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) and blood samples for determination of HMPV-specific IgM and IgG antibody titres were taken. Five staff members and 18 residents fulfilled the clinical case definition. Of those, five residents tested positive for HMPV by rRT-PCR. The combination of rRTPCR and serology identified nine confirmed cases, six probable cases, six possible cases and ruled out two persons as cases. Among residents, the outbreak of HMPV had an attack rate, ranging from 5% for laboratory- confirmed cases, to 13% for clinical cases. This outbreak investigation shows that HMPV is a potential serious pathogen for institutionalised elderly.

Evaluation of the antiviral response to zanamivir administered intravenously for treatment of critically ill patients with pandemic influenza A (H1N1) infection.

A retrospective nationwide study on the use of intravenous (IV) zanamivir in patients receiving intensive care who were pretreated with oseltamivir in the Netherlands was performed. In 6 of 13 patients with a sustained reduction of the viral load, the median time to start IV zanamivir was 9 days (range, 4-11 days) compared with 14 days (range, 6-21 days) in 7 patients without viral load reduction (P = .052). Viral load response did not influence mortality. We conclude that IV zanamivir as late add-on therapy has limited effectiveness. The effect of an immediate start with IV zanamivir monotherapy or in combination with other drugs need to be evaluated.

Quantification of viral DNA and liver enzymes in plasma improves early diagnosis and management of herpes simplex virus hepatitis.

Herpes simplex virus (HSV) hepatitis is a rare and potential life-threatening disease. The diagnosis of HSV hepatitis is hampered by its indifferent clinical presentation, which necessitates confirmatory laboratory data to identify HSV in the affected liver. However, liver biopsies are often contraindicated in the context of coagulopathy, are prone to sampling errors and have low sensitivity in mild HSV hepatitis cases. There is an unmet need for less invasive diagnostic tools. The diagnostic and therapeutic value of HSV DNA load and liver enzyme level kinetics was determined in five patients with HSV hepatitis and twenty disease controls with HSV-DNAemia without hepatitis. At time of hospitalization, patients with HSV hepatitis had a higher median (± interquartile range) HSV DNA load (6.0 × 10(6) ± 1.2 × 10(9)) compared to disease controls (171 ± 2845). Viral DNA load correlated with liver transaminase levels and disease severity. Antiviral treatment led to rapid decline of HSV DNA load and improvement of liver function of patients with HSV hepatitis. The data advocate the prompt and consecutive quantification of the HSV DNA load and liver enzyme levels in plasma of patients suspected of HSV hepatitis as well as those under antiviral treatment.

Norovirus in a Dutch tertiary care hospital (2002-2007): frequent nosocomial transmission and dominance of GIIb strains in young children.

We report a retrospective analysis of norovirus (NoV) infections occurring in patients of a tertiary care hospital during five winter seasons (2002/03 to 2006/07). Data were compared with national surveillance data and with corresponding data for rotavirus. Between July 2002 and June 2007, faecal specimens from 221 (9.0%) of 2458 hospital patients with diarrhoea tested positive for NoV. The incidence in children varied from 2.52 per 1000 admissions in 2004/05 (when testing began to be performed routinely) to 11.9 per 1000 admissions in 2006/07, while the incidence in adults remained stable (mean: 1.49 per 1000 admissions). Two genotypes predominated during the study period: GIIb strains occurred mainly in children below the age of two-and-a-half years [odds ratio (OR): 14.7; P<0.0001] whereas GII.4 strains affected all age groups. Compared with rotavirus infections, NoV infections in children were more often hospital-acquired (59% vs 39%, OR: 2.29; P<0.01). Among these cases we identified 22 clusters of NoV infection among inpatients. Twelve of 53 patients from whom follow-up samples were available demonstrated long-term virus shedding. We report a dynamic pattern of sporadic NoV infections in large hospitals, with frequent nosocomial transmission and with the predominance of GIIb-related strains in children. Effective prevention strategies are required to reduce the impact of sporadic NoV infection in vulnerable patients.

Clinical relevance of quantitative varicella-zoster virus (VZV) DNA detection in plasma after stem cell transplantation.

Detection of Varicella-Zoster virus (VZV) DNA in plasma can facilitate the early recognition of complicated VZV-infection in immunocompromised hosts. The correlation of VZV-DNA in plasma with clinical presentations of VZV-infection and subsequent aciclovir treatment in allogeneic stem cell transplant (allo-SCT) recipients was studied. In 81 consecutive VZV-IgG positive allo-SCT recipients, VZV-DNA was measured at regular time points (1, 2 and 4 months) following allo-SCT and patient records were screened for VZV-related symptoms and aciclovir treatment. Subsequently, possible VZV-cases were studied in detail for the course of VZV-DNA and treatment effects. During the initial screening, VZV-DNA was detectable in seven patients. The survey of VZV-related symptoms revealed five additional possible VZV-cases. In cases where suitable plasma samples were available (10 out of 12), VZV-DNA was present almost simultaneously with the first clinical manifestations. No evidence of a preceding phase detectable by VZV-DNA only could be observed. Treatment with aciclovir was associated with a prompt reduction of VZV-DNA load. Detection of VZV-DNA in plasma in allo-SCT recipients accurately reflected the clinical presentation of VZV-infection and treatment with aciclovir. VZV-DNA detection in plasma of allo-SCT recipients appears clinically relevant as this may support early recognition and therapeutic management of VZV-infections following allo-SCT.

Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR.

BACKGROUND: Adequate laboratory diagnosis of human rhinoviruses (hRV) and human metapneumoviruses (h MPV) requires molecular methods as viral culture lacks sensitivity. However, setting up individual PCRs for all respiratory viruses is not practical so preferentially multiplex PCRs are used. OBJECTIVES: To develop for routine diagnosis a rapid real-time PCR assay for detection of hRV and h MPV including an internal control in a single tube multiplex reaction using probes carrying different fluorophores to discriminate targets. STUDY DESIGN: The multiplex real-time RNA PCR was optimized to include the internal control virus and a total of 358 respiratory samples from 239 patients taken over a one-year period were analyzed by the multiplex assay. RESULTS: The multiplex assay with co-amplification of the internal control was as sensitive and specific as the individual assays. Application of this assay on clinical samples from 239 patients in a one-year period resulted in an incidence of hRV and h MPV of 41/239 (17.1%) and 6/239 (2.5%), respectively. Inhibition, defined as poor internal control amplification, was detected in 8 (2.2%) samples. Culture was performed on these samples and only four hRV were detected. CONCLUSIONS: This real-time PCR method enables sensitive diagnosis of these two respiratory pathogens with the potential to expand the assay as part of a full molecular respiratory viral screen.

Adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity.

Human adenoviruses (HAdV) are a frequent cause of potentially fatal infections in patients after allogeneic stem cell transplantation, especially in children. Monitoring of serum/plasma by real-time quantitative PCR is a sensitive tool for the recognition of patients at risk of a potentially fatal infection and for the evaluation of the efficacy of treatment. Data from a retrospective study and from a prospective study demonstrate that recovery of immunity after transplantation is essential for the elimination of HAdV infection. The feasibility of several approaches for the manipulation of immunity in the immunocompromised host to prevent a fatal course of the infection is discussed.

Effect of ribavirin on the plasma viral DNA load in patients with disseminating adenovirus infection.

Adenovirus (AdV) infections are an increasingly frequent and potentially fatal complication in allogeneic stem cell transplant recipients. To determine the antiviral potential of ribavirin in an unbiased way, 4 patients without immune recovery were prospectively analyzed by quantitative measurement of plasma AdV DNA load. Administration of ribavirin at the first signs of AdV dissemination was not accompanied by a decrease in the plasma AdV DNA load in any of these patients, and an increase in the AdV load was even documented in 3. These observations question the potential of ribavirin to improve the outcome for patients with disseminating AdV infection and support a critical evaluation of antiviral treatments for AdV infection that involves the kinetics of virus DNA load as an objective parameter of viral replication.

First experiences with an accelerated CMV antigenemia test: CMV Brite Turbo assay.

BACKGROUND: Cytomegalovirus disease is still a major problem in immunocompromised patients, such as bone marrow or kidney transplantation patients. The detection of viral antigen in leukocytes (antigenemia) has proven to be a clinically relevant marker of CMV activity and has found widespread application. Because most existing assays are rather time-consuming and laborious, an accelerated version (Brite Turbo) of an existing method (Brite) has been developed. The major modification is in the direct lysis of erythrocytes instead of separation by sedimentation. OBJECTIVE: In this study the Brite Turbo method has been compared with the conventional Brite method to detect CMV antigen pp65 in peripheral blood leukocytes of 107 consecutive immunocompromised patients. RESULTS: Both tests produced similar results. Discrepancies were limited to the lowest positive range and sensitivity and specificity were comparable for both tests. CONCLUSIONS: Two major advantages of the Brite Turbo method could be observed in comparison to the original method: assay-time was reduced by more than 50% and only 2 ml of blood was required. An additional advantage was the higher number of positive nuclei in the Brite Turbo method attributable to the increased number of granulocytes in the assay. Early detection of CMV infection or reactivation has become faster and easier with this modified assay.

Subacute sclerosing panencephalitis in The Netherlands--1976-1990.

Since 1976, when general immunization against measles was introduced in the Netherlands, all new cases of subacute sclerosing panencephalitis (SSPE) were registered and detailed data about immunization, epidemiology and disease progression were collected on them. Up to 1991, 99 new patients have been registered of which 81 were born in this country and 18 elsewhere. From 1981 onwards, the incidence of SSPE among those born in the Netherlands decreased gradually from 13 cases per year to one case per year. This decrease is attributed to the large scale of immunization against measles. Three SSPE patients had been immunized against measles, all of them without a history of clinical measles. Epidemiology and risk factors of SSPE did not differ from those reported in other countries. An exceptional cluster of four patients in one town, who had measles in the same year, is reported. Progression of SSPE appeared to be age related. A total of 28 patients was treated with Inosiplex; no significant effect on survival in stage 3 of the disease was found.

Comparison of allogeneic T cell-depleted peripheral blood stem cell and bone marrow transplantation: effect of stem cell source on short- and long-term outcome.

We report the results of a retrospective single-center study comparing engraftment, acute and chronic GVHD, relapse and survival in patients with malignant hematological disorders transplanted with allogeneic peripheral blood stem cells (alloPBSCT, n = 40) or bone marrow cells (alloBMT, n = 42). All transplants were T cell depleted by in vitro incubation with the Campath-1 monoclonal antibody. Primary graft failure occurred in none of the patients receiving an alloPBSCT compared with 3/42 of the recipients of an alloBMT. In addition, two patients in the alloBMT group showed no platelet engraftment. Recipients of PBSC had a more rapid recovery of neutrophils (median 14 days) compared to BM transplant recipients (median 32 days). Platelet recovery was also accelerated in PBSC recipients compared to BM recipients (11 vs 38 days). There was an increase in the incidence of grade II acute GVHD and chronic GVHD in patients after alloPBSCT (18% and 23%, respectively) compared to patients receiving alloBMT (5% and 8%, respectively). The 2-year cumulative incidence of relapse was similar in both groups (47%). At 6 months after transplantation, transplant-related mortality (TRM) was lower in PBSCT recipients than in BMT recipients. However, at a follow-up of 3 years TRM was similar in both groups. The disease-free survival rate at 3 years after transplantation did not differ between the groups (42% for PBSCT and 41% for BMT recipients). Our results indicate that T cell-depleted alloPBSCT compared to alloBMT is associated with a more rapid hematopoietic reconstitution and a decreased TRM at 6 months follow-up after transplantation. However, at a follow-up of 3 years, no sustained survival benefits were observed.

HLA-B27 as a receptor for cytomegalovirus.

Acute anterior uveitis (AAU) is strongly associated with the genetic marker and cell membrane protein HLA-B27. Although also other genetic factors must play a pathogenetic role, the HLA-class I molecule B27 is up to now the only hold. The normal task of HLA class I molecules is to present endogenous, mostly viral, peptides to receptors on cytotoxic T cells. It is possible that HLA molecules at the cell surface serve as viral receptors. Human cytomegalovirus (HCMV) particles have been found to bind beta 2m. This might promote infectivity by a binding to HLA alpha-chains on cell membranes. We studied this mechanism using mouse fibroblasts transfected for human HLA class I molecules. Susceptibility of these cells for HCMV was compared by measuring of HCMV immediate early antigen (IEA) expression. Earlier we observed that cells transfected with HLA-B27 alpha-chains and beta 2m were significantly more infected than cells expressing HLA-A2 + beta 2m or HLA-B7 or HLA-B27 without beta 2m. However, studying another four, separately transfected, cell lines, all expressing HLA-B27 and beta 2m, three of the five B27 cell lines showed low IEA levels. The degree of infectivity was independent of the degree of B27 expression. These results do not support the previous suggestion that HLA-B27 might act as an HCMV receptor.

[Meningitis caused by Toscana virus during a summer stay in Italy]. / Meningitis door Toscana-virus tijdens een zomers verblijf in Italië.

A 44-year-old woman suffered fever, headache and meningism during a summer stay in a region of central Italy where sand-fly fever is endemic. A few days after returning to the Netherlands, she appeared mentally and physically slow but had no fever. Because of the possibility of viral meningitis an examination of the cerebrospinal fluid (CSF) was carried out. This revealed a cell count of 1074/3 cells. Toscana virus antibodies of the IgG and IgM subclass were detected in two respective serum samples and in the CSF. This led to the diagnosis 'Toscana virus meningo-encephalitis'. Toscana virus is classified amongst the sandfly fever virus group. These viruses are transmitted by sand flies (Phlebotomus species) which reside in humid areas around the Mediterranean. Toscana virus is the main cause of viral meningoencephalitis in some areas of central Italy and possibly in southern Spain. The patient recovered within a few days without antiviral therapy. Toscana virus meningo-encephalitis should be included in the differential diagnosis of holidaymakers with neurological symptoms returning from the Mediterranean.