Ortwin Adams

Correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection.

Abstract Background The clinical significance of viral load and co-infections in children with respiratory infections is not clear. Objective To evaluate the correlation of viral load as well as viral and bacterial co-infections with disease severity in hospitalized children with lower respiratory tract infections (LRTIs). Study design This is a prospective study conducted in children admitted for LRTIs for two seasons. To determine viral and bacterial load of respiratory pathogens we performed multiplex real-time polymerase chain reaction and semiquantitative bacterial cultures on nasopharyngeal aspirates (NPA). Results During the study period 244 (60%) children were hospitalized for LRTI with acute virus-induced wheezing and 160 (40%) for radiologic confirmed pneumonia. In the first NPA, viruses were identified in 315 (78%) of the 404 samples and bacteria in 198 (63.3%) of 311 samples. The viral load significantly decreased between the first and second NPA sample in most single and viral co-infections, except rhinovirus and human bocavirus infections. Viral load was inversely related to CRP in RSV infections, whereas a positive correlation was observed in adenovirus infections. Duration of hospitalization was significantly longer in RSV single infections compared to rhinovirus single infections whereas in the latter, leucocytosis and use of systemic steroids was more common. In RSV viral co-infections the presence of fever, leucocytosis, and the use of antibiotics was significantly more frequent. Positive cultures of Haemophilus influenzae dominated in RSV and rhinovirus single infections and Moraxella catarrhalis in RSV viral co-infections. Conclusions Specific viral single and co-infections as well as viral load contribute to disease severity in children with LRTIs.

Frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction.

Background: Respiratory viruses are the main cause of acute respiratory tract infection (ARI) in children. Real-time polymerase chain reaction (PCR) technology is highly practicable for the rapid detection of viral pathogens. The simultaneous detection of a broad spectrum of viruses enables the diagnosis and evaluation of viral coinfection in ARI. Methods: A 1-step real-time PCR was developed for the detection of 12 respiratory viruses (10 RNA and 2 DNA viruses) in clinical samples. Clinical samples from 254 children admitted to the Departments of Pediatrics with ARI during a 10-month period were tested. Results: Respiratory syncytial virus (RSV) was the most frequently detected pathogen in 112 samples (44.1%), followed by human bocavirus (hBoV) in 49 (19.3%), and rhinovirus in 17 samples (6.7%). Viral coinfection was detected in 41 (16.1%) samples with RSV and hBoV being the most dominating combination (27 cases, 10.6%). Viral coinfection was found in 10 cases (17%) of children with bronchitis (n = 58) and in 7 cases (23%) of bronchiolitis (n = 30). In patients with pneumonia (n = 51), 17 cases (33%) were positive for 2 or more viral pathogens. Conclusions: Simultaneous testing of respiratory viruses by real-time PCR is a suitable tool for the detection of viral coinfections. In children hospitalized because of respiratory infection viral coinfection is frequently detected with RSV and hBoV being a common combination.

HFE mutations and chronic hepatitis C: H63D and C282Y heterozygosity are independent risk factors for liver fibrosis and cirrhosis

BACKGROUND/AIMS The impact of heterozygous HFE mutations on the course of chronic hepatitis C and iron indices was studied. METHODS Ferritin, transferrin saturation (TS), serum iron, C282Y and H63D mutations were determined in 401 patients with chronic hepatitis C virus (HCV) infection and 295 healthy controls. Liver histologies were available in 217 and HCV genotypes in 339 patients. RESULTS Allele frequencies of the C282Y and H63D mutation did not differ between HCV patients and healthy controls (6.95 vs. 6.2%; 14.75 vs. 16.4%; n.s.). HFE heterozygous HCV patients had higher ferritin (349+/-37 vs. 193+/-15 microg/l; P<0.0005), TS (38+/-2 vs. 32+/-1%; P<0.0005), serum iron (144+/-6 vs. 121+/-3 microg/dl; P<0.0005), semiquantitative liver iron staining (0.26+/-0.07 vs. 0.09+/-0.03; P<0.006) and fibrosis scores (1.9+/-0.2 vs. 1.4+/-0.1; P<0.003) compared to HFE wildtypes. By multivariate regression analysis odds ratios for liver cirrhosis were 5.9 (confidence interval (CI) 1.6-22.6; P<0.009) for C282Y heterozygotes and 2.9 (CI 1.0-8.4; P<0.05) for H63D heterozygotes compared to HFE wildtypes. Considering all HFE heterozygous HCV patients, odds ratios of 3.6 (CI 1.4-9.3; P<0.009) for cirrhosis and 3.1 (CI 1.3-7.3; P<0.009) for fibrosis were calculated. CONCLUSIONS C282Y or H63D heterozygosity is an independent risk factor for liver fibrosis and cirrhosis in HCV infected individuals. Screening for HFE mutations should be considered in HCV infection.

Quantitative Detection of Norovirus Excretion in Pediatric Patients With Cancer and Prolonged Gastroenteritis and Shedding of Norovirus

Although chronic courses of norovirus infection have been described in immunocompromised patients, little is known about noroviral shedding and correlation with clinical symptoms in these patients. In this report, the quantitative courses of norovirus excretion in nine pediatric patients with hematologic and oncologic disorders and prolonged gastroenteritis were investigated. In a retrospective study multiple fecal samples from nine pediatric cancer patients were examined by a one‐step real‐time PCR. Clinical data of the patients were reviewed and virological data were correlated with clinical symptoms. All nine patients presented with prolonged illness and prolonged noroviral shedding. Vomiting and diarrhea were associated with high norovirus concentrations and norovirus excretion declined slowly in the patients. Retrospectively, initial PCR‐testing for norovirus was performed with a median of 7 days after onset of symptoms. This finding hints at the difficulty of obtaining early diagnosis of the infection in these children. The patients were shedding high norovirus concentration over a long period of time. Results of sequential quantitative PCR‐testing for norovirus correlated with clinical symptoms. Both clinical symptoms and quantitative PCR‐testings help to define the severity of norovirus infection and to estimate the risk for transmission. To prevent the spread of the disease, usage of virocidal disinfectants and isolation procedures should be maintained as long as patients are positive for noroviruses. Since vomiting is frequent in pediatric patients with oncological conditions, a screening program for rapid detection of norovirus infection in this group of patients should be considered. J. Med. Virol. 80:1461–1467, 2008.

Disease course and outcome of 15 monocentrically treated natalizumab-associated progressive multifocal leukoencephalopathy patients

Objective Although the prognosis of natalizumab-associated progressive multifocal leukoencephalopathy (PML) seems to be better than HIV-associated PML, little is known about the long-term functional outcome in multiple sclerosis (MS) patients and the subsequent return of MS disease activity. We evaluated retrospectively 15 patients with natalizumab-associated PML treated at our centre. Patients and methods Fifteen MS-PML patients (nine women, six men) were referred to us from adjacent local centres. The patients had a median natalizumab exposure of 34 months at PML diagnosis. They received standardised treatment as described in previous work. Expanded Disability Status Scale (EDSS) and Karnofsky score in the year pre-PML, at PML-diagnosis (pre-immune reconstitution inflammatory syndrome (IRIS)) and post-PML were determined in 3–6 monthly intervals. Results The median follow-up of these 15 patients was 21.5 months. None of the 15 patients died. Three patients had a Karnofsky score of 80 or higher, nine patients between 50–70 and three patients of 40 or lower at latest examination. Eight of the 15 patients developed seizures during acute PML phase. Fifty percent of those patients were not seizure-free one year post PML, despite continuation of antiepileptic treatment. The median EDSS in the year pre-PML was 2.5, 4.5 at PML diagnosis, 6.5 post-IRIS and 5.5 at latest examination. CSF became virus-free in eight of the 15 patients after a median time of 4.5 months. In nine patients, disease reappeared after a median time of seven months from PML diagnosis. Conclusions Although the clinical outcome of natalizumab-treated PML patients is much better than in patients with HIV-associated PML, this may be further improved by treatment at reference centres using standardised therapy regimens and transient intensive care if needed. Systematic studies of appropriate MS immunotherapies after PML are critically needed.

Congenital Infections with Human Herpesvirus 6

Cord blood DNA was tested for the presence of human herpesvirus 6 (HHV-6) DNA by the polymerase chain reaction. Specific DNA could be detected in the specimens of 5 (1.6%) of 305 babies born to ostensibly healthy mothers, indicating that intrauterine infection had occurred. These transmissions would not have been detected by serologic methods, because no specific IgM antibody could be found in the fetal sera. These results indicate that, in addition to infections acquired in early childhood, congenital infections may account for the HHV-6 seropositivity in children.

Cerebrospinal Fluid JC Virus Antibody Index for Diagnosis of Natalizumab-Associated Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML), caused by JC virus (JCV), can occur in patients receiving natalizumab for multiple sclerosis (MS). JCV detection by quantitative polymerase chain reaction (qPCR) in cerebrospinal fluid (CSF), or brain biopsy, is required for probable or definite diagnosis of PML. However, in some patients only low levels of JCV DNA (<100 copies/ml) are present in CSF, making the diagnosis challenging. Our objective was to assess the complementary value of a CSF JCV antibody index (AIJCV) in the diagnosis of natalizumab‐associated PML.

Prospective, Comprehensive, and Effective Viral Monitoring in Children Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Major advances in the monitoring and treatment of viral infections after hematopoietic stem cell transplantation (HSCT) have been achieved over the last decade. The appropriate extent of viral monitoring and antiviral therapy remains controversial, and reports in pediatric patients receiving allogeneic unmanipulated hematopoietic stem cells (HSCs) are sparse. A total of 40 pediatric patients who underwent HSCT with either peripheral blood stem cells (PBSCs, n = 30) or bone marrow (BM; n = 10) were prospectively monitored every week for viral DNAemia (VDNA) by simultaneous detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), human adenovirus (ADV), and polyoma BK virus (BKV) using real-time TaqMan polymerase chain reaction (PCR). All patients received prophylactic acyclovir and preemptive ganciclovir (GCV) when 500 copies/microg DNA (EBV/HHV6) or >1 copy/microg DNA (CMV) were detected on 2 consecutive measurements. VDNA occurred in 25 of 40 recipients (CMV, 11/40 patients [28%]; EBV, 19/40 [48%]; HHV6, 2/40 [5%]; ADV/BKV, 1/40) and was found exclusively after neutrophil engraftment and in most cases up to day +100. Recurrent VDNA (P = .028) and (readily treatable) viral disease (P = .003) were observed predominantly in patients suffering from nonmalignant diseases, a cohort characterized by delayed lymphocyte engraftment. VDNA occurred more frequently in HLA-mismatched HSCT and in the 24 of 40 patients receiving antithymocyte globulin (ATG). The incidence of EBV, but not that of CMV, was increased in the ATG group. Yet, in these patients, viral loads of both EBV and CMV were higher, but with prompt initiation of preemptive GCV, no posttransplantation lymphoproliferative disorder or other life-threatening morbidities occurred. HHV6 was typically detected at low viral loads (<10(2) copies/microg DNA), with only 5% of HSC recipients fulfilling our HHV6 criteria for triggering GCV treatment. In multivariate analysis, ATG treatment, HLA mismatch, recipient CMV seropositivity, and stem cell source, but not severe acute graft-versus-host disease were identified as independent risk factors for VDNA. This comprehensive viral monitoring program with defined thresholds for initiation of preemptive GCV effectively prevents the development of critical viral disease, even in high-risk patients receiving ATG.

Natalizumab exerts a suppressive effect on surrogates of B cell function in blood and CSF.

Background: Natalizumab for multiple sclerosis (MS) increases the risk of progressive multifocal leukoencephalopathy (PML). Objective: We aimed to assess the effect of natalizumab on cellular composition and functional B cell parameters including patients with natalizumab-associated PML (n=37). Methods: Cellular composition by flow cytometry, levels of immunoglobulin (Ig)G/IgM by immunonephelometry, and oligoclonal bands by isoelectric focusing were studied in blood and cerebrospinal fluid. Results: In MS patients treated with natalizumab without PML (n=59) the proportion of CD19+ B cells was higher in blood, but lower in cerebrospinal fluid compared with MS patients not treated with natalizumab (n=17). The CD4/CD8-ratio in cerebrospinal fluid was lower, and IgG and IgM levels as well as the IgG index dropped in longitudinal samples during natalizumab therapy. Oligoclonal bands persisted, but the total amount of the intrathecally produced IgG fraction, and the polyclonal intrathecal IgG reactivity to measles, rubella, and zoster declined. At the time of diagnosis of PML patients with natalizumab-associated PML had low total IgG levels in blood and cerebrospinal fluid. Conclusions: Natalizumab impacts B and T cell distribution and exerts an inhibitory effect on surrogates of B cell function in periphery and in cerebrospinal fluid, potentially contributing to the increased risk of developing PML.

Role of human brain microvascular endothelial cells during central nervous system infection : Significance of indoleamine 2,3-dioxygenase in antimicrobial defence and immunoregulation

The cerebral endothelium is involved both in regulating the influx of immune cells into the brain and in modifying immunological reactions within the CNS. A number of human pathogens may cause encephalitis or meningitis when this important protective barrier is impaired. We have previously shown that interferon-gamma activated human brain microvascular endothelial cells (HBMEC) restrict the growth of bacteria and parasites. We now provide evidence that HBMEC are also capable of inhibiting viral replication after stimulation with IFN-gamma, an effect further augmented by costimulation with IL-1. This antiviral effect was completely blocked in the presence of L-tryptophan, indicating the induction of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO) to be responsible for the observed antiviral effect. Apart from exerting antimicrobial effects tryptophan depletetion has also been described as a regulatory mechanism in T cell responses to both allo- and autoantigens. We were able to demonstrate that IDO mediated degradation of L-tryptohan in HBMEC is responsible for a significant reduction in T lymphocyte proliferation. Resupplementation of L-tryptophan and restoration of initial T cell responses demonstrated the central role of this essential amino acid in the reduction of T-cell proliferation. Brain endothelial cells appear to limit microbial expansion in the CNS by local degradation of tryptophan, thus acting in concert with other IDO-positive cell populations on the parenchymal side of the blood-brain barrier such as astrocytes, microglia and neurons. Since all dietary tryptophan must cross the blood-brain barrier, the microvascular endothelial cells may play a key role in restricting tryptophan influx from the bloodstream into the brain. As deleterious effects of brain infections can often be attributed to subsequently invading immune cells, an IDO-mediated reduction of lymphocyte proliferation may be beneficial for preventing collateral brain damage.