Dieter Neumann-Haefelin

Novel Human Metapneumovirus Sublineage

In a pediatric surveillance network, 287 (5.1%) of 5,580 specimens from patients with acute respiratory infections tested positive for human metapneumovirus (HMPV). Phylogenetic analysis of N- and F-gene sequences of identified HMPV showed that 30% belonged to a novel phylogenetic cluster.

Disseminated bocavirus infection after stem cell transplant.

To the Editor: Human bocavirus (HBoV) (1) is increasingly recognized as a cause of respiratory infections worldwide. Children and infants appear to be most at risk (2–7), although HBoV’s role in immunocompromised patients remains unclear. We report on a child with disseminated HBoV infection after hematopoietic stem cell transplantation (HSCT). HBoV DNA was detected at high levels in nasopharyngeal aspirates (NPAs) and in blood and stool samples. n nA 4.5-year-old boy with dyskeratosis congenita was brought for treatment to our hospital due to severe persistent cytopenia. Allogenic HSCT was performed in August 2006 after conditioning with total body irradiation (200 cGy, day –8 before HSCT surgery), fludarabine (days –7 to –4), antithymocyte globulin (days –4 to –1), and cyclophosphamide (days –3 to –2). He received 7.16 × 108 nucleated bone marrow cells/kg body weight from a 9/10 human leukocyte antigen–matched unrelated donor. Graft-versus-host disease (GvHD) prophylaxis consisted of a short course of methotrexate and cyclosporin A. Neutrophil and platelet engraftment occurred on days 22 and 65 after surgery, respectively. Despite pre- and post-HSCT anti-infective prophylaxis with cotrimoxazole, colistin, acyclovir, and fluconazole, Enterobacter cloacae sepsis was diagnosed on day 2. After meropenem treatment, blood cultures remained negative. On day 12, fever reoccurred, elevated C-reactive protein values (229 mg/L) and reduced general health were noted, but no bacterial pathogen was isolated. During this period, the patient received antimicrobial drug therapy with meropenem, tobramycin, vancomycin, and amphotericin B. On day 16, his body temperature peaked to 40.6°C, and a cough and dyspnea without wheezing developed. Chest radiograph results suggested pneumonia with perihilar infiltrates. Reduced oxygen saturation (pO2 86%) was recorded transcutaneously, and oxygen supplementation (maximum 4 L/min) was started by face mask (Appendix Figure). An NPA sample investigated by multiplex PCR (results provided by W. Puppe and J. Weigl; www.pid-ari.net) was negative for adenovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses 1–4, influenza viruses A and B, coronavirus, reovirus, enterovirus, Clamydia pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, B. parapertussis, and Legionella pneumophila, but positive for rhinovirus RNA. Retrospectively, the same NPA sample was reanalyzed for HBoV DNA by real-time PCR (7) and showed a viral load of 4.6 × 107 copies/mL (Appendix Figure); specificity was confirmed by sequencing. n nFrom day 19 on, the patient’s general health improved and the chest radiograph results returned to normal. After neutrophil engraftment (day 22) and addition of erythromycin to the antimicrobial drug regimen, body temperature decreased and oxygen supplementation was discontinued. However, rhinitis, cough, and low-grade fever (<38.5°C) persisted until day 50 (Appendix Figure), and HBoV DNA was detected in NPAs on days 37 and 44 at 2.4 × 1011 and 1.3 × 1014 copies/mL, respectively (Appendix Figure). The NPA sample on day 37 was still rhinovirus positive. n nConcurrent with the increased HBoV load in NPAs, cytomegalovirus (CMV) reactivation was first diagnosed by PCR on day 20 and peaked (58.250 copies/mL whole blood) on day 41 despite gancyclovir therapy. Switching to foscarnet led to temporary control of CMV replication (Appendix Figure). Additionally, on day 22, acute GvHD grade I with skin manifestations developed. Treatment with steroids until day 60 led to complete resolution. n nHBoV infection in this patient was not restricted to the respiratory tract. Diarrheic stool samples obtained on day 21 and, after resolution of respiratory symptoms, on day 75 showed substantial HBoV DNA (2.5 × 106 and 6.0 × 105 copies/mg, respectively; Appendix Figure). Tests for rotavirus and adenovirus antigens were negative, and no bacterial pathogen was isolated. Moreover, HBoV DNA was detected at lower levels (3.7 × 103 to 7.8 × 104 copies/mL) in 4 EDTA plasma samples taken days 21–47. Subsequent plasma (days 61, 68, 75, 88, 219), NPA (day 219), and stool (day 219) samples were negative for HBoV DNA. However, the ability of HBoV to cause persistent infection, as do other members of the Parvovirinae subfamily, cannot be excluded. Future investigations are needed to address this hypothesis. n nHere, we report on disseminated HBoV infection in an immunocompromised patient. Whether the clinical course in this case was more severe or prolonged than it would have been for HBoV infections in non-HSCT children remains unknown due to the lack of long-term observations in immunocompetent children. The dramatic increase of HBoV load in NPAs and viral dissemination most likely resulted from progressive impairment of cellular immunity as indicated by simultaneous CMV reactivation. Moreover, the increased viral load might have also been a consequence of steroid addition to immunosuppressive therapy to control GvHD. The contribution of HBoV to respiratory disease remains ambiguous because 2 NPA samples were also rhinovirus positive. Additional studies are required to investigate the pathogenic role of HBoV in double or multiple infections. Association of HBoV with the patient’s continued diarrhea is in accordance with previous studies (8–10). Prolonged fecal shedding has important implications for isolation measures in transplantation units. More studies in immunocompromised patients are required to evaluate the spectrum of pathology caused by this emerging virus.

Complex Effects of Deletions in the 5′ Untranslated Region of Primate Foamy Virus on Viral Gene Expression and RNA Packaging

ABSTRACT Due to various advantageous features there is current interest in retroviral vectors derived from primate foamy viruses (PFVs). Two PFV cis-acting sequences have been mapped in the 5′ region of the RNA (pre-)genome and in the 3′ pol genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5′ untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5′ UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV gag gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and thegag gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5′-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentialy packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the polgene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve.

Human metapneumovirus induces more severe disease and stronger innate immune response in BALB/c mice as compared with respiratory syncytial virus

BackgroundHuman metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are members of the Pneumovirinae subfamily of Paramyxoviridae and can cause severe respiratory disease, especially in infants and young children. Some differences in the clinical course of these infections have been described, but there are few comparative data on pathogenesis in humans and animal models. In this study, HMPV and RSV were compared for replication, pathogenesis and immune induction in BALB/c mice infected with equivalent inocula of either virus.MethodsViral titers in the lungs and in the nasal turbinates of mice were determined by plaque assay. Histopathological changes in the lungs as well as weight loss and levels of airway obstruction were monitored in the infected mice to record the severity of illness. Inflammatory cells recruited to the lungs were characterized by flow cytometry and by differential staining. In the case of natural killer cells, cytotoxic activity was also measured. Cytokine levels in the BAL were determined by cytometric bead array.ResultsRSV replicated to higher titers than HMPV in the lung and in the upper respiratory tract (URT), and virus elimination from the lungs was more rapid in HMPV-infected mice. Clinical illness as determined by airway obstruction, weight loss, and histopathology was significantly more severe after HMPV infection. A comparison of the cellular immune response revealed similar recruitment of T lymphocytes with a predominance of IFN-γ-producing CD8+ T cells. By contrast, there were obvious differences in the innate immune response. After HMPV infection, more neutrophils could be detected in the airways and there were more activated NK cells than in RSV-infected mice. This correlated with higher levels of IL-6, TNF-α and MCP-1.ConclusionThis study shows important differences in HMPV and RSV pathogenesis and suggests that the pronounced innate immune response observed after HMPV infection might be instrumental in the severe pathology.

Human bocavirus DNA detected by quantitative real-time PCR in two children hospitalized for lower respiratory tract infection

Viral pathogens such as respiratory syncytial virus, human metapneumovirus, rhinovirus, adenovirus, parainfluenza virus and influenza virus are the most frequent causes of acute respiratory tract infection and hospitalization in infants and young children. However, no pathogen can be identified in about 30% of suspected respiratory infections [1]. Recently, a new human virus belonging to the Bocavirus genus of the subfamily Parvovirinae was cloned from pooled human respiratory samples and its pathogenic potential was proposed according to its association with respiratory illness [2]. Later studies suggested human bocavirus (HBoV) may be causative for lower respiratory tract disease in young children [3–6]. In this report, we describe the detection by quantitative real-time PCR of HBoV DNA in the nasopharyngeal aspirates of two German children hospitalized for pneumonia. Sequencing of an NP-1 gene fragment and phylogenetic analysis revealed high sequence identity for this HBoV strain compared with the prototype strain HBoV st1 [2] and other strains worldwide [2–6]. Our finding of a high viral load combined with symptoms of acute respiratory tract infection may support the assertion of others that HBoV is an important emerging pathogen [2–6]. In March 2004, a 32-month-old boy presented with a 3day history of high fever and cough. He was treated with corticosteroids and bronchodilators to control acute bronchoconstriction. No antibiotics were administered. Due to increasing tachypnea and dyspnea, the child was hospitalized. Clinical examination upon admission revealed a body temperature of 39°C, tachypnea and dyspnea, as well as subcostal retractions. Wheezing and dry bilateral fine rales were present upon lung auscultation. Transcutaneously measured oxygen saturation was decreased to 87% and the patient required oxygen supplementation (4 l/min) for 6 days and intravenous treatment with corticosteroids for persistent bronchoconstriction. Laboratory tests showed a leukocyte count of 7.7×10/l, a blood pH level of 7.4, base excess of −4.7 mmol/l, and a C-reactive protein value of 5.6 mg/l. Chest radiograph revealed beginning right paracardial pulmonary infiltration without pleural effusion, indicating pneumonia. Under antiobstructive therapy with sultanol and intravenous corticosteroids, the boy’s respiratory condition improved. On day 4 after admission, he developed rotavirus gastroenteritis, probably of nosocomial origin, which rapidly improved under symptomatic therapy. By discharge on day 9, his general condition was good, although a mild fever (38°C) was still present. In the same period, an 18-month-old boy presented with a 2-day history of increasing dyspnea, fever (39.5°C), and cough. Clinical examination revealed a weakened general condition, tachypnea, a body temperature of 38.8°C, and modest wheezing as well as bilateral basal rales upon auscultation. The patient required oxygen supplementation (2 l/min) for 4 days. Initial laboratory tests showed an elevated leukocyte count of 12.5×10/l, a blood pH level of Eur J Clin Microbiol Infect Dis (2007) 26:147–149 DOI 10.1007/s10096-006-0244-6

Performance of a novel microarray multiplex PCR for the detection of 23 respiratory pathogens (SYMP-ARI study)

Symptoms of acute febrile respiratory tract infection are often unspecific, but the rapid identification of pathogens allows optimised patient management. The objective of this study was to evaluate a novel multiplex polymerase chain reaction (PCR) suspension microarray which detects 19 viral and four atypical bacterial targets. A comprehensive set of sensitive monoplex real-time PCR assays was used for each pathogen as the gold standard. A panel of archived as well as 300 prospectively collected clinical samples was analysed by both methods. At least one target was detected in 165/300 (55xa0%) samples by monoplex PCR and in 140/300 (46xa0%) samples by multiplex PCR, respectively. The positivity rate was significantly higher in paediatric patients compared to adults [126/154 (82xa0%) vs. 39/146 (27xa0%) by monoplex and 114/154 (74xa0%) vs. 26/146 (18xa0%) by multiplex PCR, respectively]. Among all samples, 17/300 (5.6xa0%) were positive for atypical bacteria by monoplex and 8/300 (2.6xa0%) by multiplex PCR, respectively. Multiple detections were recorded in 35/300 (11.6xa0%) samples by monoplex and 26/300 (8.7xa0%) by multiplex PCR. For the most common pathogens, the sensitivity ranged from 57 to 93xa0% and the specificity ranged from 95 to 100xa0%. The overall concordance between both methods was 77xa0% [95xa0% confidence interval (CI) 72–81xa0%]. False-negative results by multiplex PCR were mainly due to the low target concentration. Compared to monoplex PCR, the novel microarray assay proved its principle but displayed overall lower sensitivities, potentially restricting its use to paediatric patients. For some targets, only small numbers of positive samples were available, requiring larger studies to firmly assess the sensitivity and specificity.

Persistence of human bocavirus DNA in immunocompromised children.

Human bocavirus is frequently detected in immunocompetent as well as in immunocompromised children. However, the course of infection in immunocompromised children is still poorly investigated. In the present study, we describe 4 cases of repeat human bocavirus detection in the presence of severe immunodeficiency. In the view of homologous viral sequences identified in serial samples, possible persistence and reactivation in these patients are discussed.

Detection of respiratory viruses using a multiplex real-time PCR assay in Germany, 2009/10.

The aim of this study was to determine the prevalence of respiratory viruses and to prospectively evaluate the performance of the fast-track diagnostics (FTD) respiratory pathogens multiplex PCR assay shortly after the 2009/10 influenza pandemic. Highly sensitive monoplex real-time PCR assays served as references. Discrepant results were further analyzed by the xTAG RVP Fast assay. A total of 369 respiratory samples from children and adults were collected prospectively in Germany from December 2009 until June 2010. The sensitivity and specificity of the FTD assay after resolution of discrepant results was 92.2xa0% and 99.5xa0%, respectively. Lowest specificity of the FTD assay was observed for human bocavirus. Multiple detections were recorded in 33/369 (8.9xa0%) of the samples by monoplex PCR and in 43/369 (11.7xa0%) using the FTD assay. The most prevalent viruses were respiratory syncytial virus and human metapneumovirus. Only pandemic influenza virus A/H1N1 (2009), and not seasonal influenza virus, was detected. Viruses other than influenza virus accounted for the majority of acute respiratory infections. The FTD assay can be easily implemented in general diagnostic laboratories and facilitate the optimization of patient-management schemes.

Human bocavirus DNA in paranasal sinus mucosa.

To the Editor: Human bocavirus (HBoV) is a newly described parvovirus for which pathogenic potential has not clearly been elucidated (1). Recent findings suggest that HBoV may establish persistent infection of mucosal lymphocytes or contribute to tonsillar hyperplasia in children (2). In previous reports, we described prolonged HBoV DNA detection in immunocompromised children (3,4). Partial sequencing of the VP1 gene of HBoV from bronchoalveolar lavage fluid, plasma, and sphenoid sinus samples showed 100% identity, which suggested persistence of the same HBoV strain over a 5-month period (3). It remains speculative, however, whether paranasal sinus mucosa represents a site of HBoV persistence. To clarify this, we analyzed samples of paranasal mucosal tissue and nasal polyps from patients with chronic sinusitis for respiratory viruses and atypical bacteria. n nA total of 102 tissue samples were obtained from 88 patients (median age 48.5 years, range 13.3–88.1 years) from July 2009 through September 2010 after elective surgery. Indication for surgery was established by otorhinolaryngologists. The most common indication was chronic sinusitis. No patients displayed acute respiratory symptoms at the time of surgery. To detect asymptomatic shedding in the upper respiratory tract and viremia, we collected nasal swabs and EDTA-blood samples concurrently. The study protocol was approved by the Ethics Committee of the University of Freiburg. Informed written consent was obtained from all study participants. n nApproximately 25 mg of each tissue specimen was used for nucleic acid extraction by using an RNeasy Mini Kit, as described (5) (QIAGEN, Hamburg, Germany). To provide evidence that the QIAGEN RNeasy kit is also suitable for DNA extraction, we spiked HBoV negative samples with different amounts of HBoV DNA before extraction of nucleic acids was done with either the QIAGEN RNeasy Kit or DNA Blood Kit (QIAGEN). Extracted nucleic acids were then subjected to real-time PCR by using primers specific for HBoV. Minimal differences (±1 cycle threshold [Ct] value) in the HBoV PCR were detected; the QIAGEN RNeasy Kit was therefore used throughout the study (data not shown). Nasal swabs and ETDA-blood were purified by using a QIAamp MinElute Virus Spin Kit (QIAGEN). Multiplex PCR for respiratory viruses (Fast-track Diagnostics, Junglinster, Luxembourg) was conducted to detect influenza A (including pandemic [H1N1] 2009) and B viruses; respiratory syncytial virus; human metapneumovirus; HBoV; parainfluenza virus 1–4; human coronaviruses HKU1, NL63, 229E, and OC43; human rhinoviruses; human enteroviruses and parechoviruses; and adenoviruses. Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and Bordetella pertussis were analyzed as described (6–8). n nA single virus was detected in 22/102 (21.5%) tissue specimens, with HBoV being the most frequent (18/102, 17.6%), followed by rhinovirus (2/102, 1.9%), coronavirus 229E, and influenza A pandemic (H1N1) 2009 virus (1/102, 0.9%). HBoV was detected in specimens collected during July–September (13/18) and during February and March (5/18). All positive results were confirmed by single real-time PCR (9). For 14 patients, 2 different mucosal samples were tested and gave identical results. No multiple viral infections and no bacteria were detected. Median patient age was 51.2 years (range 14.4–74.2 years) for HBoV-positive and 47.6 years (range 13.3–88.1 years) for HBoV-negative samples. Ct analysis in single real-time PCR revealed a median Ct of 31 (range 28–38), corresponding to 200 genome equivalents/106 cells (range 3–1.8 × 104 copies/106 cells). No correlation between Ct value and patients’ age was observed (r2xa0=xa00.008; data not shown). No underlying disease was diagnosed for 13/18 HBoV-positive patients, whereas 2/18 and 3/18 patients had chronic obstructive pulmonary and oncologic disease, respectively. n nNasal swabs and EDTA-blood samples were obtained from 17/18 and 7/18 HBoV-positive patients, respectively. No HBoV was detected in any swabs or EDTA-blood samples available, indicating no virus shedding in the respiratory tract and no viremia. However, the 2 patients with rhinovirus-positive samples obtained from biopsy also had rhinovirus RNA detectable in nasal swabs, suggesting rhinovirus infection. Unfortunately, no nasal swab was available from the 2 patients whose sinus biopsy samples were positive for HCoV 229E and influenza A virus. n nIn this study, we simultaneously analyzed tissue specimens of paranasal sinuses and nasal polyps, as well as nasal swabs and blood samples, for a broad panel of viruses and atypical bacteria. To avoid seasonal bias, specimens were collected over a 1-year period and exclusively obtained from patients undergoing elective surgery in the absence of acute respiratory symptoms. n nThe finding that HBoV was present as a single virus in 18/22 virus-positive biopsy samples is intriguing. Moreover, the fact that no HBoV DNA was detected in nasal swabs or EDTA-blood samples indicates no active HBoV infection. In previous studies, HBoV DNA was frequently identified in the adenoids and tonsils of children (2,5,10). However, in contrast with our findings, detection of HBoV was mostly associated with other viruses, suggesting that co-virus–induced cellular damage might contribute to bocavirus reactivation and replication (5). Our findings indicate that persistence of viral nucleic acid in sinus mucosa might be a special advantage of HBoV, although the relevance of this observation remains unclear. Whether this presence as a single virus means a dead end for HBoV infection, true latency including the potential of reactivation, or a role in the pathogenesis of clinical conditions requiring surgery warrants future studies.

Performance of 14 rubella IgG immunoassays on samples with low positive or negative haemagglutination inhibition results

BACKGROUNDnRubella IgG testing is routinely done in prenatal care and seroepidemiological studies. Recently concern was raised that seropositivity rates were decreasing questioning vaccination policies. Manufacturers of rubella IgG assays and authors of seroepidemiological studies use different cut-offs for the definition of seropositivity. As rubella virus circulation is reduced since many years, seronegativity rates might be overestimated using an inappropriate cut-off.nnnOBJECTIVESnUsing different cut-off definitions we compared fourteen current rubella IgG immunoassays for sensitivity and qualitative result concordance in samples with low positive or negative haemagglutination inhibition (HI) titre.nnnSTUDY DESIGNn150 clinical samples from patients and health care workers were included in the study. All samples were measured in 14 different rubella IgG immunoassays. Seropositivity was defined using recombinant rubella IgG immunoblot as reference standard.nnnRESULTSnThe concordance of qualitative results using the manufacturers cut-off definitions was 56.4% if grey-zone results were analysed separately and 69.8% if grey-zone results were defined as positive. Using universal cut-offs of 10 IU/ml or 15 IU/ml the concordance was 70% and 61.4% respectively. Using the different cut-off definitions up to 71 out of the 124 immunoblot-positive samples tested negative in the immunoassays. The mean coefficient of variation (CV) of quantitative results in positive samples was 51% (range 19-113%).nnnCONCLUSIONSnDetermination of rubella immunity by measurement of rubella-IgG in a population with high vaccination coverage with current assays leads to a high number of false negative results. The value of routine rubella antibody testing in countries with high vaccination coverage should be discussed.