Ramona Liza Tillmann

Prospective study of Human Bocavirus (HBoV) infection in a pediatric university hospital in Germany 2005/2006

Abstract Background Human Bocavirus (HBoV), a new species of the genus parvovirus newly detected in 2005, seems to be a worldwide distributed pathogen among children with respiratory tract infection (prevalence 2%–18%). Recently published retrospective studies and one prospective birth cohort study suggest that HBoV-primary infection occurs in infants. Methods Prospective single center study over one winter season (November 2005–May 2006) with hospitalized children without age restriction using PCR-based diagnostic methods. Results HBoV DNA was detected in 11 (2.8%) of 389 nasopharyngeal aspirates from symptomatic hospitalized children (median age 9.0 months; range: 3–17 months). RSV, HMPV, HCoV, and Influenza B were detected in 13.9% (n =54), 5.1% (n =20), 2.6% (n =10), and 1.8% (n =7), respectively. There was no influenza A DNA detected in any of the specimens. The clinical diagnoses were acute wheezing (bronchitis) in four patients, radiologically confirmed pneumonia in six patients (55%) and croup syndrome in one patient. In five to six patients with pneumonia, HBoV was the only pathogen detected. While no patient had to be mechanically ventilated, 73% needed oxygen supplementation. In four (36.4%) patients at least one other viral pathogen was found (plus RSV n =3; 27.3%; Norovirus n =1; 9.1%). Conclusion HBoV causes severe respiratory tract infections in infants and young children. Its role as a copathogen and many other open questions has to be defined in further prospective studies.

Detection of Head-to-Tail DNA Sequences of Human Bocavirus in Clinical Samples

Parvoviruses are single stranded DNA viruses that replicate in a so called “rolling-hairpin” mechanism, a variant of the rolling circle replication known for bacteriophages like ϕX174. The replication intermediates of parvoviruses thus are concatemers of head-to-head or tail-to-tail structure. Surprisingly, in case of the novel human bocavirus, neither head-to-head nor tail-to-tail DNA sequences were detected in clinical isolates; in contrast head-to-tail DNA sequences were identified by PCR and sequencing. Thereby, the head-to-tail sequences were linked by a novel sequence of 54 bp of which 20 bp also occur as conserved structures of the palindromic ends of parvovirus MVC which in turn is a close relative to human bocavirus.

Pneumocystis jirovecii Can Be Productively Cultured in Differentiated CuFi-8 Airway Cells

ABSTRACT Although Pneumocystis jirovecii is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether P. jirovecii could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with bronchoalveolar fluid that had been confirmed to be positive for P. jirovecii using PCR. Five days later, the cells and basal medium were harvested and tested for P. jirovecii using quantitative PCR (qPCR), commercially available immunofluorescence detection assays, and Grocott staining of formalin-fixed, paraffin-embedded thin sections of infected-cell cultures. We successfully productively cultivated and propagated P. jirovecii from these P. jirovecii-positive bronchoalveolar lavage fluid (BALF) samples. Furthermore, we provide evidence that P. jirovecii induced cytopathic effects on lung epithelial cells and was even invasive in cell culture. To the best of our knowledge, the cell culture system developed herein represents the first methodology to enable molecular analyses of this pathogen’s life cycle and further in vitro studies of P. jirovecii, such as assessments of drug sensitivity and resistance as well as investigations of the pathogen’s stability against environmental factors and disinfectants. IMPORTANCE This is the first report of the successful productive cultivation and propagation of Pneumocystis jirovecii, a human-pathogenic fungus of major clinical significance. These findings are groundbreaking because they will influence the field of diagnostic microbiology, facilitate the testing of antibiotics against P. jirovecii, and enable stability studies of this pathogen when exposed to the environmental factors and chemicals that hospitals are required to use for disinfection. Because productively culturing P. jirovecii has been attempted unsuccessfully for several decades, this study represents a breakthrough in this field. This is the first report of the successful productive cultivation and propagation of Pneumocystis jirovecii, a human-pathogenic fungus of major clinical significance. These findings are groundbreaking because they will influence the field of diagnostic microbiology, facilitate the testing of antibiotics against P. jirovecii, and enable stability studies of this pathogen when exposed to the environmental factors and chemicals that hospitals are required to use for disinfection. Because productively culturing P. jirovecii has been attempted unsuccessfully for several decades, this study represents a breakthrough in this field.

Novel application for isothermal nucleic acid sequence-based amplification (NASBA).

Human bocavirus (HBoV), solely based on phylogenetic analyses, was classified as the second autonomous human parvovirus. Unfortunately, neither susceptible cell cultures nor animal models were described hitherto, thus complicating studies on viral genome structure and replication steps. A novel application of nucleic acid sequence-based amplification (NASBA) revealed that in all tested samples (100%) that became positive by NASBA the negative strand of the HBoV genome was packaged. Additionally, two of those samples also contained a detectable amount of positive strand (14.3%). The data confirm the assumed single-stranded negative-sense nature of HBoV-genomes that is independent of the viral subtype while showing that NASBA is useful not only for diagnosis.

Low prevalence of human metapneumovirus and human bocavirus in adult immunocompromised high risk patients suspected to suffer from Pneumocystis pneumonia

Summary Background Novel respiratory viruses were discovered in the last years predominantly in children. Until now information on newly identified respiratory viruses in immunosuppressed adult patients is limited. Here we investigated immunocompromised adults with suspected Pneumocystis jirovecii pneumonia (PCP) for new respiratory viruses. Methods Bronchoalveolar lavage (BAL) samples of 128 patients with atypical pneumonia (HIV-infected n =50, hematological malignancy n =51, immunosuppressive treatment n =27) were prospectively evaluated for P. jirovecii and retrospectively for new respiratory viruses (HMPV, HBoV, HCoV-NL63/SARS/HKU1). Results P. jirovecii was detected in 26/128, bacteria in 10, fungi in four, Influenza A in two patients. Novel respiratory viruses were found in only two/128 patients with hematological malignancy, of those one patient with HBoV-infection and one with HMPV-infection, respectively. No pathogens were detected in 82/128 patients. The one patient with detection of hMPV and clinical diagnosis of atypical pneumonia died of pulmonary failure. Conclusion Human bocavirus and human metapneumovirus are rarely involved in atypical pneumonia in immunocompromised adult patients with suspected PCP, but may contribute to severe respiratory failure.

Polyomaviruses KI and WU in children with respiratory tract infection

The polyomaviruses KI (KIPyV) and WU (WUPyV) have recently been discovered in specimens from patients with respiratory tract infections. To analyze the frequency and clinical impact in a cohort of pediatric patients in a German University Children’s Hospital. Nasopharyngeal aspirates or bronchoalveolar lavage specimens of 229 children with acute respiratory tract infection were screened for KIPyV and WUPyV using polymerase chain reaction-based methods. KIPyV was detected in 2 (0.9%) and WUPyV in 1 (0.4%) patients, without co-infections with other respiratory viruses but with co-detection of CMV, EBV and HHV 6 in one immunocompromised patient. Only a very small proportion (1.3%) of positive samples for KIPyV and WUPyV was documented in this study; the clinical relevance of these viruses remains unclear and requires further evaluation.

Sensitive Commercial NASBA Assay for the Detection of Respiratory Syncytial Virus in Clinical Specimen

The aim of the study was to evaluate the usability of three diagnostic procedures for the detection of respiratory syncytial virus in clinical samples. Therefore, the FDA cleared CE marked NOW® RSV ELISA, the NucliSENS® EasyQ RSV A+B NASBA, and a literature based inhouse RT-PCR protocol were compared for their relative sensitivities. Thereby, NASBA turned out to be the most sensitive method with a total number of 80 RSV positive samples out of a cohort of 251 nasopharyngeal washings from patients suffering from clinical symptoms, followed by the inhouse RT-PCR (62/251) and ELISA (52/251). Thus, NASBA may serve as a rapid and highly sensitive alternative for RSV diagnostics.

Novel mutation in YMDD motif and direct neighbourhood in a child with chronic HBV-infection and clinical lamivudine and adefovir resistance - a scholarly case.

ContextChronic HBV infection is a major cause of hepatocellular carcinoma (HCC) which meanwhile has become the 5th most reason for a fatal outcome of cancer. Worldwide, approximately 350 million people are chronically HBV infected and as such of risk to develop HCC, of those an estimated high rate of children. Treatment of chronic infection is sufficient to reduce the rate of HCC but the rate of sustained virological response remains to low, not at least due to emergence of resistant virus strains. Less is known on HBV infection in children despite the extremely high rate of chronicity.Objective, Design, Setting, and PatientThe case of a nine years old male with a 6 year history of chronic HBV infection, of those 5 years with antiviral treatment is described.Interventions and Main Outcome Measure(s)Before our lab was consulted, the patient was unsuccessfully treated with interferon, an obscure drug named Hepon, which should activate antiviral immune response, and Lamivudine, the latter most likely becoming ineffective due to the mergence of resistant subpopulations (rtL180 M, rtV207 M, two strains with stop codons at position rt188 and rt198, rtM204V (YVDD), rtM204K (YKDD)). Replacement of Lamivudine by adefovir displayed no advantage despite the lack of resistance mutations, thus no decrease in viremia was observed under adefovir treatment.Results and ConclusionsNovel mutations in the YMDD motif and its direct neighbourhood were observed, both being compatible with Lamivudine resistance. No mutations were found that are associated with ADF resistance. Both, the clinical course of treatment and the genotypic resistance profile emphasize the need for systematic analyses of the HBV resistance mechanisms and structured therapy concept also for children chronically infected with HBV.

Human HepG2 cells support respiratory syncytial virus and human metapneumovirus replication

Human metapneumovirus (hMPV) and human respiratory syncytial (RSV) virus cause mild to severe infections of the respiratory tract in all age groups. So far, several cell lines derived from respiratory tissues have been identified to support replication of both viruses. Unfortunately, titers attained during replication differ between the both viruses within one cell line despite equal infection conditions, on the one hand giving raise to the assumption that the individual susceptibility may vary in dependence of the virus, and, on the other hand, making it difficult to compare results between both viruses. Low titers may cause problems in experiments such as animal trials, in which high titers in low volumes are a prerequisite for successful experiments. The advantages are described of the use of a human cell line (normally used for hepatitis viruses research) susceptible for RSV and hMPV in which both viruses replicate to comparable and high titers. It is also shown that the cell line can also be used for applications such as cell viability tests. Cell viability tests can be used as reciprocal determination tests of viral titers and therefore offer the opportunity to replace classical virological tests such as TCID(50). The cell line can be also used for high throughput applications like drug screening, making it a useful tool for screening for antiviral compound active against RSV and hMPV.