Anne Lahtinen

Clinical Assessment and Improved Diagnosis of Bocavirus-induced Wheezing in Children, Finland

Accurate diagnosis of respiratory infections requires serologic analysis and PCR of serum.

Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples: Implications for respiratory transmission and latency

Abstract Background Merkel cell polyomavirus (MCPyV) was discovered recently. It is considered a potential causative agent of Merkel cell carcinoma, a life-threatening skin cancer. Objectives To study the prevalence of MCPyV in a large number of clinical samples of various types. Most of the samples were examined also for the other newly found polyomaviruses KI (KIPyV) and WU (WUPyV). Study design Altogether 1390 samples from immunocompetent or immunocompromised patients, including (i) tonsillar tissues and sera from tonsillectomy patients; (ii) nasopharyngeal aspirates (NPAs) and sera from wheezing children and (iii) nasal swabs, sera and stools from febrile leukemic children were studied for MCPyV. The tonsils, nasal swabs and stools were also studied for KIPyV and WUPyV. Results MCPyV DNA was detected in 14 samples altogether; 8 of 229 (3.5%) tonsillar tissues, 3 of 140 (2.1%) NPAs, 2 of 106 (1.9%) nasal swabs and 1 of 840 (0.1%) sera. WUPyV and KIPyV were detected in 5 (2.2%) and 0 tonsils, 1 (0.9%) and 4 (3.8%) nasal swabs and 0 and 2 (2.7%) fecal samples, respectively. The patients carrying in tonsils MCPyV were of significantly higher age (median 42years) than those carrying WUPyV (4years, p <0.001). Conclusions MCPyV DNA occurs in tonsils more frequently in adults than in children. By contrast, WUPyV DNA is found preferentially in children. MCPyV occurs also in nasal swabs and NPAs, in a frequency similar to that of KIPyV and WUPyV. The tonsil may be an initial site of WUPyV infection and a site of MCPyV persistence.

Serodiagnosis of Primary Infections with Human Parvovirus 4, Finland

To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle–based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus–infected injection drug users.

Human parvoviruses B19, PARV4 and bocavirus in pediatric patients with allogeneic hematopoietic SCT

Among the immunocompetent, infections with parvovirus B19 (B19V) and human bocavirus (HBoV) 1 range clinically from asymptomatic to severe, while following allogeneic hematopoietic SCT (HSCT) B19V can cause a persistent severe illness. The epidemiology and clinical impact of HBoV1 and the other emerging parvovirus 4 (PARV4) among immunocompromised patients have not been established. To determine the occurrence and clinical spectrum of B19V, PARV4 and HBoV1 infections, we performed a longitudinal molecular surveillance among 53 allogeneic HSCT recipients for pre- and post-HSCT DNAemias of these parvoviruses. Quantitative real-time PCR showed B19V DNA in sera of 16 (30%) patients, at mean levels of 4.6 × 103, 9.9 × 107, 1.1 × 1010 and 1.6 × 102 B19V DNA copies/mL pre-HSCT (9/53), and at 1 (6/53), 2 (4/53) and 3 months (1/25) post HSCT, respectively. However, no clinical manifestation correlated with the presence of B19V viremia. All B19V sequences were of genotype 1. None of the sera investigated contained PARV4 or HBoV1 DNAs. Our data demonstrate B19V viremia to be frequent among pediatric allogeneic HSCT recipients, yet without apparent clinical correlates. PARV4 or HBoV1 viremias were not seen in these immunocompromised patients.

Expression and serological characterization of polyomavirus WUPyV and KIPyV structural proteins.

The polyomaviruses WUPyV and KIPyV were recently discovered. We expressed their structural proteins VP1, VP2, and VP3, and the corresponding proteins of BKV and JCV, for immunoblotting of IgG antibodies from 115 wheezing young children and 25 asymptomatic adults. Furthermore, nasopharyngeal aspirates (NPA) and sera from the children were examined by PCR for viral DNA. The overlapping minor proteins VP2 and VP3 of WUPyV and KIPyV were more reactive in immunoblots than the major protein VP1; of 100 NPA PCR-negative wheezing children aged < or = 4 y, 31 (31%) and 31 (31%) were positive for WUPyV and KIPyV VP2/VP3, compared to only 3 (3%) and 5 (5%) for VP1, respectively. For comparison, the respective WUPyV and KIPyV IgG seroprevalences as determined by immunofluorescence assay (IFA) with nondenatured VP1 were 80% and 54%, respectively, among 50 NPA PCR-negative children aged < or = 2 y. This difference shows the importance of conformational VP1 antigenicity. Of the 25 adults, 52% and 68% were IgG-positive in immunoblots for VP2/VP3 of WUPyV and KIPyV, and 8% and 12% were for VP1, respectively. Of the 192 NPA samples studied by PCR, 7 (3.6%) were positive for WUPyV, and 3 (1.5%) were positive for KIPyV DNA. Unlike the NPA samples, none of the corresponding 443 sera contained WUPyV or KIPyV DNA. Together with the high VP2/VP3 IgG prevalence, this points to a paucity or brevity of KIPyV and WUPyV viremias among immunocompetent children. Our results indicate the significance of protein conformation in immunoreactivity of VP1, and show the antigenic importance of the WUPyV and KIPyV minor proteins VP2 and VP3. The high and rapidly increasing IgG prevalence rates observed in this study for WUPyV and KIPyV support the notion that these novel polyomaviruses are widespread and are acquired early in childhood.

Absence of human bocavirus from deceased fetuses and their mothers

BACKGROUND The human bocavirus (HBoV), a newly discovered parvovirus, is closely related to the bovine parvovirus and the canine minute virus, which are known to cause adverse pregnancy outcomes. Another human parvovirus, B19, can lead to fetal hydrops, miscarriage and intrauterine fetal death (IUFD). OBJECTIVES To determine the prevalence of HBoV DNA in aborted fetuses and IUFDs. The HBoV serology of the mothers was also studied. STUDY DESIGN We retrospectively studied all available fetuses (N=535) autopsied during 7/1992-12/1995, and 1/2003-12/2005 in Helsinki, Finland. All available formalin-fixed paraffin-embedded fetal tissues - placenta, heart and liver - of 120 miscarriages, 169 IUFDs, and 246 induced abortions were studied by quantitative PCR. We also measured the HBoV IgM and IgG antibodies in the corresponding maternal sera (N=462) mostly of the first trimester. The IgM-positive sera underwent HBoV PCR. RESULTS None of the fetal tissues harbored HBoV DNA. A total of 97% (448/462) of the mothers were positive for IgG antibodies to HBoV, while only 0.9% (4/462) exhibited HBoV-specific IgM antibodies without viremia or respiratory symptoms. One IgM-positive mother had an unexplained fetal loss. CONCLUSIONS We did not find HBoV DNA in any of the deceased fetuses. Almost all pregnant women were HBoV-IgG positive.

Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.

Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.

A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4

Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes.