Michael Kleines

High Prevalence of Human Bocavirus Detected in Young Children with Severe Acute Lower Respiratory Tract Disease by Use of a Standard PCR Protocol and a Novel Real-Time PCR Protocol

ABSTRACT The human bocavirus (HBoV) was recently isolated from respiratory tract samples. Within a study collective of children with severe lower respiratory tract disease, the patients testing positive for HBoV (12.8%) had a higher rate of underlying cardiopulmonary disease. Viral loads in respiratory tract specimens varied from 102 to 1010 genome equivalents/ml.

Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression

Human TLR1 plays an important role in host defense against Mycobacterium tuberculosis. Our aim was to analyze the association of the loss of TLR1 surface expression and TLR1 SNPs with susceptibility to TB. TLR1neg and TLR1pos cells from healthy individuals were identified by flow cytometry and compared by sequencing. TLR1 expression was measured using quantitative real‐time PCR and immunoblotting. TLR1 SNP analyses of healthy individuals and TB patients from EU‐C and Ghana were performed, and association of the TLR1 genotypes with increased risk of developing TB was statistically evaluated. Lack of TLR1 surface expression accompanied by impaired function was strongly associated with TLR1 SNP G743A. Genotyping of EU‐C controls and TB patients revealed an association of TLR1 743A/1805G alleles [OR 2.37 (95% CI 1.13, 4.93), P=0.0219; OR 2.74 (95% CI 1.26, 6.05), P=0.0059] as well as TLR1neg 743AA/1805GG versus TLR1pos genotypes 743AG/1805TG [OR 4.98 (95% CI 1.64, 15.15), P=0.0034; OR 5.70 (95% CI 1.69, 20.35), P=0.0015] and 743AG + GG/1805TG + TT [OR 3.54 (95% CI 1.29, 9.90), P=0.0086; OR 4.17 (95% CI 1.52, 11.67), P=0.0025] with increased susceptibility to TB. No association of G743A with TB was found in Ghana as a result of a low frequency of genotype 743AA. Our data gain new insights in the role of TLR1 in M. tuberculosis defense and provide the first evidence that TLR1 variants are associated with susceptibility to TB in a low‐incidence country.

Expanding the spectrum of neurological disease associated with Epstein-Barr virus activity.

The purpose of this study was to delineate the spectrum of neurological diseases attributed to Epstein-Barr virus (EBV) activity. The approach was a retrospective study on patients with EBV activity proven by a positive EBV antibody-specific index (AI) and/or cerebrospinal fluid (CSF) PCR. One hundred six children and adults (AI positive = 77, AI + PCR positive = 3, PCR positive = 26) were identified, most with reactivated infections. Twenty-eight showed typical EBV-related diseases (encephalitis, neuritis, meningitis), 19 further infections (HSV encephalitis, neuroborreliosis, HIV infection, bacterial meningitis), nine immune-mediated disorders (multiple sclerosis, optic neuritis), and 50 further diseases not typical for EBV. The highest AI values occurred in patients with encephalitis. No relationship between disease category or AI values and viral loads was found. Additional reanalysis of 1,500 consecutive CSF EBV PCR studies revealed the highest positive rates among patients with further infections (n = 18/227, 7.9%) but lower rates among patients with typical EBV-related disorders (5/395; 1.3%), immune-mediated disorders (n = 2/174; 1.1%) and other conditions (n = 4/704; 0.6%). Intrathecal EBV activity is not restricted to typical EBV-related disorders, unexpectedly frequent in further CNS infections and also present in non-inflammatory conditions. Prospective studies should assess the pathogenic role of EBV in these different diseases.

Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR

ABSTRACT The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available.

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.

Failure to detect an association between aggressive periodontitis and the prevalence of herpesviruses.

BACKGROUND Herpes simplex virus type 1 (HSV-1), human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) have been suspected to play a causal role in periodontitis pathogenesis. The aim of this study was to determine the prevalence of these viruses in subgingival plaque samples of Caucasian patients with generalized aggressive periodontitis compared to periodontally healthy controls. METHODS A total of 65 patients with aggressive periodontitis and 65 unmatched controls from Germany were investigated in the study. Subgingival plaque samples were analysed for the presence of HSV-1, EBV and HCMV by quantitative real-time polymerase chain reaction assays. Viral antibody titres were determined quantitatively by immunosorbent assays. RESULTS DNA of HSV-1 and HCMV were detected in 1.5% of the patients and controls, whereas EBV DNA was present in 10.8% and 13.9% respectively. Detection rates of serum IgG against HSV-1 (76.1% versus 73.9%), EBV (98.5% versus 96.9%), HCMV (47.7% versus 46.2%) and IgM levels against HSV-1 (6.2% versus 1.5%), EBV (0% versus 0%), HCMV (0% versus 1.5%) did not significantly differ between patients and controls. CONCLUSION The data of our study do not suggest any contribution of HSV-1, EBV or HCMV to aggressive periodontitis in a German population. Ethnic and methodological aspects might have caused conflicting results of previous studies.

Application of Virus-Specific Immunoglobulin M (IgM), IgG, and IgA Antibody Detection with a Polyantigenic Enzyme-Linked Immunosorbent Assay for Diagnosis of Epstein-Barr Virus Infections in Childhood

ABSTRACT The Enzygnost anti-Epstein-Barr virus enzyme-linked immunosorbent assay (ELISA) system, which is based on a defined antigen mixture and on detection of antibodies of the immunoglobulin G (IgG), IgM, and IgA classes, was evaluated for its reliability in diagnosing Epstein-Barr virus infections in childhood. With samples from 66 children, the Epstein-Barr virus status and the infection phase were defined by indirect immunofluorescence and anticomplement fluorescence assays: 11 children were seronegative, 8 had a primary infection, 20 had a recent primary or past infection, and in 27 a reactivated Epstein-Barr virus infection was diagnosed. When applying the Enzygnost ELISAs, 15 serum samples (22.7%) were not interpretable due to indeterminate results in at least one of the assays used and were therefore excluded from further evaluation. The respective sensitivities and specificities for the diagnosis of seronegativity were 100 and 100%, those for the diagnosis of primary infection were 100 and 97%, those for the diagnosis of recent primary or past infection were 100 and 52%, and those for the diagnosis of reactivated infection were 10 and 100%. This poor performance of the Enzygnost system with reactivated infections is due to the prerequisite of an IgG antibody value of >650 IU/ml for the diagnosis of viral activity, which was fulfilled in only two of the children. Despite the high rate of indeterminate results, the Enzygnost system is useful in diagnosing acute and past Epstein-Barr virus infection in childhood. For serological diagnosis of viral activity in childhood, a supplementary assay is necessary.

Detection of Cytomegalovirus DNA in Human Specimens by LightCycler PCR: Melting Point Analysis Is Mandatory To Detect Virus Strains with Point Mutations in the Target Sequence of the Hybridization Probes

In our recently published paper we described the application of the LightCycler technology for the detection of cytomegalovirus (CMV) DNA in human plasma and urine (1). We are now able to give further information gained since September 2000, when this PCR procedure was introduced as the standard assay for the detection of CMV DNA in our diagnostic laboratory. As of June 2001, we had tested 912 specimens using this assay. According to the described test protocol, 185 specimens (20.3%) were positive for CMV DNA. A melting point (Tm )o f 59.2°C was observed for the hybridization probes with PCR amplicons of all positive specimens. Another 12 specimens (1.3%) were negative in quantitative analysis but generated a significant peak, with a Tm of 53.1°C, in the melting point analysis. Gel electrophoresis revealed a distinct PCR product of about 250 bp with these specimens that corresponds to the targeted 254-bp amplicon. DNA sequencing of the PCR products with the decreased melting point confirmed the specific amplification of CMV DNA using these specimens as a template. The decline of the melting point is caused by a point mutation in position 630 of the CMV glycoprotein B gene (GenBank accession no. A13758), resulting in a shift from cytosine to thymidine. This strain variant is not included in the databases and causes a mismatch with position 5 of the LCRed 640-labeled acceptor fluorophore probe. However, the existence of further strain variations in the amplified region cannot be excluded. The melting point analysis allows the reliable detection of CMV strain variants harboring this point mutation. Specificity of the signal should be further confirmed by gel electrophoresis. Melting point analysis is mandatory to detect virus strain variants in the target sequence of the hybridization probes with the described LightCycler CMV PCR assay.

Clinical application of viral cerebrospinal fluid PCR testing for diagnosis of central nervous system disorders: a retrospective 11-year experience ☆

The cerebrospinal fluid (CSF) polymerase chain reaction (PCR) is the gold standard to detect cerebral viral activity. As positive findings do not prove an impact on the neurological disorder, data interpretation is difficult. To better assess the impact of positive CSF PCR findings in different neurological diseases and to identify coherences facilitating CSF PCR data interpretation, we performed this retrospective analysis of CSF PCR data of 481 pediatric and 2604 adult patients, including herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and enteroviruses (EV). Nucleic acid of EBV was detected in 1.6% (39/2449), of VZV in 1.3% (34/2624), of HSV in 1.24% (37/2994), of EV in 0.4% (10/2364), of HHV-6 in 0.17% (4/2417), and of CMV in 0.2% (5/2514) of the patients. Newborns and elderly people showed highest infection rates. HSV, VZV, and EV prevailed in typical infectious central nervous system (CNS) diseases; EBV, in further inflammatory neurological diseases; HSV and EBV, in immunocompromised patients; and EBV, HSV, and HHV-6, in further non-inflammatory neurological diseases. Analysis of successive PCR studies revealed delayed viral detection for EBV (6/147) and HSV (1/217), respectively. Rapid viral clearance was typical for HSV, VZV, CMV, and EV infections, although the maximum duration of viral detection was 15days for HSV and 12days for VZV, respectively. This suggests that the detection of HSV, VZV, CMV, and EV strongly indicates symptomatic viral CNS disease. Secondary viral reactivation mostly underlies positive EBV and HHV-6 findings. Their detection does not rule out clinical impact but recommends searching for additional underlying conditions.

Low to Medium WU-Virus Titers in Young Children with Lower Respiratory Tract Infections

The WU-virus (WUV), a novel polyomavirus, has recently been recovered from respiratory tract samples. Within a study collective of children with severe lower respiratory tract disease, 3% of the patients tested WUV positive. Viral loads ranged from 5 × 102 copies/ml to 1 × 104 copies/ml. The WUV genome-positive patients did not display specific clinical or radiological characteristics to be distinguished from other respiratory tract infections.