Renate Egerer

Detection of multiple human herpes viruses by DNA microarray technology.

AbstractBackground: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. Methods: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. Results: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. Conclusions: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

Virus Diagnostics on Microarrays

Whereas the majority of microarray applications still deal with expression analysis for gathering information about levels of gene products at certain cell states, other approaches simply ask the question whether particular genes, which are usually indicative for particular microorganisms and pathogens, are present in a sample or not. Investigations that are more detailed try to evaluate the presence of particular subtypes of a given pathogen. The combination of microarray technology and virus diagnostics promises to generate an ideal platform for fast, sensitive, specific, and parallelized virus diagnostics. Performing virus diagnostics on microarrays, however, requires other basic techniques to be optimized. This is necessary in order to obtain unambiguous and reproducible results, which are compatible with the needs for clinical routine. Parameters that have to be considered include supports, coupling chemistry, chemical oligonucleotide synthesis, signal enhancement strategies, and optimal coordination of PCR reactions, hybridizations, and signal detection, as well as interpretation strategies. Finally, considerations should be given to economic aspects, one chip-one patient strategies and low integrated arrays as a custom-tailored way to fast and accurate diagnostic tools.

Low level myocardial parvovirus B19 persistence is a frequent finding in patients with heart disease but unrelated to ongoing myocardial injury

While myocardial parvovirus B19 (B19V), aside from enteroviruses (EV) and adenoviruses (ADV), has recently been found often in patients with myocarditis and idiopathic dilated cardiomyopathy (IDC), the pathogenetic significance of B19V genomes in those patients has not yet been sufficiently elucidated. In the present study, left ventricular endomyocardial biopsies from 24 patients with left ventricular ejection fraction (LVEF) below 55% due to IDC, and tissue from the right atrial appendage of 10 control patients undergoing bypass surgery with normal LVEF (>55%) were investigated for B19V, ADV, and EV genomes by specific nested polymerase chain reaction (PCR), by real time PCR or by reverse‐transcription PCR, respectively. The myocardial tissue samples from the 10 controls were analyzed each in three different virological laboratories for B19V. In the IDC group, the frequency of the myocardial virus genomes found in 54% (13/24) of the patients was as follows: B19V: 50% (12/24), EV: 8% (2/24), including one patient with B19V and EV, and ADV: 0% (0/24). For comparison, the prevalence of B19V genomes was between 30% and 60% in the control group as detected in three different laboratories, but all these control subjects were EV‐ and ADV‐negative. The number of B19V gene copies, however, was very low and similar both in the IDC and control group. In the majority of patients myocardial B19V persistence was associated with a low virus load irrespective of the underlying heart disease so that it may be of no importance in the pathogenesis of IDC. J. Med. Virol. 82:1449–1457, 2010.

Vergleich zwischen konventioneller und Real-Time RT-PCR zur Quantifizierung der jun-Protoonkogen-mRNA und Analyse der junB-mRNA-Expression in Synovialmembranen und isolierten synovialen Fibroblasten von Patienten mit rheumatoider Arthritis

Summary. AP-1 dependent genes, e.g., matrix-metallo-proteinases, are involved in the pathogenesis of rheumatoid arthritis (RA). Therefore, the transcription factor AP-1 and its subunits, proteins of the Jun and Fos proto-oncogene families, are interesting targets for analysis in RA. In this study, we analyzed the mRNA expression of junB in synovial membrane (SM) samples and isolated synovial fibroblasts of patients with RA, osteoarthritis (OA), and normal, non-inflammatory controls.To address the suitability of real-time RT-PCR for the quantitation of Jun proto-oncogene family members, conventional RTPCR and real-time PCR were comparatively applied for junD, a gene representing a major challenge because of its high GC-content (70%, increasing the probability of secondary structures interfering with the PCR) and its sequence homology to other Jun proto-oncogenes. In addition, a comparison was performed concerning the precision, reproducibility, costs, as well as labor and time consumption of the two PCR methods.Real-time RT-PCR proved superior to conventional PCR in terms of precision (mean deviation of measured from employed concentration 58% for real-time PCR vs 225% for conventional PCR), reproducibility, as well as labor and time consumption (4 times less for real-time RT-PCR). Experimental cDNA normalization for equivalent cDNA concentrations by sample dilution was more reliable than mathematical cDNA normalization. However, real-time PCR was 3.6-fold more expensive.Applying the more reliable real-time RT-PCR for the exvivo analysis of junB mRNA-expression, no significantly different expression of junB was observed in SM or isolated synovial fibroblasts from RA as compared to OA. Interestingly, however, junBmRNA expression was significantly lower in RA SM and borderline significantly lower in OA SM than in normal/non-inflammatory SM, with potential effects on the functional properties of the resulting AP-1 complexes. Immunohistochemical staining of the SM with JunB-specific antibodies showed comparable JunB protein expression in SFB (collagen III mRNA-positive) of RA and OA samples.Thus, real-time RT-PCR appears suitable and time-saving for the quantitation of jun proto-oncogene mRNA-expression in tissue and cell samples with high precision and reproducibility. Zusammenfassung. AP-1-abhängige Gene, z. B. Matrix-Metallo- Proteinasen, sind an der Pathogenese der rheumatoiden Arthritis (RA) beteiligt. Deshalb sind der Transkriptionsfaktor AP-1 und seine Untereinheiten – die Proteine der Jun- und Fos-Proto-Onkogen-Familien – interessante Ziele für die Analyse in der RA. In dieser Studie wurde die mRNA-Expression von JunB in Synovialmembran-Gewebeproben (SM) und primären synovialen Fibroblasten von Patienten mit RA und Osteoarthritis (OA), sowie normalen, nicht entzündlichen Kontrollen analysiert.Um die Eignung der Real-time PCR für die Quantifizierung von Mitgliedern der Jun-Genfamilie zu überprüfen, wurde ein Vergleich der konventionellen RT-PCR mit der Real-time PCR für das Gen junD durchgeführt. Dieses stellt aufgrund seines hohen GC-Gehaltes (70%, wodurch die mögliche Bildung von mit der Polymerase- Reaktion interferierenden Sekundärstrukturen deutlich erhöht wird) und der Sequenzhomologien zu anderen Jun-Genen eine große methodische Herausforderung dar. Außerdem wurde in die Untersuchung ein Vergleich in Hinblick auf die Präzision, die Reproduzierbarkeit, die Kosten, sowie den Arbeits- und Zeitaufwand der beiden Methoden einbezogen.Die Real-time PCR erwies sich der konventionellen PCR in den Punkten Präzision (die mittlere Abweichung der gemessenen von der eingesetzten Konzentration betrug bei der Real-time PCR 58% gegenüber 225% bei der konventionellen PCR), Reproduzierbarkeit und Arbeits-/Zeitaufwand (4-fach geringer bei der Real-time RT-PCR) überlegen. Eine experimentelle Normalisierung durch Verdünnung der untersuchten cDNA-Proben auf äquivalente cDNA-Konzentrationen stellte sich gegenüber einer rein mathematischen Normalisierung als genauer heraus. Allerdings waren die Kosten der Realtime PCR 3,6-mal so hoch wie die der konventionellen PCR.Die zuverlässigere Real-time PCR wurde anschließend zur Ex-vivo- Analyse der junB mRNA-Expression eingesetzt. Dabei konnten keine signifikanten Unterschiede zwischen den Expressionsniveaus von junB in SM oder primären synovialen Fibroblasten von RA- und OA-Patienten nachgewiesen werden.Interessanterweise wurde allerdings eine signifikant niedrigere junB-Expression in der RA-SM und eine grenzwertig signifikant niedrigere junB-Expression in der OA-SM im Vergleich zu den Normalkontrollen beobachtet, was die Funktion der resultierenden AP-1 Komplexe beeinflussen könnte. Die immunhistologische Färbung der SM mit JunB-spezifischen Antikörpern zeigte eine vergleichbare JunB Proteinexpression in SFB (Kollagen III mRNA positiv) bei RA- und OA-Proben.Insgesamt erwies sich die Realtime RT-PCR in dieser Studie als eine geeignete und zeitsparende Methode für die Quantifizierung der mRNA-Expression von jun- Proto-Onkogenen in Gewebe und Zellproben mit hoher Präzision und Reproduzierbarkeit.

Multiple viral infections after haploidentical hematopoietic stem cell transplantation in a child with acute lymphoblastic leukemia

After allogeneic hematopoietic stem cell transplantation (HSCT), viral infections/reactivations are a frequent complication, sometimes with fatal outcome. Thus, early diagnosis is recommended by screening of whole blood or plasma preparations using highly sensitive molecular techniques that test for the most common viral pathogens, such as Epstein–Barr virus, cytomegalovirus, and adenoviruses (ADVs). Despite this approach, not every reactivation/infection can be adequately detected or excluded, even with highly sensitive polymerase chain reaction. Particularly after toxic treatment, uncommon infections or infections resistant to first‐line treatment can occur, even in unusual locations. Herein, we present the case of a child with Philadelphia chromosome‐positive acute lymphoblastic leukemia after allogeneic HSCT who suffered from 5 different viral reactivations/infections, including acyclovir‐resistant herpes simplex virus type 1 esophagitis, human herpesvirus 6 encephalitis, rotavirus gastroenteritis, respiratory syncytial virus pneumonia, and ADV esophagitis, despite routinely performed blood examinations for viral pathogens remaining unrevealing at all times.

Epstein-Barr virus-associated pneumonia and bronchiolitis obliterans syndrome in a lung transplant recipient

We report the case of a 25-year-old lung and liver transplant recipient who developed respiratory failure. High levels of Epstein-Barr virus (EBV) genome copies were detectable in respiratory tract specimens, while the search for various other viral, bacterial or fungal pathogens remained empty. Post-transplant lymphoproliferative disease was excluded. Due to the rapid progression of respiratory insufficiency, a re-transplantation of the lung was performed. EBV-encoded small RNAs could be demonstrated by in situ hybridization within pneumocytes and lymphocytes of the explanted lung tissue. The clinical situation improved soon after re-transplantation, and the EBV load detected in the lower respiratory tract decreased significantly.

Analysis of an echovirus 18 outbreak in Thuringia, Germany: insights into the molecular epidemiology and evolution of several enterovirus species B members

In October and November 2010, six children and one woman were presented with symptoms of aseptic meningitis in Jena, Thuringia, Germany. Enterovirus RNA was detected in the cerebrospinal fluid of all patients by RT-PCR, and preliminary molecular typing revealed echovirus 18 (E-18) as causative agent. Virus isolates were obtained from stool samples of three patients and several contact persons. Again, most isolates were typed as E-18. In addition, coxsackievirus B5 (CV-B5) and echovirus 25 (E-25) were found to co-circulate. As only few complete E-18 sequences are available in GenBank, the entire genomes of these isolates were determined using direct RNA-sequencing technology. We did not find evidence for recombination between E-18, E-25 or CV-B5 during the outbreak. Viral protein 1 gene sequences and the cognate 3D polymerase gene sequences of each isolate and GenBank sequences were analysed in order to define type-specific recombination groups (recogroups).

Outcome predictors in dilated cardiomyopathy or myocarditis

The objective of this study was to identify parameters of prognostic relevance in patients presenting with chronic left ventricular dysfunction who underwent endomyocardial biopsy.