Rapid testing for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) of patients presenting to emergency departments (EDs) facilitates the decision for isolation on admission to hospital wards. Differences in the sensitivity of molecular assays have implications for diagnostic workflows. This study evaluated the performance of the cobas® Liat® RT-PCR, which is routinely used as the initial test for ED patients in our hospitals, compared with the eazyplex® RT-LAMP. A total of 378 oropharyngeal and nasal swabs with positive Liat® results were analysed. Residual sample aliquots were tested using NeuMoDx™, cobas® RT-PCR, and the eazyplex® assay. Patients were divided into asymptomatic (n = 157) and symptomatic (n = 221) groups according to the WHO case definition. Overall, 14% of positive Liat® results were not confirmed by RT-PCR. These samples were mainly attributed to 26.8% of asymptomatic patients, compared to 3.8% of the symptomatic group. Therefore, positive Liat® results were used to provisionally isolate patients in the ED until RT-PCR results were available. The eazyplex® assay identified 62% and 90.6% of RT-PCR-confirmed cases in asymptomatic and symptomatic patients, respectively. False-negative eazyplex® results were associated with RT-PCR Ct values > 30, and were more frequent in the asymptomatic group than in the symptomatic group (38.1% vs. 5.1%, respectively). Both the Liat® and eazyplex® assays are suitable for testing symptomatic patients. Their use in screening asymptomatic patients depends on the need to exclude any infection or identify those at high risk of transmission.

Respiratory tract infections (RTI) can take a serious course under immunosuppression. Data on the impact of the underlying pathogens are still controversial. Samples from the upper (n = 322) and lower RT (n = 169) were collected from 136 children and 355 adults; 225 among them have been immunocompromised patients. Exclusion criteria were presence of relevant cultivable microorganisms, C-reactive protein > 20 mg/dl, or procalcitonin > 2.0 ng/ml. Samples were tested by PCR for the presence of herpesviruses (HSV-1/-2; VZV; CMV; HHV6; EBV), adenoviruses, bocaviruses, entero-/rhinoviruses (HRV), parechoviruses, coronaviruses, influenza viruses (IV), parainfluenza viruses as well as for pneumoviruses (HMPV and RSV), and atypical bacteria (Mycoplasma pneumoniae, M.p.; Chlamydia pneumoniae, C.p.). Viral/bacterial genome equivalents were detected in more than two-thirds of specimens. Under immunosuppression, herpesviruses (EBV 30.9%/14.6%, p < 0.001; CMV 19.6%/7.9%, p < 0.001; HSV-1: 14.2%/7.1%, p = 0.012) were frequently observed, mainly through their reactivation in adults. Immunocompromised adults tended to present a higher RSV prevalence (6.4%/2.4%, p = 0.078). Immunocompetent patients were more frequently tested positive for IV (15.0%/5.8%, p = 0.001) and M.p. (6.4%/0.4%, p < 0.001), probably biased due to the influenza pandemic of 2009 and an M.p. epidemic in 2011. About 41.8% of samples were positive for a single pathogen, and among them EBV (19.9%) was most prevalent followed by HRV (18.2%) and IV (16.6%). HSV-2 and C.p. were not found. Marked seasonal effects were observed for HRV, IV, and RSV. Differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. The exact contribution of some herpesviruses to the development of RTI remains unclear.

BACKGROUND:Diagnostic assays for severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) that are easy to perform and produce fast results are essential for timely decision making regarding the isolation of contagious individuals. OBJECTIVE:We evaluated the CE-approved eazyplex® SARS-CoV-2, a ready-to-use real time RT-LAMP assay for identification of the SARS-CoV-2 N and ORF8 genes from swabs in less than 30 min without RNA extraction. STUDY DESIGN:Oropharyngeal and nasal swabs from 100 positive and 50 negative patients were inoculated into 0.9 % saline and tested by NeuMoDx™ RT-PCR. An aliquot was diluted fivefold in Copan sputum liquefying (SL) solution and directly analyzed by eazyplex® SARS-CoV-2. In addition, 130 patient swabs were prospectively tested with both methods in parallel. Analytical sensitivity of the assay was determined using virus stock dilutions. RESULTS:Positive percent agreement (PPA) between the eazyplex® SARS-CoV-2 and RT-PCR was 74 % for samples with Ct values < 35. When using a Ct cut-off ≤ 28 the PPA increased to 97.4 %. In the prospective part of the study overall PPA of the eazyplex® kit was 66.7 % but increased to 100 % when only Ct values ≤ 28 were considered. There were no false positive results. The median time to positivity was 12.5 min for the N gene and 16.75 min for ORF8. Analytical sensitivity was 3.75 TCID50/mL. 105 virus copies/mL were reproducibly detected. CONCLUSION:The eazyplex® SARS-CoV-2 is a rapid assay that accurately identifies samples with high viral loads. It may be useful for near-patient testing outside of a molecular diagnostic laboratory.

Background: Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. Objectives: This study was designed to evaluate the clinical performance of the CE-labeled variplex (TM) real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. Study design: RNA extracted from pharyngeal swabs was tested by variplex (TM) RT-LAMP and Corman's LightMix (TM) E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID19) and negative control patients were analyzed by variplex (TM) without RNA extraction and tested in parallel with the Allplex (TM) and VIASURE BD MAX RT-PCRs. Results: Using isolated RNA variplex (TM) RT-LAMP showed a sensitivity of 75 % compared to LightMix E gene RTPCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex (TM) RT-LAMP conducted on unprocessed samples and Allplex (TM) and VIASURE RT-PCRs (Cohen's kappa ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3 % for variplex (TM), 84.2 % for Allplex (TM) and 68.4 % for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100 %. Conclusion: The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.

The Epstein-Barr virus (EBV) produces different microRNAs (miRNA) with distinct regulatory functions within the infectious cycle. These viral miRNAs regulate the expression of viral and host genes and have been discussed as potential diagnostic markers or even therapeutic targets, provided that the expression profile can be unambiguously correlated to a specific stage of infection or a specific EBV-induced disorder. In this context, miRNA profiling becomes more important since the roles of these miRNAs in the pathogenesis of infections and malignancies are not fully understood. Studies of EBV miRNA expression profiles are sparse and have mainly focused on associated malignancies. This study is the first to examine the miRNA profiles of EBV reactivation and to use a correction step with seronegative patients as a reference. Between 2012 and 2017, we examined the expression profiles of 11 selected EBV miRNAs in 129 whole blood samples from primary infection, reactivation, healthy carriers and EBV seronegative patients. Three of the miRNAs could not be detected in any sample. Other miRNAs showed significantly higher expression levels and prevalence during primary infection than in other stages; miR-BHRF1-1 was the most abundant. The expression profiles from reactivation differed slightly but not significantly from those of healthy carriers, but a specific marker miRNA for each stage could not be identified within the selected EBV miRNA targets.

BACKGROUND:The objective of this study was to identify parameters of prognostic relevance in patients presenting with chronic left ventricular dysfunction who underwent endomyocardial biopsy. MATERIALS AND METHODS:A total of 351 consecutive patients (age 47·7 ± 12·6 years, 281 male) with a chronic left ventricular dysfunction were enrolled. Endomyocardial biopsies were analysed by histopathology according to Dallas criteria and immunohistological WHO criteria. Virus genome was detected by polymerase chain reaction. The combined end point was time to death or heart transplantation. RESULTS:About 19% of patients (n = 67) showed positive Dallas criteria and 39% (n = 118) immunohistochemical signs of inflammation. Viral genome was present in 58% (n = 155). During follow-up, 25% (n = 89; 76 death, 13 HTx) reached the end point. Dallas-positive histopathology (hazard ratio: 0·42; 95% CI: 0·29-0·84, P = 0·031), ejection fraction (hazard ratio: 0·97; 95% CI: 0·94-0·99, P = 0·019) and β-blocker therapy (hazard ratio: 0·41; 95% CI: 0·23-0·69, P = 0·003) were independent outcome predictors. For patients under β-blocker therapy, Dallas-positive histopathology (hazard ratio: 0·37; 95% CI: 0·25-0·76, P = 0·009) and NYHA class III and class IV (hazard ratio: 2·11; 95% CI: 1·04-3·12, P = 0·006) were independent predictors. CONCLUSIONS:For patients with a chronic left ventricular dysfunction, Dallas-positive histopathology, β-blocker therapy and left ventricular ejection fraction are the most striking parameters for outcome prediction.

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).

A case of primary Epstein–Barr virus (EBV) infection/parvovirus B19 reactivation fulfilling five of eight criteria for hemophagocytic lymphohistiocytosis (HLH) is presented. Despite two coinciding viral infections, massive splenomegaly, and fulminant hepatitis, the patient had a good clinical outcome, probably due to an early onset form of HLH with normal leukocyte count, normal natural killer (NK) cell function, and a lack of hemophagocytosis.

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.

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.

Hemorrhagic cystitis (HC) is a serious complication after hematopoietic stem cell transplantation (HSCT). The pathogenesis of HC in adults is not fully understood and may be influenced by BK virus infection, type of transplant, conditioning regimen, stem cell source, and graft-versus-host disease. Little is known about the development of HC in children after HCST, especially about the association with BK virus infection. Therefore, we retrospectively analyzed the incidence, risk factors and BK virus association of HC in 165 consecutive children (median age, 12 years) who underwent peripheral blood stem cell (n=97; T-cell depleted: n=48) or bone marrow transplantation (n=68) between 1/2000 and 12/2006 in a single center. Fifty nine patients received autologous HSCT and 106 patients underwent allogeneic HSCT. Nineteen of the 165 patients (11.5%) developed HC after a median of 33 days (range, 1–98 days). All 19 patients with HC underwent allogeneic HSCT and showed BK viruria after transplantation. An acute graft-versus-host disease was significantly more frequent in children with HC (P < .001). Significant risk factors in univariate binary logistic analyses were age > 12 years (OR, 3.275; P < .031), use of busulfan (OR, 3.514; P < .013), use of busulfan and cyclophosphamide in combination (OR, 4.935; P < .002), and an unrelated donor (OR, 3.309; P < .043). Independent risk factors in multivariate binary logistic analyses were age > 12 years and the combination of busulfan and cyclophosphamide. We suggest that cyclophosphamide is toxic to the urinary bladder and busulfan enhances this effect. Furthermore, we analyzed the BK virus load in urine by real-time polymerase chain reaction. We found in patients without HC a significantly increased number of BK virus copies in urine in children older than 12 years (P < .009) and in children who received antithymocyte globulin (P < .001). In addition, BK virus load in urine was significantly increased in children who suffered from HC. Thirteen of 14 children with HC had a BK virus load in urine >107 copies/mL (P < .001). We observed in individual BK virus profiles an increase of BK virus copies in urine before the onset of HC. We conclude that HC in children is a disease of multiple etiologies. Allogeneic HSCT, the combination of busulfan and cyclophosphamide, age > 12 years, and an unrelated donor are risk factors for the development of HC in childhood. Increased BK virus load in urine of more than 107 copies/mL may lead to HC. Therefore, it is useful to quantify BK virus in urine in those children with above mentioned risk factors to initiate early treatment or to prevent the development of HC.

Background The presence of viral genome in the myocardium of patients with dilated cardiomyopathy (DCM) has been suggested as causative for the underlying cardiac disease. Nevertheless, the results of present studies are conflicting regarding the natural course of heart diseases associated with detection of viral genome. This study was undertaken to determine if the defection of viral genome in the myocardium of patients with DCM is of functional and prognostic relevance under modern treatment strategies of heart insufficiency.Methods In 197 patients with DCM, left ventricular endomyocardial biopsies were performed. Analysis for genome of adenovirus, enterovirus (EV), and parvovirus B19 as well as enteroviral replication and immunohistology was performed.Results The increase in ejection fraction (EF) was 14.5 +/- 12.4% in the EV-positive group compared with 11.1 +/- 14.2 in the EV-negative group (P = not significant [NS]) after a mean follow-up (FU) of 19.5. and 17.6 months. The increase in EF in the virus-positive group (positive for EV, adenovirus, or parvovirus B19) was 15.3 +/- 13.3% compared with 12.3 +/- 11.9% in the virus-negative group (P = NS) after a mean FU of 17.6 and 11.5 months. There was no significant difference in the change of EF between the EV-positive and virus-negative groups. Detection of enteroviral RNA replication (detection of EV minus-strand RNA) did not result in a deterioration of left ventricular function compared with the virus-negative group (P = NS) after mean FU of 11.2 and 12.0 months. The transplantation-free survival of the patients was not influenced by detection of viral genome.Conclusions Our results favor the view that the presence of viral genome in the myocardium of patients with DCM is of no functional and prognostic relevance.

The primary infection with human polyomavirus BK (BKV) occurs in early childhood and leads to viral latency within the urogenital tract. Up to 90% of the adult population are seropositive. In immunosuppressed patients, the BKV may be reactivated resulting in typical disease patterns like hemorrhagic cystitis and tubulointerstitial nephritis. Based on serological and molecular methods, BKV isolates were classified into four subtypes previously. Sixty specimens obtained from German renal and bone marrow transplant recipients were analyzed to gain data on the prevalence of BKV subtypes in Germany. With 90.9%, BKV subtype I was found to be predominant in both patient groups. 6.1% of BKV strains were classified as subtype IV. This pattern of phylogenetic distribution is similar to that demonstrated previously in England, Tanzania, the United States and Japan. Remarkably, there was one German BKV virus with a sequence which clusters together with strain SB in subtype II. The BKV subtype I was found to consist of at least three subgroups designated as Ia, Ib, and Ic. While the majority of the German sequences represent subgroup Ic, most of the Japanese sequences are clearly distinct. These findings support the hypothesis of distinct geographical prevalence of BKV subgroups. For the genotyping region, a relationship of BKV subgroups to disease patterns like hemorrhagic cystitis or tubulointerstitial nephritis could not be demonstrated.

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different 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 on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5′ ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.

Aside from enteroviruses and other viruses, e.g., adenoviruses, which are known to be associated with idiopathic dilated cardiomyopathy (IDC), a cardiac tropism is also attributed to parvovirus B19 (PVB19). The purpose of the present study was to determine the prevalence of enterovirus, adenovirus and PVB19 genomes in the myocardium of adult patients with IDC and to analyze the significance of PVB19 with regard to the course of the disease, as compared to the other cardiotropic viruses. In 52 adult patients with IDC and 10 control patients with normal left ventricular ejection fraction (≥55%) undergoing coronary artery bypass surgery, myocardial tissue samples were investigated for enteroviral RNA using polymerase chain reaction (PCR) and Southern blot hybridization of the PCR product. Specific nested PCR was used to assess the prevalence of adenovirus and PVB19 DNA, in addition to sequencing of the latter. The clinical and echocardiographic course of the disease was followed for a mean (± SD) period of 21.1±9.5 months. Fourteen of the 52 patients (27%) were enterovirus-positive, 2/52 (4%) patients were adenovirus-positive, 14/52 (27%) patients were PVB19-positive, 8/52 (15%) patients were enterovirus plus PVB19-positive, and in 14/52 (27%) patients no viral genomes were found. Six patients died during the follow-up period, without any significant difference between the patient groups: 1/14 (7%) in the enterovirus-positive, 0/2 (0%) in the adenovirus-positive, 2/14 (14%) in the PVB19-positive, 1/8 (12.5%) in the enterovirus plus PVB19-positive, and 2/14 (14%) in the virus-negative group. PVB19 genome was found in 4 of the 10 (40%) control patients, but no enterovirus or adenovirus genomes were detected in these patients. In conclusion, in the myocardium of patients with IDC, PVB19 is detectable as frequently as enteroviral genome. PVB19-positive patients with IDC have a rather favorable prognosis and do not differ significantly from the other virus-positive or virus-negative patient groups with respect to survival. Finally, the pathogenetic and prognostic significance of PVB19 in IDC still remains unclear.

Background: 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.

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 ex vivo 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.

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.

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 Jun B 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 jun D 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 jun B mRNA-Expression eingesetzt. Dabei konnten keine signifikanten Unterschiede zwischen den Expressionsniveaus von jun B in SM oder primären synovialen Fibroblasten von RA- und OA-Patienten nachgewiesen werden. Interessanterweise wurde allerdings eine signifikant niedrigere jun B-Expression in der RA-SM und eine grenzwertig signifikant niedrigere jun B-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 Jun B-spezifischen Antikörpern zeigte eine vergleichbare Jun B 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.