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Background: Determination of the hepatitis C virus (HCV) genotype and discrimination between HCV subtypes 1a and 1b is still mandatory prior to anti-HCV treatment initiation. The aim of this study was to evaluate the performance of the recently introduced cobas® HCV GT assay (Roche) and to compare it to two comparator assays., Methods: The cobas® HCV GT assay is based on primer-specific real-time polymerase chain reaction (PCR). For comparison, the TRUGENE® HCV 5'NC Genotyping Kit (Siemens) and the VERSANT® HCV Genotype 2.0 Assay (Siemens) were employed. Accuracy of the new assay was determined using proficiency panels. For clinical evaluation, 183 residual clinical samples obtained from patients with chronic hepatitis C infection were included., Results: When accuracy was tested, panel members containing HCV subtypes 1a, 1b, and 3a were identified as expected; however, the new assay failed to identify low titer panel members containing HCV subtype 5a correctly. Of 183 clinical samples, 160 gave concordant results. For seven samples, an indeterminate result was reported with the cobas® HCV GT assay and the remaining 16 samples were found discordant with one of the comparator assays. When time-to-results of the assays were compared, the new assay showed shorter total time and similar hands-on time per sample., Conclusions: The cobas® HCV GT assay showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Due to the high level of automation, fast and reliable results are obtained with short hands-on time.

Background: There is growing evidence of an interaction between HIV-infection, anti-retroviral therapy (ART) and cardiovascular diseases (CVD). Epidemiological studies in Europe and North America have been observing a shift towards an increased incidence of coronary heart disease and acute myocardial infarctions in HIV-infected populations compared to the general population even after adjusting for traditional cardiovascular risk factors. Despite South Africa (and sub-Saharan Africa, SSA) being regarded as the epicentre of the global HIV epidemic, very little is known about the prevalence of cardiovascular risk factors and precursors of vascular disease in HIV-infected populations in this region. The knowledge gap is further widened by the paucity of data from prospective studies. We present the rationale, objectives and key methodological features of the EndoAfrica study, which aims to determine whether HIV-infection and ART are associated with altered cardiovascular risk and changes in vascular endothelial structure and function in adults living in the Western Cape Province of South Africa., Methods: In this longitudinal study, comprehensive cardiovascular assessments of HIV-negative and HIV-positive (with and without ART) study participants are performed by clinical and biochemical screening for traditional cardiovascular risk factors and biomarkers of CVD. Vascular and endothelial function is determined by brachial artery flow-mediated dilatation (FMD), carotid-intima-thickness (IMT) measurements and quantitative retinal blood vessel analyses, complemented by vascular endothelial biomarker assays. Finally, we aim to statistically determine whether HIV-infection and/or ART are associated with increased cardiovascular risk and vascular endothelial dysfunction, and determine whether there is progression/regression in these endpoints 18 months after the baseline assessments., Discussion: The EndoAfrica study provides a unique opportunity to recruit a cohort of HIV-infected patients and HIV-negative controls who will be comprehensively and longitudinally assessed for cardiovascular risk and disease profile with vascular endothelial function as a potentially important intermediate cardiovascular phenotype. To our knowledge, it is the first time that such a systematic study has been established in the context of SSA and South Africa.

Objectives: Despite the input of microbiome research, a group of 20 bacteria continues to be the focus of periodontal diagnostics and therapy. The aim of this study was to compare three commercial kits and laboratory-developed primer pairs for effectiveness in detecting such periodontopathogens., Materials and Methods: Fourteen bacterial mock communities, consisting of 16 randomly assembled bacterial strains, were used as reference standard for testing kits and primers. Extracted DNA from mock communities was analyzed by PCR in-house with specific primers and forwarded for analysis to the manufacturer's laboratory of each of the following kits: ParoCheck®Kit 20, micro-IDent®plus11, and Carpegen® Perio Diagnostik., Results: The kits accurately detected Fusobacterium nucleatum, Prevotella intermedia/Prevotella nigrescens, Parvimonas micra, Aggregatibacter actinomycetemcomitans, Campylobacter rectus/showae, Streptococcus mitis, Streptococcus mutans, and Veillonella parvula. The in-house primers for F.nucleatum were highly specific to subtypes of the respective periopathogen. Other primers repeatedly detected oral pathogens not present in the mock communities, indicating reduced specificity., Conclusions: The commercial kits used in this study are reliable tools to support periodontal diagnostics. Whereas the detection profile of the kits is fixed at a general specificity level, the design of primers can be adjusted to differentiate between highly specific strains. In-house primers are more error-prone. Bacterial mock communities can be established as a reference standard for any similar testing., Clinical Relevance: The tested kits render good results with selected bacterial species. Primers appear to be less useful for routine clinical diagnostics and of limited applicability in research. Basic information about the periodontopathogens identified in this study supports clinical decision-making., Competing Interests: Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Funding This study was supported by the Hygiene Fund of the Institute of Hygiene, Microbiology and Environmental Medicine at the Medical University of Graz, Austria. Informed consent For this type of study, formal consent is not required. Conflict of interest The authors declare that they have no conflict of interest.

To gain deeper insight into the epidemiology of HIV-1 transmission in South-East Austria we performed a retrospective analysis of 259 HIV-1 partial pol sequences obtained from unique individuals newly diagnosed with HIV infection in South-East Austria from 2008 through 2014. After quality filtering, putative transmission linkages were inferred when two sequences were ≤1.5% genetically different. Multiple linkages were resolved into putative transmission clusters. Further phylogenetic analyses were performed using BEAST v1.8.1. Finally, we investigated putative links between the 259 sequences from South-East Austria and all publicly available HIV polymerase sequences in the Los Alamos National Laboratory HIV sequence database. We found that 45.6% (118/259) of the sampled sequences were genetically linked with at least one other sequence from South-East Austria forming putative transmission clusters. Clustering individuals were more likely to be men who have sex with men (MSM; p<0.001), infected with subtype B (p<0.001) or subtype F (p = 0.02). Among clustered males who reported only heterosexual (HSX) sex as an HIV risk, 47% clustered closely with MSM (either as pairs or within larger MSM clusters). One hundred and seven of the 259 sequences (41.3%) from South-East Austria had at least one putative inferred linkage with sequences from a total of 69 other countries. In conclusion, analysis of HIV-1 sequences from newly diagnosed individuals residing in South-East Austria revealed a high degree of national and international clustering mainly within MSM. Interestingly, we found that a high number of heterosexual males clustered within MSM networks, suggesting either linkage between risk groups or misrepresentation of sexual risk behaviors by subjects.

Although amateur sports have become increasingly competitive within recent decades, there are as yet few studies on the possible health risks for athletes. This study aims to determine the impact of ultra-endurance exercise-induced stress on the number and function of circulating hematopoietic progenitor cells (CPCs) and hematological, inflammatory, clinical, metabolic, and stress parameters in moderately trained amateur athletes. Following ultra-endurance exercise, there were significant increases in leukocytes, platelets, interleukin-6, fibrinogen, tissue enzymes, blood lactate, serum cortisol, and matrix metalloproteinase-9. Ultra-endurance exercise did not influence the number of CPCs but resulted in a highly significant decline of CPC functionality after the competition. Furthermore, Epstein-Barr virus was seen to be reactivated in one of seven athletes. The link between exercise-induced stress and decline of CPC functionality is supported by a negative correlation between cortisol and CPC function. We conclude that ultra-endurance exercise induces metabolic stress and an inflammatory response that affects not only mature hematopoietic cells but also the function of the immature hematopoietic stem and progenitor cell fraction, which make up the immune system and provide for regeneration., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

Background: The COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring., Objectives: The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS(®) TaqMan(®) HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies., Study Design: Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples., Results: All quantifiable WHO dilutions were within ±0.3log10IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5log10IU/mL. When low viral load results (25-3500IU/mL) were compared, values obtained by the ART assay were significantly lower (p<0.0001) than those obtained with the CAP/CTM2., Conclusions: The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Quantitation of EBV DNA has been shown to be a useful tool to identify and monitor patients with immunosuppression and high risk for EBV-associated disease. In this study, the analytical and clinical performance of the new Realquality RS-EBV Kit (AB Analitica, Padova, Italy) was investigated. The clinical performance was compared to that of the EBV R-gene (bioMerieux, Varilhes, France) assay. When the accuracy of the new assay was tested, all results except of one were found to be within ±0.5log10 unit of the expected panel results. Determination of linearity showed a quasilinear curve, the between day imprecision ranged from 18% to 88% and the within run imprecision from 16% to 53%. When 96 clinical EDTA whole blood samples were tested, 77 concordant and 19 discordant results were obtained. When the results for the 69 samples quantifiable with both assays were compared, the new assay revealed a mean 0.31log10 unit higher measurement. The new assay proved to be suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. The variation between quantitative results obtained by the assays used in this study reinforces the use of calibrators traceable to the existing international WHO standard making different assays better comparable., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Broad-range PCR has become a valuable tool for the identification of microorganisms in the clinical laboratory over the last years. It was primarily used to identify slow-growing and fastidious microorganisms with poor biochemical activity. Nowadays, it is also used to identify microorganisms directly from clinical samples such as blood or punctuates from primarily sterile body sites. In these specimens, the usage of broad-range PCR is challenging regarding contamination and standardization. To overcome these problems, a new test system, the SepsiTest™, was introduced recently employing broad-range PCR for the identification of microorganisms in septic patients. In this chapter, the test system is described and the equipment necessary listed.

The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600-573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10-99 and reverse transcriptase (RT) codons 1-251 were generated using molecular barcoded primers. 454-UDS was performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1364 reads per position (IQR=809-2065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1-25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1 and 10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the method. HIV-1 drug resistance testing using 454 ultra-deep pyrosequencing results in an accurate and highly reproducible, albeit complex, approach to the analysis of HIV-1 mutant spectra, even at frequencies well below those detected by routine population sequencing., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Testing for (1→3)-beta-D-glucan (BDG) is used for detection of invasive fungal infection. However, current assays lack automation and the ability to conduct rapid single-sample testing. The Fungitell assay was adopted for automation and evaluated using clinical samples from patients with culture-proven candidemia and from culture-negative controls in duplicate. A comparison with the standard assay protocol was made in order to establish analytical specifications. With the automated protocol, the analytical measuring range was 8-2500 pg/ml of BDG, and precision testing resulted in coefficients of variation that ranged from 3.0% to 5.5%. Samples from 15 patients with culture-proven candidemia and 94 culture-negative samples were evaluated. All culture-proven samples showed BDG values >80 pg/ml (mean 1247 pg/ml; range, 116-2990 pg/ml), which were considered positive. Of the 94 culture-negative samples, 92 had BDG values <60 pg/ml (mean, 28 pg/ml), which were considered to be negative, and 2 samples were false-positive (≥80 pg/ml; up to 124 pg/ml). Results could be obtained within 45 min and showed excellent agreement with results obtained with the standard assay protocol. The automated Fungitell assay proved to be reliable and rapid for diagnosis of candidemia. It was demonstrated to be feasible and cost efficient for both single-sample and large-scale testing of serum BDG. Its 1-h time-to-result will allow better support for clinicians in the management of antifungal therapy., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Reliable and rapid diagnosis of influenza A H1N1 is essential to initiate the appropriate antiviral therapy and preventive measures. As PCR assays are time-consuming and rapid antigen tests have a limited sensitivity, official influenza case definitions are used in many clinical settings. These, however, are based exclusively on clinical criteria and have only a moderate potential to differentiate between influenza and other febrile diseases. Only limited data on the differences in clinical and laboratory parameters between influenza and non-influenza febrile diseases are available to date., Methods: This was a retrospective case-negative control series that was conducted in Styria, southeast Austria. We analyzed the differences in clinical presentation and laboratory admission parameters between patients with PCR-confirmed H1N1 influenza infection (n = 199) and those with influenza-like disease and negative influenza PCR results (ILD group; n = 252)., Results: In the multivariable analysis lower C-reactive protein (CRP) level, lower white blood cell (WBC) count, fever, wheezing, cough, and the absence of nausea or sudden onset remained significant predictors of H1N1 influenza in adult patients (n = 263). Lower CRP level, lower WBC count, and cough remained significant predictors in pediatric patients (<16 years; n = 188)., Conclusion: Lower CRP level, lower WBC count, and cough were significant predictors of H1N1 in both the adult and pediatric patient group. These data may help to develop an improved case definition for suspected H1N1 infection which combines clinical findings and easily available laboratory parameters.

Reliable and rapid diagnosis of influenza A H1N1 is essential to initiate appropriate antiviral therapy and preventive measures. We analysed the differences in clinical presentation and laboratory parameters between emergency department patients with PCR-confirmed H1N1 influenza infection (n = 199) and those with PCR-negative influenza-like illness (ILI; n = 252). Cough, wheezing, leucopenia, eosinopenia and a lower C-reactive protein remained significant predictors of H1N1 influenza. Proposed combinations of clinical symptoms with simple laboratory parameters (e.g. reported or measured fever and either cough or leucocytes <8.5 × 10(9) /L) were clearly superior to currently used official ILI case definitions that use clinical criteria alone., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Accurate quantitation of hepatitis C virus (HCV) RNA is mandatory for the management of anti-HCV therapy., Objectives: The genotype-dependent performance of the new commercially available VERSANT HCV RNA 1.0 Assay (kPCR) and the COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test, version 2.0 was investigated., Study Design: The molecular assays for quantitation of HCV RNA were performed according to the manufacturer's package insert instructions. HCV genotypes/subtypes/isolates, and mutations in the 5'NCR were detected by direct sequencing., Results: When members of a worldwide HCV performance panel including HCV subtypes 1a, 1b, 2a, 3b, and 4a were tested with the Siemens assay and the results were compared with those obtained by the Roche assay, the mean log10 unit differences for members containing HCV subtypes 1a, 1b, 3b, and 4a were found to be within ±0.5 log(10) units. For the panel member containing HCV subtype 2a, the HCV RNA concentration was found to be >0.5 log(10) units lower with the Siemens assay. When clinical samples were tested, the HCV RNA concentration of all samples containing HCV subtype 2a were found to be >0.5 log(10) units lower with the Siemens assay while that of certain HCV subtype 3a and 4a isolates were found to be >1.0 log(10) units lower., Conclusion: The VERSANT HCV RNA 1.0 Assay substantially underestimates HCV RNA concentrations in HCV subtype 2a samples and in HCV subtype 3a and 4a samples containing certain isolates. This may be caused by mismatches with the target sequences due to the primer and/or probe design., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this small study, the LightCycler® SeptiFast, and the SepsiTest™ were compared with blood culture. The SeptiFast showed a higher sensitivity (42.9%) and specificity (88.2%) when compared to blood culture than the SepsiTest™ (28.6 and 85.3%). The SeptiFast provides more species specific results, although the identification panel is smaller., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background. Adherence to hygiene guidelines is of utmost importance for healthcare professionals. The aim of this study was to evaluate the knowledge on and the adherence to hygiene guidelines among medical students in Austria. Additionally, a possible difference between female and male students was investigated. Methods. An open paper-based survey among third-year medical students at the Medical University of Graz was conducted. The questionnaire consisted of 20 single-choice questions covering compliance with basic hygiene standards, self-rated knowledge of hygiene guidelines, and satisfaction with current hygiene education, equipment, and quality standards. Results. Of 192 medical students, 70% judged their knowledge of hygiene standards as "excellent" or "good"; however, only 49% reported adherence to hygiene guidelines and only 43% performed hygienic hand disinfection according to WHO guidelines. Of the respondents, 79% voted for a mandatory course on hygiene standards in medical education. No significant gender differences were observed. Conclusion. While the knowledge on hygiene guidelines appears to be good among medical students, adherence is limited and requires improvement. The need for an optimum education in hygiene is high.

Nocardiosis is an infrequent but serious pulmonary infection caused by Gram-positive aerobic actinomycetes. In this paper, we report on a 48-year-old patient with pleuropulmonary nocardiosis and cirrhosis due to chronic hepatitis C virus infection treated with triple antiviral treatment complicated by prolonged neutropenia.

Background: Nucleic acid extraction has a major impact on the reliability of results in routine molecular diagnostics. Optimal isolation of nucleic acids and removal of inhibitors are essential., Objectives: This study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples., Study Design: Norovirus positive stool samples and CMV positive plasma samples were aliquoted and analyzed using five different automated platforms (easyMAG, bioMerieux; m2000sp, Abbott; MagNA Pure LC 2.0, Roche; QiaSymphony, Qiagen; sample preparation module of the VERSANT kPCR Molecular System, Siemens). Similar sample input and output volumes, the identical real-time PCR cycler, and the identical assays for amplification and detection for norovirus RNA and CMV DNA, respectively, were chosen., Results: Of 39 stool samples, 36 tested positive for norovirus RNA with all extraction platforms. The three discrepant samples showed inhibition after extraction with at least one platform. Only with the VERSANT platform all samples tested positive for both the target RNA and the internal controls. Of 42 plasma samples, 27 gave quantifiable results for CMV DNA with all extraction platforms. There was significant variance between viral concentrations when different extraction platforms were compared. The majority of the 15 discrepant samples showed low viral concentrations. The internal control of the CMV assay gave positive results for all samples tested below the limit of quantification., Conclusions: The five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: In hemodialysis (HD) patients, a decrease of serum HCV RNA concentration during HD has been reported., Objectives: To evaluate the effect of two different extracorporeal blood treatments, HD and hemodiafiltration (HDF), on HCV RNA concentration under standardized conditions., Study Design: Eleven chronic HD patients with chronic hepatitis C (CHC) and thirty-three non-uremic patients with CHC as controls were studied. Blood samples were collected at baseline (t=0 min), 30 min (t=30), and 180 min (t=180) after start of HD or HDF. HCV RNA concentrations were determined by a real-time PCR assay. Values obtained 30 min and 180 min after start of HD or HDF were adjusted according to the ultrafiltration-induced hemoconcentration., Results: Baseline HCV RNA concentrations were found to be similar in dialysis patients and controls (2.9E+06 vs. 5.8E+06 IU/ml). After adjustment for hemoconcentration, no significant differences of HCV RNA concentrations were observed when HD versus HDF treatments and blood samples collected pre versus those collected post membrane were compared. Adjusted HCV RNA concentrations increased by 13% (not significant) at 30 min and by 56% (p<0.001) at 180 min after start of HD or HDF. Inhibitory effects on PCR through heparin and uremic toxins could be excluded., Conclusions: In contrast to recent publications, a significant increase of serum HCV RNA within 180 min after start of HD or HDF was observed. Changes in serum HCV RNA concentration are independent from HD and HDF procedures, dialysis membrane, heparin concentration, and uremic toxins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Molecular diagnostics has become one of the dominant platforms in clinical laboratory medicine. Technological improvements, from automated sample preparation to real time amplification technology, provide the possibility to develop and run assays for a growing number of clinical questions. However, quality assurance and quality control issues have often remained underdeveloped but are still critical. To relate patient results to prior results or to absolute values in clinical practice guidelines, those results need to be comparable across time and methods. This may be achieved either by producing the identical value across methods and test versions or by using reliable and stable conversions. The establishment of international standards and reference materials is thus of paramount importance. This review focuses on general and specific issues relevant for quality assurance and quality control in the routine molecular diagnostics laboratory.

Genotypic HIV-1 drug resistance testing with standard Sanger sequencing is limited to the detection of mutations with >20% prevalence. A new protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer (Roche 454 Life Sciences, Branford, CT) was evaluated. The new technology was compared with the standard Sanger sequencing method. For accuracy testing, genotype B samples obtained from proficiency panels were examined with ultra-deep sequencing. Reproducibility was determined by repeat GS-FLX sequencing of 21 clinical samples. Clinical performance was evaluated with 44 samples and the results were compared to the TRUGENE HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics, Tarrytown, NY). Sequences generated with both protocols were analyzed using the Stanford University HIV drug resistance database. When accuracy was tested, 316 of 317 mutation codons included in the analysis of proficiency panels could be identified correctly with ultra-deep sequencing. Reproducibility testing resulted in a correlation value of R(2)=0.969. Analysis of 44 routine clinical samples with the Stanford University HIV drug resistance database revealed a total number of 269 and 171 mutations by the ultra-deep and standard Sanger sequencing, respectively. Drug resistance interpretations showed differences for 11 samples. With ultra-deep sequencing, total time to result was four times longer in comparison to standard Sanger sequencing. Manual work was increased significantly using the new protocol. The ultra-deep sequencing protocol showed good accuracy and reproducibility. However, automation and shorter time to obtain results are essential for use in the routine diagnostic laboratory., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH). We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation. FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes. Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia. General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix), Firmicutes (LGC354 A-C; hereafter LGCmix), and Bacteroidetes (Bac303). In addition, specific probes binding to Streptococcus mutans (MUT590) and Porphyromonas gingivalis (POGI) were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle. Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

Background & Aims: Polymorphisms of the IL28B gene (rs12979860 and rs8099917) are associated with high sustained virological response (SVR) rates in HCV genotype 1 patients. This study analyzes the impact of these IL28B polymorphisms on early treatment response (weeks 2 and 4) and SVR in HCV genotype 3 patients., Methods: rs12979860 and rs8099917 were analyzed by the Step-OnePlus Real-time PCR system in 71 out of 72 Caucasian HCV genotype 3 patients participating, at our center, in a randomized study comparing 400mg with 800 mg ribavirin/day. HCV RNA was determined at weeks 2 and 4 of 180 μg/week peginterferon alfa-2a/ribavirin treatment. Sixty-nine patients completed the treatment and follow-up., Results: rs12979860 genotyping revealed that 27 (37.5%) patients had C/C, 39 (54.2%) T/C, and 5 (6.9%) T/T. Thirteen patients (18.1%) became HCV RNA negative at week 2 and an additional 30 (41.7%) at week 4 (rapid virologic response; RVR); thus a total of 43 had a RVR (C/C: 77.8%; T/C or T/T: 50.0%). Irrespective of the ribavirin dose, the viral load decline was larger than in those with the T allele (T/C or T/T) (week 2: 4.46; [0.36-6.02] median; [range] vs. 3.50; [0.14-5.62]; log IU HCV-RNA/ml; p<0.001; week 4: 4.97; [1.21-6.20] vs. 4.49; [1.16-6.23]; p=0.003). Despite the faster initial viral response in C/C carriers, SVR rates were not different compared to T-allele carriers. Results of the SNP in the rs8099917 region were similar., Conclusions: IL28B polymorphisms modulate early virologic response to peginterferon/ribavirin treatment. In contrast to HCV genotype 1 patients, no effect on SVR rates was observed in genotype 3 patients. The clinical relevance of an earlier viral decline in C/C patients needs to be determined., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Single nucleotide polymorphisms (SNPs) in the gene that encodes interleukin (IL)-28B predict response of patients with chronic hepatitis C to antiviral therapy. We investigated the roles of polymorphisms rs12979860 and rs8099917 on the early virologic response of treatment-naïve patients., Methods: SNPs were identified by real-time polymerase chain reaction analysis of samples from 682 patients (genotype [GT]1=372, GT2/3=208, GT4=102) who were treated with 180 μg pegylated interferon-α2a and 400 or 800 mg (GT2/3, depending on the protocol) or 1000-1200 mg (GT1/4) ribavirin/day. The duration of treatment was 24 (GT2/3) or 24-72 weeks (GT1/4)., Results: Patients with C/C also had higher rates of rapid virologic response (RVR) (GT1, 38.3% vs 11.6%; GT4, 76.5% vs 23.5%; both P<.001) and sustained virologic responses (SVRs) (GT1, 79.1% vs 43.2%; GT4, 85.3% vs 44.1%; both P<.001). In patients with GT2/3, the RVR was more frequent in carriers of C/C (75.3% vs 52.6%, P<.01), but SVR rates were similar between those with C/C and T (80.5% vs 74.4%, P=.31). Results for rs8099917 were comparable. The positive predictive value of rs12979860 C/C for SVR was higher than that of rs8099917 T/T (80.5% vs 71.6%). Overall, RVR was the best predictor of SVR. In patients who did not have GT1, IL28B polymorphisms did not affect the SVR if RVR data were included in the multivariate analysis., Conclusions: An early virologic response to pegylated interferon and ribavirin is more likely among carriers of rs12979860 C/C and rs8099917 T/T, which might underlie their high rates of SVR. Determination of the IL28B genotype and whether patients have an RVR might be used in future studies of patients with hepatitis C virus genotype 1 or 4., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In routine molecular diagnostics, detection of herpesviruses has made a major impact. Infection with herpesviruses is indicated by demonstrating the presence of the virus in selected specimens. Rapid and reliable detection of herpesvirus DNA helps to decrease the lethality as well as the sequelae of herpesvirus infection in patients at risk. This chapter discusses specimen types and both laboratory-developed and commercially available assays useful for molecular detection of herpesviruses. To meet the need for reliable laboratory results, it is advisable to employ maximum automated and standardized kits based on reagents and standards of reproducible high quality. In the routine diagnostic laboratory, introduction of IVD/CE and/or FDA-labeled tests is preferred.

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) are associated with various infectious pathogens. We analyzed the presence of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis DNA in 47 nongastrointestinal and 14 gastrointestinal MALT lymphomas, 37 nonmalignant control samples, and 27 autoimmune precursor lesions by polymerase chain reaction amplification and direct sequencing. In 47 nongastrointestinal MALT lymphomas, 13 (28%) were positive for C psittaci DNA compared with 4 (11%) of 37 nonmalignant control samples (P = .09). C psittaci was detected at variable frequencies in MALT lymphomas of different sites: lung, 100% (5/5; P < .01); thyroid gland, 30% (3/10; P > .05); salivary gland, 13% (2/15; P > .05); ocular adnexa, 15% (2/13); and skin, 25% (1/4). Of 27 autoimmune precursor lesions (11 Hashimoto thyroiditis and 16 Sjögren syndrome), 11 (41%) contained C psittaci DNA. Only 1 (7%) of 14 gastrointestinal MALT lymphomas was positive for C psittaci. All specimens were negative for C trachomatis and C pneumoniae. Besides ocular adnexal lymphomas, C psittaci infection is associated with nongastrointestinal MALT lymphomas and autoimmune precursor lesions, suggesting possible involvement of C psittaci-induced antigenic-driven MALT lymphomagenesis.

Quantitation of cytomegalovirus (CMV) DNA in whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical performance of the artus™ CMV RG PCR kit in conjunction with automated sample preparation on the new QIAsymphony™ SP instrument was evaluated. Clinically referred samples were tested and results compared to those obtained with the routinely used molecular test system. Accuracy testing showed results to be within ±0.2 log(10) unit of the expected panel results. The assay was linear (R = 0.9972) from a lower quantification limit of 148 copies/ml to 1.3 × 10(7) copies/ml. The between-day imprecision CV was 8-63%, and the within-run imprecision CV was 16-61%. When 100 clinically referred samples were tested, results obtained with the new test system showed an acceptable concordance with those obtained with the routinely used easyMAG™ sample preparation and CMV HHV6,7,8 R-gene™ test system. Discrepant results were found with low-titer samples containing CMV DNA concentrations under the lower limit of quantification or within half a log unit above. The time to results was comparable for both test systems. The QIAsymphony™ sample preparation and artus™ CMV RG PCR test system allows for a rapid, sensitive, precise, and accurate high-throughput quantitation of CMV DNA in whole blood in the routine diagnostic laboratory.

Background: Detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA have gained significance because of their widespread community transmission., Objective: To study the accuracy and the performance of four molecular assays for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA in the routine diagnostic laboratory., Study Design: The accuracy of the molecular assays was determined with reference material. For evaluation of the performance, 104 clinical specimens were studied. Sample preparation was done on a fully automated extraction instrument. For amplification and detection of influenza RNA, all molecular assays evaluated were based on real-time PCR., Results: When the accuracy was tested, the majority of assays yielded results as expected. When clinical samples were analyzed, 94 samples gave concordant results with all assays. One of the assays showed one false-negative result and another assay 10 false-negatives., Conclusion: The majority of assays evaluated in this study proved suitable for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA. All assays are easy to handle and provide results rapidly., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The TRUGENE HIV-1 Genotyping Kit for HIV-1 drug resistance testing is limited by the need for samples with HIV-1 RNA loads of at least 1000 copies/mL. In order to enable sequencing of clinical samples with viral loads under 1000 copies/mL, an optimized automated sample preparation protocol on the VERSANT kPCR Sample Preparation (SP) Module was evaluated. In order to prove the concept of successful sequencing of low-titer clinical samples with the optimized protocol, a dilution series of a routine clinical sample was analyzed. Furthermore, 57 routine clinical samples with viral loads below 1000 HIV-1 RNA copies/mL were tested. Finally, samples obtained from two patients with low viral loads were tested retrospectively for HIV-1 drug resistances and results were compared with those of the preceding and the subsequent sample. The dilution containing 92 HIV-1 RNA copies/mL was the lowest yielding an analyzable sequence with the optimized protocol. When routine clinical samples with viral loads between 100 and 1000 HIV-1 RNA copies/mL were tested, a sequence was obtained in 90.5%. Samples with low viral load of two patients that could not be analyzed with the routine protocol showed identical drug mutations in both, the low viral-load and the subsequent samples. Together with the optimized automated sample preparation protocol, the TRUGENE HIV-1 Genotyping Kit allows successful sequencing of the majority of samples with HIV-1 RNA loads between 100 and 1000 copies/mL. Detection of resistance mutations in low viral-load samples may lead to an earlier optimized antiretroviral therapy., (Copyright 2010 Elsevier B.V. All rights reserved.)

Idiopathic peripheral facial nerve palsy has been associated with the reactivation of herpes simplex virus type 1 (HSV-1) or varicella zoster virus (VZV). In recent studies, detection rates were found to vary strongly which may be caused by the use of different oral fluid collection devices in combination with molecular assays lacking standardization. In this single-center pilot study, liquid phase-based and absorption-based oral fluid collection was compared. Samples were collected with both systems from 10 patients with acute idiopathic peripheral facial nerve palsy, 10 with herpes labialis or with Ramsay Hunt syndrome, and 10 healthy controls. Commercially available IVD/CE-labeled molecular assays based on fully automated DNA extraction and real-time PCR were employed. With the liquid phase-based oral fluid collection system, three patients with idiopathic peripheral facial nerve palsy tested positive for HSV-1 DNA and another two tested positive for VZV DNA. All patients with herpes labialis tested positive for HSV-1 DNA and all patients with Ramsay Hunt syndrome tested positive for VZV DNA. With the absorption-based oral fluid collection system, detections rates and viral loads were found to be significantly lower when compared to those obtained with the liquid phase-based collection system. Collection of oral fluid with a liquid phase-based system and the use of automated and standardized molecular methods allow early and reliable detection of HSV-1 and VZV DNAs in patients with acute idiopathic peripheral facial nerve palsy and may provide a valuable decision support regarding start of antiviral treatment at the first clinical visit.

Background: Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples has gained significance in the routine diagnostic laboratory., Methods: In this study, the analytical and clinical performance of the artus EBV RG PCR kit in conjunction with automated sample preparation on the QIAsymphony SP instrument was evaluated., Results: When the accuracy of the new test system was tested, all results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity showed a quasilinear curve over 4 log units. The lower limit of detection was determined to be 391 EBV DNA copies/mL in EDTA whole blood. The between day imprecision ranged from 18% to 66%, and the within run imprecision ranged from 11% to 50%. When clinical samples were tested and the results compared with those obtained with the routinely used easyMAG sample preparation and EBV R-gene test system, 60 samples tested positive and 31 samples tested negative by both assays. Nineteen samples were found to be positive using the QIAsymphony sample preparation and artus EBV RG PCR test system only, and no samples tested positive with the routinely used test system only., Conclusions: The QIAsymphony sample preparation and artus EBV RG PCR test system is suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory.

Background: The VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of HIV-1 RNA has recently been introduced., Objectives: In this study, the performance of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0., Study Design: Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 196 routine clinical samples including a high number of HIV-1 subtype non-B samples were investigated., Results: When accuracy of the new kit was tested, all of the quantifiable results were found to be within -0.5log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve up to the initial concentration of 3.4x10(5)copies/mL. The interassay variation ranged from 12 to 20%, and the intra-assay variation ranged from 8 to 16%. When clinical samples were tested by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, the results for 95% of all samples with positive results by both tests were found to be within +/-1.0log(10) unit. The viral loads for all samples measured by the Siemens and Roche assays showed a high correlation (R(2)=0.94); quantitative results obtained by the Siemens assay were usually found to be lower than those obtained by the Roche assay., Conclusions: The new VERSANT HIV-1 RNA 1.0 Assay (kPCR) proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.

Hepatitis C virus (HCV) infection represents a substantial risk to both, health care workers and patients. It is of major importance to detect health care workers with HCV infection and to establish regulations how to deal with infected individuals working in specific health care settings. Currently, there are no consistent recommendations, regulations or guidelines concerning prevention of health care worker-to-patient transmission of HCV. Questions arising include: Should health care workers be screened or tested individually on HCV infection and what kind of assay(s) should be used? When and how often should health care workers be tested? How should health care workers with HCV infection be managed? Based on these questions, this article reviews the most relevant published literature. Furthermore, suggestions for establishing a future common regulatory framework are provided.

The early diagnosis of human cytomegalovirus (CMV) infection in immunosuppressed patients has been improved by molecular detection of CMV DNA. In this study, corresponding EDTA whole blood and EDTA plasma specimens were obtained from 42 bone marrow transplant recipients. For detection of CMV DNA, two commercially available assays, the CMV HHV6,7,8 R-gene (ARGENE), and the artus CMV LC PCR Kit (QIAGEN), were employed. The linearity of both assays was determined by using a clinical EDTA whole blood sample with high CMV DNA load. With the CMV HHV6,7,8 R-gene test, CMV DNA was detected in 40 EDTA whole blood and in 19 EDTA plasma samples, while the artus CMV LC PCR Kit test detected CMV DNA in 27 EDTA whole blood and in 30 EDTA plasma samples. In conclusion, EDTA whole blood samples were found to be the superior material when using the CMV HHV6,7,8 R-gene test. However, this benefit may not exist when employing alternative assays.

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.

Objective: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA)., Study Design: A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits., Results: In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used., Conclusion: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

The presence of Chlamydia (C.) psittaci, C. pneumoniae and C. trachomatis DNAs in MALT lymphomas of ocular adnexals from 13 Austrian patients were studied. Gastrointestinal MALT lymphomas and gastritis specimens served as controls. Of 13 MALT lymphomas of the ocular adnexals, seven were positive for C. psittaci DNA. In contrast, one of 17 gastrointestinal specimens tested positive. All specimens were negative for C. trachomatis and C. pneumoniae DNAs. In conclusion, C. psittaci infection was observed in the majority of MALT lymphomas of ocular adnexals in Austrian patients.

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.

Background: Standardization in collection and testing of oral fluid is lacking., Methods: A novel standardized collection and quantification system for oral fluid testing was evaluated. Sample collection volumes were determined. For determination of the saliva content in oral fluid samples, tartrazine, which is an integral part of the saliva extraction solution serving as an internal standard, was used. Results were compared with those obtained by employing glucose as a 'supplemental internal standard' for determination. Analytical performance of the new system for collection and quantification of oral fluid was evaluated. Calcium and magnesium analyte concentrations in oral fluid samples were measured with a routine laboratory method and compared to a reference method., Results: Volumes of oral fluid samples collected ranged from 4.9 to 10.5 mL. The mean saliva content in oral fluid samples was found to be 65.5 percent by volume (vol.-%) when determined through the tartrazine concentration and 65.0 vol.-% when determined through the glucose concentration. Evaluation of analytical performance revealed interassay imprecision coefficients of variation (CVs) ranging from 1.5% to 3.2% and intraassay imprecision CVs from 1.1% to 2.5%. When linearity was tested, a quasi-linear curve was observed (R2=0.99). Comparison of two different methods for determination of calcium and magnesium concentrations showed correlations of R2=0.96 for calcium and R2=0.97 for magnesium., Conclusions: The new system for collection and quantification of oral fluid helps to improve standardization of pre-analytics in oral fluid testing and to provide reliable and accurate quantification of analytes in oral fluid samples.

Background: There is currently no consensus about the significance of therapeutic drug monitoring (TDM) in the management of human immunodeficiency virus (HIV) infection., Objectives: In this study, single drug levels from routine clinical samples were determined in order to add further data on the therapeutic plasma level range and on drug levels under anti-HIV therapy in general., Materials and Methods: A total of 215 plasma samples obtained from patients with HIV infection for whom TDM was requested for the first time were evaluated. Plasma values were determined for abacavir and zidovudine between 1 and 3 h after drug intake. Lamivudine, efavirenz, nevirapine, lopinavir and nelfinavir plasma values were determined between 1 and 12 h after drug intake. Drug levels were determined with a home-brewed assay based on high-performance liquid chromatography., Results: Plasma concentrations of HIV drugs showed a large variability. Plasma concentrations of abacavir, zidovudine and lamivudine were found to be similar to those reported recently. For efavirenz and nevirapine, 37 and 61% of the samples did not meet the minimum trough levels suggested. For lopinavir and nelfinavir, the majority of the samples (78 and 80%) were found to be within the therapeutic plasma level range., Conclusion: Despite a large variation of anti-HIV drug plasma concentrations, single-measurement TDM may be sufficient in the routine management of the majority of patients., (Copyright 2008 S. Karger AG, Basel.)

Currently available anti-hepatitis C virus (HCV) therapy is effective in only half of infected patients and is limited by adverse effects that often necessitate discontinuation. Therefore, new treatments are being developed, including optimization of current standard treatment with peginterferon plus ribavirin, specifically targeted antiviral therapy for HCV, novel immunomodulatory agents and treatments aimed at reducing fibrosis. This review focuses on novel anti-HCV drugs that are currently in an advanced stage of clinical development. Albinterferon-alpha-2b, a fusion molecule of albumin and interferon-alpha-2b, has a longer half-life than peginterferon, which enables a bi-weekly administration interval. Preliminary data indicate similar response rates for albinterferon-alpha-2b plus ribavirin compared with peginterferon-alpha-2b plus ribavirin, but possible benefits with respect to quality of life. Telaprevir, a NS3/4 protease inhibitor, demonstrated a rapid and profound antiviral effect in phase I trials that was synergistic with that of peginterferon-alpha-2a. Recently completed phase II trials on triple combination treatment with telaprevir, peginterferon-alpha-2a and ribavirin given for 12-24 weeks reported sustained virological response in up to 68% of patients with treatment-naive HCV genotype 1 infection.

This review summarizes major issues of verification and validation procedures and describes minimum requirements for verification and validation of diagnostic assays in clinical virology including instructions for CE/IVD-labeled as well as for self-developed ("home-brewed") tests or test systems. It covers techniques useful for detection of virus specific antibodies, for detection of viral antigens, for detection of viral nucleic acids, and for isolation of viruses on cell cultures in the routine virology laboratory.

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/mul, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/mul, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.

Background: The Abbott RealTime HCV assay for quantitative detection of HCV RNA has recently been introduced., Objectives: In this study, the performance of the Abbott RealTime HCV assay was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HCV test., Study Design: Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 243 routine clinical samples were investigated., Results: When accuracy of the new assay was tested, the majority of results were found to be within +/-0.5 log(10) unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve up to 1.0 x 10(6)IU/ml. The interassay variation ranged from 15% to 32%, and the intra-assay variation ranged from 5% to 8%. When clinical samples were tested by the Abbott RealTime HCV assay and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test, the results for 93% of all samples with positive results by both tests were found to be within +/-1.0 log(10) unit. The viral loads for all patients measured by the Abbott and Roche assays showed a high correlation (R(2)=0.93); quantitative results obtained by the Abbott assay were found to be lower than those obtained by the Roche assay., Conclusions: The Abbott RealTime HCV assay proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.

Background: Hepatitis C virus (HCV) genotyping and accurate subtyping is becoming increasingly relevant to epidemiological studies, clinical management, pathogenicity, and vaccine development., Methods: The TRUGENE HCV 5'NC Genotyping Kit, the new VERSANT HCV Genotype 2.0 Assay (LiPA), and a new laboratory-developed HCV NS5b sequencing assay designed for automated sequencing of the HCV NS5b region were used. Clinical samples and a molecular diagnostics HCV genotyping proficiency program panel were used to determine accuracy and differentiate performance characteristics of the three methods., Results: All amplified samples from among the members of a HCV genotyping proficiency program panel that contained a single HCV genotype were subtyped correctly using all three HCV genotyping assays. With the TRUGENE HCV 5'NC Genotyping Kit, the HCV subtype was determined in 357 of 441 of routine clinical samples. When the 84 samples with only genotype results were retested with the VERSANT HCV Genotype 2.0 Assay (LiPA), 61 could be further subtyped accurately. With the new laboratory-developed HCV NS5b sequencing assay, all 84 could be subtyped accurately., Conclusions: The two new methods show advantages over the routinely used TRUGENE HCV 5'NC Genotyping Kit in terms of genotyping and subtyping accuracy by utilizing part of the HCV core region and NS5b region, respectively.

Background: Analysis of HIV-1 subtypes and genotypic resistance have been shown to be relevant for epidemiologic and therapeutic studies or for vaccine development. In Europe, the majority of HIV-1 isolates belong to subtype B. Due to migration an increasing incidence for additional subtypes and complex recombinant forms are expected., Objectives and Study Design: To evaluate the prevalence of HIV-1 subtypes in Austria, 188 plasma samples of treatment experienced patients were investigated. For phylogenetic analysis protease and reverse transcriptase genes were amplified and sequenced. Subtypes were determined by comparing reference sequences. For genotypic resistance determination, the Resistance-Algorithm-Comparison from Stanford University was used., Results: Non-B subtypes were found in 20.2% of all patients with a dominant prevalence (50%) in the Southern provinces of Austria. With 85% CRF01&#95;AE and CRF02&#95;AG are the predominant circulating recombinant forms in Austria. When resistance mutations were analyzed, 57.4% of all patients were susceptible to all three groups of antiretroviral drugs, whereas in 12.2% resistance against all three classes of antiretroviral drugs was found., Conclusion: HIV-1 subtype B is still dominant in major parts of Austria. However, a significantly increasing percentage of non-B subtypes and recombinant forms are observed in the Southern provinces.

In routine molecular diagnostics, real-time PCR has made a major impact because of the faster time to the result; decreased hands-on time; and virtual elimination of the major issue in the early days of PCR, sample contamination from previously amplified DNA. The shorter time to the result for a real-time PCR assay means that a new diagnostic application may be developed relatively quickly. This chapter discusses the fluorescent chemistries typically used in molecular diagnostic applications and the additional features, such as internal controls, which are highly desirable in these assays. After an assay has been developed, the assay's performance must also be evaluated before the assay can be implemented in the testing laboratory.