Jörg Berg

Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run

BACKGROUND Human herpes viruses cause a spectrum of diseases that are usually self-limiting but can be reactive during immuno-suppression and may then lead to severe or even life-threatening diseases. The LightCycler technology allows rapid polymerase chain reaction (PCR) including product analysis within a closed system. This approach has been demonstrated to be suitable for routine diagnostic virus detection. Several LightCycler PCR assays have been established to the detection of human herpes viruses. The assays vary in their detection formats and PCR cycling protocols. So, they cannot be performed within a single LightCycler run. OBJECTIVES Development of four LightCycler PCR assays for parallel detection of DNA derived from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) in a single run. STUDY DESIGN Primers and hybridization probes were tailored to suit one LightCycler PCR program. LightCycler PCRs were established, detection limits were determined, and clinical samples were evaluated. RESULTS With quantified herpes virus type specific DNA spiked into cerebrospinal fluid, serum or EDTA plasma the detection limits were found either at 500 or 250 viral DNA copies per ml depending on the virus DNA specific PCR and on the specimen type used. The applicability of the new LightCycler assays for routine molecular testing was evaluated by testing 96 clinical samples. CONCLUSION The developed set of LightCycler PCRs permits parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV in a single LightCycler run. The new molecular assays can easily be used to the rapid, simple, and convenient detection of herpes virus DNA in cerebrospinal fluid, serum and EDTA plasma in the routine diagnostic laboratory.

Single-Run, Parallel Detection of DNA from Three Pneumonia-Producing Bacteria by Real-Time Polymerase Chain Reaction

A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.

Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments

ABSTRACT We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.

Lymphocyte imbalance in vitiligo patients indicated by elevated CD4+/CD8+ T-cell ratio.

ZusammenfassungDie Vitiligo stellt eine Pigmentierungsstörung mit einem assoziierten autoimmun bedingten Verlust von Melanozyten der Epidermis dar. Die Beteiligung humoraler wie auch zellulärer immunologischer Vorgänge wurde untersucht. Wir evaluierten die Rolle proinflammatorischer Cytokine und von Lymphozyten-Subtypen im peripheren Blut bei einer Gruppe österreichischer Patienten mit Vitiligo. Morgens wurden Blutproben von 40 Patienten mit Vitiligo gesammelt. 21 Patienten wiesen eine aktive und 19 eine stabile Krankheitsform auf. Bei allen Patienten bestand eine nicht-segmentale Krankheitsform in unterschiedlicher Ausprägung. 16 Patienten hatten zusätzlich eine autoimmun bedingte Schilddrüsenerkrankung. Um eine Beteiligung proinflammatorischer Cytokine bei Vitiligo zu beurteilen, bestimmten wir sTNF-RI, IL-6 und zusätzlich CIC (zirkulierende Immunkomplexe). Wir verglichen diese Ergebnisse mit den Daten von Normalpersonen. Zur Beurteilung von zellulären Immunvorgängen wurden ein Differentialblutbild und eine Lymphozytentypisierung mittel Durchflusszytometrie erstellt. Die Serum-werte von sTNF-RI, IL-6 und CIC lagen im Normbereich. Der mediane Wert von sTNF-RI in der Patientengruppe betrug 1,5 ng/ml und von CIC 35,2 µg/ml, ein statistisch signifikanter Unterschied zur Kontrollgruppe bestand nicht. Der mediane Wert von IL-6 bei Vitiligopatienten war 2,7 pg/ml und im normalen Bereich gelegen – aber höher als der mediane Wert von 0,5 pg/ml, der bei den Normalpersonen festgestellt wurde (p < 0,001). Absolutund Verhältniswerte der Lymphozytensubtypen waren normal. Das Verhältnis von CD4+/CD8+ T-Zellen wies median einen erhöhten Werte von 2,6 [Quartilabstand 2,0; 3,1] auf. 61 % der Vitiligopatienten hatten einen erhöhten Verhältniswert ober halb des Grenzwertes von 2,4. Bei den meisten Patienten mit Vitiligo erwies sich die Balance von zytotoxischen Suppressorzellen und Helfer T-Zellen im peripheren Blut als gestört, was zu einer Verteilungsstörung der T-Zell-Subtypen am intrakutanen Locus des autoimmunen Melanozytenverlusts führen könnte.SummaryVitiligo is a disorder of pigmentation associated with an autoimmune-mediated loss of melanocytes from the epidermis. Humoral immunity and the involvement of cellular immunity have been investigated in the pathogenesis of vitiligo. We evaluated the role of pro-inflammatory cytokines and lymphocyte fractions in peripheral blood in a cohort of Austrian patients with vitiligo. Morning blood samples from 40 patients with vitiligo were collected. Twenty-one patients had active and 19 had stable vitiligo disease. All patients were suffering from non-segmental vitiligo at different stages of the disease. Sixteen persons presented with an additional autoimmune thyroid disease. To evaluate a possible involvement of proinflammatory cytokines in vitiligo we measured sTNF-RI (soluble tumour necrosis factor receptor I), IL-6 and additionally CIC (circulating immune complexes). We compared these findings to the data from matched normal persons. To investigate the mechanisms of cellular immunity, peripheral blood cell count and lymphocyte subtype analysis by flow cytometry were done. sTNF-RI, IL-6 and CIC serum levels were in the normal range. In the patient group median sTNF-RI level was 1.5 ng/ml and median CIC level was 35.2 µg/ml, and no statistically significant differences to the control group were observed. Median IL-6 level in vitiligo patients was 2.7 pg/ml and in the normal range–but higher than the median level of 0.5 pg/ml observed in normal persons (p < 0.001). Absolute and relative counts of lymphocyte subtypes were normal. The ratio of CD4+/CD8+ T-cells had an elevated median value of 2.6 [quartiles 2.0; 3.1]. 61% of the vitiligo patients had a ratio higher than 2.4, which was the normal cut-off point. In most vitiligo patients the balance of cytotoxic/suppressor and helper/inducer T-cells in peripheral blood is disturbed which might lead to a predominance of T-cell subtypes in the intracutaneous site of autoimmune melanocyte loss.

Automated detection of five human herpes virus DNAs by a set of LightCycler PCRs complemented with a single multiple internal control

BACKGROUND Herpes viruses represent important causes of morbidity and mortality especially in immuno-compromised patients. To assist in rapid diagnosis real-time PCR assays have been developed for the detection of herpes virus DNA in patient specimens. A recently described set of real-time PCR assays using LightCycler technology enabled parallel detection of DNA from cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 and 2 (HSV-1/-2), and varicella-zoster virus (VZV) by using a single LightCycler program [J. Clin. Virol. 26 (2003) 85]. The set of assays lacked automation of DNA purification and of PCR mixture preparation, and was not furnished with measures to monitor for sample adequacy. OBJECTIVES Development of a set of automated LightCycler-PCR assays for the detection CMV-, EBV-, HSV-1/-2- and VZV-DNA in plasma samples and complementation of the assays with internal amplification controls (ICs). STUDY DESIGN The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. A single multiple IC-DNA specific for all four herpes virus type-specific PCRs was generated and used in all four LightCycler assays. Detection limits were determined and clinical samples were evaluated. RESULTS With quantified herpes virus type-specific reference DNA spiked into EDTA plasma, the detection limits were found at 250 copies/ml of CMV-, EBV-, HSV-1/-2-DNA and at 500 copies/ml of VZV-DNA. The novel set of assays was evaluated by testing 112 EDTA plasma samples. The use of the IC led to the detection of PCR-inhibited samples. CONCLUSION The set of automated LightCycler assays was found rapid, markedly labour saving and suitable for the routine diagnostic laboratory. The use of the one internal control molecule simplified the assay protocol and allowed monitoring for sample adequacy.

Genotypic Prediction of Human Immunodeficiency Virus Type 1 Tropism from Plasma and Peripheral Blood Mononuclear Cells in the Clinical Routine Laboratory

ABSTRACT We developed a sequencing assay for genotypic HIV-1 tropism determination. The assay allows examination of HIV RNA from plasma and HIV DNA from peripheral blood mononuclear cells (PBMC), including PBMC samples from patients with undetectable viral loads. Assessment of 100 pairs of plasma and PBMC samples showed a high concordance of 90%. With the limitations of population-based sequencing, the assay was found to be robust and suitable for the routine clinical laboratory.

Entirely Automated Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma by Using the Ultrasensitive COBAS AMPLICOR HIV-1 Monitor Test and RNA Purification on the MagNA Pure LC Instrument

ABSTRACT The ultrasensitive COBAS AMPLICOR HIV-1 Monitor test was complemented with automated RNA purification on the MagNA Pure LC instrument. This enabled entirely automated ultrasensitive assessment of viral loads in human immunodeficiency virus type 1 (HIV-1)-infected individuals. The detection limit of the fully automated assay and the viral load measurements in 80 clinical samples were found to be in good agreement with those of the conventional ultrasensitive COBAS AMPLICOR HIV-1 Monitor test. The fully automated assay showed markedly reduced hands-on time and was found to be suitable for the routine assessment of HIV-1 viral loads in a clinical diagnostic laboratory.

Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

Immunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ.It has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ-chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ-chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.

Genotypic Antiretroviral Resistance Testing for Human Immunodeficiency Virus Type 1 Integrase Inhibitors by Use of the TruGene Sequencing System

ABSTRACT A sequencing assay for detection of mutations conferring resistance to human immunodeficiency virus type 1 (HIV-1) integrase inhibitors raltegravir and elvitegravir was developed using the automated TruGene sequencing system. The assay returned clear sequencing results for samples with ≥500 RNA copies/ml for mutation detection and HIV-1 subtyping across a spectrum of HIV-1 subtypes.

HeLa-LAV, an epithelial cell line stably infected with HIV-1

An HeLa-LAV cell line was established by infecting and subcloning previously described CD4-expressing HeLa cells with HIV-1. Cells of this line stably synthesize all major HIV proteins, release infectious particles of HIV-1, but do not die even after long term culture. More than 90% of the cells express the envelope protein gp120 on the surface. The cells can be easily and efficiently labeled with 51chromium, and exhibit a low spontaneous release. Because they are susceptible to killing by allogeneic cytotoxic T cells (CTL) when targeted to gp120, they ought to be a useful source of target cells in any kind of HIV-specific killing assays. The cells may also help studies on HIV replication in non-lymphatic/non-monocytic cells. The HeLa-LAV cell line will be freely available from the AIDS Research and Reference Reagent Program.