Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material

Predictors for non- and slow progression in human immunodeficiency virus (HIV) type 1 infection: low viral RNA copy numbers in serum and maintenance of high HIV-1 p24-specific but not V3-specific antibody levels

To gain insight into determinants that define the duration of the asymptomatic period preceding AIDS, groups of long-term asymptomatic (LTA) person (> 7 years of follow-up) and slow and rapid progressors of human immunodeficiency virus infection were studied. LTAs had no clinical manifestations of AIDS or immunologic abnormalities in 7 years of follow-up. RNA copy numbers, gag- and env-specific, and neutralizing antibody titers in serum were determined 1 and 5 years after seroconversion or entry into the cohort. Early in infection, before immunologic markers or clinical manifestations allowed group discrimination, subjects who were later classified as LTAs had significantly less serum viral RNA than progressors. No significant increase in virus load was found in progressors, indicating that the initial load defines clinical outcome. In slow progressors, high virus load was associated with high p24-specific antibody titers, suggesting that delay of clinical manifestations of AIDS may be related to the presence of high levels of p24-specific but not V3-specific antibodie

The value of an artificial neural network in the decision-making for prostate biopsies

Purpose: In majority of patients who are subjected to prostate biopsies, no prostate cancer (PCa) is found. It is important to prevent unnecessary biopsies since serious complications may occur. An artificial neural network (ANN) may be able to predict the risk of the presence of PCa. Methods: Included were all patients, who underwent transrectal ultrasound-guided prostate biopsies between June 2006 and June 2007 with a total PSA (tPSA) level between 2 and 20 μg/l. The patients were divided into two groups according to their tPSA level (2-10 μg/l and 10-20 μg/l). The ANN Prostataclass of the Universitätsklinikum Charité in Berlin was used. The predictions of the ANN were compared to the pathology results of the biopsies. Results: Overall 165 patients were included. PCa was diagnosed in 53 patients, whereas the ANN predicted "no risk" in 19 of these patients (36%). The ANN output receiver operator characteristic (ROC) plots for the range of tPSA 2-10 μg/l and tPSA 10-20 μg/l showed an area under the curve (AUC) of 63 and 88% for the initial biopsy group, versus 69 and 57%, respectively, for the repeat biopsy group. Conclusions: The ANN resulted in a false negative rate of 36%, missing PCa in 19 patients. For use in an outpatient-clinical setting, this ANN is insufficient to predict the risk of presence of PCa reliably. © Springer-Verlag 2009

One-Tube Real-Time Isothermal Amplification Assay To Identify and Distinguish Human Immunodeficiency Virus Type 1 Subtypes A, B, and C and Circulating Recombinant Forms AE and AG

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the “gold standard.” In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies

The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean +/- SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 +/- 0.02. A progressive and significant (P <.0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 +/- 0.1, +1 hour: 1.3 +/- 0.9, +2 hours: 4.1 +/- 0.9), peaking at +3 hours (10 +/- 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge. (Blood. 2000;96:554-559

Plasma concentration of von Willebrand factor predicts mortality in patients on chronic renal replacement therapy

Clinical epidemiolog

A first study on the usability and feasibility of four subtypes of suicidality in emergency mental health care

BACKGROUND: Based on clinical experience, a (hypothetical) four-type model of suicidality that differentiates between subtypes with a unique pathway to entrapment ((h)4ME)was developed. The subtypes are: 1) perceptual disintegration (PD), 2) primary depressive cognition (PDC), 3) psychosocial turmoil (PT) and 4) inadequate communication/coping (IC). This study was carried out to examine the usability and feasibility of the subtypes in an absolute and dimensional way with the SUICIDI-2 instrument. OBJECTIVE: A first step was to examine the model and the SUICIDI-2 instrument for usability and feasibility in clinical practice. We aim to investigate the'real life' practical application of the model and hope the feedback we get after practical use of the model will help us with improvements for the model and the SUICIDI-2 instrument. METHODS: Discharge letters to general practitioners of 25 cases of anonymized suicidal emergency patients were independently reviewed by three psychiatrists and three nurses. Using the SUICIDI-2 instrument, describing the proposed subtypes, cases were classified by the psychiatrists and nurses. Intraclass Correlation Coefficients (ICC) for absolute/discrete and dimensional ratings were calculated to examine the model's usability and the instrument's feasibility. The study was approved by the ethical board. RESULTS: All raters were able to recognize and classify the cases in subtypes. We found an average measure of good reliability for absolute/(discrete) subtypes. For dimensional scores, we found excellent average measures for the subtype PDC, and good average measures for the subtypes PD, PT and IC. The reliability of dimensional score for the SUICIDI-2 was relatively lower than an alternative dimensional rating, but had good ICC values for all subtypes. After reviewing the results though, we found some inconsistently assessment between raters. This was ground to narrow down the criteria per subtype to describe the subtypes more precisely. This resulted in adjusted formulations for subtypes PD and IC and agreement was achieved about formulations in the revised SUICIDI-3. CONCLUSIONS: The hypothetical model of entrapment leading to suicidality shows promising results for both the usability and feasibility of the SUICIDI instrument. Follow up studies with participants with a more diverse background may show consistency and validity for the model