Development and validation of a fully automated laboratory-developed test for detection of HSV-1/2 and VZV in clinical samples run on the Panther Fusion® system.

The purpose of this study was to develop a laboratory developed test (LDT) for HSV1/2 and VZV to run on fully automated Hologic Panther Fusion® System. The Panther Fusion System is a fully automated walkaway system, providing end-to-end workflow from sample input to DNA/RNA extraction, amplification, automated analysis, and reporting to a laboratory information system (LIS). The LDT was developed and validated on 230 clinical and 20 reference samples (n = 250) and compared to a commercially available kit. Assessment of the analytical and diagnostic performances of the LDT revealed >98% accuracy, sensitivity, and specificity, which is consistent with or better than many of the commercial or laboratory-developed tests available. The advantage of this LDT is that it is designed to perform a single-run full female health screening in parallel with 4 commercially available Hologic kits for Chlamydia trachomatis/Neisseria gonorrhea (CT/NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), and bacterial vaginosis (BV).

Measuring human immunodeficiency virus type 1 RNA loads in dried blood spot specimens using NucliSENS EasyQ HIV-1 v2.0.

BACKGROUND: HIV-1 RNA plasma level is a key parameter for anti-viral treatment monitoring in HIV-1 infected individuals. Plasma stability and accurate measurement of clinical state is at risk when transporting from remote areas. Dried blood spot (DBS) testing can reduce this risk. OBJECTIVES: Determine the performance of NucliSENS EasyQ HIV-1 v2.0 for DBS. STUDY DESIGN: 100 HIV-1 negative, and 129 HIV-1 spiked blood specimens (2180 copies/ml) were used for diagnostic specificity and system robustness. Analytical performance was tested in the range 50-85,000,000 copies/ml. Clinical reactivity was measured with specimens obtained from 224 HIV-1 infected individuals. HIV-1 RNA stability was analyzed after applying several different storage conditions. RESULTS: Diagnostic specificity was 100% and system robustness was demonstrated by 100% detection rate without invalids. Limit of detection (95% detection rate) was 800 copies/ml. Linear results were obtained over the whole range tested. For clinical specimens, percentage positive results were comparable for DBS (57%) and plasma (58%). DBS quantification was on average 0.36log10 lower as compared to plasma. Specimen stability was demonstrated for 1 week at 55 degrees C/60% humidity, 3 weeks at 37 degrees C/80% humidity, 9 weeks at 37 degrees C/40% humidity, 3 months at -20 degrees C/70% humidity, 3 weeks at 4 degrees C/100% humidity, 9 months at room temperature (15-30 degrees C), and 9 weeks shipment simulation. CONCLUSION: Results obtained fully support the use of DBS for the NucliSENS EasyQ HIV-1 v2.0 assay. These findings are especially of importance in cases that plasma stability is currently at risk due to for example, long transport routes from remote areas under less controlled conditions.

NucliSENS EasyQ HPV v1 test - Testing for oncogenic activity of human papillomaviruses.

BACKGROUND: Analytical sensitivity of DNA-based assays to detect infection with human papillomaviruses is very high, but clinical specificity for cervical cancer strongly depends on the age of the patient and case classification. To solve the dilemma between sensitivity and specificity, a new generation of assays focuses on the pathogenic factors that underlie the development of HPV-associated tumors: the expression of the viral oncogenes E6 and E7. Demonstration of persistent expression of these mRNAs or expression in the context of relevant clinical symptoms has a strong positive predictive value for the development of HPV-associated carcinomas and strongly warrants further diagnostic action. OBJECTIVES: The NucliSENS EasyQ HPV v1 test was designed to test cervical scrapes for the expression of the oncogenic E6/E7 mRNA from the five most common carcinogenic HPV types (16, 18, 31, 33 and 45). The test can be used for confirmation and risk stratification of individuals with proven infection with high risk papillomaviruses. STUDY DESIGN: In order to establish performance of the assay, sensitivity, specificity, repeatability, and reproducibility were determined with artificial and clinical specimens. Further, a total of 420 cervical scrapes were tested and the results directly compared to the CE-market device PreTect HPV-Proofer assay (NorChip, Klokkarstua, Norway). For arbitration of discordant clinical results, the specimens were rated according to Pap-classification and the presence of HPV DNA was determined. RESULTS: The limit of detection for the five HPV types 16, 18, 31, 33, and 45 ranged from 2.3x10(2) to 3.0x10(4) copies/mL on a background of 5x10(3) HPV-negative HS67 cells. No cross-reactivity for other viral, bacterial, or fungal agents known to be potentially present in cervical fluids was detected. Repeatability and reproducibility were shown by testing panels of HPV-spiked artificial and clinical samples. A comparative analysis of 420 cervical scrapes demonstrated an overall concordance of >90% between the NucliSENS EasyQ HPV test and the technologically related PreTect HPV-Proofer assay.

Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons.

For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay.