Homozygous deletion of HFE produces a phenotype similar to the HFE p.C282Y/p.C282Y genotype.

Hemochromatosis is predominantly associated with the HFE p.C282Y homozygous genotype, which is carried by approximately 1 person in 200 in Northern European populations. However, p.C282Y homozygosity is often characterized by incomplete penetrance. Here, we describe the case of a woman who had a major structural alteration in the HFE gene. Molecular characterization revealed an Alu-mediated recombination leading to the loss of the entire HFE gene sequence. Although homozygous for the HFE deleted allele, the woman had a phenotype similar to that seen in most women homozygous for the common p.C282Y mutation. Contrasting with previously reported results in Hfe knockout and Hfe knockin mice, our report gives further evidence that progression of the disease depends on modifying factors.

Analytical evaluation of the PapilloCheck test, a new commercial DNA chip for detection and genotyping of human papillomavirus.

Recently, a commercially available HPV DNA chip, the PapilloCheck test, developed by Greiner Bio-One, has become available for human papillomavirus (HPV) genotyping. The PapilloCheck test is a PCR-based test using a new consensus primer set targeting the E1 HPV gene. HPV oligoprobes immobilized on a DNA chip allow for the identification of 24 HPV types from the amplified product. In the present study, the analytical performance of the PapilloCheck test is compared to the Linear Array HPV genotyping test (Roche Diagnostics). Cervical specimens collected in PreservCyt (Cytyc) solution and obtained from women who presented abnormal cytological findings were tested primarily by the Hybrid Capture 2 High-Risk assay (HC2-HR, QIAGEN). A total of 144 samples were selected according to the signal intensity obtained with the HC2-HR test, expressed as RLU/CO value, and divided into 4 groups as follows: [0-1] RLU/CO (negative HC2-HR result, 34 samples); [1-5] RLU/CO (positive HC2-HR result, 30 samples); [5-40] RLU/CO (positive HC2-HR result, 40 samples); >40 RLU/CO (positive HC2-HR result, 40 samples). The concordance levels between the HC2-HR test and each of the genotyping assays was similar (88.8%) and the crude agreement between these assays was considered as "good". The detailed analysis of the discrepant results confirmed a possibly high rate of false positive results of HC2-HR test in the 1-5 RLU/CO grey zone. Genotype-specific comparison analysis was limited to the 23 HPV types detected by both genotyping assays (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 70, 73 and 82). Of the 135 samples available for comparison, 91 (67.4%) showed absolute agreement between the assays (concordant genotype-specific results), 34 (25.1%) showed correspondence for some but not all genotypes detected by both assays (compatible genotype-specific results), and the remaining 10 (7.4%) samples did not show any similarity between the tests (discordant results). The majority of discordances were found in samples containing multiple HPV types and in samples harboring low amounts of HPV. For some HPV genotypes, there were slight differences in the detection rate between the two genotyping methods. The Linear Array test seemed to be more sensitive to detect HPV type 53 whereas PapilloCheck test seemed to be more sensitive to detect HPV type 56. For the other genotypes, including HPV types 16 and 18, the results obtained by the two methods did not differ significantly. In conclusion, this study shows that the PapilloCheck test and the Linear Array test give comparable results for detecting HPV in cervical specimens. However, these results also suggest that there is a need to standardize the type-specific sensitivity of genotyping methods and to evaluate their accuracy to detect multiple HPV infections. This would be a prerequisite for the use of genotyping assays in cervical cancer screening algorithms.

Rapid screening for HLA-B27 by a TaqMan-PCR assay using sequence-specific primers and a minor groove binder probe, a novel type of TaqMan trade mark probe.

HLA-B27 is strongly associated with ankylosing spondylitis (AS). As typing for HLA-B27 is routinely performed by serological methods, false-positive results can be generated. Therefore, several more accurate molecular methods have been developed for HLA-B27 genotyping. We describe a real-time PCR method for the detection of the HLA-B27 allele using sequence-specific primers (SSP) combined with a fluorogenic MGB probe (minor groove binder probe), a novel type of TaqMan probe. The MGB increases the melting temperature (T(m)) of the probe, allowing the use of shorter probes. Moreover, the use of a non-fluorescent quencher (NFQ) attached to the MGB probe improves the efficiency of fluorescence quenching, thus providing a very low fluorescent background. We tested this method on 150 subjects (41 HLA-B27 positive and 109 HLA-B27 negative) who underwent routine HLA-B27 serological testing by flow cytometry (FC). Serology and our TaqMan assay gave identical results in all cases and no false positive or negative results were observed. A graphical representation of fluorescence and normalized reporter signal (DeltaRn) values demonstrated that HLA-B27 positive and HLA-B27 negative samples formed two tight clusters making it possible to clearly differentiate between HLA-B27 positive and negative samples. This single tube PCR method for the detection of HLA-B27 should be particularly suitable for the routine analysis of large numbers of samples in the laboratory.

Evaluation of the COBAS AmpliPrep-total nucleic acid isolation-COBAS TaqMan hepatitis B virus (HBV) quantitative test and comparison to the VERSANT HBV DNA 3.0 assay.

Quantitative detection of hepatitis B virus (HBV) in serum or plasma has become the most direct and reliable method for monitoring chronic hepatitis B. Here, we report the performance characteristics of a real-time PCR hepatitis B DNA quantitative assay, the COBAS TaqMan (CTM) HBV test (Roche Diagnostics, Meylan, France), in combination with an automated DNA extraction on the COBAS AmpliPrep (CAP) instrument using the total nucleic acid isolation kit (TNAI kit), a generic reagent for nucleic acid isolation (both from Roche Diagnostics). The linearity, accuracy, and specificity of the CAP-TNAI-CTM HBV test were evaluated using various reference panels and standards (HBV panel 2004 from Quality Control for Molecular Diagnostics, OptiQuant HBV panel from AcroMetrix, WHO International Standard for HBV, and Teragenix hepatitis B genotype panel). Quantitative results show that the CAP-TNAI-CTM HBV test performed well with respect to linearity, accuracy, and reproducibility from at least 100 to 500,000 HBV DNA IU/ml. Based on the log(10) IU of HBV DNA/ml measured, the intra-assay variation ranged from 2.49% to 8.46% and the interassay variation ranged from 1.88% to 7.83%. The test was extremely sensitive and could detect samples containing HBV DNA below the reported quantification threshold (<30 IU/ml). All HBV genotypes were correctly amplified, and no cross-contamination occurred during the automated sample preparation. In addition, 402 human serum samples were tested comparatively to the VERSANT HBV DNA 3.0 assay (bDNA; Bayer Diagnostics, Puteaux, France). The viral load results of the CAP-TNAI-CTM test and bDNA were significantly correlated, but the agreement between the two tests was poor, with large differences between results for individual samples. The hands-on time was estimated to be reduced from 2.30 h with bDNA to 45 min with the CAP-TNAI-CTM test, and up to 84 samples were completely processed within a working day. Overall, the performance characteristics of the CAP-TNAI-CTM test demonstrated that it provides a high-throughput sensitive and reliable method for quantitation of HBV DNA levels in the routine molecular laboratory.

PreservCyt transport medium used for the ThinPrep Pap test is a suitable medium for detection of Chlamydia trachomatis by the COBAS Amplicor CT/NG test: results of a preliminary study and future implications.

The commercial COBAS Amplicor CT/NG test (Roche Diagnostic Systems, Meylan, France) is a sensitive and specific method for detection of Chlamydia trachomatis infections. This test currently consists of using a nucleic acid amplification method to detect C. trachomatis in first-void urine specimens and in endocervical swabs collected in 2-sucrose-phosphate (2SP) transport medium. We conducted a prospective study to determine whether the automated COBAS Amplicor CT/NG test can detect C. trachomatis in cervical specimens collected in PreservCyt transport medium (ThinPrep Pap Test; Cytyc Corporation, Boxborough, Mass.). PreservCyt medium is used to preserve cervical samples before the preparation of ThinPrep slides. We collected 1,000 cervical specimens from young women (age range, 15 to 25 years) during routine Pap smear tests. Only specimens with normal cytology and in which the gynecologist found no clinical evidence of urogenital infections were selected. The samples were stored in PreservCyt transport medium at 15 to 20 degrees C. C. trachomatis was detected in 22 of the 1,000 cervical specimens that had been stored in PreservCyt. To confirm the positive samples, the test was repeated on new endocervical swab specimens collected in 2SP transport medium. Only 9 of the 22 positive patients agreed to undergo this control, but all 9 retested positive. To evaluate the influence of storage conditions on the sensitivity of the C. trachomatis PCR test, all of the positive samples were stored at 15 to 20 degrees C in PreservCyt transport medium and were retested every 2 weeks for 6 weeks. C. trachomatis was successfully amplified from all 22 specimens for the whole 6-week period. The prevalence of C. trachomatis infection was 2.2% in our study population. These results demonstrate that PreservCyt transport medium is a suitable transport medium for detection of C. trachomatis by the COBAS Amplicor CT/NG test. The ThinPrep Pap Test may enable gynecologists to monitor for both cervical lesions and C. trachomatis infections with a single endocervical specimen.