Irena J. Marin

Hybrid Capture II HPV Test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail

BACKGROUND Hybrid Capture II HPV Test (HCII) (Digene Corporation, Gaithersburg, MD) is a signal amplified hybridization microplate-based assay designated to detect 18 human papillomavirus (HPV) genotypes using two probe cocktails, for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 and low-risk HPV genotypes 6, 11, 42, 43 and 44. At present, HCII is the only commercially available HPV assay with sufficient scientific data to support its performance in the clinical setting. OBJECTIVES To determine the specificity and accuracy of HCII high-risk probe cocktail for detection of 13 HPV genotypes included in the high-risk probe cocktail. STUDY DESIGN Cervical samples obtained from 325 women recognized as HPV positive using the HCII high-risk probe cocktail were included in the study. HPV genotypes were determined by restriction fragment analysis of PGMY09/PGMY11 polymerase chain reaction (PCR) products using seven restriction endonucleases. RESULTS A 450 bp fragment of HPV L1 gene was successfully amplified from 312 out of 325 samples. Of the 312 PCR-positive samples, 280 samples were associated with the expected high-risk HPV genotypes and 32 samples with the HPV genotypes not included in the HCII high-risk probe cocktail. Thus, HPV53 was detected in 8 samples, HPV66 in 4 samples, HPV54 in 3 samples, HPV6, HPV26, HPV70 each in 2 samples, and HPV11, HPV40, HPV42, HPV61, HPV73, HPV81, MM4, IS39, CP6108 each in 1 sample. In 2 samples, we were not able to determine the HPV genotype. CONCLUSIONS Our study shows that HCII high-risk probe cocktail detects at least 15 HPV genotypes not included in the current HCII high-risk probe cocktail. The potential impact of HCII high-risk probe cocktail cross-reactivity with phylogenetically related and unrelated HPV genotypes, including genotypes currently considered to be low-risk HPVs, remains to be determined.

Human telomerase catalytic subunit gene re‐expression is an early event in oral carcinogenesis

Aims:  Detection of telomerase catalytic subunit (hTERT) mRNA has been used as a surrogate marker for estimation of telomerase activity. The exact role and timing of telomerase re‐activation, a key enzyme implicated in cellular immortalization and transformation, in the multistep process of oral carcinogenesis is still unknown. The aim was to test the hypothesis that (i) quantitative rather than qualitative differences exist in the level of hTERT mRNA expression between normal oral mucosa, different grades of oral epithelial abnormalities and squamous cell carcinomas of the oral cavity, and that (ii) hTERT gene re‐expression is an important, probably early event in oral carcinogenesis.

Telomerase Reactivation Is an Early Event in Laryngeal Carcinogenesis

The exact role and timing of reactivation of telomerase, a key enzyme implicated in cellular immortalization and transformation in the multistep process of laryngeal carcinogenesis, is still unknown. We attempted to (1) determine that quantitative differences exist in the levels of telomerase catalytic subunit (hTERT) mRNA expression among different grades of laryngeal epithelial abnormalities classified according to the Ljubljana classification; (2) determine that telomerase reactivation is an important, most probably early event in laryngeal carcinogenesis; and (3) analyze whether the relative quantity of hTERT mRNA can be used as a molecular biomarker in the early detection of precancerous lesions. The relative quantity of hTERT mRNA, expressed as an hTERT index, was analyzed in 140 frozen laryngeal tissue specimens representing different morphological stages of laryngeal carcinogenesis by using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. The presence and relative quantity of hTERT mRNA in laryngeal epithelium increases progressively with the degree of epithelial abnormalities. hTERT mRNA was detectable in 1/15 normal laryngeal epithelia (7%, mean hTERT index 0.02), 3/15 simple hyperplasias (20%, mean hTERT index 0.09), 10/27 abnormal hyperplasias (37%, mean hTERT index 0.18), 9/12 atypical hyperplasias (75%, mean hTERT index 0.74), 8/9 intraepithelial carcinomas (89%, mean hTERT index 1.82), and 53/62 invasive laryngeal squamous cell carcinomas (85%, mean hTERT index 2.51). Statistical analysis revealed two groups of laryngeal epithelial changes with significant differences in the levels of hTERT mRNA expression (P < .0033): (1) normal and reactive hyperplastic laryngeal epithelium (simple and abnormal hyperplasia) and (2) atypical hyperplasia (precancerous lesion), intraepithelial and invasive laryngeal squamous cell carcinoma. The results of the present study suggest that telomerase reactivation is an early event in laryngeal carcinogenesis, detectable already at the stage of precancerous laryngeal epithelial changes. Nevertheless, other genetic abnormalities appear to be necessary for progression of these epithelial abnormalities toward invasive laryngeal squamous cell carcinoma.

Comparative Evaluation of Semiautomated COBAS AMPLICOR Hepatitis B Virus (HBV) MONITOR Test and Manual Microwell Plate-Based AMPLICOR HBV MONITOR Test

ABSTRACT Comparative evaluation of the semiautomated COBAS AMPLICOR hepatitis B virus (HBV) MONITOR Test (COBAS-HBV) and manual AMPLICOR HBV MONITOR Test (AMPLICOR-HBV) on 208 serum samples revealed no significant difference in the sensitivities of the two assays. Twenty samples tested HBV DNA negative and 183 samples tested HBV DNA positive by both assays. Three samples tested positive by COBAS-HBV only and two samples tested positive by AMPLICOR-HBV only. HBV DNA concentrations determined by the two assays were significantly related (n = 183, r = 0.97,P < 0.0001), which indicates that COBAS-HBV could replace AMPLICOR-HBV. The major inconvenience of COBAS-HBV is the required performance of appropriate predilutions of high-titer samples in order to extend the narrow dynamic range of the assay.

Second-generation Hybrid capture test and Amplicor monitor test generate highly correlated hepatitis B virus DNA levels

The performance of the Digene Hybrid Capture II HBV DNA Test HC II and the Roche Cobas Amplicor Monitor Test (Cobas-HBV) was evaluated on 252 serum samples. One hundred and seventy-three samples were HBV DNA positive and 75 HBV DNA negative by both assays. Four samples were HBV DNA positive by Cobas-HBV only. Linear regression analysis showed that the HBV DNA concentrations obtained from both assays were significantly related (n=173, r=0.976, P<0.0001). The results of the study show that Hybrid capture II and Cobas-HBV could be used equally in the management for patients with chronic HBV infection.

Frequency of the 32-base pair deletion in the chemokine receptor CCR5 gene is not increased in hepatitis C patients.

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Seroprevalence of HIV-1 subtypes A-E among HIV-1 infected individuals from Slovenia.

To investigate the prevalence of HIV-1 subtypes A-E in Slovenia, 82 HIV-1 infected individuals were tested for the presence of HIV-1 subtype specific antibodies using a research competitive peptide enzyme immuno assay supplied by Boehringer Mannheim. In 74 individuals unambiguous results were obtained. As in other European countries, the majority of Slovenian HIV-1 infected individuals (86.5%) were infected with subtype B. Infections with subtypes C, A, D and E were detected in 8.1%, 2.7%, 1.3% and 1.3% individuals, respectively.