Katja Seme

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 papillomavirus infection in esophageal carcinomas: A study of 121 lesions using multiple broad-spectrum polymerase chain reactions and literature review

To elucidate the putative role of human papillomavirus (HPV) infection in the etiology of esophageal cancer, 121 formalin-fixed, paraffin-embedded specimens originating from a non-high-incidence area for this carcinoma, from Slovenia, were screened for HPV infection using eight different polymerase chain reactions (PCR). Three different HPV consensus primer sets and four primer sets specific for HPV types 6, 16, and 18 failed to detect HPV DNA sequences in any of the tumor samples. Fragments of human beta-globin gene that served as internal controls were successfully amplified from 120 of 121 specimens. Our study confirms the opinion that most esophageal cancers originating from non-high-incidence geographic areas of this cancer are not associated with HPV infection. According to the studies reviewed, it is likely that HPV infection plays a much more significant role in esophageal carcinogenesis in those areas of the world with a high incidence of ESCC.

Pediatric tick-borne encephalitis in 371 cases from an endemic region in Slovenia, 1959 to 2000

Background. With the exception of Lyme borreliosis, tick-borne encephalitis (TBE) is the most prevalent tick-transmitted disease in Europe. Here we report clinical and epidemiologic features of the largest number of children with TBE reported to date and the longest (i.e. 42-year) retrospective survey of pediatric TBE cases from one geographic region. Methods. Case records of 371 patients, age 0 to 15 years, with serologically confirmed TBE and hospitalized between 1959 and 2000 at the Department of Infectious Diseases of the General Hospital Celje, Slovenia were reviewed and analyzed. Results. Children represented 23.5% of 1578 confirmed TBE cases in the study period. Children were admitted to hospital throughout the year, but the majority were treated during summer months. In 178 (47.9%) children, a tick bite was noticed before admission. A biphasic course of illness occurred in 249 (67.1%) patients. The most common symptoms and signs of TBE were raised body temperature [>38°C (n = 371)], headache and meningeal signs (n = 346), fatigue (n = 337) and vomiting (n = 327). Meningitis was diagnosed in 232 (62.5%) children, and meningoencephalitis was diagnosed in 139 (37.5%). There was a tendency for greater severity of TBE with increasing age. None of the children with TBE died, and none had permanent sequelae. Conclusions. The results of our study indicate that pediatric TBE is relatively mild disease with favorable outcome.

Comparison of Clinical and Analytical Performance of the Abbott RealTime High Risk HPV Test to the Performance of Hybrid Capture 2 in Population-Based Cervical Cancer Screening

ABSTRACT The clinical performance of the Abbott RealTime High Risk HPV (human papillomavirus) test (RealTime) and that of the Hybrid Capture 2 HPV DNA test (hc2) were prospectively compared in the population-based cervical cancer screening setting. In women >30 years old (n = 3,129), the clinical sensitivity of RealTime for detection of cervical intraepithelial neoplasia of grade 2 (CIN2) or worse (38 cases) and its clinical specificity for lesions of less than CIN2 (3,091 controls) were 100% and 93.3%, respectively, and those of hc2 were 97.4% and 91.8%, respectively. A noninferiority score test showed that the clinical specificity (P < 0.0001) and clinical sensitivity (P = 0.011) of RealTime were noninferior to those of hc2 at the recommended thresholds of 98% and 90%. In the total study population (women 20 to 64 years old; n = 4,432; 57 cases, 4,375 controls), the clinical sensitivity and specificity of RealTime were 98.2% and 89.5%, and those of hc2 were 94.7% and 87.7%, respectively. The analytical sensitivity and analytical specificity of RealTime in detecting targeted HPV types evaluated with the largest sample collection to date (4,479 samples) were 94.8% and 99.8%, and those of hc2 were 93.4% and 97.8%, respectively. Excellent analytical agreement between the two assays was obtained (kappa value, 0.84), while the analytical accuracy of RealTime was significantly higher than that of hc2. RealTime demonstrated high intralaboratory reproducibility and interlaboratory agreement with 500 samples retested 61 to 226 days after initial testing in two different laboratories. RealTime can be considered to be a reliable and robust HPV assay clinically comparable to hc2 for the detection of CIN2+ lesions in a population-based cervical cancer screening setting.

Antibiotic susceptibility profiles of oral pathogens.

Periodontitis is a bacterial disease that can be treated with systemic antibiotics. The aim of this study was to establish the antibiotic susceptibility profiles of five periodontal pathogens to six commonly used antibiotics in periodontics. A total of 247 periodontal bacterial isolates were tested for susceptibility to the six antibiotics using the Etest method. MIC(50) and MIC(90) values (minimum inhibitory concentrations for 50% and 90% of the organisms, respectively) were calculated. Both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints were used in the study to interpret results. β-Lactamase production was tested when amoxicillin resistance was found. MIC(90) values of the anaerobic bacteria were all well below breakpoint values, except for three isolates of Prevotella intermedia and one isolate of Fusobacterium nucleatum that were resistant to amoxicillin (CLSI breakpoints); these isolates were β-lactamase-positive. Two isolates of the capnophilic Aggregatibacter actinomycetemcomitans appeared to be amoxicillin-resistant but failed to show β-lactamase activity. Comparison with a previous study from The Netherlands showed minor differences in susceptibility profiles, but the MIC(90) values of A. actinomycetemcomitans for amoxicillin, clindamycin, azithromycin and tetracycline were higher. Geographical differences in the susceptibility profiles of Porphyromonas gingivalis and A. actinomycetemcomitans between European countries were noted. Comparison of European susceptibility profiles with that of a South American country (Colombia) revealed a much higher resistance in the latter. Owing to these differences in susceptibility profiles, it is of concern to regularly perform surveillance studies on antibiotic resistance.

DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods.

The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa‐stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze–thaw method, boiling in 10% Chelex‐100 resin solution, proteinase K/Tween 20/NP‐40 method coupled with simplified phenol/chloroform/isoamyl alcohol protocol or salting‐out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen, Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa‐stained bone marrow slides.

Detection of Human Papillomaviruses in Tissue Specimens

Summary During the past decade, molecular methods based on the detection of viral DNA have become a key tool for the detection of human papillomaviruses (HPVs) in tissue. The methods can be divided into two groups: those in which tissue destruction is unavoidable for the detection of HPV DNA, and those in which the detection of viral DNA is performed in a way that allows tissue morphology preservation. Polymerase chain reaction is currently the most sensitive method for HPV detection and an excellent research tool. However, because of frequent contamination problems and lack of standardization, it is not readily applicable to diagnostic laboratories. The recent improvements in in situ hybridization have made it possible for this method to become the most appropriate method for routine detection of HPVs in tissue. At present, however, the use of at least two independent HPV DNA detection methods is indispensable for accurate determination of HPVs.

Commercially available molecular tests for human papillomaviruses (HPV): 2015 update.

Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests.

Rapid detection and typing of human papillomaviruses by consensus polymerase chain reaction and enzyme-linked immunosorbent assay

A novel method for the detection and typing of human papillomaviruses (HPV) based on consensus polymerase chain reaction (PCR) using MY09/MY11 primers followed by detection of PCR products in a standard microtiter plate format using a recently developed commercially available standardised PCR ELISA kit (Boehringer Mannheim, Germany) was developed. The reliability and feasibility of the method were evaluated on 140 HPV-positive and 85 HPV-negative DNA samples extracted from different archival clinical specimens. Virtually complete agreement between the results of this novel method and the results of previous in-house PCRs and typing method was obtained. The sensitivity level of the novel method, determined by serial log-dilutions of SiHa cells, is about 50 copies of HPV 16. The PCR-ELISA provides the potential for an automated, simple, rapid and accurate test for detection and typing of HPV in diagnostic virological laboratories.

Tumor‐specific and gender‐specific pre‐vaccination distribution of human papillomavirus types 6 and 11 in anogenital warts and laryngeal papillomas: A study on 574 tissue specimens

Anogenital warts and laryngeal papillomas are two most important benign tumors etiologically linked with HPV. In the study, which included both the largest number of laryngeal papilloma tissue specimens (152 specimens from 152 patients) to date and the largest number of prospectively collected and histologically confirmed tissue specimens of anogenital warts obtained from both genders (422 specimens from 315 patients), HPV DNA was detected in 413/422 (97.9%) of anogenital warts and 139/152 (91.4%) of laryngeal papillomas. HPV‐6 and/or HPV‐11 were detected in 291/315 (92.4%) patients with anogenital warts and in 138/152 (90.8%) patients with laryngeal papillomas, indicating that the great majority of both tumors could be prevented with prophylactic quadrivalent vaccine. The HPV‐6 gender‐specific distribution in both anogenital warts and laryngeal papillomas was not statistically significant. In contrast, HPV‐11 was found almost three times more often in males than in females with anogenital warts (16.5% vs. 6.3%; P = 0.008), with a gender neutral HPV‐11 type distribution in laryngeal papillomas. The overall HPV DNA prevalence in anogenital warts was significantly different from that in laryngeal papillomas (97.1% vs. 91.4%; P = 0.01). In the first comparison of the HPV‐6/HPV‐11 type‐specific distribution between patients suffering from anogenital warts and laryngeal papillomas with the same geographic and ethnic background, a significant imbalance in tumor‐specific distribution of HPV‐6 and HPV‐11 was identified: HPV‐6 was statistically more often present in anogenital warts than in laryngeal papillomas (79.0% vs. 59.2%; P = 0.000013), whereas HPV‐11 was statistically more frequent in laryngeal papillomas than in anogenital warts (28.9% vs. 12.4%; P = 0.00003). J. Med. Virol. 84: 1233–1241, 2012.