Siegfried Burggraf

Type-specific detection of human papillomaviruses in a routine laboratory setting: Improved sensitivity and specificity of PCR and sequence analysis compared to direct hybridisation

Abstract Human papillomaviruses (HPV) are known to cause cervical dysplasia and cervical carcinoma. We used a 3-step PCR protocol that allows rapid type-specific HPV testing in a routine laboratory setting: HPV-16-positive samples were determined using a specific Light-Cycler PCR; HPV-16-negative samples were amplified by nested PCR and typed by sequence analysis. During a period of 7 months, 1275 PCR-based HPV tests were performed. Of the 1275 samples, 829 samples tested negative for HPV and 446 tested positive, including 124 positives found in the initial HPV-16-specific Light-Cycler assay. Sequence analysis of 132 samples detected 18 HPV types that are not included in the widely used Hybrid Capture II assay. For comparison, the first 100 cervical specimens were tested in parallel using PCR and direct hybridisation (Hybrid Capture II assay). PCR detected HPV DNA in 23 samples that tested negative in the Hybrid Capture assay. Four out of 37 samples that tested positive for HPV in the Hybrid Capture test may be false positives, because sequence analysis detected HPV types not included in the probe mixtures. As rare and novel HPV types may also confer an oncogenic risk, highly sensitive and specific PCR assays will help in understanding cervical HPV infection and cervical cancer of unknown causes.

Comparison of an Internally Controlled, Large-Volume LightCycler Assay for Detection of Mycobacterium tuberculosis in Clinical Samples with the COBAS AMPLICOR Assay

ABSTRACT We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-μl glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.

Rapid and Specific Detection of Bordetella pertussis in Clinical Specimens by LightCycler PCR

The bacterium Bordetella pertussis is the causative agent of whooping cough, which is an infectious disease occurring worldwide with a high incidence among young, unvaccinated infants. Related species which may also cause pertussis syndrome are B. parapertussis (milder respiratory infection) and B. bronchiseptica (primarily infecting animals). The striking and unique presentation of classical pertussis does not usually present a clinical diagnostic dilemma. Atypical pertussis, however, which may occur with mild or absent symptoms in adults or previously vaccinated children, offers a greater diagnostic challenge to the clinician. It has been shown that atypical illness in adults is common, endemic, and usually unrecognized [1]. The epidemiological implications of unrecognized pertussis are that exposure of unimmunized infants to individuals with pertussis places them at high risk and that pertussis remains endemic in society. The recent increase in the incidence of pertussis in the United States [2] and the fact that other agents (such as adenovirus, cytomegalovirus, or chlamydiae) are thought to cause pertussis syndrome underscore the need for rapid and accurate diagnostic methods to guide therapeutic and preventive interventions.

Correlation of HPV16 L1 Capsid Protein Expression in Cervical Dysplasia with HPV16 DNA Concentration, HPV16 E6*I mRNA and Histologic Outcome

OBJECTIVE To determine whether the absence ofHPV1i6 LI capsid protein is a prognostic parameter for the histologic outcome of cervical intraepithelial neoplasia (CIN) 2+ in cytologically diagnosed cervical dysplasia. STUDY DESIGN Papanicolaou-stained microscopic slides of 95 HPV16-positive cervical samples that had a cytologic diagnosis of cervical dysplasia or borderline cytology were immunostained using an HPV16-specific anti-L1 viral capsid antibody. In parallel to the cytologic examination, HPV16 DNA and E6*I mRNA were quantitated using real-time polymerase chain reaction. Expression of L1 protein was correlated with relative levels of HPV16 DNA and E6*I mRNA as well as histologic diagnoses/cytologic follow-up. RESULTS Thirty-five cases with a histologic diagnosis of CIN 2+ (61%) were negative for HPV16 L1 protein; 22 (39%) were positive. Of the cases that either were CIN 1 or regressed to normal cytology, 10 cases (26%) were positive for HPV16 L1 protein, while 28 (74%) were negative. L1-negative and L1-positive cases showed no statistically significant difference (p = 0.22) in their histologic diagnosis/cytologic follow-up. The positive predictive value for CIN 2+ was 56% if L1 protein was absent; the negative predictivre value for CIN 1/regression to normal cytology was 31% if L1 protein was present. HPV16 Ll-positive cases had significantly higher HPV16 DNA concentrations than L1-negative cases (p < 0.001), while levels of HPV16 E6*I mRNA were comparable in both types of cases (p = 0.36). CONCLUSION The expression of HPV16 L1 capsid protein is correlated with viral DNA load but does not predict the histologic outcome of HPV16-positive cervical dysplasia.

Genotyping of the Methionine-Valine Polymorphism at Codon 129 of the Human Prion Protein by Melting Point Analysis of Fluorescently Labeled Hybridization Probes

Human prion diseases include Creutzfeldt-Jakob disease (CJD), the GerstmannStraussler-Scheinker syndrome, fatal familial insomnia, and kuru. Since 1996 a new variant of CJD (vCJD) has been known, which is associated with the bovine spongiform encephalopathy (BSE). Prion diseases are characterized by the accumulation of abnormal prion protein deposits within the brain. The inherited forms are caused by mutations in the human prion protein gene on chromosome 20. In addition, a common methionine/valine polymorphism at codon 129 of the prion protein gene modulates disease susceptibility and phenotypic variability of human prion diseases [1–4]. Several studies have shown that methionine homozygocity at codon 129 is significantly more frequent in sporadic and iatrogenic CJD compared with the genotype distribution of the Caucasian population and confers an increased risk of developing CJD [1, 5–7]. Interestingly, all vCJD patients tested were methionine homozygotes at codon 129 [8, 9].

Rapid Screening for Five Major Cystic Fibrosis Mutations by Melting Peak Analysis Using Fluorogenic Hybridization Probes

Cystic fibrosis (CF), also known as mucoviscidosis, is a disorder of the exocrine glands [1]. Symptoms of CF are secretion of abnormally viscous mucus and elevated sweat electrolytes. The accumulation of thick mucus in the epithelium of the respiratory system and digestive tract causes progressive respiratory and gastrointestinal problems, including liver disease and diabetes mellitus, resulting in high morbidity and reduced life expectancy of CF patients. Variations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate regulated chloride channel, are responsible for CF [2]. CF is one of the most common autosomal single gene disorders in Caucasian populations. The CFTR gene has a size of 250,000 bp separated into 27 exons, most of which are not larger than 200 bp [3]. So far, almost 1,000 CF-causing mutations, more or less scattered over the whole CFTR gene, have been registered by the Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/cftr/). Only persons with homozygote or compound heterozygote CFTR gene mutations suffer from CF. The prevalence of CF in North America and Europe is between 1/2,400 and 1/1,600 and 4%–5% of the population are healthy carriers of one CF mutation.