ABSTRACT
We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence.
Subject(s)
Capsid Proteins/genetics , DNA, Viral/isolation & purification , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/virology , RNA, Viral/isolation & purification , Sequence Analysis, DNA , Sequence Analysis, RNA , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , Warts/virology , Automation, Laboratory , Cervix Uteri/virology , Colposcopy , Databases, Genetic , Female , Genotype , Humans , Papillomavirus Infections/diagnosis , Polymerase Chain Reaction , Skin/virology , Uterine Cervical Neoplasms/diagnosis , Warts/diagnosis , Uterine Cervical Dysplasia/diagnosisABSTRACT
OBJECTIVE: To evaluate the application of ThinPrep liquid-based cytology (LBC) and present our experience using LBC in the diagnosis of metastatic tumors in cerebrospinal fluid (CSF) samples. STUDY DESIGN: We examined 38 cytologic specimens of CSF, processed by ThinPrep technique. Of these, 18 presented with a previously diagnosed primary malignancy. Various immunocytochemical markers were performed. RESULTS: ThinPrep technology provided preservation of cytomorphologic features, high cellularity per slide and clear background. Analysis revealed 8 breast carcinomas, 5 lung carcinomas, 4 lymphomas, 3 adenocarcinomas of the gastrointestinal tract, 1 squamous cell carcinoma of uterine cervix and 1 urinary bladder carcinoma. Fifteen samples were negative for malignancy. CONCLUSION: CSF cytology is the only examination that verifies the presence of malignancy. Thin monolayer technology is suggested as an appropriate diagnostic method for metastatic tumors in CSF in everyday routine and seems to be a valuable tool for further management and planning of treatment.
Subject(s)
Biomarkers, Tumor/analysis , Cerebrospinal Fluid/cytology , Cytodiagnosis , Cytological Techniques/methods , Neoplasm Metastasis/diagnosis , Adult , Aged , Antibodies, Monoclonal/metabolism , Avidin/metabolism , Biotin/metabolism , Cytological Techniques/instrumentation , Female , Humans , Male , Middle Aged , Neoplasm Metastasis/pathology , Retrospective Studies , Vaginal SmearsABSTRACT
We infected HeLa cells with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers and compared the resulting human papilloma virus (HPV), retinoic acid receptor alpha subunit (RARalpha) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content of surviving infected hosts with that of their uninfected precursors by semi-quantitative reverse transcriptase/polymerase chain reaction amplification (RT/PCR). This comparison revealed a moderate and drastic dependence of HPV and RARalpha mRNA content, respectively, but a complete independence of GAPDH mRNA expression on viral titer. A mechanism of adoptive replacement of tolerable cellular with viral gene expression was proposed to explain these findings. We conclude that the reported ability of influenza B viruses to specifically target and eliminate the cervical adenocarcinoma HeLa cell line studied may find practical applications in biological cancer management.
Subject(s)
Adenocarcinoma/virology , Influenza B virus/pathogenicity , Oncolytic Virotherapy , Oncolytic Viruses/pathogenicity , Uterine Cervical Neoplasms/virology , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/therapy , Antigens, Viral/metabolism , Apoptosis , Cell Survival , Female , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Viral , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , HeLa Cells , Human papillomavirus 18/genetics , Human papillomavirus 18/metabolism , Humans , Influenza B virus/genetics , Influenza B virus/immunology , Oncolytic Viruses/genetics , Oncolytic Viruses/immunology , Phenotype , RNA, Messenger/metabolism , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinoic Acid Receptor alpha , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/therapyABSTRACT
Dirofilariasis is a parasitic disease, which if treated inappropriately due to misdiagnosis, can cause unwanted complications particularly when the infection is located in the breast. The numerous obstacles that can cause misdiagnosis of dirofilariases by standard morphological procedures prompted the development of a Dirofilaria repens-specific direct polymerase chain reaction (PCR)-based diagnostic approach using freshly infected dog blood. Reliable amplification of nematode DNA from formalin-fixed infected human specimens by this method is only possible from relatively fresh biological material, preserved in the fixative for up to 20 days. We report here our first case of dirofilariasis since the development of PCR genotyping, where the pathogen was morphologically unrecognizable and the diagnosis was based exclusively on DNA amplification. We complete our methodological contribution to the clinical laboratory diagnosis of dirofilariasis by presenting two more cases, where the primary genotypic assignment of infection by D. repens was further confirmed by conventional morphological means.
