Using the polymerase chain reaction to genotype human papillomavirus DNAs in samples containing multiple HPVs may produce inaccurate results.

Compared with other laboratory techniques, the polymerase chain reaction (PCR) is a simple, rapid, sensitive method for detecting human papillomavirus (HPV) DNA in cervical samples. However, since many cervical samples contain multiple HPV types, we decided to investigate whether PCR results from such samples accurately reflected the relative amounts of each HPV type present. Theoretical calculations of product accumulation when multiple DNAs with different amplification efficiencies are present in the same sample were done. In addition a set of samples in which cloned HPV DNAs were mixed in varying proportions prior to PCR was tested. Finally, four clinical samples containing multiple HPV types by hybridization assays were subjected to PCR, using two different primer sets. Each of these lines of investigation showed that selective amplification of one HPV DNA over another can occur when mixed HPV types are present. This effect may lead to inaccurate information regarding both types and relative amounts of HPV DNAs in samples containing multiple HPV types. A protocol to avoid this problem is described.

Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18.

BACKGROUND: Quantitation of human papillomavirus (HPV) DNA in clinical samples may yield important clinical information. METHODS AND RESULTS: We developed a 5' exonuclease fluorescent probe assay for HPV quantitation that uses real-time PCR. The assay was optimized for HPV types 6 (HPV-6), -11, -16, and -18. A multiplex format was developed to quantify a cellular target of known iteration simultaneously with HPV quantitation, which controls for the amount of input DNA. Dilution series of target and heterologous templates were used to verify the assay. The assay was successfully used on fresh and PreservCyt-fixed cell lines, as well as cervical samples. The linear range of the assay is from 10 to 10 million copies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures. CONCLUSION: The method is accurate over a large copy number range, reproducible, type specific, normalized for input DNA quantity, and applicable to PreservCyt-fixed material.

Tricorrectional bunionectomy for surgical repair of juvenile hallux valgus.

The authors propose the use of the tricorrectional bunionectomy as an alternate correction of severe deformity in juvenile hallux valgus. In the past, hallux valgus surgery in juveniles has been avoided. A follow-up study of the tricorrectional bunionectomy as the surgical treatment for juvenile bunion deformity in seven patients is presented.

A case-control study of human papillomavirus and cervical squamous intraepithelial lesions (SIL) in Harris County, Texas: differences among racial/ethnic groups.

We conducted a case-control study of the association between SIL and HPV among whites (W), African Americans (AA), and Hispanics (H) in Harris County, Texas. Cases were identified at M.D. Anderson Cancer Center Colposcopy Clinic. Controls were identified among women obtaining routine Pap screening at two Harris County Health Department Clinics. HPV was detected by a PCR-based fluorescent assay. Dichotomous and polytomous logistic regression models were used to estimate adjusted odd ratios (AOR) and 95% confidence intervals (CI) for SIL among racial/ethnic groups and grade of disease. Prevalence of HPV infection was 64% in low grade SIL (LSIL), 84% in high grade SIL (HSIL), and 19% in controls. Risk of SIL was higher in H than in W and AA, AOR 29.5 (12.4-70.5), 15.3 (6.0-33.8), and 5.8 (2.6-12.6), respectively. Similarly, racial/ethnic differences were observed for both LSIL (AOR = 16.6, 7.7, and 4.3, respectively) and HSIL (AOR = 78.6, 34.6, and 14.2, respectively). Findings support the association between SIL and HPV and differences in the strength of the association with LSILs and HSILs. Data also suggest a higher risk for H and a lower risk for AA.

Drugs and liver disease: a tabular compilation of drugs and the histopathological changes that can occur in the liver.

A compilation of drugs and the histopathological changes that can occur in the liver is presented. The purpose of this review is to provide the reader with a comprehensive and reliable source of information on various drugs that have been documented by liver biopsy to cause hepatocellular damage. The morphologic terms used in the tables have been chosen based on past publications dealing with this subject. This review is intended as a concise guide to aid in the identification of drug-induced liver diseases.

Parenteral-verapamil-induced sustained hypotension.

The incidence of oral-verapamil-induced hypotension in the presence of concomitant beta-adrenergic blockade by the oral route is quite rare. The major contraindication to the use of intravenous verapamil is recent administration of intravenous beta-blocking agents (i.e., within the past two hours). We report a case of sustained hypotension after administration of parenteral verapamil. Vasopressors were required to support blood pressure for approximately five hours. The patient had been on oral propranolol for the past five years and had taken his last dose of propranolol approximately ten hours before the administration of parenteral verapamil for treatment of a supraventricular tachycardia documented by an electrocardiogram.

Acalculous cholecystitis and fever related to total parenteral nutrition.

Over the past several years, clinicians have become aware of the importance of maintaining a positive nitrogen balance in hospitalized patients. This has led to the widespread use of total parenteral nutrition (TPN). However, with increased experience with this form of nutrition, numerous potential complications have been uncovered. One of the complications demonstrated with increased frequency is that of abnormal liver function, manifested by elevated serum liver enzymes. This report describes a 44-year-old woman with rectal abscesses and possible inflammatory bowel disease who developed severe right upper quadrant pain, abnormally elevated liver enzymes, and elevated body temperature during her course of TPN therapy. These problems possibly were related to the TPN regimen. Once TPN therapy was discontinued, the patient's liver enzyme values and elevated body temperature began to return to baseline. She subsequently was discharged from the hospital. A follow-up visit to the physician's office revealed that all liver enzyme values had returned to normal, the pain had resolved, and she was recovering well.

Nucleotide and amino acid sequence variation in the L1 and E7 open reading frames of human papillomavirus type 6 and type 16.

Human papillomavirus (HPV) type 6 and type 16 DNA sequence variants were found by partially sequencing the L1 and E7 open reading frames, using templates generated with the polymerase chain reaction. Identical variants were found in patients from widely separated locations, such as the United States, the Philippines, and India. The same sequence variants of HPV 16 were found in women with invasive cervical carcinoma and in women with no evidence of disease. Variation in the predicted amino acid sequences of the HPV 16 L1 and E7 proteins was found. A single nucleotide change at position 6433 was found in about 50% of the HPV 16 DNAs, resulting in a change in predicted amino acid sequence from threonine to alanine at the equivalent position in the L1 protein. Predicted amino acid changes were found in the HPV 16 E7 proteins at amino acid positions 28, 29, and 47. Variation at these positions could affect known properties of the E7 protein, including binding to the retinoblastoma protein.

A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA.

A simple method for the detection of a number of human papillomavirus (HPV) genotypes associated with cervical cancer has been developed. The assay exploits the 5'-->3' exonucleolytic activity of Taq DNA polymerase to increase the signal from fluorescent dyes by releasing them from genotype-specific probes during PCR. The probes are oligonucleotides with a 5' reporter dye (6-carboxyfluorescein), a quencher dye (6-carboxy-tetramethyl-rhodamine), and a phosphate-blocked 3' end. In the intact probe, the proximity of the reporter and the quencher results in suppression of reporter fluorescence by Förster-type energy transfer (V. T. Förster. Ann. Phys. 2:55-75, 1948). If the probe is bound downstream of either primer during PCR, the 5'-->3' exonucleolytic activity of Taq polymerase degrades it, allowing the reporter to diffuse away from the quencher, which results in an increase in reporter fluorescence. The increased fluorescence is directly related to the amount of target DNA and can be detected with an automated fluorometer. Probes for the L1 region of the cervical-cancer-associated HPV types 16, 18, 31, 33, and 35 were synthesized and the assays were optimized. The most sensitive assay can detect as few as two copies of HPV DNA in human cervical specimens.

A computer controlled system for multiple site microcirculatory measurements.

A system has been developed for intravital microscopy studies that records optical signals at different tissue sites together with X-Y coordinates within an area of up to 100 cm2. Z axis coordinates can also be recorded. The system utilizes a computer and a motor-driven microscope stage to rapidly and repetitively move between selected sites. The system can also be used to map topological features and geometry. The application of the system to study spatial heterogeneity of changes in tissue metabolism with time and to map vascular network architecture is described.

Genotypes and sequence variants of human papillomavirus DNAs from human immunodeficiency virus type 1-infected women with cervical intraepithelial neoplasia.

Human papillomavirus (HPV) DNA was found in cervicovaginal lavage fluids from 9 of 11 human immunodeficiency virus type 1 (HIV-1)-seropositive female prostitutes with cervical intraepithelial neoplasia (CIN) in Kinshasa, Zaire. Since 7 yielded complex nucleic acid hybridization results consistent with mixed HPV infections, limited sequencing of HPV DNA was used to identify the HPVs present. Three of HPV 16 and 1 each of HPV 18, 31, 33, and 56 and ME180-HPV were identified by sequencing in 8 samples. Each of these genotypes has been found in specimens from HIV-1-seronegative women with CIN. Some DNAs had nucleic acid and amino acid sequence variations relative to the reference HPVs, but the variants were closely related to variants that have been found in HIV-1-seronegative women. Variant amino acids were found predominantly at three positions in one 40-amino-acid segment of the L1 open reading frame sequenced. The predominant HPV 16 variant observed has been found rarely in other countries.

Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type.

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.