The performance of human papillomavirus high-risk DNA testing in the screening and diagnostic settings.

OBJECTIVE: We sought to evaluate the performance of the human papillomavirus high-risk DNA test in patients 30 years and older. MATERIALS AND METHODS: Screening (n=835) and diagnosis (n=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II (HCII) test result with the worst histologic report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR-), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear. RESULTS: For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR- were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR- were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing. CONCLUSIONS: The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older.

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

Detection and sequence analysis of a nickel-induced mutation in a retroviral model system

We have developed a novel way to assess the mutagenicity of environmentally important metal carcinogens, such as nickel, by creating a positive selection system based upon the conditional expression of a retroviral transforming gene. The target gene is the v-mos gene in MuSVts110, a murine retrovirus possessing a growth temperature dependent defect in expression of the transforming gene due to viral RNA splicing. In normal rat kidney cells infected with MuSVts110 (6m2 cells), splicing of the MuSVts110 RNA to form the mRNA from which the transforming protein, p85$\sp{\rm gag-mos}$, is translated is growth-temperature dependent, occurring at 33 C and below but not at 39 C and above. This splicing "defect" is mediated by cis-acting viral sequences. Nickel chloride treatment of 6m2 cells followed by growth at 39 C, allowed the selection of "revertant" cells which constitutively express p85$\sp{\rm gag-mos}$ due to stable changes in the viral RNA splicing phenotype, suggesting that nickel, a carcinogen whose mutagenicity has not been well established, could induce mutations in mammalian genes. We also show by direct sequencing of PCR-amplified integrated MuSVts110 DNA from a 6m2 nickel-revertant cell line that the nickel-induced mutation affecting the splicing phenotype is a cis-acting 70-base duplication of a region of the viral DNA surrounding the 3$\sp\prime$ splice site. These findings provide the first example of the molecular basis for a nickel-induced DNA lesion and establish the mutagenicity of this potent carcinogen. ^

Detection of enterotoxigenic Escherichia coli in foods by polymerase chain reaction

Enterotoxigenic Escherichia coli (ETEC) causes significant morbidity and mortality in infants of developing countries and is the most common cause of diarrhea in travelers to these areas. Enterotoxigenic Escherichia coli infections are commonly caused by ingestion of fecally contaminated food. A timely method for the detection of ETEC in foods would be important in the prevention of this disease. A multiplex polymerase chain reaction (PCR) assay which has been successful in detecting the heat-labile and heat-stable toxins of ETEC in stool was examined to determine its utility in foods. This PCR assay, preceded by a glass matrix and chaotropic DNA extraction, was effective in detecting high numbers of ETEC in a variety of foods. Ninety percent of 121 spiked food samples yielded positive results. Samples of salsa from Guadalajara, Mexico and Houston, Texas were collected and underwent DNA extraction and PCR. All samples yielded negative results for both the heat-labile and heat-stable toxins. Samples were also subjected to oligonucleotide probe analysis and resulted in 5 samples positive for ETEC. Upon dilution testing, it was found that positive PCR results only occurred when 12,000 to 1,000,000 bacteria were present in 200 mg of food. Although the DNA extraction and PCR method has been shown to be both sensitive and specific in stool, similar results were not obtained in food samples. ^

DNA nucleotide excision repair gene single nucleotide polymorphisms and hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease caused by germline mutations in DNA mismatch repair(MMR) genes. The nucleotide excision repair(NER) pathway plays a very important role in cancer development. We systematically studied interactions between NER and MMR genes to identify NER gene single nucleotide polymorphism (SNP) risk factors that modify the effect of MMR mutations on risk for cancer in HNPCC. We analyzed data from polymorphisms in 10 NER genes that had been genotyped in HNPCC patients that carry MSH2 and MLH1 gene mutations. The influence of the NER gene SNPs on time to onset of colorectal cancer (CRC) was assessed using survival analysis and a semiparametric proportional hazard model. We found the median age of onset for CRC among MMR mutation carriers with the ERCC1 mutation was 3.9 years earlier than patients with wildtype ERCC1(median 47.7 vs 51.6, log-rank test p=0.035). The influence of Rad23B A249V SNP on age of onset of HNPCC is age dependent (likelihood ratio test p=0.0056). Interestingly, using the likelihood ratio test, we also found evidence of genetic interactions between the MMR gene mutations and SNPs in ERCC1 gene(C8092A) and XPG/ERCC5 gene(D1104H) with p-values of 0.004 and 0.042, respectively. An assessment using tree structured survival analysis (TSSA) showed distinct gene interactions in MLH1 mutation carriers and MSH2 mutation carriers. ERCC1 SNP genotypes greatly modified the age onset of HNPCC in MSH2 mutation carriers, while no effect was detected in MLH1 mutation carriers. Given the NER genes in this study play different roles in NER pathway, they may have distinct influences on the development of HNPCC. The findings of this study are very important for elucidation of the molecular mechanism of colon cancer development and for understanding why some mutation carriers of the MSH2 and MLH1 gene develop CRC early and others never develop CRC. Overall, the findings also have important implications for the development of early detection strategies and prevention as well as understanding the mechanism of colorectal carcinogenesis in HNPCC. ^

A PCR based assay for the detection of enteric pathogens from Hemoccult(RTM) cards

Background. Large field studies in travelers' diarrhea (TD) in multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport and storage of fecal specimens that does not require immediate processing, refrigeration and is stable for months would be advantageous. ^ Objectives. Determine if enteric pathogen bacterial DNA can be identified in cards routinely used for evaluation of fecal occult blood. ^ Methods. U.S. students traveling to Mexico in 2005-07 were followed for occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard method. Cards were then stored at room temperature prior to DNA extraction. A multiplex fecal PCR was performed to identify enterotoxigenic Escherichia coli and enteroaggregative E. coli (EAEC) in DNA extracted from stools and occult blood cards. ^ Results. Significantly more EAEC cases were identified by PCR done in DNA extracted from cards (49%) or from frozen feces (40%) than by culture followed by HEp-2 adherence assays (13%). Similarly more ETEC cases were detected in card DNA (38%) than fecal DNA (30%) or culture followed by hybridization (10%). Sensitivity and specificity of the card test was 75% and 62%, respectively, and 50% and 63%, respectively, when compared to EAEC and ETEC culture, respectively, and 53% and 51%, respectively compared to EAEC multiplex fecal PCR and 56% and 70%, respectively, compared to ETEC multiplex fecal PCR. ^ Conclusions. DNA extracted from fecal cards used for detection of occult blood is of use in detecting enteric pathogens. ^