HR-HPV E6/E7 mRNA In Situ Hybridization: Validation Against PCR, DNA In Situ Hybridization, and p16 Immunohistochemistry in 102 Samples of Cervical, Vulvar, Anal, and Head and Neck Neoplasia.

Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types ("HR-RISH," aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases.

Clinical microbiology costs for methods of active surveillance for Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae.

OBJECTIVE: To compare direct laboratory costs of different methods for perirectal screening for carbapenemase-producing Enterobacteriaceae (CPE) colonization. DESIGN: Cost-benefit analysis. SETTING: A university hospital and affiliated long-term acute care hospital (LTACH). PARTICIPANTS: Inpatients from the hospital or LTACH. METHODS: Perirectal samples were collected from inpatients at risk for exposure to CPE. In 2009, we compared the accuracy of the Centers for Disease Control and Prevention (CDC)-recommended CPE screening method with similar methods incorporating a chromogenic agar (CA). We then performed a cost projection analysis using 2012 screening results for the CA method, the CDC method, and a molecular assay with wholesale pricing based on the 2009 analysis. Comparisons of turnaround and personnel time were also performed. RESULTS: A total of 185 (2.7%) of 6,860 samples were confirmed as CPE positive during 2012. We previously found that the CDC protocol had a lower sensitivity than the CA method and predicted that the CDC protocol would have missed 92 of the CPE-positive screening results, whereas the modified protocol using CA would have missed 26, assuming similar prevalence and performance. Turnaround time was 3 days using the CDC and CA-modified protocols compared with 1 day for molecular testing. The estimated annual total program cost and total technologist's hours would be the following: CA-modified protocol, $37,441 and 376 hours; CDC protocol, $22,818 and 482 hours; and molecular testing, $224,596 and 343 hours. CONCLUSIONS: The CDC screening protocol appeared to be the least expensive perirectal screening method. However, expense must be weighed against a lower sensitivity and extra labor needed for additional work-up of non-CPE isolates. The molecular test has the shortest turnaround time but the greatest expense.

Development and characterization of xenograft model systems for adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common malignancies to arise in human salivary glands, and it also arises in the glandular tissue of other organ systems. To address the paucity of experimental model systems for this tumor type, we have undertaken a program of transplanting tissue samples of human ACC into immunodeficient nu/nu mice to create xenograft model systems. In 17 of 23 attempts (74%), xenograft tumors were successfully grown. In all cases, the histologic appearance of the donating tumor was recapitulated in the subsequent xenograft. Characterization of a subset of xenograft models by immunohistochemical biomarkers and by RNA transcript microarray analysis showed good fidelity in the recapitulation of gene expression patterns in the xenograft tumors compared with the human donor tumors. As ACC is known to frequently contain a t(6;9) translocation that fuses the MYB and NFIB genes, fluorescence in situ hybridization (FISH) of 12 ACC xenograft models was performed that assayed MYB locus break-apart and MYB-NFIB locus fusion. Of 12 xenograft models, 11 (92%) revealed MYB locus rearrangement and 10 (83%) showed evidence of fusion of the MYB and NFIB loci. The two related xenograft models (derived from primary and metastatic tumors, respectively, of the same human subject) were karyotyped, showing a t(1;6) translocation, suggesting MYB translocation to a novel fusion partner gene. Overall, our results indicate that ACC is amenable to xenografting and that ACC xenograft models recapitulate the molecular and morphologic characteristics of human tumors, suggesting utility as valid experimental and preclinical model systems for this disease.

Determination of Echinocandin MICs for Candida species in less than 8 hours: comparison of the rapid susceptibility assay with the Clinical and Laboratory Standards Institute's broth microdilution assay.

The echinocandins prevent fungal cell wall synthesis by inhibiting beta-1,3-glucan synthesis, a significant glucose-consuming process. Previous studies suggested that echinocandin inhibitory activity is evident within 1 h of exposure. We hypothesized that a susceptibility assay based on glucose consumption may provide clinically useful MICs rapidly. The rapid susceptibility assay (RSA), which provides MICs in less than 8 h, was compared with the standard broth microdilution susceptibility assay (Clinical and Laboratory Standards Institute, document M27-A3, 2008) for 56 Candida species strains. Variables which are known to influence MICs determined by the M27-A3 method were also assessed for their effects on the RSA results. Excellent agreement (>90%) between the results of the RSA and M27-A3 methods was achieved for all three FDA-approved echinocandins (micafungin, caspofungin, and anidulafungin). Candida lusitaniae strains were responsible for most of the discordant results. Assay variables such as the test medium, the age of the inoculum culture, and the presence of human serum affected MIC results from the RSA and the M27-A3 method similarly. The RSA is equivalent to the standard M27-A3 method for determining echinocandin MICs for Candida species. The RSA provides MIC results in less than 8 h and can be applied to old and young yeast colonies. The assay could potentially provide clinically useful MICs on the same day that yeast growth from a specimen is first detected on solid medium.