There is earlier engagement of innate and adaptive immunity in infected subjects with previous vaccination, possibly explaining suppressed viremia in vaccine breakthrough infections. Although breakthrough infections occur in partially protected vaccine recipients, vaccination likely contributes to early control of replication, limiting immune activation and preventing development of clinically significant acute and chronic HBV infection.
We compared the ligase chain reaction (LCR) assay to cell culture for diagnosis of genitourinary chlamydial infections in women using swab specimens obtained by clinicians from the endocervix and by patients from their own vaginas. Specimens from 40 (12.9%) of 309 patients were positive for chlamydial infection by culture, while the specimens of 50 (16.2%) patients were positive by LCR. Chlamydia trachomatis infection was verified for 9 of 10 patients whose LCR specimens were positive but whose cultures were negative. Vaginal and cervical swab specimens were positive by LCR for 46 (93.9%) and 44 (89.8%) of 49 chlamydia-infected patients, respectively. These data suggest that LCR testing for chlamydia with vaginal swab specimens obtained by patients themselves is as sensitive as cervical LCR and more sensitive than cell culture.
Purpose: Patients with glioblastoma (GBM) have a poor prognosis and are in desperate need of better therapies. As therapeutic decisions are increasingly guided by biomarkers, and EGFR abnormalities are common in GBM, thus representing a potential therapeutic target, we systematically evaluated methods of assessing EGFR amplification by multiple assays. Specifically, we evaluated correlation among fluorescence in situ hybridization (FISH), a standard assay for detecting EGFR amplification, with other methods. Experimental Design: Formalin-fixed, paraffin-embedded tumor samples were used for all assays. EGFR amplification was detected using FISH (N ¼ 206) and whole-exome sequencing (WES, N ¼ 74). EGFR mRNA expression was measured using reverse transcription-polymerase chain reaction (RT-PCR, N ¼ 206) and transcriptome profiling (RNAseq, N ¼ 64). EGFR protein expression was determined by immunohistochemistry (IHC, N ¼ 34). Significant correlations among various methods were determined using Cohen's kappa (k ¼ 0.61-0.80 defines substantial agreement) or R 2 statistics. Results: EGFR mRNA expression levels by RNA sequencing (RNAseq) and RT-PCR were highly correlated with EGFR amplification assessed by FISH (k ¼ 0.702). High concordance was also observed when comparing FISH to WES (k ¼ 0.739). RNA expression was superior to protein expression in delineating EGFR amplification. Conclusions: Methods for assessing EGFR mRNA expression (RT-PCR, RNAseq) and copy number (WES), but not protein expression (IHC), can be used as surrogates for EGFR amplification (FISH) in GBM. Collectively, our results provide enhanced understanding of available screening options for patients, which may help guide EGFR-targeted therapeutic approaches.
Molecular beacons are single-stranded, fluorophore-labeled nucleic acid probes that are capable of generating a fluorescent signal in the presence of target, but are dark in the absence of target. Molecular beacons allow multiplex detection of PCR products in real time in a homogeneous assay format. Real time detection is inherently quantitative and affords a greater dynamic range than end-point detection methods. Reactions in a homogeneous assay format are sealed before amplification takes place, providing improved contamination control. A single cycler/reader instrument, coupled with automated sample preparation, results in higher throughput and greater ease of use. A multiplex qualitative assay that detects Chlamydia trachomatis and Neisseria gonorrhoeae, along with an internal control, has been developed. High specificity is achieved through careful selection of primers, probes and assay conditions. Quantitative HIV, HCV, and HBV viral load assays, with sensitivities of 50 copies/ml, 20 IU/ml, and 50 copies/ml, respectively, are achievable. The viral load assays are designed to quantitate all subtype and genotype specimens equivalently. A molecular beacon assay has been designed to detect a single nucleotide polymorphism in the beta2 adrenergic receptor gene.
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