ObjectiveRecently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA.DesignWe developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case–control study comparing results with those obtained with the Abbott RealTime HCV test.ResultsThe PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country.ConclusionWe report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions.Trial registration numberNCT02992184.

The human PRL-inducible protein (PIP)/gross cystic disease fluid protein-15 is expressed in pathological conditions of the mammary gland and in several exocrine tissues, such as the lacrimal, salivary, and sweat glands. In human breast cancer cells, the expression of PIP/gross cystic disease fluid protein-15 is stimulated by androgen and PRL, and inhibited by estrogen. However, it is not known whether the expression of PIP in other tissues is under similar hormonal regulation. In the present study we employed reverse transcriptase-polymerase chain reaction followed by rapid amplification of complementary DNA (cDNA) ends to amplify the PIP cDNA homolog, the submaxillary gland protein (SMGP) in the mouse. The mouse PIP/SMGP cDNA encodes a putative secreted peptide of 144 amino acids with a 51% identity with human PIP. Using the mouse PIP/SMGP cDNA as a probe, we examined the tissue- and cell-specific expression of PIP/SMGP messenger RNA by in situ hybridization and Northern blot analysis of mouse and rat tissues. Hormonal regulation was also studied in the rat. PIP/SMGP messenger RNA expression was only detected in the lacrimal and submaxillary glands of the rodents. In the rat submaxillary gland, PIP/SMGP gene expression was confined to the acinar cells. In the male rat lacrimal gland, castration resulted in an increase in expression, and in both male and female rats, androgen replacement abolished PIP/SMGP gene expression. This pattern of regulation was not observed in the submaxillary gland and was actually reversed in human breast cancer cells. PRL had no effect on the regulation of PIP/SMGP in either salivary or lacrimal glands. Our study indicates that tissue-specific factors are important in determining the hormone responsiveness of the PIP/SMGP gene. Regulation of the PIP/SMGP gene in vivo may provide a useful model system to study the mechanism of down-regulation of expression by androgen in a tissue-specific manner.

The timely detection of viremia in HIV-infected patients receiving antiviral treatment is key to ensuring effective therapy and preventing the emergence of drug resistance. In high HIV burden settings, the cost and complexity of diagnostics limit their availability. We have developed a novel complementary metal-oxide semiconductor (CMOS) chip based, pH-mediated, point-of-care HIV-1 viral load monitoring assay that simultaneously amplifies and detects HIV-1 RNA. A novel low-buffer HIV-1 pH-LAMP (loop-mediated isothermal amplification) assay was optimised and incorporated into a pH sensitive CMOS chip. Screening of 991 clinical samples (164 on the chip) yielded a sensitivity of 95% (in vitro) and 88.8% (on-chip) at >1000 RNA copies/reaction across a broad spectrum of HIV-1 viral clades. Median time to detection was 20.8 minutes in samples with >1000 copies RNA. The sensitivity, specificity and reproducibility are close to that required to produce a point-of-care device which would be of benefit in resource poor regions, and could be performed on an USB stick or similar low power device.

The ability to assess and monitor target engagement is crucial for informing early drug development decisions. High vascularisation of the hair follicle, frequent epithelial origin of tumors, and high degree of congruence of expression in hair of pathways dysregulated in cancers, makes the cellular bulb on plucked human scalp hair an excellent surrogate tissue for non-invasive monitoring of PD effects in clinical trials. Disruption of the p53-HDM2 interaction with small molecules has demonstrated single agent anti-tumor activity in preclinical models and represents an attractive treatment strategy in oncology. Development of a peripheral tissue based gene expression signature of inhibition of the p53-HDM2 interaction could facilitate the early development of these compounds. To develop a peripheral PD biomarker of antagonism of this interaction, we used two selective p53-HDM2 antagonists, Nutlin-3 and Sanofi's SAR405838 and applied our plucked hair biomarker platform to develop a gene expression signature indicative of compound exposure. SAR405838 displays potent activity in vitro and in vivo against p53 WT cell lines / xenograft models, but not in the p53 mutant context. Three hairs from each of four healthy donors were exposed to either SAR405838 or Nutlin-3 over a range of compound concentrations for 6hr or 24hr in our proprietary ex vivo cultures. We extracted RNA from the cellular bulb of individual hairs and assessed the transcriptome by microarray analysis. Biological enrichment analysis of genes differentially expressed revealed strong correlation to activated P53 signaling pathways. Further analysis revealed a core set of congruent genes as candidate PD biomarkers of p53-HDM2 antagonism, one of which was MIC-1, a secreted plasma protein that demonstrates a strong PK/PD relationship in patients treated with p53-MDM2 antagonists in Phase 1 trials. In ex vivo plucked hair, we have demonstrated biologically relevant differential expression of a panel of transcriptional markers exhibiting common response to Nutlin-3 and SAR405838. These reflect compound mechanism of action, and can provide further evidence of target engagement in addition to plasma MIC-1 levels. While the temporal and kinetic relationship between gene expression changes, toxicity, and clinical efficacy remains to be determined, the genes identified in this study may be used to provide further MOA information in clinical settings to monitor PD responses in plucked scalp hair obtained from patients exposed to SAR405838. Citation Format: Gino Miele, Elliot Harrison, Tim Mefo, Jo Read, Lydia Meyer Turkson, Alan Murdoch, Michael Teufel, Laurent Debussche, Donald Bergstrom, James Watters. Plucked hair as a biomarker platform for monitoring transcriptional consequences of clinical exposure to antagonism of the HDM2/P53 interaction in tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3350. doi:10.1158/1538-7445.AM2013-3350

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