Latest Advances in Arbovirus Diagnostics.

Arboviruses are a diverse family of vector-borne pathogens that include members of the Flaviviridae, Togaviridae, Phenuviridae, Peribunyaviridae, Reoviridae, Asfarviridae, Rhabdoviridae, Orthomyxoviridae and Poxviridae families. It is thought that new world arboviruses such as yellow fever virus emerged in the 16th century due to the slave trade from Africa to America. Severe disease-causing viruses in humans include Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Crimean-Congo hemorrhagic fever virus (CCHFV), severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV). Numerous methods have been developed to detect the presence of these pathogens in clinical samples, including enzyme-linked immunosorbent assays (ELISAs), lateral flow assays (LFAs) and reverse transcriptase-polymerase chain reaction (RT-PCR). Most of these assays are performed in centralized laboratories due to the need for specialized equipment, such as PCR thermal cyclers and dedicated infrastructure. More recently, molecular methods have been developed which can be performed at a constant temperature, termed isothermal amplification, negating the need for expensive thermal cycling equipment. In most cases, isothermal amplification can now be carried out in as little as 5-20 min. These methods can potentially be used as inexpensive point of care (POC) tests and in-field deployable applications, thus decentralizing the molecular diagnosis of arboviral disease. This review focuses on the latest developments in isothermal amplification technology and detection techniques that have been applied to arboviral diagnostics and highlights future applications of these new technologies.

Hypermethylation of the inhibin alpha-subunit gene in prostate carcinoma.

Inhibin is composed of an alpha- and a beta-subunit. Transgenic studies assigned a tumor-suppressive role to the inhibin alpha-subunit, and in human prostate cancer inhibin alpha-subunit gene expression was down-regulated. This study examined the inhibin alpha-subunit gene promoter and gene locus to determine whether promoter hypermethylation or LOH occurred in DNA from prostate cancer. The 5'-untranslated region of the human inhibin alpha-subunit gene was sequenced and shown to be highly homologous to the bovine, rat, and mouse inhibin alpha-subunit promoter sequences. A 135-bp region of the human promoter sequence that continued a cluster of CpG sites was analyzed for hypermethylation. Significant (P < 0.001) hypermethylation of the inhibin alpha-subunit gene promoter occurred in DNA from Gleason pattern 3, 4, and 5 carcinomas compared with nonmalignant tissue samples. A subset of the carcinomas with a cribriform pattern were unmethylated. LOH at 2q32-36, the chromosomal region harboring the inhibin alpha-subunit gene, was observed in 42% of prostate carcinomas. These data provide the first demonstration that promoter hypermethylation and LOH are associated with the inhibin alpha-subunit gene and gene locus in prostate cancer.

Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels.

We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.

Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells.

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples.