Management of Viral Central Nervous System Infections: A Primer for Clinicians

Viruses are a common cause of central nervous system (CNS) infections with many host, agent, and environmental factors influencing the expression of viral diseases. Viruses can be responsible for CNS disease through a variety of mechanisms including direct infection and replication within the CNS resulting in encephalitis, infection limited to the meninges, or immune-related processes such as acute disseminated encephalomyelitis. Common pathogens including herpes simplex virus, varicella zoster, and enterovirus are responsible for the greatest number of cases in immunocompetent hosts. Other herpes viruses (eg, cytomegalovirus, John Cunningham virus) are more common in immunocompromised hosts. Arboviruses such as Japanese encephalitis virus and Zika virus are important pathogens globally, but the prevalence varies significantly by geographic region and often season. Early diagnosis from radiographic evidence and molecular (eg, rapid) diagnostics is important for targeted therapy. Antivirals may be used effectively against some pathogens, although several viruses have no effective treatment. This article provides a review of epidemiology, diagnostics, and management of common viral pathogens in CNS disease

Ultra-fast vitrification of patient-derived circulating tumor cell lines

Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs

Molecular analysis of vitrified CTCs.

<p>(A) RINs were obtained for fresh (control) and vitrified BRx142 cells to determine whether RNA integrity was compromised. No significant loss in RNA integrity was observed following cryogenic storage (control n = 6, vitrified n = 7). (B) Gene expression signatures were further evaluated for the BRx142 cells using qPCR to detect changes in common breast cancer biomarkers. No significant loss was observed.</p

Vitrification of patient-derived CTCs.

<p>Five patient-derived CTC cell lines, BRx42, 50, 68, 82 and 142, were selected for this study. (A) The selected CTC cell lines demonstrate variation in size, growth as singles or clusters, and EpCAM intensity. (B) The microcapillaries used in this study are 200 μm in diameter and hold approximately 2 μL cell suspension. Insert shows capillary loaded with BRx42 cells (imaged with a 40x objective). (C) Each CTC cell line was vitrified using our standardized procedure. Excellent viability was observed after thawing where 55.7–86.4% of CTCs were viable.</p

Cell culture of vitrified CTCs.

<p>CTC growth in culture was characterized for fresh and vitrified cells. Each cell line was monitored on Day 1, 3 and 5, with the exception of BRx68 which was measured on Day 1 and 8 only due to the slow doubling rate.</p

Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics