Impression cytology is a non-invasive and effective method for ocular cell obtention from babies with Congenital Zika Syndrome: perspectives in OMIC studies
Authors: Barbosa Raquel Hora , Santos Maria Luiza B. dos , Silva Thiago P. , Rosa-Fernandes Liva , Pinto Ana , Spínola Pricila S. , Bonvicino Cibele R. , Fernandes Priscila V. , Lucena Evandro , Palmisano Giuseppe , Melo Rossana C. N. , Cardoso Claudete , Lemos Bernardo 
Published: March 2019

IMPORTANCE

Noninvasive techniques for obtaining ocular surface cells (neuroepithelial) from babies with Congenital Zika Syndrome CZS - resulting from infection by zika virus (ZIKV) during gestational period (malformations include ocular abnormalities and microcephaly) - remain to be determined.

OBJECTIVES

The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from babies with CZS in satisfactory amounts and quality to enable the application in the context of genome approaches well as morphological and molecular evaluations.

DESIGN, SETTINGS AND PARTICIPANTS

In this observational study, ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. Samples were collected with an authorized informed consent from both eyes of eight ZIKV infected babies according to the CZS diagnostic criteria (4 babies with positive PCR for Zika virus in gestation and presence of clinical signs which included ocular abnormalities and microcephaly and 4 babies with positive PCR for Zika virus during gestation but no clinical signs identified) and four unaffected babies (control samples / negative PCR, without clinical signs). Cells were used for microscopy analyses, transcriptomic and proteomic experiments and molecular procedure.

MAIN OUTCOMES AND MEASURES

The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures, including viability evaluation. In parallel, molecular approaches were performed.

RESULTS

On impression cytology, a considerable amount of viable cells were captured. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed clear morphological alterations (cell keratinization, piknosis, karyolysis, anucleation and vacuolization). Genomic DNA and RNA were successfully isolated from all samples and allowed the establishment of transcriptomic and proteomic studies. Transcriptome analysis showed 8582 transcripts quantified in all samples and 63 differentially expressed genes in ocular cells from the exposed babies. Proteomics analysis allowed the identification of 2080, 2085 and 2086 high confident and unique proteins with at least one unique peptide in the unaffected, exposed to ZIKV and asymptomatic and CZS babies, respectively, being 2062 in common. Multivariate supervised analysis using the total quantitative protein features revealed a clear discrimination between the groups.

CONCLUSIONS AND RELEVANCE

Our method proved to be a suitable, fast, and non-invasive tool for detailed and precise morphological analyses with a perspective of application in OMIC studies for clinical and research studies of CZS.

Key points

Question

Are the ocular surface cells of babies with Congenital Zika Syndrome viable to investigate the association between Zika virus infection during embryogenesis and ocular impairment?

Findings

To this date, this is the first study using an approach with perspectives in morphological, molecular and “OMICs” research from ocular samples captured by impression cytology of ZIKV infected babies during embryogenesis. The microscopic features of the conjunctival epithelial cells from all ZIKV infected babies showed clear morphological alterations.

Meaning

Ocular cell surface capture offers a powerful model for studying the pathways involved in ocular diseases associated with ZIKV.