Abstract
Purpose :
The conjunctival microbiome may potentially contribute to dry eye symptoms and eye infections. However, there is limited literature on the conjunctival microbiome, which is known to be low biomass and difficult to sample. We evaluated the feasibility of using 16S rRNA gene-based broad-coverage qPCR to quantify the conjunctival microbiome using various swabbing protocols.
Methods :
A factorial design was used in a two-stage study sampling 48 eyes from healthy volunteers without current eye infection, allergy to drops, or recent ocular antibiotic use. Test conditions included swab type (calcium alginate [CA], Isohelix [I], Floq [F]), numbing drops (Proparacaine 0.5% [P] versus no numbing [NP]) and swab condition (saline wetted [W] versus dry [D] swab). In the first stage, participants were randomized to ten protocols: CA/NP/D, CA/P/D, CA/P/W, CA/NP/W, I/NP/D, I/NP/W, I/P/D, I/P/W, F/P/W, and F/P/D. In the second stage, participants were randomized to conditions that yielded positive results in the first stage. Participants were swabbed once per eye with modest pressure across the lower fornix. Sterile surgical gloves were used to prevent contamination. Samples were placed in sterile tubes, stored in a -80 C cryo freezer, and shipped to the laboratory for 16S rRNA sequencing.
Results :
Six participants yielded 24 swabs to the first stage and 12 participants contributed 24 swabs to the second. From the first 24 samples, only 2 swabs yielded quantifiable microbial DNA from conditions CA/NP/D and I/NP/D. From the second 24 swabs, an additional 2 swabs yielded quantifiable microbial DNA from conditions I/P/D and I/NP/D. Only the use of topical proparacaine appeared to influence detectability of microbial DNA. Table 1 shows the relative abundance of the top 15 taxa for the four participants with analyzable data. Relative abundances were markedly different, which may be a product of the difficulty sequencing such low biomass samples.
Conclusions :
Results from the study suggest that conjunctival microbial DNA sufficient for reliable quantification by 16S rRNA PCR is challenging to obtain and the low biomass leads to increased risk of contamination and unreliable sequencing results. The difficulty quantifying conjunctival microbiome may also suggest that the conjunctiva is not a likely source of many infections and ocular issues.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.