@article {166, title = {Dual-target, real-time PCR for the diagnosis of intraocular infections.}, journal = {Br J Ophthalmol}, volume = {103}, year = {2019}, month = {2019 04}, pages = {569-572}, abstract = {

BACKGROUND: is the most common infectious cause of posterior uveitis worldwide. Two multicopy targets (B1 and Rep529) are commonly used in PCR assays, but studies evaluating these targets in ocular fluid samples are limited. Herein, we determine the analytical characteristics of a single-reaction, internally controlled, dual-target, real-time PCR and evaluate the clinical performance of this assay in intraocular fluid samples obtained at a reference ophthalmologic centre in the USA.

METHODS: Lower limits of detection for the B1 and Rep529 components of the dual-target assay were determined using serial dilutions of cultured strain Z185. The dual-target assay was then used to test 148 archived intraocular samples (132 vitreous,16 aqueous humour) collected at the Francis I. Proctor Foundation between January 2010 and December 2015 for testing by a nested, conventional PCR targeting the B1 gene.

RESULTS: The 95\% lower limits of detection for the dual-target assay was determined to be 1.05 tachyzoites/mL for B1 and 0.83 tachyzoites/mL for Rep529. Using archived clinical intraocular specimens, the dual-target assay demonstrated 97.2\% positive per cent agreement (n=35/36; 95\% CI 83.7\% to 99.9\%) and 99.1\% negative per cent agreement (n=111/112; 95\% CI 94.4\% to 100\%) compared with the nested, conventional B1 PCR.

CONCLUSION: This single-reaction, internally controlled, dual-target (B1, Rep529) real-time PCR for the detection of DNA in intraocular specimens demonstrated excellent agreement with nested, conventional, B1 PCR. The dual-target design may ensure detection when variation is present in one of two target regions.

}, keywords = {Animals, Aqueous Humor, DNA, Protozoan, Eye Infections, Parasitic, Humans, Real-Time Polymerase Chain Reaction, Toxoplasma, Toxoplasmosis, Ocular, Uveitis, Posterior}, issn = {1468-2079}, doi = {10.1136/bjophthalmol-2018-313064}, author = {Gomez, Carlos A and Sahoo, Malaya K and Kahn, Ghazala Yasmeen and Zhong, Lina and Montoya, Jos{\'e} G and Pinsky, Benjamin A and Doan, Thuy} } @article {256, title = {Paucibacterial Microbiome and Resident DNA Virome of the Healthy Conjunctiva.}, journal = {Invest Ophthalmol Vis Sci}, volume = {57}, year = {2016}, month = {2016 Oct 01}, pages = {5116-5126}, abstract = {

Purpose: To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques.

Methods: Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses.

Results: Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65\% of all conjunctiva samples tested.

Conclusions: Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.).

}, keywords = {Adult, Bacteria, Conjunctiva, DNA, Bacterial, Eye Infections, Bacterial, Female, Healthy Volunteers, High-Throughput Nucleotide Sequencing, Humans, Male, Microbiota, Middle Aged, Prospective Studies, Real-Time Polymerase Chain Reaction, Sequence Analysis, DNA}, issn = {1552-5783}, doi = {10.1167/iovs.16-19803}, author = {Doan, Thuy and Akileswaran, Lakshmi and Andersen, Dallin and Johnson, Benjamin and Ko, Narae and Shrestha, Angira and Shestopalov, Valery and Lee, Cecilia S and Lee, Aaron Y and Van Gelder, Russell N} }