Abstract
Purpose :
Ocular TB (OTB) is a form of extrapulmonary TB that causes inflammation and visual morbidity. Its diagnosis is challenging due to the paucibacillary nature, difficulties in obtaining ocular tissue, tests with poor sensitivity and specificity, and variable clinical presentations. Recent studies suggested the use of altered host transcripts as potential biomarkers for TB. Hence, it is proposed that studying the alterations in host transcripts particularly at the site of infection in the vitreous fluid of OTB patients will identify signatures that can be used for molecular diagnosis of OTB.
Methods :
Vitreous fluid samples were collected from TB uveitis (n=6) and non-TB uveitis patients (n=3). Multiple methods of isolation of good quality RNA from vitreous samples were carried out. Finally, a method involving isolation of vitreous cells was used. The isolated cells were used for RNA sequencing (RNASeq) on Illumina by using Takara’s SMART-Seq Stranded kit which can be used for even 100-1000 cells. RNASeq reads were aligned to human reference genome and differentially expressed genes were identified using DESEQ software (v1.40.1).
Results :
The yield of fluorescent cRNA, isolated from vitreous fluid by TRIzol method was unsuitable for microarray. Alternatively, REPLI-G WTA kit was used to amplify cDNA from low amounts of vitreous RNA followed by fluorescent cRNA synthesis. However, none of the samples passed QC for sequencing. Therefore, another method using the vitreous cells directly for RNASeq yielded libraries of high quality and proceeded for downstream analysis. RNASeq data revealed 273 upregulated and 117 downregulated genes with fold change cutoff ≥+2 and ≤-2. Further on considering the significance at FDR <0.3 and p-value <0.05, 47 genes were differentially regulated, with 27 upregulated and 20 downregulated genes in OTB patients. MIR581, RN7SKP14, MIR4762, SPPR3, MIR3181 were the top upregulated genes that were expressed in at least 5/6OTB samples. Notably, RN7SKP14 was present in all 6 TB samples, while MIR581 and MIR4762 were detected in 5 TB samples and none of the non-TB samples. This indicated their specificity towards OTB. Further studies are going on to validate these transcripts as biomarkers for OTB.
Conclusions :
MIR581, MIR4762 and RN7SKP14 have potential to be used as biomarkers in OTB and can be exploited to develop a molecular diagnostic test for ocular TB as an adjunct to clinical workup.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.