Effectiveness of real-time PCR for diagnosis and prognosis of varicella-zoster virus keratitis

Kandori-Inoue

et al. 2021

IJMS

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Senescence, sterile inflammation, and infection cause dysfunction of corneal endothelial cells, leading to visual morbidity that may require corneal transplantation. With increasing age, the extracellular matrix is modified by non-enzymatic glycation forming advanced glycation end products (AGEs). The modifications are primarily sensed by the receptors for the AGEs (RAGE) and are manifested as a type I interferon response. Interestingly, in our study, human corneal endothelial cells (HCEn) cells did not respond to the typical RAGE ligands, including the AGEs, high mobility group box 1 (HMGB1), and serum amyloid-A (SAA). Instead, HCEn cells responded exclusively to the CpG DNA, which is possessed by typical corneal pathogen, herpes simplex virus-1 (HSV-1). Upon HSV-1 infection, the surface expression of RAGE was increased, and endocytosed HSV-1 was associated with RAGE and CpG DNA receptor, TLR9. RAGE DNA transfection markedly increased interferon-β secretion by CpG DNA or HSV-1 infection. HSV-1 infection-induced interferon-β secretion was abolished by TLR9 inhibition and partially by RAGE inhibition. Global transcriptional response analysis confirmed that RAGE and TLR9 were both significantly involved in type I interferon responses. We conclude that RAGE is a sensor of HSV-1 infection and provokes a type I interferon response.

Section: Discussionmentioning

confidence: 99%

Role Played by Receptors for Advanced Glycosylation End Products in Corneal Endothelial Cells after HSV-1 Infection

Kandori-Inoue

et al. 2021

IJMS

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“…For the exclusion of HSV infections, a history of dendritic lesions or recurring stromal keratitis should be considered. A history of herpes zoster ophthalmicus highly suggests the presence of ocular VZV infection [ 16 ] [ 17 ]. Thus, the exclusion of HSV and VZV may also be achieved by the history, and DNA detection is not required.…”

Section: Discussionmentioning

confidence: 99%

Diagnostic efficacy of real-time PCR for ocular cytomegalovirus infections

Graefes Arch Clin Exp Ophthalmol

et al. 2018

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PurposeThe aim of this study is to determine the efficacy of quantitative real-time PCR (qPCR) and clinical characteristics to diagnose ocular cytomegalovirus (CMV) infections.MethodsThe technical factors were assessed by the outcomes of the qPCR assay at five institutions in Japan using the WHO International Standard of cytomegalovirus. The clinical factors were assessed by examining the aqueous humor samples of 197 eyes of 197 consecutive patients suspected of CMV using the receiver operating characteristics (ROCs).ResultsAll of the institutions had excellent detection efficacy, although the copy number ranged from 0.82 to 4.66 copies/IU. In the clinical samples, CMV was detected in 51 eyes, and the amount of CMV DNA was significantly higher for CMV retinitis. In corneal diseases, the amount of CMV DNA was significantly associated with frequency of recurrences and IOP elevations. The sensitivity and specificity of qPCR for the diagnosis was 90.0 and 98.7%, respectively. For the corneal and anterior uveitis types of CMV diseases, the area under the curve (AUC) of qPCR was 0.95 and 0.96, followed by frequency of recurrences with AUC of 0.89 and 0.82, and IOP elevations with AUC of 0.78 and 0.76. Unclassified cytomegalovirus detection, which did not meet diagnostic criteria of CMV corneal endotheliitis, anterior uveitis, or retinitis, was 4.6%, and it was significantly associated with corneal diseases and history of corneal transplantation.ConclusionsqPCR with standardization is specific and accurate; however, the inclusion and knowledge of the clinical characteristics improve the diagnostic efficacy.

“…The number of 16S r-DNA copies was calculated using a standard curve generated by serial dilutions of cloned DNA fragments of 16S r-DNA [12]. The number of 16S r-DNA copies of the bacterial DNA was converted to logarithmic (log 10 ) units, and it was adjusted by the logarithm of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) copy numbers [13].…”

Section: Broad-range Quantitative Real-time Pcrmentioning

confidence: 99%

Effectiveness of 16S ribosomal DNA real-time PCR and sequencing for diagnosing bacterial keratitis

Graefes Arch Clin Exp Ophthalmol

Ehara

et al. 2019

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Purpose To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis. Methods We studied 272 eyes of 272 patients with keratitis. Eyes with keratitis were classified as "definite" (N = 118), "likely" (N = 71), or "non-bacterial" (N = 83) to have bacterial keratitis. The diagnostic efficacy of real-time PCR and conventional testing was determined by receiver operating characteristic analysis. The copy numbers of bacterial DNA and clinical characteristics were retrospectively analyzed for association with concordant culture results in the "definite" cases. Results The level of bacterial DNA was significantly associated with the diagnostic probability of the three diagnostic categories. The level of bacterial DNA had comparable diagnostic efficacy with the area under the curve (AUC) at 0.67, by culture at 0.65, and by smear testing at 0.73. The efficacy was significantly improved by combining the DNA level with the conventional culture testing with an AUC of 0.81. Analysis of the "definite" cases showed culture positivity in 51.8% (58 eyes), and of these, 41 eyes (70.7%) were higher than the cutoff PCR values and 40 eyes were identified by 16S r-DNA sequencing. In the culture-negative eyes, the level of bacterial DNA was significantly lower (P = 0.0008). Eyes with higher bacterial DNA levels had significantly concordant outcomes with sequencing and culture results (P = 0.006). Previous antibiotic treatments decreased the bacterial DNA amount by 0.09-fold, and it was a significant factor for discordance (P = 0.006). Conclusion Quantification of the bacterial DNA level and conventional testing improves the diagnostic efficacy of infectious bacterial keratitis.