Polymerase Chain Reaction and Goldmann-Witmer Coefficient to Examine the Role of Epstein-Barr Virus in Uveitis

Purpose: To use polymerase chain reaction (PCR) and Goldmann-Witmer Coefficient (GWC) calculation to search for evidence that Epstein-Barr virus (EBV) causes uveitis.

Methods: A prospective cross-sectional study where participants with positive multiplex EBV PCR results were further investigated by: 1) real-time PCR for EBV viral loads (VL) and 2) EBV GWC.

Results: Eleven of 106 consecutive uveitis patients (10.4%) had positive multiplex PCR for EBV on aqueous humor sampling and 7/11 (63.6%) were HIV-positive. Only 4/10 (40%) cases had detectable intraocular EBV VLs which were always lower than the blood or plasma VL. EBV GWC was negative in all 10 cases tested. In 9/11 (81.8%) of these cases an alternative, more plausible cause of uveitis was identified.

Conclusion: We found no evidence of active intraocular replication or antibody production to prove that EBV caused uveitis in these cases. In most cases an alternative treatable cause of uveitis was identified.     

Diagnostic Utility of Quantitative Polymerase Chain Reaction versus Culture in Endophthalmitis and Uveitis

Purpose: To compare genetic testing for microbes in infectious endophthalmitis or uveitis to culture.Methods: This was a retrospective, single-center case series that enrolled patients with clinically suspected endophthalmitis or uveitis of unknown etiology. Aqueous humor or vitreous was collected and sent for routine cultures and genetic testing.Results: In total, 46 patients were enrolled. Genetic testing was positive in 32/46 (70%) cases and culture 6/46 cases (13%). Five of 16 uveitis cases had a final clinical diagnosis of infectious uveitis, and polymerase chain reaction (PCR) was positive in 4/5 cases (80%), versus 0% for culture. In uveitis cases, PCR was 80% sensitive and 82% specific, and culture had 0% sensitivity. The overall sensitivity and specificity of PCR for all cases were 85% and 67%, respectively, compared with 17% and 100% for culture.Conclusion: Genetic assays are inexpensive ($25/case) and more sensitive than culture for identifying intraocular pathogens in endophthalmitis and uveitis.     

Real-Time Multiplex PCR Analysis in Infectious Uveitis

ABSTRACT

Introduction: Infectious uveitis is a serious inflammatory condition that often causes grave ocular morbidity including permanent vision loss and damage to the structures of the eye. The most common causes of infectious uveitis include herpesviruses and Toxoplasma gondii. Traditionally, these infections have been identified and differentiated based on characteristic clinical examination findings; however, there is often overlap between these presentations and the unique cause of a given patient’s infection is not always clear. Therefore, a reliable and fast method for definitively diagnosing infectious uveitis would be helpful and potentially sight-saving. Several groups have recently found experimental success with real-time multiplex polymerase chain reaction (PCR) techniques.

Methods: A comprehensive review of the literature was undertaken to further understand the current state of real-time multiplex PCR and its clinical use. Search terms including “real time multiplex PCR”, “infectious uveitis”, and “uveitis diagnosis” were used. Appropriate English-language articles were included in this review.

Results: Publications from four main groups (two from the United States, one from Japan, and one from India) citing success with real-time multiplex PCR were compared and contrasted. All four groups used the same technique to develop a highly sensitive and specific multiplex PCR analysis and found that their tests maintained high sensitivity and specificity during validation testing. These tests confirmed clinical suspicions in the majority of cases of infectious uveitis, but there were also cases of clinical misdiagnosis that were corrected based on molecular pathogen detection. These patients were then initiated on appropriate antimicrobial therapy with subsequent clinical improvement.

Discussion: Real-time multiplex PCR is a highly sensitive and specific laboratory assay that allows for rapid and reliable molecular diagnosis of causative agents in infectious uveitis. This in turn facilitates swift initiation of effective therapy and prevents long-term ocular damage and vision loss.     

Applications of the Polymerase Chain Reaction to Diagnosis of Ophthalmic Disease

The polymerase chain reaction (PCR) is a powerful molecular biologic technique for the analysis of very small amounts of DNA. This technique has found increasing use in the past 10 years for the detection of pathogenic organisms associated with many forms of ocular inflammatory and infectious disease. PCR has shown utility in the diagnosis of viral uveitis, infectious endophthalmitis, and parasitic eye disease. The strengths and weaknesses of this diagnostic technique are discussed. Additionally, uses of PCR in linking known pathogens to disease, and to discovering novel pathogens, are addressed.     

Immunoblot and Polymerase Chain Reaction to Diagnose Ocular Syphilis and Neurosyphilis in HIV-positive and HIV-negative Patients

ABSTRACT

Purpose: To evaluate immunoblot (IB) and polymerase chain reaction (PCR) to diagnose ocular- and neurosyphilis.

Methods: Prospective cross-sectional study. Aqueous humor (AH) and cerebrospinal fluid (CSF) samples were tested for treponemal DNA or antibodies to treponemal antigens.

Results: Thirteen of 106 cases had positive syphilis serology of which 69.2% were HIV+ (median CD4+ = 181 cells/µL). Four cases met CDC criteria for neurosyphilis (3 confirmed, 1 probable) and 2 additional cases required neurosyphilis treatment according to UpToDate algorithms. All AH and CSF samples tested PCR negative. Five cases were CSF IB+ and 3 cases AH IB+. Using our classification, eight patients had confirmed neurosyphilis, one had probable neurosyphilis, three had confirmed ocular syphilis and nine had probable ocular syphilis.

Conclusion: Our findings suggest that IB of AH and CSF provides additional evidence to diagnose ocular and neurosyphilis and allows us to classify them as probable or confirmed.     

Patterns of Uveitis at Two University-Based Referral Centres in Cape Town,South Africa

Purpose: To analyze the pattern of uveitis at two tertiary hospitals in South Africa which has a high prevalence of HIV, TB and syphilis.

Methods: Data of 198 patients were obtained retrospectively between August 2014 and August 2016, including patient demographics, clinical examination, special investigations and final diagnosis.

Results: Infectious uveitis was the most common aetiological category (47%), followed by idiopathic (34.8%) and non-infectious (18.2%). Syphilis was the most common identifiable cause (16.2%). Other important causes were toxoplasmosis, herpes viruses, tuberculosis and HLA-B27. HIV positive patients, who constituted 40% of the study population, were more likely to present with a posterior or panuveitis (relative risk 1.50, 95% CI 1.19–1.89) and more likely to have an infectious cause compared to HIV negative patients (relative risk 2.47, 95% CI 1.82–3.35).

Conclusions: This study emphasizes the importance of HIV testing and investigations for infectious causes of uveitis, especially syphilis, in this population.     

The Etiology of Intraocular Inflammation in HIV Positive and HIV Negative Adults at a Tertiary Hospital in Cape Town,South Africa

Purpose: To describe the patterns of uveitis in South Africa.

Methods: Prospective cross-sectional study.

Results: One hundred and six patients were enrolled and 37.7% had human immune-deficiency virus (HIV) infection. Anterior and panuveitis occurred most frequently. Infectious, non-infectious and idiopathic uveitis were diagnosed in 66.0%, 17.0% and 17.0% of all cases, respectively. Eighty percent of HIV+ cases had infectious uveitis. Overall, intraocular tuberculosis (IOTB), herpetic and syphilitic uveitis were the commonest infectious causes. Sarcoidosis and HLA-B27-associated uveitis were the commonest non-infectious causes. In anterior uveitis, HIV+ cases most frequently had probable IOTB, syphilitic or idiopathic uveitis while HIV- cases had possible IOTB, idiopathic or HLA-B27-associated uveitis. In panuveitis, HIV+ cases mostly had syphilis, probable IOTB, toxoplasma and varicella-zoster virus whereas HIV- cases mostly had possible IOTB, sarcoidosis and idiopathic uveitis.

Conclusion: Infectious uveitis is common in South Africa, especially amongst HIV+ patients. Causes of anterior and panuveitis differ between HIV+ and HIV- patients.     

The Collaborative Ocular Tuberculosis Study (COTS)-1 Report 3: Polymerase Chain Reaction in the Diagnosis and Management of Tubercular Uveitis: Global Trends

Purpose: To analyze the role of polymerase chain reaction (PCR) of ocular fluids in management of tubercular (TB) anterior, intermediate, posterior, and panuveitis.

Methods: In Collaborative Ocular Tuberculosis Study (COTS)-1 (25 centers, n = 962), patients with TB-related uveitis were included. 59 patients undergoing PCR of intraocular fluids (18 females; 53 Asian Indians) were included.

Results: 59 (6.13%) of COTS-1 underwent PCR analysis. PCR was positive for Mycobacterium TB in 33 patients (23 males; all Asian Indians). 26 patients were PCR negative (18 males). Eight patients with negative PCR had systemic TB. Anti-TB therapy was given in 18 negative and 31 PCR cases. At 1-year follow-up, five patients with positive PCR (15.15%) and three with negative PCR (11.54%) had persistence/worsening of inflammation.

Conclusions: Data from COTS-1 suggest that PCR is not commonly done for diagnosing intraocular TB and positive/negative results may not influence management or treatment outcomes in the real world scenario.     

Liver Function Testing Is Not Useful in the Diagnosis of Sarcoidosis in Patients Presenting with Uveitis

Purpose: To elucidate the usefulness of abnormal liver function tests in the diagnosis of sarcoidosis in patients presenting with ocular inflammation.

Methods: Retrospective comparison of sample populations of 100 patients each with sarcoidosis-associated uveitis (SAU) and non-sarcoid uveitis controls.

Results: Number of abnormal results between SAU and control groups were: (1) raised alkaline phosphatase 6:2; (2) raised alanine aminotransferase 21:19; (3) raised total protein 14:5; (4) hypoalbuminemia 0:7; (5) raised bilirubin 1:2. The only parameters reaching statistical significance were in (3), using any elevated result; and (4), the greater risk being in controls.

Conclusions: There is no evidence that abnormal liver function tests are an indicator of sarcoidosis in new patients presenting with uveitis.     

Estimating Community Prevalence of Ocular Chlamydia trachomatis Infection using Pooled Polymerase Chain Reaction Testing

Abstract

Purpose: Trachoma is the leading cause of blindness from infection worldwide. Treatment programs require accurate Chlamydia trachomatis infection prevalence rates to guide decision making. The use of clinical examination is by far the most common way to monitor activity, but may yield overestimates of infection prevalence. Laboratory testing on individual specimens such as polymerase chain reaction (PCR) is highly sensitive and specific, but prohibitively expensive. Here we demonstrate simulations of pooled PCR results may estimate infection prevalence of an entire community yielding substantial cost savings if pool size is chosen correctly.

Methods: Community infection prevalence was estimated using maximum likelihood estimation with data collected from a previously described study. Simulations for communities were performed to determine the accuracy of prevalence estimation using pooled results. The root mean squared error was then used to determine an acceptable inaccuracy in estimates allowing for a pooling strategy to be formed.

Results: Results from simulations and empirical data suggest optimum pooling strategies to estimate community infection prevalence while keeping the root mean squared error of the estimate below 2%. Reduction of PCR testing which permits cost savings is shown to be between 5 and 80% given a community infection prevalence below 60%.

Conclusions: Pooling specimens for PCR testing often provides enough data to accurately estimate infection prevalence at the community level.     

Polymerase Chain Reaction and its Correlation with Clinical Features and Treatment Response in Tubercular Uveitis

Purpose: Correlation of results of polymerase chain reaction for Mycobacterium tuberculosis (MTB PCR) with clinical features and treatment response in tubercular uveitis.

Methods: Retrospective case study.

Results: Among 56 patients, 31 (55.3%) had acute and 25 (44.6%) had chronic uveitis. Uveitis was unilateral in 40 (71.4%) and bilateral in the remaining 16 (28.6%). Anatomical subtypes of uveitis were: anterior in 10 (13.9%) eyes, intermediate in 9 (12.5%), posterior in 17 (23.6%), and pan uveitis in 36 (50%) eyes. MTB PCR was positive in 24 patients. There was an 80% correlation between clinical response to antitubercular therapy (ATT) and PCR results. Twenty-two patients with positive PCR had a good clinical response. The sensitivity and specificity was 73.3% and 92.3%, respectively.

Conclusions: The diagnosis of intraocular TB requires strong clinical suspicion with corroborative laboratory and radiological evidence. A positive PCR is reliable whereas negative results should be correlated with clinical features. An adequate response to ATT supports PCR results.     

Achromobacter xylosoxidans Endophthalmitis Diagnosed by Polymerase Chain Reaction and Gene Sequencing

Purpose: To report a patient with Achromobacter xylosoxidansendophthalmitis that was diagnosed using polymerase chain reaction and gene sequencing. Methods: Case report. A patient with culture-negative endophthalmitis underwent an anterior chamber tap. Polymerase chain reaction was performed on the aqueous sample using the 16S ribosomal DNA primer set to detect and amplify bacterial DNA. The amplified DNA was sequenced and compared to archived sequences in a gene library using the BLAST search program. Results: A 214-base pair gene sequence was amplified and matched with the gene sequence for A. xylosoxidans. Antimicrobial treatment was instituted with resolution of hypopyon, anterior chamber cells, and vitreous cells one month after treatment. Conclusions: Polymerase chain reaction of 16S rDNA combined with gene sequencing may be an alternative method of diagnosing culture-negative bacterial endophthalmitis.     

Comparative Quantification of Plasma TDRD7 mRNA in Cataract Patients by Real-time Polymerase Chain Reaction

Purpose

To investigate the relationship between plasma TDRD7 mRNA and lens transparency, and to evaluate plasma TDRD7 mRNA as a potential marker for cataracts and its sub-type by quantitatively analyzing human peripheral blood.

Methods

Plasma RNA was extracted from 40 patients with cataracts, and 30 normal controls of matched age and gender. Blood cholesterol and fasting glucose were measured, and the RNA extracted from the sample was synthesized into cDNA. After polymerase chain reaction, the results were compared after quantifying the TDRD7 mRNA using ABL1 mRNA for normalization. We analyzed the relative gene expression data via the ΔΔCt method.

Results

The normalized 2^-ΔΔCt of plasma TDRD7 mRNA based on ABL1 mRNA was 1.52 ± 0.63 in the case of the control group and 1.05 ± 0.34 in the case of the cataract patients, and the TDRD7 expression level of the cataract patients was lower than that of the control group (p = 0.048). The comparison of the genetic values of different types of cataracts demonstrated that the TDRD7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017).

Conclusions

Human cataracts and the TDRD7 gene loss-of-function mutations are strongly causally related, as the expression level of plasma TDRD7 mRNA in patients with cataracts was statistically significantly lower than in the normal control group.     

Polymerase chain reaction in the diagnosis of bacterial endophthalmitis

BACKGROUND—Microbiological investigations of vitreous fluid (VF) and aqueous humour (AH) specimens have often failed to detect the infecting agent in infectious endophthalmitis, resulting in a clinical dilemma regarding therapy. In this study, the polymerase chain reaction (PCR) was evaluated in the diagnosis of bacterial and Propionibacterium acnes endophthalmitis.
METHODS—58 intraocular specimens (30 VF and 28 AH) from 55 cases of endophthalmitis and 20 specimens (14 VF and 6 AH) as controls from non-infective disorders were processed for microbiological investigations. Nested PCR directed at the 16S rDNA using universal primers for eubacterial genome was done. PCR for P acnes was performed on specimens microbiologically negative by conventional techniques but eubacterial genome positive.
RESULTS—Of the 20 controls from non-infective cases, one (5%) was positive using eubacterial primers and none with P acnes primers. PCR for eubacterial genome showed 100% correlation with 20 (34.5%) bacteriologically positive specimens. Eubacterial genome, was detected in 17 (44.7%) of 38 bacteriologically negative specimens and nine (52.9%) out of the 17 were positive for P acnes genome. Among the 21 eubacterial PCR negative specimens, seven were fungus positive. By inclusion of PCR, microbiologically positive specimens increased from 46.5% to 75.8%. PCR on AH was as sensitive as that on VF for the detection of both eubacterial and the P acnes genome.
CONCLUSION—PCR performed on AH and VF is a reliable tool for the diagnosis of bacterial and P acnes endophthalmitis particularly in smear and culture negative specimens.

Keywords: polymerase chain reaction; bacterial endophthalmitis; infectious endophthalmitis     

聚合酶链反应在感染性葡萄膜炎诊断中的应用

聚合酶链反应（polymerase chain reaction,PCR）是一种有力的分子生物学工具,检测所需标本量少,耗时短,敏感性高,对于某些不典型的感染性葡萄膜炎,PCR能够在感染早期从极少量眼内液中检测出病原体的复制数量,提高诊断的效率。与传统的血清学抗体检测、病原微生物培养相比,PCR在辅助诊断方面表现出更大的优势。     

Correlation of Clinical Outcomes with Quantitative Polymerase Chain Reaction DNA Copy Number in Patients with Acute Retinal Necrosis

Purpose: To correlate visual acuity outcomes and clinical features with quantitative PCR DNA copy number in patients with acute retinal necrosis (ARN).

Methods: Retrospective, consecutive case series.

Results: In total, 14 eyes of 13 patients were diagnosed with ARN, based on the American Uveitis Society criteria, and were followed for a mean of 324.5 days (median 250.5 days, SD ± 214 days). Anterior chamber fluid analyzed by quantitative PCR identified viral DNA in 11 of 14 eyes (78.5%). Varicella zoster virus (VZV) was identified in seven eyes (50%) and herpes simplex virus (HSV) in four eyes (28.5%). Mean DNA copy number was 7.9 × 10⁶/mL (median 2.10 × 10⁶/mL, range: 0–5.60 × 10⁷/mL). Eyes with quantitative PCR DNA copy number of ≥5.0 × 10⁶/mL (n = 6 eyes) had worse baseline visual acuity (logMAR 1.48 ± 0.71 vs 0.94 ± 0.76, p = 0.196) and final visual acuity (logMAR 2.10 ± 0.60 vs 0.82 ± 0.81, p = 0.007) compared with patients with a DNA copy number <5.0 × 10⁶/mL (n = 8 eyes). Patients with a DNA copy number of ≥5.0 × 10⁶/mL were more likely to have at least 5 clock hours of retinitis on funduscopic exam (p = 0.03) and developed retinal detachment more frequently (p = 0.08).

Conclusions: Quantitative DNA copy number of ≥5.0 × 10⁶/mL is associated with more extensive retinitis, worse visual acuity, and development of retinal detachment in patients with acute retinal necrosis.     

Comparative Analysis of Polymerase Chain Reaction Assay for Herpes Simplex Virus 1 Detection in Tear

Purpose

To comparatively analyze the methodological efficacy of the polymerase chain reaction (PCR) assay for herpes simplex virus 1 (HSV) detection in tears.

Methods

This retrospective study reviewed the medical records of 115 patients who were clinically diagnosed with herpes keratitis, and their tear samples were collected for HSV detection. PCR positive rates were analyzed for their dependence on the PCR primers used (conventional PCR primer vs. nested PCR primer), the tear collecting method used (micropipetting vs. collection with schirmer strip), the disease manifestation and the patient''s previous medication history.

Results

HSV DNA was detected in 23 out of 115 (20%) tear samples. The PCR positive rate in tear samples did not differ depending on the PCR primer or tear collection method used. Typical epithelial lesions showed a higher positive rate (31.4%) than atypical epithelial lesions (10.9%). The previous history of the antiviral agent seemed to affect the PCR positive rate.

Conclusions

Although the PCR positive rate was not dependent on the tear collection method or primers, HSV detection in tears using PCR was shown to be a supplementary diagnostic test in typical and atypical herpes epithelitis.     

用聚合酶链反应检测单疱病毒性角膜炎静止期角膜内HSV基因

目的:单疱病毒性角膜炎是主要的致盲眼病之一,近年来研究认为HSV可在角膜内潜伏,但未获结论性证据,本研究目的为明确角膜是否为HSV的另一潜伏地。方法:选择18例已确认为HSK病例,所有病例角膜病变静止3个月以上(平均3.7年),行部分穿透性角膜移植术取下角膜片,用聚合酶链反应方法(引物为P15’CATCACCGACC-CGGAGAGGGAC,P2 5’GGGCCAGGCGCTTGGTGTA)检测角膜片内HSV基因。结果:18例静止期HSK病例角膜片中,用聚合酶链反应方法在17例病例中检测出HSV基因,1例阴性。结论:单疱病毒性角膜炎静止期角膜内有HSV基因存留,角膜可能为HSV的另一潜伏地,用聚合酶链反应检测角膜内HSV DNA方法简单,敏感性高。眼科学报 1995;11:183—185。