scholarly journals Relationships between the clinical characteristics and copy numbers of DNA of cytomegalovirus determined by real-time PCR

2020 ◽  
Vol 40 (9) ◽  
pp. 2297-2305
Author(s):  
Ayano Yoshimura ◽  
Kaoru Araki-Sasaki ◽  
Noriko Toyokawa ◽  
Rho Fujiwara ◽  
Nobuo Jho ◽  
...  
Keyword(s):  
Real Time ◽  
Aqueous Humor ◽  
Clinical Characteristics ◽  
Topical Steroid ◽  
Corneal Edema ◽  
Copy Numbers ◽  
Keratic Precipitates ◽  
Oral Valganciclovir ◽  
Clinical Phenomenon ◽  
Corneal Endotheliitis

Abstract Purpose To determine whether there is a correlation between the clinicals characteristics including various types of keratic precipitates and the copy numbers of the DNA of cytomegalovirus (CMV) in eyes with CMV corneal endotheliitis. Methods We reviewed the medical charts of four cases of corneal endotheliitis that were CMV-positive. We have classified types of clinical phenomenon into four types: coin-shaped KPs, sectoral corneal edema with or without Khodadoust line-like KPs, mutton-fat KPs, and fine KPs and have graded their severity. We also determined the copy numbers of the DNA of CMV in the aqueous humor by real-time polymerase chain reaction before and during the treatment. We evaluated the correlation between the patterns of clinical characteristics and copy number of the DNA of CMV. Results There were clinical improvements in all eyes following topical ganciclovir in conjunction with low dose of topical steroid treatment, with or without oral valganciclovir. The clinical characteristics and the copy numbers of the DNA of CMV varied during the treatment period. The presence of coin-shaped KPs was correlated with high copy numbers (105–103 copies/ml) of the DNA of CMV. The copy numbers of the DNA of CMV with sectoral corneal edema with or without Khodadoust line-like KPs ranged from 104 to 102 copies/ml, and it was occasionally accompanied by high intraocular pressure. Mutton-fat KPs were observed inferiorly, sometimes together with coin-shaped KPs and sectoral corneal edema, or solely. The copy numbers in eyes with mutton-fat KPs varied and occasionally less than the cutoff level. Fine-pigmented KPs were observed after the resolution of the endotheliitis, and no DNA of CMV was detected in the aqueous humor. Conclusions Careful observations of the clinical characteristics such as the KPs and corneal edema might be helpful in estimating the amount of the DNA of CMV in eyes with corneal endotheliitis.

scholarly journals Diagnosis of cytomegalovirus corneal endotheliitis using surgically removed Descemet’s membrane and endothelium despite negative results with aqueous humor PCR: a case report

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Suguru Nakagawa ◽  
Hitoha Ishii ◽  
Mitsuko Takamoto ◽  
Toshikatsu Kaburaki ◽  
Kiyoshi Ishii ◽  
...  
Keyword(s):  
Case Report ◽  
Aqueous Humor ◽  
Clinical Manifestations ◽  
Corneal Edema ◽  
Bullous Keratopathy ◽  
Varicella Zoster ◽  
Negative Results ◽  
Keratic Precipitates ◽  
The Right ◽  
Corneal Endotheliitis

Abstract Background Cytomegalovirus (CMV) has been known to cause unilateral corneal endotheliitis with keratic precipitates and localized corneal edema, iridocyclitis, and secondary glaucoma. CMV endotheliitis is diagnosed based on clinical manifestations and viral examination using qualitative polymerase chain reaction (PCR) of the aqueous humor. Case presentation An 80-year-old woman was referred to our department for bullous keratopathy. Pigmented keratic precipitates were found in the right eye without significant anterior chamber inflammation. After 8 months there was inflammation relapse with mutton fat keratic precipitates and PCR on aqueous humor was performed, with negative results for CMV, herpes simplex virus, and varicella zoster virus. Keratic precipitates disappeared with steroid instillation, and Descemet-stripping automated endothelial keratoplasty (DSAEK) was performed for the right eye. CMV-DNA was positive at 6.0 × 102 copies/ GAPDH 105 copies in real time PCR of corneal endothelial specimen removed during DSAEK with negative results for all the other human herpes viruses. After diagnosis of CMV corneal endotheliitis, treatment with systemic and topical ganciclovir was initiated and there was resolution of symptoms. No recurrence of iridocyclitis or corneal endotheliitis was observed at 6 months follow up. Conclusions This case report suggests that PCR should be performed using the endothelium removed during DSAEK for bullous keratopathy of an unknown cause, even if PCR for aqueous humor yields negative results.

scholarly journals Aqueous Angiography: Real-Time and Physiologic Aqueous Humor Outflow Imaging

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0147176 ◽  
Author(s):  
Sindhu Saraswathy ◽  
James C. H. Tan ◽  
Fei Yu ◽  
Brian A. Francis ◽  
David R. Hinton ◽  
...  
Keyword(s):  
Real Time ◽  
Aqueous Humor ◽  
Aqueous Humor Outflow

scholarly journals Should Calibration Curve Retire From Real-Time PCR Assays?

2021 ◽  
Author(s):  
David An
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Dyes ◽  
Historical Data ◽  
Genetic Disorder ◽  
Calibration Curves ◽  
Copy Numbers ◽  
Biological Technique ◽  
Pcr Assays ◽  
Number Of Cycles

Abstract Real-time polymerase chain reaction (real-time PCR) is a biological technique that collects data of target nucleotides as PCR occurs by integrating fluorescent dyes as visual indicators into the amplification cycles. This enables the detection and quantification of the DNA segments in a sample through measurements of the fluorescent's intensity. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to pass a specified threshold and is inverse to the copy number, the initial number of nucleotides in the sample. Calibration curves are commonly used to approximate the copy numbers of experimental samples using standards with known copy numbers. This study is a retrospective review of historical data to help evaluate the efficacy and accuracy of calibration curves in a real-time PCR assay which have been used for screening of a genetic disorder in laboratories. The hypothesis is that including calibration curves in real-time PCR assays may decrease the screening specificity and accuracy, resulting in more false positives and additional retests. Three different scenarios were designed to replay the historical data and evaluate the relative accuracy of assays without calibration curves. The outcomes of all the scenarios conclude that calibration curves are not helpful for detecting target DNA fragments with low copy numbers, suggesting a reconsideration of their implantation in real-time PCR assays.

scholarly journals Peritoneal Effluent MicroRNA Profile in Encapsulating Peritoneal Sclerosis

2021 ◽  
Author(s):  
Kun-Lin Wu ◽  
Che-Yi Chou ◽  
An-Lun Li ◽  
Chien-Lung Chen ◽  
Jen-chieh Tsai ◽  
...  
Keyword(s):  
Real Time ◽  
Mirna Expression ◽  
Real Time Pcr ◽  
Clinical Characteristics ◽  
Area Under The Curve ◽  
Noninvasive Test ◽  
Candidate Mirnas ◽  
Microrna Profile ◽  
Auc Value ◽  
Diagnostic Technologies

Abstract Encapsulating peritoneal sclerosis (EPS) is a catastrophic complication of chronic peritoneal dialysis (PD). Late diagnosis is associated with high mortality. With the advancement of new diagnostic technologies, such as microRNA (miRNA), we attempted to develop a noninvasive test to assist in the diagnosis of EPS. The eight-hour PD effluents were collected from 71 non-EPS and 56 EPS patients. The screening set included 28 samples (20 of non-EPS vs. 8 of EPS). After analyzing the ratio values of two miRNA expression levels from the high-throughput real-time PCR-array of 377 miRNAs, eight candidate miRNAs were selected. The prediction model was conducted using 127 samples (71 of non-EPS vs 56 of EPS) to produce an area under the curve (AUC) value of the miRNA classifier. Candidate miRNAs were also verified by single real-time PCR. The ratios of the five miRNAs with the top five ROC values were selected to calculate the combined AUC by multiple logistic regression. The AUC value to detect EPS with the five miRNA ratios was 0.8929 with an accuracy of 78.7%. The accuracy of the EPS diagnosis was further optimized to 94.1% after considering clinical characteristics (AUC value 0.9931). A signature-based model of clinical characteristics and miRNA expression in PD effluents can efficiently assist in the diagnosis of EPS, thus preventing the catastrophic prognosis.

scholarly journals Co-infection of bovine papillomavirus type-1 and -10 in teat warts of dairy cattle

2013 ◽  
Vol 58 (No. 12) ◽  
pp. 605-608
Author(s):  
P. Kumar ◽  
BL Jangir ◽  
G. Saikumar ◽  
R. Somvanshi
Keyword(s):  
Dairy Cattle ◽  
Real Time ◽  
Real Time Pcr ◽  
Rice Grain ◽  
Bovine Papillomavirus ◽  
Viral Dna ◽  
Specific Primers ◽  
Copy Numbers ◽  
Pcr Techniques

The present study was carried out to investigate the involvement of different bovine papillomaviruses in the teat warts of cattle. A total of 11 teat wart samples showing rice grain-like and small, sessile elevated greyish or flesh-like growths were collected from dairy cattle. DNA was extracted from these teat wart samples and PCR and real time PCR techniques were applied using specific primers for BPV-1 and -10 to detect the presence of viral nucleic acid. PCR revealed the presence of viral DNA of BPV-1 and -10 in three and seven samples, respectively. Quantification using real time PCR revealed that the copy numbers of the viral DNA of BPV-1 and -10 DNA varied from 1.12E + 04 to 2.99E + 04 and 3.56E + 02 to 5.23E + 06, respectively. From the present study it can be concluded that BPV-1 and -10 are involved in production of rice grain-like and sessile elevated growths on the teats of cattle.

scholarly journals Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System

2020 ◽  
Vol 8 (1) ◽  
pp. 14
Author(s):  
Shengwen Calvin Li ◽  
Kara J. Sparks ◽  
Leonard S. Sender
Keyword(s):  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Range ◽  
Measurement Range ◽  
Quantitative Detection ◽  
Clinical Settings ◽  
Stem Cell Therapies ◽  
Cell Therapies ◽  
Copy Numbers

Allogenic stem-cell therapies benefit patients in the treatment of multiple diseases; however, the side effects of stem-cell therapies (SCT) derived from the concomitant use of immune suppression agents often include triggering infection diseases. Thus, analysis is required to improve the detection of pathogen infections in SCT. We develop a polymerase chain reaction (PCR)-based methodology for the qualitative real-time DNA detection of cytomegalovirus (CMV), with reference to herpes simplex virus types 1 (HSVI), Epstein–Barr virus (EBV), and varicella-zoster virus (VZV) in blood, urine, solid tissues, and cerebrospinal fluid. This real-time PCR of 96-well plate format provides a rapid framework as required by the Food and Drug Administration (FDA) for clinical settings, including the processing of specimens, reagent handling, special safety precautions, quality control criteria and analytical accuracy, precisely reportable range (analyst measurement range), reference range, limit of detection (LOD), analytical specificity established by interference study, and analyte stability. Specifically, we determined the reportable range (analyst measurement range) with the following criteria: CMV copies ≥200 copies/mL; report copy/mL value; CMV copies ≤199 copies/mL; report detected but below quantitative range; CMV copies = 0 with report <200 copies/mL. That is, with reference range, copy numbers (CN) per milliliter (mL) of the LOD were determined by standard curves that correlated Ct value and calibrated standard DNA panels. The three repeats determined that the measuring range was 1E2~1E6 copies/mL. The standard curves show the slopes were within the range −2.99 to −3.65 with R2 ≥ 0.98. High copy (HC) controls were within 0.17–0.18 log differences of DNA copy numbers; (2) low copy (LC) controls were within 0.17–0.18 log differences; (3) LOD was within 0.14–0.15 log differences. As such, we set up a fast, simple, inexpensive, sensitive, and reliable molecular approach for the qualitative detection of CMV pathogens. Conclusion: This real-time PCR of the 96-well plate format provides a rapid framework as required by the FDA for clinical settings.

scholarly journals Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

2012 ◽  
Vol 78 (10) ◽  
pp. 3630-3637 ◽  
Author(s):  
Karol Krak ◽  
Martina Janoušková ◽  
Petra Caklová ◽  
Miroslav Vosátka ◽  
Helena Štorchová
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Glomus Intraradices ◽  
Large Subunit ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Content Type ◽  
Copy Numbers ◽  
Pcr Assays

ABSTRACTReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates ofGlomus intraradicessensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

scholarly journals Photo-Induced Electron Transfer Real-Time PCR for Detection ofPlasmodium falciparum plasmepsin 2Gene Copy Number

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Samaly Santos Souza ◽  
Mariangela L'Episcopia ◽  
Carlo Severini ◽  
Venkatachalam Udhayakumar ◽  
Naomi W. Lucchi
Keyword(s):  
Electron Transfer ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Gene Copy ◽  
Content Type ◽  
Copy Numbers ◽  
Ofplasmodium Falciparum ◽  
Photo Induced Electron Transfer ◽  
Gene Copy Numbers

ABSTRACTPiperaquine is an important partner drug used in artemisinin-based combination therapies (ACTs). An increase in theplasmepsin 2and3gene copy numbers has been associated with decreased susceptibility ofPlasmodium falciparumto piperaquine in Cambodia. Here, we developed a photo-induced electron transfer real-time PCR (PET-PCR) assay to quantify the copy number of theP. falciparumplasmepsin 2gene (PfPM2) that can be used in countries whereP. falciparumis endemic to enhance molecular surveillance.

scholarly journals Clinical analysis of microbiologically proven fungal keratitis according to prior topical steroid use: a retrospective study in South Korea

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Chan-Ho Cho ◽  
Sang-Bumm Lee
Keyword(s):  
Risk Factors ◽  
Treatment Failure ◽  
Treatment Outcomes ◽  
Clinical Characteristics ◽  
Significant Risk ◽  
Topical Steroid ◽  
Clinical Analysis ◽  
Fungal Keratitis ◽  
Topical Steroids ◽  
Deep Infiltration

Abstract Background To compare the clinical characteristics and treatment outcomes of microbiologically proven fungal keratitis between users and non-users of prior topical steroids (PS and NPS, respectively). Methods Eighty-three cases with microbiologically proven fungal keratitis between January 2000 and December 2016 were reviewed retrospectively. Diagnosis of fungal keratitis was made through potassium hydroxide smear, culture, PCR, or biopsy. Baseline epidemiology, predisposing factors, clinical characteristics, microbiological profiles, and treatment outcomes were evaluated and compared between the PS and NPS groups. Treatment failure was defined as any case with complications or requiring surgery. The risk factors for treatment failure were evaluated using multivariate logistic regression in the overall cohort. Results A total of 30 cases with PS group and 53 cases with NPS group were included. Of these, sixteen fungal isolates were identified in the PS group and 14 isolates in the NPS group. Candida was the most common organism in both groups (6 cases, respectively), while Aspergillus (4 cases) was found only in the PS group (p = 0.103). No significant differences were observed in the mean age, sex, occupational distribution, epithelial defect size, hypopyon, and presenting best-corrected visual acuity (BCVA) between the two groups. Differences were observed between the PS and NPS groups in terms of previous ocular surface disease (OSD) (43.3% vs. 22.6%, p = 0.048) and deep infiltration (53.3% vs. 32.1%, p = 0.057). Regarding treatment outcomes, final BCVA < 0.1 (60% vs. 44.2%, p = 0.133), the use of voriconazole (topical 10% vs. 0%, p = 0.044; systemic 23.3% vs. 1.9%, p = 0.003), surgical intervention (43.3% vs. 20.8%, p = 0.029), and treatment failure (46.7% vs. 22.6%, p = 0.023) were more common in the PS group than in the NPS group. The significant risk factors for treatment failure were hypopyon (odds ratio [OR] 6.01, p = 0.005) and deep infiltration (OR 4.38, p = 0.013). Conclusions Previous OSD and deep infiltration were more common in the PS group compared to the NPS group. The PS group also experienced worse disease progression and treatment outcomes. These results highlight the need for paying attention to the use of steroids in clinical practice.

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