Cycle Threshold Data Research Articles

Examining Cycle Threshold values in Repeat SARS-CoV-2 Cases: A Retrospective Analysis in Ireland

Abstract Background Effective public health management during the COVID-19 pandemic requires accurate differentiation between re-infections and residual viral RNA detections in repeat positive cases. This study investigates cycle threshold (CT) trends in repeat SARS-CoV-2 cases to discern between persistent viral remnants and active infections. Collaboration between contact tracing and laboratories is essential for shaping robust public health strategies. Methods A retrospective analysis was conducted on 427 individuals with repeat positive PCR tests. Data was collected at a contact tracing centre from March to August 2021. Key variables including age, gender, and biomedical markers (test intervals, CT values) were examined. Regression and correlation techniques assessed CT trends and their implications. Results Younger individuals (<60 years) occasionally exhibited significantly lower CT values in subsequent tests, suggesting potential re-infections, while older adults generally displayed higher CT values indicative of residual RNA. These age-specific CT trends were statistically significant and instrumental in refining testing protocols and isolation guidelines. Further analysis is expected to strengthen these findings Conclusions Collaborative approaches between contact tracing and laboratories are vital in shaping robust public health strategies for distinguishing between types of repeat infections. Revision of health policies is imperative to prevent unnecessary isolation and optimise healthcare workforce allocation during ongoing transmission periods. Key messages • Decisive decision-making skills are crucial in managing repeat SARS-CoV-2 infections. • Revised health policies align testing and isolation practices with empirical CT data, enhancing public health efficacy.

Administration of Clinical COVID-19 Mouthwashing Protocol and Potential Modulation of Pediatric Oral Bacterial Prevalence of Selenomonas noxia: A Pilot Study.

Dental office protocols to combat the SARS-CoV-2 (COVID-19) pandemic include mouth washing for an extended 60 s, thereby reducing detectable oral virus. However, it is unclear whether this protocol has any effects on the newly identified periodontal pathogen and obesity-related bacterium often found among pediatric patients, Selenomonas noxia. To determine if the mouthwash protocol has any measurable effect on S. noxia amongst pediatric patients, clinical pediatric saliva samples were obtained from pediatric patients during routine visits for clinical care and treatment. Using an approved protocol, two saliva samples were collected on the same visit before and after chlorhexidine mouthwash (Sample A, Sample B). The third sample (Sample C) was taken at the recall appointment-usually between two and eight weeks later. A total of n = 97 pre-mouthwash samples, and an equal number of matching post-mouthwash samples (n = 97) were collected, with a small number of matching recall samples (n = 36) that were subsequently collected and identified. The demographic composition of the study sample was analyzed using Chi square statistics. Sample DNA from the matching pre-, post-, and recall collections (Sample A, Sample B, and Sample C) was isolated and screened using qPCR and validated primers, which revealed that 11.1% (n = 4/36) from Sample A tested positive for S. noxia with 0% (n = 0/36) of Sample B testing positive and 13.9% (n = 5/36) of the recall (Sample C) testing positive. In addition, comparative analysis of the qPCR cycle threshold data revealed relatively lower expression (quantity) of S. noxia DNA among the recall samples, as determined by two-tailed t-tests (p=0.004). These data and results provide new evidence for the oral prevalence of S. noxia among pediatric patients, while also demonstrating that the COVID-19 protocol of mouth washing prior to clinical treatment for periods extending up to 60 s may be sufficient to reduce the levels of detectable S. noxia-at least temporarily. More research will be needed to determine whether these effects may be limited to the short- or may exhibit more lasting effects in the long-term.

Abstract WP137: Mirna 206 Expression Is Associated With Perihematomal Edema Volume And Functional Outcomes After Intracerebral Hemorrhage

Introduction: Circulating micro-RNAs (miR) are reported in the pathophysiology of intracerebral hemorrhage. Previous reports suggested that miR-206, miR-486, miR-150 and miR-146a are associated with ICH outcomes. In this, study we explored the associated of miRNA expression with radiological predictors of outcome hematoma expansion (HE) and perihematomal edema volume (PHEV) and functional outcomes. Methods: Patients (n=96) with acute spontaneous ICH were consented and blood samples were collected within 48 hours of ICH onset. Plasma miR were used to screen 752 well-characterized miR using locked nucleic acid (LNA) probe-based real-time PCR (ver3.3.5). Cycle threshold data were obtained using the regression method and global mean normalization to a universally expressed miR-92a was applied. Significance levels were assessed using the Mann-Whitney’s U-test and the Welch’s t-test (GenEx). Demographic, clinical data and Modified Rankin Score (MRS) were collected with follow-up calls at 30, 90, and 365-days from ICH onset. Hematoma and perihematomal edema volumes were calculated based on the ABC/2 formula at presentation, 24hrs, and 96hrs from ICH onset. Spearman’s rank correlation was used to determine the association of miRNA expression with HE (>30% increase in volume) and PHEV at onset, 24hrs and 96hrs. Simple logistic regression was used to determine the odds of survival at 30-days and favorable outcome at 90- and 365-days, defined as MRS < 3. Results: Overall, cohort mean age 60.2years (IQR 52-73), 57.1% were deep nuclei (thalamic and basal ganglia ICH), mean ICH vol. 38.4 (SD23.2). We observed a positive correlation with miR-206 and PHEV at 48-96hrs (rho 0.451, p=0.003). Expression of miR-206 was associated with a higher odds of survival at 30-days (OR 1.149 95% CI 1.016-1.299 p=0.0266) and a favorable outcome at 365-days (OR 1.135 95% CI 1.007-1.278 p=0.038). There was no association of miR-206 expression with HE. miR-486, miR-150, and miR-146a were not associated with radiological or functional outcomes. Conclusion: In our cohort, miR-206 was associated with PHEV at 96hrs, survival at 30days and favorable outcome at 1 year. Further studies are ongoing to further delineate the role of miR206 in ICH related biological mechanisms.

Evaluation of potential COVID-19 recurrence in patients with late repeat positive SARS-CoV-2 testing.

BackgroundSARS-CoV-2 reinfection and reactivation has mostly been described in case reports. We therefore investigated the epidemiology of recurrent COVID-19 SARS-CoV-2.MethodsAmong patients testing positive for SARS-CoV-2 between March 11 and July 31, 2020 within an integrated healthcare system, we identified patients with a recurrent positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) assay ≥60 days after an initial positive test. To assign an overall likelihood of COVID-19 recurrence, we combined quantitative data from initial and recurrent positive RT-PCR cycle thresholds—a value inversely correlated with viral RNA burden— with a clinical recurrence likelihood assigned based on independent, standardized case review by two physicians. “Probable” or “possible” recurrence by clinical assessment was confirmed as the final recurrence likelihood only if a cycle threshold value obtained ≥60 days after initial testing was lower than its preceding cycle threshold or if the patient had an interval negative RT-PCR.ResultsAmong 23,176 patients testing positive for SARS-CoV-2, 1,301 (5.6%) had at least one additional SARS-CoV-2 RT-PCRs assay ≥60 days later. Of 122 testing positive, 114 had sufficient data for evaluation. The median interval to the recurrent positive RT-PCR was 85.5 (IQR 74–107) days. After combining clinical and RT-PCR cycle threshold data, four patients (3.5%) met criteria for probable COVID-19 recurrence. All four exhibited symptoms at recurrence and three required a higher level of medical care compared to their initial diagnosis. After including six additional patients (5.3%) with possible recurrence, recurrence incidence was 4.3 (95% CI 2.1–7.9) cases per 10,000 COVID-19 patients.ConclusionsOnly 0.04% of all COVID-19 patients in our health system experienced probable or possible recurrence; 90% of repeat positive SARS-CoV-2 RT-PCRs were not consistent with true recurrence. Our pragmatic approach combining clinical and quantitative RT-PCR data could aid assessment of COVID-19 reinfection or reactivation by clinicians and public health personnel.

Cycle Thresholds Among Solid Organ Transplant Recipients Testing Positive for SARS-CoV-2.

The optimal duration of transmission-based precautions among immunocompromised patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. Retrospective review of patients with solid organ transplant with positive SARS-CoV-2 polymerase chain reaction result from nasopharyngeal specimens admitted to the hospital between March 13, 2020 and May 15, 2020. Twenty-one percent of solid organ transplant recipients with positive SARS-CoV-2 polymerase chain reaction detected ≥20 d after symptom onset (or after first positive test among asymptomatic individuals) had a low cycle threshold (ie, high viral load). The majority of these patients were asymptomatic or symptomatically improved. Solid organ transplant recipients may have prolonged high viral burden of SARS-CoV-2. Further data are needed to understand whether cycle threshold data can help inform strategies for prevention of healthcare-associated transmission of SARS-CoV-2 and for appropriate discontinuation of transmission-based precautions.

Predicting TB treatment outcomes using baseline risk and treatment response markers: developing the PredictTB early treatment completion criteria

Standard treatment of drug-sensitive pulmonary tuberculosis requires six months of treatment. Several randomized clinical trials have attempted to shorten treatment to four months using various strategies but thus far all have failed. The PredictTB trial is an ongoing international randomized clinical trial testing a treatment shortening strategy whereby only drug-sensitive pulmonary TB patients who meet the study early treatment completion criteria are randomized to four vs. six months of treatment. The PredictTB early treatment completion criteria were developed based on a cohort of 92 pulmonary tuberculosis patients treated programmatically through the local tuberculosis treatment program in Cape Town, South Africa, with FDG-PET/CT scans also performed at baseline and week 4 of treatment. Patients were followed for one year after the end of therapy for programmatic treatment outcomes. This methodology paper describes how the PET/CT scans and GeneXpert cycle threshold data of this cohort were analyzed to develop the early treatment completion algorithm currently being used in the PredictTB trial.

Measuring the Cost of Overtesting and Overdiagnosis of Clostridioides difficile Infection

Background:C. difficile is the leading healthcare-associated pathogen. The C. difficile real-time polymerase chain reaction (PCR) stool test, used by >70% of hospitals, is highly sensitive but cannot differentiate colonization from infection. Inappropriate C. difficile testing may result in overdiagnosis and unnecessary treatment. Healthcare costs attributed to C. difficile are substantial, but the economic burden associated with C. difficile false positives in colonized patients is poorly understood. C. difficile PCR cycle threshold (CT) is as an inverse proxy for organism burden; high CT (≥30.9) has a high (>98%) negative predictive value compared to the reference gold standard, thus is a marker of colonization. Conversely, a low CT (≤28.0) suggests high organism burden and high specificity for true infection. Methods: A propensity score matching model for cost per hospitalization was developed to determine the costs of a hospital stay associated with C. difficile and to isolate the financial impacts of both true C. difficile infection and false positives. Relevant predictors of C. difficile positivity used in the model were age, Charlson comorbidity index, white blood cell count, and creatinine. We used CT data to identify and compare 3 inpatient groups: (1) true CDI, (2) C. difficile colonization, and (3) C. difficile negative. Results: A diagnosis of C. difficile adds significantly (>$3,000) to unadjusted hospital cost compared to a negative result. Propensity-adjusted analyses demonstrated that C. difficile colonization was associated with significantly increased (median, $5,000) hospital cost whereas any positive or true diagnoses of C. difficile were not associated with increased cost. Colonized patients also had significantly higher lengths of stay (1 day) and cost per length of stay ($218 per day). Conclusions:This is the first C. difficile cost analysis to utilize PCR CT data to differentiate colonization. Surprisingly, patients with a high CT had disproportionately higher hospital costs compared to matched C. difficile–negative patients, which was not seen among patients with a low CT or with any positive result. We hypothesize that this unexpected finding may be due to misdiagnosis and mistreatment of diarrhea not caused by C. difficile or unadjusted factors associated with high cost and non–C. difficile diarrhea. In addition, the discrepantly high cost attributed to C. difficile diagnosis cited in the literature ($3,000–11,000 per hospitalized case) could be explained by the common use of administrative data to identify C. difficile cases and controls as opposed to our study, which directly linked cost data to C. difficile–positive and –negative test results.Funding: NoneDisclosures: None

Abstract B32: Longitudinal changes in plasma miRNA in patients with benign and malignant colorectal neoplasia

Abstract Introduction: The adenoma-carcinoma sequence is well-recognized as a stepwise pattern of mutational activation of oncogenes and inactivation of tumor suppressor genes resulting in sporadic colorectal cancer (CRC) development from colorectal adenomas (CRA) within the chromosomal instability pathway. MicroRNAs (miRNA) play an important role in oncogenesis by regulating gene expression and are known to be actively released from cells. They are found in body fluids such as plasma, saliva, feces and urine. We have previously demonstrated differential expression of plasma miRNAs in patients with CRC as compared to that of controls. Methods: Plasma was isolated from 10 patients: 5 patients with advanced colorectal adenoma (>0.6cm diameter) and 5 patients with stage II or III colorectal cancer prior to treatment and 4-6 weeks following endoscopic removal or surgical resection, respectively. RNA was extracted from plasma (Qiagen® miRNeasy), RT-PCR performed and 768 miRNAs were screened using microfluidic array technology (Applied BioSystems®) in pre- and post-treatment samples for each patient. Data were analyzed using paired t-tests after normalizing raw cycle threshold data to endogenous RNU6 for pre- and post-treatment samples. In addition, miRNA expression data from both pre- and post-treatment CRA and CRC samples were compared to that of plasma from 10 individuals without neoplasia (controls) (ANOVA). Results: Significant differential expression of plasma miRNAs was observed between pre- and post-treatment samples in both CRA and CRC groups. Interestingly, there was generalized miRNA upregulation in CRA when comparing pre-treatment samples to post-treatment samples. Conversely there was miRNA down-regulation in pre-treatment CRC plasma as compared to post-resection samples. Utilizing significantly dysregulated miRNAs, as well as p-values, AUC, fold-change and biological significance, miRNA panels were selected to facilitate detection of CRA recurrence (following complex polypectomy for large polyps) or for detection of recurrent CRC following resection. The resulting panel was able to differentiate between pre- and post-treatment samples for CRA (miR-186 and miR-623) with AUC 0.94 (95% CI 0.76 – 1.00) and for CRC (miR-324-5p, miR-30d and miR-766) with AUC 0.88 (95% CI 0.63 – 1.00). In addition, while miRNA expression of pre-treatment CRA and CRC samples differed significantly from controls, post-treatment CRA and CRC samples did not. Conclusion: Comparison of pre- and post-treatment plasma samples reveals differing patterns of changes in miRNA expression in benign colorectal neoplasms (CRA) as opposed to invasive neoplasia (CRC). The observed miRNA down-regulation prior to surgical resection of CRC might indicate decreased expression of miRNAs inhibiting oncogenes prior to cancer treatment, while the miRNA upregulation seen in pre-treatment CRA plasma could indicate inhibition of tumor suppressors. Such observations will perhaps elucidate the role of miRNA in carcinogenesis and may provide for a relatively non-invasive method of detecting such lesions. Further validation is needed to develop a miRNA panel that will allow monitoring for evidence of recurrent disease. Citation Format: Jane Carter, Vanessa States, Uri Netz, Jianmin Pan, Shesh Rai, Susan Galandiuk. Longitudinal changes in plasma miRNA in patients with benign and malignant colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B32.

Linear Methods for Analysis and Quality Control of Relative Expression Ratios from Quantitative Real-Time Polymerase Chain Reaction Experiments

Relative expression quantitative real-time polymerase chain reaction (RT-qPCR) experiments are a common means of estimating transcript abundances across biological groups and experimental treatments. One of the most frequently used expression measures that results from such experiments is the relative expression ratio (RE), which describes expression in experimental samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to one or more experimental or nonbaseline condition) in terms of fold change relative to calibrator samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to a control or baseline condition). Over the past decade, several models of RE have been proposed, and it is now clear that endogenous reference gene stability and amplification efficiency must be assessed in order to ensure that estimates of RE are valid. In this review, we summarize key issues associated with estimating RE from cycle threshold data. In addition, we describe several methods based on linear modeling that enable researchers to estimate model parameters and conduct quality control procedures that assess whether model assumptions have been violated.

Chemical Induction of Misfolded Prion Protein Conformers in Cell Culture

Prion-infected cells accumulate a heterogeneous population of aberrantly folded PrP conformers, including the disease-causing isoform (PrP(Sc)). We found that specific chemicals can modulate the levels of various PrP conformers in cultured cells. Positively charged polyamidoamines (dendrimers) eliminated protease-resistant (r) PrP(Sc) from prion-infected cells and induced the formation of insoluble, protease-sensitive PrP aggregates (designated PrP(A)). Larger, positively charged polyamidoamines more efficaciously induced the formation of PrP(A) and cleared rPrP(Sc), whereas negatively charged polyamidoamines neither induced PrP(A) nor cleared rPrP(Sc). Although the biochemical properties of PrP(A) were shown to be similar to protease-sensitive (s) PrP(Sc), bioassays of PrP(A) indicated that it is not infectious. Our studies argue that PrP(A) represents an aggregated PrP species that is off-pathway relative to the formation of rPrP(Sc). It remains to be established whether the formation of PrP(A) inhibits the formation of rPrP(Sc) by sequestering PrP(C) in the form of benign, insoluble aggregates.

Mitochondrial Genome Deletion Aids in the Identification of False- and True-Negative Prostate Needle Core Biopsy Specimens

We report the usefulness of a 3.4-kb mitochondrial genome deletion (3.4 mtdelta) for molecular definition of benign, malignant, and proximal to malignant (PTM) prostate needle biopsy specimens. The 3.4 mtdelta was identified through long-extension polymerase chain reaction (PCR) analysis of frozen prostate cancer samples. A quantitative PCR assay was developed to measure the levels of the 3.4 mtdelta in clinical samples. For normalization, amplifications of a nuclear target and total mitochondrial DNA were included. Cycle threshold data from these targets were used to calculate a score for each biopsy sample. In a pilot study of 38 benign, 29 malignant, and 41 PTM biopsy specimens, the difference between benign and malignant core biopsy specimens was well differentiated (P & .0001), with PTM indistinguishable from malignant samples (P = .833). Results of a larger study were identical. In comparison with histopathologic examination for benign and malignant samples, the sensitivity and specificity were 80% and 71%, respectively, and the area under a receiver operating characteristic (ROC) curve was 0.83 for the deletion. In a blinded external validation study, the sensitivity and specificity were 83% and 79%, and the area under the ROC curve was 0.87. The 3.4 mtdelta may be useful in defining malignant, benign, and PTM prostate tissues.

Calibration Curves for Real-Time PCR

Despite the increasing use of real-time PCR in the diagnosis and management of viral infections, there are no published studies adequately addressing the optimum number of calibrators, the number of replicates of each calibrator, and the frequency with which calibration needs to be repeated. This study was designed to address these issues. Cycle threshold data (ABI 7700) was collected from >50 consecutive real-time PCR runs for hepatitis B and Epstein-Barr viruses. Our routine calibration curve made from serial 10-fold dilutions run in duplicate was compared with alternative options, including duplicate 100-fold dilutions, inclusion of a low-copy calibrator, and omission of the duplicate determination. Control data were used to examine the use of an average calibration curve made from multiple runs. Use of duplicate serial 10-fold dilutions led to the least imprecision, duplicate 100-fold dilutions had slightly higher imprecision, and calibration curves obtained with singlet measurements showed the greatest imprecision. For patient data, the duplicate 100-fold dilution calibration curve produced results that best matched those from the routine calibration curve. Use of singlet dilutions or inclusion of a low-copy calibrator produced poorer agreement. Variability in controls was lower with a daily calibration curve than with an average calibration curve. Duplicate 100-fold dilution calibration curves produced equivalent results and the same imprecision as curves with more calibrators, and thus are a valid alternative. Laboratories should carefully evaluate the variability resulting from the use of average calibration curves before adopting this approach.

Genogroup I and II Noroviruses Detected in Stool Samples by Real-Time Reverse Transcription-PCR Using Highly Degenerate Universal Primers

Genogroup I noroviruses from five genetic clusters and genogroup II noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse transcription-PCR (RT-PCR) with SYBR Green detection. Two degenerate primer sets, designated MON 431-433 and MON 432-434, were designed from consensus sequences from the major clusters of norovirus based on the RNA-dependent RNA polymerase region of the norovirus genome. Viruses were extracted from stool samples within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semiquantitative results by means of the cycle threshold data and dilution endpoint standard curves. Presumptive product verification was achieved by evaluation of first-derivative melt graphs. Multiple clusters of noroviruses were identified simultaneously in a multiplex fashion by virtue of slight differences in melting temperature. The detection of 13 different genetic clusters suggests that the MON primers may serve as universal primers for most, if not all, of the noroviruses in a multiplex assay. Our technique provides a framework for broad application of real-time RT-PCR in clinical, environmental, and food testing laboratories for a wide range of noroviruses.

Vascular Bone Morphogenetic Protein (BMP) expression decreases after mouse carotid ligation

BMPs have been previously identified in atherosclerotic lesions. The purpose of this study was to characterize the temporal changes in BMP expression following acute alterations in blood flow using a mouse model of arterial remodeling. Methods. We evaluated bmp expression in mouse carotid vessels after acute ligation of the common carotid artery at its bifurcation. Male C57/Bl6 mice weighing 19–21 g underwent left carotid ligation followed by harvest at 3 ( n = 7) and 7 ( n = 7) days. Carotid vessels from unoperated mice were harvested and used as controls ( n = 5). Vessels were snap-frozen in liquid nitrogen prior to RNA extraction and cDNA syntheses using reverse transcriptase. Expression of bmp2, bmp4, bmp7, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was then examined using quantitative real-time polymerase chain reaction (qRT-PCR). Product identities were confirmed using melt curve analysis and sequencing. Baseline subtracted cycle threshold data were analyzed using the 2 −(ΔCT) formula to quantify bmp expression relative to GAPDH for each specimen. Copy numbers relative to GAPDH were analyzed using analysis of variance (ANOVA). Results. Expression of bmp4 decreased by 71% of control at 3 days postligation (ANOVA, P = 0.04) and returned to baseline levels by 7 days. A similar trend was observed for bmp2 expression (ANOVA, P = 0.008). However, no change in bmp7 mRNA levels was identified in this series of experiments. Conclusion. Acute flow changes caused by carotid ligation produce a temporary decrease in bmp4 and bmp2 mRNA levels at 3 days with a return to baseline levels by day 7. This data suggest that BMPs may be involved in the vascular remodeling that occurs in response to changes in blood flow.