Mean CT Values Research Articles

427. Variation in SARS-CoV-2 molecular diagnostic test performance in symptomatic versus asymptomatic populations

BackgroundGrowing recognition of the importance of asymptomatic and pre-symptomatic transmission for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to a substantial expansion of testing from symptomatic to asymptomatic patients, and particularly those with risk factors for infection. Viral burden in asymptomatic individuals can differ from symptomatic patients, which can impact test performance. We therefore evaluated the impact of expanded testing indications upon the sensitivity and specificity of molecular diagnostic assays for SARS-CoV-2.MethodsWe performed a retrospective review of laboratory results from 5,122 emergency room patients and inpatients tested for SARS-CoV-2 between 05/03/2020 and 06/13/2020 using the Hologic Panther Fusion and the Cepheid Xpert assays at the Brigham & Women’s Hospital in Boston, MA. Descriptive analyses were performed for trends in testing volume, rates of positivity and cycle thresholds (Cts) over time based on symptom status. We calculated the proportion of new diagnoses made on a patient’s first test as an indirect measure of sensitivity. We calculated the proportion of first tests that are positive with a Ct value < 35 as an indirect measure of specificity.ResultsThe overall rate of positivity over the study period was 8.7% (599/7,510 tests; 440/4,795 people) and declined by 1.8% (95% CI -2.2% - -1.4%, P< 0.0001) each week. Relative to tests in symptomatic people, the asymptomatic population had a higher mean Ct value (35.1 vs 32.3; P < 0.0001). Ct values increased by 0.7 (95% CI -0.1 - +1.4, P=0.07) and 0.8 (95% CI +0.3 - +1.4; p=0.01), sensitivity declined by 4% (95% CI -9% - +1%, P=0.08) and 12% (95% CI -20% - -5%, P-0.01) and specificity declined by 8% (95% CI -3% to 20%; P=0.13) and 9% (95% CI 7% - 11%; P=0.0002), over the time period of the study for asymptomatic and symptomatic patients, respectively.Figure 1: Trends in Ct values by symptoms Figure 2: Trends in diagnosis by first versus second test by symptoms Figure 3: Trends in proportion of people with their first test having Ct < 35 by symptoms ConclusionWe show that the proportion of patients with low SARS-CoV-2 viral loads has increased as testing has expanded to the asymptomatic population and as transmission wanes in the community. This negatively impacts the performance of molecular assays by increasing the risk of false negatives and the detection of non-viable virus. Decision algorithms based on molecular assay results may need re-evaluation in light of these dynamics.Disclosures All Authors: No reported disclosures

658. Effect of HIV Status on Tuberculosis Load as Detected by Xpert MTB/RIF in Sputum vs. Saliva Samples

BackgroundTuberculosis (TB) is the most common co-infection among people living with HIV, but HIV positivity is associated with a lower Mycobacterium tuberculosis (MTB) bacillary load in sputum, making TB often difficult to diagnose with current diagnostic solutions. GeneXpert MTB/RIF (Cepheid, USA), a rapid, molecular diagnostic assay, has transformed the TB diagnostic landscape and can be used to diagnose TB and limited drug resistance in HIV patients from direct clinical samples in < 2 hours, but results can be significantly affected by sample bacterial load, which is quantified by the GeneXpert MTB/RIF (Xpert) instrument using Ct values. Our primary objective was to assess how a patient’s HIV status affected their MTB bacterial load in sputum vs. saliva samples submitted for Xpert diagnosis of TB.MethodsWe completed a retrospective analysis of >4,000 patient records from the Myanmar National TB Program captured as part of a nation-wide electronic reporting system developed with the assistance of FIND (Geneva). De-identified records included HIV status, Xpert testing results, and for a subset of patients, specimen type. With this diagnostic information, we compared the distribution of MTB load (quantified by Xpert Ct values) in sputum and saliva in HIV positive vs. HIV negative patients using STATA.ResultsBased on mean Ct value comparison independent of HIV status, saliva samples (mean Ct = 22.7) contained a significantly lower bacterial load of MTB as compared to sputum samples (mean Ct=19.2, p < .001).Within saliva samples, a lower bacterial load was also detected in HIV positive patients (mean Ct = 26.9) compared to HIV negative patients (mean Ct = 22.3, p< .05). Similarly, in sputum samples, a lower bacterial load was detected in HIV positive patients (mean Ct = 21.6) compared to HIV negative patients (mean Ct = 19.0, p < .001) (Figure 1).Figure 1. Ct Values by HIV Status and Specimen Type ConclusionSputum samples have a significantly higher bacterial load on average compared to saliva samples independent of HIV status. Additionally, when looking at both saliva and sputum as sample types, HIV positive patients have significantly lower bacterial load than individuals who are HIV negative. Based on these results, sputum is the optimal sample type for Xpert TB detection, especially in people living with HIV.Disclosures All Authors: No reported disclosures

543. SARS-CoV-2 Viral Dynamics For Symptomatic People Living with HIV Requiring Hospitalization For COVID-19

BackgroundOver 8 million people have been infected with severe acute respiratory syndrome 2 (SARS-CoV-2). People living with HIV (PWH) affected by SARS-CoV-2 represent a small proportion of patients admitted to the hospital for COVID-19 disease. Viral dynamics in this subpopulation are still unknown.MethodsWe conducted a retrospective cohort study from a large, urban academic center in New Haven, CT of patients consecutively admitted with laboratory confirmed SARS-CoV-2 infection through May 31, 2020. Main outcome measure was the ribonucleic acid (RNA) viral load (VL) detected in respiratory samples by cycle threshold (Ct) values and trend over time as a measure of nucleic acid concentration and replication. Epidemiological, clinical, laboratory results, ART treatment, COVID treatment and outcomes were abstracted from patient records. We evaluated the relationship between virologic data and disease severity.ResultsAmong 19 PWH hospitalized with covid19, 84% were >50 years of age, 53% were women, 63% were black, 95% were on antiretrovirals, 95% with undetectable HIV VL (< 50 copies/ml), and median CD4 count of 827.9 cells/mm3. Symptom duration was 2–14 days. Median length of stay was 12 days. There was no in-hospital mortality. A total of 62 respiratory samples were collected at various time points and evaluated for SARS-CoV-2 RNA viral load. Eight patients had 1 specimen. Patient with more severe disease had higher baseline SARS-CoV-2 viral loads. Mean Ct values for N1 and N2 on admission were 21.6 and 23.7, respectively.ConclusionPWH with COVID19 represents only a small percentage of hospitalized patients and viral dynamics are not well defined. SARS-CoV-2 VL was higher among PWH with advanced disease at the presentation of illness. Overall, PWH with COVID-19 had favorable outcomes.Disclosures All Authors: No reported disclosures

Upper respiratory viral load in asymptomatic individuals and mildly symptomatic patients with SARS-CoV-2 infection

BackgroundAsymptomatic individuals with SARS-CoV-2 infection have viable viral loads and have been linked to several transmission cases. However, data on the viral loads in such individuals are lacking. We assessed...

Metal artifacts reduction in computed tomography: A phantom study to compare the effectiveness of metal artifact reduction algorithm, model-based iterative reconstruction, and virtual monochromatic imaging.

The purpose of this study was to compare the effectiveness of a metal artifact reduction algorithm (MAR), model-based iterative reconstruction (MBIR), and virtual monochromatic imaging (VMI) for reducing metal artifacts in CT imaging.A phantom study was performed for quantitatively evaluating the dark bands and fine streak artifacts generated by unilateral hip prostheses. Images were obtained by conventional scanning at 120 kilovolt peak, and reconstructed using filtered back projection, MAR, and MBIR. Furthermore, virtual monochromatic images (VMIs) at 70 kilo-electron volts (keV) and 140 keV with/without use of MAR were obtained by dual-energy CT. The extents and mean CT values of the dark bands and the differences in the standard deviations and location parameters of the fine streak artifacts evaluated by the Gumbel method in the images obtained by each of the methods were statistically compared by analyses of variance.Significant reduction of the extent of the dark bands was observed in the images reconstructed using MAR than in those not reconstructed using MAR (all, P < .01). Images obtained by VMI at 70 keV and 140 keV with use of MAR showed significantly increased mean CT values of the dark bands as compared to those obtained by reconstructions without use of MAR (all, <.01). Significant reduction of the difference in the standard deviations used to evaluate fine streak artifacts was observed in each of the image sets obtained with VMI at 140 keV with/without MAR and conventional CT with MBIR as compared to the images obtained using other methods (all, P < .05), except between VMI at 140 keV without MAR and conventional CT with MAR. The location parameter to evaluate fine streak artifacts was significantly reduced in CT images obtained using MBIR and in images obtained by VMI at 140 keV with/without MAR as compared to those obtained using other reconstruction methods (all, P < .01).In our present study, MAR appeared to be the most effective reconstruction method for reducing dark bands in CT images, and MBIR and VMI at 140 keV appeared to the most effective for reducing streak artifacts.

Self-Sampling of Oropharyngeal Swabs Among Healthcare Workers for Molecular Detection of Respiratory Viruses: A Valuable Approach for Epidemiological Studies and Surveillance Programs.

This study aimed at assessing the validity of self-collected (self-sampled) oropharyngeal (OP) swabs among healthcare workers compared to those collected by trained sentinel general practitioners (GP-sampled) from individuals with influenza-like illness (ILI), to be implemented in epidemiological studies and/or surveillance programs of viral pathogens involved in community respiratory infections. In our study, OP swabs were collected from adults (>18 years) with ILI during the 2018–2019 influenza season. Two groups of samples were considered: group 1−131 self-sampled OP swabs collected by healthcare workers after being trained on the sampling procedure; group 2−131 GP-sampled OP swabs collected from outpatients by sentinel GPs operating within the Italian Influenza Surveillance Network. To assess swabbing quality, following RNA extraction, each sample was tested for the presence of the human ribonuclease P gene (RNP) by in-house real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Samples with a cycle threshold (Ct) <35 were considered adequate for further virological analysis. Influenza viruses (IVs), respiratory syncytial virus (RSV), and rhinovirus (RV) genomes were detected by in-house real-time RT-PCR. All samples were positive to RNP detection with Ct <35. The mean Ct value was similar in the two groups (group 1 vs. group 2: 25.93 ± 2.22 vs. 25.46 ± 2.40; p = 0.10). IVs, RSV, and RV positivity rates were 26.7 vs. 52.7% (p < 0.01), 7.6 vs. 9.9% (p = 0.52), and 21.4 vs. 19.9% (p = 0.76), respectively. Self-sampled OP swabs resulted as valid as GP-sampled OP swabs for molecular detection of respiratory viruses. Self-swabbing can thus be a worthwhile strategy for sample collection to implement molecular surveillance of respiratory pathogens and carry out epidemiological studies, easily reaching a larger population size.

Identification of Field Tolerance and Resistance to Mal Secco Disease in a Citrus Germplasm Collection in Sicily

Mal secco is a tracheomycotic disease caused by the fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley that has caused severe damage and loss of yield in the citrus industry in the Mediterranean area, for 100 years. While the disease can affect different cultivated citrus species, lemon (C. × limon var. limon (L.) Burm. f.) and citron are the most susceptible. The identification of resistant or field-tolerant clones and hybrids is a major goal for lemon growers and breeders. To identify sources of resistance or tolerance to the disease, we performed a phenotypic survey on a lemon and lemon-like open-field germplasm planted at CREA (Research Centre for Olive, Fruit and Citrus Crops), Italy, in an area with high pathogen pressure. Phenotyping was performed visually, four times, for three consecutive years, on a total of 50 accessions, with two or three replicate trees per accession. Moreover, molecular screening based on real-time PCR was performed, for two consecutive years, on twigs, young leaves, and mature leaves of all plants, to detect the pathogen in the absence of clear symptoms. The accessions were categorized into seven groups based on the presence of visual symptoms, real-time PCR pathogen detection, and canopy volume. The results revealed sources of tolerance in lemon and citron hybrids. The molecular screening identified P. tracheiphilus in all lemon clones, with mean Ct values ranging from 17 to 39. The screening also identified P. tracheiphilus in clones without clear symptoms, indicating their ability to tolerate the disease. Moreover, a strong negative correlation was found between the Ct values in twigs and symptom severity (r = −0.72). This indicates that the DNA from twigs is the most appropriate for use in performing reliable phenotyping of mal secco susceptibility in adult plants. An autotetraploid lemon (Doppio Lentini) seems to be immune to the disease, under natural pressure, since P. tracheiphilus was not detected by real-time PCR and visual screening. Overall, the data obtained are a valuable resource for identifying both the most tolerant lemon varieties suitable for areas with high pathogen pressure and the best breeding parents for the introgression of resistance genes into lemon genotypes.

Characterization of thrombus composition with multimodality CT-based imaging: an in-vitro study

BackgroundCT is the most commonly used imaging modality for acute ischemic stroke evaluation. There is growing interest to use pre-operative imaging to characterize clot composition in stroke. We performed an...

Asian citrus psyllid adults inoculate huanglongbing bacterium more efficiently than nymphs when this bacterium is acquired by early instar nymphs

The Asian citrus psyllid (Diaphorina citri) transmits the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), which causes huanglongbing (citrus greening) disease, in a circulative-propagative manner. We compared CLas inoculation efficiency of D. citri nymphs and adults into healthy (uninfected) citron leaves when both vector stages were reared from eggs on infected plants. The proportion of CLas-positive leaves was 2.5% for nymphs and 36.3% for adults. CLas acquisition by early instar nymphs followed by dissections of adults and 4th instar nymphs revealed that CLas bacterium had moved into the head-thorax section (containing the salivary glands) in 26.7–30.0% of nymphs and 37–45% of adults. Mean Ct values in these sections were 31.6–32.9 and 26.8–27.0 for nymphs and adults, respectively. Therefore, CLas incidence and titer were higher in the head-thorax of adults than in nymphs. Our results suggest that following acquisition of CLas by early instar D. citri nymphs, emerging adults inoculate the bacteria into citrus more efficiently than nymphs because adults are afforded a longer latent period necessary for multiplication and/or translocation of CLas into the salivary glands of the vector. We propose that CLas uses D. citri nymphs mainly for pathogen acquisition and multiplication, and their adults mainly for pathogen inoculation and spread.

Repeated Coronavirus Disease 2019 Molecular Testing: Correlation of Severe Acute Respiratory Syndrome Coronavirus 2 Culture With Molecular Assays and Cycle Thresholds.

BackgroundRepeated coronavirus disease 2019 (COVID-19) molecular testing can lead to positive test results after negative results and to multiple positive results over time. The association between positive test results and infectious virus is important to quantify.MethodsA 2-month cohort of retrospective data and consecutively collected specimens from patients with COVID-19 or patients under investigation were used to understand the correlation between prolonged viral RNA positive test results, cycle threshold (Ct) values and growth of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cell culture. Whole-genome sequencing was used to confirm virus genotype in patients with prolonged viral RNA detection. Droplet digital polymerase chain reaction was used to assess the rate of false-negative COVID-19 diagnostic test results.ResultsIn 2 months, 29 686 specimens were tested and 2194 patients underwent repeated testing. Virus recovery in cell culture was noted in specimens with a mean Ct value of 18.8 (3.4) for SARS-CoV-2 target genes. Prolonged viral RNA shedding was associated with positive virus growth in culture in specimens collected up to 21 days after the first positive result but mostly in individuals symptomatic at the time of sample collection. Whole-genome sequencing provided evidence the same virus was carried over time. Positive test results following negative results had Ct values >29.5 and were not associated with virus culture. Droplet digital polymerase chain reaction results were positive in 5.6% of negative specimens collected from patients with confirmed or clinically suspected COVID-19.ConclusionsLow Ct values in SARS-CoV-2 diagnostic tests were associated with virus growth in cell culture. Symptomatic patients with prolonged viral RNA shedding can also be infectious.

Convolutional neural network-based automatic liver delineation on contrast-enhanced and non-contrast-enhanced CT images for radiotherapy planning.

This study evaluated a convolutional neural network (CNN) for automatically delineating the liver on contrast-enhanced or non-contrast-enhanced CT, making comparisons with a commercial automated technique (MIM Maestro®). Intensity-modulated radiation therapy requires careful labor-intensive planning involving delineation of the target and organs on CT or MR images to ensure delivery of the effective dose to the target while avoiding organs at risk. Contrast-enhanced planning CT images from 101 pancreatic cancer cases and accompanying mask images showing manually-delineated liver contours were used to train the CNN to segment the liver. The trained CNN then performed liver segmentation on a further 20 contrast-enhanced and 15 non-contrastenhanced CT image sets, producing three-dimensional mask images of the liver. For both contrast-enhanced and non-contrast-enhanced images, the mean Dice similarity coefficients between CNN segmentations and ground-truth manual segmentations were significantly higher than those between ground-truth and MIM Maestro software (p < 0.001). Although mean CT values of the liver were higher on contrast-enhanced than on non-contrast-enhanced CT, there were no significant differences in the Hausdorff distances of the CNN segmentations, indicating that the CNN could successfully segment the liver on both image types, despite being trained only on contrast-enhanced images. Our results suggest that a CNN can perform highly accurate automated delineation of the liver on CT images, irrespective of whether the CT images are contrast-enhanced or not.

Topical lignocaine anaesthesia for oropharyngeal sampling for COVID-19.

ObjectivesTo ascertain if topical lignocaine application in oropharynx prior to swab sampling to test for COVID-19 improves a patient’s comfort and to assess its effect on the swab sample taken to conduct the RT-PCR.MethodsAdult patients testing positive on the RT-PCR COVID-19 test were sampled again within 48 h after administering topical oropharyngeal anaesthesia. Patients were asked to rate their discomfort on a visual analog scale (VAS) for both sample A and B. A qualitative real-time RT-PCR for detection of SARS-CoV-2 RNA, was performed, and the cycle threshold value (Ct), used as a surrogate marker for the viral load, was measured for the sample taken without lignocaine (sample A) and the sample taken post-lignocaine application (sample B). The difference in Ct values of both the groups was checked for any statistical significance using paired t-test. Wilcoxon signed rank test was used on VAS scores to determine any significant decrease in discomfort.ResultsForty patients were included in the study. Twenty-nine patients (72.5%) reported the procedure to be more comfortable post-lignocaine application. Median (IQR) discomfort on VAS decreased from 7 (1) to 5 (2) after lignocaine use, which was statistically significant (p < 0.05). Mean Ct value for sample A was 17.21 ± 5.25 and for sample B was 18.44 ± 4.8 (p > 0.05), indicating a non-significant effect of lignocaine on SARS-CoV-2 concentration in the sample.ConclusionTopical lignocaine, while improving the comfort of the procedure of oropharyngeal sampling for patient did not alter the SARS-CoV-2 viral load that was detected in nasal and oropharyngeal samples taken together.

Can different Catphan phantoms be used in a multi-centre audit of radiotherapy CT image quality?

To determine the variation between Catphan image quality CT phantoms, specifically for use in a future multi-centre image quality audit. 14 Catphan phantoms (models 503, 504 and 604) were scanned on a Canon Aquilion Prime CT scanner using a single scan protocol. Measurements were made of noise in the uniformity section, visibility of low contrast targets and contrast, x-ray attenuation and CT number for 5 materials in the sensitometry section. Scans were also acquired using one phantom and varying reconstruction field of view, image slice thickness, effective tube-current-time product and iterative reconstruction settings to determine how the degree of inter-phantom variability compared with the magnitude of changes from scan parameter alteration. Across all phantoms the mean CT value in the uniformity section was 7.0 (SD 0.9) range: 4.9-8.1 HU. For the different materials the CT numbers were air: -1004±5, Polymethylpentene: -190±2, Polystyrene: -42±2, Delrin: 321±5 and Teflon: 898±8 HU. Consistency of low contrast targets through visual scoring was good. Measured contrast was lower (p<0.001) with more variability for 504 versus 604 models. All phantoms produced identical tube current settings with x-ray tube current modulation, indicating no x-ray attenuation differences. The degree of change in image quality metrics between phantoms was small compared with results when scan parameters were varied. Catphan phantoms model 604 showed minimal differences and will be used for multi-centre inter-comparison work, with the consistency between phantoms appropriate for measuring possible variations in image quality.

Diagnostic Challenges in Canine Parvovirus 2c in Vaccine Failure Cases.

In this study, three different diagnostic tests for parvovirus were compared with vaccination status and parvovirus genotype in suspected canine parvovirus cases. Faecal samples from vaccinated (N17) and unvaccinated or unknown vaccination status (N41) dogs that had clinical signs of parvovirus infection were tested using three different assays of antigen tests, conventional and quantitative PCR tests. The genotype of each sample was determined by sequencing. In addition to the suspected parvovirus samples, 21 faecal samples from apparently healthy dogs were tested in three diagnostic tests to evaluate the sensitivity and specificity of the tests. The antigen test was positive in 41.2% of vaccinated dogs and 73.2% of unvaccinated diseased dogs. Conventional PCR and qPCR were positive for canine parvovirus (CPV) in 82.4% of vaccinated dogs and 92.7% of unvaccinated dogs. CPV type-2c (CPV-2c) was detected in 82.75% of dogs (12 vaccinated and 36 unvaccinated dogs), CPV-2b was detected in 5.17% dogs (one vaccinated and two unvaccinated) and CPV-2a in 1.72% vaccinated dog. Mean Ct values in qPCR for vaccinated dogs were higher than the unvaccinated dogs (p = 0.049), suggesting that vaccinated dogs shed less virus, even in clinical forms of CPV. CPV-2c was the dominant subtype infecting dogs in both vaccinated and unvaccinated cases. Faecal antigen testing failed to identify a substantial proportion of CPV-2c infected dogs, likely due to low sensitivity. The faecal samples from apparently healthy dogs (n = 21) showed negative results in all three tests. Negative CPV faecal antigen results should be viewed with caution until they are confirmed by molecular methods.

Pathogen flows from on-site sanitation systems in low-income urban neighborhoods, Dhaka: A quantitative environmental assessment.

Despite wide usage of on-site sanitation, there is limited field-based evidence on the removal or release of pathogens from septic tanks and other primary treatment systems, such as anaerobic baffled reactors (ABR). In two low-income areas in Dhaka, we conducted a cross-sectional study to explore pathogen loads discharged from commonly used on-site sanitation-systems and their transport in nearby drains and waterways. We collected samples of drain water, drain sediment, canal water, and floodwater from April-October 2019. Sludge, supernatant, and effluent samples were also collected from septic tanks and ABRs. We investigated the presence and concentration of selected enteric pathogens (Shigella, Vibrio cholerae (V. cholerae), Salmonella Typhi (S. Typhi), Norovirus Genogroup-II (NoV-GII), and Giardia) and presence of Cryptosporidium in these samples using quantitative polymerase chain reaction (qPCR).The equivalent genome copies (EGC) of individual pathogens were estimated in each sample by interpolation of the mean Ct value to the corresponding standard curve and the dilution factor for each sample type. Absolute quantification was expressed as log10 EGC per 100mL for the water samples and log10 EGC per gram for the sediment samples. Among all samples tested (N=151), 89% were contaminated with Shigella, 68% with V. cholerae and NoV-GII, 32% with Giardia, 17% with S. Typhi and 6% with Cryptosporidium. A wide range of concentration of pathogens [range: mean log10 concentration of Giardia=0.74 EGC/100mL in drain ultrafiltration samples to mean log10 concentration of NoV-GII and Giardia=7.11 EGC/100mL in ABR sludge] was found in all environmental samples. The highest pathogen concentrations were detected in open drains [range: mean log10 concentration=2.50-4.94 EGC/100mL], septic tank effluent [range: mean log10 concentration=3.32-4.65 EGC/100mL], and ABR effluent [range: mean log10 concentration=2.72-5.13 EGC/100mL]. High concentrations of pathogens (particularly NoV-GII, V.cholerae and Shigella) were frequently detected in environmental samples from two low-income urban neighbourhoods of Dhaka city. The numerous environmental exposure pathways for children and adults make these findings of public health concern. These results should prompt rethinking of how to achieve safe sanitation solutions that protect public health in dense low-income areas. In particular, improved management and maintenance regimes, further treatment of liquid effluent from primary treatment processes, and appropriate application of onsite, decentralised and offsite sanitation systems given the local context.

Evaluation of SARS-CoV-2 in Tears of Patients with Moderate to Severe COVID-19

PurposeTo investigate the presence of SARS-CoV-2 RNA in tears of patients with moderate to severe coronavirus disease 2019 (COVID-19).DesignCross-sectional study.ParticipantsPatients with laboratory-proven moderate to severe COVID-19.MethodsTears were collected within 48 hours of laboratory confirmation using 3 methods: conjunctival swab plus Schirmer’s test strips (group 1), conjunctival swab (group 2), and Schirmer’s test strips (group 3). Samples from both the eyes of each patient were transported in a single viral transport media for real-time RT-PCR. Detailed demographic profiles, systemic symptoms, comorbidities, and ocular manifestations were noted.Main Outcome MeasuresViral load of a sample was determined using cycle threshold (Ct) value of E gene. A specimen was considered to show positive results if the amplification curve for the E gene crossed the threshold line within 35 cycles and if it showed positive results on an RNA-dependent RNA polymerase or open reading frame 1b gene assay.ResultsOf the 78 patients enrolled in the study, samples from 3 patients were found to be inadequate for analysis. Thirty-six patients (48%) had moderate disease, whereas 39 patients (52%) had severe disease, with no ocular involvement in any patient. In the 75 patients, RT-PCR analysis of tears showed positive results in 18 patients (24%), and 29 of 225 samples (12.9%) showed positive results. Positive results were found in 11 (14.7%), 11 (14.7%), and 7 (9.3%) patients in groups 1, 2, and 3, respectively (P = 0.3105). Mean Ct values in groups 1, 2, and 3 were 28.36 ± 6.15, 29.00 ± 5.58, and 27.86 ± 6.46 (P = 0.92), respectively. Five patients showed positive RT-PCR results by all 3 methods (mean Ct value, 25.24 ± 6.33), and 12 patients showed positive results by any of the 3 methods (mean Ct value, 32.16 ± 1.94), the difference in Ct values being statistically significant (P = 0.029). The median value of symptomatology in patients with positive RT-PCR results from tears was 5 days (range, 4–9 days).ConclusionsSARS-CoV-2 RNA was detected in tears of 24% of patients with laboratory-proven moderate to severe COVID-19. Conjunctival swab remains the gold standard of tear collection for RT-PCR assay. A significantly higher possibility of viral transmission exists through tears in patients with moderate to severe COVID-19.

Validation of Dried Tube Sample Format Quality Controls for the Monitoring of Viral Load and Blood Screening Assays

HIV viral load (VL) and donor screening assays experience variation and require quaity assurance (QA). NRL sought to confirm a dried tube sample format (HIVDTS) sample type for use in quality control (QC) programs for HIV molecular testing.50 μL of HIV supernatant at 1 × 105 copies per millilitre (copies/mL)) was dried for 48 hours at room temperature. Post-production and shipped integrity studies were undertaken. Dried HIVDTS was reconstituted in PBS buffer and tested in HIV VL (six participants) or blood screening assays (four participants). Results were entered into NRL’s QC monitoring software (EDCNet™) for analysis.The mean of 224 VL results when HIVDTS QCs were tested in Biocentric HIV GENERIC Charge Virale assay was 4.54 log10 copies/mL, with the percentage coefficient of variation (CV%) ranging from 1.75 to 13.20%. The mean Ct value for HIVDTS QCs tested on Roche Cobas MPX assay results was 28.71 (range 28.33 to 29.14), with CV% ranging from 1.56 to 3.98%.The study confirms HIVDTS QCs can effectively monitor the performance of HIV molecular testing and offers a cheaper alternative to commercial QC samples that require cold-chain shipping on dry ice and UN3373 conditions.

Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic has resulted in significant shortages of RT-PCR testing supplies including RNA extraction kits. The goal of our study was to determine if a simplified heat-RNA release method would provide comparable detection of SARS-CoV-2 without the need for nucleic acid extraction. RT-PCR results using the ChromaCode HDPCR™ SARS-CoV-2 were compared using the heat-RNA release method and an automated RNA extraction system (EMAG). The heat-RNA release method correctly identified 94 % (81/86 nasopharyngeal samples) that were positive for SARS-CoV-2. Five samples that were missed by heat-RNA release method had a mean Ct value: 35 using the automated extraction instrument, indicating a very low viral load. Our findings show that a simple heat-RNA release method is a reasonable alternative for the majority of COVID-19 positive patients and can help overcome the cost and availability issues of RNA extraction reagents.

Comprehensive evaluation of 68Ga-PSMA-11 PET/CT parameters for discriminating pathological characteristics in primary clear-cell renal cell carcinoma.

To evaluate parameters derived from 68Ga-PSMA-11 PET/CT images for discriminating pathological characteristics in primary clear-cell renal cell carcinoma (ccRCC). The study retrospectively examined data for 36 ccRCC patients with preoperative 68Ga-PSMA-11 PET/CT scan and surgical specimens. Radiological parameters including maximal tumor diameter, mean CT value, and maximal standard uptake value (SUVmax) were derived from PET/CT images. Pathological characteristics included WHO/ISUP grade and adverse pathology (tumor necrosis or sarcomatoid or rhabdoid feature). Values of radiological parameters were compared within subgroups of pathological characteristics. Receiver operating characteristic (ROC) curve analysis was used for the effectiveness of radiological parameters in differentiating pathological characteristics, estimating area under the ROC curve (AUC) and 95% confidence intervals (CIs). The WHO/ISUP grade distribution for 36 tumors was grade 1, 9 (25.0%); grade 2, 12 (33.3%); grade 3, 9 (25.0%); and grade 4, 6 (16.7%). Adverse pathology was positive for 15 (41.7%). Radiological tumor diameter and SUVmax significantly differed by WHO/ISUP grade, pT stage, and adverse pathology (all P < 0.05), with no difference by CT value. Tumor diameter demonstrated sensitivity 86% and specificity 88% for pT stage, with cutoff 6.70 and AUC 0.91 (95% CI, 0.79-1.00, P< 0.001). SUVmax could effectively differentiate WHO/ISUP grade (3-4 vs. 1-2) and adverse pathology (positive vs. negative), with AUC 0.89 (95% CI, 0.81-0.98, P< 0.001), cutoff 16.4, sensitivity 100%, and specificity 71% and AUC 0.92 (95% CI, 0.85-0.99, P< 0.001), cutoff 18.5, sensitivity 94%, and specificity 87%, respectively. 68Ga-PSMA-11 PET/CT could effectively identify aggressive pathological features of ccRCC.

Lower nasopharyngeal viral load during the latest phase of COVID-19 pandemic in a Northern Italy University Hospital.

A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been anecdotally reported over the latest phase of COVID-19 pandemic in Italy. Several factors may contribute to this observation, including the effect of lockdown, social distancing, lower humidity, lower air pollution, and potential changes in the intrinsic pathogenicity of the virus. In this regard, the clinical severity of COVID-19 could be attenuated by mutations in SARS-CoV-2 genome that decrease its virulence, as well as by lower virus inocula. In this pilot study, we compared the reverse transcription polymerase chain reaction (RT-PCR) amplification profile of 100 nasopharyngeal swabs consecutively collected in April, during the peak of SARS-CoV-2 epidemic, to that of 100 swabs collected using the same procedure in May. The mean Ct value of positive samples collected in May was significantly higher than that of samples collected in the previous period (ORF 1a/b gene: 31.85±0.32 vs. 28.37±0.5, p<0.001; E gene: 33.76±0.38 vs. 29.79±0.63, p<0.001), suggesting a lower viral load at the time of sampling. No significant differences were observed between male and females in the two periods, whilst higher viral loads were found in (i)patients over 60-years old, and (ii) patients that experienced severe COVID-19 during the early stages of the pandemic. This pilot study prompts further investigation on the correlation between SARS-CoV-2 load and different clinical manifestation of COVID-19 during different phases of the pandemic. Laboratories should consider reporting quantitative viral load data in the molecular diagnosis of SARS-CoV-2 infection.