Ct Values Research Articles

Performance of the Flash10 COVID-19 point-of-care molecular test

After the COVID-19 pandemic, fever clinics urgently require rapid nucleic acid tests to enhance their capacity for timely pathogen detection. This study evaluated the analytical performance and clinical utility of the Flash10 SARS-CoV-2 point-of-care test (Flash10 POCT) for detecting SARS-CoV-2 in patients with fever in the adult fever clinic in Beijing Tsinghua Changgung Hospital from August 1 to August 30, 2023. The analytical performance and clinical utility of the Flash10 POCT for detecting SARS-CoV-2 were assessed in 125 patients with fever syndrome in the adult fever clinic. The Flash10 POCT demonstrated an analytical precision of 3.1% for the Ct values of the ORF1ab gene and 2.9% for the Ct values of the N gene in SARS-CoV-2 nucleic acid testing. Furthermore, the Flash10 POCT demonstrated a lower limit of detection (LoD) of 100 copies/mL, with no detected aerosol contamination leakage. Of the 125 patients (median age 61.9 years, 52% male and 48% female), both the Flash10 POCT and RT-PCR tests yielded positive results for 100 patients and negative results for 25 patients (Fisher’s exact test, p < 0.0001). The median turn-around-time for the Flash10 POCT was significantly shorter, at 1.05 h, compared to 16.15 h required for RT-PCR tests (Wilcoxon signed rank test, p < 0.0001). The Flash10 POCT showed high analytical performance, achieving a 100% detection rate for SARS-CoV-2 compared to RT-PCR tests, while also exhibiting a significantly shorter turn-around-time. Implementing the Flash10 POCT had the potential to expedite the care of adults presenting with fever.

Determining diagnostic sensitivity loss limits for sample pooling in duplex rtPCR surveillance testing: Theileria orientalis and Anaplasma marginale.

To expand surveillance testing capacity through sample pooling, a thorough understanding is needed of how sample dilution through pooling affects the sensitivity of candidate assays. We validated a robust and representative framework for assessing the dilution effect of sample pooling using duplex rtPCR surveillance of Theileria orientalis and Anaplasma marginale, both of which are causative agents of severe anemia in cattle and a serious threat to the cattle industry in Virginia and many other states. We used 200 known-positive samples with Ct values representative of typical surveillance results in a series of pools in which we re-tested each sample individually, followed by each sample diluted in equal volumes with negative samples to make pools of 2, 4, 6, 8, and 10 total samples. We compared the Ct values of the individual positives with the Ct values of each pool size to determine if Ct values increase past the limit of detection in the 45-cycle assay. We observed a maximum of 2% sensitivity loss (no more than 2 of 100 samples returned a false-negative result) for both T. orientalis and A. marginale during the pooling series, with lower-than-expected average Ct increase and sensitivity loss. We conclude that pooling up to 10 samples would be acceptable for regional surveillance of T. orientalis and A. marginale using our rtPCR assay. The described strategy is applicable to validate pooling for a wide range of single and duplex rtPCR assays, which could expand efficient disease surveillance.

A systematic approach for identifying unique genomic sequences for Fusarium oxysporum f. sp. lactucae race 1 and development of molecular diagnostic tools

Fusarium oxysporum f. sp. lactucae (FOLac) is a soil- and seedborne fungal pathogen that causes Fusarium wilt of lettuce, an important disease threatening global lettuce production. Based on pathogenicity on differential lettuce cultivars, four races (1-4) have been identified, with race 1 the only race detected in the United States, and the closely related, emerging race 4 known only in Europe. The development of race-specific diagnostic tools is hindered by insufficient genomic data to distinguish between the two races and FOLac from other F. oxysporum formae speciales and nonpathogenic isolates. Here, we describe a systematic approach for developing diagnostic markers for FOLac race 1 that utilized a comprehensive sequence database of F. oxysporum to identify 15 unique genomic sequences. Marker specificity was validated through an exhaustive screening process against genomic data from 797 F. oxysporum isolates representing 64 formae speciales and various plants and non-plant substrates. One of the unique sequences was used to develop a TaqMan quantitative polymerase chain reaction assay and a recombinase polymerase amplification assay, both exhibiting 100% sensitivity and specificity when tested against purified DNA from 171 F. oxysporum isolates and 69 lettuce samples. The relationship between qPCR Ct values and colony forming units (CFU)/g values was also determined. This study not only introduces a new marker for FOLac race 1 diagnostics and soil quantitation, but also underscores the value of an extensive genomic database and screening software pipeline for developing molecular diagnostics for F. oxysporum formae speciales and other fungal taxa.

Evaluation of Fecal Sample Pooling for Real-Time RT-PCR Testing SARS-CoV-2 in Animals

During the COVID-19 pandemic, veterinary diagnostic laboratories tested both human and animal samples and needed to ensure that they could accurately perform large numbers of diagnostic tests in a timely manner. Sample pooling, a methodology used effectively for over 80 years as a surveillance tool for screening large numbers of potentially infected individuals, was employed. Given its sensitivity, real-time polymerase chain reaction (PCR) is more suitable for employing this strategy, as compared to other less sensitive testing methods. In this study, we evaluated the capability of detecting SARS-CoV-2 in both 5-sample and 10-sample pools of feces using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) as well as determined the level of sensitivity. A blinded method test (BMT) by an independent laboratory was conducted to assess the five-sample fecal pool. To complement detection capability, the stability of the genome within a PBS fecal suspension was measured under various time and temperature conditions across a 28-day period. Our results showed that the limit of detection for 5-sample and 10-sample fecal pools is 12.8 and 6.4 genome copies in a 25 µL PCR, respectively. The 5-sample and 10-sample pooling resulted in a cycle threshold (Ct) value loss of 2.35 and 3.45, as compared to Ct values of known positive individual samples, but consistent detection was still achieved in pools containing positive samples with an original Ct below 36 and 34, respectively. The simulation of clinical five-sample pooling showed that all positive samples could be detected regardless of the number (1–3) of positive samples in each pool. The BMT results demonstrated excellent sensitivity (100 copies/reaction) in five-sample pools for the detection of SARS-CoV-2 RNA even though a fecal matrix effect was observed. Finally, our results show that the SARS-CoV-2 genome remains stable over a wide range of time and temperature variations. Overall, our findings provide solid data to scale up SARS-CoV-2 testing capacity in veterinary diagnostic laboratories.

Neither increasing the frequency of sow feedings nor decreasing the interval between feedings prior to farrowing reduced piglet stillbirths

Abstract Farrowing durations that exceed 240 min cause stillborn rates to increase. Therefore, feeding strategies in late gestation have been studied to mitigate the negative consequences of extended farrowing durations. A total of 1,501 sows (PIC 1050 Camborough) were used for this study at two individual farms near Carthage, Illinois. Farm 1 (758 sows) was porcine reproductive and respiratory virus (PRRSv) stable (previously experienced a PRRSv outbreak and currently vaccinated for PRRSv) with an older parity structure (3.67). Farm 2 (743 sows) was PRRSv positive (sows with pigs demonstrating Ct values &amp;lt; 36 determined by pig processing fluids) and had a younger parity structure (2.96). Sows were moved into farrowing rooms at approximately d 112 of gestation and started on their respective treatment. Treatment 1 sows were fed 1 meal of 2.27 kg per day at 0600 h. Treatment 2 sows were fed 2 equal meals of 1.13 kg (2.27 kg total) at 0600 and 1400 h. Treatment 3 sows were fed 2 equal meals of 1.13 kg (2.27 kg total) at 0600 and 1800 h. Treatment 4 sows were fed 3 equal meals of 0.77 kg (2.27 kg total) at 0600, 1400, and 2200 h. Treatments were assigned to farrowing rooms in a replicated 4×4 Latin Square arrangement of treatments where each treatment was fed in each room one time at each farm. Daily feed intakes were recorded from the time sows were introduced to the farrowing room until 5 days after farrowing. The total number of pigs born, pigs born alive, stillbirths, and mummies were recorded for each litter within 24h of farrowing. Live pigs were weighed as a group to record litter birth weight. Pig mortality and morbidities were recorded until 5 days after farrowing. The number of total pigs born, and pigs born alive were not different (P ≥ 0.59) among treatments. The number of stillborn pigs were not different (P = 0.33) among treatment 1(1.15 ±1.41), treatment 2 (1.20 ±1.36), treatment 3 (1.30 ±1.46), and treatment 4 (1.14 ±1.28). Sows fed 3 times per d at 8 h intervals tended (P = 0.08) to reduce the percentage of sows farrowed under supervision compared with sows fed once a d. Sows fed twice per d at 12 h intervals reduced (P = 0.01) the percentage of sows provided assistance compared with feeding sows once per d. Feeding a sow one meal of 2.27 kg, two meals (2.27 kg total), or 3 meals (2.27 kg total) of feed a day before farrowing did not reduce the number of stillborn piglets regardless of farm health status or parity structure.

Evaluating the efficacy of probiotics and ascorbic acid as anti-stress agents against heat stress in broiler chickens

Heat stress poses a substantial challenge to poultry production worldwide, highlighting the urgent need for effective management strategies. This study investigated the efficacy of probiotics (Saccharomyces cerevisiae) and ascorbic acid as antistress agents using cloacal and body surface temperatures (CT and BST) as heat stress biomarkers in broiler chickens. A total of 56 broiler chicks were used for the experiment and were divided into four distinct groups: control, probiotics (1 g/kg of feed), ascorbic acid (200 mg/kg of feed) and the combination of probiotics and ascorbic acid (1 g/kg and 200 mg/kg of feed, respectively). The study lasted 35 days; measurements were taken for ambient temperature (AT), CT, and BST. The ambient temperature in the pens consistently exceeded the thermoneutral zone (TNZ) established for broiler chickens. The CT values for broiler chickens in the probiotic group were significantly lower (p &amp;lt; 0.05) compared to the control group. Additionally, the BST values in the probiotic and probiotic + ascorbic acid groups were significantly lower (p &amp;lt; 0.05) than those in the control group. The findings suggest that incorporating probiotics, with or without ascorbic acid, can effectively reduce CT and BST values in broiler chickens thereby, enhancing thermoregulation when compared to the control group. This implies that using probiotics in poultry diets may enhance health and growth performance, potentially leading to better feed efficiency and reduced reliance on antibiotics. Implementing these dietary strategies could improve the productivity and welfare of broiler chickens in commercial settings.

Presence of Soya in Industrial and Homemade Sausage Production in Kosovo and Its Reflection on Fatty Acid Profile

In the present study, we investigated to what extent soybean was used in industrial and homemade sausage produced in Kosovo, as well as its potential impact on the fatty acid profile. In total, 63 samples, 42 industrial and 21 traditional sausages, were collected either from the market or the production site. All samples were tested by means of real-time PCR for the detection and quantification of soy content, as well as the GC-FID to determine the potential reflection in the fatty acid profile. The presence of soy DNA was detected in 54 out of 63 samples. In total, 41 out of 42 industrial sausage samples were positive for soy DNA, with an average ct value of 22.60 and a standard deviation (SD) of 4.28, whereas 13 out of 21 traditional sausage samples were positive for soy DNA, with a mean ct value of 23.36 and a standard deviation (SD) of 4.56. There was a statistically significant difference in means between industrial sausage and traditional sausage, with p ≤ 0.001 based on CT values (ANOVA test). We investigated the correlation between ct values in real-time PCR for soy DNA with each fatty acid content. There is a moderate correlation of soy DNA ct values with C16:0 palmitin (decrease), C18:0 stearic acid (decrease), C18:1 oleic acid (increase), and overall saturated fatty acids (decrease). With the exception of C14:0 Myristin, C18:1 oleic, and C20:0 Arachin acids and monosaturated fats, the ANOVA test reveals a significant difference in means between groups for the majority of the fatty acids between industrial sausages and traditional sausages. The current study demonstrates that the fatty acid composition of sausages is influenced by the amount of soy present in them. The extent to which other components affect the fatty acid profile is unknown. However, an increase in oleic acid and a decrease in stearic and overall saturated fatty acids are expected.

Feasibility study in quantitative identification of fresh and old osteoporotic vertebral compression fracture by vertebral CT value and CT value difference

BackgroundThis study aims to assess the diagnostic utility of vertebral CT value and CT value difference in distinguishing between fresh and old osteoporotic vertebral compression fractures (OVCF).MethodsA retrospective analysis was conducted on 118 patients with OVCF who underwent both MRI and CT examinations at our hospital. The nature of the fractured vertebra was determined according to MRI. The CT value of the fractured vertebrae and the mean CT value of the adjacent normal vertebrae were measured separately, and the differences between these values were calculated. Independent samples t-tests were used to compare CT value and CT value difference among all groups. The receiver operating characteristic (ROC) curve was employed to determine the optimal cut-off value for both CT value and CT value difference in differentiating fresh and old fractures.ResultsThe study included a total of 163 fractured vertebrae from 118 patients. The CT value of fresh fractured vertebrae was significantly higher than those of adjacent normal vertebrae, which was statistically different (P < 0.001). In contrast, the difference between CT value of old fractured vertebrae and those of adjacent normal vertebrae was not statistically significant (P > 0.05). There were significant differences in CT value and CT value difference between fresh fractured vertebrae and old fractured vertebrae (P < 0.001). The ROC curve analysis showed that the optimal cut-off value of CT value for fresh fractures and old fractures was 103.40 HU. The optimal cut-off value of CT value difference was 39.81 HU.ConclusionVertebral CT value and CT value difference offer a certain reference value for distinguishing between fresh and old OVCF. These parameters can serve as a rapid diagnostic tool when MRI is unavailable or impractical, aiding in the timely assessment of OVCF.

Comparison of different iterative reconstruction algorithms with contrast-enhancement boost technique on the image quality of CT pulmonary angiography for obese patients

ObjectiveTo evaluate the effect of the contrast-enhancement-boost (CE-boost) postprocessing technique on improving the image quality of obese patients in computed tomography pulmonary angiography (CTPA) compared to hybrid iterative reconstruction (HIR) and model-based iterative reconstruction (MBIR) algorithms.MethodsThis prospective study was conducted on 100 patients who underwent CTPA for suspected pulmonary embolism. Non-obese patients with a body mass index (BMI) under 25 were designated as group 1, while obese patients (group 2) had a BMI exceeding 25. The CE-boost images were generated by subtracting non-contrast HIR images from contrast-enhanced HIR images to improve the visibility of pulmonary arteries further. The CT value, image noise, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) were quantitatively assessed. Two chest radiologists independently reviewed the CT images (5, best; 1, worst) across three subjective characteristics including diagnostic confidence, subjective image noise, and vascular contrast. The Friedman test and Dunn-Bonferroni correction were used for statistical analysis.ResultsThe CE-boost had significantly higher CT values than HIR and MBIR in both groups (all p < 0.001). The MBIR yielded the lowest image noise compared with HIR and CE-boost (all p < 0.001). The SNR and CNR of main pulmonary artery (MPA) were significantly higher in CE-boost than in MBIR (all p < 0.05), with HIR showing the lowest values (all p < 0.001). Group 2 MBIR received significantly better subjective image noise scores, while the diagnostic confidence and vascular contrast scored highest with the group 2 CE-boost (all p < 0.05).ConclusionCompared to the HIR algorithm, both the CE-boost technique and the MBIR algorithm can improve the image quality of CTPA in obese patients. CE-boost had the greatest potential in increasing the visualization of pulmonary artery and its branches.

SARS-CoV-2 variant survey: Comparison of RT-PCR screening with TGS and variant distribution across two divisions of Bangladesh.

The widespread increase in multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) since 2020 is causing significant health concerns worldwide. While whole-genome sequencing (WGS) has played a leading role in surveillance programs, many local laboratories lack the expertise and resources. Thus, we aimed to investigate the circulating SARS-CoV-2 variants and evaluate the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) for screening and monitoring the emergence of new SARS-CoV-2 variants in Bangladesh. A total of 600 confirmed SARS-CoV-2-positive cases were enrolled either prospectively or retrospectively from two divisions of Bangladesh. The samples were screened by variant RT-PCR targeting five mutations of the spike gene (N501Y, P681R, L452R, E484K, E484Q). A subsample of the study population was also selected for third-generation sequencing (TGS) and the results were compared to the variant RT-PCR screening. An in-depth comparison was made between the two methods in terms of congruence and cost-benefit. Seven variants were detected among samples, with similar distributions of the variants across both divisions. Variant RT-PCR for the targeted mutations lead to a 98.5% call rate; only nine samples failed to be determined. No association was found regarding the demographic features, clinical criteria, or routine RT-PCR Ct values across the variants. The clade diversity of the sequenced subpopulation (n = 99) exhibited similar distributions across the two study sites and other epidemiologic variables. Variant RT-PCR successfully distinguished variants of concern (VOCs) and variants of interest (VOIs); however, 8% discrepancy was observed for the closest lineages. Moreover, the variant RT-PCR represented an ideal balance of cost, time, and accuracy that outweigh their limitations. Based on the strong agreement of variant RT-PCR with TGS, such rapid, easily accessible approaches of rapid strain typing are essential in the context of pandemic responses to guide both treatment decisions and public health measures.

Cycle Threshold Values of SARS-CoV-2 RT-PCR during Outbreaks in Nursing Homes: A Retrospective Cohort Study.

a retrospective cohort design with Poisson regression and multinomial logistic regression were used. We studied four nursing home SARS-CoV-2 outbreaks, and the average infection rate, reinfection rate, and case fatality were 72.7%, 19.9%, and 5.5%, respectively; 98.9% of residents were vaccinated with three doses of a mRNA SARS-CoV-2 vaccine. Ct values for first infections and reinfections were 27.1 ± 6.6 and 31.9 ± 5.4 (p = 0.000). Considering Ct values ≥ 30 versus <30, residents with reinfections had Ct values higher than residents with a first infection, an adjusted relative risk of 1.66 (95% Confidence interval 1.10-2.51). A sensitivity analysis confirmed these results. Reinfection and SARS-CoV-2 vaccination (hybrid immunity) could protect against severe disease better than vaccination alone. High Ct values suggest lower transmission and severity. Its value can be useful for surveillance and forecasting future SARS-CoV-2 epidemics.

Novel Approach for Detection of BRAF and KRAS Mutations Using ARMS (amplification refractory mutation system) Primers for Colon Cancer Therapeutic Response Prediction

Abstract Introduction/Objective KRAS and BRAF V600E mutations are confirmed predictive biomarkers for anti-EGFR monoclonal antibody therapy response and prognosis for colorectal cancer and often considered mutually exclusive with few exceptions (0.05% of mCRC cases). We have developed a multiplex PCR assay utilizing ARMS (amplification refractory mutation system) Primers and Probes for screening and further testing of BRAF mutation status in a 2 step process. Methods/Case Report The Multiplex PCR Assay or BRAF/KRAS mutation was designed as a 5 color assay. Primers and Probes consisted of Wild Type Kras and wild and mutant (V600E/V600K) type BRAF primers and probes. For specimen, Positive and negative control standards made of engineered positive control plasmids for wild type KRAS &amp; BRAF and mutant type V600E &amp; V600K BRAF as well as internal control of bet actin and 2 Patient samples with known BRAF V600E mutant status were run. Mutant type V600E &amp; V600K BRAF were mixed with wild type V600 controls in different ratios as well. Results were interpreted as BRAF wild KRAS wild/ BRAF wild KRAS mutant/ BRAF V600E KRAS wild/BRAF V600K KRAS wild type and NGS was recommended in cases of concurrent BRAF and KRAS mutations. Results (if a Case Study enter NA) Positive/negative/internal control standards for wild type KRAS &amp; BRAF and mutant type V600E &amp; V600K BRAF showed expected results in both CFX 96 and ABI 7500 runs in triplicates. Graphs were able to determine separate Ct values and sigmoidal amplification plots that readily differentiated KRAS wild/BRAF wild/ BRAF V600E types. Mutant type V600E &amp; V600K BRAF that were mixed with wild type V600 controls in different ratios all showed expected results and were able to differentiate between wild and mutant types with accuracy. Also, the 2 patient samples with known (Sanger sequencing-diagnosed) V600E mutation status showed positive Ct values for KRAs wild and V600E probes and negative CT values for V600 wild/V600K probes. No invalid runs or unexpected control results were observed. Conclusion The ARMS BRAF/KRAS multiplex test is a efficient, accurate 2 step assay. In the patients and positive and negative controls prepared, it was able to reliably diagnose KRAS and BRAF mutation status in a matter of 4 hours running time. Given the fact that this is a cost effective assay that can utilize most commonly used Real time PCR machines this test may be clinically implemented if larger clinical trials utilizing tissue samples from a larger number of colon cancer patients are conducted.

Prevalence, Radiological, and Pathological Findings of Ossification and Calcification of the Lumbar Spinal Ligamentum Flavum: A Comparative Study with Thoracic Lesions.

This study aimed to determine the prevalence of lumbar ligamentum flavum lesions and identify correlations between radiological and pathological findings. We conducted an observational cross-sectional study of 349 patients (lumbar: n = 296, thoracic: n = 39, lumbar and thoracic: n = 14, mean age: 69 ± 12 years, male: 74%) who underwent posterior surgery for thoracolumbar spinal canal stenosis between January 2008 and April 2023 at our hospital.Computed tomography (CT) revealed that the prevalence of ligamentum flavum lesions defined as a high-density area with a CT value of 200 Hounsfield Unit or higher in the lumbar and thoracic spine was 47% (147/310) and 85% (45/53), respectively. CT showed that most patients had radiologically suspected ossification in the lumbar (90%) and thoracic spine (98%) than radiologically suspected calcification. Lumbar lesions were thinner than the thoracic lesions (2.5 vs 3.7 mm, p < 0.01). Pathological examinations were performed in specimens collected from 34 cases (lumbar: n = 13, thoracic: n = 21), and ossification was found in 62% (8/13) and 95% (20/21) of lumbar and thoracic lesions (p = 0.02), respectively. Lastly, ossification was confirmed pathologically in 72% (8/11) and 95% (19/20) of lumbar and thoracic lesions that showed ossification on imaging (p = 0.13), respectively. The literature review revealed that the prevalence of the lumbar ligamentum flavum lesions varied from 1.5 to 35% and the patient population was mostly asymptomatic.Collectively, we found that the prevalence of lumbar ligamentum flavum lesions in symptomatic patients was greater than previously reported. Histologically confirmed ossification was less common in lumbar lesions than in thoracic lesions.

Association of COVID-19 with acute and post-acute risk of multiple different complications and mortality in patients infected with omicron variant stratified by initial disease severity: a cohort study in Hong Kong

BackgroundFew studies have attempted to use clinical and laboratory parameters to stratify COVID-19 patients with severe versus non-severe initial disease and evaluate age-specific differences in developing multiple different COVID-19-associated disease outcomes.MethodsA retrospective cohort included patients from the electronic health database of Hong Kong Hospital Authority between 1 January 2022 and 15 August 2022 until 15 November 2022. The cohort was divided into three cohorts by age (≤ 40, 41–64, and ≥ 65 years old). Each age cohort was stratified into four groups: (1) COVID-19 critically exposed group (ICU admission, mechanical ventilation support, CRP > 80 mg/L, or D-dimer > 2 g/mL), (2) severely exposed group (CRP 30–80 mg/L, D-dimer 0.5–2 g/mL, or CT value < 20), (3) mildly–moderately exposed group (COVID-19 positive-tested but not fulfilling the criteria for the aforementioned critically and severely exposed groups), and (4) unexposed group (without COVID-19). The characteristics between groups were adjusted with propensity score-based marginal mean weighting through stratification. Cox regression was conducted to determine the association of COVID-19 disease severity with disease outcomes and mortality in the acute and post-acute phase (< 30 and ≥ 30 days from COVID-19 infection) in each age group.ResultsA total of 286,114, 320,304 and 194,227 patients with mild–moderate COVID-19 infection; 18,419, 23,678 and 31,505 patients with severe COVID-19 infection; 1,168, 2,261 and 10,178 patients with critical COVID-19 infection, and 1,143,510, 1,369,365 and 1,012,177 uninfected people were identified in aged ≤ 40, 40–64, and ≥ 65 groups, respectively. Compared to the unexposed group, a general trend tending towards an increase in risks of multiple different disease outcomes as COVID-19 disease severity increases, with advancing age, was identified in both the acute and post-acute phases. Notably, the mildly–moderately exposed group were associated with either insignificant risks (aged ≤ 40) or the lowest risks (aged > 40) for the disease outcomes in the acute phase of infection (e.g., mortality risk HR (aged ≤ 40): 1.0 (95%CI: 0.5,2.0), HR (aged 41–64): 2.1 (95%CI: 1.8, 2.6), HR (aged > 65): 4.8 (95%CI: 4.6, 5.1)); while in the post-acute phase, these risks were largely insignificant in those aged < 65, remaining significant only in the elderly (age ≥ 65) (e.g., mortality risk HR (aged ≤ 40): 0.8 (95%CI: (0.5, 1.0)), HR (aged 41–64): 1.1 (95%CI: 1.0,1.2), HR (aged > 65): 1.5 (95%CI: 1.5,1.6)). Fully vaccinated patients were associated with lower risks of disease outcomes than those receiving less than two doses of vaccination.ConclusionsThe risk of multiple different disease outcomes in both acute and post-acute phases increased significantly with the increasing severity of acute COVID-19 illness, specifically among the elderly. Moreover, future studies could improve by risk-stratifying patients based on universally accepted thresholds for clinical parameters, particularly biomarkers, using biological evidence from immunological studies.

Oropharyngeal swab sampling for PRRSV detection in large-scale pig farms: a convenient and reliable method for mass sampling

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) has significant productivity and economic impacts in swine herds. Accurately determining the PRRSV status at the herd level is crucial for producers and veterinarians to implement strategies to control and eliminate the virus from infected herds. This study collected oropharyngeal swabs (OSs), nasal swabs (NSs), oral fluid swabs (OFs), rectal swabs (RSs), and serum samples continuously from PRRSV challenged pigs under experimental conditions and growing pigs under field conditions. Additionally, OSs and serum samples were collected from individual sows from 50 large-scale breeding farms, and the collection of OSs does not require the sows to be restrained. Ct values of PRRSV were detected in all samples using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR).ResultsIn PRRSV challenged pigs, OSs showed a higher PRRSV-positive rate until the end of the observation period. The Ct values of OSs were significantly lower than those of NSs, OFs, and RSs at 2, 8, 12, 14 and 20 days post-challenge (DPC) (P < 0.05). For growing pigs, the positivity rate of PRRSV in OSs was higher than that in other sample types at 30, 70, and 110 days of age. In sows, 24,718 OSs and 6259 serum samples were collected, with PRRSV-positive rate in OSs (9.4%) being significantly higher than in serum (4.1%) (P < 0.05). However, the Ct values of PRRSV RNA in serum were significantly lower than those in OSs (P < 0.001).ConclusionsThe OSs sample type yielded higher PRRSV-positive rates for longer periods compared to NSs, RSs, OFs and serum samples for PRRSV detection in infected pigs. Therefore, OSs has a good potential to be a convenient, practical, and reliable sample type for implementing mass sampling and testing of PRRSV in large-scale pig farms.

Correlation of COVID-19 vaccination and RT-PCR ct value among cases in Addis Ababa, Ethiopia: implication for future preparedness

BackgroundThe COVID-19 disease requires accurate diagnosis to effectively manage infection rates and disease progression. The study aims to assess the relationship between vaccination status and RT-PCR cycle threshold (Ct) values by comparing clinical, RDT and RT-PCR results.MethodsA total of 453 suspected COVID-19 cases were included in this study. Nasopharyngeal swabs were collected for both RDT and RT-PCR testing, with RDTs conducted on-site and RT-PCR at the Ethiopian Public Health Institute (EPHI) genomics laboratory. Detailed clinical, RDT, and RT-PCR results were analyzed. Data analysis included descriptive statistics, cross-tabulation, and Chi-Square tests to investigate the connections between diagnostic outcomes and vaccination status, with a focusing on Ct values.ResultsRDT results showed 34.0% negative and 65.8% positive, while RT-PCR results indicated 35.8% negative and 64.2% positive cases. The discrepancies between RDT and RT-PCR results emphasize the importance of thorough testing. No significant association was found between vaccination status and viral load, as indicated by Ct values. Among RT-PCR positive cases, 49.8% had been vaccinated, suggesting challenges in interpreting results among vaccinated individuals. Further analysis revealed that vaccination (first or second dose) had minimal impact on Ct values, indicating limited influence of vaccination status on viral load dynamics in infected individuals.ConclusionsThe study highlights the significant differences between RDT and RT-PCR outcomes, underscoring the need for a comprehensive testing approach. Additionally, the findings suggest that vaccination status does not significantly impact RT-PCR Ct values, complicating the interpretation of diagnostic results in vaccinated individuals, especially in breakthrough infections and potential false positives.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

Exploring the causes of variability in quality of oropharyngeal swab sampling for SARS-CoV-2 nucleic acid testing and proposed improvement measures: a multicenter, double-blind study.

Although coronavirus disease 2019 (COVID-19) has not been considered a public health emergency of international concern since last year, intermittent regional impacts still persist, and accurate testing remains crucial. Ribonuclease P protein subunit P30 (RPP30) RNA, known for its broad and stable expression in tissue cells, was used to evaluate samples from 10 hospitals with over 3,000 negative nucleic acid tests. The results revealed that the overall pass rate for the collected samples was consistently low and exhibited significant heterogeneity. After reassessing the evaluative effectiveness of RPP30 RNA Ct values from the samples of 132 positive individuals under quarantine observation, it was used to identify factors affecting sampling quality. These factors included different stages ranging from sample collection to PCR processing, various characteristics of both samplers and individuals being sampled, as well as sampling season and location. The results indicated that post-sampling handling had minimal impact, winter and fever clinic samples showed higher quality, whereas children's samples had lower quality. The key finding was that the characteristics of samplers were closely related to sampling quality, emphasizing the role of subjectivity. Quality control warnings led to substantial improvements, confirming this finding. Consequently, although there are various factors during the testing process, the most critical aspect is to improve, supervise, and maintain standardized practices among sampling staff.IMPORTANCEThis study further confirmed the reliability of internal references (IRs) in assessing sample quality, and utilized a large sample IR data to comprehensively and multidimensionally identify significant interference factors in nucleic acid test results. By further reminding and intervening in the subjective practices of specimen collectors, good results could be achieved.

Assessing infectiousness and the impact of effective treatment to guide isolation recommendations for people with pulmonary tuberculosis.

Determining the extent and duration of infectiousness of individuals with pulmonary tuberculosis (TB) is critical for various aspects of TB care, including decisions regarding isolation. Studies suggest considerable heterogeneity in infectiousness of people with pulmonary TB. Pre-treatment, measures of bacillary burden including sputum smear microscopy, culture time-to-positivity, and Xpert MTB/RIF cycle threshold (Ct) value, predict the risk of transmission to contacts. Index patients with smear negative disease pose lower infectious risk than those who have smear-positive disease, and household contact infection is more likely with index patients who have lower Xpert Ct values. Newer tools that enable detection of Mycobacterium tuberculosis complex (Mtb complex) from cough aerosol sampling and face mask sampling may be better predictors of contact infection risk. Clinical factors such as cough strength and frequency, and presence of cavitation on chest imaging, may also assist with risk prediction. Post-treatment, smear and culture status are poor predictors of infectiousness. While the exact duration of infectiousness post treatment initiation remains uncertain, data from human-to-guinea pig transmission studies and clinical studies suggest effective treatment results in a rapid decline in infectiousness, irrespective of smear or culture conversion. This is largely supported by early bactericidal activity and transcriptomic studies, and cough aerosol sampling studies, although a subset of patients may have persistent cough aerosol positivity. These findings can enable a more nuanced approach to isolation decision making, while further research studies are awaited.

Characterizing best practices for tonsil-oral-scrubbing (TOSc) collection for PRRSV RNA detection in sows

BackgroundA Tonsil-Oral-Scrubbing (TOSc) method was developed to sample the sow’s oropharyngeal and tonsillar area without snaring and has shown comparable porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection rates with tonsil scraping in infected sows. This study investigated the effect of specific TOSc collection factors on the PRRSV RT-rtPCR results (detection rates and Ct values). Those factors include whether the sow was snared or not snared at TOSc collection (“snared” vs. “not snared”); whether the sow was laying down or standing at collection (“laying down” vs. “standing”); and type of collectors used for TOSc collection (“TOSc prototype” vs. “Spiral-headed AI catheter (SHAC)”). Volume of fluid was compared between “snared” and “not snared” groups, and collection time was compared between “laying down” and “standing” groups as well.ResultsThe effect for each factor was assessed in three independent studies following the same design: TOSc was collected twice from each studied sow, once with the baseline level for a factor (“not snared”, or “standing”, or “TOSc prototype”), and another time followed by the other level of the paired factor (“snared”, “laying down”, or “SHAC”, correspondingly). Results showed that “not snared” TOSc had numerically higher PRRSV RNA detection rate (60.7% vs. 52.5%, p = 0.11), significantly lower median Ct values (31.9 vs. 32.3, p < 0.01), and significantly higher volume of fluid than “snared” samples (1.8 mL vs. 1.2 mL, p < 0.01); “laying down” TOSc samples did not differ statistically (60.7% vs. 60.7%) in the PRRSV RNA detection rate, obtained numerically lower median Ct values (30.9 vs. 31.3, p = 0.19), but took 40% less collection time compared to “standing” TOSc samples; samples collected using the “TOSc prototype” had numerically higher PRRSV RNA detection rate (91.7% vs. 88.3%, p = 0.27) and significantly lower median Ct values (32.8 vs. 34.5, p < 0.01) than that from “SHAC”.ConclusionsUnder the conditions of this study best practices for TOSc collection aiming higher detection rate of PRRSV RNA while minimizing time for collection were suggested to be sampling TOSc without snaring, when sows are laying down, and using a prototype TOSc collector.