Average CT Value Research Articles

A high scale SARS-CoV-2 profiling by its whole-genome sequencing using Oxford Nanopore Technology in Kazakhstan.

Severe acute respiratory syndrome (SARS-CoV-2) is responsible for the worldwide pandemic, COVID-19. The original viral whole-genome was sequenced by a high-throughput sequencing approach from the samples obtained from Wuhan, China. Real-time gene sequencing is the main parameter to manage viral outbreaks because it expands our understanding of virus proliferation, spread, and evolution. Whole-genome sequencing is critical for SARS-CoV-2 variant surveillance, the development of new vaccines and boosters, and the representation of epidemiological situations in the country. A significant increase in the number of COVID-19 cases confirmed in August 2021 in Kazakhstan facilitated a need to establish an effective and proficient system for further study of SARS-CoV-2 genetic variants and the development of future Kazakhstan’s genomic surveillance program. The SARS-CoV-2 whole-genome was sequenced according to SARS-CoV-2 ARTIC protocol (EXP-MRT001) by Oxford Nanopore Technologies at the National Laboratory Astana, Kazakhstan to track viral variants circulating in the country. The 500 samples kindly provided by the Republican Diagnostic Center (UMC-NU) and private laboratory KDL “Olymp” were collected from individuals in Nur-Sultan city diagnosed with COVID-19 from August 2021 to May 2022 using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). All samples had a cycle threshold (Ct) value below 20 with an average Ct value of 17.03. The overall average value of sequencing depth coverage for samples is 244X. 341 whole-genome sequences that passed quality control were deposited in the Global initiative on sharing all influenza data (GISAID). The BA.1.1 (n = 189), BA.1 (n = 15), BA.2 (n = 3), BA.1.15 (n = 1), BA.1.17.2 (n = 1) omicron lineages, AY.122 (n = 119), B.1.617.2 (n = 8), AY.111 (n = 2), AY.126 (n = 1), AY.4 (n = 1) delta lineages, one sample B.1.1.7 (n = 1) belongs to alpha lineage, and one sample B.1.637 (n = 1) belongs to small sublineage were detected in this study. This is the first study of SARS-CoV-2 whole-genome sequencing by the ONT approach in Kazakhstan, which can be expanded for the investigation of other emerging viral or bacterial infections on the country level.

Feasibility of an ultra-low dose contrast media protocol for coronary CT angiography

To evaluate the feasibility of an ultra-low volume contrast media (CM) protocol for coronary computed tomography angiography (CTA). In total, 214 patients receiving coronary CTA were enrolled prospectively and divided into group A (n=107) receiving a conventional dose of CM and group B (n=107) receiving an ultra-low dose. CT values of the right coronary artery (RCA), left anterior descending artery (LAD), and left circumflex artery (LCX) were measured and radiation doses recorded. The image quality was compared between the groups. Changes in renal function indices and proteinuria before, 24, and 72 hours after coronary CTA among those with chronic kidney disease (CKD) were also assessed. There were significant differences in CT values and radiation doses between groups A and B. In group A, the average RCA, LAD, and LCX CT values were 412.5±79.2, 423.5±73.7, and 422.0±88.1 HU, respectively. In group B, the average RCA, LAD, and LCX CT values were 275.2±16.2, 277.8±16.4, and 278.9±16.5 HU, respectively. The radiation dose in the ultra-low protocol recipients (118.70±18.52 mGy·cm) was significantly lower than that used in conventional coronary CTA (131.75±20.96 mGy·cm). The image quality of group B was comparable to that of group A, satisfying the diagnostic requirement. In patients with mild CKD, there were no significant differences in renal functions after coronary CTA. An ultra-low CM protocol was established for coronary CTA, providing comparable image quality and diagnostic yields but significantly lower radiation dose compared with a conventional protocol. This new protocol might be applicable to patients with mild CKD.

Diagnostic Efficacy of CT Radiomic Features in Pulmonary Invasive Mucinous Adenocarcinoma.

In order to solve the problem of the effect of CT images on the diagnosis of lungs, the authors proposed a method for the diagnosis of invasive mucinous adenocarcinoma of the lungs based on CT radiomic features, and the modified method is found by reviewing past cases: among the 34 cases of primary pulmonary lymphoma, 12 cases were nodular mass type, 19 cases were nonnodular mass type, and 3 cases were mixed type; 13 cases involved bilateral lung lobes, 7 cases involved right lung, and 4 cases involved left lung example. There were 17 cases of tumor consolidation density shadow, 17 cases of mixed density shadow, the average CT value was about 32HU, 15 cases of cavitation sign, 6 cases of cavity, 9 cases of angiography sign, 30 cases of air bronchus sign, 22 cases of bronchiectasis, bronchial stenosis or amputation in 8 cases, pleural effusion in 12 cases, lymph node enlargement in 15 cases, and pleural metastasis in 2 cases. The final pathological results included 24 cases of membrane-associated lymphoid tissue (MALT) lymphoma, 9 cases of diffuse large B-cell lymphoma (DLBCL), and 1 case of T-cell lymphoma. The CT manifestations of primary pulmonary lymphoma (PPL) are diverse and do not have obvious specificity, the imaging manifestations are correlated with pathological types, and air bronchial signs, bronchiectasis, angiography signs, and other signs are used for the diagnosis of PPL. This is of great significance for the diagnosis of PPL.

Spaceflight validation of one-step Gene Sampling tool for genetic analysis on the International Space Station

• The Gene Sampling tool enables rapid RNA purification for genetic analysis on ISS. • The technology is non-destructive to the sample and allows repeated testing. • The tool was successfully used for plant gene expression analysis on orbit. • The average Ct values of the ground control and the ISS tests were 17.5 and 15.2.

Reporting of RT-PCR cycle threshold (Ct) values during the first wave of COVID-19 in Qatar improved result interpretation in clinical and public health settings.

Introduction. The cycle threshold (Ct) value in real-time PCR (RT-PCR) is where a target-specific amplification signal becomes detectable and can infer viral load, risk of transmission and recovery. Use of Ct values in routine practice is uncommon.Gap Statement. There is a lack of routine use of Ct values when reporting RT-PCR results in routine practice.Aim. To automatically insert Ct values and interpretive comments when reporting SARS-CoV-2 RT-PCR to improve patient management.Methodology. Routine Ct values across three different RT-PCR platforms were reviewed for concordance at presentation and clearance in patients with COVID-19. An indicative threshold (IT) linked to viral clearance kinetics was defined at Ct30 to categorize Ct values as low and high, reflecting high and low viral loads respectively.Results. The different gene targets of each platform showed high correlation and kappa score agreement (P<0.001). Average Ct values were automatically generated with values ≤Ct30 reported as positive and >Ct30 as reactive; interpretive comments were added to all reports. The new reporting algorithm impacted on: physician interpretation of SARS-CoV-2 results; patient management and transfer; staff surveillance; length of stay in quarantine; and redefinition of patient recovery.Conclusion. Incorporation of Ct values into routine practice is possible across different RT-PCR platforms and adds useful information for patient management. The use of an IT with interpretive comments improves clinical interpretation and could be a model for reporting other respiratory infections. Withholding Ct values wastes useful clinical data and should be reviewed by the profession, accreditation bodies and regulators.

SARS-CoV-2 breakthrough infections among hospitalized patients in southeastern Michigan

Background: As of January 2022, more than 57 million cases of COVID-19 have been reported in the United States. Three primary COVID-19 vaccines are widely available: Pfizer (BNT162b2), Moderna (mRNA-1273), and Johnson &amp; Johnson’s-Janssen (JNJ-78436735). The vaccines are effective but do not prevent all infections. We investigated trends in type of vaccine receipt, demographic characteristics, and disease outcomes in COVID-19 breakthrough infections among hospitalized patients. Methods: A breakthrough case is defined as the detection of SARS-CoV-2 ≥14 days after completion of all doses of an FDA-authorized COVID-19 vaccine. An electronic medical record report in EPIC EHR software identified 85 fully vaccinated patients with a documented positive SARS-CoV-2 result between February and September 2021 at 2 hospitals in southeastern Michigan. Demographic information and hospitalization characteristics, including length of stay and oxygen requirements, were collected from the report. Patients were classified into disease severity categories: nonsevere, severe, or critical. A case was considered severe if the patient’s oxygen saturation level (SpO2) was ≤94% on room air or if the patient required supplemental oxygen. Illness was considered critical if the patient developed respiratory failure, including mechanical ventilation or extracorporeal membrane oxygenation. All other cases were classified as nonsevere. Cycle threshold (Ct) values, the number of PCR cycles required to reach a threshold of SARS-CoV-2 genomic material, were collected from the hospital microbiology lab. Results: We identified 85 breakthrough infections (Fig. 1). The average patient age was 69.9±15.7 years, and 44 (51.8%) were female. Severe disease was most common (n = 73, 85.9%) followed by nonsevere disease (n = 7, 8.24%), and 9 patients (10.6%) in this cohort died. Most patients received either the Moderna (n = 35, 41.2%) or Pfizer (n = 38, 44.7%) vaccines. Pfizer vaccine receipt was most common among patients with severe illness (n = 33 of 73, 45.2%), and Moderna vaccine receipt was most common among patients with critical illness (n = 4 of 5, 80.0%). Average time from last vaccination to positive test was longest among Moderna vaccine recipients (181.9±43.1 days) and shortest among J&amp;J vaccine recipients (91.0±61.1 days). The average Ct value was 23.8±7.5 and ranged from 13.0 to 41.3. There were no appreciable differences in the average Ct value by vaccine manufacturer. Conclusions: Breakthrough infections among hospitalized patients were uncommon, but incidence increased with time after vaccine receipt in all vaccines. Further study is needed to examine differences and severity in breakthrough infections by vaccine type and in individuals who completed booster vaccines.Funding: NoneDisclosures: None

Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay: sample pooling increases testing throughput

During the COVID-19 pandemic, sample pooling has proven an effective strategy to overcome the limitations of reagent shortages and expand laboratory testing capacity. The inclusion of influenza and respiratory syncytial virus (RSV) in a multiplex tandem PCR platform with SARS-CoV-2 provides useful diagnostic and infection control information. This study aimed to evaluate the performance of the influenza and RSV targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay, including the effect of pooling samples on target detection.RSV target detection in clinical samples was compared to the Cepheid Xpert Xpress Flu/RSV assay as a reference standard. Samples were then tested in pools of four and detection rates were compared. Owing to the unavailability of clinical samples for influenza, only the effect of sample pooling on simulated samples was evaluated for these targets.RSV was detected in neat clinical samples with a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.5% compared to the reference standard, demonstrating 99.7% agreement. This study demonstrates that sample pooling by four increases the average Ct value by 2.24, 2.29, 2.20 and 1.91 cycles for the target's influenza A, influenza A typing, influenza B and RSV, respectively. The commercial AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay was able to detect influenza and RSV at an intermediate concentration within the limit of detection of the assay. Further studies to explore the applicability of sample pooling at the lower limit of detection of the assay is needed. Nevertheless, sample pooling has shown to be a viable strategy to increase testing throughput and reduce reagent usage. In addition, the multiplexed platform targeting various respiratory viruses assists with public health and infection control responses, clinical care, and patient management.

Automatic quantitative evaluation of normal pancreas based on deep learning in a Chinese adult population.

To develop a 3D U-Net-based model for the automatic segmentation of the pancreas using the diameters, volume, and density of normal pancreases among Chinese adults. A total of 2778 pancreas images (dataset 1) were retrospectively collected and randomly divided into training (n = 2252), validation (n = 245), and test (n = 281) datasets. The segmentation model for the pancreas was constructed through cascaded application of two 3D U-Net networks. The segmentation efficiency for the pancreas was evaluated by the Dice similarity coefficient (DSC). Another dataset of 3189 normal pancreas CT images (dataset 2) was obtained for external validation, including 1063 non-contrast images, 1063 arterial phase images, and 1063 portal venous phase images. The pancreas segmentation in dataset 2 was assessed objectively and manually revised by two radiologists. Then, the pancreatic volume, diameters, and average CT value for each phase of pancreas images in dataset 2 were calculated. The relationships between pancreas volume and age, sex, height, and weight were analyzed. In dataset 1, a mean DSC of 0.94 for the test dataset was achieved. In dataset 2, the objective assessment yielded a 90% satisfaction rate for the automatic segmentation of the pancreas as external validation. The diameters of the pancreas were 43.71-44.28mm, 67.40-68.15mm, and 114.53-117.06mm, respectively. The average pancreatic volume was 63,969.06-65,247.75 mm3, which was greatest at the age of 18-38 and then decreased to a minimum at the age of 69-85. The CT value of the pancreas also decreased with age, from a maximum value of 38.87 ± 9.70 HU to a minimum of 27.72 ± 10.85 HU. The pancreas segmentation tool based on deep learning can segment the pancreas on CT images and measure its normal diameter, volume, and CT value accurately and effectively.

The Application Value of Spiral CT Lung Densitometry Software in the Diagnosis of Radiation-Induced Lung Injury.

In order to study the application value of spiral CT lung density measurement software in the diagnosis of radioactive lung injury, the average CT values of lung apex, hilum, and diaphragm were measured by Pulmo automatic evaluation software of 16-slice spiral CT in 96 patients with different types of radiation lung injury diagnosed by conventional CT and 80 healthy subjects. The radiation lung injury on CT slices was classified, and the lung density was measured. In 96 patients with different types of radiation lung injury, 56 patients had different degrees of increase in average lung density, which was most obvious in the type of air insufficiency and chronic fibrosis. CT values of lung density in the ground glass stage and patch stage of acute radiation pneumonia had little influence due to the range and time of exposure. The lung density of 35 patients with radiation injury was measured in the normal range. There was a significant difference between normal lung density and abnormal lung density in different types of radiation lung injury (X2 = 56.718, P < 0.001). The mean lung density of 68 cases was normal and that of 12 cases was abnormal. There was a significant difference in lung density between the lung injury group and the normal group (X2 = 18.027, P < 0.001). Spiral CT lung density measurement can accurately evaluate the lung density values of different types of radiation lung injury and judge the correlation between lung density and different types of radiation lung injury. It is of great value to diagnose, locate, and master the radiation dose of different types of radiation lung injury.

43. SARS-CoV-2 Cycle threshold (Ct) values predict future COVID-19 cases

BackgroundThe threat of surging COVID-19 cases prompted many hospitals in the United States to preemptively suspend elective procedures throughout the pandemic. Utilizing samples from a large hospital in Los Angeles, we sought to determine if temporal trends in SARS-CoV-2 Cycle threshold (Ct) values (proxy for viral RNA loads) were predictive for the number of future COVID-19 cases.MethodsNasopharyngeal specimens on symptomatic patients and asymptomatic admissions were tested using the Xpert Xpress SARS-CoV-2 and SARS-CoV-2/Flu/RSV assays (Cepheid). Ct values for all SARS-CoV-2 detections between October 2020 to March 2021 were compiled for analysis.ResultsA total of 2,114 SARS-CoV-2-positive samples were included. The number of tests performed per week increased dramatically in December peaking the first week of January before returning to pre-surge numbers by Mid-February. Ct values fell during this same period with values in December and January (25.6±7.8 and 27±7.9, respectively) significantly lower than those of the other months (30±9.3 to 37.7±6.3). Average weekly Ct values for all patients were significantly, negatively correlated with the number of tests run the following week (R= -0.71, P< 0.001) and two weeks later (R= -0.75, P< 0.0001). Ct values for patients who were asymptomatic at the time of testing most strongly correlated with total number of tests performed one month later (R= -0.86, P< 0.0001). Average weekly Ct values and number of test run As cases (light grey) increased during December and January, there was a significant decrease in Ct values (dark grey) during that same time period.Average Ct values are a leading indicator of cases Average weekly Ct values for all patients (light grey) were significantly, negatively correlated with the number of tests run the following week (R= -0.71, P<0.001) and two weeks later (R= -0.75, P<0.0001). Ct values for patients who were asymptomatic at the time of testing (dark grey) most strongly correlated with total number of tests performed one month later (R= -0.86, P<0.0001).ConclusionLower Ct values, representing higher levels of viral RNA, have been associated with risk of intubation and infectivity. During the winter surge, we observed significantly lower Ct values suggesting that the increased transmission and morbidity of COVID-19 was temporarily associated with higher viral loads. Interestingly, Ct values for asymptomatic patients were most strongly associated with number of cases observed 1 months in the future, suggesting that asymptomatic viral load may be a leading indicator for forthcoming outbreaks. Given this association, Ct values may be a useful tool for predicting regional outbreaks of COVID-19 and more judicious cessation of elective procedures.Disclosures All Authors: No reported disclosures

Why get tested? Linking Intentions for Seeking SARS-CoV-2 Testing with RT-PCR Results and Cycle Threshold Values at Community Testing Sites

During the SARS-CoV-2 pandemic, community-based testing sites have provided free COVID testing to millions of people who would have otherwise faced substantial barriers. In one metropolitan area, over 700,000 tests were performed from September 2020 through February 2021. To make appointments online, people not only provided basic demographic information such as age, gender, and address but also free-text reasons for seeking testing. We sought to examine how reason for testing, coded as free text, in combination with other collected demographic information, correlated with rates of SARS-CoV-2 positivity and measures of infectiousness such as the Real-Time Polymerase Chain Reaction (RT-PCR) Cycle Threshold (Ct) value. Data from the scheduling software used to coordinate the on site testing workflow was extracted and linked to SARS-CoV-2 PCR assay metadata stored in a laboratory data warehouse, including Ct values. We categorized individual text responses into 3 separate categories: symptomatic, exposed but asymptomatic, and asymptomatic without exposure. Symptomatic individuals were identified based on the presence of common keywords and phrases like “symptom”, “fever”, “cough”, and “sore throat.” Similarly, exposed individuals were identified by keywords such as “exposed”, “exposure”, and “roommate.” All others were categorized as asymptomatic. We found that symptomatic individuals had the highest frequency of testing positive at 11.6% (21,150/181,868). Exposed individuals and asymptomatic individuals had lower rates of detection with frequencies of 7.4% (10,266/138,721) and 4.9% (8,987/185,026) respectively. Repeat testing represented 29.9% of the results and positivity rates were calculated using the first result for each individual. In addition, among those who tested positive, symptomatic individuals exhibited the lowest average Ct values, while those who are asymptomatic exhibit the highest average Ct values. Among those who self-reported duration of symptoms, recent onset of symptoms was strongly correlated with lower Ct values. Across more than 700,000 results, self-reported symptomatic people, based on free text responses, have a higher likelihood of testing positive for SARS-CoV-2. People who claim a more recent onset of symptoms are more likely to exhibit higher concentrations of viral RNA.

Disinfection Kinetics of Free Chlorine, Monochloramines and Chlorine Dioxide on Ammonia-Oxidizing Bacterium Inactivation in Drinking Water

With the widespread use of chloramines disinfection, nitrification has become a problem that cannot be ignored. In order to control nitrification in the drinking water distribution system (DWDS), the inactivation effect of free chlorine, monochloramine and chlorine dioxide on ammonia-oxidizing bacterium (AOB) was studied under different temperature (8 °C, 26 °C and 35 °C) and pH (6.0, 7.0 and 8.7) conditions. The inactivation effect of Nitrosomonas europaea (a kind of AOB) by the three disinfectants increases with increasing temperature. As for the raised pH, the inactivation effect of free chlorine and monochloramine on AOB decreased, while that of chlorine dioxide increased. Last, but certainly not least, the experimental data of the disinfection were calculated to develop the N. europaea inactivation kinetic model, which was based on the first order Chick–Watson equation. The proposed model in this study took the two variables, pH and temperature, into consideration simultaneously, which were used to evaluate the average Ct value (multiplying the concentration of the residual disinfectant by the time of contact with N. europaea) required for different disinfectants when they produced the ideal inactivation effect on N. europaea.

Evaluation of observed and unobserved self-collection of saline gargle samples for the detection of SARS-CoV-2 in outpatients

The diagnostic sensitivity of observed and unobserved self-collected saline gargle samples for the molecular detection of SARS-CoV-2 in adults and school-aged children was evaluated against a reference standard of health care worker collected nasopharyngeal flocked swab.A total of 46 participants had a positive nasopharyngeal swab sample; of these, 10 were in the observed phase and 36 were in the unobserved phase. Only one matching saline gargle sample tested negative and this was in the unobserved phase, giving an overall sensitivity of 98%. Average viral target Ct values were higher in the saline gargle samples. RNaseP Ct values were lower in unobserved collected samples compared to observed collected samples.Unobserved self-collection of saline gargle samples is a promising outpatient testing method for COVID-19 diagnosis. The self-collection method has potential to simplify the diagnostic cycle and facilitate implementation of COVID-19 testing, particularly in settings with limited access to health care workers.

Research on Individualized Phantom Based on 3D Printing for Radiotherapy Dose Verification

Dose verification is carried out on the individualized three-dimensional phantom based on 3D printing technology, which simulates the anatomical structure of human body, contour shape, tumor anatomical structure and other dangerous organs to the greatest extent, and produces a reasonable and effective dose validation phantom. According to the need to obtain effective patient data, import Mimics software to reconstruct the parts of the body and its surrounding tissues and organs that need to be measured, and make them into three-dimensional shell components. The 3D printing is used to assemble and fill the equivalent tissue, and then the body phantom is made. The phantom was scanned by CT and the data was transmitted to TPS system. The previously completed treatment plan was transplanted to the phantom. The phantom was placed according to the patient's location information, irradiated and measured data. The three-dimensional shell assembly is completely reconstructed according to the patient's data, and the contour difference is not significant. The shell is filled with tissue radiation equivalent material whose CT value is the same as the average CT value of the shell volume. The CT image data show that the radiation equivalence of the phantom is similar to the actual tissue of the patient, and the equivalent dose distribution conforms to the conventional treatment range. It can provide a reliable means of dose verification for the accurate design of intensity modulated radiation therapy.

A preliminary evaluation study of applying a deep learning image reconstruction algorithm in low-kilovolt scanning of upper abdomen.

To investigate feasibility of applying deep learning image reconstruction (DLIR) algorithm in a low-kilovolt enhanced scan of the upper abdomen. A total of 64 patients (BMI<28) are selected for the enhanced upper abdomen scan and divided evenly into two groups. The tube voltages in Group A are 100kV in arterial phase and 80kV in venous phase, while tube voltages are 120kV during two phases in Group B. Image reconstruction algorithms used in Group A include the filtered back projection (FBP) algorithm, the adaptive statistical iterative reconstruction-Veo (ASIR-V 40% and 80%) algorithm, and the DLIR algorithm (DL-L, DL-M, DL-H). Image reconstruction algorithm used in Group B is ASIR-V40%. The different reconstruction algorithm images are used to measure the common hepatic artery, liver, renal cortex, erector spinae, and subcutaneous adipose in the arterial phase and the average CT value and standard deviation of the portal vein, liver, spleen, erector spinae, and subcutaneous adipose in the portal phase. The signal-to-noise ratio (SNR) is calculated, and the images are also scored subjectively. In Group A, noise in the aorta, liver, portal vein (the portal phase), spleen (the portal phase), renal cortex, retroperitoneal adipose, and muscle is significantly lower in both the DL-H and ASIR-V80% images, and the SNR is significantly higher than those in the remaining groups (P<0.05). The SNR of each tissue and organ in Group B is not significantly different from that in DL-M, DL-L, and ASIR-V40% in Group A (P>0.05). The subjective image quality scores in the DL-H and B groups are higher than those in the other groups, and the FBP group has significantly lower image quality than the remaining groups (P<0.05). For upper abdominal low-kilovolt enhanced scan data, the DLIR-H gear yields a more satisfactory image quality than the FBP and ASIR-V.

Drainage Capillary Pressure Distribution and Fluid Displacement in a Heterogeneous Laminated Sandstone

AbstractWe applied three‐dimensional X‐ray microtomography to image a capillary drainage process (0–1,000 kPa) in a cm‐scale heterogeneous laminated sandstone containing three distinct regions with different pore sizes to study the capillary pressure. We used differential imaging to distinguish solid, macropore, and five levels of subresolution pore phases associated with each region. The brine saturation distribution was computed based on average CT values. The nonwetting phase displaced the wetting phase in order of pore size and connectivity. The drainage capillary pressure in the highly heterogeneous rock was dependent on the capillary pressures in the individual regions as well as distance to the boundary between regions. The complex capillary pressure distribution has important implications for accurate water saturation estimation, gas and/or oil migration and the capillary rise of water in heterogeneous aquifers.

Comparison and Sensitivity Evaluation of Three Different Commercial Real-Time Quantitative PCR Kits for SARS-CoV-2 Detection.

Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most sensitive and specific assay and, therefore, is the “gold standard” diagnostic method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to compare and analyze the detection performance of three different commercially available SARS-CoV-2 nucleic acid detection kits: Sansure Biotech, GeneFinderTM, and TaqPathTM on 354 randomly selected samples from hospitalized COVID-19 patients. All PCR reactions were performed using the same RNA isolates and one real-time PCR machine. The final result of the three evaluated kits was not statistically different (p = 0.107), and also had a strong positive association and high Cohen’s κ coefficient. In contrast, the average Ct values that refer to the ORF1ab and N gene amplification were significantly different (p < 0.001 and p < 0.001, respectively), with the lowest obtained by the TaqPathTM for the ORF1ab and by the Sansure Biotech for the N gene. The results show a high similarity in the analytical sensitivities for SARS-CoV-2 detection, which indicates that the diagnostic accuracy of the three assays is comparable. However, the SanSure Biotech kit showed a bit better diagnostic performance. Our findings suggest that the imperative for improvement should address the determination of cut-off Ct values and rapid modification of the primer sets along with the appearance of new variants.

Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae).

Three molecular protocols using qPCR TaqMan probe, SYBR Green, and loop-mediated isothermal amplification (LAMP) methods were set up for the identification of larvae and adults of an African invasive moth, Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae). The DNA extracts from larval and adult samples of T. leucotreta were perfectly amplified with an average Ct value of 19.47 ± 2.63. All assays were demonstrated to be inclusive for T. leucotreta and exclusive for the nontarget species tested; the absence of false positives for nontarget species showed a 100% of diagnostic specificity and diagnostic sensitivity for all assays. With the SYBR Green protocol, the Cq values were only considered for values less than 22 (cutoff value) to prevent false-positive results caused by the late amplification of nonspecific amplicons. The limit of detection (LoD) for the qPCR probe protocol was equal to 0.02 pg/µl while a value equal to 0.128 pg/µl for the qPCR SYBR Green assay and LAMP method were established, respectively. The intrarun variabilities of reproducibility and repeatability in all the assays evaluated as CV%, ranged between 0.21 and 6.14, and between 0.33 and 9.52, respectively; the LAMP values were slightly higher than other assays, indicating a very low interrun variability. In order for an operator to choose the most desirable method, several parameters were considered and discussed. For future development of these assays, it is possible to hypothesize the setup of a diagnostic kit including all the three methods combined, to empower the test reliability and robustness.

Comparative analysis of various clinical specimens in detection of SARS-CoV-2 using rRT-PCR in new and follow up cases of COVID-19 infection: Quest for the best choice.

An appropriate specimen is of paramount importance in Real Time reverse transcription-polymerase chain reaction (rRT-PCR) based diagnosis of novel coronavirus (nCoV) disease (COVID-19). Thus, it's pertinent to evaluate various diversified clinical specimens' diagnostic utility in both diagnosis and follow-up of COVID-19. A total of 924 initial specimens from 130 COVID-19 symptomatic cases before initiation of treatment and 665 follow up specimens from 15 randomly selected cases comprising of equal number of nasopharyngeal swab (NPS), oropharyngeal swab (OPS), combined NPS and OPS (Combined swab), sputum, plasma, serum and urine were evaluated by rRT-PCR. Demographic analysis showed males (86) twice more affected by COVID-19 than females (44) (p = 0.00001). Combined swabs showed a positivity rate of 100% followed by NPS (91.5%), OPS (72.3%), sputum (63%), while nCoV was found undetected in urine, plasma and serum specimens. The lowest cycle threshold (Ct) values of targeted genes E, ORF1b and RdRP are 10.56, 10.14 and 12.26 respectively and their lowest average Ct values were found in combined swab which indicates high viral load in combined swab among all other specimen types. Analysis of 665 follow-up multi-varied specimens also showed combined swab as the last specimen among all specimen types to become negative, after an average 6.6 (range 4-10) days post-treatment, having lowest (15.48) and average (29.96) Ct values of ORF1b respectively indicating posterior nasopharyngeal tract as primary nCoV afflicted site with high viral load. The combined swab may be recommended as a more appropriate specimen for both diagnosis and monitoring of COVID-19 treatment by rRT-PCR for assessing virus clearance to help physicians in taking evidence-based decision before discharging patients. Implementing combined swabs globally will definitely help in management and control of the pandemic, as it is the need of the hour.

Short Communication: Detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products using Real-Time PCR Multiplex Method

Abstract. Sophian A, Purwaningsih R, Igirisa RPJ, Amirullah ML, Lukita BL, Fitri RA. 2020. Short Communication: Detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products using Real-Time PCR Multiplex Method. Asian J Nad Prod Biochem 21: 17-20. The detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products was carried out using Multiplex Real-Time PCR (qPCR) in the Microbiology and Molecular Biology Laboratory at the Indonesian Food and Drug Authority in Gorontalo. The purpose of this study was to provide alternative testing methods for food products circulating in the market. The sample consisted of 25 samples of processed meat products spike with Salmonella typhimurium ATCC 14028 phase 2 and Listeria monocytogenes ATCC 7644 phase 2. The method used in the study was qPCR analysis using the SYBR Green method, while DNA isolation used the direct PCR method. Data analysis was carried out based on Cycle threshold and Melting temperature based on two main criteria. Cycle threshold (Ct) analysis determines the Ct value of the sample and comparing it with the control. Melting temperature (Tm) analysis determines the temperature at which 50% of double-stranded DNA changed to a single standard and comparing it with the melting temperature of positive control. The results showed Salmonella typhimurium ATCC 14028 in the processed meat was detected at an average Ct value of 10.34, and a Tm value of 85.70. The presence of Listeria monocytogenes ATCC 7644 in the samples was recognized at an average Ct value of 14.04, and an average Tm value of 80.07. It can be concluded that the real-time multiplex PCR method can be used to detect Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 by using the melting curve (Tm) analysis.