High Ct Values Research Articles

SARS-CoV2 RT-PCR assays: In vitro\ufeff comparison of 4 WHO approved protocols on clinical specimens and its implications for real laboratory practice through variant emergence

IntroductionRT-PCR testing on nasopharyngeal swabs is a key component in the COVID-19 fighting, provided to use sensitive and specific SARS-CoV2 genome targets. In this study, we aimed to evaluate and to compare 4 widely used WHO approved RT-PCR protocols on real clinical specimens, to decrypt the reasons of the diverging results and to propose recommendations for the choice of the genome targets.Methods260 nasopharyngeal samples were randomly selected among the samples tested between Week-16, 2020 and week-16 2021, in the Institut Pasteur de Tunis, Tunisia, one of the referent laboratories of COVID-19 in Tunisia. All samples were tested by Charité, Berlin protocol (singleplex envelop (E) and singleplex RNA-dependent RNA polymerase (RdRp)), Hong Kong Universiy, China protocol (singleplex nucleoprotein (N) and singleplex Open reading frame Orf1b), commercial test DAAN Gene® (using the CDC China protocol), (triplex N, Orf1ab with internal control) and Institut Pasteur Paris protocol (IPP) (triplex IP2(nsp9) and IP4 (nsp12) with internal control). For IPP, a selection from samples positive by IP2 but negative with IP4 was re-tested by exactly the same protocol but this time in singleplex. New results were described and analyzed.ResultsIn vitro analysis showed discordant results in 29.2% of cases (76 out of 260). The most discordant protocol is DAAN Gene® due to the false positive late signals with N target. Discordant results between the two protocol’s targets are more frequent when viral load are low (high Ct values). Our results demonstrated that the multiplexing has worsened the sensitivity of the IP4 target.ConclusionWe provide concise recommendations for the choice of the genome targets, the interpretation of the results and the alarm signals which makes suspect a gene mutation.

Rapid Antigen Test LumiraDxTM vs. Real Time Polymerase Chain Reaction for the Diagnosis of SARS-CoV-2 Infection: A Retrospective Cohort Study.

Background: Real time reverse transcription polymerase chain reaction (real time RT-PCR) testing is the gold standard for the diagnosis of SARS-CoV-2 infections. However, to expand the testing capacity, new SARS-CoV-2 rapid antigen tests (Ag-RDTs) have been implemented. Ag-RDTs are more rapid, but less reliable in terms of sensitivity, and real-life data on their performance in comparison with the real time RT-PCR test are lacking. Methods: We aimed at assessing the diagnostic performance of the third-generation antigenic swab LumiraDx™ compared with real time RT-PCR in a retrospective cohort study at the Infectious Diseases Unit of Padua. All of the patients who were consecutively tested for SARS-CoV-2 in our centre (by both real time RT-PCR and Ag-RTD LumiraDxTM) from 19 January to 30 May 2021, were included. Cycle-threshold (Ct) values of positive real time RT-PCR were recorded as well as the number of days from symptoms’ onset to testing. Results: Among the 282 patients included, 80.9% (N = 228) tested positive to real time RT-PCR, and among these, 174 tested positive also to LumiraDx™. Compared with real time RT-PCR, which is considered as the gold standard for the assessment of the presence/absence of SARS-CoV-2 infection, LumiraDx™ showed an overall sensitivity of 76.3% and specificity of 94.4%. Sensitivity increased to 91% when testing was performed <10 days from symptoms’ onset, and to 95% when considering Ct < 25. Multivariable binomial logistic regression showed that false negative LumiraDx™ results were significantly associated with high Ct values, and with further testing from symptoms’ onset. Conclusions: The results of our study suggested that the LumiraDx™ SARS-CoV-2 antigen assay may be appropriate for the detection of SARS-CoV-2 infection, especially in its early phase when the test largely meets the performance requirements of the European Centre for Disease Prevention and Control (ECDC).

Effect of insilico predicted and designed potential siRNAs on inhibition of SARS-CoV-2 in HEK-293 cells

ObjectivesThe COVID-19 was identified for the first time from the sea food market, Wuhan city, China in 2019 and the pathogenic organism was identified as SARS-CoV-2. Currently, this virus has spread to 223 countries and territories and known as a serious issue for the global human community. Many vaccines have been developed and used for immunization. MethodsWe have reported the insilico prediction, designing, secondary structure prediction, molecular docking analysis, and in vitro assessment of siRNAs against SARS-CoV-2. The online bioinformatic approach was used for siRNAs selection and designing. The selected siRNAs were evaluated for antiviral efficacy by using Lipofectamine 2000 as delivery agent to HEK-293 cells. The MTT assay was used for cytotoxicity determination. The antiviral efficacy of potential siRNAs was determined based on the Ct value of q-RT-PCR and the data analysis was done by Prism-GraphPad software. ResultsThe analyzed data resulted in the selection of only three siRNAs out of twenty-six siRNAs generated by online software. The secondary structure prediction and molecular docking analysis of siRNAs revealed the efficient binding to the target. There was no cellular toxicity observed in the HEK-293 cells at any tested concentrations of siRNAs. The purification of RNA was completed from inoculated cells and subjected to q-RT-PCR. The highest Ct value was observed in siRNA 3 than the others. The results offered valuable evidence and invigorated us to assess the potency of siRNAs by using alone or in combination in other human cells. ConclusionThe data generated from this study indicates the significance of in silico prediction and narrow down the potential siRNA' against SARS-CoV-2, and molecular docking investigation offered the effective siRNAs binding with the target. Finally, it is concluded that the online bioinformatics approach provided the prediction and selection of siRNAs with better antiviral efficacy. The siRNA-3 was observed to be the best for reduction of viral RNA in cells.

Evaluation of the Panbio™ COVID-19 ag rapid test at an emergency room in a hospital in São Paulo, Brazil

BackgroundThe performance characteristics of the Panbio™ COVID-19 Ag test was evaluated at an emergency room setting against RT-PCR, considered the gold-standard for the detection of SARS-CoV-2, in São Paulo, Brazil. The study aimed to determine the sensitivity, specificity, Positive Percent Agreement (PPA), and Negative Percent Agreement (NPA) as compared to RT-PCR. MethodsSpecimens from 127 suspected patients were tested by both the Panbio™ COVID-19 Ag test and by RT-PCR. ResultsIn relation to RT-PCR using Ct values ≤ 40 as the upper limit for positivity, the Panbio™ COVID-19 Ag test showed an overall sensitivity of 84.3% (95% CI 75‒93.8%) and 98.2% (95% CI 96‒98.8%) overall specificity. For Ct values ≤ 25 (n = 37), the Panbio™ COVID-19 Ag test showed 97% sensitivity. DiscussionThe concordance between the Panbio™ COVID-19 Ag test and RT-PCR was 97% at Ct values below 25 but decreased at higher Ct values. For disease control, it is very important to identify infected individuals who present COVID-19 symptoms and also those who are suspected of infection due to contact with infected individuals. ConclusionThe Panbio™ COVID-19 Ag test is suitable for use as a diagnostic test for rapid screening of patients presenting COVID-19 symptoms, or those suspected of being infected, prior to being admitted to hospital.

Natural SARS-CoV-2 infection in two cats in Spain

PurposeSARS-CoV-2 is a zoonotic pathogen able to infect humans, pets and other animal species, mustelids and felids being highly susceptible. Experimentally, it has been shown that SARS-CoV-2 replicates efficiently in cats, inducing humoral immunity and causing a variable clinical presentation. Here we report the case of two cats (G1 and G2) naturally infected with SARS-CoV-2 after contact with their COVID-19 positive owner diagnosed in September 2020.Methods & MaterialsOral swabs soaked in PBS were collected daily from 1 to 16 days after the owner tested positive for SARS-CoV-2. Urine and feces were taken several times during this period. Samples were analyzed by in-house real-time RT-PCR (based on CDC 2019-nCoV_N1). Serum samples, collected at day 30, were tested by IDVet multi-species ELISA. Vero E6 cells was used for virus isolation. Partial spike gene was sequenced by Sanger and compared with the owner sequence and the reference Wuhan 2019 (NC_045512.2).ResultsG1 showed apathy, anorexia, lack of grooming and mild respiratory symptoms (tearing and sneezing) for 5 days and then recovered gradually. G2 remained clinically asymptomatic. Viral RNA peak in oral swabs was detected in both cats at day 2 showing similar Ct value (25). SARS-CoV-2 was consistently shedding in G1 for 9 days, after sporadically detected until day 16. Viral shedding in G2 decreased quickly during the first 6 days. Faeces were positive to SARS-CoV-2 until 9 (G1) or 12 (G2) days post-viral detection. Viral RNA in urine was sporadically detected in G2 with high Ct values. Specific antibodies were detected in both cats one month after infection. Virus isolation was not successful. Cats and owner sequences of the spike protein were homologous with two mutations (A222V and D614G) that classify the virus within the 20A.EU1 clade.ConclusionSARS-CoV-2 natural infection of two cats occurred after close contact with their infected owner. Both cats developed humoral immune response but they showed different clinical presentation and viral shedding pattern. Oral swabs and feces are proper samples for SARS-CoV-2 detection in cats. Variant 20A.EU1 detected in this study emerged in early summer 2020, presumably in Spain, and subsequently spread throughout Europe.

Detection of SARS-CoV2 by a Commercial RNA Detection Kit: a Public Health Laboratory Experience

PurposeThere is a critical shortage of the commercial SARS-CoV2 detection kits and a major problem for testing capacity in Bangladesh since December, 2019 to combat the Covid19 pandemic. So this study was aimed to assess SARS-CoV2 detection with a commercial RNA detection kit by the qualitative real time PCR from respiratory specimens (nasal or throat swab) of Covid19 suspected cases.Methods & MaterialsThis cross sectional study was done by convenience sampling from Munshiganj district and four different hospitals in Dhaka city from 28 November 2020 to 4 December 2020. Nasal or throat swab samples were collected from Covid19 suspected cases and sent to NIPSOM RT-PCR lab, National Institute of Preventive and Social Medicine (a public health laboratory), NIPSOM, Mohakhali, Dhaka, Bangladesh. A commercial kit [Adaltis MOLGen SARS-CoV-2 (3Genes) RealTime PCR, Italy] containing the primer and probe set in fluorimeter channel was used to detect three genes of SARS-CoV-2: RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) with endogenous internal control (IC). Threshold cycle (Ct) value was selected at ≤40 for each positive gene and detection of at least two different genes (RdRp and E gene or N gene) was interpreted as SARS-CoV2 positive.ResultsA total of 1061 samples of Covid19 suspected cases within 4 to 98 years of age were included in this study and male female ratio was 1.33:1. Among 1061 samples, 299 (28.18%) were tested positive for SARS-CoV2 with mean age of 43.33 years and male was found predominantly. Of them, 94.98% were positive for RdRp, E and N genes whereas all (100%) were found positive for only RdRp and E gene. Here, the Ct value of IC ranged within <20 to 40 and 44.48% was found at Ct 30–35 followed by 36.79% at Ct 25–30 and 10.37% at Ct 35–40. For individual RdRp gene, E gene and N gene, highest Ct values were at 30–35 with 26.42%, 30.43% and 28.87% respectively.ConclusionTo address accurate and safely diagnosis of SARS CoV-2, this commercial RNA detection kit will help during the COVID-19 pandemic.

Development of Duplex and Multiplex Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) Assays for Clinical Diagnosis of SARS-COV-2 in Sri Lanka

PurposeDespite the rollout of several vaccines targeting SARS-CoV-2, attainment of near-universal vaccination is a challenging task, particularly for low- and middle-income nations such as Sri Lanka. Rapid, reliable diagnostics for the detection of the virus is of vital importance for the predominantly export- and tourism-based economy of the country. Herein, we report the development of a RT-LAMP assay as an alternative to the gold-standard RT-qPCR method for diagnostic laboratories in Sri Lanka in a cost-effective and highly reliable manner.Methods & MaterialsAbout 313 nasopharyngeal and oropharyngeal samples from the community were collected and subjected to RNA purification and subjected to simultaneous RT-qPCR and RT-LAMP experiments by using previously published primers in a thermocycler. Duplex (containing N and E gene primers) and multiplex (containing N, E and ORF1ab gene primers) RT-LAMP assay results were compared with standard RT-qPCR results using an agreement attribute statistical test. The effect of guanidine hydrochloride was also analyzed.ResultsThe limit of detection for the duplex assay was found to be 10 copies µL-1 at a constant temperature of 63°C, and 5 copies µL-1 for multiplex assays at 66.4°C. Both types of RT-LAMP assay were specific only for the SARS-COV-2 virus, successfully distinguishing it from multiple other human viruses. Attribute agreement analysis between duplex- and multiplex RT-LAMP vs RT-qPCR yielded 93% and 96.5% scores, respectively. Moreover, both RT-LAMP assays showed 100% agreement with RT-qPCR when Ct was <25 in positive samples and showed 100% (duplex) or 97.22% (multiplex) at 35≥ Ct ≥25. The discrepancy between agreements at higher Ct values was attributed to the higher sensitivity of the multiplex RT-LAMP assay. The addition of guanidine hydrochloride increased the sensitivity and decreased detection time significantly for both the duplex and multiplex assays.ConclusionOverall, we have demonstrated a potentially rapidly deployable diagnostic test kit not only for widespread community use but particularly for high-risk locations such as ports of entry or manufacturing facilities to mitigate the effects of the SARS-CoV-2 virus in Sri Lanka.

Effect of heat inactivation for the detection of severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) with reverse transcription real time polymerase chain reaction (rRT-PCR): evidence from Ethiopian study

BackgroundCoronavirus disease 2019 (COVID-19) has been a major public health importance and its specimen needs to be handled safely due to concerns of potential transmissibility to health care workers. Heat inactivation of the sample before nucleic acid isolation might permit safe testing processes. Hence, it is important to assess the effect of heat inactivation on SARS-CoV-2 RT-PCR detection in resource limited settings.MethodsAn experimental study was conducted at Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing. One batch of the sample was inactivated at 56 °C heat for 30 min, and the other batch was stored at 4 °C for a similar period of time. RNA extraction and detection were done by DAAN Gene kit protocols. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. p-value < 0.05 was considered as statistically significant.ResultsOut of 188 total samples, 119 (63.3%) were positive and 69 (36.7%) were negative in the non-inactivated group. While, 115 (61.2%) of samples were positive and 73 (38.8) were negative in heat inactivated sample batch. Rate of positivity between groups did not have statistically significant difference (p > 0.05). The mean Cycle threshold (Ct) value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95% CI − 0.247–0.331; t = 0.28; p = 0.774) and 0.38 (95% CI 0.097–0.682; t = 2.638; p = 0.010) respectively.ConclusionHeat inactivation at 56 °C for 30 min did not affect the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding showed that there was statistically significant Ct value increment after heat inactivation compared to untreated samples. Therefore, false-negative results for high Ct value (Ct > 35) samples were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.

False positive results seen at high CT values with the Xpert Xpress SARS-CoV-2 test

From early-2020 to mid-2021, Singapore had very low community prevalence of SARS-CoV-2. During this time, there were several high CT results with the Xpert Xpress SARS-CoV-2 PCR test in our laboratory from low epidemiological risk patients, who were negative on repeat testing. This raised concerns that such results could be false positive.

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels.

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

Setting-Up a Rapid SARS-CoV-2 Genome Assessment by Next-Generation Sequencing in an Academic Hospital Center (LPCE, Louis Pasteur Hospital, Nice, France).

Introduction: Aside from the reverse transcription-PCR tests for the diagnosis of the COVID-19 in routine clinical care and population-scale screening, there is an urgent need to increase the number and the efficiency for full viral genome sequencing to detect the variants of SARS-CoV-2. SARS-CoV-2 variants assessment should be easily, rapidly, and routinely available in any academic hospital.Materials and Methods: SARS-CoV-2 full genome sequencing was performed retrospectively in a single laboratory (LPCE, Louis Pasteur Hospital, Nice, France) in 103 SARS-CoV-2 positive individuals. An automated workflow used the Ion Ampliseq SARS-CoV-2 panel on the Genexus Sequencer. The analyses were made from nasopharyngeal swab (NSP) (n = 64) and/or saliva (n = 39) samples. All samples were collected in the metropolitan area of the Nice city (France) from September 2020 to March 2021.Results: The mean turnaround time between RNA extraction and result reports was 30 h for each run of 15 samples. A strong correlation was noted for the results obtained between NSP and saliva paired samples, regardless of low viral load and high (>28) Ct values. After repeated sequencing runs, complete failure of obtaining a valid sequencing result was observed in 4% of samples. Besides the European strain (B.1.160), various variants were identified, including one variant of concern (B.1.1.7), and different variants under monitoring.Discussion: Our data highlight the current feasibility of developing the SARS-CoV-2 next-generation sequencing approach in a single hospital center. Moreover, these data showed that using the Ion Ampliseq SARS-CoV-2 Assay, the SARS-CoV-2 genome sequencing is rapid and efficient not only in NSP but also in saliva samples with a low viral load. The advantages and limitations of this setup are discussed.

Molecular methods for diagnosing novel coronavirus infection: comparison of loop-mediated isothermal amplification and polymerase chain reaction

Currently, the basis for molecular diagnostics of most infections is the use of reverse transcription polymerase chain reaction (RT-PCR). Technologies based on reverse transcription isothermal loop amplification (RT-LAMP) can be used as an alternative to RT-PCR for diagnostic purposes. In this study, we compared the RTLAMP and RT-PCR methods in order to analyze both the advantages and disadvantages of the two approaches. For the study, we used reagent kits based on RT-PCR and RT-LAMP. The biological material obtained by taking swabs from the mucous membrane of the oropharynx and nasopharynx in patients with symptoms of a new coronavirus infection was used. We tested 381 RNA samples of the SARS-CoV-2 virus (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) from various patients. The obtained values of the threshold cycle (Ct) for RT-PCR averaged 20.0 ± 3.7 s (1530 ± 300 s), and for RT-LAMP 12.8 ± 3.7 s (550 ± 160 s). Proceeding from the theoretical assumptions, a linear relationship between values obtained in two kits was proposed as a hypothesis; the correlation coefficient was approximately 0.827. At the same time, for samples with a low viral load (VL), the higher Ct values in RT-LAMP did not always correlated with those obtained in RT-PCR. We noted a significant gain in time for analysis using RT-LAMP compared to RT-PCR, which can be important in the context of testing a large number of samples. Being easy to use and boasting short turnaround time, RT-LAMP-based test systems can be used for mass screening in order to identify persons with medium and high VLs who pose the greatest threat of the spread of SARS-CoV-2, while RT-PCR-based diagnostic methods are also suitable for estimation of VL and its dynamics in patients withCOVID-19.

Fluctuating RT-PCR CT values: A case series

Real-Time Reverse Transcriptase Polymerase Chain Reaction (r RT-PCR) has been the main diagnostic tool for SARS-CoV-2 infection since the early stages of the COVID-19 pandemic and a positive test enables the clinicians and public health professionals to quickly isolate the patient and prevent spread of the disease. The presence of viral RNA confirms SARS CoV-2 and the Cycle Threshold (Ct) values may give a rough estimate of viral load. Theoretically, the Ct value is inversely proportional to the amount of genetic material (RNA) in the starting sample and lower Ct values is generally associated with high viral load. Some experts assume that high viral load is directly correlated with increased infectiousness and severity of disease and suggest using Ct value or calculating viral load in decision-making. As per the policy, dated 14th June 2020, National Health Strategic Command Group in Qatar recommends using RT-PCR Ct value as a key determinant for decision on admission, discharge and isolation. The objective of this case series is to show that Ct values vary as the course of infection progresses, hence cannot be used as a reliable marker to take clinical decisions or even public health actions such as quarantine/ isolation. All six confirmed COVID19 cases showed fluctuating RT-PCR Ct values throughout the course of illness. The Ct value was higher in the beginning of the course of infection, depicting low viral load and later in the course the Ct value dropped indicating higher viral load. The Ct value decreased as symptoms developed or worsened. There is a high probability that patients in early symptomatic stage may show a high Ct value which may subsequently change and thus management will change. In such cases, the high Ct values will give a false sense of security and thus have an impact on the containment of spread of infection to others. Hence, it is not recommended to rely on numerical Ct values for determining infectiousness of COVID-19 patients and deciding patient management protocols since the Ct value an indirect marker of viral load, changes over the course of infection. Keywords: fluctuating CT values; COVID-19; SARS CoV-2; RT-PCR CT values.

Genotyping and evaluation of interleukin-10 and soluble HLA-G in abortion due to toxoplasmosis and HSV-2 infections.

Abortion is a condition that occurs due to one of the pathological injuries, often one of the members of the TORCH is the real cause. The current study aimed to investigate the impact of toxoplasmosis, HSV-2 infections with abortion, and also, the identification of immunogenetics marker (interleukin-10) that may be associated with abortion. Anti-Toxoplasma IgG, IgM, Herpes simplex virus-2 IgM, human soluble leukocyte antigen class I-G and interleukin-10 were estimated by ELISA technique, while the expression of IL-10 gene was investigated by using the real-time PCR. The results showed that among aborted women the rate of anti-Toxoplasma and HSV-2 IgM antibodies occurred within the age groups (21-30) years and (31-40) years 32(100.0%) and 36(100.0% ), respectively. A significant relationship was found between IL-10 and cases with a P=0.005. The pattern of distribution of HLA-G in the studied groups showed that there was a significant relationship between HLA-G and cases with a P=0.005. Regarding the IL-10 rs gene, the results revealed an amplified product of 377 bp and there was a high Ct value for patients and controls with a high Ct value of templates, preoperational to the gene concentration. We concluded that there was a significant relationship between human leukocyte antigen-G and the cases. It was found that there was a high Ct value for patients and controls with a high Ct value for templates.

Technical Notes No Inhibitory Effect of Heparinized Blood on Real-Time PCR Analysis of Thrombophilic Mutations

We compared the efficiency of real-time PCR analysis of FII (c.*97G&gt;A, G20210A) and FV Leiden (c.1601G&gt;A) thrombophilic mutations in the samples obtained from venous blood treated with various anti­coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalate-treated samples: 18.5 and 18.9, respectively. Signi­ficantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P &lt; 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P &lt; 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.

A One Man Army- TrueNat Testing for the Identification of COVID-19 in Firozabad, Uttar Pradesh, India

Introduction: The Coronavirus Disease 2019 (COVID-19) pandemic has affected the entire world. The need of timely detection of the virus has been of prime importance and the efforts to develop sensitive, specific, rapid, portable and cost effective diagnostic methods promoted the indigenous development of TrueNat testing for viral load in COVID-19 detection which had been previously designed for detection of Tuberculosis and other infectious organisms. Aim: To see the importance of TrueNat testing among symptomatic and asymptomatic cases in different age groups and gender. Materials and Methods: In this retrospective study conducted in the Department of Microbiology, Autonomous State Medical College and SNM Hospital, Firozabad, Uttar Pradesh, India, from June 2020 - May 2021, a total of 4,659 samples were collected from patients (Influenza Like Illness (ILI), Severe Acute Respiratory Illness (SARI), symptomatic, asymptomatic, those seeking hospitalisation, emergency), contacts and travellers and were subjected to testing by TrueNat (Molbio Quattro). The cases were divided into group A of patients who presented with symptoms ≤7 days; group B of patients who presented with signs and symptoms &gt;7 days and group C comprised of asymptomatic patients. The symptoms of patients were associated with the Cycle threshold (Ct) values of the Envelope (E) gene and the RNAdependent RNA polymerase gene (RdRp) gene. The Chi-square test was done to test the statistical significance of association of symptomatic patients with the outcome of the test. Results: The maximum number of positive cases was found in the age group 20-39 years (p-value &lt;0.05). The least positivity was found in the higher (80 years) and lower (below 9 years) age groups. The positivity rates had no significant impact on the gender. The percentage positivity as detected by Truenat testing was 3.3% and maximum positive patients were found in the group having symptoms &lt;7 days (p&lt;0.05). On association of the Ct values of E gene and RdRp gene with the symptoms it was found that 28.1% and 27.2% of the patients were in the high Ct value group. Conclusion: TrueNat was found to be a portable and easy to perform test which did not require special laboratory set up. The use of Viral Lysis Medium (VLM) reduced the time of RNA extraction which not only rendered it safer to perform but expedited the results.

Simulated Flock-Level Shedding Characteristics of Turkeys in Ten Thousand Bird Houses Infected with H7 Low Pathogenicity Avian Influenza Virus Strains.

Understanding the amount of virus shed at the flock level by birds infected with low pathogenicity avian influenza virus (LPAIV) over time can help inform the type and timing of activities performed in response to a confirmed LPAIV-positive premises. To this end, we developed a mathematical model which allows us to estimate viral shedding by 10,000 turkey toms raised in commercial turkey production in the United States, and infected by H7 LPAIV strains. We simulated the amount of virus shed orally and from the cloaca over time, as well as the amount of virus in manure. In addition, we simulated the threshold cycle value (Ct) of pooled oropharyngeal swabs from birds in the infected flock tested by real-time reverse transcription polymerase chain reaction. The simulation model predicted that little to no shedding would occur once the highest threshold of seroconversion was reached. Substantial amounts of virus in manure (median and ; 50% egg infectious dose) were predicted at the peak. Lastly, the model results suggested that higher Ct values, indicating less viral shedding, are more likely to be observed later in the infection process as the flock approaches recovery.

495. Evaluation of Antigen Testing for Detection of COVID-19 Vaccine Breakthrough Cases in Long-Term Care

BackgroundLong-term care facilities (LTCFs) are at high risk for severe COVID-19 outbreaks due to their congregate nature and vulnerable population. Oregon Health Authority (OHA) deployed point-of-care antigen (Ag) tests to promptly identify COVID-19 cases in LTCFs. However, their performance in identifying vaccine breakthrough cases has not been evaluated.MethodsDuring 2/25/21–5/25/21, OHA supported testing of residents and staff for two outbreaks at a single LTCF. Paired nasal swabs were collected and tested for SARS-CoV-2 by CDC Influenza SARS-CoV-2 Multiplex PCR Assay (molecular test) and Abbott BinaxNOW COVID-19 Ag Card (Ag test) twice weekly during the outbreaks. Participants were considered fully vaccinated if ≥ 14 days had passed since completion of a vaccine series; all others were deemed unvaccinated. A vaccine breakthrough case was defined as a positive Ag or molecular test from a fully vaccinated person’s specimen. Performance characteristics of the Ag test were assessed, with molecular test as the reference standard. Cycle threshold (Ct) values were compared by one-sided independent t-tests.Results94 unvaccinated residents and staff provided 563 paired samples; SARS-CoV-2 was detected in 21 (12 by Ag and molecular test, 6 by molecular test only, 3 by Ag test only), yielding Ag test sensitivity of 66.7% (95% CI: 43.8–83.7%) and specificity of 99.4% (95% CI: 98.4–99.8%). Mean Ct values were higher for specimens positive by PCR but negative by Ag than those positive by both (30.0 vs. 20.7, P < .01). 81 vaccinated persons provided 925 paired samples; SARS-CoV-2 was detected in 5 (1 by Ag and molecular test, 4 by molecular test only), yielding Ag test sensitivity of 20% (95% CI: 3.6–62.5%) and specificity of 100% (95% CI: 99.6–100%). Mean Ct values for specimens from vaccinated cases were higher than those from unvaccinated cases (30.2 vs. 23.8, P < .05). The lone Ag-positive breakthrough case had a Ct of 20; all others had Ct > 29.ConclusionAg test performance and reduced sensitivity on specimens with high Ct values found in this population are consistent with published data. Molecular testing maximizes identification of vaccine breakthrough cases. More studies are needed to estimate the proportion of breakthrough cases missed by Ag testing and their risk of transmitting the virus in LTCFs.Disclosures All Authors: No reported disclosures

Performance of Abbott ID-Now rapid nucleic amplification test for laboratory identification of COVID-19 in asymptomatic emergency department patients.

ObjectiveWe sought to evaluate the test characteristics of Abbott ID‐Now as a screening tool compared to polymerase chain reaction (PCR) testing for identification of COVID in an asymptomatic emergency department population.MethodsWe performed a prospective study enrolling a convenience sample of asymptomatic patients presenting to a single academic emergency department (ED) who received simultaneous testing with ID‐Now and PCR per standardized ED protocols. Sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of ID‐Now were calculated compared to PCR. Stratified analysis by cycle threshold (Ct) values was also performed, defined as high viral load (Ct < 33) and low viral load (Ct ≥ 33).ResultsA total of 3121 patients were enrolled, of whom 2895 had valid results for ID‐Now and PCR. COVID prevalence was 2.6%. ID‐Now had a sensitivity of 85.1% (95% CI 75.9% to 92.7%) and a specificity of 99.7% (99.5% to 99.9%). PPV and NPV were high at 87.5% (83.1% to 96.1%) and 99.6% (99.3% to 99.8%). Stratified analysis by low and high Ct values demonstrated reduction in sensitivity in patients with low viral loads: 91.7% (81.6% to 97.2%) in low Ct value patients versus 58.3% (27.7% to 84.8%) in high Ct value patients.ConclusionsID‐Now had excellent performance in asymptomatic ED patients with a low rate of false positives. Cycle threshold analysis suggests a relationship between viral load and ID‐Now sensitivity. Given its speed and performance in this population, ID‐Now should be considered an excellent tool to support clinical decision‐making in ED populations.

Identifying Inconclusive Data in the SARS-CoV-2 Molecular Diagnostic Using Nucleocapsid Phosphoprotein Gene as a Target.

The gold standard test to identify the presence of SARS-CoV-2 in COVID-19 patients is the real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), but inconclusive data and false-positive diagnosis remain the major problem of this approach. To compare the fitness of 2 primer sets to the SARS-CoV-2 nucleocapsid phosphoprotein gene (NP) in the molecular diagnosis of COVID-19, we verified the inconclusive data and confidence of high cycle threshold (Ct) values in SARS-CoV-2 detection. The 970 patient samples were tested by using United States Centers for Disease Control and Prevention protocol. We compared the fitness of 2 primer sets to 2 different regions of the NP gene. In addition, we checked the consistency of positive samples with high Ct values by retesting extracted SARS-CoV-2 RNA or by second testing of patients. N1 and N2 displayed similar fitness during testing, with no differences between Ct values. Then, we verified security range Ct values related to positive diagnostics, with Ct values above 34 failing in 21 of 32 cases (65.6%) after retesting of samples. The patient samples with Ct values above 34.89 that were doubly positive revealed a low sensitivity (52.4%) and specificity (63.6%) of the test in samples with Ct values above 34. It is safe to use 1 primer set for the NP gene to identify SARS-CoV-2 in samples. However, samples with high Ct values may be considered inconclusive and retested to avoid false-positive diagnosis.