What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes.

Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955-0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

Effectiveness of rapid immunochromatographic tests to the detection of feline Coronavirus

Feline coronavirus (FCoV) belongs among pathogens with common occurrence in the cats population in the whole world. FCoV is ubiquitous in environments with a higher concentration of cats, e.g. in shelters, multicat households and kennels. FCoV primarily attacks the digestive feline tract, replicates in its cells and is excreted in the feces to surroundings of permanently or transiently infected cats. The aim of the study was the detection of FCoV in the feces of newly admitted cats to the shelter by the qPCR method and by means of commercial rapid immunochromatographic (antigen) tests from three different producers. For each of the antigen tests, sensitivity and specifity were determined by comparison with the qPCR analysis result. Out of 70 examined fecal samples, viral RNA was by the qPCR analysis identified in 44 samples (62.9%). Neither the age nor the gender of cats played a significant role in the viral excretion. Found sensitivity of the antigen tests was at a low (< 35%;tests A and C) to a satisfactory level (> 50%, test B). The number of viral particles in the samples determined by the qPCR method did not correlate significantly with the result of the antigen tests. The results of this study suggest that the use of rapid antigen tests for routine screening of FCoV shedding in feline shelters is limited due to the high rate of false-negative results.

COVID-19: Test, Test and Test.

A new virus was identified in late December 2019 when China reported the first cases of pneumonia in Wuhan, and a global COVID-19 pandemic followed. The world was not late to respond, with a number of sweeping measures ranging from social distancing protocols, stringent hygienic practices, and nation-wide lockdowns, as well as COVID-19 testing campaigns in an attempt to prevent the transmission of the disease and contain the pandemic. Currently, different types of diagnostic testing have been adopted globally, such as nucleic acid detection tests, immunological tests and imaging approaches; however, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) remains the "gold standard" for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pre-analytical factors, such as specimen selection and collection, are crucial for RT-PCR, and any suboptimal collection may contribute to false-negative results. Herein, we address some of the specimen types that have been used in molecular detection methods for COVID-19. However, the pandemic is still evolving, and information might change as more studies are conducted.

Molecular Diagnostic Tools against SARS-CoV-2 in Poland in 2022.

The most effective way to stop the spread of COVID-19 (coronavirus disease 2019) is to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and isolate those infected as soon as possible. More than 1000 types of molecular and antigen-based immunoassay tests to detect SARS-CoV-2 are now commercially available worldwide. In this review, we present the possibilities of molecular diagnostics available in Poland in 2022. We provide a description of what samples have proven useful to confirm SARS-CoV-2 infection, we describe what methods are used, as well as what safeguards can and should be used to prevent false-negative and false-positive results, and finally we review the products that diagnostic laboratories have to choose from. We also describe diagnostic problems associated with the mutation of the virus.

Optimization, validation and analysis of a 2019-nCoV nucleic acid detection kit. (Special Issue: Spatio-temporal dynamics analysis, risk assessment and emergency management for COVID-19 epidemic.) [Chinese]

Objective: To optimize the sensitivity and specificity of a 2019-nCoV nucleic acid detection kit, so as to improve the positive detection rate and provide guidance for clinical use by comparison with different kits.

A case of Parvovirus B19 infection diagnosed as COVID-19 due to false PCR positivity

Although PCR is the most reliable test for the diagnosis of Covid-19 infection, false negative and positive results can also occur. Our case is a 32-year-old laboratory technician who applied with headache and malaise and her first Covid-19 PCR test was negative. It was repeated two days later, and the result was positive. Additional tests were also performed, and Parvovirus IgM antibody was also found to be positive. Four weeks later, while Covid-19 IgM and IgG test results were negative, the Parvovirus B19 IgG and IgM test results were positive. The Covid-19 PCR test was evaluated as false positive. We aimed to emphasize the need to consider other viral infections in the differential diagnosis even under pandemic conditions.

Effect of sampling positions in nucleic acid test on the test result: a report from 47 confirmed COVID-19 cases in Jinan City

Objective: To investigate the effect of sampling positions in nucleic acid test for COVID-19 on test results.

Sensitivity of SARS-CoV-2 antigen tests for large scale testing

Aim: The test, based on the detection of SARS-CoV-2 antigen, is often seen as an alternative to the PCR method in connection with the need for screening of larger populations. In order to assess the suitability of such an approach, we evaluated the sensitivity of two antigenic assays on a group of individuals, including both patients with symptoms of covid-19 and asymptomatic and healthy individuals.

Review of COVID-19 testing and diagnostic methods.

More than six billion tests for COVID-19 has been already performed in the world. The testing for SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) virus and corresponding human antibodies is essential not only for diagnostics and treatment of the infection by medical institutions, but also as a pre-requisite for major semi-normal economic and social activities such as international flights, off line work and study in offices, access to malls, sport and social events. Accuracy, sensitivity, specificity, time to results and cost per test are essential parameters of those tests and even minimal improvement in any of them may have noticeable impact on life in the many countries of the world. We described, analyzed and compared methods of COVID-19 detection, while representing their parameters in 22 tables. Also, we compared test performance of some FDA approved test kits with clinical performance of some non-FDA approved methods just described in scientific literature. RT-PCR still remains a golden standard in detection of the virus, but a pressing need for alternative less expensive, more rapid, point of care methods is evident. Those methods that may eventually get developed to satisfy this need are explained, discussed, quantitatively compared. The review has a bioanalytical chemistry prospective, but it may be interesting for a broader circle of readers who are interested in understanding and improvement of COVID-19 testing, helping eventually to leave COVID-19 pandemic in the past.

How to take a nasopharyngeal swab: nuanced modifications aiding reduction in false negative results and auxiliary training

It is well known that nasopharyngeal a n d o r o p h a r y n g e a l s w a b s g i v e significant false negative results for SARS-CoV-2 detection. Thus, it becomes all the more imperative to take a proper NPS sample to avoid missing cases, and more importantly to decrease the patient's discomfort. There is a need for training and re-training the frontline workers involved in NPS sampling. Based on the above observation, this article recommends the following modification for NPS sample collection: (1) Seat the patient straight with head straight and a fixed support behind (2) Insert the swab horizontally in the nasal cavity to a distance equal to that between nostril and opening of outer ear.

The effect of normal mouthwash and teeth whitening mouthwash on COVID-19 patient viral load in mouth and PCR results

COVID-19 is a life-threatening viral infection that considers airborne viruses and it's very hard to control the separation of such a pathogen and early diagnosis and treatment can play important role in directly affects patients prognosis, so the period of infection and diagnosis of COVID-19 with good treatment according to patient vital singe is very important. The current diagnosis of COVID-19 has yet to be further improved. To investigate the effect of normalMouthwashand Teeth Whiting Mouthwash onSARS COV-2 patient Viral load in the oral cavity(Lu et al, 2019). Mouth wash has been done on SARS COV-2 patients for 1 min by normal mouth wash (ganger) group 1 and then mouth swap for RT-PCR of SARS-Cov-2 before and after 30 min of mouth washing and other mouth wash has been done on SARS COV-2 patients for 1 min by whiting mouth wash (Oxiactive) group 2 and then mouth swap for RT-PCR of SARS-Cov-2 to know the variation in CT PCR level pre and post mouth wash and to compare the effectiveness between them. The effectiveness of normal mouth wash of COVID-19 patients reducing the viral load, which is considered as an impotent way to protect from the separation of viral shedding (the 14 patients were COVID -19 PCR positive and then after washing became PCR negative). The effectiveness of whitening mouth wash of COVID-19 patients strong reducing the viral load and most cases are becoming negative PCR, which is considered as an important way to protect from the separation of viral shedding and maximum lowering 14 CT or (16000 folds). Mouth wash according to the result could be the best way to decrease the contamination and infection between dentist and patient. Reducing the nosocomial infection by applying mouth wash for several minutes. The PCR false-negative result could be caused by mouth wash the effect the truth of laboratory tests.

Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference.

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%-95.26%) and specificity (83.72%-98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.

False-Negative Results of Real-Time Reverse-Transcriptase Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2: Role of Deep-Learning-Based CT Diagnosis and Insights from Two Cases.

The epidemic of 2019 novel coronavirus, later named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still gradually spreading worldwide. The nucleic acid test or genetic sequencing serves as the gold standard method for confirmation of infection, yet several recent studies have reported false-negative results of real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Here, we report two representative false-negative cases and discuss the supplementary role of clinical data with rRT-PCR, including laboratory examination results and computed tomography features. Coinfection with SARS-COV-2 and other viruses has been discussed as well.

Digital PCR is a sensitive new technique for SARS-CoV-2 detection in clinical applications.

The global coronavirus disease 2019 (COVID-19) pandemic has posed great challenges in people's daily lives. Highly sensitive laboratory techniques played a critical role in clinical COVID-19 diagnosis and management. In this study the feasibility of using a new digital PCR-based detection assay for clinical COVID-19 diagnosis was investigated by comparing its performance with that of RT-PCR. Clinical patient samples and samples obtained from potentially contaminated environments were analyzed. The study included 10 patients with confirmed COVID-19 diagnoses, 32 validated samples of various types derived from different clinical timepoints and sites, and 148 environmentally derived samples. SARS-CoV-2 nucleic acids were more readily detected in respiratory tract samples (35.0%). In analyses of environmentally derived samples, the positivity rate of air samples was higher than that of surface samples, probably due to differences in virus concentrations. Digital PCR detected SARS-CoV-2 in several samples that had previously been deemed negative, including 3 patient-derived samples and 5 environmentally derived samples. In this study digital PCR exhibited higher sensitivity than conventional RT-PCR, suggesting that it may be a useful new method for clinical SARS-CoV-2 detection. Improvement of SARS-CoV-2 detection would substantially reduce the rates of false-negative COVID-19 test results, in particular those pertaining to asymptomatic carriers.