Decreasing parvovirus B19 and hepatitis A nucleic acid test positivity rates in Canadian plasma donors following the initiation of COVID-19 restriction in March 2020.

BACKGROUND AND OBJECTIVES: In Canada, plasma sent for fractionation is tested for both parvovirus B19 (B19V) and hepatitis A virus (HAV). This study compared positivity rates of B19 and HAV nucleic acid tests (NATs) in Canadian plasma samples for the pre-COVID-19 restriction era (2015 to end of February 2020 [Q1] 2020) and the post-COVID-19 restriction era. MATERIALS AND METHODS: Pooled EDTA plasma specimens were tested within 24 months of blood draw using the Procleix Panther System (Grifols Diagnostic Solutions Inc, San Diego, CA, USA) for B19V and HAV detection. Reactive pools were resolved by individual specimen testing. RESULTS: Between 1 January 2015, and 31 March 2022, 3,928,619 specimens from Canadian plasma donors were tested for B19V. For the same period, 3,922,954 specimens were tested for HAV. To account for a lag in specimen testing for up to 24 months, the data were divided into: (1) a pre-pandemic period (1 January 2015-31 March 2020; B19V tested n = 2,412,701, B19V NAT-positive n = 240 [0.01%], HAV tested n = 2,407,036, HAV NAT-positive n = 26 [0.001%]); (2) a two-year mixed-impact period (1 April 2020-31 March 2022; B19V tested n = 968,250, B19V NAT-positive n = 14 [0.001%], HAV tested n = 968,250, HAV NAT-positive n = 2 [0.0002%]); and (3) a pandemic-impact period (1 April 2022-31 March, 2023; B19V tested n = 597,668, B19V NAT-positive n = 3 [0.0005%], HAV tested n = 597,668, HAV NAT-positive n = 1 [0.0002%]). CONCLUSION: The percentage of B19V- and HAV-positive donations was significantly reduced from the pre-pandemic period to the pandemic-impact period.

Clinical evaluation of a modified SARS-CoV-2 rapid molecular assay, ID NOW ™ COVID-19 2.0.

In the diagnosis of coronavirus disease 2019 (COVID-19), several types of instruments and reagents for SARS-CoV-2 nucleic acid testing have been introduced to meet clinical needs. We evaluated the clinical performances of ID NOW™ COVID-19 2.0 (ID NOW™ 2.0), which is capable of detecting SARS-CoV-2 within 12 min as part of point-of-care testing (POCT). Patients who displayed COVID-19 related symptoms, and who were tested for screening purposes, were recruited to this study. Two nasopharyngeal swabs were collected and tested using the ID NOW™ 2.0 test. Reference testing was performed using the cobas 8800 or 6800 (reagents: cobas SARS-CoV-2 and Flu A/B). A total of 38 samples and 46 samples were tested positive and negative, respectively, by the reference test. The ID NOW™ 2.0 showed a sensitivity of 94.7% (95% CI: 82.3-99.4) and a specificity of 100% (95% CI: 92.3-100). Samples that were positive by reference testing had cycle threshold (Ct) values ranging from 11.90 to 35.41. Among these reference positive samples, two samples were negative by ID NOW™ 2.0 with Ct values of 35.25 and 35.41. For samples with Ct values < 35, the sensitivity of ID NOW™ 2.0 was 100%. In Japan, the restrictions related to COVID-19 have been relaxed, however the COVID-19 epidemic still continues. ID NOW™ 2.0 is expected to be used as a rapid and reliable alternative to laboratory-based RT-PCR methods.

Validation of a Parvovirus B19 NAT Assay for Screening of Umbilical Cord Blood for Allogenic Hematopoietic Stem Cell Donation.

Introduction: Parvovirus B19 transmitted by umbilical cord blood (UCB) products may cause severe disease in allogenic hematopoietic stem cell transplant recipients. Thus, commercially available nucleic acid test (NAT) assays for highly sensitive detection of parvovirus B19 DNA validated for the specimen cord blood plasma (CBP) are required to avoid parvovirus B19 transmission by umbilical hematopoietic stem cell preparations. Methods: The multiplex cobas DPX NAT assay was validated for detection of parvovirus B19 DNA in CBP derived from citrate anticoagulated UCB units which have been processed by the Rubinstein method. In total, 363 retained CBP samples pretested negative for parvovirus B19 DNA were prepared for analyzing sensitivity, specificity, and interference of that NAT assay. The 3rd WHO International Standard for parvovirus B19 DNA was used for determining the 95% limit of detection (LOD95) by probit analysis. Results: The validation of the parvovirus B19 NAT assay for CBP demonstrated high sensitivity, specificity, intra- and inter-assay precision. Dilution series and replicate analyses showed a high linearity of the assay with a coefficient of determination above 0.99 and revealed a LOD95 of 17 International Units (IU)/mL (95% confidence interval, 14-44 IU/mL) for parvovirus B19 DNA in CBP samples. Conclusion: The validation of a commercially available parvovirus B19 NAT assay for the specimen CBP demonstrated a high assay performance fulfilling German guidelines and international regulations.

Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Nucleic Acid Point-of-Care Testing to Improve Diagnostic Preparedness.

Testing programs for severe acute respiratory syndrome coronavirus 2 have relied on high-throughput polymerase chain reaction laboratory tests and rapid antigen assays to meet diagnostic needs. Both technologies are essential; however, issues of cost, accessibility, manufacturing delays, and performance have limited their use in low-resource settings and contributed to the global inequity in coronavirus disease 2019 testing. Emerging low-cost, multidisease point-of-care nucleic acid tests may address these limitations and strengthen pandemic preparedness, especially within primary healthcare where most cases of disease first present. Widespread deployment of these novel technologies will also help close long-standing test access gaps for other diseases, including tuberculosis, human immunodeficiency virus, cervical cancer, viral hepatitis, and sexually transmitted infections. We propose a more optimized testing framework based on greater use of point-of-care nucleic acid tests together with rapid immunologic assays and high-throughput laboratory molecular tests to improve the diagnosis of priority endemic and epidemic diseases, as well as strengthen the overall delivery of primary healthcare services.

Potential Life-Cycle Environmental Impacts of the COVID-19 Nucleic Acid Test.

Massive diagnostic testing has been performed for appropriate screening and identification of COVID-19 cases in the ongoing global pandemic. However, the environmental impacts of COVID-19 diagnostics have been least considered. In this paper, the environmental impacts of the COVID-19 nucleic acid diagnostics were assessed by following a full cradle-to-grave life-cycle approach. The corresponding life-cycle anthology was established to provide quantitative analysis. Moreover, three alternative scenarios, i.e., material substitution, improved waste treatment, and electric vehicle (EV)-based transportation, were further proposed to discuss the potential environmental mitigation and conservation strategies. It was estimated that the life cycle of a single COVID-19 nucleic acid diagnostic test in China would lead to the emission of 612.9 g CO2 equiv global warming potential. Waste treatment, as a step of life cycle, worsen the environmental impacts such as global warming potential, eutrophication, and ecotoxicity. Meanwhile, diesel-driven transportation was considered as the major contributor to particulate air. Even though COVID-19 diagnostics are of the greatest importance to end the pandemic, their environmental impacts should not be ignored. It is suggested that improved approaches for waste treatment, low-carbon transportation, and a reliable pool sampling strategy are critical for the achievement of sustainable and green diagnostics.

Results of a SARS-CoV-2 virus genome detection external quality assessment round focusing on sensitivity of assays and pooling of samples.

OBJECTIVES: Results of earlier external quality assessment (EQA) rounds suggested remarkable differences in the sensitivity of SARS-CoV PCR assays. Although the test systems are intended to detect SARS-CoV-2 in individual samples, screening is often applied to sample pools to increase efficiency and decrease costs. However, it is unknown to what extent these tests actually meet the manufacturer's specifications for sensitivity and how they perform when testing sample pools. METHODS: The sensitivity of assays in routine use was evaluated with a panel of positive samples in a round of a SARS-CoV-2 virus genome detection EQA scheme. The panel consisted of samples at or near the lower limit of detection ("weakly positive"). Laboratories that routinely test sample pools were asked to also analyze the pooled EQA samples according to their usual pool size and dilution method. RESULTS: All participants could detect a highly positive patient-derived sample (>106 copies/mL). Most (96%) of the test systems could detect at least 1,000 copies/mL, meeting the minimum acceptable benchmark, and many (94%) detected the vRNA in a sample with lower concentration (500 copies/mL). The false negative ratio increased to 16 and 26% for samples with 100 and 50 copies/mL, respectively. CONCLUSIONS: The performance of most assays met or exceeded their specification on sensitivity. If assays are to be used to analyze sample pools, the sensitivity of the assay and the number of pooled samples must be balanced.

PCR to CRISPR: Role of Nucleic Acid Tests (NAT) in detection of COVID-19.

COVID-19 infection has emerged as an unparalleled pandemic with morbidity and mortality tolls challenging diagnostic approaches and therapeutic interventions, and raising serious questions for healthcare policy-makers. From the diagnostic perspective, Reverse transcriptase polymerase chain reaction remains the gold standard. However, issues associated with gene primer variation in different countries, low analytical sensitivity, cross-reactivity with certain human coronaviruses have raised serious concerns within the scientific community. Alongside longer turnaround times, requirements of sophisticated equipment and trained technicians are the other challenges for conventional reverse transcriptase polymerase chain reaction testing. The recent biotechnological boom has now allowed newer nucleic acid testing options for diagnosing severe acute respiratory syndrome Coronovairus 2 (SARS-CoV2) with much better diagnostic efficiency, reduced turnaround times and possible benefit for use as a point-of-care test. Isothermal techniques with simple equipment requirements along with uniform temperature for analysis have emerged to be more sensitive and specific with turnaround times as low as 10-15 minutes. Similarly, Cluster Regularly Interspaced Short Palindromic Repeats have also been seen to play a very decisive role in COVID-19 diagnostics with much superior diagnostic efficiency and feasibility as a point-of-care test and its possible use for sequencing. The current narrative review was planned to consolidate data for all possible nucleic acid testing options under research/clinical use, and to provide a comparative assessment from the perspective of both the clinician and the laboratory.

EVALUATION OF SEROLOGICAL SCREENING AND PCR-AMPLIFICATION OF HEPATITIS B VIRUS DNA AMONG IRAQI BLOOD DONORS.

OBJECTIVE: The aim: Infection with the hepatitis B virus (HBV) caused by blood transfusion is a big problem throughout the world. The aim of study is to determine the faster and more accurate methods for detection of hepatitis B infections by serological screening and PCR- amplification. PATIENTS AND METHODS: Materials and methods: A total of 140528 donors were tested for HBsAg and total anti-HBc from January to October 2021 in Iraq's National Blood Transfusion Center; however, only 100 samples with HBsAg (-) and anti-HBc (+) were collected and tested for HBV DNA using quantitative real-time PCR. RESULTS: Results: From 2015 to 2021, the percentage of HBsAg positive donors was 0.33 percent in 2015, 0.32 percent in 2016, 0.30 percent in 2017, 0.28 percent in 2018, 0.23 percent in 2019, 0.22 percent in 2020, and 0.27 percent in 2021. Between January and October of 2021, the overall anti-HBc rate among the (140528) donors was 4.42 percent. According to our findings, only 7% of blood samples from NBTC donors with HBsAg (-) anti-HBc (+) were positive for HBV DNA. The results showed no significant change in HBs Ag (+) and total anti-HBc rates among blood donors between 2015 and 2021. CONCLUSION: Conclusions: HBV infection could be transmitted from a blood donor with OBI. PCR (RT PCR) is substantially more sensitive and effective. Despite this the use of an anti-HBc test for blood donors could be seen as a second choice to control HBV from spreading during blood transfusions.

Transplantation of Kidneys From Hepatitis C Virus-Infected Donors to Hepatitis C Virus-Negative Recipients: One-Year Kidney Allograft Outcomes.

RATIONALE & OBJECTIVE: Transplant centers in the United States are increasingly willing to transplant kidneys from hepatitis C virus (HCV)-infected (HCV+) donors into HCV- recipients. We studied the association between donor HCV infection status and kidney allograft function and posttransplantation allograft biopsy findings. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: We examined 65 HCV- recipients who received a kidney from a HCV+ donor and 59 HCV- recipients who received a kidney from a HCV- donor during 2018 at a single transplant center. EXPOSURE: Predictor(s) of donor infection with HCV. OUTCOMES: Kidney allograft function and allograft biopsy findings during the first year following transplantation. ANALYTICAL APPROACH: We compared estimated glomerular filtration rate (eGFR), findings on for-cause and surveillance protocol biopsies, development of de novo donor-specific antibodies (DSAs), and patient and allograft outcomes during the first year following transplantation between recipients of HCV+ and HCV- kidneys. We used linear regression to estimate the independent association between allograft function and HCV viremic status of the kidney donor. RESULTS: The mean age of recipients was 52 ± 11 (SD) years, 43% were female, 19% and 80% of recipients were White and Black, respectively. Baseline characteristics were similar between the HCV+ and HCV- groups. There were no statistically significant differences between the HCV+ and HCV- groups in delayed graft function rates (12% vs 8%, respectively); eGFRs at 3, 6, 9, and 12 months post-transplantation; proportions of patients with cellular rejection (6% vs 7%, respectively); and proportions with antibody-mediated rejection (7% vs 10%, respectively) or de novo DSAs (31% vs 20%, respectively). HCV viremic status was not associated with eGFR at 3, 6, 9, or 12 months. LIMITATIONS: Generalizability from a single-center study and small sample size was limited. CONCLUSIONS: Recipients of kidneys from donors infected with HCV had similar kidney allograft function and probability of rejection in the first year after transplantation compared to those who received kidneys from donors without HCV infection.

Analysis of the positive rate of 4254 cases of COVID-19 nucleic acid tests in different aites in Wuhan, China.

There's an outbreak of coronavirus diesase 2019 (COVID-19) since December 2019, first in Wuhan. It has caused huge medical challenges to Hubei Province with currently more than 67 thousand confirmed cases till 8th March 2020. Identification, there is no clinically effective drug. Isolation and masks are essential to limit human-to-human transmission initially. The nucleic acid test (NAT) of COVID-19 currently was the most reliable established laboratory diagnosis method in clinical. From 8th February to 7th March 2020, 4254 cases were collected for analysis at six nucleic acid collection sites in the community medical team of Hubei Provincial Hospital of Traditional Chinese Medicine, which cover almost all groups who need NAT in Wuhan. Distribution of positive rates in different sites by genders, ages, or occupations were compared. The positive rates of different sites from high to low were: hospital wards (24.71%) > fever clinics (16.57%) > nursing homes (5.51%) > isolation hotels (5.30%) > rehabilitation stations (1.36%) >close contact sites (0.17%). The confirmed patients in isolation hotels, hospital ward, and fever clinical were mainly middle-aged and elderly, and most of them were women. The positive rate in isolation hotels and fever clinics gradually decreased over time. There were no significant differences between genders among those six nucleic acid collection sites (P < .05). The hospital wards have the highest positive rate; however, close contact sites have lowest one. Patients who are discharged from hospitals may still have potential risks. Middle-aged and older people remain the focus of epidemic prevention and control.

Rapid point-of-care testing for SARS-CoV-2 virus nucleic acid detection by an isothermal and nonenzymatic Signal amplification system coupled with a lateral flow immunoassay strip.

An outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in December 2019. Accurate, rapid, convenient, and relatively inexpensive diagnostic methods for SARS-CoV-2 infection are important for public health and optimal clinical care. The current gold standard for diagnosing SARS-CoV-2 infection is reverse transcription-polymerase chain reaction (RT-PCR). However, RTPCR assays are designed for use in well-equipped laboratories with sophisticated laboratory infrastructure and highly trained technicians, and are unsuitable for use in under-equipped laboratories and in the field. In this study, we report the development of an accurate, rapid, and easy-to-implement isothermal and nonenzymatic signal amplification system (a catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method that can detect SARSCoV-2 in oropharyngeal swab samples. Our method avoids RNA isolation, PCR amplification, and elaborate result analysis, which typically takes 6-8 h. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 min. Such methods are simple and require no expensive equipment, only a simple thermostatically controlled water bath and a fluorescence reader device. We validated our method using synthetic oligonucleotides and clinical samples from 15 patients with SARS-CoV-2 infection and 15 healthy individuals. Our detection method provides a fast, simple, and sensitive (with a limit of detection (LoD) of 2000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100 % positive and negative predictive agreements.

Clinical testing for COVID-19.

As the novel coronavirus severe acute respiratory syndrome coronavirus 2 caused coronavirus disease 2019 cases in the United States, the initial test was developed and performed at the Centers for Disease Control and Prevention. As the number of cases increased, the demand for tests multiplied, leading the Centers for Disease Control and Prevention to use the Emergency Utilization Authorization to allow clinical and commercial laboratories to develop tests to detect the presence of the virus. Many nucleic acid tests based on RT-PCR were developed, each with different techniques, specifications, and turnaround time. As the illnesses turned into a pandemic, testing became more crucial. The test supply became inadequate to meet the need and so it had to be prioritized according to guidance. For surveillance, the need for serologic tests emerged. Here, we review the timeline of test development, the turnaround times, and the various approved tests, and compare them as regards the genes they detect. We concentrate on the point-of-care tests and discuss the basis for new serologic tests. We discuss the testing guidance for prioritization and their application in a hospital setting.

[Application of anti-HCV and HCV RNA detection in intravenous drug users].

Objective: To explore the diagnostic value of anti-HCV and HCV RNA so as to provide an accurate and efficient detection strategy for the diagnosis of HCV in intravenous drug users. Methods: 527 plasma samples from intravenous drug users were collected, and preliminary anti-HCV ELISA screening test was performed. A recombinant immunoblot assay (RIBA) was used as confirmatory assay for reactive antibody samples. All samples were tested for HCV RNA, followed by analysis of anti-HCV screening test, RIBA and HCV nucleic acid test results. Results: Anti-HCV ELISA results were reactive in 386 out of 527 intravenous drug users and non-reactive in 141. Among the 386 reactive antibody samples detected by RIBA, 370 cases were anti-HCV positive, 6 cases were anti-HCV indeterminate and 10 cases were anti-HCV negative. Anti-HCV ELISA and RIBA positive coincidence detection rate was 95.85% (370/386), and 70.21% (370/527) among intravenous drug users. HCV RNA was negative in all 10 anti-HCV RIBA non-reactive samples. 376 anti-HCV RIBA-positive and indeterminate samples were tested for HCV RNA, of which 56.93% (300/527) were current HCV infection, and 14.42% (76/527) were past HCV infection. Among 141 anti-HCV ELISA negative samples, the residual risk by anti-HCV ELISA screening for HCV RNA was 1.52% (8/527). HCV viral load distribution among intravenous drug users showed that the high viral load value (>10(7) IU/ml) and low viral load values (< 10(2) IU/ml) accounted for 1.95% and 2.27%, respectively, while the samples with viral load value of 1×10(2) ~ 1×10(7) IU/ mL accounted for 95.78% (295/308), and were mainly distributed in 1×10(5) ~ 1×10(6) IU/ml (37.99%). ELISA + RIBA + NAT assay detection strategies had differentiated 300 cases of current HCV infection, 76 cases of past HCV infection and 10 cases of false positive anti-HCV results, while ELISA+NAT assay detection strategies had only detected 300 cases of current HCV infection. However, of the 386 positive subjects screened for antibodies, 10 (2.59%) were undifferentiated false positives. Conclusion: Intravenous drug users are the high-risk population of HCV infection with high prevalence and high viral load. Anti-HCV screening for intravenous drug users will have a certain degree of residual risk. Therefore, anti-HCV ELISA screening and nucleic acid detection strategy can accurately diagnose the current infected patients; however, it cannot distinguish the false positive results of antibody screening.

Clinical Performance of the Novel GenMark Dx ePlex Blood Culture ID Gram-Positive Panel.

Rapid identification from positive blood cultures is standard of care (SOC) in many clinical microbiology laboratories. The GenMark Dx ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a multiplex nucleic acid amplification assay based on competitive DNA hybridization and electrochemical detection using eSensor technology. This multicenter study compared the investigational-use-only (IUO) BCID-GP Panel to other methods of identification of 20 Gram-positive bacteria, four antimicrobial resistance genes, and both Pan Candida and Pan Gram-Negative targets that are unique to the BCID-GP Panel. Ten microbiology laboratories throughout the United States collected residual, deidentified positive blood culture samples for analysis. Five laboratories tested both clinical and contrived samples with the BCID-GP Panel. Comparator identification methods included each laboratory's SOC, which included matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and automated identification systems as well as targeted PCR/analytically validated real-time PCR (qPCR) with bidirectional sequencing. A total of 2,342 evaluable samples (1,777 clinical and 565 contrived) were tested with the BCID-GP Panel. The overall sample accuracy for on-panel organisms was 89% before resolution of discordant results. For pathogenic Gram-positive targets (Bacillus cereus group, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Listeria spp., Listeria monocytogenes, Streptococcus spp., Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus pneumoniae, and Streptococcus pyogenes), positive percent agreement (PPA) and negative percent agreement (NPA) ranged from 93.1% to 100% and 98.8% to 100%, respectively. For contamination rule-out targets (Bacillus subtilis group, Corynebacterium, Cutibacterium acnes, Lactobacillus, and Micrococcus), PPA and NPA ranged from 84.5% to 100% and 99.9% to 100%, respectively. Positive percent agreement and NPA for the Pan Candida and Pan Gram-Negative targets were 92.4% and 95.7% for the former and 99.9% and 99.6% for the latter. The PPAs for resistance markers were as follows: mecA, 97.2%; mecC, 100%; vanA, 96.8%; and vanB, 100%. Negative percent agreement ranged from 96.6% to 100%. In conclusion, the ePlex BCID-GP Panel compares favorably to SOC and targeted molecular methods for the identification of 20 Gram-positive pathogens and four antimicrobial resistance genes in positive blood culture bottles. This panel detects a broad range of pathogens and mixed infections with yeast and Gram-negative organisms from the same positive blood culture bottle.

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. METHODS: We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. RESULTS: Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. CONCLUSION: Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Positive result of Sars-Cov-2 in faeces and sputum from discharged patients with COVID-19 in Yiwu, China.

BACKGROUND: With the effective prevention and control of COVID-19 in China, the number of cured cases has increased significantly. Further monitoring of the disease prognosis and effective control of the "relapse" of the epidemic has become the next focus of work. This study analysed the clinical prognosis of discharged COVID-19 patients by monitoring their SAR-CoV-2 nucleic acid status, which provided a theoretical basis for medical institutions to formulate discharge standards and follow-up management for COVID-19 patients. METHODS: We included 13 discharged COVID-19 patients who were quarantined for 4 weeks at home. The patient's daily clinical signs were recorded and sputum and faecal specimens were regularly sent for detection of SARS-CoV-2 nucleic acid. RESULTS: The time between initial symptoms and meeting discharge criteria was 18 to 44 days with an average of 25 ± 6 days. The faecal samples of two patients still tested positive after meeting the discharge criteria and the sputum samples of four patients returned positive 5 to 14 days after discharge. The rate of the recurring positive test result in samples from the respiratory system was 31% (4/13). CONCLUSION: Under the present discharge criteria, the high presence of SARS-CoV-2 nucleic acid in faecal and respiratory samples of discharged COVID-19 patients indicates potential infectivity. Therefore, we suggest that faecal virus nucleic acid should be tested as a routine monitoring index for COVID-19 and a negative result be added to the criteria. Simultaneously, we should strengthen the regular follow-up of discharged patients with continuous monitoring of the recurrence of viral nucleic acid.

Cross-priming isothermal amplification combined with nucleic acid test strips for detection of meat species.

Adulteration of high-quality meat with their cheaper counterparts can be minimized by rapid and reliable methods for detecting meat species. Here an isothermal cross-primer amplification (CPA) technique combined with colloidal gold nucleic acid test strips (CPA strips) was developed to differentiate cow, sheep, arctic fox, and pig meat. A simple primer design for multiplex differentiation using a universal single-labeled CPA primer system and four detection-level species-specific labeling primers were analyzed by colloidal gold-based test strip assay. Moreover, simultaneous detection of fox and pig meat on a double-test line strip was feasible. The CPA strip assay indicated a lower amounts sensitivity of 0.3 ng DNA when one targeted species was tested and a detection limit of 1% when arctic fox meat was detected in the meat mixtures. Using a minimal set of primers, this study provides a promising tool for detecting the species of different types of meat using a constant temperature amplification technology.

Prevalence of hepatitis E viraemia among blood donors: a systematic review.

BACKGROUND: Hepatitis E virus (HEV) is usually transmitted by faecal-oral route. Recent reports have documented HEV viraemia in donated blood units and HEV transmission through blood transfusion. This systematic review summarizes the available data on prevalence of HEV viraemia in blood donors. METHODS: Electronic databases were searched on 17 December 2018 to identify full-text English papers reporting original data on prevalence of HEV RNA in donated blood units. Two authors independently extracted the relevant data, which were pooled using simple aggregation as well as a random-effects meta-analysis; heterogeneity was assessed using the I2 method. RESULTS: In all, 59 data sets from 28 countries were identified. The available data showed marked heterogeneity. Of a total of 2 127 832 units studied, 561 (263·6 [95% confidence intervals = 242·7-286·4] per million units) tested positive for HEV RNA. On random-effects meta-analysis, the pooled prevalence was 60·9 [6·7-155·4] per million units. In the viraemic units, HEV RNA titre varied by nearly one million-fold, and most had genotype 3 HEV. The prevalence was higher in blood units with anti-HEV antibodies or elevated alanine aminotransferase. Only nearly one-fourth of viraemic units had anti-HEV antibodies. CONCLUSIONS: The prevalence of HEV viraemia among healthy blood donors is low, though the available data had limited geographical representation and marked heterogeneity. There is a need for further data on HEV viraemia in blood donors from areas with non-3 HEV genotype preponderance.

Recurrence of SARS-CoV-2 nucleic acid positive test in patients with COVID-19: a report of two cases.

BACKGROUND: The recurrence of positive SARS-CoV-2 nucleic acid test results in patients with COVID-19 is becoming more important and warrants more attention. CASE PRESENTATION: This study reports 2 cases, a child with mild COVID-19 and an adult female with moderate COVID-19, who were discharged after three consecutive negative nucleic acid tests and were later readmitted to the hospital for recurrence of SARS-CoV-2 nucleic acid positivity. By tracking the patients' symptoms, serum antibodies, and imaging manifestations after readmission, we found that they showed a trend of gradual improvement and recovery throughout treatment. They were cured without additional treatment, with the appearance of antibodies and the recovery of immune functions. CONCLUSIONS: It is deemed extremely necessary to improve the discharge standard of care. At the same time, nucleic acid detection is recommended to increase the dynamic monitoring of serum antibodies and imaging, strengthen the management of discharged patients, and appropriately extend the home or centralized isolation time.