A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing

The outbreak of virus-induced infectious diseases poses a global public-health challenge.Nucleic acid amplification testing(NAAT)enables early detection of pandemic viruses and plays a vital role in preventing onward transmission.However,the requirement of skilled operators,expensive instrumentation,and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients.Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive,specific,and rapid viral nucleic acid testing.The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification(RT-LAMP)were integrated into the reaction units of a microfluidic disc.The whole processing steps such as injection of reagents,fluid actuation by rotation,heating and temperature control,and detection of fluorescence signals were carried out automatically by a customized instrument.We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)armored RNA particles.The estimated limit of detection for armored RNA particles is 2 copies per reaction,the throughput is 21 reactions per disc,and the assay sample-to-answer time is approximately 70 min.This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol,and can be readily adapted for virus detection outside the diagnostic laboratory.

Development and evaluation of a thermostatic nucleic acid testing device based on magnesium pyrophosphate precipitation for detecting Enterocytozoon hepatopenaei

Enterocytozoon hepatopenaei(EHP)infection has seriously affected prawn culture globally.The symptoms of the infection are not apparent,and traditional detection methods are time consuming and low in accuracy.We developed a new onsite rapid testing device(size 18.8×16.7×6.6 cm^(3))for EHP based on magnesium pyrophosphate precipitation and facilitated by loop mediated isothermal amplification(LAMP).The design and fabrication of the device enables efficient light absorbance.The device has a highly sensitive detector,high-precision thermal controller,and humanized touch screen.The temperature control precision of the device is 0.2-0.3℃ at 60℃,63℃,and 65℃.The coefficients of variation values(CVV)of the luminous power in one channel at light on and off were found to be 0.0097 and 0.0014,respectively,within 1 h.The CVV of the background,luminous power,and values of eight PCR tubes filled with pure water were all less than 5%.In the EHP experiment,eight samples(including seven positive and one negative)confirmed the effectiveness of the device,and four positive and four negative samples verified whether cross-contamination exists.Among them,the rise time of the curve was about 15 min.These results assert that the developed device exhibits enhanced stability and uniformity and has excellent performance with high sensitivity,good specificity,and low testing time.Moreover,the optimal and minimum absorbance range was 555-655 nm for monitoring the production of LAMP.

A brief review of novel nucleic acid test biosensors and their application prospects for salmonids viral diseases detection

Viral diseases represent one of the major threats for salmonids aquaculture.Early detection and identification of viral pathogens is the main prerequisite prior to undertaking effective prevention and control measures.Rapid,sensitive,efficient and portable detection method is highly essential for fish viral diseases detection.Biosensor strategies are highly prevalent and fulfill the expanding demands of on-site detection with fast response,cost-effectiveness,high sensitivity,and selectivity.With the development of material science,the nucleic acid biosensors fabricated by semiconductor have shown great potential in rapid and early detection or screening for diseases at salmonids fisheries.This paper reviews the current detection development of salmonids viral diseases.The present limitations and challenges of salmonids virus diseases surveillance and early detection are presented.Novel nucleic acid semiconductor biosensors are briefly reviewed.The perspective and potential application of biosensors in the on-site detection of salmonids diseases are discussed.

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The ”first generation“ antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now < 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

Turnaround times for molecular testing of pediatric viral cerebrospinal fluid samples in United Kingdom laboratories

BACKGROUND Rapid molecular testing has revolutionized the management of suspected viral meningitis and encephalitis by providing an etiological diagnosis in<90 min with potential to improve outcomes and shorten inpatient stays.However,use of molecular assays can vary widely.AIM To evaluate current practice for molecular testing of pediatric cerebrospinal fluid(CSF)samples across the United Kingdom using a structured questionnaire.METHODS A structured telephone questionnaire survey was conducted between July and August 2020.Data was collected on the availability of viral CSF nucleic acid amplification testing(NAAT),criteria used for testing and turnaround times including the impact of the coronavirus disease 2019 pandemic.RESULTS Of 196/212(92%)microbiology laboratories responded;63/196(32%)were excluded from final analysis as they had no on-site microbiology laboratory and outsourced their samples.Of 133 Laboratories included in the study,47/133(35%)had onsite facilities for viral CSF NAAT.Hospitals currently undertaking onsite NAAT(n=47)had much faster turnaround times with 39 centers(83%)providing results in≤24 h as compared to those referring samples to neighboring laboratories(5/86;6%).CONCLUSION Onsite/near-patient rapid NAAT(including polymerase chain reaction)is recommended wherever possible to optimize patient management in the acute setting.

The point-of-care-testing of nucleic acids by chip, cartridge and paper sensors

Point-of-care nucleic acid testing(POCNAT) has played an important role in the outbreak of infectious diseases(e.g., COVID-19) over recent years. POCNAT aims to realize the rapid, simple and automatic detection of nucleic acid. Thanks to the development of manufacturing technology, electronic information technology, artificial intelligence technology, and biological information technology in recent years, the development of the POCNAT device has led to significant advancement. Instead of the normal nucleic acid detection methods used in the laboratory, some novel experimental carriers have been applied, such as chips, cartridges and papers. The application of these experimental carriers has realized the automation and integration of nucleic acid detection. The entire process of nucleic acid detection is normally divided into three steps(nucleic acid extraction, target amplification and signal detection). All of the reagents required by the process can be pre-stored on these experimental carriers, without unnecessary manual operation. Furthermore, all of the processes are carried out in this experimental carrier, with the assistance of a specific control device. Although they are complicated to manufacture and precise in design,their application provides a significant step forwards in nucleic acid detection and realizes the integration of nucleic acid detection. This technology has great potential in the field of point-of-care molecular diagnostics in the future. This paper focuses on the relevant content of these experimental carriers.

Evaluation of factors contributing to variability of qualitative and quantitative proficiency testing for SARS-CoV-2 nucleic acid detection

The pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has led to unprecedented social and economic disruption.Many nucleic acid testing(NAT)laboratories in China have been established to control the epidemic better.This proficiency testing(PT)aims to evaluate the participants’performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities.Two different concentrations of RNA samples(A,B)were used for quantitative PT.Pseudovirus samples D,E(different concentrations)and negative sample(F)were used for qualitative PT.50 data sets were reported for qualitative PT,of which 74.00%were entirely correct for all samples.Fortytwo laboratories participated in the quantitative PT.37 submitted all gene results,of which only 56.76%were satisfactory.For qualitative detection,it is suggested that laboratories should strengthen personnel training,select qualified detection kits,and reduce cross-contamination to improve detection accuracy.For quantitative detection,the results of the reverse transcription digital PCR(RT-dPCR)method were more comparable and reliable than those of reverse transcription quantitative PCR(RT-qPCR).The copy number concentration of ORF1ab and N in samples A and B scattered in 85,223,50,and 106 folds,respectively.The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills.Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing,95.65%of the laboratories with satisfactory quantitative results also judged the qualitative results correctly,while 85.71%of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments.Therefore,the quantitative ability is the basis of qualitative judgment.Overall,participants from hospitals reported more satisfactory results than those from enterprises and universities.Therefore,surveillance,daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma.The management of HCV infection should not only be focus on therapy,but also to screen carrier individuals in order to prevent transmission.In the present,molecular detection and quantification of HCV genome by real time polymerase chain reaction(PCR)represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens.However,real time PCR is a complicated approach and of limited distribution.On the other hand,isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care.In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.

Current knowledge on the laboratory diagnosis of Clostridium difficile infection

Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from selflimited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.

Positive SARS-Cov-2 test in a woman with COVID-19 at 22 days after hospital discharge:A case report

Background:In a few discharged patients with coronavirus disease 2019(COVID-19),the nucleic acid test shows positive results again.Whether this is due to relapse of the disease,reinfection by the virus,or a false-positive result at hospital discharge is worth exploring.Case presentation:A woman with COVID-19 was discharged from the hospital after integrative treatment with traditional Chinese and Western medicine because she met the discharge standards.However,she obtained positive results on a nucleic acid test 22 days later.Conclusion:Based on this positive test result in a discharged patient with COVID-19,anal tests and coronavirus antibody tests should be combined with throat swab tests to further develop the diagnosis and discharge standards for patients with COVID-19.

Suspected SARS-CoV-2 infection with fever and coronary heart disease:A case report

BACKGROUND The coronavirus disease 2019(COVID-19)is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Suspected cases accounted for a large proportion in the early stage of the COVID-19 outbreak.The deviation of the nucleic acid test by throat swab(the current gold standard of COVID-19)caused by variation in sampling techniques and reagent kits and coupled with nonspecific clinical manifestations make confirmation of the suspected cases difficult.Proper management of the suspected cases of COVID-19 is crucial for disease control.CASE SUMMARY A 65-year-old male presented with fever,lymphopenia,and chest computed tomography(CT)images similar to COVID-19 after percutaneous coronary intervention.The patient was diagnosed as having bacterial pneumonia with cardiogenic pulmonary edema instead of COVID-19.This was based on four negative results for throat swab detection of SARS-CoV-2 nucleic acid using reverse transcriptase-polymerase chain reaction assay and one negative result for serological antibody of SARS-CoV-2 with the serological assay.Additionally,the distribution of ground-glass opacities and thickened blood vessels from the CT images differed from COVID-19 features,which further supported the exclusion of COVID-19.CONCLUSION Distinguishing COVID-19 patients from those with bacterial pneumonia with cardiogenic pulmonary edema can be difficult.Therefore,it requires serious identification.

An outlook on coronavirus disease 2019 detection methods

Diagnostic testing plays a fundamental role in the mitigation and containment of coronavirus disease 2019(COVID-19),as it enables immediate quarantine of those who are infected and contagious and is essential for the epidemiological characterization of the virus and estimating the number of infected cases worldwide.Confirmation of viral infections,such as COVID-19,can be achieved through two general approaches:nucleic acid amplification tests(NAATs)or molecular tests,and serological or antibody-based tests.The genetic material of the pathogen is detected in NAAT,and in serological tests,host antibodies produced in response to the pathogen are identified.Other methods of diagnosing COVID-19 include radiological imaging of the lungs and in vitro detection of viral antigens.This review covers different approaches available to diagnosing COVID-19 by outlining their advantages and shortcomings,as well as appropriate indications for more accurate testing.

Determination of the predictive factors for diagnostic positivity of nucleic acid amplification tests for diagnosing pulmonary tuberculosis

Background:Tuberculosis(TB)remains a major threat to human health,and TB diagnostic methods remain unsatisfactory.Nucleic acid amplification tests(NAATs)show higher sensitivity compared with culture for the diagnosis of pulmonary TB(PTB).However,NAATs are expensive and cannot be easily implemented outside major medical centers.To improve the sensitivity of NAATs for PTB diagnosis,we investigated the predictive factors that might optimize NAAT utilization.Methods:A total of 1263 patients with suspected PTB were enrolled for evaluation.The sensitivity,specificity,and accuracy of methods including smear-microbiology,culture of Mtb and NAAT for Mycobacterium tuberculosis(Mtb)detection in sputum and bronchoalveolar lavage fluid samples were compared.Odds ratios and 95%confidence intervals were used to assess variables that might be associated with positive NAAT results for sputum and bronchoalveolar lavage fluid from patients with suspected PTB.Results:NAAT showed higher sensitivity for Mtb detection(61.1%)when compared with smear(9.0%)and Mtb culture(47.8%).We found that an elevated erythrocyte sedimentation rate,the presence of cavities,and positive interferon-𝛾release assay(IGRA)results were indicative of positive Mtb detection by NAAT.Moreover,individuals who had all three of these characteristics showed an 86%diagnostic positivity for PTB from Mtb detection by NAAT.Conclusions:Our study suggests that an elevated erythrocyte sedimentation rate,a positive IGRA result,and the presence of pulmonary cavities are helpful factors for predicting positive Mtb detection by NAAT.Patients with the three positive clinical markers should undergo NAAT for Mtb detection because they are the most likely individuals to be bacteriologically confirmed as having TB.

Assessing the effectiveness of the intervention measures of COVID-19 in China based on dynamical method

Normalized interventions were implemented in different cities in China to contain the outbreak of COVID-19 before December 2022.However,the differences in the intensity and timeliness of the implementations lead to differences in final size of the infections.Taking the outbreak of COVID-19 in three representative cities Xi'an,Zhengzhou and Yuzhou in January 2022,as examples,we develop a compartmental model to describe the spread of novel coronavirus and implementation of interventions to assess concretely the effectiveness of Chinese interventions and explore their impact on epidemic patterns.After applying reported human confirmed cases to verify the rationality of the model,we apply the model to speculate transmission trend and length of concealed period at the initial spread phase of the epidemic(they are estimated as 10.5,7.8,8.2 days,respectively),to estimate the range of basic reproduction number(2.9,0.7,1.6),and to define two indexes(transmission rate vt and controlled rate vc)to evaluate the overall effect of the interventions.It is shown that for Zhengzhou,vc is always more than v t with regular interventions,and Xi'an take 8 days to achieve vc>v t twice as long as Yuzhou,which can interpret the fact that the epidemic situation in Xi'an was more severe.By carrying out parameter values,it is concluded that in the early stage,strengthening the precision of close contact tracking and frequency of large-scale nucleic acid testing of non-quarantined population are the most effective on controlling the outbreaks and reducing final size.And,if the close contact tracking strategy is sufficiently implemented,at the late stage largescale nucleic acid testing of non-quarantined population is not essential.

The first local case of mpox caused by an imported case in the Chinese mainland

Monkeypox (mpox) is a zoonotic disease caused by the mpox virus (MPXV) that has been primarily limited to Central and West African nations since its discovery. The recent spread of the West African lineage of MPXV in historically unaffected countries has raised concerns for global public health. Despite a significant decrease in global mpox cases, there is still a risk of a global resurgence. This study reports the first local case of mpox caused by an imported case in the Chinese mainland. Polymerase chain reaction (PCR) diagnosed the two cases, and the viral genomes were obtained by next-generation sequencing. Genomic analysis revealed that the two strains shared an identical genome sequence and belonged to the B.1.3 branch of the West African lineage, which is the first local case of mpox caused by an imported case in the Chinese mainland, highlighting the potential threat of mpox in China and the immediate need for adequate surveillance measures.

Laboratory diagnosis of nonpolio enteroviruses:A review of the current literature

Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management.Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques,classical virus-isolation techniques are still used in refer-ence laboratories.Other techniques,such as antigen detection and serology,are becoming obsolete and rarely used in diagnosis.An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently,full-genome next-generation sequencing.The latter allows the typing of all EVs,better investigation of EV outbreaks,detection of coinfec-tion,and identification of severity markers in the EV genome.

Double-Stranded DNA Matrix for Photosensitization Switching

Photosensitization,originated from the activation of triplet states,is the basis of many photodynamic applications,but often competes with a series of nonradiative processes.Herein,we communicate a new function of double-stranded DNA(dsDNA)for label-free photosensitization switching.Up to∼70-fold singlet oxygen generation boosting was observed for SYBR Green I(SG)upon binding with dsDNA.Detailed photophysical and theoretical studies have revealed the role of dsDNA as a matrix,which could efficiently suppress the nonradiative transitions of SG.

Expert Consensus by Qilu Hospital of Shandong University on the diagnosis,management,and treatment of suspected COVID-19 cases(English version)

We devised a protocol to establish a standardized method of screening,diagnosing,and managing suspected cases of coronavirus disease(COVID-19)and to enhance the management of COVID-19 suspected cases.The protocol that included diagnostic criteria,preventive measures,and control measures against COVID-19 was developed based on new evidence regarding the epidemiological and clinical characteristics of COVID-19.A consensus document was subsequently formulated.The consensus focused on the clinical management of patients with suspected fever and reviewed the procedure for undergoing severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)nucleic acid testing.This consensus will contribute to the ongoing efforts worldwide for the prevention and control of COVID-19.

Serologic Response to SARS-CoV-2 in COVID-19 Patients with Different Severity

The immense patient number caused by coronavirus disease 2019(COVID-19)global pandemic brings the urge for more knowledge about its immunological features,including the profile of basic immune parameters.In this study,eighty-eight reported COVID-19 patients in Wuhan were recruited from January to February,2020,including 32 severe/critical cases and56 mild/moderate cases.Their mean age was 56.43 years(range 17–83)and gender ratio(male/female)was 43:45.We tested SARS-CoV-2 RNA with commercial kits,investigated the level of serologic IgM and IgG antibodies against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)using magnetic particle chemiluminescence immunoassays,and compared the results of serologic tests and nucleic acid test(NAT).Among 88 patients,95.45%were confirmed as positive by the combination of NAT and antibody test,which was significantly higher(P<0.001)than by single nucleic acid test(73.86%)or serologic test(65.91%).Then the correlation between temporal profile and the level of antibody response was analyzed.It showed that seroconversion started on day 5 after disease onset and IgG level was rose earlier than IgM.Comparison between patients with different disease severity suggested early seroconversion and high antibody titer were linked with less severe clinical symptoms.These results supported the combination of serologic testing and NAT in routine COVID-19 diagnosis and provided evidence on the temporal profile of antibody response in patients with different disease severity.