Virus Nucleic Acid Amplification Research Articles

An international review of the characteristics of viral nucleic acid-amplification testing (NAT) reveals a trend towards the use of smaller pool sizes and individual donation NAT.

Nucleic acid-amplification testing (NAT) is used for screening blood donations/donors for blood-borne viruses. We reviewed global viral NAT characteristics and NAT-yield confirmatory testing used by blood operators. NAT characteristics and NAT-yield confirmatory testing used during 2019 was surveyed internationally by the International Society of Blood Transfusion Working Party Transfusion-Transmitted Infectious Diseases. Reported characteristics are presented herein. NAT was mainly performed under government mandate. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) NAT was performed on all donors and donation types, while selective testing was reported for West Nile virus, hepatitis E virus (HEV), and Zika virus. Individual donation NAT was used for HIV, HCV and HBV by ~50% of responders, while HEV was screened in mini-pools by 83% of responders performing HEV NAT. Confirmatory testing for NAT-yield samples was generally performed by NAT on a sample from the same donation or by NAT and serology on samples from the same donation and a follow-up sample. In the last decade, there has been a trend towards use of smaller pool sizes or individual donation NAT. We captured characteristics of NAT internationally in 2019 and provide insights into confirmatory testing approaches used for NAT-yields, potentially benefitting blood operators seeking to implement NAT.

Nucleic acid amplification testing (NAT) impact on blood safety compared to Immunoassays in blood banks: A Review

Blood transfusions are fundamental to clinical procedures; however, many people worldwide cannot access safe blood. Blood product safety must be essential in each country’s national health policies. Several aspects of the blood donation process are carefully performed, including laboratory testing comprising blood type determination, antigen-antibody analyses, and nucleic acid amplification testing (NAT); however, NAT is not mandatory in all countries. The traditional screening method is based on antigen-antibody binding techniques, such as ELISA (enzyme-linked immunosorbent assay), with high sensitivity and specificity. Nevertheless, these methods have a seroconversion window period (WP), in which antigen-antibody testing cannot detect the pathogen and has not caused any symptoms yet. NAT is a sensitive molecular method based on viral nucleic acid amplification and detection. Moreover, its use in blood banks is increasing worldwide because it narrows the window period. For example, Huang et al. in 2017 reported the detection of 22 samples reactive only by nucleic acid testing for either HIV, HBV, or HCV compared with ELISA. The present article shows how blood safety has improved by implementing NAT as a routine method for viral nucleic acid detection, highlighting the importance of this technique as evidenced by the findings presented herein. Moreover, these results are highly significant, demonstrating the relevance of NAT and advocating for its application on a global scale in blood management protocols. This development could be particularly beneficial for regions with a high viral infection prevalence, including many countries. Keywords: Nucleic acid amplification, Immunoassay, viral infection, blood bank.

2721. Respiratory Virus Infections Following CAR T-cell Therapy: Risk Factors and Outcomes

Abstract Background Chimeric antigen receptor (CAR) T-cell therapy is a novel cancer therapy which functions by engineering patients’ own cells to target a malignancy. The preparative lymphodepleting chemotherapy and cell versus tumor effect create a novel, dynamic state of immunocompromise. Respiratory viruses have been found to be the predominant late infectious complication, but little is known about specific risk factors and outcomes of these infections. Methods We retrospectively identified adult patients who received CD19-directed CAR T-cell therapy at our center and followed them up to two years post-infusion (or until disease progression, death, or February 2022 — whichever came first). We undertook structured chart review using REDCap. Descriptive statistics were used where indicated. An adapted ordinal scale was used to assess the severity of respiratory virus infections (RVIs) (PMCID: PMC9278199). Results We found 73 patients who met search criteria. Of those, 23 patients (32%) developed an RVI, and their baseline characteristics are listed in Table 1. There were 38 episodes of RVI with an average of 1.65 episodes per affected patient. The median time from infusion to RVI was 180 days. The etiologic agent for the majority of RVIs was either unknown or rhino/enterovirus (Figure 1). The median ordinal score for RVI severity was 1, corresponding to ambulatory management for most (Figure 2). However, 24% of patients required a higher level of care. In patients where serial NAATs were performed, the duration of viral shedding was a median of 33 days (IQR 32 – 39). Immediately prior to infection the median IgG was 370 mg/dl (IQR 292 – 566), ANC was 1690 cells/μL (IQR 873 – 3165), and ALC was 480 cells/μL (IQR 285 – 703). 10 (43%) patients were treated with IVIG and 4 (17%) patients received antivirals. Many patients did not seek care for their RVIs or sought care outside of the Hematology/Oncology clinic. The RVI diagnosis relied on the impression of the treating physician at the time the patient presented with symptoms, or when the patient described past symptoms for which they did not present to the Hematology/Oncology clinic for care. Additionally, sometimes a full panel of respiratory viral nucleic acid amplification testing was done and negative (possibly false negative due to a collection issue or non-tested virus). Other times only COVID/FLU/RSV testing was done. Other times no respiratory virus panel was done. “Unknown” is a clinical syndrome of respiratory viral infection encompassing patients either without nucleic acid amplification testing being performed or with negative testing. The Ordinal Scale for EHR Use* was used to rank the severity of each instance of RVI. Possible rankings included 1, 3, 5, 7, 9, or 11. None of the RVIs met the criteria for 9 (organ support) or 11 (death). *Khodaverdi M, Price BS, Porterfield JZ, Bunnell HT, Vest MT, Anzalone AJ, Harper J, Kimble WD, Moradi H, Hendricks B, Santangelo SL, Hodder SL; N3C Consortium Collaborators. An ordinal severity scale for COVID-19 retrospective studies using Electronic Health Record data. JAMIA Open. 2022 Jul 9;5(3):ooac066. doi: 10.1093/jamiaopen/ooac066. PMID: 35911666; PMCID: PMC9278199. Conclusion There is a high burden of delayed RVI in patients who receive CAR T-cell therapy, and these infections can lead to severe illness requiring hospitalization. Clearance of virus was delayed, with duration of shedding lasting at least one month for most. Many CAR T-cell therapy patients had lower immunoglobulin G and lymphocyte counts prior to RVI. Further research is needed to understand the best treatment and prevention strategies for RVIs in this population. Disclosures All Authors: No reported disclosures

Evaluating direct amplification from viral transport medium for SARS-CoV-2 detection, strain typing, and angiotensin-converting enzyme genotyping and expression assays.

The aim of this study was to compare the performance of direct amplification of viral nucleic acid from transport medium to extracted nucleic acid for polymerase chain reaction (PCR), sequencing, and genotyping applications. XpressAmp lysate and extracted total nucleic acid from viral transport medium containing nasopharyngeal specimens were evaluated across different molecular applications to determine performance characteristics. SARS-CoV-2 quantitative PCR and angiotensin-converting enzyme (ACE) genotyping assays worked well with XpressAmp lysate, almost equal with or better than extracted nucleic acid in some specimens. However, XpressAmp completely failed to perform in next-generation sequencing for strain typing. Both protocols failed to detect ACE2 expression in viral transport medium. Direct amplification of viral nucleic acid from viral transport medium containing nasopharyngeal specimen works well for molecular assays with low thresholds of quality; however, it does have limitations with assays that require high quality nucleic acid for input. Use of the XpressAmp protocol significantly improves turnaround time and allows for easy ramp-up of PCR and genotyping assays.

Prediction of clinical outcomes in patients with coronavirus disease 2019 using high-sensitive troponin I and N-terminal pro-B-type natriuretic peptide

Abstract Background Several comorbidities, including cardiovascular diseases or myocardial injury, are reported to be associated with poor prognosis in patients with Coronavirus disease 2019 (COVID-19). However, detailed prognostic analysis of myocardial injury by various biomarkers in COVID-19 patients is limited. Purpose This study aims to explore the prognostic values of high-sensitive Troponin I (hsTnI) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) for COVID-19 patients using Japanese real-world data. Methods The COVID-MI study is a retrospective cohort study that enrolls consecutive laboratory-confirmed COVID-19 patients admitted to the hospital from July 2020 to September 2021. We collected clinical data, including cardiac biomarker values, by chart review. If the prespecified biomarkers in concern were not available, we measured them using the institutional serum blood bank, which enrolled patients prospectively from July 2020. Patients with available biomarkers were analyzed according to the values of hsTnI or NT-proBNP, using the clinically relevant thresholds (hsTnI: 5 ng/L and 99th percentile of the upper reference limit [99%ile URL], and NT-proBNP: 125 pg/mL and 900 pg/mL). The primary outcome measure was all-cause death. Secondary outcome measures included acute respiratory distress syndrome, myocardial infarction, myocarditis/pericarditis, venous thromboembolism, cerebral infarction, and bleeding events. Results We enrolled 917 patients with COVID-19 confirmed by viral nucleic acid amplification test. The mean age was 61 years, and 591 patients (64%) were men. On admission, the number of patients classified as severe or critical COVID-19 was 515 (56%) and 85 (8.7%), respectively. Among the 544 patients with hsTnI values, 365 (67%) patients had elevated hsTnI of ≥5 ng/L, and 134 patients (25%) had TnI of ≥99%ile URL. Besides, among 546 patients with NT-proBNP values, 295 patients (54%) had elevated NT-pro-BNP of ≥125 pg/mL, and 93 patients (17%) had NT-proBNP of ≥900 pg/mL. The median follow-up period was 31 days (interquartile range: 11–90 days). In cumulative incidence analysis, higher levels of hsTnI and NT-proBNP were associated with significantly higher mortality (hsTnI: <5 ng/L group; 8.8%, 5 ng/L to 99%ile URL group; 19%, and ≥99%ile URL group; 37%, P<0.001, and NT-proBNP: <125 pg/mL group; 7.8%, 125 to 900 pg/mL group; 21%, and ≥900 pg/mL group; 45%, P<0.001). The adjusted risk for all-cause death remained significant for each threshold of cardiac biomarkers (hsTnI ≥99%ile URL: hazard ratio [HR] 1.98, 95% confidence interval [CI] 1.11–3.54, P=0.02, and NT-proBNP ≥900 pg/mL: HR 3.60, 95% CI 1.86–6.98, P<0.001). Conclusion Elevation of hsTnI or NT-proBNP was associated with poor prognosis in the current relatively severely ill COVID-19 patients. Measuring hsTnI or NT-proBNP can be an attractive option for risk stratification and deciding appropriate management in patients with COVID-19. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Institutional Research Fund at Kobe City Medical Center General Hospital

Immunomolecular assay based on selective virion capture by spike antibody and viral nucleic acid amplification for detecting intact SARS-CoV-2 particles

BackgroundEffective therapeutics and vaccines for coronavirus disease 2019 (COVID-19) are currently lacking because of the mutation and immune escape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on the propagation characteristics of SARS-CoV-2, rapid and accurate detection of complete virions from clinical samples and the environment is critical for assessing infection risk and containing further COVID-19 outbreaks. However, currently applicable methods cannot achieve large-scale clinical application due to factors such as the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods. In this study, we developed an immuno molecular detection method combining capture of the viral spike glycoprotein with monoclonal antibodies and nucleic acid amplification via quantitative reverse transcription PCR to rapidly and accurately detect complete virions.ResultsAfter constructing a novel pseudovirus, screening for specific antibodies, and optimizing the detection parameters, the assay achieved a limit of detection of 9 × 102 transduction units/mL of viral titer with high confidence (~ 95%) and excellent stability against human serum and common virus/pseudovirus. The coefficients of variation were 1.0 ~ 2.0% for intra-assay and inter-assay analyses, respectively. Compared with reverse transcription-PCR, the immunomolecular method more accurately quantified complete virions. SARS-CoV-2/pseudovirus was more stable on plastic and paper compared with aluminum and copper in the detection of SARS-CoV-2 pseudovirus under different conditions. Complete virions were detected up to 96 h after they were applied to these surfaces (except for copper), although the titer of the virions was greatly reduced.ConclusionConvenient, inexpensive, and accurate complete virus detection can be applied to many fields, including monitoring the infectivity of convalescent and post-discharge patients and assessing high-risk environments (isolation rooms, operating rooms, patient living environments, and cold chain logistics). This method can also be used to detect intact virions, including Hepatitis B and C viruses, human immunodeficiency virus, influenza, and the partial pulmonary virus, which may further improve the accuracy of diagnoses and facilitate individualized and precise treatments.Graphical

CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles

The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials.Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs.In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.

Viral Hepatitis - The Road Traveled and the Journey Remaining.

Hepatitis is defined as inflammation of the liver and is commonly due to infection with The hepatotropic viruses – hepatitis A, B, C, D and E. Hepatitis carries one of the highest disease burdens globally and has caused significant morbidity and mortality among different patient populations. Clinical presentation varies from asymptomatic or acute flu-like illness to acute liver failure or chronic liver disease, characterized by jaundice, hepatomegaly and ascites among many other signs. Eventually, this can lead to fibrosis (cirrhosis) of the liver parenchyma and carries a risk of development into hepatocellular carcinoma. Hepatitis B and C are most notorious for causing liver cirrhosis; in 2019, an estimated 296 million people worldwide had chronic hepatitis B infection and 58 million are currently estimated to have chronic hepatitis C, with 1.5 million new infections of both hepatitis B and C, occurring annually. With the help of latest serological biomarkers and viral nucleic acid amplification tests, it has become rather simple to efficiently screen, diagnose and monitor patients with hepatitis, and to commence with appropriate antiviral treatment. More importantly, the development of vaccinations against some of these viruses has greatly helped to curb the infection rates. Whilst there has been exceptional progress over the years in the management of viral hepatitis, many hurdles still remain which must be addressed in order to proceed towards a hepatitis-free world. This review will shed light on the origin and discovery of the hepatitis viruses, the global epidemiology and clinical symptoms, diagnostic modalities, currently available treatment options, the importance of prevention, and the journey needed to move forward towards the eradication of its global disease burden.

Investigation on HTLV Infection among Voluntary Blood Donors in Wuzhou City

Objective: To understand the infection of HTLV among voluntary blood donors in Wuzhou City, and to provide reference for the national health administrative department to formulate blood safety screening strategies. Methods: The HTLV double-antigen sandwich ELISA reagent was used to screen the blood samples of unpaid blood donors, and the reactive samples in the initial screening were subjected to a double-well retest; Specimens that were still reactive in the retest were further confirmed by viral nucleic acid amplification test (PCR) and western blotting (WB). Results: A total of 9 of 20,222 unpaid blood donation samples were screened to be reactive, and the screening response rate was 0.04%; Two samples of HTLV-1 nucleic acid and western blotting (WB) were confirmed to be positive, and the other seven samples were negative; The confirmed positive rate was 0.01%. Conclusion: There was a certain positive rate of HTLV-1 serological screening among the non remunerated blood donors in Wuzhou City, and the confirmation test confirmed that there was a certain risk of HTLV infection; In order to further understand the HTLV infection of blood donors in this city, we should further increase the number of screening samples, so as to obtain more reliable and accurate data in this region, and provide data and reference for the health administration department to formulate HTLV screening strategies for blood donors.

Development and Efficacy of Lateral Flow Point-of-Care Testing Devices for Rapid and Mass COVID-19 Diagnosis by the Detections of SARS-CoV-2 Antigen and Anti-SARS-CoV-2 Antibodies.

The COVID-19 pandemic is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020–2021. COVID-19 is becoming one of the most fatal pandemics in history and brings a huge challenge to the global healthcare system. Opportune detection, confinement, and early treatment of infected cases present the first step in combating COVID-19. Diagnosis via viral nucleic acid amplification tests (NAATs) is frequently employed and considered the standard procedure. However, with an increasing urge for point-of-care tests, rapid and cheaper immunoassays are widely utilized, such as lateral flow immunoassay (LFIA), which can be used for rapid, early, and large-scale detection of SARS-CoV-2 infection. In this narrative review, the principle and technique of LFIA applied in COVID-19 antigen and antibody detection are introduced. The diagnostic sensitivity and specificity of the commercial LFIA tests are outlined and compared. Generally, LFIA antigen tests for SARS-CoV-2 are less sensitive than viral NAATs, the “gold standard” for clinical COVID-19 diagnosis. However, antigen tests can be used for rapid and mass testing in high-risk congregate housing to quickly identify people with COVID-19, implementing infection prevention and control measures, thus preventing transmission. LFIA anti-SARS-CoV-2 antibody tests, IgM and/or IgG, known as serology tests, are used for identification if a person has previously been exposed to the virus or vaccine immunization. Notably, advanced techniques, such as LFT-based CRISPR-Cas9 and surface-enhanced Raman spectroscopy (SERS), have added new dimensions to the COVID-19 diagnosis and are also discussed in this review.

High concentrations of hypochlorous acid-based disinfectant in the environment reduced the load of SARS-CoV-2 in nucleic acid amplification testing.

During the severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) pandemic, chlorine‐containing disinfectants have been widely used in nucleic acid amplification testing laboratories. Whether the use of disinfectants affect the results of viral nucleic acid amplification is unknown. We examined the impact of different hypochlorous acid (HOCl) concentrations on the quantitative results of SARS‐CoV‐2 by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR). We also explored the mechanisms and models of action of chlorine‐containing disinfectants that affected the detection of SARS‐CoV‐2. The results showed that different HOCl concentrations and different action times had an impact on the SARS‐CoV‐2 results. High concentrations of ambient HOCl have a greater impact than low concentrations, and this effect will increase with the extension of the action time and with the increase in ambient humidity. Compared with the enzymes or the extracted RNA required for RT‐PCR, the impact of HOCl on the SARS‐CoV‐2 detection is more likely to be caused by damage to primers and probes in the PCR system. The false negative result still existed after changing the ambient disinfectant to ethanol but not peracetic acid. The use of HOCl in the environment will have an unpredictable impact on the nucleic acid test results of SARS‐CoV‐2. In order to reduce the possibility of false negative of SARS‐CoV‐2 nucleic acid test and prevent the spread of epidemic disease, environmental disinfectants should be used at the beginning and end of the experiment rather than during the experimental operation.

Epidemiological and clinical characteristics of coronavirus disease (COVID-19) cases at a screening clinic during the early outbreak period: a single-centre study.

Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by Severe Acute Respiratory Corona Virus-2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019–2020 corona virus pandemic. SARS-CoV-2 is closely related to the original SARS-CoV. It is thought to have a zoonotic origin. The virus is primarily spread between people during close contact, often via small droplets produced by coughing, sneezing or talking. People may also become infected by touching a contaminated surface and then touching their face. COVID-19 patients currently remain the primary source of infection. An epidemiological survey indicated that the general population is susceptible to SARS-CoV-2. The spectrum of this disease ranges from mild to life-threatening. Fever is the most common symptom, although older people and those with comorbidities may experience fever later in the disease. Other common symptoms include cough, loss of appetite, fatigue, shortness of breath, sputum production, and muscle and joint pains. Symptoms such as nausea, vomiting and diarrhea have been observed in varying percentages. Some cases might progress promptly to acute respiratory distress syndrome (ARDS) and/or multiple organ function failure. Asymptomatic carriers and those in the incubation period may also be infectious.Aim. To determine the epidemiological and clinical characteristics of patients presenting with COVID-19 at the screening clinic of a tertiary care hospital in Peshawar, Pakistan.Methodology. In this descriptive study, we analysed data of patients presenting to a newly established Covid-19 screening clinic in Rehman Medical Institute. Anyone who reported with new onset fever and/or cough was tested for SARS-CoV-2 in the screening clinic. We documented and analysed demographic, epidemiological and clinical characteristics, which included age, sex, travel history, clinical features, comorbidities and laboratory data of patients confirmed by real-time reverse-transcription (RT)-PCR at Rehman Medical Institute, Peshawar, Pakistan from 15 March till 21 April 2020. Paired specimens of throat swabs and nasal swabs were obtained from 845 patients, ribonucleic acid (RNA) was extracted and tested for SARS-CoV-2 by the RT-PCR assay.Results. A total of 845 specimens were taken as described above. The positive rate for SARS-CoV-2 was about 14.3%. Male and older population had a significantly higher positive rate. Of the 121 patients infected with SARS-CoV-2, the mean age was 43.19 years (sd, 17.57) and the infections were more frequent among male gender accounting for 85 (70.25 %) patients. Common symptoms included fever (88 patients, 72 %), cough (72 patients, 59.5 %) and shortness of breath (69 patients, 57 %). Twenty-two (18 %) patients had recent travel history outside Pakistan in the previous 14 days, the majority of whom had returned back from Saudi Arabia.Conclusion. In this single-centre, prospective, descriptive study, fever, cough and shortness of breath were the most common symptoms. Old age (>50 years), chronic underlying comorbidities and travel history may be risk factors. Therefore, we concluded that viral nucleic acid amplification tests (NAAT) played an important role in identifying SARS-CoV-2 infection in a screening clinic, which helped with isolation and cohorting of these patients.

Zika virus RNA and IgM persistence in blood compartments and body fluids: a prospective observational study

Characterisation of the dynamics of Zika virus persistence following acute infection is needed to inform blood donor and diagnostic testing policies and understand the natural history of Zika virus infection. We aimed to characterise the natural history, persistence, and clinical outcomes of Zika virus infection through a prospective study in initially asymptomatic Zika virus RNA-positive blood donors. Zika virus-infected blood donors identified through Zika virus nucleic acid amplification test (NAAT) screening at three blood collection organisations in the USA were enrolled into a 1-year follow-up study, with blood and body fluid samples and detailed symptom data collected at up to seven visits. All samples were tested for Zika virus RNA by real-time PCR (rtPCR); follow-up plasma, whole blood, and urine were also tested by replicate NAAT. Plasma was tested for flavivirus-specific IgM and IgG by ELISA. Zika virus RNA persistence for each assay or sample type and plasma antibody persistence from estimated date of plasma NAAT-detectable infection were calculated from follow-up data using survival statistical methods. Between July 6, 2016 and March 7, 2017, we enrolled 53 participants. From the estimated date of plasma NAAT-detectable infection, Zika virus RNA was detectable in plasma for 9·9 days (95% CI 8·1-12·0), in red blood cells for 95·4 days (62·8-129·1), and in whole blood for 73·5 days (39·8-107·5). Replicate NAATs (one or more of eight replicates positive) extended detection of Zika virus RNA in plasma to 34·8 days (19·9-56·2) and in whole blood (at least one of two tests positive) to 104·8 days (76·7-129·9). Urine was rtPCR reactive up to 14·5 days (10·5-20·3) and saliva up to 26·4 days (19·7-38·7). Zika virus IgM persisted for 237·7 days (128·7-459·5) from estimated time since plasma NAAT-detectable infection. Zika virus RNA fell below detectable limits more rapidly in the saliva of participants with pre-existing dengue virus IgG than in those without. Of 25 donors identified pre-seroconversion with symptom data at the first or second study visit, 16 (64%) developed multiple Zika virus-related symptoms after asymptomatic index donations, compared with nine (36%) of 25 donors detected after seroconversion. Determination of viral marker persistence is enhanced by follow-up of blood donors who are pre-symptomatic or asymptomatic, Zika virus RNA-positive, and antibody negative. Zika virus RNA persists in red blood cells for several months following clearance from plasma and body fluids, and replicate, highly sensitive NAATs extend RNA detection in all compartments. Whole blood testing can extend detection of acute infection for diagnostics and monitoring of pregnant women, sexual partners, and travellers. National Heart, Lung, and Blood Institute, Biomedical Advanced Research and Development Authority.

Rapid and sensitive detection of viral nucleic acids using silicon microchips.

Clinical laboratory-based nucleic acid amplification tests (NAT) play an important role in diagnosing viral infections. However, laboratory infrastructure requirements and their failure to diagnose at the point-of-need (PON) limit their clinical utility in both resource-rich and -limited clinical settings. The development of fast and sensitive PON viral NAT may overcome these limitations. The scalability of silicon microchip manufacturing combined with advances in silicon microfluidics present an opportunity for development of rapid and sensitive PON NAT on silicon microchips. In the present study, we present rapid and sensitive NAT for a number of RNA and DNA viruses on the same silicon microchip platform. We first developed sensitive (4 copies per reaction) one-step RT-qPCR and qPCR assays detecting HCV, HIV, Zika, HPV 16, and HPV 18 on a benchtop real-time PCR instrument. A silicon microchip was designed with an etched 1.3 μL meandering microreactor, integrated aluminum heaters, thermal insulation trenches and microfluidic channels; this chip was used in all on-chip experiments. Melting curve analysis confirmed precise and localized heating of the microreactor. Following minimal optimization of reaction conditions, the bench-scale assays were successfully transferred to 1.3 μL silicon microreactors with reaction times of 25 min with no reduction in sensitivity, reproducibility, or reaction efficiencies. Taken together, these results demonstrate that rapid and sensitive detection of multiple viruses on the same silicon microchip platform is feasible. Further development of this technology, coupled with silicon microchip-based nucleic acid extraction solutions, could potentially shift viral nucleic acid detection and diagnosis from centralized clinical laboratories to the PON.

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

BackgroundNext-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools.MethodsIn this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform.ResultsTwenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25–30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2–3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample.ConclusionsThis work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.

Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus.

Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R2 = 0.99; secondary standard, Y = 1.04X - 0.26, R2 = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log10; copies/ml, -0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.

Metagenomic characterization of viral communities in corals: mining biological signal from methodological noise.

Reef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities and requires specialized methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this minireview, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double- and single-stranded DNA and RNA viruses. The identified viruses are dominated by double-stranded DNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral and other eukaryotes in close association.

Metagenomic characterisation of viral communities in corals: Mining biological signal from methodological noise

Summary Reef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet, knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities, and requires specialised methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this mini-review, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double- and single-stranded DNA and RNA viruses. The identified viruses are dominated by dsDNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral, and other eukaryotes in close association.

Hepatitis B virus nucleic acid amplification testing of Australian blood donors highlights the complexity of confirming occult hepatitis B virus infection

We present an analysis of the first 2 years of hepatitis B virus (HBV) nucleic acid testing (NAT) of the Australian donor population. Between July 5, 2010, and July 4, 2012, all blood donations were screened for HBV DNA and hepatitis B surface antigen (HBsAg). Donors who tested HBsAg negative but HBV NAT positive were assessed as occult hepatitis B infections (OBI) if reactive for antibodies to HBV core antigen (anti-HBc). Donors who were anti-HBc reactive but with nonrepeatable or nondiscriminated NAT results were assessed as HBV inconclusive pending follow-up testing. During the study period a total of 2,673,521 donations were screened for HBV. Forty-two chronic OBI infections (5.55/100,000 donors) were identified compared to eight acute serologic window period infections (1.06/100,000 donors). Of the 42 OBI cases, 23 (54.8%) were detected the first time they were screened for HBV DNA while 19 (45.2%) gave one or more HBV NAT-nonreactive results before detection. Of 68 donors initially assessed as HBV inconclusive and available for follow-up, 10 later confirmed as OBI cases while 51 were NAT nonreactive but remained anti-HBc reactive and OBI could not be excluded. This study demonstrated a substantially higher prevalence of OBI compared to acute serologic window period HBV infections in Australian blood donors. Follow-up testing of OBI cases indicates that HBV DNA is often only intermittently detectable in OBI, highlighting the importance of including anti-HBc to optimize the HBV testing algorithm.

Correlation analysis of HPV and cervical cancer

Objective To investigate the correlation between HPV positive rate and incidence rate of cervical cancer.Methods 438 women,who reveived cervical Pap smears and biopsy,were included in the present study.Human papilloma virus nucleic acid amplification genotyping assay kit was used to detect 21 kinds of HPV subtypes.Results There were 102 patients with cervical cancer (23.29％) and HPV-positive 94 patients (21.46％),which had no significance difference (x2 =0.421,P =0.517).There were 56 patients HPV16 positive (54.90％),19 patients HPV58-positive (18.63％),12 patients HPV18 positive (11.76％) and 8 patients HPV52 positive (7.84％),which were significantly higher than those of other types of HPV-positive rate(all P ＜ 0.05).The proportion of the patients with invasive cervical cancer in HPV i6,HPV58,HPV18,or HPV52-positive was significantly higher than that of intraepithelial lesions and carcinoma in situ (all P ＜ 0.05).Conclusion HPV16,HPV58,HPV18,and HPV52 have high correlation with uterine cancer. Key words: Uterine cervical neoplasms ; Papilloma virus, human ; Correlation