Comparative analysis of next-generation sequencing methods for whole-genome sequencing of seasonal influenza viruses.

Insights into the structure of RNPs from segmented negative-sense RNA viruses

Sex differences in HIV-1 viral load and progression to AIDS

The current epizootic of H5N1 highly pathogenic avian influenza (HPAI) in poultry is unprecedented in its virulence, extent and longevity, raising global concern that the virus could mutate into a form easily transmitted between humans and initiate an influenza pandemic. The ability to rapidly and accurately diagnose infections with novel influenza subtypes is crucial to minimizing morbidity and mortality in humans and reducing the potential for a pandemic. However, questions remain about how to ensure validity of the currently available diagnostics, optimize their availability and the potential offered by new technologies. To address these questions, during 19–20 February 2007, more than 40 scientists, clinicians, researchers and industry representatives from around the world came together for the first World Health Organization (WHO) Consultation on Diagnosis of H5N1 Avian Influenza Infections in Humans (summary available at http://www.who.int/csr/disease/avian_influenza/guidelines/diagnosis_consultation/en/index.html). The meeting was co-organized by the WHO Global Influenza Programme (GIP), the International Society for Influenza and other Respiratory Viruses (ISIRV) and the Foundation for Innovative New Diagnostics (FIND). This marked the first time public and private sectors met at length to discuss this important issue. An 'open forum' meeting style was adopted, and substantial time was allotted for discussion. Overall, the consultation addressed: The 'state of the art' for H5N1 diagnostics in humans. Considerations and gaps related to H5N1 diagnostic capacity. Collaborative ways forward and the roles of WHO, private industry and other stakeholders. This meeting summary will present the discussions and recommendations generally agreed by the consultation participants. Diagnostic tests (to identify influenza virus in clinical material, containing cells and secretions and tissues) are based either on growth of virus in culture or by direct detection of virus antigen or RNA. Virus may be amplified in embryonated chicken eggs or mammalian cell culture, and then subjected to further testing for identification. Serological techniques [e.g. haemagglutination inhibition (HI) or microneutralization (MN)] may also be used to identify the presence of antibody in the serum of exposed individuals, providing indirect evidence of infection. These basic techniques can be used for diagnosing infections both in humans and in animals. In general, antigenic or molecular screening is used to first identify influenza virus type (A or B). Then the specific subtype is identified based on either serological reactivity of two viral surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA), or on molecular characterization of the genes coding for these two proteins. There are 16 recognized HA and nine recognized NA subtypes of influenza A viruses. Wild waterfowl are considered the natural reservoir for influenza A viruses, and all HA and NA subtypes of influenza A have been identified in birds. Currently, only two influenza A subtypes (H1N1 and H3N2) are circulating or appearing in humans, causing recurring human seasonal influenza epidemics. Since the start of the current H5N1 HPAI epizootic in 2003, the virus has caused disease in poultry and wild birds in at least 59 countries in Asia, Africa, and Europe (http://www.oie.int). Although to date H5N1 remains an avian virus, it can cross the species barrier, and human infections with the avian H5N1 virus have now been confirmed in 12 countries.† † http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_07_25/en/index.html In addition to global concern about disease and deaths in humans, there is also concern that the virus will mutate into a form easily transmitted between humans, initiating a pandemic. The ongoing exposure of humans in countries experiencing disease in animals and ensuing global pandemic concern have highlighted some gaps and challenges in human influenza diagnostics. Appropriate clinical management, including timely treatment of human H5N1 cases‡ ‡ http://www.who.int/medicines/publications/WHO_PSM_PAR_2006.6.pdf , as well as plans for containing an emerging influenza pandemic,§ § http://www.who.int/csr/disease/avian_influenza/guidelines/draftprotocol/en/index.html rely on the ability to rapidly and accurately diagnose the virus in humans. Ensuring that effective influenza diagnostic systems are in place globally could be extremely cost effective. For example, it has been shown that although laboratory diagnosis represents a small percentage of medical centre costs, it leverages 60–70% of all critical decisions, e.g. admission, discharge and drug therapy.1Diagnosis of H5N1 in humans is not yet achievable in the vast majority of diagnostic laboratories. One challenge to rapid and accurate diagnosis is the continual evolution of influenza viruses.2 The eight RNA gene segments of influenza A viruses mutate at different rates.3 Specifically, the HA and NA genes, on which diagnostics depend, have high mutation rates compared to the other genes. This rapid evolution in the H5N1 viruses isolated since 1997 has resulted in the emergence of genetically and antigenically distinct lineages (http://www.WHOweblink.org). The circulating H5N1 viruses can currently be grouped into many different clades with four clades including viruses that have infected humans in the following countries:4 Clade 1 Thailand, Vietnam, Cambodia, China Clade 2.1 Indonesia Clade 2.2 China, Iraq, Azerbaijan, Turkey, Egypt, Nigeria, Djibouti Clade 2.3 China, Laos, Vietnam A second major challenge to global diagnostic capability is the availability of healthcare infrastructure to rapidly diagnose H5N1 infection at the initial point of care (POC), as the virus is circulating in many regions that lack existing diagnostic capacity, even for seasonal influenza. In practice, diagnosis of viral infections is conducted in several different environments, each having specific features, and therefore having somewhat different test requirements (Table 1). The third challenge is the uncertainty about the demand for tests for emerging influenza strains over the next months and years. Because the course of the H5N1 epizootic in animals and associated infections in humans cannot be predicted, it is possible that demand will decrease if the epizootic begins to be controlled in animals. It is also possible that demand will increase rapidly if there is suspected human-to-human transmission and the pandemic phase increases. Therefore, questions of stockpiling, reagent/kit shelf life, production times, etc. must be considered. The actual technical 'know how' for influenza diagnosis is fairly advanced, though this has not yet translated into significant innovation in rapid detection in field settings. Improvements are continually being made in both antigenic and molecular techniques for antigen and antibody detection, including development of increasingly simple-to-use tests (e.g. dipstick tests). Simpler techniques are required for routine diagnostic screening and sero-epidemiological studies in the field. Despite technological advances, however, the accuracy of H5N1 diagnoses relies heavily on the quality of the specimens collected and their preparation. If samples are not collected from patients early in the course of their infection and/or from sites where the viral load is high, or if samples are not handled, stored, and transported appropriately, false-negative tests may result irrespective of the validity of the test used. Approaches to collecting, preserving and shipping specimen for the diagnosis of avian influenza A (H5N1) have been summarized in a WHO document previously and are available at http://www.who.int/csr/resources/publications/surveillance/WHO_CDS_EPR_ARO_2006_1/en/. The basic diagnostic approaches, including benefits and constraints, are described below. Virus culture in eggs is traditionally regarded as the gold standard for amplifying and detecting avian influenza viruses. Cell culture can also be used for amplification with several lines (e.g. primary monkey kidney, MDCK, HeLa, MRC-5 or LLC-MK2) available, using tube culture, shell vial or multi-well plates. The cytopathic effect in cell culture to identify positives is not always distinctive; sensitivity of cell lines can vary for different strains, and there can be variation in the relative diagnostic yield from different techniques. Once cultured, virus can be easily detected and identified using techniques such as haemadsorption, antigen detection by immunofluorescence, other immunossays or haemagglutination (http://www.diagnosticdocweblink.org). Increasingly, polymerase chain reaction (PCR) is being used directly on original clinical samples, eliminating this virus isolation step for the purpose of diagnosis (see below). However, virus isolation as part of the diagnostic approach has the additional benefit of providing strains for further characterization, and vaccine development. The need for BSL-3 containment (BSL-3 enhanced or BSL-4 in some countries) for isolation and/or amplification of the HPAI H5N1 viruses constrains the use of virus isolation for diagnosis of this virus in many laboratories. The MN assay remains the gold standard for serological diagnosis of H5N1 infection in humans.4 Other methods include HI with use of horse red blood cells, complement fixation, single-radial haemolysis and enzyme immuno assay. Conventional HI tests that use turkey or chicken RBC have poor sensitivity for the detection of antibodies to avian influenza viruses including H5N1. However, the HI assay using horse red blood cells may be a suitable alternative for sero-diagnosis of some avian viruses (e.g. H5N1) but this may not apply to all avian influenza subtypes, highlighting the fact that significant strain/subtype differences exist. The international body of knowledge for serological diagnosis of H5 subtype infections is growing but information on other subtypes (e.g. H7) is limited. Although the methods for serological diagnosis differ in various laboratories, WHO does provide a set of standard criteria for serological diagnosis of human infection of avian influenza infection, i.e. a person meeting clinical definition of H5N1 case and one of the following:¶ ¶ http://www.who.int/csr/disease/avian_influenza/guidelines/case_definition2006_08_29/en/index.html Serological confirmation with appropriately timed paired sera. Greater than fourfold rise in neutralization antibody titre for H5N1. An MN antibody titre for H5N1 ≥1:80. A positive result using a different serological assay (e.g. A horse RBC HI titre of ≥1:160 or greater or H5-specific western blot positive result). There can be considerable variability in results on consecutive serological testing. Thus, negative and positive controls must always be included and samples/studies with low titre cut-off points should be interpreted with caution. Nonspecific reactivity of samples can be a problem. Modification techniques (e.g. serum adsorption) may be necessary to remove cross-reactive antibodies, especially when human infection with a novel avian subtype (such as H5) is reported. Nonspecific cross reactivity in patients 60–70 years of age can be seen when using the MN test.5 It remains unclear whether the cross-reactivity might be associated with some degree of protection in humans.6 Novel serological assays based on the use of engineered viruses with H5 antigen may allow 'neutralization' of H5N1 viruses to be carried out in a BSL-2 setting.7 As antibody response to H5N1 virus appears only in the second week of illness, serological tests cannot be used to detect early stages of influenza infection. Current serological tests are therefore most useful to identify mild or asymptomatic infections and epidemiologically assess populations at risk of exposure, such as family members and contacts of H5N1 case-patients, healthcare workers or co-workers and individuals exposed to infected domestic or wild birds. However, there is not much sero-epidemiological information being systematically collected globally. Follow-up investigations on specific outbreaks have yielded some data8, 9 but the extent of human exposure to H5N1 remains largely unknown. Immunofluorescence assays (both direct and indirect) can be used for detection of H5N1 antigen in samples, but rely heavily on specimen quality. While rapid, these methods are also dependent on the quality of fluorescence reagents and the expertise of the person interpreting the results of the tests and have inherently low sensitivity. Enzyme immunoassays in a micro-plate format are not widely used for human influenza diagnostics but the immuno-assay principle has been adapted for rapid antigen detection (rapid diagnostic tests) by flow-through or lateral flow devices. Sensitivity and specificity of antigenic tests depend not only on the test technique, but also on factors like type of specimen analysed, quality of specimen and timing of specimen collection (related to viral shedding).10 Based on published data, sensitivities for detection of human influenza H1N1 or H3N2 in rapid diagnostic tests are approximately 70–75% while specificities are approximately 90–99%. It should be noted that sensitivity of such methods for direct detection of H5N1 has been disappointing so far. The analytical sensitivity of currently available antigen detection test kits for influenza A remains too low for reliable use as POC tests for direct detection of H5N1 virus in clinical specimens. But if the sensitivity of such methods can be enhanced, they may become useful for H5N1 rapid testing.11 The use of molecular techniques to identify specific gene sequences provides a sensitive method for diagnosis. Furthermore, their use can potentially reveal the genetic sequence of the virus which is useful for molecular epidemiology and provides other important characteristics of the virus, including antiviral resistance status, occurrence of genetic reassortment or presence of key virulence mutations. While some of this information can be obtained by direct sequencing of PCR-amplified viral cDNA, more detailed molecular analysis typically requires prior virus amplification by culture. PCR is used widely now, with thermocyclers and other requisite equipment available in many national laboratories throughout affected regions although maintenance of the assays requires regular update of generic information. The multiple test steps (extraction, amplification, detection) and reagent preparation are highly sensitive to minor changes and requires experienced personal working within good quality systems. In particular, the amplification reaction of viral nucleic acids makes it susceptible to cross-contamination, unless stringent measures to avoid such contamination are in place.12 'Chip technology', which includes miniaturized approaches to genetic sequence detection may also allow simple, automated, rapid and economical PCR testing on a large scale, but automated systems are still expensive, and availability of a POC chip platform is at least 4 years away. Numerous sophisticated chip approaches to detection are available but all ultimately depend upon binding to specified virus sequences. As the viral mutation rate is high, it is important for all these approaches that constant surveillance of viral genetic sequence variations occurs, allowing adjustments to primers and probes. PCR can also be performed in a multiplex format for a panel of respiratory pathogens that is relevant to the differential diagnosis of AI and viral pneumonia (e.g. influenza B, parainfluenza 1, 2 and 3, respiratory syncytial virus, metapneumovirus, adenovirus, coronaviruses, mycoplasma and chlamydiae). A clinically and/or epidemiologically credible alternative diagnosis is useful in excluding AI. Closed tube real-time (RT) PCR systems that utilize fluorescent detectors are now widely available in a variety of formats including portable ones easily used in the field or for POC analysis. These show promise, but remain expensive for provincial or local laboratories and even though off the shelf reagents are available for detection of H5N1 strains, training of personnel and suitable laboratory environments are still crucial. Other molecular strategies are under development for rapid identification of influenza infections. For example, microarray and proteomic analysis of peripheral blood leucocytes or serum, respectively, may, in future, identify host response markers (e.g. gene response profiles, acute phase proteins, cytokines or other immune regulators) that may provide useful diagnostic signatures characteristic of groups of aetiological agents. During the consultation, a myriad of technical, political, economic and cultural issues were discussed. The following three general points emerged as being key to optimizing H5N1 diagnostics globally. In general, current technologies are adequate for the detection and characterization of diagnostic samples at the reference laboratory level, though advances in speed and miniaturization are occurring. There is however an acute need for field and POC tests that are relatively simple, sensitive and specific enough for use at referral hospitals and primary healthcare facilities. Such tests need to be to detect and between currently circulating strains of both avian influenza and seasonal influenza and enough to genetic changes in the For POC screening the sensitivity should be as high as possible to and tests should be The sensitivity of currently used rapid tests for H5N1 disease is from in the 1997 to in the and sensitivity does not always clinical sensitivity of diagnostic However, the poor clinical sensitivity of current POC tests for detecting H5N1 is not to a poor sensitivity for detecting H5N1 virus to human influenza but the poor analytical sensitivity for detecting influenza viral antigen in Furthermore, the of test also on the of the disease for test sensitivity and the positive for test will be and negative will be when influenza is rapid POC diagnostic with high sensitivity tests must be where it is for and shipping specimens to laboratories in the This may new techniques to be that into the challenges at many POC in affected In general, the ability to rapidly and accurately including human influenza has in though issues remain that the of many techniques. Appropriate collection may be including viral collection and and The specimens for virus detection have been summarized in the relevant WHO load in different clinical specimens in patients with H5N1 disease that are to and that respiratory specimens (e.g. are to be than respiratory specimens. There are with of specimens as well as A chain may be in and of and systems may not have been previously and the may not be may to to lack of and uncertainty of are to expensive when available, and may with a shelf and kits may or protection from which cannot be and may be of high (e.g. become or when may when have an even more shelf for may be In there is specific national to the reagents and There may be a lack of experienced lack of for training and a lack of training There may not be of the various assays and their use and (e.g. including of the different rapid detection While these may be and other emerging disease and public the need for diagnostic for influenza. of equipment is of an than is the lack of infrastructure to including for of the equipment and technical as well as international and may be in some and may not be possible in some laboratories, risk of of samples and risk of human human protection equipment may not be available, or may be used to training or (e.g. of protection level, of to and other equipment may be for the of new tests and reference strains for their quality are on a global level, from industry to assays and diagnostic As an international standard for H5N1 diagnostic test though has not yet been a relatively influenza gene (such as the infections with influenza A subtype can still be identified even in the of ongoing virus However, for identification of virus the reagents in diagnostic tests on either molecular sequences or must be continually to the currently circulating false-negative results can be and kits must therefore be easily to changes to allow detection of emerged strains and reagents should be continually identified by and be available WHO Influenza testing for both genes (e.g. to detect all influenza A strains with subtype specific tests the haemagglutinin of human and avian subtypes, one can avoid false-negative results of variations in the viral However, timely availability of viral and genetic sequence is a major to the and of reference reagents and Thus, ongoing surveillance of H5N1 viruses in animals and humans and global of are ultimately crucial to diagnostic test development and the validity of tests used. The Influenza influenza in countries and a of and laboratories by Currently, the H5N1 controls H5 H5 RNA and H5N1 has that one set and is not suitable for all and some diagnostic have specific Therefore, within Europe it is that different of reference reagents should be available, and primers and must be on each Currently, the WHO for Influenza at the for and in the provides domestic for its PCR influenza including of assays to laboratories and to other public laboratories, and of positive H5N1 to public laboratories in the at The and reagents are also available to international public laboratories. for human diagnostic test differ countries and from and to also vary different countries and some countries more than a to new diagnostic techniques. countries and/or for their although these may be time and expensive to especially for new technologies. International of requirements for could countries by providing both and industry a set of recognized should be based on risk and to public and be and International for including can be by following studies different laboratories. The of WHO International for avian influenza diagnosis should be to and subtype it may not be possible to international for H5N1 reagents and the of may need to be Serological test results are highly between laboratories. In to be to H5N1 results from different assays or laboratories, assays an standard may be more than an response can be and variation could be compared and Currently, the WHO is with including and on a virus neutralization to between laboratories H5 from show that the laboratories using and HI assays to test for H3N2 of the laboratories could not than results in In a of a quality for influenza virus detection and was by for Diagnostics in with the for Diagnostics of and some national reference laboratories. from within various sectors (e.g. reference laboratories, laboratories, and public were from that positives were Other challenges remain in detecting and of influenza virus, in influenza and influenza quality remain crucial to and document adequate and should be An important is the of positive H5N1 clinical samples for test from other for and should be and the various of samples, use of considered. International should be As the of avian influenza infections in humans it is important to that H5N1 remains a disease of animals. Although the for influenza testing are somewhat the and of diagnostic test techniques are for and human In the currently circulating strains in animals are still that will most as the virus has not yet adapted to humans. Therefore, the of of tests and as well as technical personnel in human and diagnostic laboratories should be As diagnosis of AI in animals is made on specimens where viral load is high and a only requires a animals from a to be confirmed as AI for relevant the sensitivity of POC tests is stringent that it is for diagnosis of human infection. surveillance and is to the risk factors for human infection with ongoing of the public sectors with the sectors and studies at the (e.g. birds in where H5N1 has poultry workers and poultry at should be national should not on laboratory but should in the collection of the surveillance in to strategies of and to the and of WHO and the public in to direct its and development for influenza diagnostics in the of the uncertainty of for H5 and influenza diagnostics. is considerable time and in new and diagnostic approaches and technologies for from use of to of methods to large or multiplex assay The public benefits when industry is in to ongoing and can by the required diagnostic analytical sensitivity and other clinical It should also be recognized that for of diagnostic clinical specimens of viral load to be in clinical are important in test and may in fact be to clinical specimens from patients which are a and and may be with multiple specimen should be to from such clinical specimens for test under the WHO International Health will be to rapid detection of human infections with influenza viruses in the now influenza reference laboratories, with of culture and influenza could diagnostic testing where national is The WHO Global Influenza Programme and its of Influenza and can a in this in by providing training and technical and global public benefit from of the following the of local and the for influenza testing at POC and in referral hospitals in regions and at risk development and of rapid, sensitive and specific POC screening tests for H5N1 infections in humans. collection of virus from animals and humans and their to reference laboratories in to be to currently circulating influenza strains and update tests the of reference laboratories in providing technical kits and reference should be between public and and available international of reagents and clinical are to from such specimens for test a global of avian influenza viruses in with development of the international of the of international for H5N1 diagnostic criteria and for all for new and including use of samples requirements a WHO working to the next steps in of reagents global of avian influenza viruses and for of H5N1 diagnostic tests

To the Editors HIV transmission is rare at viral loads (VL) up to several thousand copies.1 However, persons with low VL are at risk for sexually transmitted disease (STD) if they engage in unprotected sex, which may be related to beliefs about lower HIV infectiousness.2 Among persons with HIV, coinfection with another STD is common3 but has not been studied in a population-based group that is generally assumed unlikely to transmit HIV.4 We investigated STD prevalence among persons with different HIV VL levels, focusing on the subpopulation with low VL, by matching HIV and STD registry data. The New York City (NYC) HIV/AIDS surveillance registry contains data on New Yorkers diagnosed as having AIDS since 1981 and HIV infection or disease since 2000. Since 2005, it has received HIV-related laboratory results on persons living with HIV/AIDS (PLWHA) in NYC. The NYC STD surveillance registry contains data on New Yorkers diagnosed and reported to have any of 7 notifiable STD. A cross-registry match included 138,235 PLWHA as of March 30, 2011, excluding persons who died before January 1, 2000, and STD cases diagnosed January 1, 2000 to June 30, 2010. Viral load test dates and results were obtained from the HIV registry as of June 30, 2011. Among 69,418 PLWHA in 2009 who had 1 or more VL tests in 2009 and survived 31 days or longer after VL measurement (62.8% of all 2009 PLWHA), we calculated the proportion of those with a diagnosis of early syphilis, gonorrhea, chlamydia, or lymphogranuloma venereum within 31 days before or after the last VL in 2009 and characterized demographic and clinical characteristics and coinfection rates of PLWHA whose last VL was low (defined as 0–3499 copies/mL).1 Last VL was 0 to 3499 for 55,558 persons, 80.0% of PLWHA in the analysis, among whom last VL was less than 1000 copies/mL for 92.9% and less than 400 copies/mL for 87.3%. Of the 55,558, 478 (0.9%) had an STD diagnosis within 31 days of their low VL, with the highest prevalence among persons aged 20 to 29 years (3.1%) and 30 to 39 years (1.9%) and men who have sex with men (MSM; 1.8%) (Table 1). Early syphilis (43.1%) and chlamydia (36.3%) were the most frequent STD among PLWHA with low VL, followed by gonorrhea (19.7%) and lymphogranuloma venereum (0.8%). Most persons with STD and low VL were male (91.4%) and, specifically, MSM (73.8%). Sexually transmitted disease prevalence was higher among PLWHA with higher last VL (1.9% among 3500–9999; 2.0% among 10,000–99,999; and 2.2% among ≥100,000 [P trend <0.0001]).TABLE 1: STD Diagnosis Within 31 Days Among PLWHA With a Low (<3500) VL, by Demographics, Transmission Risk, and Clinical Characteristics, for All Persons and MSM, NYC 2009Overall, bacterial STD was rare (<1%) among PLWHA who, on the basis of their low VL, are assumed unlikely to transmit HIV. Still, these STD prevalence data suggest ongoing risk behavior (unprotected sex) among some persons with low VL and reinforce the value of STD screening and risk reduction services, even for persons with low HIV VL. Subgroups such as young adults and MSM had a higher STD prevalence that would be even higher if the highly prevalent genital herpes simplex virus were reportable also. Previous studies have found disproportionate STD rates among MSM PLWHA locally5,6 and nationally,7 and a recent mathematical model suggested that increased unprotected sex among MSM has diminished the population-level benefits of antiretroviral treatment for HIV prevention.8 Our findings reinforce the value of STD screening and risk reduction counseling among persons with low HIV VL. Ellen W. Wiewel, MHS Sarah L. Braunstein, PhD, MPH Preeti Pathela, DrPH, MPH Qiang Xia, MD, MPH Lucia V. Torian, PhD New York City Department of Health and Mental Hygiene New York, NY

Impact of admission viral load on respiratory outcomes in hospitalized SARS-CoV-2 infected patients with cancer and without cancer: A 2-, 4- and 6-months follow-up prospective study

ABSTRACTTools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.

BackgroundPatients hospitalized with coronavirus disease 2019 (COVID-19) frequently require mechanical ventilation and have high mortality rates. However, the impact of viral burden on these outcomes is unknown.MethodsWe conducted a retrospective cohort study of patients hospitalized with COVID-19 from 30 March 2020 to 30 April 2020 at 2 hospitals in New York City. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load was assessed using cycle threshold (Ct) values from a reverse transcription-polymerase chain reaction assay applied to nasopharyngeal swab samples. We compared characteristics and outcomes of patients with high, medium, and low admission viral loads and assessed whether viral load was independently associated with intubation and in-hospital mortality.ResultsWe evaluated 678 patients with COVID-19. Higher viral load was associated with increased age, comorbidities, smoking status, and recent chemotherapy. In-hospital mortality was 35.0% (Ct <25; n = 220), 17.6% (Ct 25–30; n = 216), and 6.2% (Ct >30; n = 242) with high, medium, and low viral loads, respectively (P < .001). The risk of intubation was also higher in patients with a high viral load (29.1%) compared with those with a medium (20.8%) or low viral load (14.9%; P < .001). High viral load was independently associated with mortality (adjusted odds ratio [aOR], 6.05; 95% confidence interval [CI], 2.92–12.52) and intubation (aOR, 2.73; 95% CI, 1.68–4.44).ConclusionsAdmission SARS-CoV-2 viral load among hospitalized patients with COVID-19 independently correlates with the risk of intubation and in-hospital mortality. Providing this information to clinicians could potentially be used to guide patient care.

Although segmented negative-sense RNA viruses (SNSRVs) have been frequently discovered in various fungi, most SNSRVs reported only the large segments. In this study, we investigated the diversity of the mycoviruses in the phytopathogenic fungus Fusarium asiaticum using the metatranscriptomic technique. We identified 17 fungal single-stranded RNA (ssRNA) viruses including nine viruses within Mitoviridae, one each in Narnaviridae, Botourmiaviridae, Hypoviridae, Fusariviridae, and Narliviridae, two in Mymonaviridae, and one trisegmented virus temporarily named Fusarium asiaticum mycobunyavirus 1 (FaMBV1). The FaMBV1 genome comprises three RNA segments, large (L), medium (M), and small (S) with 6,468, 2,639, and 1,420 nucleotides, respectively. These L, M, and S segments putatively encode the L protein, glycoprotein, and nucleocapsid, respectively. Phylogenetic analysis based on the L protein showed that FaMBV1 is phylogenetically clustered with Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2) and Sclerotinia sclerotiorum negative-stranded RNA virus 5 (SsNSRV5) but distantly related to the members of the family Phenuiviridae. FaMBV1 could be vertically transmitted by asexual spores with lower efficiency (16.7%, 2/42). Comparison between FaMBV1-free and -infected fungal strains revealed that FaMBV1 has little effect on hyphal growth, pathogenicity, and conidium production, and its M segment is dispensable for viral replication and lost during subculture and asexual conidiation. The M and S segments of AtNSRV2 and SsNSRV5 were found using bioinformatics methods, indicating that the two fungal NSRVs harbor trisegmented genomes. Our results provide a new example of the existence and evolution of the segmented negative-sense RNA viruses in fungi. IMPORTANCE Fungal segmented negative-sense RNA viruses (SNSRVs) have been frequently found. Only the large segment encoding RNA-dependent RNA polymerase (RdRp) has been reported in most fungal SNSRVs, except for a few fungal SNSRVs reported to encode nucleocapsids, nonstructural proteins, or movement proteins. Virome analysis of the Fusarium spp. that cause Fusarium head blight discovered a novel virus, Fusarium asiaticum mycobunyavirus 1 (FaMBV1), representing a novel lineage of the family Phenuiviridae. FaMBV1 harbors a trisegmented genome that putatively encodes RdRp, glycoproteins, and nucleocapsids. The putative glycoprotein was first described in fungal SNSRVs and shared homology with glycoprotein of animal phenuivirus but was dispensable for its replication in F. asiaticum. Two other trisegmented fungal SNSRVs that also encode glycoproteins were discovered, implying that three-segment bunyavirus infections may be common in fungi. These findings provide new insights into the ecology and evolution of SNSRVs, particularly those infecting fungi.

Objective: To investigate the threshold of cytomegalovirus (CMV) DNAemia for preemptive antiviral therapy in patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Viral load between 1×10(3) copies/ml and 5×10(3) copies/ml was defined as low viral load by real time Q-PCR. Clinical data and outcome were collected. Results: A total of 95 allo-HSCT recipients with low viral load from September 2014 to February 2015 were recruited in this study. The control group included 37 patients who received preemptive initial antiviral therapy. The other 58 patients didn't received antiviral treatment after positive viremia was confirmed. During monitoring, CMV viremia was cleared spontaneously in 17 patients of study group. Among 41 patients with continuous positive viremia in study group, 26 patients received antiviral therapy after second positivity including 18 with viral load >5×10(3) copies/ml, 2 with fever but still low viral load, 2 with hemorrhagic cystitis and low viral load, 4 with continuous low viral load. Eleven patients received antiviral therapy after the third positivity including 5 with viral load >5×10(3) copies/ml, 1 low viral load patient with fever and diarrhea, 5 with continuous low viral load. Only 4 patients received antiviral therapy after the fourth positivity of >5×10(3) copies/ml. In the study group, 35 cases received ganciclovir and 6 cases received foscarnet. The incidence of neutropenia did not differ significantly between study and control groups [minimum of neutrophil count: (1.63±0.41)×10(9)/L vs. (1.58±0.36)×10(9)/L]. The proportion of viral load greater than 5×10(3) copies/ml in the first week was comparable in two groups. Successful viral clearance rate was not statistically different (P=0.87). Of all 95 patients, no CMV diseases developed, neither did patient die of CMV infection. Conclusions: Spontaneous clearance of viremia occurs in some patients receiving allo-HSCT with low CMV viral load. Delayed antiviral treatment of continuous positive viremia does not prolong the whole treatment duration, neither contributes to the progression of CMV diseases.

BackgroundBackground. Understanding the viral load and potential infectivity of individuals in nursing homes (NH) with repeat positive SARS-CoV-2 tests ≥ 90 days after initial infection has important implications for safety related to transmission in this high-risk setting.MethodsMethods. We collected epidemiologic data by reviewing records of a convenience sample of NH residents and staff with respiratory specimens who had positive SARS-CoV-2 rRT-PCR test results from July 2020 through March 2021 and had a SARS-CoV-2 infection diagnosed ≥ 90 days prior. No fully vaccinated individuals were included. Each contributed one repeat positive specimen ≥ 90 days after initial, which was sent to CDC and retested using rRT-PCR. Specimens were assessed for replication-competent virus in cell culture if Cycle threshold (Ct) < 34 and sequenced if Ct < 30. Using Ct values as a proxy for viral RNA load, specimens were categorized as high (Ct < 30) or low (if Ct ≥ 30 or rRT-PCR negative at retesting). Continuous variables were compared using Wilcoxon signed-rank tests. Proportions were compared using Chi-squared or Fisher’s exact tests. ResultsResults. Of 64 unvaccinated individuals with specimens from 61 unique NHs, 14 (22%) were sent for culture and sequencing. Ten of 64 (16%) had a high viral RNA load, of which four (6%) were culture positive and none were known variants of interest or concern (Figure 1). Median days to repeat positive test result were 122 (Interquartile range (IQR): 103–229) and 201 (IQR: 139–254), respectively, for high versus low viral load specimens (p=0.13). More individuals with high viral loads (5/10, 50%) reported COVID-19 symptoms than with a low viral load (1/27, 4%, p=0.003). Most individuals (46/58, 79%) were tested following known or suspected exposures, with no significant differences between high and low viral load (p=0.18). ConclusionIn this study, nearly 1 in 6 NH residents and staff with repeat positive tests after 90 days demonstrated high viral RNA loads and viable virus, indicating possible infectivity. While individuals with high RNA viral load may be more likely to be symptomatic, distinguishing asymptomatic individuals who have high viral loads may be difficult with timing since initial infection, other test results, or exposure history alone. Disclosures John A. Jernigan, MD, MS, Nothing to disclose.

BackgroundGenotyping drug resistance testing methods for HIV-1 are continually evolving, as exemplified by Next Generation Sequencing (NGS) technology, which is gradually replacing Sanger sequencing (SS) in clinical diagnostics. A comparison...

Negative sense RNA viruses (NSV) include some of the most detrimental human pathogens, including the influenza, Ebola and measles viruses. NSV genomes consist of one or multiple single-stranded RNA molecules that are encapsidated into one or more ribonucleoprotein (RNP) complexes. These RNPs consist of viral RNA, a viral RNA polymerase, and many copies of the viral nucleoprotein (NP). Current evolutionary relationships within the NSV phylum are based on alignment of conserved RNA-directed RNA polymerase (RdRp) domain amino acid sequences. However, the RdRp domain-based phylogeny does not address whether NP, the other core protein in the NSV genome, evolved along the same trajectory or whether several RdRp-NP pairs evolved through convergent evolution in the segmented and non-segmented NSV genomes architectures. Addressing how NP and the RdRp domain evolved may help us better understand NSV diversity. Since NP sequences are too short to infer robust phylogenetic relationships, we here used experimentally-obtained and AlphaFold 2.0-predicted NP structures to probe whether evolutionary relationships can be estimated using NSV NP sequences. Following flexible structure alignments of modeled structures, we find that the structural homology of the NSV NPs reveals phylogenetic clusters that are consistent with RdRp-based clustering. In addition, we were able to assign viruses for which RdRp sequences are currently missing to phylogenetic clusters based on the available NP sequence. Both our RdRp-based and NP-based relationships deviate from the current NSV classification of the segmented Naedrevirales, which cluster with the other segmented NSVs in our analysis. Overall, our results suggest that the NSV RdRp and NP genes largely evolved along similar trajectories and that even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

Negative sense RNA viruses (NSV) include some of the most detrimental human pathogens, including the influenza, Ebola, and measles viruses. NSV genomes consist of one or multiple single-stranded RNA molecules that are encapsidated into one or more ribonucleoprotein (RNP) complexes. These RNPs consist of viral RNA, a viral RNA polymerase, and many copies of the viral nucleoprotein (NP). Current evolutionary relationships within the NSV phylum are based on the alignment of conserved RNA-dependent RNA polymerase (RdRp) domain amino acid sequences. However, the RdRp domain-based phylogeny does not address whether NP, the other core protein in the NSV genome, evolved along the same trajectory or whether several RdRp-NP pairs evolved through convergent evolution in the segmented and non-segmented NSV genome architectures. Addressing how NP and the RdRp domain evolved may help us better understand NSV diversity. Since NP sequences are too short to infer robust phylogenetic relationships, we here used experimentally obtained and AlphaFold 2.0-predicted NP structures to probe whether evolutionary relationships can be estimated using NSV NP sequences. Following flexible structure alignments of modeled structures, we find that the structural homology of the NSV NPs reveals phylogenetic clusters that are consistent with RdRp-based clustering. In addition, we were able to assign viruses for which RdRp sequences are currently missing to phylogenetic clusters based on the available NP sequence. Both our RdRp-based and NP-based relationships deviate from the current NSV classification of the segmented Naedrevirales, which cluster with the other segmented NSVs in our analysis. Overall, our results suggest that the NSV RdRp and NP genes largely evolved along similar trajectories and even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging-especially in the case of respiratory viruses-due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2,000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/µL of SARS-CoV-2 or 625.1 copies/µL of influenza A virus. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/µL, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.IMPORTANCETarget capture sequencing has been developed and applied for direct genome sequencing of viruses in clinical specimens to overcome the low detection sensitivity of metagenomic next-generation sequencing. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A, focusing on clinical specimens containing low copy numbers of viral genomes. Our results showed that the target capture sequencing yielded dramatically higher read counts than metagenomic sequencing for both viruses. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with other viruses, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.