Application, Verification, and Implementation of SARS-CoV-2 Serologic Assays with Emergency Use Authorization.

Interest continues to grow regarding the role of serologic assays for the detection of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The U.S. Food and Drug Administration (FDA) has granted emergency use authorization (EUA) status to many SARS-CoV-2 serologic assays. In this document, expert recommendations from clinical microbiologist members of the American Society for Microbiology (ASM) concerning detailed verification strategies for SARS-CoV-2 serologic assays with FDA EUA are provided, as are insights into assay limitations and reporting considerations for laboratories. Assessments concerning single-antibody and multiantibody isotype detection assays, which may provide either differentiated or nondifferentiated (i.e., total antibody) antibody class results, are addressed. Additional considerations prior to assay implementation are also discussed, including biosafety, quality control, and proficiency testing strategies. As the landscape of SARS-CoV-2 serologic testing is rapidly changing, this document provides updated guidance for laboratorians on application of these assays.

A large-scale study of respiratory virus infection over 2 years using the Luminex xTAGRVP assay.

Traditional methods of detecting and identifying respiratory viruses like cell culture and immunofluorescence are labor intensive, often slow, and are dependent on specimen viability. As a result, there has been a shift in laboratory practices from these methods to molecular-based techniques such as polymerase chain reaction, which can be faster, more sensitive, and less labor intensive than traditional methods. The Food and Drug Administration approved version of the Luminex xTAG respiratory viral panel (RVP) assay detects 12 respiratory viruses simultaneously. We evaluated the performance of the RVP assay, on over 8,000 nasopharyngeal specimens during a 2-year period. Approximately 70% of all specimens tested were positive for at least one respiratory virus. Influenza A (Inf A) was the most prevalent, followed by respiratory syncytial virus. The RVP assay also detected the newly emerging Inf A porcine H1N1 that started to circulate in 2008. However, it could not identify it to subtype level and required further confirmatory tests. This study shows that the RVP assay is an invaluable tool in monitoring seasonal outbreaks and pandemic events. It not only detects newly emerging influenza strains, but also allows the throughput of thousands of clinical specimens in a timely manner, reducing the turnaround time from weeks to days, when compared to cell culture.

Antimicrobial resistance surveillance in the AFHSC-GEIS network.

International infectious disease surveillance has been conducted by the United States (U.S.) Department of Defense (DoD) for many years and has been consolidated within the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) since 1998. This includes activities that monitor the presence of antimicrobial resistance among pathogens. AFHSC-GEIS partners work within DoD military treatment facilities and collaborate with host-nation civilian and military clinics, hospitals and university systems. The goals of these activities are to foster military force health protection and medical diplomacy. Surveillance activities include both community-acquired and health care-associated infections and have promoted the development of surveillance networks, centers of excellence and referral laboratories. Information technology applications have been utilized increasingly to aid in DoD-wide global surveillance for diseases significant to force health protection and global public health. This section documents the accomplishments and activities of the network through AFHSC-GEIS partners in 2009.

A growing global network's role in outbreak response: AFHSC-GEIS 2008-2009.

A cornerstone of effective disease surveillance programs comprises the early identification of infectious threats and the subsequent rapid response to prevent further spread. Effectively identifying, tracking and responding to these threats is often difficult and requires international cooperation due to the rapidity with which diseases cross national borders and spread throughout the global community as a result of travel and migration by humans and animals. From Oct.1, 2008 to Sept. 30, 2009, the United States Department of Defense's (DoD) Armed Forces Health Surveillance Center Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) identified 76 outbreaks in 53 countries. Emerging infectious disease outbreaks were identified by the global network and included a wide spectrum of support activities in collaboration with host country partners, several of which were in direct support of the World Health Organization's (WHO) International Health Regulations (IHR) (2005). The network also supported military forces around the world affected by the novel influenza A/H1N1 pandemic of 2009. With IHR (2005) as the guiding framework for action, the AFHSC-GEIS network of international partners and overseas research laboratories continues to develop into a far-reaching system for identifying, analyzing and responding to emerging disease threats.

A hantavirus pulmonary syndrome (HPS) DNA vaccine delivered using a spring-powered jet injector elicits a potent neutralizing antibody response in rabbits and nonhuman primates.

Sin Nombre virus (SNV) and Andes virus (ANDV) cause most of the hantavirus pulmonary syndrome (HPS) cases in North and South America, respectively. The chances of a patient surviving HPS are only two in three. Previously, we demonstrated that SNV and ANDV DNA vaccines encoding the virus envelope glycoproteins elicit high-titer neutralizing antibodies in laboratory animals, and (for ANDV) in nonhuman primates (NHPs). In those studies, the vaccines were delivered by gene gun or muscle electroporation. Here, we tested whether a combined SNV/ANDV DNA vaccine (HPS DNA vaccine) could be delivered effectively using a disposable syringe jet injection (DSJI) system (PharmaJet, Inc). PharmaJet intramuscular (IM) and intradermal (ID) needle-free devices are FDA 510(k)-cleared, simple to use, and do not require electricity or pressurized gas. First, we tested the SNV DNA vaccine delivered by PharmaJet IM or ID devices in rabbits and NHPs. Both IM and ID devices produced high-titer anti-SNV neutralizing antibody responses in rabbits and NHPs. However, the ID device required at least two vaccinations in NHP to detect neutralizing antibodies in most animals, whereas all animals vaccinated once with the IM device seroconverted. Because the IM device was more effective in NHP, the Stratis(®) (PharmaJet IM device) was selected for follow-up studies. We evaluated the HPS DNA vaccine delivered using Stratis(®) and found that it produced high-titer anti-SNV and anti-ANDV neutralizing antibodies in rabbits (n=8/group) as measured by a classic plaque reduction neutralization test and a new pseudovirion neutralization assay. We were interested in determining if the differences between DSJI delivery (e.g., high-velocity liquid penetration through tissue) and other methods of vaccine injection, such as needle/syringe, might result in a more immunogenic DNA vaccine. To accomplish this, we compared the HPS DNA vaccine delivered by DSJI versus needle/syringe in NHPs (n=8/group). We found that both the anti-SNV and anti-ANDV neutralizing antibody titers were significantly higher (p-value 0.0115) in the DSJI-vaccinated groups than the needle/syringe group. For example, the anti-SNV and anti-ANDV PRNT50 geometric mean titers (GMTs) were 1,974 and 349 in the DSJI-vaccinated group versus 87 and 42 in the needle/syringe group. These data demonstrate, for the first time, that a spring-powered DSJI device is capable of effectively delivering a DNA vaccine to NHPs. Whether this HPS DNA vaccine, or any DNA vaccine, delivered by spring-powered DSJI will elicit a strong immune response in humans, requires clinical trials.

Severe influenza outbreak in Western Ukraine in 2009--a molecular-epidemiological study.

INTRODUCTION: In the autumn of 2009 the authors participated in a humanitarian operation in Western Ukraine by undertaking an epidemiological investigation of an influenza-like-illness (ILI) in the L'viv Oblast region. Mobile biological survey teams took samples from civilian patients with severe acute respiratory distress syndrome, rapid transportation of the samples, and their molecular analysis in Poland to provide accurate results. OBJECTIVE: The aim of the study was the molecular and epidemiological analysis of the biological samples collected. MATERIAL AND METHODS: Real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), multiplex PCR techniques, traditional Sanger Sequencing and classical viral culture methods were used. RESULTS: Among the 124 influenza-like illness cases, ~50% (58) were positive for influenza A virus in WHO-CDC molecular assay, including subtyping. The specimens were further analyzed to confirm results and determine the genetic sequence. Phylogenetically, the nucleotide similarity of both the Ukraine specimens and reference A/California/7/2009 (pH1N1) was 99.2-99.3%. Oseltamivir resistance was not registered. HA1 region characterization showed an overall protein identity of 98.5-99.4%. CONCLUSIONS: An unexpected high contribution of influenza A was confirmed among ILI patients, as well as a very limited number of other detected viruses, indicate that the 2009 epidemic in western Ukraine was strongly related to novel influenza A/H1N1. The importance of swift sharing of information and reference laboratories networking in surveillance, as well as serving governments and international agencies in pursuing adequate actions, should be stressed.

Comparative analysis of eight Arthrobacter plasmids.

Despite the prevalence of Arthrobacter in the environment little is known about their plasmids, or the capacity of Arthrobacter plasmids to mediate horizontal gene transfer. In this study, we compared eight plasmids from five Arthrobacter strains in order to identify putative core maintenance genes for replication, segregation, and conjugation. Iteron like sequences were identified on some of the plasmids; however, no genes with obvious similarity to known replication sequences such as an origin of replication, or rep genes were identified. All eight plasmids contained a putative conjugation system. Genes with similarity to a relaxase, coupling protein, and various components of a type IV secretion system were identified on each plasmid; it appears that three different systems may be present. Putative parA partitioning genes were found in all of the plasmids. Each of the Arthrobacter strains examined contained a putative parB gene; however, of the three plasmids in Arthrobacter strain FB24 only one plasmid had a putative parB gene. Cluster analysis of many of the Arthrobacter genes suggested that they often formed branches within existing families of plasmid maintenance genes. Comparison of a concatenation of all the maintenance genes from each plasmid suggests that the eight Arthrobacter plasmids represent multiple evolutionary pathways.