CHAPTER 5 Wallace H. Greene1, Marilyn A. Menegus2, and Allan L. Truant3 1 Pennsylvania State University College of Medicine, Hershey, PA, USA 2 University of Rochester School of Medicine, Rochester, NY, USA 3 Temple University Hospital and Lewis Katz School of Medicine, Philadelphia, PA, USA Diagnostic virology has experienced a great evolution over the past decade. When the first edition of this book was published, there was a limited menu of molecular assays for the detection and identification of viruses. Cell culture remained the “gold standard” for most viral assays. Due to the time required to grow most viruses and the technical expertise this method requires, rapid antigen detection tests (RADT) were being developed at a fast pace and becoming very popular as adjunct detection assays to cell culture. These included numerous monoclonal antibody reagents for immunofluorescence analyses, direct and indirect fluorescent antibody assays (DFA and IFA) for direct specimen testing as well as cell culture confirmation. In addition, many enzyme immunoassay (EIA) kits were being developed in both manual and automated formats. The diagnostic virology literature contained many studies evaluating these new assays, most often using cell culture as the gold standard. Over the past decade there has been a surge in the development of molecular-based diagnostic methods for all viral pathogens. These assays were relatively expensive in the beginning and required technical skills and expertise that were not available in many diagnostic laboratories. In the past several years as more economical, simpler, and automated molecular assays have become available, their use in diagnostic laboratories has become much more common, with many laboratories adopting molecular assays as their primary or exclusive testing method. As the improved analytical sensitivity of the molecular assays became apparent, they gradually replaced cell culture as the gold standard. Many rapid antigen detection assays were found to be significantly less sensitive when compared to molecular tests rather than cell culture. Currently, rapid antigen assays are primarily used as rapid screening tests where negative results are further evaluated by the more sensitive molecular assays. A rapid assay that can be performed in outpatient clinics, or in laboratories lacking adequate staff or resources to provide molecular testing 24 h a day, with immediate results often available before the patient leaves the clinic can impact treatment decisions. RADTs are simple to perform and give results in 15–30 min, but are limited to detecting influenza A and B, respiratory syncytial virus (RSV), and a few others [24, 30, 36]. DFA can be completed in 30–60 min and can detect influenza A and B, RSV, parainfluenza types 1, 2, and 3, adenovirus, and human metapneumovirus [36]. DFA techniques require a higher level of expertise to interpret and equipment and may not be available outside larger hospitals and reference laboratories. Because of this, RADTs are still used as the “front-line” diagnostic tests in many smaller laboratories, clinics and physician offices. However, the lack of sensitivity (and specificity in some instances) greatly limits the actual clinical utility of many of these tests [5, 7, 13, 14, 19, 24, 29]. Reported sensitivities for influenza viruses and RSV range from 10–85% and 50–98% respectively [24]. The broad range in sensitivity is due to numerous factors, including sample type, age of the patient, the time of specimen collection after the onset of symptoms, transport and storage of the specimen prior to testing, strain variations in the virus being tested for, and the particular assay used for testing and for comparison [31, 33]. In general, RADTs require 105 to 106 virus particles to give a positive result. Cell culture and molecular assays may detect as few as 10 virus particles [32]. Nasopharyngeal (NP) flocked swabs have been shown to provide improved specimens for the detection of respiratory viruses due to their ability to collect and release respiratory epithelial cells [1, 8, 35]. (Figure 5.1) It is important that specimens be collected within 3–5 days from the start of symptoms then be transported and stored refrigerated to improve test performance [31, 33]. In the past couple of years, new antigen detection assays have become available that utilize inexpensive instruments to evaluate test results. These assays have been demonstrated to have increased sensitivity when compared to the earlier assays [23, 28]. Figure 5.1 Copan flocked swab versus traditional swab. (Courtesy of Copan Diagnostics.) This chapter will review current literature evaluating the more common commercially available reagents and kits that have Food and Drug Administration (FDA) clearance as in vitro diagnostic (IVD) assays. Studies that compare the performance of these assays to molecular methods will be included when available. Numerous testing strategies are used in these assays, some are Clinical Laboratory Improvement Amendments (CLIA) waived and can be used as point-of-care (POC) tests. New pooled antibody reagents labeled with different fluorescent dyes, and improved specimen collection devices using flocked swabs have aided in improving the performance of the rapid methods. Molecular methods for detecting virus are reviewed in this manual in a separate chapter. Due to the continual development on new assays, reviewing current literature and references such as those provided by the American Society for Microbiology, the Centers for Disease Control and Prevention (CDC) and the FDA website (http://www.accessdata.fda.gov/scripts/cdrh/devicesatfda/index.cfm) for the most up-to-date listing of IVD assays is strongly recommended. The Participant Summaries from the College of American Pathologists (CAP) Proficiency Testing Surveys provide excellent comparisons of the most frequently used assays in a broad multilaboratory “real-world” setting. Simply reviewing an assay’s performance in literature published years ago or reading package inserts may not give an accurate indication of how the assay will perform in your laboratory. Although most new diagnostic assays are directed at molecular testing, new and improved assays for direct antigen detection are continually becoming available and reviewing the resources listed above is useful in selecting the “best” assay for your laboratory’s needs. Influenza viruses are enveloped, single-stranded RNA viruses. They belong to the family Orthomyxoviridae and they are divided into three different groups (A, B, and C) based on the antigenic reactivity of the group-specific antigens found on the nucleocapsid and matrix proteins. The influenza A group is further subtyped based on the antigenic reactivity of the hemagglutinin and neuraminidase proteins found on the viral envelope (e.g., H1N1). The influenza genome is segmented, with A and B having eight segments and C seven segments. These segments may reassort in vivo, which may result in the appearance of new strains of influenza. This may result in the development of an influenza pandemic, as occurred recently in 2009. In addition to reassorting the RNA segments, the genome is subject to frequent gene mutations, which may circumvent a patient’s immunity plus reduce the sensitivity of antibody-based assays [2]. Influenza virus outbreaks occur every year, usually during the winter months. The 2009 pandemic was unusual due to the outbreak occurring during the summer and fall. Initially, the columnar epithelial cells of the upper respiratory tract are infected, commonly producing illness characterized by fever, pharyngitis, and myalgias. More severe disease results from the virus spreading to the lower respiratory tract resulting in tracheobronchitis and pneumonia. Frequently, pneumonia is the result of a secondary bacterial infection. Other complications of influenza virus infection include encephalopathy, myocarditis, pericarditis, and myositis. In the distant past, serology and cell culture were the only laboratory methods for detecting an infection with influenza virus. Virus isolation may take 1–2 days up to 2 weeks [2]. To address this long delay in virus detection, numerous commercially developed kits utilizing monoclonal antibodies became available in the mid-1980s. These kits were based on either the immunofluorescence of directly stained cells observed microscopically, or numerous enzyme-linked immunosorbent assay (ELISA) methods to detect viral antigens. Cell culture, immunofluorescence and ELISA assays have remained the primary methods for detecting respiratory viruses, however, these assays are gradually being replaced by molecular assays due to the greater sensitivity and the shorter turn-around times (TAT) when compared to cell culture [20, 37]. The commercially available FDA-approved assays are listed in Table 5.1. The sensitivity and specificity of these assays vary greatly depending on the type of assay they are compared to. Those compared to cell culture had much better performance characteristics than when they were compared to molecular assays. When investigating an assay, review the most recent literature to determine how it performs against a molecular assay in detecting the current strains. Table 5.1 Rapid diagnostic tests for influenza virus * CLIA-waived test that can be used in any office setting. Figure 5.2 BinaxNOW Influenza A+B card. (Courtesy of Alere, Inc.) Figure 5.3 BD Directigen EZ Flu A+B kit. (Courtesy and © Becton, Dickinson and Company.) Figure 5.4 (a) QuickVue Influenza A+B Kit. (b) Quidel QuickVue Influenza A+B test strips. (Courtesy of Quidel Corporation.) Test performance is often affected by the appearance of new strains of influenza. Simple point mutations and gene rearrangements can greatly affect the performance of rapid detection assays that rely on specific antibodies to detect the virus. RADTs, DFA, and cell culture assays were all demonstrated to have suboptimal performance in detecting the new Flu A H1N1 strain [4, 15, 19, 29]. At the beginning of the 2009 pandemic, the CDC evaluated three widely used RADTs. Sensitivities ranged from 40 to 69% for the new H1N1 strain compared to 60 to 83% for the seasonal H1N1 strain. Following the pandemic, the CDC released a guidance report for using rapid diagnostic testing for influenza to highlight many of these issues (http://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm). In the report, 11 commercially available Rapid Influenza Diagnostic Tests, United States 2011–2012 were evaluated and summarized (Morbidity and Mortality Weekly Report (MMWR) November 2, 2012; Vol. 61, No. 43). The sensitivities for detecting 23 different strains of influenza A and B were very diverse. Assays with relatively good sensitivities for some strains had much lower sensitivities for detecting other strains. Regardless of the sensitivity and specificity issues with the RADTs, they remain the most commonly used assays due to their ease of use, rapid TAT and relative low cost compared to molecular methods. For many laboratories and clinics, lack of experienced staff and funds dictate these assays are the only tests available. Educating clinicians of the diagnostic performance issues of these tests is an important yet challenging responsibility of the laboratory director and staff. More recently, the FDA has approved three new RADT systems for detecting influenza A and B. These lateral flow assays are unique in that they use readers to evaluate the results, increasing sensitivity and reducing the subjectivity being interpreted by technologists. In addition, both systems provide printouts of the results that may be kept as a record of the patients test. This is a CLIA waived assay that detects and differentiates influenza A and B in approximately 10 min (Figure 5.5
Rapid Antigen Devices and Instruments for the Detection and Identification of Viruses
5.1 Influenza viruses
Procedure
(manufacturer/distributor)
Influenza virus types detected
Approved specimens
Test time (min)
3M Rapid Detection
(3M)
A and B
NP swab/aspirate
Nasal wash/aspirate
15
BD Veritor System for Rapid Detection of Flu A&B*
(Becton Dickinson)
A and B
NP swab/nasal swab
10
BinaxNOW Influenza A&B
(Alere)
(Figure 5.2)
A and B
NP swab
Nasal aspirate/swab/wash
15
BioSign Flu A+B*
(Princeton BioMedTech)
A and B
NP aspirate/swab/wash, nasal swab
15
Clearview Exact Influenza A&B
(Alere)
A and B
Nasal swab
15
Directigen EZ Flu A+B
Becton Dickinson)
(Figure 5.3)
A and B
NP aspirate/swab/wash
Throat swab
15
OSOM Influenza A&B
(Sekisui Diagnostics )
A and B
Nasal swab
10
QuickVue Influenza Test*
(Quidel)
A or B
Nasal
aspirate/swab/wash
10
QuickVue Influenza A+B*
(Quidel)
(Figure 5.4a and b)
A and B
NP swab
Nasal aspirate/swab/wash
10
SAS FluAlert A&B*
(SA Scientific)
A and B
NP swab
Nasal aspirate/swab/wash
15
SAS FluAlert A*
(SA Scientific)
A only
Nasal wash/aspirate
15
SAS FluAlert B
(SA Scientific)
B only
Nasal wash/aspirate
15
Sofia Influenza A+B
(Quidel)
A and B
NP aspirate/swab/wash
Nasal wash
15
TRU FLU
(Meridian Bioscience)
A and B
NP aspirate/swab
Nasal wash
15
XPECT Flu A&B
(Remel/ThermoFisher)
A and B
Nasal wash/swab
Throat swab
15
5.1.1 BD Veritor System for rapid detection of influenza A and B
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
