6: Molecular Tests for the Identification of Viruses

Molecular Tests for the Identification of Viruses

Scott Duong1 and Christine C. Ginocchio2

1 Department of Pathology and Laboratory Medicine, Northwell Health System, Hofstra Northwell School of Medicine, Lake Success, NY, USA

2 Hofstra Northwell School of Medicine, Hempstead, NY, USA

6.1 Introduction

The transition of nucleic acid based technologies from basic research tools to everyday clinical diagnostic tests has greatly enhanced many aspects of laboratory medicine. However, few fields have changed more than infectious disease diagnostics, especially in the area of clinical virology. In particular, nucleic acid amplification tests (NAAT) can achieve levels of sensitivity for critically important diagnoses that often cannot be obtained using traditional antigen- or culture-based methods.

Careful assay design allows NAATs to be highly specific for a targeted virus, or conversely enables the detection of different strains within a family of viruses or not yet identified viral pathogens. Using NAATs, the laboratory can detect viruses that cannot be grown in traditional cell culture (e.g., hepatitis B virus [HBV]) or viruses simply too hazardous to routinely grow in the laboratory (e.g., variola). Importantly for patient care, NAATs can detect a virus within hours compared to days or weeks typically needed for traditional culture methods [130].

NAATs are used for a variety of applications in clinical virology. Qualitative assays are primarily used for diagnosis of common viral pathogens (e.g. herpes simplex virus [HSV]) as well as emerging viral pathogens such as the severe acute respiratory syndrome (SARS) coronavirus (CoV) in 2003, and for screening blood products (e.g., human immunodeficiency virus [HIV]) [60,92,199]. Quantitative viral assays, on the other hand, have become indispensable laboratory tests for predicting patient prognosis, monitoring response to antiviral therapy, and for the determination of antiviral resistance in patients with HIV, viral hepatitis, influenza, and organ transplantation [16,18,28,161]. NAATs are available as single analyte assays (monoplex) or as syndromic panels (multiplex) for viral pathogens causing respiratory tract, gastrointestinal tract, genital tract, and central nervous system infections. The relatively limited number of known clinically relevant viruses, and the capability to accurately quantify viruses in patient samples has contributed to the broad acceptance of NAATs as the new “gold standard” in clinical virology [60,70,213,216]. The increasing acceptance and utilization of NAATs not only in large university hospital and commercial laboratories, but also in small community hospital laboratories has been due in large part to the growing number and types of US Food and Drug Administration (US FDA) cleared/approved in vitro diagnostic (IVD) NAATs, increased automation, and decreased test complexity.

This chapter is divided into several sections: 6.26.6 will discuss commercially available molecular testing for viruses that cause respiratory, gastrointestinal, central nervous system, and genital tract disease, as well as post-transplant monitoring. The final section will consider requirements for assay implementation, verification/validation, and continued quality assurance.

6.2 Respiratory viral infections

6.2.1 Background

Viruses are the most common cause of respiratory tract infections (RTI) in children and adults [65,153,218]. RTIs are one of the major reasons for absenteeism at school and work as well as visits to clinics and emergency departments. RTIs are traditionally divided into upper respiratory infections (URI) and lower respiratory infections (LRI). URIs include nasopharyngitis, also known as the common cold, as well as sinusitis, pharyngitis, and tracheitis. LRIs involve the lungs and the respiratory tract below the trachea. Examples of LRI include bronchitis, bronchiolitis, and pneumonia. In infants and children, most cases of community-acquired pneumonia (CAP) are viral [111,196]. Viral infections decline during adulthood to about one-third of pneumonia cases, but still constitute the second most common cause of CAP in adults (after the bacterial pathogen Streptococcus pneumoniae) [64,196]. In older patients viral pneumonia again increases in frequency [134,215]. Children, the elderly, and immunocompromised patients may also experience more severe RTIs and shed virus for longer periods of time [35,68,95].

Respiratory viruses are diverse with different seasonal activity, targets, clinical presentations, severity, response to treatment, and predilection for mutation. The characteristics of RTI viruses are summarized in Table 6.1. The main causes of RTIs (see Table 6.1) are adenovirus (ADV), bocavirus (BoV), coronaviruses (CoV-229E, NL63, OC43, HKU-1), human metapneumovirus (HMPV), human rhinoviruses (HRV), influenza A (FluA), influenza B (FluB), parainfluenza viruses 1–4 (PIV-1, PIV-2, PIV-3, PIV-4), and respiratory syncytial virus (RSV). Mixed viral infections are present in about 5–20% of RTIs [19,59,60,85,171,207]. Furthermore, immunocompromised patients are vulnerable to more atypical respiratory infections with viruses such as HSV or cytomegalovirus (CMV) in addition to common respiratory virus [113].

Table 6.1 Respiratory virus summary


* Seasonal activity for the Northern Hemisphere. Viral activity patterns will differ in other regions of the world.

Determination of the cause of RTIs is based on a number of factors including patient’s signs and symptoms, age, medical history, immune status, sick contacts, time of the year and the related seasonality of viral infections, as well as awareness of the prevalence of circulating viruses in the community at the time of presentation. Though these factors can narrow the differential to certain pathogens, clinical symptoms can overlap significantly, making an accurate etiologic diagnosis challenging and often inaccurate [144,172]. Differentiation between a viral, bacterial, or fungal RTI is necessary to appropriately treat the patient. For example, an outpatient with a viral URI would not benefit from antibiotic therapy. Unfortunately, it is estimated that about half of ambulatory visits to clinicians for URIs result in an antibiotic prescription being given despite the lack of efficacy against viruses, the most common cause of URIs [21,86,104,170]. Furthermore, excessive and unnecessary antibiotic treatment increases selective pressure for antimicrobial drug resistance in healthcare institutions and the community [141,234]. Although currently FDA approved antiviral treatments are available only for influenza and RSV, antiviral compounds that target other common respiratory viruses are under development. Prompt identification (within 48 h of symptom onset) is crucial for guiding treatment [66,112]. Finally, viral strain typing can be important in therapy selection. For example, prior to the emergence of the 2009 influenza A H1N1 pandemic strain, the circulating seasonal influenza A H1N1 strain was resistant to the neuraminidase inhibitor oseltamivir, but was susceptible to zanamivir. In contrast, the subsequent 2009 influenza A H1N1 pandemic strain was sensitive to oseltamivir, so identification of the strain could be used as a proxy for susceptibility [66,110]. In addition to treatment, viral respiratory pathogens are often highly infectious and can cause outbreaks that are a threat to public health, best exemplified by the emergence and spread of SARS in 2003 [92]. Nosocomial spread of viral RTIs among patients in healthcare settings increases morbidity, mortality, and duration of hospitalization [206]. Therefore, early and accurate identification of the pathogen(s) can aid in appropriate isolation or cohorting of patients with similar infections thus avoiding further spread to both patients and staff.

Immunocompromised patients who develop respiratory viral infections often have more severe disease and shed virus for a longer period of time [113]. These patients can develop significant RTIs from viral pathogens not usually associated with RTIs in immunocompetent persons, including CMV, HSV, and varicella zoster virus (VZV) [225].

6.2.2 Detection of respiratory viruses

Conventional tests for the detection of respiratory viruses include rapid antigen direct tests (RADT), direct fluorescent antibody (DFA), and cell culture. A large variety of RADTs are available to detect FluA, FluB, and RSV.

In general, NAATs have better sensitivity than RADT’s, DFA, and viral cell culture and a faster time to results when compared to cell culture [104,145,159]. In addition, NAATs can detect viruses such as BoV, CoV, HMPV, and PIV-4 that do not grow in cell culture [6,83,103]. Studies have demonstrated that the sensitivities of detecting a virus in respiratory samples vary significantly based on test method. For example, during the 2009 influenza A H1N1 pandemic, NAAT identified viruses in 64% of the samples, compared to 17% for RADTs, 22% for DFA and 36% for cell culture [82].

The performances of all test methods are affected to varying degrees by multiple factors, including appropriate sample collection, storage, and transport. Influenza and other respiratory viruses have high levels of replication in the columnar cells of the nasopharynx (NP) [139]. Therefore, samples collected from the nasopharynx are superior to samples from the nares or the oropharynx alone [133]. Therefore, NP swabs, NP aspirates, and NP washes are recommended specimens. However, variations can occur depending on the particular virus. For example, a combination of both NP and oropharyngeal (OP) swabs placed in a single viral transport media are the recommended sample for avian influenza [49]. Lower respiratory tract samples were found to be positive in intensive care unit (ICU) patients with acute 2009 influenza A H1N1, yet NP swabs were negative [128]. Appropriate specimens for LRIs include sputum, bronchial alveolar lavage (BAL), and bronchial washings. Synthetic flocked swabs have been shown to increase recovery of epithelial cells and virus compared to traditional fiber swabs [106,228]. Swab samples should be placed into an appropriate transport media that will maintain nucleic acid stability during storage and transport to the laboratory. The laboratory should consult the manufacturer’s package insert for acceptable sample types and the variety of transport media that are approved for the molecular assays. Sample types and transport media other than those recommended by the manufacturer can be used as long as the laboratory performs an in-house validation to demonstrate that the alternative transport media have comparable performance and results.

In general, respiratory specimens for NAAT should be transported on wet ice and stored 2–8°C if testing is to be performed within 48 h. If testing is delayed beyond 48 h, specimens should be stored at ≤ −70°C to preserve specimen integrity [83]. Finally, the timing of sample collection also has a significant effect on the detection of respiratory pathogens [83,130]. Respiratory virus shedding is highest a few days after the onset of symptoms, so generally specimens should be collected within 3 days after onset of symptoms in adult patients and 5 days in pediatric patients. The larger time window for respiratory specimen collection from pediatric reflects the higher viral levels and longer shedding periods in children [97,106,128]. Since NAATs are not dependent on viable virus, only the presence of nucleic acids, the recovery of virus nucleic acids is not as susceptible to timing or transport delay as other traditional test methods.

6.2.3 Commercially available in vitro diagnostic tests

Currently there are over a dozen FDA-cleared IVD NAATs for the detection of a variety of respiratory viruses. The characteristics of each NAAT are listed in Table 6.2. Currently these tests can detect as few as a single viral target (e.g., RSV) to up to 18 viral targets in one panel. Assay formats vary from all-inclusive unit-based tests to assays that require multiple instruments and multiple steps. Acceptable sample types for each test are also listed in the table. Technical hands-on-time varies from minutes to hours and time to results range from 1 to 10 h. All assays contain an internal control (IC).

Table 6.2 Respiratory virus FDA-cleared/approved NAAT assays. Information in this table was current as of the time of writing


* NA: none available; ADV: adenovirus; CoV: coronavirus; FluA: all influenza A types; H1: seasonal H1N1; H3: seasonal H3N2; 2009-H1N1: 2009 pandemic influenza A (H1N1); H5N1: avian influenza A H5N1: Flu B: influenza B; hMPV: human metapneumovirus: EV: enterovirus; HRV: human rhinovirus; PIV: parainfluenza virus; RSV; respiratory syncytial virus.

NPS; nasopharyngeal swab; NPW: nasopharyngeal wash; NPA: nasopharyngeal aspirate; NS: nasal swab; TS: throat swab; NA: nasal aspirate; NW: nasal wash; NPS/TS: dual specimen consisting of nasopharyngeal swab and throat swab. These specimen types are specified in product package inserts cleared by the US Food and Drug Administration.

Many assays use standard formats consisting of nucleic acid extraction and amplification/detection on generic real-time platforms. Excluding NA extraction (if required), many of these assays can have results available within 75 min. Assay formats are conducive for low- to high-volume batch testing. Tests by Hologic/Gen-Probe/Prodesse (San Diego, CA) include ProFlu+ (FluA, FluB, RSV), ProFluFAST (FluA H1, FluAH3, FluA 2009-H1N1 typing), ProAdeno+, ProPara+ (PIV-1,-2,-3) and ProhMPV+ [129,138,203]. Nucleic acid (NA) extraction is performed using the NucliSENS easyMAG (bioMérieux, France), Magna Pure or Magna Pure LC (Roche Molecular Diagnostics, Pleasanton, CA) platforms and real-time amplification/detection performed on the SmartCycler II (Cepheid). Each assay uses fluorogenic probes to detect the specific viral targets [129,138,203]. Tests by Focus Diagnostics (Cyprus, CA) include the Simplexa influenza A H1N1 and the Simplexa influenza A/B and RSV tests, both of which require initial NA extraction using either the NucliSENS easyMag or Magna Pure LC [4]. Real-time amplification/detection is performed on the rapid 3M Integrated Cycler (3M, St. Paul, MN), using bifunctional fluorescent probe-primers together with corresponding reverse primers and internal control RNA. The Simplexa influenza A/B & RSV Direct Test is a version of the assay that is also performed on the 3M Integrated cycler, however, an upfront NA extraction step is not required as samples are loaded directly into direct amplification discs. The Qiagen artus Infl A/B RG RT-PCR Kit (Qiagen, Valencia, CA) requires a separate NA extraction followed by amplification and fluorescent detection of the FluA and FluB targets and an internal control, using the Rotor-gene Q MDx instrument (Qiagen) [77]. Quidel (San Diego, CA) currently has two respiratory virus assays, one that detects and differentiates FluA and FluB and one that detects HPMV. Both Quidel assays require NA extraction using the NucliSENS easyMAG and real-time detection on SmartCycler II or ABI 7500 Fast DX (Applied Biosystems, Foster City, CA). Additional selected platforms with unique characteristics, varying formats and levels of complexity will be described below.

The Luminex xTAG Respiratory Viral Panel Version 1 (xTAG RVP v1) (Luminex, Austin, TX) is approved for the detection of ADV, FluA (pre-2009 H1, H3 and 2009 H1), subtyping of pre-2009 FluA H1 andFluA H3, FluB, HMPV, HRV, PIV-1, PIV-2, PIV-3, and RSV [15,38,69,78,151,163,164]. The Conformité Européenne (CE) marked version of the assay also includes the CoVs (OC43, NL63, 229E, HKU-1), PIV-4, and enterovirus (EV, grouped with HRV). Purified viral nucleic acids are amplified by multiplex reverse transcription polymerase chain reaction (RT-PCR), followed by target specific primer extension. The 5’ ends of the primers contain a unique Tag sequence that allows the amplicon to bind to a complementary anti-Tag sequence on a target-specific color coded microbead. During the primer extension process, biotinylated deoxycytidine triphosphates (dCTP) are incorporated into the amplicons, which will in turn bind via streptavidin to a phycoerythrin reporter dye moiety. The Luminex 100/200 detection platforms (Figure 6.1) use one laser to identify the target-specific bead and a second laser to detect if the bead fluoresces, hence indicating the presence of target amplicons on the beads. The xTAG RVP Fast version of the assay reduces the number of steps to four and the amount of “hands-on” time; the turnaround time is improved from 10 to 6 h as compared to xTAG RVP v1 (204). However, there are fewer targets available in the Fast version of the assay (ADV, FluA generic, pre-2009 FluA H1, FluA H3, HMPV, HRV, RSV) and some targets have reduced sensitivity as compared to xTAG RVP v1 [14,165]. Assays that require the opening of samples with amplified products such as the xTAG, necessitate separation of testing areas and careful attention to procedures to prevent contamination. This assay is best suited for medium- to high-volume testing since the assay is performed using microwell plates and must be batched [143].

Photo displaying the Luminex 100/200 detection platforms.

Figure 6.1 The Luminex 100/200 detection platforms.

(Images provided by Luminex Corporation)

The GenMark Respiratory Virus Panel (GenMark Dx, Carlsbad, CA) assay consists of four separate steps, nucleic acid extraction, conventional end-point RT-PCR, amplicon denaturation and hybridization, and then detection using the eSensor platform (Figure 6.2). Single stranded DNA products are hybridized to ferrocene-labeled signal probes specific for the different viral targets and added to the detection cartridge. Cartridges are then loaded onto the eSensor XT-8 instrument for data acquisition and automated analysis based on electrochemical detection. The eSensor platform consists of a base module and up to three processing towers that contain eight cartridge slots each, allowing up to 24 samples to be analyzed simultaneously. Total hands-on-time is less than 60 min and over 3.5 h of walk-away time. The assay detects ADV (B, C, E) CoV (229E, HKU-1, OC43, NL63); FluA with subtype determination, FluB, HMPV, HRV, PIV-1, PIV-2, PIV-3, PIV-4, and RSV (A, B) [175].

Photo displaying the GenMark e‐sensor platform.

Figure 6.2 GenMark e-sensor platform.

(Image provided courtesy of GenMark Diagnostics)

The Verigene Respiratory Plus Test (Nanosphere, Northbrook, IL) is a closed modular system and consists of a nucleic acid extraction and amplification Processor SP module and a Verigene Reader unit. Extraction tray, amplification tray, tip-holder assembly, and test cartridges are loaded into the Processor SP module and the sample is added into the test cartridge. Within the processor nucleic acids are extracted, amplified, and amplicons are captured by sequence-specific oligonucleotides immobilized onto glass slides. Sequence-specific mediator oligonucleotides attached to gold particles hybridize to the immobilized amplicons. The glass slide array is then removed from the cartridge and loaded into the Verigene Reader, which detects the target gold particles through scattered light. Time from sample loading to results is approximately 3 h. The Respiratory Plus assay can identify generic FluA, pre-2009 FluA H1, FluA 2009 H1N1, FluB, RSV (A, B), and the FluA H275Y mutation that confers oseltamivir resistance [4].

The FilmArray Respiratory Panel (Biofire Diagnostics, Salt Lake City, UT) is a pouch based, highly multiplex, real-time nested PCR assay which runs on the FilmArray System. The assay can detect and identify ADV, CoVs (OC43, NL63, 229E, HKU-1), HMPV, FluA (H1, H3, 2009-H1N1), FluB, HRV/EV, PIV-1, PIV-2, PIV-3, PIV-4, and RSV (A, B) as well as the pathogens Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Bordetella pertussis. Sample and hydration solution are added to the reagent pouch and the pouch is loaded into the Film Array instrument (Figures 6.3 and 6.4). Inside the pouch, nucleic acids are extracted and undergo a two-step nested PCR; first a multiplex PCR then the amplified products are diluted and separated into multiple reaction chambers where secondary monoplex PCRs are performed. Target amplicons are fluorescently labeled by SYBR green and the identity is confirmed by melt-curve analysis. Currently, one pouch with one patient sample can be processed on a FilmArray instrument with time to results approximately 1 h [138,174,180,184].

Photo displaying the Biofire FilmArray pouch.

Figure 6.3 Biofire FilmArray pouch.

(Courtesy of Biofire Diagnostics, Inc.)

Photo displaying the Biofire FilmArray instrument.

Figure 6.4 Biofire FilmArray instrument.

(Courtesy of Biofire Diagnostics, Inc.)

The Xpert Flu (Cepheid, Sunnyvale, CA) is a cartridge based, random access, totally integrated multiplex, real time RT-PCR assay which runs on the GeneXpert system (Figure 6.5). The Xpert Flu assay detects generic FluA, 2009 FluA, H1N1, and FluB. The patient sample and an elution reagent are added to the disposable cartridge, which is then manually loaded into a single bay of the GeneXpert system. The GeneXpert Systems have different capacity models that include slots for 1, 2, 4, and 16 cartridges. Alternatively, the expandable walk-away Infinity system can load and test up to 80 cartridges simultaneously. The Xpert Flu test takes about 75 min to complete [117,160,197].

Photo displaying the Xpert Flu instrument.

Figure 6.5 Xpert Flu.

(Courtesy of Cepheid.)

The Liat is a cartridge based, multiplex, real time PCR system manufactured by Iquum (Marlborough, MA). The cartridge consists of a tube containing all assay reagents needed for extraction, amplification, and fluorescent detection prepacked in tube segments, separated by removable seals. The analyzer is small, lightweight, and portable (battery or AC powered). The Liat Influenza A/B assay is FDA cleared and can detect FluA and FluB in just 20 min.

The PLEX-ID Flu (Abbott, Chicago, IL) assay utilizes RT-PCR to amplify target nucleic acids for FluA (H1, H3, 2009-H1N1) and FluB [45]. Following amplification the samples undergo post-PCR desalting, purification, and then analysis by using electrospray ionization mass spectrometry (ESI/MS). Spectral analysis determines the nucleotide base composition of the single-stranded oligonucleotides complementary to the initial target and compares the spectra to a database of viral targets. The PLEX-ID Flu is FDA-cleared.

6.2.4 Laboratory developed, investigational, and research-use only tests

Many new assays and platforms are currently in development for the detection of respiratory viruses. A few selected assays are described below and in Table 6.3. The Icubate 2.0 (Icubate, Huntsville, AL) is a modular cartridge-based system. The processor unit prepares the sample, performs ARM (amplicon rescued multiplex)-PCR, hybridization, and washing procedures. The processor can run one to foiur cassettes in a random access fashion and up to 12 units can be linked. The cassette contains all the reagents necessary to perform multiplex amplification and detection. Cassettes are transferred to a reader containing a high-speed rotating plate, laser, and photomultiplier tube, which allow the rapid acquisition of data from each cassette. The proposed targets in the assay will include ADV-3, ADV-4, ADV-7, BoV, CoV (NL63, 229E, OC43 HKU-1), coxsackievirus (A, B), echovirus, FluA, 2009 FluA H1N1, H274Y mutation, FluA H3, FluB, HMPV, HRV, PIV-1, PIV-2, PIV-3, PIV-4, and RSV (A, B). An influenza typing assay will differentiate FluA, FluB, pré-2009 FluA H1, 2009 FluA H1N1, FluA H3, FluA H5, FluA N1, and FluA N2.

Table 6.3 Respiratory virus non-FDA-cleared/approved NAAT assays. Information in this table was current as of the time of writing


* NA: none available; ADV: adenovirus; CoV: coronavirus; FluA: all influenza A types; H1: seasonal H1N1; H3: seasonal H3N2; 2009-H1N1: 2009 pandemic influenza A (H1N1); H5N1: avian influenza A H5N1: Flu B: influenza B; hMPV: human metapneumovirus: EV: enterovirus; HRV: human rhinovirus; PIV: parainfluenza virus; RSV; respiratory syncytial virus.

NPS; nasopharyngeal swab; NPW: nasopharyngeal wash; NPA: nasopharyngeal aspirate; NS: nasal swab; TS: throat swab; NA: nasal aspirate; NW: nasal wash; NPS/TS: dual specimen consisting of nasopharyngeal swab and throat swab.

The Qiagen Resplex II (Qiagen, Valencia, CA) assay detects ADV (B, E), BoV, CoV (229E, NL63, 229E, HKU-1), coxsackievirus/echovirus, FluA, FluB, HMPV, HRV, PIV-1, PIV-2, PIV-3, PIV-4, and RSV (A, B) [15,78,100,132,146,229]. After a separate NA extraction step, amplification is performed using TEM (target-enriched multiplex)-PCR. For each viral target, two sets of nested gene-specific primers at very low concentration are used to enrich for the target in the first few cycles. The inner nested primer has a Tag sequence recognized by universal super primers (biotin labeled), at a concentration for exponential amplification. Allele-specific detection probes are attached to the Luminex bead and detection is performed using the Luminex 100/200 instruments, as previously described [132,151].

The Infiniti RVP System (Autogenomics, Vista, CA) (Figure 6.6) requires a separate NA extraction and then uses RT-PCR to amplify ADV (A, B, C, E), CoV (229E, NL63, 229E, HKU-1), EV (A, B, C, D), FluA, FluA 2009-H1N1, FluB, HMPV (A, B), HRV (A, B), PIV-1, PIV-2, PIV-3, PIV-4, and RSV (A, B). Detection of the target is performed using the INFINITI analyzer, which measures fluorescence signals of the labeled DNA target hybridized to BioFilmChip microarrays via molecular zip codes (attached to array) and anti-zip codes (attached to amplified target) [7,187].

Photo displaying the Infiniti RVP system.

Figure 6.6 Infiniti RVP System.

(Reproduced by permission of Autogenomics, Inc.)

The next generation Multicode-PLx by Luminex (formerly Eragen) uses standard and asymmetric PCR, and high-resolution melt. Each module consists of two independent bays that can run up to six individual tests per bay. The technology is based upon the unique MultiCode synthetic base pairs, isoC and isoG, with properties that enable site-specific incorporation of the isobases during amplification. MultiCode-RTx primers include a fluorescent reporter in close proximity to an isoC base on the 5′ end. During the amplification reaction, the MultiCode isoG base that is covalently attached to a quencher present in the reaction mix is incorporated opposite to the isoC. This site-specific interaction of the reporter-labeled isoC and the quencher-labeled isoG results in a decrease in fluorescence. Since the reporter quenching is reversible, melting-curve analysis can be used to confirm the presence of target. The panel detects FluA, FluB, ADV, PIV-1, PIV-2, PIV-3, PIV-4, RSV (A,B), HMPV, HRV, and CoV (229E, NL63, 229E, HKU-1) [15].

The PLEX-ID Respiratory Virus Assay (Abbott, Chicago, IL) [45] was formerly the Ibis T5000 Respiratory Virus Surveillance II kit by Ibis Biosciences, now a part of Abbott. The assay utilizes RT-PCR to amplify target nucleic acids for ADV (A-H), BoV, CoV, FluA, FluB, HMPV, PIV-1, PIV-2, PIV-3, PIV-4, and RSV in a microwell plate [217]. Following amplification the samples undergo post-PCR desalting, purification, and then analysis by using ESI/MS on the Ibis T5000 universal biosensor platform. Spectral analysis determines the nucleotide base composition of the single-stranded oligonucleotides complementary to the initial target and compares the spectra to a database of pathogens including viruses. In addition, the relative concentration of the target or targets present can be obtained by comparing the peak heights with the internal PCR calibration internal mass standard present in every well [39,53,68].

6.3 Enteric viruses

6.3.1 Background

The most common causes of viral gastroenteritis worldwide include rotaviruses, noroviruses, sapoviruses, astroviruses, and the enteric ADVs 40 and 41 and are summarized in Table 6.4 [24,127,232]. Rotaviruses are the most common global causes of pediatric gastroenteritis and responsible for the deaths of over 500,000 children < 5 years old annually [232,236]. Although rotavirus disease tends to be longer in duration and more severe than the other enteric viruses, all are responsible for significant morbidity and mortality. Noroviruses are genetically diverse and are associated with foodborne infections, large outbreaks (e.g., cruise ships) causing moderate to severe diarrhea [155]. Sapoviruses, first identified in 1977 in a cluster of gastroenteritis in Sapporo, Japan, primarily cause outbreaks of diarrhea in children, but have also been associated with institutional outbreaks [155,169]. Astroviruses primarily affect children < 2 years old and the elderly and generally cause milder and more limited disease than rotaviruses or noroviruses [232]. Astroviruses can be detected in asymptomatic children [147]. Astroviruses are prevalent in the winter months in temperate regions and during the rainy season in tropical regions [147]. Adenoviruses can also cause gastroenteritis especially in children. Serotypes 40 and 41 account for a majority of ADV associated gastroenteritis in children and may be responsible for anywhere from 3 to 20% of pediatric gastroenteritis in the United States [127]. Other viruses associated with sporadic gastroenteritis are BoV, CoV, toroviruses, and CMV in immunocompromised patients [6,24,109,127].

Table 6.4 Enteric virus summary

Virus Family Genome Age Clinical significance
Adenovirus 40 and 41 Adenoviridae DNA, double stranded Children Usually mild diarrhea compared to other enteric viruses, but have been associated with large outbreaks and can cause severe disease in immune-suppressed patients
Astrovirus Astroviridae RNA, single stranded Children and elderly Generally more mild than rotavirus or norovirus. Associated with outbreaks in nursing homes
Cytomegalovirus Herpesviridae DNA, double stranded Immunocompromised Gastroenteritis in immunocompromised patients such as HIV and transplant patients
Norovirus Caliciviridae RNA, single stranded All ages Nausea and diarrhea. Associated with food borne outbreaks and clusters in schools and long-term care facilities
Rotavirus Reoviridae RNA, segmented double stranded Infants Associated with outbreaks of severe diarrhea in infants and children worldwide. Estimated to cause > 500,000 deaths among children < 5 years old annual worldwide
Sapovirus Caliciviridae RNA, single stranded Children and elderly Gastroenteritis primarily in children, but recently found to cause outbreaks in nursing homes

References: 24, 87, 109, 127, 147, 155, 167, 169, 232, 236.

Transmission is usually through a fecal–oral route including food and water that have been contaminated during production, transport, or preparation [24]. Outbreaks of viral gastroenteritis can occur at restaurants, schools, day care centers, nursing homes, and other long-term care facilities [24,167]. Viral gastroenteritis is also a major concern in regions with inadequate potable water and sanitation, such as sites of natural disasters, refugee camps, and other areas of extreme need. Clinical presentation can include fever, nausea, vomiting, abdominal pain, and diarrhea that can range from mild self-limited diarrhea to life threatening dehydration, particularly in infants and young children. Immunocompromised patients may experience more severe disease and can shed viruses in their stool for an extended period of time. Although viral gastroenteritis is generally managed with oral or intravenous hydration fluid to replace electrolytes and water, laboratory testing can still have significant clinical utility. Confirmation of an enteric virus can prevent unnecessary antibiotic treatment and reduce testing for other causes of diarrhea. Pathogen identification in an outbreak is important for determining the source, halting the spread of disease, and preventing future outbreaks [24].

6.3.2 Molecular assays for enteric viruses

In general, the enteric viruses are difficult to grow in cell culture, therefore diagnostic methods have been limited to either rapid antigen tests or laboratory developed NAATs [155,232]. Consequently, the cause of most cases of viral enteritis is never established. Recently, highly multiplexed commercial NAATs have been developed to detect the enteric viruses, in addition to the common bacterial and parasitic pathogens. One assay, the Luminex xTAG GGP panel, is FDA cleared (see details below) but there are also a number of assays in clinical trials or reagents available as analyte-specific reagents (ASRs). Ideally a comprehensive enteric panel consisting of viral, bacterial, and parasitic targets would be able to replace the need for multiple tests such as antigen detection, stool culture, and microscopic examination. In addition, an acceptable test would have to amply address challenging matrix issues in stool, in particular the presence of amplification inhibitors and the instability of nucleic acid targets, which can lead to false negative results. Robust extraction procedures and the use of an internal control that goes through the entire testing process are necessary to guarantee assay performance.

The xTAG gastrointestinal pathogen panel (xTAG GPP, Luminex) is a highly multiplexed PCR assay that is FDA cleared for the detection of rotavirus A and norovirus GI/GII, in addition to Campylobacter, Clostridium difficile toxin A/B, enterotoxigenic Escherichia coli (ETEC) LT/ST, E. coli O157, Shiga-like toxin producing E. coli STEC (stx 1/stx 2), Salmonella, Shigella, Giardia, and Cryptosporidium from fresh stool [135,136,224]. Positive results should be considered presumptive and additional confirmation testing is required. The CE marked version of GPP also detects ADV 40/41, Vibrio cholerae, Yersinia entercolitica, and Entamoeba histolytica. Assay format is similar to the xTAG RVP Fast (see section 6.2), with results available in 5 h including extraction. Detection platforms include the Luminex 100/200 instruments and in Europe and Canada the small compact MAGPIX instrument (Luminex) (Figure 6.7).

Photo displaying the Luminex Magpix instrument.

Figure 6.7 Luminex Magpix.

(Image provided by Luminex Corporation)

The Verigene Enteric Pathogens assay (Nanosphere) uses the same test format and instruments as the Verigene RVP assay. This assay is not currently FDA cleared at the time of writing. The assay detects ADV 40/41, noroviruses, and rotaviruses plus the nonviral targets: Campylobacter, Salmonella, Shigella, Y. entercolitica, Vibrio, STEC Shiga Toxin Gene 1 (stx1), and Shiga Toxin Gene 2 (stx2).

The FilmArray GI Panel (Biofire) is a pouch-based, highly multiplex, real-time nested PCR assay which runs on the FilmArray System [135,136]. The test system and assay format is the same as their Respiratory Virus Panel, previously described. The assay can detect adenovirus types 40/41, astroviruses, noroviruses GI/GII, rotavirus A, and sapoviruses. Nonviral targets included in the FilmArray GI Panel are Aeromonas, Campylobacter, Clostridium difficile, ETEC, enteropathogenic E. coli (EPEC), STEC, enteroinvasive E. coli (EIEC), enteroaggregative Escherichia coli (EAEC), E. coli O157, Plesiomonas shigelloides, Salmonella, Shigella spp, Shigella dysenteriae, Vibrio spp, Yersinia enterocolitica, Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, and Giardia lamblia. Fresh stool and stool in Cary-Blair transport media are both acceptable specimen types. After the sample and hydration solution are added to the reagent pouch, results are available in approximately 1 h [135,136].

There are two Argene/bioMérieux PCR assays (bioMérieux, Marcy l’Etoile, France) for enteric viruses, the Adenovirus Consensus PCR kit and the Calici/Astrovirus Consensus RT-PCR kit, which detect noroviruses, sapoviruses, and astroviruses [119]. These assays require NA extraction using the NucliSENS easyMAG, standard RT-PCR, and end-point detection using biotinylated probes. Both assays are not FDA cleared but are CE marked at the time of writing.

6.4 Enterovirus and parechovirus

6.4.1 Background

Enteroviruses (EV) and parechoviruses (PeV) are nonenveloped, RNA members of the Picornaviridae virus family (for the most current categorization: http://www.picornaviridae.com/). Parechoviruses were formerly classified as enteroviruses until nucleic acid sequencing determined that they were divergent enough from other enteroviruses to be placed into a separate genus within the Picornaviridae virus family [99]. Poliovirus, the causative agent of poliomyelitis, is limited to a few unvaccinated regions in the world and is targeted for global elimination by the Global Polio Eradication Initiative [12]. Enterovirus and parechoviruses infections are ubiquitous worldwide and commonly present as nonspecific fevers or as asymptomatic infections in children < 5 years of age [99,195,198]. EVs/PeVs are the most frequent (85–95%) cause of aseptic meningitis in the United States and may also cause encephalitis, especially in children [194,195]. Additionally, EV/PeV can cause paralysis, pericarditis, myocarditis, conjunctivitis, herpangina, respiratory infections, and exanthems. Newborns, born to mothers with active EV/PeV infections, can develop an EV/PeV sepsis-like syndrome, with or without meningitis [233]. Enterovirus 71 has been associated with outbreaks of severe rhombencephalitis in children in Southeast Asia in recent years [212]. Both EVs and PeVs are usually transmitted through a fecal–oral route as the virus is shed through the intestinal tract [198]. In temperate regions, EV activity tends to be seasonal with a higher number of cases and outbreaks during the summer and fall [198].

While some EV/PeV infections can be diagnosed clinically or just treated symptomatically, diagnosis of EV/PeV-associated meningitis and encephalitis requires laboratory testing for proper patient management. Although EV and PeV are the most common cause of meningitis, initial clinical presentation can be difficult to differentiate from bacterial meningitis or HSV meningitis/encephalitis. Early EV/PeV meningitis can present with neutrophil predominance in the cerebrospinal fluid (CSF) resembling a bacterial meningitis [156]. A diagnosis can be particularly difficult in infants or immunocompromised patients who may lack CSF pleocytosis during infection [48,201]. Therefore, differentiation of usually benign EV/PeV meningitis from serious bacterial meningitis or HSV encephalitis is critical to ensure prompt appropriate treatment with antibiotics or acyclovir. Conversely, EV/PeV meningitis can be managed with supportive care and often does not require hospitalization [191,198].

6.4.2 Assays for enterovirus and parechovirus

Specimens for the detection of EV and PeV infections can include CSF, throat swabs, rectal swabs, pericardial fluid, and blood. Traditionally EV was isolated and identified using cell culture, requiring multiple cell lines for optimal recovery due to strain diversity [41,130]. Growth usually requires 4–8 days [133] and commercial panenterovirus DFA reagents are used to confirm cytopathogenic effect (CPE). However, some enteroviruses, Coxsackie A viruses and PeV-3 in particular, grow poorly in cell culture and therefore are rarely recovered. In general, due to low concentration of virus in the CSF, the sensitivity of culture for EV is less than 50% for CSF in cases of acute meningitis or encephalitis [51,88,159].

Therefore in suspected cases of viral meningitis or neonatal EV/PeV sepsis NAATs are the standard of care. NAATs have both sufficient sensitivity and rapid enough turnaround time (1–6 h) to clinically manage patients [80,124]. The 5’ UTR region of EV and PeV are the standard target for NAATs [157,194]. Rhinoviruses share sequence homology with EVs in this region and there is a risk of cross-reactivity with HRVs in EV assays [31]. However, since HRVs are not typical CSF pathogens, this is not a significant problem in testing CSF. On the other hand, inference of an EV meningitis/encephalitis based on an EV NAAT positive throat or rectal swab must be interpreted cautiously as the NAAT may be detecting HRV and not EV. There are only two FDA-cleared NAATs for the qualitative detection of EV in CSF and only LDTs for the detection of PeV’s. The NucliSENS EasyQ Enterovirus assay (bioMérieux, France) uses NASBA and real-time molecular beacon fluorescent detection [126]. The NA extraction is performed using either the NucliSENS miniMAG or easyMAG platforms and amplification and detection steps are performed simultaneously on the NucliSENS EasyQ instrument (bioMérieux, France). The assay shows minimal cross-reactivity with HRV (only at very high titers) and takes approximately 4 h to complete [31,80,126]. The format of the assay is amenable to batch testing. The sensitivity and specificity of the molecular beacon FDA version of the assay are 95.5 and 100%, respectively, compared to the original NucliSENS EasyQ detection by electrochemiluminescence (ECL) version, which in turn was 30% more sensitive than viral cell culture [31,80,124–126]. The Xpert EV (Cepheid) is a cartridge-based, multiplex, real-time RT-PCR assay which runs on the GeneXpert system (described in section 6.2) [48,205]. The assay takes approximately 2.5 h to complete and is more amenable to random-access rapid testing. However, no residual extracted nucleic acid sample remains after the testing, whereas a separate extraction step in the NucliSENS EV assay allows for additional CSF testing for other viruses such as HSV. This can be especially beneficial when limited CSF volume is available and multiple NAATs are requested. The Xpert EV assay’s sensitivity was 94–97% and specificity was 100% for EV isolates [120,158].

There are no FDA-cleared assays for the detection of EV in non-CSF specimens such as blood, respiratory, or stool. Enterovirus R-gene and parechovirus R-gene ASRs are available from bioMérieux [176]. Both assays use real-time RT-PCR with detection by Taqman probes and require a separate NA extraction step; the assays are performed on a variety of instruments. The Seeplex Meningitis ACE Detection Kit (Seegene, Gaithersburg, MD) is a RT-PCR kit that can detect five bacterial targets (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Streptococcus agalactiae

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Dec 10, 2017 | Posted by in MICROBIOLOGY | Comments Off on 6: Molecular Tests for the Identification of Viruses

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