Aids in diagnosing enterovirus infections
Real-Time Polymerase Chain Reaction (PCR)/RNA Probe Hybridization
Enterovirus (Coxsackievirus and Echovirus)
Hand, Foot, and Mouth Disease
Submit a raw clinical sample (not a culture isolate) for enterovirus PCR. This test will detect enterovirus, but will not differentiate viruses in this family or provide serotyping information.
Collection Container/Tube: Lavender top (EDTA)
Submission Container/Tube: Screw-capped, sterile container
Specimen Volume: 1 mL
Collection Instructions: Spin down promptly.
If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.
|Specimen Type||Temperature||Time||Special Container|
|Plasma EDTA||Refrigerated (preferred)||7 days|
Aids in diagnosing enterovirus infections
Enteroviruses are positive-sense RNA viruses in the Picornaviridae family. These viruses were initially classified by serotype as polioviruses (3 types), echoviruses (31 types, including types 22 and 23, which are now classified as parechoviruses), coxsackievirus A (23 types), and coxsackievirus B (6 types). However, genomic studies have demonstrated that there is significant overlap in the biological characteristics of different serotypes and, more recently, isolated enteroviruses are now named with consecutive numbers (eg, EV68, EV69).
The normal site of enterovirus replication is the gastrointestinal tract where the infection is typically subclinical. However, in a proportion of cases, the virus spreads to other organs, causing systemic manifestations, including mild respiratory disease (eg, common cold); conjunctivitis; hand, foot, and mouth disease; aseptic meningitis; myocarditis; and acute flaccid paralysis. Collectively, enteroviruses are the most common cause of upper respiratory tract disease in children. In addition, the enteroviruses are the most common cause of central nervous system (CNS) disease; they account for almost all viruses recovered in culture from spinal fluid. Differentiation of enteroviruses from other viruses and bacteria that cause CNS disease is important for the appropriate medical management of these patients.
Traditional cell culture methods require 6 days, on average, for enterovirus detection. In comparison, real-time PCR allows same-day detection. Detection of enterovirus nucleic acid by PCR is also the most sensitive diagnostic method for the diagnosis of CNS infection caused by these viruses.
A positive result indicates the presence of enterovirus RNA in the specimen.
A negative result does not rule out the possibility of enterovirus infection. This assay may detect virus from a variety of specimen types in asymptomatic individuals. This assay should only be used for patients with a clinical history and symptoms consistent with enterovirus infection, and must be interpreted in the context of the clinical picture. This test should not be used to screen asymptomatic patients.
Accuracy/Diagnostic Sensitivity and Specificity:
We compared the generic detection of enteroviruses from spinal fluid by conventional tube cell culture (MCR-5) and by LightCycler PCR. Of 715 specimens tested, enteroviruses were detected in 65 (9%) by conventional cell culture and 82 (11%) by LightCycler PCR. Twenty-two of 82 (27%) were exclusively positive by PCR; whereas, only 5 of 65 (8%) were exclusively positive by conventional cell cultures.
Supplemental Data (Spiking Studies):
To supplement the data above, 30 or more negative specimens of each specimen type (cerebrospinal fluid/sterile body fluid, dermal/ocular/rectal swabs, plasma, and upper and lower respiratory specimens) were spiked with enterovirus culture control at approximately 10 to 50 targets/mcL (the approximate limit of detection). The spiked specimens were run in a blinded manner along with negative (nonspiked) specimens of each specimen type. Of the spiked specimens, 97% to 100% were positive, and 100% of the nonspiked specimens were negative. A total of 489 spiked and nonspiked specimens were tested.
The assay detected all 64 members of an enterovirus panel, consisting of coxsackieviruses, polio viruses, echoviruses, and other enteroviruses.
Analytical Specificity/Limit of Detection (LoD):
The lower LoD of this assay is approximately 10 to 50 RNA target copies/mcL. This was confirmed in all specimen types accepted for this assay.
The assay did not crossreact with a specificity panel containing other RNA-containing viruses (rhinovirus; reovirus; influenza virus, types A and B; respiratory syncytial virus; and parainfluenza virus) and DNA-containing viruses (herpes simplex, Epstein-Barr virus, varicella-zoster virus, and cytomegalovirus).
This is a qualitative assay and results are reported a either negative or positive for targeted enterovirus RNA.
1. Enterovirus surveillance-United States, 1970-2005. MMWR Morb Mortal Wkly Rep Sept 15 2006;55(SS08):1-20
2. Foray S, Pailloud F, Thouvenot D, et al: Evaluation of combining upper respiratory tract swab samples with cerebrospinal fluid examination for the diagnosis of enteroviral meningitis in children. J Med Virology 1999;57(2):193-197
3. Furione M, Zavattoni M, Gatti M, et al: Rapid detection of enteroviral RNA in cerebrospinal fluid (CSF) from patients with aseptic meningitis by reverse transcription-nested polymerase chain reaction. New Microbiol 1998;21(4):343-351
For this real-time reverse-transcription laboratory-developed PCR assay, viral nucleic acid is extracted from specimens using the MagNA Pure automated instrument (Roche Applied Science), followed by amplification and detection on the Roche LightCycler 2.0 instrument. This PCR assay has been optimized to detect a target sequence in the polyprotein region. Primers amplify a 193-bp product.
Enterovirus genomic RNA is first transcribed to cDNA by reverse transcriptase, followed by amplification of the cDNA product. The LightCycler instrument can rapidly (30-40 minutes) detect amplicon development through stringent air-controlled temperature cycling in capillary cuvettes. The detection of amplified products is based on the fluorescence resonance energy transfer (FRET) principle. For FRET product detection, a hybridization probe with a donor fluorophore, fluorescein, on the 3'-end is excited by an external light source and emits light that is absorbed by a second hybridization probe with an acceptor fluorophore, LC-Red 640, at the 5'-end. The acceptor fluorophore then emits a light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. FRET (with subsequent production of a detectable fluorescent signal) only occurs when the probes have specifically annealed to the target sequence of the amplicon.
Melting curve analysis is performed following PCR amplification and is the detection phase of the assay, since it offers greater sensitivity than the amplification phase and maintains high specificity.
The melting phase of the assay occurs as follows:
Starting at 45 degrees Celsius, which allows the probes to bind to the amplified product, the temperature in the thermal chamber is then slowly raised to 80 degrees Celsius and the fluorescence is measured at frequent intervals to determine the point where half of the fluorescence is lost as the probes are denatured (ie, "melt") off of the target. This is called the melting temperature (Tm) of that virus. Analysis of the PCR amplification and probe melting curves is accomplished through the use of LightCycler software.(Cockerill FR III, Uhl JR: Applications and challenges of real-time PCR for the clinical microbiology laboratory. In Rapid Cycle Real-Time PCR Methods and Applications. Edited by U Reischl, et al.
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This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.