General Principles
Specimen selection depends on the specific disease syndrome, viral etiologies suspected, and time of year. Selecting a specimen based on disease is confusing, because most viruses enter through the upper respiratory tract and infect tissues that may produce symptoms distant from the primary inoculation site. For example, aseptic meningitis, caused by infection with various types of enterovirus, may be identified by detecting virus in throat, rectal swab, or cerebrospinal fluid (CSF) specimens. Pharyngitis and gastrointestinal symptoms may not be included in the patient’s complaints.
Specimen selection based on the suspected viral etiology is complicated by the fact that similar clinical syndromes can be caused by many different viruses. When specimens required for identification of a specific virus are collected without thorough consideration of other possible viral agents, additional important etiologic agents may be missed. For example, testing smears of nasal secretions from an infant using fluorescence staining or enzyme immunoassay to detect RSV does not allow for diagnosis of similar disease resulting from infection with influenza virus, parainfluenza virus, or metapneumovirus.
Selection of the appropriate type of specimen is one of the keys to a correct test result. Selection should include the proper specimen source and the correct sample volume and timing of collection. This information should be reviewed institutionally on an annual basis and made available to clinicians.
Appropriate specimen selection dictates that the specimen type and suspected viruses should be included on the requisition. The laboratory should always be notified if rare agents representing a danger to laboratory workers are suspected (e.g., SARS coronavirus, H5N1 avian influenza virus, hemorrhagic fever viruses). Serum for serologic testing may be necessary, and some viral diseases should be considered during specific months. Table 1 presents specimens for the diagnosis of viral diseases, noting trends in seasonality.
Table1. Specimens for Diagnosis of Viral Diseases *
Table2. Specimens for Diagnosis of Viral Diseases—cont’d
Specimens for the detection of virus should be collected as early as possible after the onset of symptomatic disease. Virus may no longer be present as early as 2 days after the appearance of symptoms. However, other factors, such as the patient’s immune status or age, the type of virus, and the amount of systemic involvement, may play a role in the length of time viral shedding is evident, allowing effective laboratory detection. Certain viruses, such as West Nile virus, produce a brief, low viremia and undetectable levels at the onset of symptoms. Recommendations for collection of various specimens are summarized in this section.
In addition to the type of specimen and collection method, validated devices or containers can enhance the recovery and detection of the viral agent. Swab specimens should not contain chemicals or other compounds that may be toxic to cultured cells and therefore are unsuitable for viral specimen collection. Calcium alginate swabs interfere with PCR, the recovery of some enveloped viruses, and fluorescent-antibody tests and therefore should not be used.
Throat, Nasopharyngeal Swab, or Aspirate
In general, nasopharyngeal aspirates are superior to throat or nasopharyngeal swabs for recovering viruses; however, swabs are considerably more convenient. Throat swabs are acceptable for the recovery of enteroviruses, adenoviruses, and HSV, whereas nasopharyngeal swab or aspirate specimens are preferred for the detection of RSV and influenza and parainfluenza viruses. Rhinovirus detection requires a nasal specimen. Throat specimens are collected with a dry, sterile swab by passing the swab over the inflamed, vesiculated, or purulent areas on the posterior pharynx. The swab should not be touched to the tongue, buccal mucosa, teeth, or gums. Nasopharyngeal secretion specimens are collected by inserting a swab with a flexible shaft through the nostril into the nasopharynx or by washing and collecting the secretions by rinsing with a bulb syringe and 3 to 7 mL of buffered saline. The saline is squirted into the nose by squeezing the bulb and aspirated with a small tubing inserted into the other nostril when the bulb or suction is released.
All respiratory specimens are acceptable for culture of most viruses. However, respiratory and oral samples often are contaminated with bacteria. Contaminants may be removed by concentrating the sample through centrifugation. However, this process may also result in removal of virus-infected cells and reduce the recovery of viral agents from the sample.
Bronchial and Bronchoalveolar Washes
Washings and lavage fluid collected during bronchos copy are excellent specimens for detecting viruses that infect the lower respiratory tract, especially influenza viruses and adenoviruses.
Rectal Swabs and Stool Specimens
Stool and rectal swabs of fecal specimens are used to detect rotavirus, enteric adenoviruses (serotypes 40 and 41), and enteroviruses. Many agents of viral gastroenteritis do not grow in cell culture and require PCR or electron microscopy for detection (these are discussed later in the chapter). In general, stool specimens are preferable to rectal swabs and should be required for rotavirus and enteric adenovirus testing. Rectal swabs are acceptable for detecting enteroviruses in patients suspected of having an enteroviral disease, such as aseptic meningitis. The rectal swab is inserted 3 to 5 cm into the rectum and rotated against the mucosa to obtain feces. The swab should then be placed in appropriate transport media. A stool sample is preferred over a rectal swab because of the potential for decreased viral recovery from a small sample size. Five to 10 mL of freshly passed diarrheal stool or stool collected in a diaper from young infants is sufficient and preferred for rotavirus and enteric adeno virus detection.
Urine
CMV; mumps, rubella, and measles viruses; polyomaviruses; and adenoviruses can be detected in urine. Virus often is shed intermittently or in low numbers. Viral recovery may be increased by processing multiple (two to three) specimens. Improved recovery results with a minimum specimen volume of 10 mL from a clean-catch first-morning urine. The urine pH and contaminating bacteria may interfere with viral replication. Virus recovery is improved by centrifugation or filtering to remove contaminants and neutralizing the pH with a 7.5% solution of sodium bicarbonate.
Skin and Mucous Membrane Lesions
Enteroviruses, HSV, VZV, and in rare cases CMV or pox viruses can be detected in vesicular lesions of the skin and mucous membranes. Once the vesicle has ulcerated or crusted, detection of the virus is difficult.
Collection of specimens from cutaneous vesicles for detection of HSV or VZV may require a Tzanck smear if PCR testing is not available. Tzanck smears are prepared by carefully unroofing the vesicle. The procedure is as follows: If a tuberculin syringe is used, a small “drop” of vesicle fluid should be aspirated first and held for further use in the event a viral or bacterial culture is needed. The needle is flushed with a viral transport medium, and phosphate buffered saline or viral support media (EMEM) is added to the viral transport tube. With the roof of the vesicle folded back, excess fluid is carefully removed by dabbing with sterile gauze. A clean glass microscope slide is pressed against the base of the ulcer. The slide is lifted, moved slightly, and pressed again. Cells from the base of the ulcer stick to the slide, making an “impression smear” of infected and uninfected cells. Additional smears can be made from other vesicles. The slides are sent to the laboratory for fixation and staining. As an alternative, vesicle fluid and cells scraped from the base of an unroofed vesicle can be added to 2 to 3 mL of viral transport medium. Smears can be prepared in the laboratory with cytocentrifugation of fluid medium, or PCR can be performed from the specimen in the viral transport medium.
Sterile Body Fluids Other Than Blood
Sterile body fluids, especially CSF and pericardial and pleural fluids, may contain enteroviruses, HSV, VZV, influenza viruses, or CMV. These specimens are collected aseptically by the physician and sent to the laboratory for processing.
Blood
Viral culture of blood is used primarily to detect CMV; however, HSV, VZV, enteroviruses and adenovirus occasionally may be encountered. CMV viremia is associated with peripheral blood leucocytes. Five to 10 mL of anticoagulated blood collected in a whole blood tube is needed. Heparinized, citrated, or ethylenediaminetetraacetic acid (EDTA) anticoagulated blood is accept able for CMV detection. Citrated blood should be used when other viruses are being considered. EDTA should be used for samples collected for nucleic acid testing, because other anticoagulants may interfere with the enzyme functions required for PCR amplification. Serum may be used for serologic tests and nucleic acid assays.
Bone Marrow
Bone marrow for virus detection should be added to a sterile tube with anticoagulant. Heparin, citrate, or EDTA anticoagulants are acceptable. As previously described for blood, EDTA should be used if the specimen is intended for nucleic acid testing. Specimens are collected by aspiration. Except for parvovirus B19, most viruses are detected more readily from sites other than bone marrow.
Tissue
Tissue specimens are especially useful for detecting viruses that commonly infect the lungs (CMV, influenza virus, adenovirus, sin nombre virus), brain (HSV), and gastrointestinal tract (CMV). Specimens are collected during surgical procedures. Fresh tissue is preferred for nucleic acid assays, but formalin-fixed and paraffin embedded tissues may be used after removal of the paraffin (deparaffinization) and extraction.
Genital Specimens
Genital specimens often are required for detection of HSV and human papillomavirus (HPV). Genital swabs should be used for ulcerations and placed in appropriate viral transport media. Cervical specimens may be collected using a swab or brush and placed in viral transport media. Some manufactured endocervical or liquid-based cytology devices are appropriate for nucleic acid testing. Following the manufacturer’s recommended protocols is essential when processing such specimens.
Serum for Antibody Testing
Acute and convalescent serum specimens may be needed to detect antibody to specific viruses. Acute specimens should be collected as soon as possible after the appearance of symptoms. The convalescent specimen is collected a minimum of 2 to 3 weeks after the acute specimen. In both cases, an appropriate specimen is 3 to 5 mL of serum collected by venipuncture.
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