Specimen Collection and Handling Procedures

Virology Section

The Virology section currently offers a variety of Laboratory services not routinely available in the average hospital microbiology laboratory for assisting in the diagnosis in patients suffering from acute viral or nonviral infections. The section uses several methods for the detection and identification of viral infections:

1. Processing clinical specimens (e.g. throat swabs, stool specimens) in order to detect and identify viruses by cell culture;
2. Detection of IgG and or IgM antibody in single serum for diagnosis or demonstrating a significant four-fold or greater rise in antibody titer in acute and convalescent-phase sera to a known agent;
3. Out of State premarital testing and diagnosis of Syphilis on Serum and Cerebrospinal Fluid.
4. Antigen testing for Cryptococcus neoformans on serum or spinal fluid.
5. Screening and confirmation of HIV-1 in serum and oral fluid
6. Detecting microbial antigens in clinical specimens by immunofluorescence using specific conjugates to the agents. This last procedure is the most rapid but is routinely used only for a few infections at the present time; rabies (in another section), herpes, and encephalitis.

Immune screening is offered for antibodies to agents capable of infecting the fetus in pregnant women including screening for cytomegalovirus, herpes simplex virus, and toxoplasmosis. In addition, immune screening is performed for rubella, rubeola, mumps, Varicella zoster (chickenpox), and Hepatitis B and C.

Virus Isolation
Virus isolation involves the testing of clinical specimens from patients (throat swabs, rectal swabs, vesicle fluid, cerebrospinal fluid), which are processed, treated with antibiotics and use to inoculate a cell culture (cynomolgus monkey kidney or diploid human lung fibroblasts). The inoculated cell cultures are examined for evidence of cytopathic effects for a period of six days ( Herpes simplex only), 10 days (other suspect viruses), or 21 days (cytomegalovirus and Varicella zoster ), before a report is sent. Cultures which exhibit viral growth are identified as specifically as possible with existing reagents.

Collection of specimens for culture
The collection of clinical material at the proper time, from the proper site and its handling will influence the recovery rate when specimens are received at the Laboratory. The following recommendations have been proposed for optimum recovery of the more labile viruses:

1) Specimens should be collected promptly, usually within the first 3 days and no longer than 7 days after onset of illness;

2) Almost any type of commercial swab can be used for most specimens. Calcium alginate fiber tips can inactivate some viruses and should not be used if another swab is available, (e.g. cotton or Dacron-tipped swab).

3) Dry swabs used to collect clinical material should be immersed in a small volume of transport medium (1-2 ml) such as tryptose phosphate or nutrient broth. Commercial swabs containing medium for collection of virus specimens are also acceptable. Media containing charcoal or animal sera are not recommended, since the former ingredient may affect the rate of virus recovery and the latter may contain other viruses or filterable microorganisms.

4) The test specimen will yield more recoverable virus if it is stored at refrigerator temperature (2-8 o C). If necessary, the specimen can be refrigerated for a couple of days prior to shipment. Freezing specimens at -20oC rapidly loses the infectivity of many viruses. Freezing is not recommended prior to shipment.

5) The specimen should be shipped in a foam container with a frozen gel pack. DO NOT USE DRY ICE! The package should be sent at the earliest possible time following current regulations for the shipping of diagnostic material.

Throat, Nasal and Nasopharyngeal Cultures
Swabs of the throat, oral cavity and nasal passages have been reported to yield the widest variety of viruses when taken in the early stage of illness (no later than 5-7 days after onset). A dry cotton swab is used to collect throat secretions by rubbing the back of the throat and any area of inflammation, ulceration or exudation. The swab is then broken or cut into a leak proof tube containing a small volume of medium (e.g. tryptose-phosphate broth with 0.5% gelatin). Nasal cultures are obtained by taking a cotton tip applicator moistened with broth and gently rotating it in a circular fashion after inserting the tip about 3/4 of an inch into the nares and then placing the applicator in broth and storing at 2-8 o C. Nasopharyngeal specimens are obtained by taking a steel wire applicator and gently passing it through the nose to the posterior nasopharynx, leaving it for a few seconds and then removing it. Swabs tend to yield a lower recovery rate of virus than washings or aspirates but are more convenient to collect.

Vesicle fluids and skin scrapings
Specimens of vesicle fluids and cellular material from the base of lesions are best collected during the first 3 days of eruption when the recovery rate is highest. Vesicle fluid can be aspirated using a 26 or 27 gauge needle attached to a tuberculin syringe or with a capillary pipette. The collected fluid should be rinsed into a small volume of transport medium. Open lesions can be swabbed to obtain both fluid and cells from the lesion base. The swab is then broken off into a vial of transport medium to preserve viable virus in the sample.

Eye swab
Any exudate or pus present in the eye should be carefully removed with a sterile swab. A second swab moistened with medium or saline should be used to rub the affected conjunctiva. This swab should be broken off into a vial of transport medium

Cervical swab
As with the eye swab, the first swab is used to clean the cervix of mucus and is discarded. A second swab is inserted about 1/4 inch into the cervical canal and rotated. If any lesions are seen, these should be collected on a swab and then the swab should be broken off into a vial of transport medium. Commercially purchased viral collection swabs are also acceptable. Be sure to break the ampoule at the base. 

Stool and rectal swabs
Small amounts (1-4 g) of stool can be collected and placed in a small leak proof container. No transport medium should be added. Inserting a dry swab 1-2 inches past the anal sphincter, rotating it and then removing it obtain a rectal swab. The swab should be broken off into a vial of transport medium and refrigerated. Commercially purchased viral collection swabs are also acceptable. Be sure to break the ampoule at the base. 

Cerebrospinal fluid for Culture
At least 2 ml should be collected into a sterile leak proof tube or vial. At least 1 ml should be obtained from infants. The specimen should not be diluted and should be kept in the refrigerator (2-8 o C) until sent to the Laboratory.

Urine
Clean-voided specimens (3-5 ml) should be stored at refrigerator temperature in sterile, leak-proof containers. Urine specimens are most useful for the isolation of cytomegalovirus in newborns and the best recovery rates occur if the specimen is held at 2-8 o C and processed within a few hours after collection.

Autopsy and biopsy specimens
Fresh tissue should be collected and placed in separate labeled sterile containers. Autopsy specimens in 1-2 gram portions are usually sufficient. Acceptable tissues from suspected cases include brain, lung, heart, lymph node and kidney. 1-2 ml of transport media should be added.

Collection of cerebrospinal fluid
CSF for Syphilis or Cryptococcus antigen is the same as for culture. Do not send CSF in serum separator tubes with gel separator inside. The gel separator interferes with results. Also CSF contaminated with serum or red cells is unsatisfactory.

Serological testing
Blood specimens should be collected without anticoagulants or preservatives. Routinely, a volume of 5 ml from an adult or 3 ml from a pediatric patient is sufficient for obtaining enough sera for testing.
Allow the blood specimen to clot at room temperature and the clot then to retract for about one hour. The specimen is then centrifuged for 10-15 minutes (at 1000-1300 X g) to separate the serum from the blood clot and any loose erythrocytes. We provide blood collection tubes with a gel separator, which will physically separate the blood clot from the serum after centrifugation. The serum can either be refrigerated at 4-8 o C or frozen for antibody testing.
Hemolysis or lysis of erythrocytes generally will render the serum specimen unsuitable for most serological tests. It is important not to freeze whole blood specimens as this will result in hemolysis. Some laboratories have recommended that the blood specimens not be centrifuged until the clot has been allowed to form and retract either at room temperature or in the refrigerator. It is best to physically separate the serum from the blood clot within 24 hours of collection.
In general, a serological diagnosis of a recent infection requires the demonstration of a four-fold or higher antibody rise in paired serum specimens taken from the same patient in the acute and convalescent-phase of illness. The acute-phase serum is drawn as early as possible after onset while the convalescent-phase serum should be taken 10-14 days later. In cryptococcosis, the diagnosis of this fungal infection causing meningitis is made through the detection of the antigen in serum or cerebrospinal fluid and not through the formation of serum antibodies. In infections such as rubeola and rubella the culture procedure is inferior to the serologic procedure and is not recommended for routine use.