Virology

Introduction

Virological tests are usually costly and labour-intensive, therefore they should be undertaken only when good clinical indications for doing so exist, and after thoughtful consideration of the types of tests to request for.

Virological tests fall into three categories:

• Culture of virus from infected body fluids or tissues
• Antibody detection in blood and, where appropriate, CSF
• Demonstration of the presence of viral antigen or viral nucleic acid in infected tissues or body fluids.

Sample Labelling & Completion of Request Forms

All samples must be clearly labelled with the patient’s name and unique identification number, e.g. NRIC number, as well as the nature, source and date of collection of the sample.

Each sample must be accompanied by a request form that is completed legibly with the patient’s name, unique identification number, location, relevant history, findings and clinical diagnosis, the test(s) required, the nature, source and date of collection of the sample, and the name and for urgent requests, the contact number of the requesting doctor. The form must be signed by the requestor.

It should be ensured that these requirements are met to avoid rejection of the sample by the laboratory or a delay in reporting.

General Guidelines for Virus Isolation

1. For most viral infections, the causative agents can only be isolated during the first 3 – 4 days of the acute illness.
2. Some viruses are slow growing. Results are available from 1 day to 3 weeks.
3. Viruses are usually very labile, so samples should reach the laboratory within one hour of collection. If this is not possible, the sample must be refrigerated at 4°C (for a maximum period of 48 hours) until transport. Do not freeze samples as many viruses are inactivated at –20°C. Within SGH, the telelift should not be used for culture samples, as this will result in considerable delay in their reaching the laboratory. For samples collected after office hours, refrigerate at 4°C until transport to the laboratory. Do not freeze.
4. Transport the sample with an ice pack or in a bag of ice cubes. First seal the sample in a plastic bag, then place this bag inside an outer plastic bag containing the ice pack or ice cubes. On no account should the ice cubes come into direct contact with the sample bottle as contamination will occur. Place the accompanying request form into another plastic bag to keep the form dry.
5. All swabs and tissue samples for virus isolation must be transported in virus transport medium (VTM). Dry swabs will not be accepted by the laboratory because the virus would probably have died. Blood, CSF, stools and effusions may be sent without VTM. For urine, see special instructions given below.
6. Swabs used for sampling should be made of cotton, dacron or rayon, not of calcium alginate, and the shaft of the swab stick should preferably be made of plastic.

Collection Methods for Virus Isolation by Sample Type

Throat Swabs

1. Swab the pharynx and both tonsils vigorously.
2. If the patient has an associated rhinitis, collect a nasal swab at the same time, using a separate swab. Place both swabs in the same bottle of VTM.

Nasopharyngeal Aspirates

Nasopharyngeal aspirates are superior to throat swabs for the recovery of viruses. Further, immunofluorescence microscopy can be carried out on exfoliated cells in the aspirate for rapid diagnosis.

1. Attach a disposable polythene catheter (French catheter 8 for infants) to a mucus trap and a suction pump.
2. Insert the catheter through the nose for a distance equal to that from the tip of the nose to the angle of the jaw.
3. Using the suction pump, aspirate mucus into the trap. If secretions are thick and trapped in the catheter, dislodge the mucus by sucking up 1 – 3 mL of VTM.
4. Disconnect the trap when collection is complete and empty contents into a bottle of VTM. (It may be necessary to pour the VTM into the mucus trap, swirl the contents, then pour it back into the VTM bottle).
5. If a suction pump is not available, attach the catheter to a 20 mL syringe, aspirate secretions, then expel secretions into VTM.

Saliva

For mumps isolation, swab the buccal mucosa opposite the upper molars where Stensen’s ducts open, then swab the floor of the mouth at the openings of the submandibular gland ducts. For cytomegalovirus (CMV) isolation, swab the buccal mucosa or aspirate saliva into a mucous trap and send in VTM.

Vesicular Lesions

Sample fresh skin vesicles during the first three days following the appearance of the eruption. Crusted lesions have a lower chance of yielding viable virus. Macules or papules should not be sampled.

1. Gently clean the surface of the vesicle with sterile saline. Do not use alcohol.
2. If the vesicle is intact, use a sterile needle to lift off the roof of the vesicle.
3. Using a sterile swab, soak up all the fluid from the vesicle, then swab the base of the lesion vigorously to dislodge cells on the base (which contain the virus) onto the swab.
4. Using the same swab, repeat the procedure with a number of vesicles, if present, in order to increase the yield of virus.
5. Insert the swab into a bottle of VTM, break off the stick and screw-cap the bottle tightly.

Open Lesions

Clean open lesions on the skin and genitalia with sterile saline to remove any pus, then swab firmly to sample the basal cells. For oral lesions, swab the base of the lesions. Insert the swab into a bottle of VTM, break off the stick and screw-cap the bottle tightly.

In the case of keratoconjunctivitis, send scrapings from lesions for virus isolation and/ or immunofluorescence.

Conjunctival Swabs

Using a swab moistened with sterile saline, pull down the lower lid and swab the conjunctiva firmly, then evert the upper lid and swab similarly. Insert the swab into a bottle of VTM, break off the stick and screw-cap the bottle tightly.

For keratoconjunctivitis, scrapings from lesions must be sent for virus isolation and/ or immunofluorescence.

Cervical Swabs

1. Use a speculum during collection.
2. Use one swab to clean the cervix of mucus and discard.
3. Insert a second swab about 1 cm into the cervical canal.
4. Rotate and leave for a few seconds to absorb the secretions.
5. If vesicles or open lesions are present, sample as described above in Collection Methods for Virus Isolation by Sample Type (Virology Section, Vesicular Lesions and Open Lesions).
6. Transport in VTM.

Biopsy and Autopsy Samples

Send fresh samples for virus isolation. Formalinised or fixed tissues cannot be used, as the virus would have been destroyed. Place a piece of the sample about 1 cm in size (or smaller if necessary) directly into the bottle of VTM, and send immediately to the laboratory on ice.

Urine

For adults, instruct the patient to clean the urethral/vulval and perineal areas with soap and water, then to collect a mid-stream sample in a sterile bottle. For infants and young children, clean the perineum and genitalia and collect the sample in a sterile bottle. Transfer the urine into a urine culture bottle up to the mark indicated (about 10 mL). This bottle, which contains antibiotics, can be obtained from the Client Services Section of the Department of Pathology.

Stools

Stools should be sent for the diagnosis of enteroviral infections. In enteroviral infections, virus is excreted in the faeces for several weeks, so stools can be collected after the first week of illness if a diagnosis is required at that stage.

Most of the gastroenteritis-causing viruses such as rotavirus, the enteric adenoviruses and the caliciviruses cannot be cultured in tissue cultures at present, and virus isolation is usually non-productive for viral gastroenteritis.

With CMV enteritis, stool cultures are usually negative and an intestinal biopsy sample is required for virus isolation or antigen detection.

Stools in amount equivalent to the tip of the little finger should be collected in a sterile bottle without VTM.

Rectal Swabs

Collect from cases with suspected enteroviral infections only when stools are difficult to collect, as rectal swabs are inferior to stools.

Insert a sterile swab into the anal orifice, (at least 3 cm deep in an adult) and rotate to ensure collection of faeces. Place the swab in a bottle of VTM, break off the swab stick and screw cap of bottle firmly.

Blood

Viruses may be isolated from leucocytes and/or plasma so unclotted blood must be sent.

1. Collect 6 mL blood into EDTA tubes. (EDTA is to be preferred to heparin as an anticoagulant because some viruses may be inactivated by heparin).
2. Mix the sample gently to prevent clotting and lysis of cells.
3. Do not freeze the sample during transport as this will cause cell lysis.

CSF

1 – 2 mL CSF should be collected in a sterile bottle without VTM.

Effusions

Collect 3 – 4 mL of fluid in a sterile bottle without VTM.

General Guidelines for Virus Serology

1. Collect 3 – 5 mL of blood for serology (6 – 8 mL if a panel of tests is being ordered) in a plain sterile tube without anticoagulants, and send the sample to the laboratory inside a sealed plastic bag. If any delay is anticipated, the sample must be refrigerated at 4°C until transport. Do not freeze as this will cause lysis of the cells, resulting in possible interference with some serological tests.
2. To demonstrate a significant rise in antibody levels, the acute sample should be collected as soon as possible after the onset and the second sample 10 days to two weeks later, and not earlier than seven days. When sending the second sample, label the sample as “second”. The two samples will then be tested in parallel in the same test run.
3. Tests must be requested by name as well as methodology, if more than one type of test is offered (e.g. rubella EIA* or rubella HI†). If in doubt about what to ask for, consult the laboratory.
4. When an acute viral infection is suspected, the first serum can be tested for IgM antibody. However, even IgM antibody may be absent in a sample collected within 1 – 3 days of the onset, and a second sample taken seven days later will be required to test for IgM. False negative IgM results can also occur, especially in infants with congenital infections (e.g. CMV). Conversely, false positive IgM antibody results can occur in patients with rheumatoid factor, collagen diseases or, sometimes, with other viral infections.
5. Great care must be taken not to send a traumatised, blood-stained CSF sample, as interpretation will be difficult. The antibody detected may merely represent that present in the blood. When sending CSF, always collect a blood sample at the same time, as this will be useful for determining the significance of any antibody detected in the CSF.
6. For immunity screening, a single sample will suffice. Results are given as antibody “Present” or “Absent”, or in the case of hepatitis B and rubella antibody, in international units. The complement fixation test (CFT) should not be requested for immunity screening because complement fixing antibody is not long lasting.
7. Serology for enteroviral infections is not available from this laboratory. For diagnosis, isolation must be carried out (see under Enterovirus Isolation).
8. For Rubella Serology on female patients suspected of having acute rubella or who are contacts of rubella cases, an additional form giving clinical details and LMP (last menstrual period) dates must be filled in.

* EIA: Enzyme Immunoassay
† HI: Haemagglutination-inhibition

General Guidelines for Viral Antigen Detection

1. The direct detection of viral antigen in secretions and tissues is very useful for the rapid diagnosis of viral infections, as direct antigen detection may give results within one and a half to two hours. However, a negative result by antigen detection may still be followed by a positive result by culture. It is therefore useful to send samples in viral transport medium, so that both antigen detection and isolation can be carried out at the same time.
2. Where both culture and antigen detection are required, samples must be sent in Virus Transport Medium (VTM). If only antigen detection is required, the sample can be sent in a sterile bottle with 1 – 2 mL of sterile saline added. Do not send dry swab. Place the container in a sealed plastic bag for despatch to the laboratory. If delay is anticipated, refrigerate at 4°C but do not freeze as this will cause cell lysis, making antigen detection impossible.
3. The types of samples that can be sent for antigen detection include:
   1. respiratory secretions such as nasopharyngeal aspirates, bronchoalveolar lavage and sputum (throat swabs are unsuitable because of the sparsity of respiratory epithelial cells in such samples)
   2. fluids from skin vesicles, pericardial and pleural fluid
   3. scrapings from the base of vesicle or open skin lesions or the conjunctivae
   4. body fluids such as urine and CSF
   5. white cells in blood (for CMV)
   6. biopsy or autopsy tissue samples

A Summary of Tests and Samples to Collect for Viral Infections

Presentation Possible	Pathogens	Samples for isolation	Samples for serology	Samples for antigen detection
Respiratory infection	Influenza, RSV, rhinovirus, adenovirus, parainfluenza, CMV, VZV, HSV, enteroviruses, SARS-CoV, hMPV	TS, NPA or BAL, lung biopsy, ETTA in VTM. Pleural fluid without VTM	Paired sera for CFT (all except rhinoviruses, enteroviruses, SARS-CoV, hMPV). Serum for SARS-CoV total antibody EIA, and IgM and IgG	NPA or BAL (all except rhinovirus, enterovirus, SARS-CoV)
Neurological diseases (meningitis, encephalitis, paralysis)	Enteroviruses, mumps, measles, HSV, VZV, JE, HIV	CSF, stool, NPA, brain biopsy. (VTM for NPA and biopsy sample)	Paired sera and CSF for CFT, (excluding HIV & enteroviruses), acute serum for IgM (except JE, entero), one serum for HIV and HTLV	NPA for measles, mumps; Vesicle swab for HSV, VZV; Brain biopsy for HSV, VZV, measles, mumps
Conjunctivitis, keratocon- junctivitis	Adenovirus, coxsackie A24, enterovirus 70, HSV, VZV	Conjunctival swab or scraping in VTM	Single serum for VZV IgM; paired sera for VZV, HSV and Adeno CFT	Conjunctival scraping for HSV, VZV, adenovirus
Viral STD	HSV, HIV	Penile, vaginal or cervical swab in VTM for HSV	Paired sera for CFT for HSV. Single serum for HIV EIA	Swab or scraping from lesion in saline for HSV
Maculopapular rashes	Measles, rubella, dengue, chikungunya, HIV, enteroviruses, parvovirus B19, CMV, EBV	Enteroviruses: throat swab in VTM and stools. Dengue: Blood Measles: NPA, TS, sputum in VTM, urine	Measles and rubella: Acute serum for IgM and paired sera for HI or CFT. Dengue: see under “Dengue” heading below. HIV: EIA or PA Parvo. B19: single serum for IgG and IgM EIA	Measles: NPA for antigen detection
Vesicular skin lesions	HSV, VZV, enteroviruses	Swab and fluid from lesion in VTM	Paired sera for CFT (HSV and VZV only)	Swab or scraping from lesion for HSV and VZV
Cardiovascular: myo- or pericarditis, pleurodynia	Enteroviruses, influenza	Throat swabs and NPA in VTM, stools (enteroviruses only), periocardial fluid	For influenza: paired sera for CFT	For influenza: NPA for antigen
Hepatitis	HAV, HBV, HCV, HDV, HEV, CMV, EBV	CMV: as below	CMV: as below. EBV: one serum for IgM. Rest: one serum for EIA	CMV: as below
Gastroenteritis	Rotavirus, norovirus, adenovirus, CMV, enterovirus (infants) and others	Intestinal biopsy for CMV in VTM. Stool for enteroviruses, adenoviruses	Paired sera for CFT (CMV and adenovirus)	Stool for rotavirus and norovirus antigen
Parotitis	Mumps virus	Saliva, urine	Single serum for IgM. Paired sera for CFT	NPA, urine or saliva for mumps antigen
CMV Infection	CMV	Saliva, NPA, BAL, biopsy in VTM, blood, urine	Paired sera for CFT. Acute serum for IgM	Blood, BAL, biopsy tissue, or urine
Dengue	Dengue types  1 – 4	Blood	Serum for IgM EIA	Blood

Note: Bold type indicates preferred samples and test

BAL : broncho-alveolar lavage
CFT : complement fixation test
CMV : cytomegalovirus
EBV : Epstein-Barr virus
EIA : enzyme immunoassay
ETTA : endotracheal tube aspirate
HAV : hepatitis A virus
HBV : hepatitis B virus
HCV : hepatitis C virus
HDV : hepatitis D (or delta) virus
HEV : hepatitis E virus
HI : haemagglutination-inhibition
HIV : human immunodeficiency virus
hMPV : human metapneumovirus
HSV : herpes simplex virus
HTLV : human T-lymphotropic virus
IF : immunofluorescence
JE : Japanese encephalitis virus
LCM : lymphocytic choriomeningitis virus
NPA : nasopharyngeal aspirate
RSV : respiratory syncytial virus
SARS-CoV : severe acute respiratory syndrome coronavirus
STD : sexually transmitted diseases
TS : Throat swab
VTM : viral transport medium
VZV : varicella-zoster virus


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