Medical Microbiology II Lecture 9

MEDICAL MICROBIOLOGY II

Lesson 8

Laboratory Methods in Diagnostic Virology

Collection of Specimens for Virology

Specimens should be collected during the acute stage of illness when viruses are shed in large numbers

Specimens such as swabs or tissues should not be allowed to dry before reaching the laboratory should be placed in about 3 - 4 mL of a viral transport medium (VTM) so that the viruses do not die due to dehydration


The VTM has a buffering system to maintain the pH between 7.2 and 7.4, a low concentration of protective proteins and antibiotics to inhibit bacterial and fungal contaminants

Various types of VTM are commercially available

Viral Transport Medium


A simple VTM can be prepared:

Hanks balanced salt solution 100 mL

Foetal bovine serum 2 mL

Gentamicin 10 mg

Amphoterin B 50 g


Commonly used specimens include:

1. Throat swab

2. Respiratory aspiration

3. Nasal washings

4. Mucous membrane swabs

5. Conjunctival swabs

6. Vesicle fluid

7. Cerebrospinal, pericardial and pleural fluid

8. Saliva

9. Urine

10. Stool

11. Tissue

12. Blood

13. Postmortem specimens


1. Throat swabs

Swab the tonsillar area and the posterior wall of the pharynx with a cotton tipped sterile swab

Place it in 3 - 4 mL of VTM, immediately delivery to laboratory


2. Respiratory aspiration

Collect the respiratory secretions into a plastic disposable suction aspirator using a fine gauge rubber catheter

In babies, pass it through the nose; in adults through posterior pharynx


3. Nasal washings

Instill about 5 mL saline into each nostril and collect into a screw-capped bottle

A Dacron or rayon swab may also be used; it should be left in the nostril

4. Mucous membrane swabs

Swab the mouth, lips or genital areas and put into VTM


5. Conjunctival swabs

Collect the swabs using dry Dacron or rayon swabs

Roll a dry cotton swab gently along the lower conjunctival surface and collect into VTM


6. Vesicle fluid

Aspirate several vesicles using a tuberculin syringe and a 25 gauge needle

If possible, choose fresh, plump vesicles

Send the vesicle fluid in a sterile container to the laboratory


7. Cerebrospinal, pericardial and pleural fluid

Place about 1 mL of fluid in a dry, sterile container

8. Saliva, urine and stool

These specimens should be collected in sterile containers

Rectal swab may be used if stool cannot be obtained


9. Tissue

It should be collected from an appropriate part of the organ, placed in a sterile container containing 3 - 4 mL of VTM and kept at 4 C until it reaches the virology laboratory

10.Blood

Collect 10 mL of blood in a sterile container containing heparin, EDTA or citrate

For serology, collect blood in a plain sterile tube


11.Post-mortem specimens

These should be collected from the suspected sites of disease and transported to the laboratory as soon as possible

Transport and Storage of Specimens for Virology

Specimens for viral diagnosis must be considered INFECTIOUS and should be handled with great care

In the laboratory, the specimens should only be handled in safety cabinets without releasing aerosols

Every virus isolation specimen should be treated as URGENT and transported to the virus laboratory IMMEDIATELY


If must wait for transport, the specimen in a VTM should be kept at 4 C, but not at 0 C or in an ice-box

Care must be taken during transport and storage of specimens that the virus in the specimen should survive


Factors which can destroy viruses are:

1. Dehydration

2. Heat

3. Freezing at temperatures near 0 C

4. Sudden pH changes

5. Oxidising agents

6. Ultraviolet light


Aspirates, fluids and tissues should be sent to the laboratory in a sterile, leak-proof container

Swabs should never be left to dry and should be placed in VTM immediately

In the laboratory, the specimens should be refrigerated (4 C) until they are inoculated into cell cultures


A specimen may be kept at 4 C for up to 96 hours

If the delay is longer, it should be stored at -70 C

Specimens expected to contain viruses such as enteroviruses, adenoviruses or poxviruses should be stored at -20 C

Cultivation of Viruses

Viruses CANNOT grow on inanimate media

They are obligate intracellular parasites

They need LIVING cells for replication

There are 3 methods:

1. Animal inoculation

2. Inoculation of embryonated eggs

3. Inoculation of organs, tissue fragments or cell monolayers

Animal Inoculation

In the past, the only known method of cultivation of viruses were by inoculation of human volunteers

Then, in the past few decades, animal inoculation has been employed for virus isolation

Laboratory animals include monkeys, rabbits, guinea pigs, rats, hamsters and mice

Animal Inoculation

The choice of animals and route of inoculation (intracerebral, intraperitoneal, subcutaneous, intradermal or intraocular) depends on the type of virus to be isolated

Animal inoculation can also be used to observe pathogenesis, immune response, epidemiology and oncogenesis.

Growth of a virus in the inoculated animal may be indicated by visible lesions, disease or death

Animal Inoculation

Sometimes, serial passage into animals may be required to obtain visible evidence of viral growth

This method requires special experience especially in the handling of animals and inoculation into the various routes

Inoculation of Embryonated Eggs

An embryonated egg provides an aseptic environment that can contain various types of viruses

A large number of viruses can be grown in the different areas of the embryonated egg

The inoculation of eggs can be performed with relatively simple equipment


For inoculation, an 8 - 11 days old hens egg, preferably with a white shell, is used

Duck eggs can be used in some cases

After inoculation, virus replication takes 2 - 7 days

The contents of the egg are harvested and inspected for evidence of virus growth


Most viruses either:

produce morphological changes at the site of inoculation

kill the embryo

produce haemagglutinins

Egg inoculation is also useful for cultivation of chlamydiae and rickettsiae


Involves 4 steps:

1. Candling

2. Drilling the egg shell

3. Inoculation

4. Harvesting the fluids


1. Candling

Is the inspection of an egg over a lamp

A special candling lamp or egg may be held over a strong incandescent lamp

Egg are candled after 3 - 5 days of laying to check if the egg is fertile or not

Infertile eggs which shows a dead embryo are discarded

Eggs are candled again on the 10th or 11th


The air space is marked with a pencil

Then, a suitable site for inoculation is selected depending on the type of virus

Candling


2. Drilling the egg shell

The egg shell is disinfected by wiping with dilute alcoholic antiseptic solution

A small hole is cut at the selected site using a sterile motor driven flexible shaft or dental handpiece

Care MUST be taken not to damage the EGG MEMBRANE


3. Inoculation

The inoculum is injected through the hole on the shell onto the desired site, using a fine needle

The inoculum has to be injected slowly to avoid spillage of the inoculum

The opening of the inoculation is sealed with a sealing mixture prepared by 2 parts of molten paraffin (55 C) and 1 part of petroleum jelly

The egg is incubated at 36 C


Routes of inoculation: The egg can be inoculated by 4 routes

a) Intra-amniotic inoculation

A hole is drilled just above the amniotic cavity, located by candling

The needle is then inserted slowly until the amniotic sac moves


The needle is thrust through the amniotic membrane and the fluid is injected slowly

A small inoculum (0.1 mL) is used

The opening is sealed immediately

This route is useful for influenza, parainfluenza and mumps viruses


b) Intra-allantoic inoculation

A site above the allantoic cavity is selected by candling

The same techniques are followed as for the intra-amniotic cavity

This is the simplest method of inoculation with a relatively large yield and is suitable for the preparation of vaccines

Influenza and paramyxoviruses grow well in the allantoic cavity


c) Yolk-sac inoculation

The position of the embryo is determined by candling

A hole is drilled in the shell at the centre of the air space at the blunt end

A long needle is inserted and the inoculum is deposited just below the centre of the egg

To ensure correct position, pull back the plunger until the yolk sac is pulled up with it


The opening of the inoculation is sealed

Yolk sac inoculation is also useful for cultivation of fastidious groups of bacteria such as Chlamydia and Rickettsia species which do not grow on inanimate media


d) Chorio-allantoic membrane (CAM) inoculation

A small triangle is marked at a site where there are no major blood vessels

This can be located by candling

The shell along the triangle is cut to expose the CAM

A new air is prepared at the site of inoculation by reducing the original air sac

A small hole is cut at the blunt end above the air sac


The egg is placed horizontally

A gentle suction is applied with a rubber teat

A new air space develops at the top of the horizontal egg at the site of the triangular cut

A pipette is filled with about 1 mL inoculum and inoculated through the gap in the CAM and the shell

The egg is rocked gently to disperse the inoculum evenly over the membrane

Both the openings are sealed by replacing the cut triangles with the sealing mixture


4. Harvesting the fluids

In order to harvest the virus infected fluids and other structures, the shell must be opened with great care to avoid unwanted dissemination of the virus

The use of safety cabinets is recommended

Never use a drill to open infected eggs because it will create aerosols


After the desired incubation period, the air sac portion at the blunt end, which is already marked during candling is opened

Forceps or a pair of scissors are used for cutting

The contents of the egg is collected in a sterile petri dish

The fluid from the inoculated area is aspirated with a syringe


After removing the contents of the egg, pull out the CAM gently with tweezers

Wash the CAM in saline 2 - 3 times

Inspect it for the presence of lesions or pocks

The harvested fluids is tested by direct method or other methods for the detection and identification of the virus

Inoculation of organs, tissue fragments or cell monolayers

The 1st application of tissue culture in virology was by Steinhardt et. al. in 1913

They used it for the maintenance of vaccinia virus in the fragments of rabbit cornea

The major obstacle in the development of tissue culture was contamination by bacteria

It was overcome when antibiotics became available


After that, major progress was achieved by Enders and others in 1949 by growing polio virus in the tissues of non-neural origin

Since then, a large number of human viruses have been grown and maintained in tissue cultures


There are 3 types of tissue cultures:

1. Organ culture

2. Explant culture

3. Cell culture


1. Organ culture

Essentially cultured tissue pieces in which the architecture and physiology of the tissue is retained

Such small bits of organs can be maintained in vitro for a few days

Organ cultures are necessary for the growth of some fastidious viruses, e.g. ferret trachea can be used for the isolation of some rhinoviruses and coronaviruses


2. Explant culture

This technique is particularly important for the isolation of viruses in the latent stage

The fragments of the tissue are placed in a test tube or a petri dish in a drop of plasma or fibrin clot

These explants are then covered with growth medium

Adenoid tissue explant cultures were used for the isolation of adenoviruses


3. Cell culture

This is the most widely used technique for growing viruses

By the action of proteolytic enzymes such as trypsin, a tissue is dissociated into its component cells

After washing, cells are suspended in growth medium containing essential amino acids, vitamins, salts, glucose and a buffer


Serum such as foetal calf or newborn bovine serum is added as supplement

Antibiotics are also added to prevent bacterial contamination

A change in pH is indicated by phenol red incorporated in the medium

The cells suspension in growth medium is dispensed in flasks or tubes

The cells adhere to the surface of the flasks


When incubated under appropriate conditions, they divide and redivide to form a confluent sheet of cells in a single layer - monolayer

It is achieved by a mechanism known as contact inhibition occurs when cells come in contact with surrounding cells which inhibits further multiplication

Formation of monolayer to cover the surface of the culture vessel usually takes 3 - 7 days depending on the type of cells used

Types of Cell Culture

3 types of cell culture are used in virology depending on their origin, chromosomal characters and the number of generations they can be maintained

1. Primary cell cultures

2. Diploid cell cultures (semi-continuous cell culture or cell strains)

3. Continuous cell lines


1. Primary cell culture

Prepared from an organ which is minced into small pieces, treated with an enzyme and then used for the preparation of a monolayer

Capable of only limited growth in culture

Only a few serial passages can be made

Advantage: Large number of cultures can be prepared if sufficient animal organs are available


Disadvantage: Primary cell cultures can contain latent viruses in the donor animal

The commonly used primary cell cultures are monkey kidney, human embryonic kidney, human amnion and chick embryo cell cultures

Useful for isolation of viruses such as enteroviruses, myxo- and paramyxoviruses

Used for large scale production of vaccines


2. Diploid cell cultures

These cultures mostly consist of a single type of cells which may undergo 30 - 50 passages before senescence or death of the culture

The human embryonic lung or kidney fibroblasts make excellent diploid cell strains, e.g. MRC5, W138 are fibroblast strains

Diploid cultures are maintained by creating a pool of frozen cells stored in liquid nitrogen


Serial passages of primary cell culture are made and large portions of the initial 8 - 10 passages are stored in liquid nitrogen

When the diploid cell strains start dying, the subcultures can be restarted from the frozen portions cells from the same source can be used for many years giving constant results

Used for selective isolation of some viruses and preparation of vaccines herpes simplex, cytomegalovirus, varicella-zoster, and rhinovirus


3. Continuous cell lines

These cells may be serially sub-cultured indefinitely

These cells have a malignant character, and the number of chromosomes is different from that of the original host

They have very fast growth rate and contact inhibition is absent


Commonly used cell lines are HeLa (human cervical cancer), HEp2 (human epithelial), BHK21 (baby hamster kidney) RK13 (rabbit kidney) and Vero (African green monkey kidney)

HeLa, HEp2 and Vero cells: Cultivation of poliovirus, coxsackie virus, adenovirus and herpes simplex virus

RK13 and BHK21: Isolation and propagation of rubella virus


Cannot be used for preparation of vaccines because vaccines grown in cancer cells are not considered to be safe

Diagnostic Methods

There are several methods by which viral disease can be diagnosed in the clinical lab

There are 3 categories:

I. Direct methods - electron microscopy, immune electron microscopy, immunological methods and nucleic acid hybridisation

II. Isolation and identification of the causative agent

III. Serological diagnosis

Serological Diagnosis

The procedures commonly used include:

1. Indirect immunofluorescence (IF) test

2. Enzyme linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA)

3. Neutralisation (NT) test

4. Complement fixation (CF) test

They are employed for the identification of viral isolates by using a known antiserum against the suspected virus

Serological Diagnosis

Serodiagnosis of viral infections involves detection of antibodies to viral antigens in patients serum in sufficiently high titres

The antibody can be titrated by using a range of dilutions

IF and ELISA are the most commonly used because they are sensitive, easy to perform and less time consuming than other tests such as CF and NT

THE END

Medical Microbiology II Lecture 9

Documents

virology specimens

used specimens

vtmcollection of specimens

gcollection of specimens

storage of specimens

postmortem specimens

large numbers specimens

sterile swab place