laboratory diagnosis of smallpox virus

8/9/2019 Laboratory Diagnosis of Smallpox Virus

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Laboratory Diagnosis ofSmallpox Viral Infection

Presented by

Walid Fouad, Dr. Ahmad Moussa, Hamd AllahMohammad, Ahmad Ali El Tayeb, Akram

Fathy, Ramy Ali, Moemen Gamil, and

Mahrous Sayed

Microbiology Department, Medical ResearchInstitute Alexandria University


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Variola Virus

1. One of the largest known viruses in size

(200 x 250 nm).

2. Belongs to family Poxviridae, and genus

Orthopoxvirus.

3. Enveloped and having a one piece of a double-

stranded DNA genome.

4. Causes the smallpox disease - a systemic viral

infection with fever and a characteristic skin rash thatresulted in high mortality rates in the developing

countries.


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Smallpox Disease:

5. In 1980, the World Health Organization (WHO)

General Assembly ratified the declaration of success

made by the Global Commission for the Certification

ofSmallpoxEradication.

6. Despite the eradication of naturally occurring

smallpox and the availability of a vaccine, the

potential "weaponization" of variola virus continuesto pose a military threat.


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Diagnosing a smallpox viral infectionA. CLINICAL DIAGNOSIS

B. LABORATORY DIAGNOSIS:

1. Sample Collection

2. Light Microscopy and Histopathology

exam of stained smears3. Electron Microscopy of pustular fluid

4. Tissue Culture

5. Serological/Immunological Tests

a. ELISA

b. Immunofluroscence Test

c. Virus Neutralization Testd. Complement Fixation Test

e. Heamagglutination-inhibition Test

6. Molecular Diagnostic Techniques

- PCR


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A. Clinical DiagnosisInitial diagnosis of a smallpoxinfection is most likely based on ahistory and physical examinationfindings.

Smallpox is an illnesscharacterized by:

a) An acute onset offever

(> 38.3C).

b) A rash that usually follows the fever, and is characterized byfirm, deep seated vesicles orpustules (blisters filled with fluid) inthe same stage of development.


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Other symptoms may include:

Severe headache Backache Fatigue

Malaise Vomiting Diarrhea Excessive bleeding (in some cases)

** Before a diagnosis of smallpox is made, the doctor will consider otherillnesses that can mimic the signs and symptoms of smallpox.Some of these illnesses include:

Chickenpox, Monkeypox, Herpes Zoster (Shingles), and Impetigo.


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Smallpox vs Chickenpox:

A disease that can still be confused with smallpox is

varicella(chickenpox).

Chickenpox is caused by the varicella-zoster virus, aDNA virus that belongs to the family of Herpesviridae.

Similar to smallpox, chickenpox is transmitted via

respiratory secretions or contact with skin lesions, and

usually presents with an abrupt onset of a pruritic rash,

low-grade fever, and malaise.


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Differential Diagnosis:Chickenpox and smallpox can

be distinguished by several

methods, including the clinical

course and the characteristic

appearance of the skin

lesions.

(1) Unlike smallpox, the

distribution of varicella lesions

is centripetal with a greaterconcentration on the trunk.

Chickenpox does not usually

affect the palms and soles.


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Differential diagnosis (cont.:

(2) Additionally, chickenpox pustulesare of varying size due to variations

in the timing of pustule eruption (asyn-chronousprogression), where newlesions appear in crops every few days

and are seen in different stages ofmaturation.

Smallpox pustules, on the contrary,are all very nearly the same sizesince the viral effect progresses moreuniformly (synchronous progression).

Once a clinical case of a smallpoxinfection is observed, it is important toconfirm it using laboratory tests.


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Differentiation between Smallpox and Chickenpox:


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B. Laboratoy Diagnosis1. Collection of a specimen:

Eitherpustular fluid orscabs can serve

as specimens for laboratory evaluation.

For suspected cases of hemorrhagic

smallpox, the doctor may sample fluid

from a spinal tap (lumbar puncture).

To collect specimens, use forceps to

pick off a scab, the blunt edge of a

scalpel to open vesicles or pustules, and

a cotton swab to absorb the fluid.

Deposit specimens in a blood collection

test tube with a rubber stopper, seal the

stopper with adhesive tape, and enclose

the tube in a watertight container.


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Sample Collection:Specimens should be collected

only by a recently vaccinated

individual wearing latex gloves

and a surgical face mask.

Laboratory examination must

be performed in a Biosafety

level IV facility.

It should be also performedonly in designated laboratories

with the appropriate training

and equipment.


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2.Light Microscopy of stained smears:

Microscopically, poxviruses produce characteristic cytoplasmicinclusions, known as Guarnieri bodies (also called B-typeinclusions) that can be seen by a light microscope.

Guarnieri bodies are characterized by being:

a. Eosinophilic intracytoplasmic viral inclusions.

b. Sites of viral replication in the host cell.

c. Variable in size, paranuclear(along side the nucleus).

c. Usually found upon microscopic inspection of epithelial cellsof patients suspected of having a poxvirus infection

(e.g. smallpox, monkeypox or vaccinia).


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Guarnieri bodies under light microscope:Histological sections or smears ofclinical specimens on glass slide arestained with Hematoxylin andEosin (H&E), and Guarnieri bodiesusually appear as pink blobs in thecytoplasm of affected epithelial cells.

Unlike A-type inclusions (which aremore strongly eosinophilic and onlyfound in infections with certainpoxviruses), Guarnieri bodies arefound in virtually all poxvirusinfections, but the absence ofGuarnieri bodies cannot be used to

rule out smallpox.Examining affectedcells for Guarnieri bodies can nothelp in differentiating a smallpoxviral infection from other poxvirusinfections.


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3.Electron Microscopy of pustular fluid:The diagnosis of an orthopoxvirus infectioncan also be made rapidly by electronmicroscopic examination of pustularfluid or scabs. EM can distinguish orthopoxviruses from other viral agents.

However, all orthopoxviruses exhibitidentical, smooth, rounded "brick-shaped" virionsby electron microscopy.

Thus, electron microscopy of vesicularscraping will not help to differentiatebetween variola, vaccinia, cowpox ormonkeypox viruses.

The definitive diagnosis and identificationof species of orthopoxvirus rests on viralculture and characterization of the virusby various biologic assays, includingPCR and restriction fragment-lengthpolymorphisms


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4.Tissue culture technique

and examining pock morphology on CAMs:Using tissue culture techniques, asmallpox infection can be definitelydiagnosed by:

1. Growing the virus onChorio-

Allantoic Membrane - CAM (part of achicken embryo), and

2. Examining the resulting pocklesions under defined temperatureconditions.

This method is very helpful in thedifferential diagnosis between asmallpox (variola) virus, and othermembers of the genus Orthopoxvirus(such as: monkeypox, vaccina, andcowpox viruses).


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Viral Pock Morphology:

When a chorioallantoic membrane of 12-day-old developingchicken embryos are inoculated with orthopoxviruses andincubated at 35 C for 72 hours, morphologicallycharacteristic pocks typical of each virus will beobserved as shown in the following figures:

a) Variola (smallpox) virus produces small, grey-white pocks.

b) Vaccinia virus produces larger, opaque, whitepocks with pale hemorrhagic ulcer.

c) Monkeypox virus produces small hemorrhagiculcer.

d) Cowpox virus produces hemorrhagic pocks.


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5.Serological/Immunological Tests:

I. Enzyme linked immunosorbent assay (ELISA):

a) ELISA can be used to detect variola virus-specific immunoglobulins.

b) It may also used to detect a specific viral antigen by using the double antibodySandwich technique.

II. Immunofluroscence Test:

a) Relatively simple and rapid.

b) Used fordetecting viral (antigen) particles in suspected samples.

c) In the indirect immunofluroscence technique, an immune rabbit serum is used to treat thesample smears. This is followed by the addition of an anti-rabbit serum "coupled" with afluorescent dye.A fluoresencent sample means a positive result.

c) The indirect technique gives more reliable results than the direct one.


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ELISA procedures:


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Serological Tests (cont.:

III. Viral Neutralization Test (VNT):

a) Definition - Neutralization of a virus is defined as the loss of infectivity through reaction of the viruswith its specific antibody.

b) Purpose - Neutralization test is used to determine the antiviral activity of the serum of a suspected

case. (I.e.In this test, the serum of the patient is tested for its power to "neutralize" variola virus in tissue cultureor on CAM).

C) Test Steps -1) Mixing the virus with the serum to be tested (which is supposed to contain virus-neutralizing antibodies) under appropriate condition, and then 2) Inoculating that mixture into cell culture,eggs or animals. Neutralization test, therefore, requires that an active suspension of that virus should be athand.


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**Disadvantages of Neutralization Test:

a) The test is rathermore time-consumingthan complement-fixation or heamagglutination inhibition.

b) The results are not always easy to interpretif the patient has been previously vaccinated against

smallpox. Neutralizing antibodies may persist in serum for years after smallpox vaccination, and usually

fade in about 10 years.

IV. Complement Fixation Test (CFT):

The complement fixation test is an immunological medical test that can be used to detect the presence

of either specific antibody or specific antigen in a patient's serum.


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Methodology of the Complement Fixation Test:


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V. Heamagglutination Inhibition Test (HIT):

A wide variety of different viruses possess the ability to agglutinate

erythrocytes. This is the outcome of the haemagglutinin part of the viral protein

binding to receptors on the membrane of red blood cells. The linking together of the

red blood cells by the viral particles results in clumping. This clumping is known ashaemagglutination, and is visible macroscopically and is the basis of

haemagglutination tests to detect the presence of viral particles.

Antibodies against the viral protein responsible for haemagglutination can prevent

haemagglutination; this is the basis behind the haemagglutination-inhibition test

(HAI).


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The HAI test is simple to

perform.

Serial dilutions of patient's sera

are allowed to react with a fixeddose of viral haemagglutinin,

followed by the addition of

agglutinable erythrocytes.

In the presence of antibody, theability of the virus to agglutinate

the erythrocytes is inhibited.


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6.Molecular Diagnostic Techniques:

Polymerase Chain Reaction (PCR):

a) PCR is a relatively simple, rapid, sensitive, and accurate method for detectionand differential diagnosis of several different orthopoxvirus infections.

b) PCR helps in diagnosing a smallpox infection through the identification ofspecies-specific DNA sequences.

d) Various polymerase chain reaction techniques:

- Essential conserved genes (E9L, A25R) - difficult to discriminate among

species of orthopoxviruses.- Nonessential, variable genes (HA, HTI, crmB) - species specific.

laboratory diagnosis of smallpox virus

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