SAMPLE COLLECTION AND

LABORATORY DIAGNOSIS
OF VIRAL DISEASES
 VARIOUS SPECIMENS FOR VIRAL DETCTION

Swabbing of lesion sites (e.g. skin, throat)

Scrapping of lesions to obtain infected cells (e.g. bases of vesicles,
corneal ulcers)
Aspirates of secretions or exudates (e.g. from posterior
nasopharynx, conjunctiva, cervix)
Excreta such as urine, faeces
Biopsy specimens obtain by needle aspiration
Blood sample after centrifugation take serum for serological testing.
 SPECIMEN COLLECTION & TRANSPORT

Throat Swab-
 Vigorously rub tonsils and posterior pharynx with cotton-wool swab
moistened with VTM or sterile physiological saline.
 Break off swab head into VTM and send sample to laboratory as
soon as possible.
 SPECIMEN COLLECTION & TRANSPORT

Nasopharyngeal Swab-
 It obtained under direct vision with the thumb of one hand, gently
elevate the tip of the nose. Moisten the tip of a small flexible
nasopharyngeal wire swab with sterile water or saline and gently
insert it into one of the nares.
 Guide the swab backward and upward along the nasal septum until a
distinct feel of resistance indicates that the posterior pharynx has
been reached. Gently remove the swab. Put immediately in VTM with
 SPECIMEN COLLECTION & TRANSPORT

Conjunctival Swab-
 Moisten a sterile cotton-wool swab with virus transport medium or
sterile physiological saline.
 Pull down lower eyelids and swab lower conjunctiva to collect cells.
 After sampling, swab head placed in the VTM container.
 SPECIMEN COLLECTION & TRANSPORT

Cervical swab-
 Visualize the cervix with vaginal speculum, Collect cervical cells by
rolling a fresh cotton-wool swab around the transitional zone of
endocervix.
 Break off swab head into viral/chlamydial transport medium and
transport to laboratory as soon as possible.
 SPECIMEN COLLECTION & TRANSPORT

Urethral swab-
 Insert a fine Aluminium shafted cotton-wool or Dacron swab 2-4 cm
into urethra.
 Rotate swab 2-3 times and remove.
 Place swab in VTM. Transport to laboratory immediately, if possible.
 SPECIMEN COLLECTION & TRANSPORT

Vesicle fluid-
 Select a freshly erupted vesicle, preferably containing clear fluid.
 Carefully puncture vesicle and collect the fluid on a cotton-wool
swab.
 Rub the base of lesion with same swab.
 Break off head of swab into VTM and transport to laboratory as
soon as possible.
 SPECIMEN COLLECTION & TRANSPORT

Stool-
 Collect walnut-sized stool specimen into a suitable clean container.
 Refrigerate specimen to retard growth of bacteria and transport to
laboratory as soon as possible.
 LABORATORY DIAGNOSIS OF VIRAL DISEASES

 Direct Demonstration of Virus

 Electron microscopy
Immunoelectron microscopy
Fluorescent microscopy
Light microscopy
Detection of Viral Antigens
By various formats such as ELISA, direct IF, ICT, flow through assays.
 LABORATORY DIAGNOSIS OF VIRAL DISEASES

 Detection of Specific Antibodies

Conventional techniques such as HAI, neutralization test and CFT
Newer diagnostic formats such as ELISA, ICT, flow through assays.
Molecular Methods to Detect Viral Genes
Nucleic acid probe—for detection of DNA or RNA by hybridization
PCR—for DNA detection by amplification
Reverse transcriptase-PCR—for RNA detection
Real time PCR—for DNA quantification
Real time RT-PCR—for RNA quantification.
 LABORATORY DIAGNOSIS OF VIRAL DISEASES

Isolation of Virus by,

Animal inoculation
Embryonated egg inoculation
Tissue cultures: Organ culture, explant culture, cell line culture (primary,
secondary and continuous cell lines).
 LIGHT MICROSCOPY

 Inclusion bodies:

 Histopathological staining of tissue sections may be useful for detection of inclusion

bodies which helps in the diagnosis of certain viral infections .
 Immunoperoxidase staining:

 Tissue sections or cells coated with viral antigens are stained using antibodies tagged
with horse radish peroxidase following which hydrogen peroxide and a coloring agent
(benzidine derivative) are added. The color complex formed can be viewed under light
microscope
 DETECTION OF VIRAL ANTIGENS

 Some important antigen detection tests include:

 HBsAg and HbeAg antigen detection for hepatitis B infection from serum.

 NS1 antigen detection for dengue virus infection from serum

 p24 antigen detection for HIV from serum

 Rotavirus antigen detection from diarrheic stool

 CMV specific pp65 antigen detection (serum)

 DETECTION OF VIRAL ANTIBODIES

 Antibody detection from serum is one of the most commonly used

method in diagnostic virology.
 Appearance of IgM antibody or a four-fold rise of titer of IgG antibody
indicates recent infection.
 Presence of IgG antibody (without a recent rise) indicates chronic or
past infection.
 DETECTION OF VIRAL ANTIBODIES

 Newer diagnostic formats such as ELISA, ICT, flow through assays

are widely used for antibody detection against most of the viral
infections such as:

 Anti-HBc, Anti-HBs and Anti-HBe antibodies for Hepatitis B infection

 Anti-Hepatitis C antibodies

 Antibodies against HIV-1 and HIV-2 antigens from serum

 Anti-Dengue IgM/IgG antibodies from serum.

 VIRAL TRANSPORT MEDIUM (VTM)

 It consist of,

 Hank’s balanced salt solution

 10% bovine albumin solution

 Sodium bicarbonate

 Penicillin/streptomycin solution in phosphate-buffered saline

solution A (PBSA)
 0.4 % phenol red
 VIRAL TRANSPORT MEDIUM (VTM)

 Uses-
 It allows safe transfer of viruses, chlamydia and
mycoplasma for further research, including
conventional cell culture methods, diagnostic
tests, and molecular b iology techniques.