MICROBIOLOGY AND PARASITOLOGY

LABORATORY NOTES/ ACTIVITIES

FINALS 2: VIRUS
SPECIMEN COLLECTION AND TRANSPORT

In most laboratories, processing with other microbiology specimens allows viral cultures to be
processed 7 days a week. If delays must occur, specimens should be stored in a viral transport medium at
4°C as described previously. Delay in the processing of fluid specimens requires dilution in a transport
medium (1:2 to 1:5) before storage. In addition to patient identification and demographics, each specimen
for virus isolation should be accompanied by a requisition that provides
1) The source of the specimen;
2) The clinical history or viruses suspected; and
3) The date and time of specimen collection. If this information is not available, a call for additional
details should be made to the requesting physician or to the person caring for the patient.

1. Time of collection - Specimens for viral identification are best collected during the time of first
presentation of symptoms

A. Virus titers (concentration of virus) are usually highest in the early part of the illness.
B. Serologically, a fourfold rise in the antibody titer between acute and convalescent sera has been
used to identify a particular infectious agent as the cause of a recent disease.

2. Viral culture - The type of viral illness and the disease symptoms influence the specimens of choice for
viral culture. Viral specimens are typically divided into non sterile sites and sterile sites.

A. Non sterile sites include the following: Viral transport medium, saline, or trypticase soy broth can
be added to sterile containers to keep tissues from drying. Several viral transport systems are
commercially available. Most transport media consist of a buffered isotonic solution with a protein
such as albumin, gelatin, or serum to protect less stable viruses. Often, antibacterial and antifungal
agents are added to transport systems to inhibit contaminating microorganisms

a. Conjunctival
b. Skin (e.g., mouth, lips)
c. Vesicular Nasal (aspirates or washes)
d. Throat/upper respiratory tract
e. Sputum
f. Urine
g. Genital (cervical, penile)
h. Stool/rectal swab
B. Sterile sites include the following: The transport container should also be unbreakable and able
to withstand freezing and thawing
a. Autopsy
b. Biopsy
c. CSF
d. Fine-needle aspirate
e. Blood
f. Bronchial alveolar (wash)
g. Pleural fluid

3. The procedure for specimen collection is vital for recovery of the infectious virus.
 Specimens collected with a Dacron or rayon swab (cotton swabs are not recommended for
collection of viral samples because they are toxic to viruses) must not dry out during transport. The
swab can be placed in a modified Stuart transport medium for transport to the laboratory.
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LABORATORY NOTES/ ACTIVITIES
 Non sterile specimens that are not liquid, vesicular fluid, fine needle aspirates, or tissue biopsy
specimens should be collected into viral transport media, such as Hanks’ balanced salt solution, 0.2
M sucrose-phosphate, or 2% fetal calf serum with Eagle’s minimum essential medium. Some
transport mediums contain the following ingredients:
1) Antibiotics to inhibit growth of normal bacterial or fungal flora
2)Gelatin media to stabilize protein

 Urine for viral culture should not be collected into containers with preservatives.
 Blood for viral culture should be collected into heparin or ethylenediamine tetra acetic acid tubes.
 Stool specimens for viral culture should be collected into a clean container.

4. All specimens submitted for viral recovery regardless of source should be transported at refrigerated
temperatures (0◦C–4◦C).
5. If the inoculation of the specimen into tissue culture media will exceed 5 days, the specimen should be
quickly frozen at −70◦C.

VIRAL DETECTION METHODS

1. Cytology and Histology
A readily available technique for detecting virus is cytologic or histologic examination for characteristic viral
inclusions. This involves the morphologic study of cells or tissue, respectively. Inclusions resulting from
infection with CMV, adenovirus, parvovirus, papillomavirus, and molluscum contagiosum virus are
detected by histologic examination of tissue stained with hematoxylin and eosin or Pap. Cytology and
histology are less sensitive than culture but are especially helpful for viruses that are difficult or dangerous
to isolate in the laboratory, such as parvovirus and rabies virus, respectively.
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2. Electron Microscopy
Very few laboratories use electron microscopes (EM) to detect viruses, because it is labor intensive and
relatively insensitive. In the clinical virology laboratory, EM is most useful for detecting gastroenteritis
viruses that cannot be detected by other methods (e.g., astroviruses) and encephalitis-causing viruses that
are undetectable with cell culture (HSV, measles virus, and JC polyomavirus).

3. Immunodiagnosis (Antigen Detection)

High-quality, commercially available viral antibody reagents have led to the development of
fluorescentantibody, enzyme immunoassay, latex agglutination, and immunoperoxidase tests that detect
viral antigen in patient specimens. The most useful immunofluorescent stains in the clinical virology
laboratory are those for RSV, influenza and parainfluenza viruses, adenovirus, HSV, VZV, and CMV.

 Direct immunofluorescence is best suited to situations in which large quantities of virus are suspected or
when high- quality, concentrated monoclonal antibodies are used, such as for the detection of RSV in a
patient specimen or the identification of viruses growing in cell culture.
 Indirect immunofluorescence should be used when lower quantities of virus are suspected, such as
detection of respiratory viruses in specimens from adult patients. High-quality monoclonal antibodies
improve the sensitivity and specificity of immunofluorescence testing.
 Enzyme immunoassay methods used in clinical virology include solid-phase enzyme-linked
immunosorbent assay (solid-phase ELISA) and the membrane-bound enzyme-linked immunosorbent
assay (membrane ELISA). The most used enzyme immunoassays for antigen detection are those for RSV
(solid-phase and membrane), rotavirus (solid- phase and membrane), and influenza viruses (membrane).

4. Molecular Detection Using Nucleic Acid Probes and Polymerase Chain Reaction Assays
 DNA probe test used to detect papillomavirus DNA in a smear of cervical cells.
 Nucleic acid probes are most useful when the amount of virus is relatively abundant; viral culture is slow
or not possible; and immunoassays lack sensitivity or specificity
 Rapid PCR testing, referred to as real-time PCR can be used to amplify and detect RNA viruses by
enzyme reverse transcriptase (RT).

5. Cell Culture
 Viruses are strict intracellular parasites, requiring a living cell for multiplication and reproduction. To
detect virus
using living cells, suitable host cells, cell culture media, and techniques in cell culture maintenance are
necessary.
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ANTIVIRAL AGENTS
NON-RETROVIRAL AGENTS
ANTIHERPES AGENT
DRUG/S USES/PROPERTIES
Acyclovir DOC for the ff:
 HSV encephalitis
 Neonatal HSV infection
 CMV
Valacyclovir Can be used for the treatment of:
 Herpes labialis/ Cold sores
 Herpes zoster/ Shingles
 Herpes simplex/ Genital herpes
Famciclovir Herpes zoster/ Shingles
Docosanol Herpes labialis/ Cold sores
Trifuridine Used for the treatment of Primary keratoconjunctivitis and recurrent epithelial keratitis
Ganciclovir CMV retinitis in immunocompromised patients
Valganciclovir CMV retinitis
Foscarnet End-organ CMV
• Ganciclovir/ Acyclovir resistant CMV
Cidofovir CMV retinitis

ANTIVIRAL AGENTS
RETROVIRAL AGENTS
NUCLEOSIDE REVERSE NON- NUCLEOSIDE REVERSE PROTEASE INHIBITOR
TRANSCRIPTASE INHIBITORS (NRTI) TRANSCRIPTASE INHIBITORS
DRUG/S ABBREVIATION/S (NNRTI)
Abacavir ABC Delavirdine – First generation Atazanavir
(teratogen)
Didanosine ddl Efavirenz – First generation Darunavir - co- administered with
ritonavir
Emtricitabine FTC Etravirine – Second generation Fosamprenavir
Lamivudine 3TC Nevirapine – Second generation Indinavir
Stavudine d4T Rilpivirine – Second generation Lopinavir
Tenofovir Nelfinavir - A/e: Diarrhea and
disoproxil flatulence
fumarate
Tenofovir Ritonavir
alafenamide
Zidovudine AZT Saquinavir
Tipranavir
FUSION INHIBITORS ENTRY INHIBITORS INTEGREASE INHIBITORS
Enfuvirtide Maraviroc Dolutegavir
The only parenteral antiretroviral drug Approved for use in combination w/ Elvitegravir
other ARV in adult patients infected Raltegravir
only with HIV
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LABORATORY NOTES/ ACTIVITIES

NAME: DATE:

COURSE/YEAR/SECTION: SCORE:

FINALS 2: VIRUS
1. Draw and label the structure of the following viruses:
a) Tobacco Mosaic virus

b) Adenovirus
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c) Influenza virus

d) Bacteriophage
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2. Define the following:
a) Virus Polymerase

b) Reverse Transcriptase

c) HIV Protease

d) RNA Antigen
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LABORATORY NOTES/ ACTIVITIES
e) Haemagglutinin Protein

f) Neuraminidase Inhibitor

g) Cell defense Proteins

3. As an allied medical student, what is the importance of studying about helminthes and protozoa
including the proper medication for various infections?