Laboratory diagnosis of viral infection

Laboratory diagnosis of viral infection

1. Laboratory diagnosis Laboratory diagnosis
of viral infectionsof viral infections
Dr.Yogita Mistry
GMC,Surat

2. ObjectivesObjectives
Introduction
Why to diagnose viral infections?
Precaution before viral diagnostic
procedure
Different diagnostic methods
Commonly used diagnostic methods for
common viral infections

3. Introduction:Introduction:
Viruses are now emerging and
reemerging to cause many diseases in the
human than previous decades.
So diagnosis of virus infection is much
more important now.

4. Why to diagnose a viral diseases?Why to diagnose a viral diseases?
Screening of viral infection (blood donors,
pregnant mothers,elder women, TB patients)
For diagnosis of etiological agent of viral
infection
For instituting proper (HIV, Herpetic
encephalitis) antiviral therapy
Detection and prediction of etiological agent
of Epidemics
Identification of antigenic variation of virus-
which helps in vaccine preparation, control
of out breaks
Helps in suspecting a new viral infection

5. Various diagnostic methods:Various diagnostic methods:
Microscopy
Antigen detection
Antibody detection
Nucleic acid detection
Virus isolation
Cytology
Histology

6. It is important for diagnosis of viral It is important for diagnosis of viral
infection …..infection …..
Proper
 Sample collection- proper site, proper time(CD4),
quantity(saliva), swabs
Transportation (VTM- protein, antibiotic ,pH indicator, not
reqiured for sterile body fluids or CSF, should not be diluted)
 Preservation(>72 hours, avoid repeated freezing & thawing)
 Test (HIV in infant)
Method (Ag, antibody detection, Viral culture)
Kits (higher sensitivity for screening of HIV)

7. –Dacron, rayon or nylon swabs are used because
they are dry swab.
–Calcium alginate, cotton swabs or wooden stick
swabs are not to be used because they are toxic
for the enveloped viruses.
–Sample collected with this swab are not
suitable for PCR, DFAT or EIA.
–Wooden shafted swab are not recommonded for
culture because it contain toxins and
formaldehyde.

8. Specimen Collection procedure
Vesicular lesion Fluid & material from base of vesicles.
place in VTM
Urine, stool, sputum, BAL, Body fluidsSterile container
Throat swab, nasopharyngeal swabVTM
Eye swab/ scrapping material VTM
Bone marrow(2 ml) in EDTA or heparinised vacuttainer
Tissue VTM

9. Viral Transport MediumViral Transport Medium
Cell culture medium
Stuart medium
Leibovitz agar charcoal medium (CVTM)
Modified Leibovitz-Emory medium
Skimmed milk based medium
HH medium
Bentonite containing media
SPG (Sucrose phosphate glutamate) medium
Tryptose phosphate broth
Bartel’s viral transport medium
Richards viral transport medium

10. Ideal VTM should have better Ideal VTM should have better
preservation of virus, suitable preservation of virus, suitable
for all test, long shelf life, readily for all test, long shelf life, readily
available, inexpensive.available, inexpensive.

11. Various viral diagnostic
methods in details

12. MicroscopyMicroscopy
Light microscopy
Electron microscopy
Fluorescence microscopy

13. Light microscopyLight microscopy
Uses:
To see the inclusion bodies
To observe the cytopathic effects
produced by growing viral in the cell lines
To see the cell lines
 To see the compactness of cell
 Rounding of cell after treatment with trypsin
For Histopathological slides examination
 After immunohistochemical staining etc

14. Inverted microscopesInverted microscopes

15. Inclusion bodiesInclusion bodies
Viral infected cell showing altered staining behavior
Dense aggregates of stainable substances of viruses,
usually proteins.
Produced due to developmental / maturation process
of virus or in some viruses seen only after viral
multiplication.
If virus assembled in the nucleus, it will found in
nucleus. If virus assembled in cytoplasm , then it will be
found in cytoplasm.

16. Examples:Examples:
Intracytoplasmic:
Rabies- Negribodies
Small pox- Gaunier bodies
Fowl pox- Bollinger bodies
Mollascum contagiosum- Henderson peterson
bodies

17. Intranuclear:
 Cowdry Type A:
Variable in size, granular in
appearance
Seen in Herpes, Yellow
fever

18. Cowdry Type B:
Multiple, more circumscribed
 Adenovirus(basophilic)
Poliovirus(acidophilic)
Owl eye: CMV
Both intranuclear and
intracytoplasmic:
Warthin finkeldey bodies-measles

19. Disadvantage: Although highly specific , less
sensitive than other diagnostic methods

20. Electron MicroscopyElectron Microscopy
Although many rapid diagnostic
methods , molecular methods are
available but still the electrone
microscopy has its own role in diagnostic
methods specially in detecting new and
unusual outbreaks.

21. Examples:Examples:
Ebola outbreak in 1976 in zaire- only electrone
microscope was able to find the virus.
Henipavirus( Hendra & Nipah) outbreaks in Asia and
Australia –was first described by using EM.
LCM (Lymphocytic coriomeningitis virus) in transplant
patients
SARS (Sub acute respiratory syndrome virus) agent –
identified and classified in a corona virus.
Monkey pox virus

22. Advantage:Advantage:
Not require a live/ intact virus-so can be useful to
identify virus after decades from the samples
Only after having a visual look on any unsuspected
newer pathogen, other diagnostic test can be possible
to make
Rapid procedure- specially in surveillance of virus that
might be used as terrorist.
Useful in elucidating viral attachment and replication
which can be useful in discovery and design of antiviral
agent and vaccines.

23. Disadvantage:Disadvantage:
Handling with universal precautions is must.
A separate room
Require electrons
Extremely expensive
Sample preparation is often much more elaborate. It is often
necessary to coat the specimen with a very thin layer of metal
(such as gold).
The sample must be completely dry. This makes it impossible to
observe living specimens.
It is not possible to observe moving specimens (they are dead).
The image is only black/white.

24. Current uses:Current uses:
In body fluids specially stool and urine
Other fluids like tears ,CSF ,BAL
(bronchoalveolar lavage)
Stool samples because:
Most gastroenteritis viruses do not grow in tissue
culture maintained by routine viral culture laboratories
and molecular and immunological reagents do not exist
for all.
So EM is the only modality to catch all these agents if
present in sufficient number.

25. Urine sample:
Because > 90% adults have been exposed
to polyoma viruses( SV-40) and would
most likely to give false positive PCR
results in urine
So for diagnosis of polyoma virus in bone
marrow/ transplant patient EM is used.

26. Fluorescence Microscopy:Fluorescence Microscopy:
Uses
Mycoplasma contamination diagnosis in cell
culture- because it can affect viral interference on cell line
After viral culture in cell lines- used for viruses
which do not produces cytopathic effects, even for rapid
diagnosis of viruses which produces cytopathic effects very
late
In herpes infection from direct lesions
Rabies diagnosis from various samples
Respiratory viral infections

27. FAT on brain smearFAT on brain smear

28. HSV in skin smearHSV in skin smear

29. Advantage:
Rapid
Sensitive
Simple
Disadvantage:
Requires fluorescence microscope
Specific antiviral antibodies tagged with fluorescence dyes
Experienced technician to interpret true positive result
Results are affected by quality of sample, staining procedure, smear
preparation

30. Virus cultivation & isolation :Virus cultivation & isolation :
Viruses are obligate intracellular parasites
so they depend on host for their survival.
They cannot be grown in non-living
culture media or on agar plates alone,
they must require living cells to support
their replication.

31. Although virus cultivation is laborious
and time consuming , it is still useful in:
To isolate and identify viruses in clinical
samples.
To do research on viral structure, replication,
genetics and effects on host cell.
To prepare viruses for vaccine production, in-
house diagnostic kit production

32. Methods for cultivation:
Animal inoculation
Egg inoculation
Tissue culture

33. Animal inoculation:Animal inoculation:
It is the earliest method for the cultivation of
viruses causing human diseases.
Reed & colleagues in 1900 used human volunteers
for their work on yellow fever.
In 1909, Landsteiner & Popper used monkeys to
isolate Polio virus.
Theiter used white mice which extended the scope
of animal inoculation greatly.

34. Animal experiments always have serious ethical and
welfare implications.
The 3Rs have been defined as "all procedures which can
completely replace the need for animal experiments,
reduce the numbers of animals required, or reduce the
amount of pain or distress suffered by animals in
meeting the essential needs of man and other animals”
(Smythe, 1978).

35. Most commonly used in virus cultivation are
 Rabbit,
Guinea pig,
Mouse,
Rat
Different animals are used for different viruses
& also there exists different routes to
inoculation.

36. There are seven routes of inoculation
that may be used and include:
Intracerebral (i/c)
Intraperitonial (i/p)
Intramuscular (i/m)
Subcutaneous (s/c)
Intradermal (i/d)
Intravenous (i/v)

37. Factors affecting the outcome of use of
experimental animal
Choice of animals:
The most susceptible animal must be selected
for a particular sample. animal should be free
of disease.
Age of animals:
The older the animal the less susceptible they
become.
Route of inoculation:
The intracerebral route is the most common
route for experimental infection.
Time of sample inoculation:
The fresher the sample the more likely a virus
will be isolated

38. Advantages of Animal Inoculation
To study the pathogenesis and clinical symptoms
of viral infection
For diagnosis of viral infection
To study the immune response by viral
infection( humoral/cell mediated)
Production of antibodies can be identified
Primary isolation of certain viruses
 To study viral replication
Source of different cell lines
Source of RBC for hemagglutination test for
different virus

39. Disadvantages of Animal
Inoculation
Expensive
Difficulties in maintenance of animals.
Difficulty in choosing of animals for particular
virus
Issues related to animal welfare systems.
Animal may interfere with viral growth and that
animals often harbor latent viruses.

40. Transmission of Rabies Virus from an Organ Donor to Four
Transplant Recipients.
 Engl J Med 2005; 352:1103-1111March 17, 2005DOI: 10.1056/NEJMoa043018.
Rabies is an acute encephalitis . The primary mode of transmission is through
the bite of an infected animal, most commonly a bat in the United
States.Although transmission of rabies virus from corneal transplants has
previously been described,to our knowledge, no cases ascribed to organ or
vascular-tissue transplants have been reported.
In May 2004, physicians at a hospital in Texas diagnosed encephalitis in three
recipients of a liver and two kidneys from a common organ donor. It was
later discovered that encephalitis also developed in a fourth patient, who had
received a vascular graft from the same donor during liver transplantation. All
four patients became progressively obtunded, lapsed into coma, and died
within 50 days after transplantation. The initial diagnostic evaluation revealed
no cause of the encephalitis.

41. Animal Uses
Rabbit: •Culture of Rabies virus ,Pox virus
Infant
mouse:
•For culture of coxsackie virus
•Source of adrenal cell line , embryonic swiss mouse fibroblast (3T3) cell line
•Culture of Rabies virus
Rat: •RBCs for hemagglutination of Adenovirus
•Source of A-10 cell line
Guinea pigs• RBCs for viral hemagglutination of influenza and para influenza viruses
Hamster: •Source of BHK-21 (baby hamster kidney)Cell line , CHO(chinese hamster
ovary) cell line
Sheep: •Erythrocytes for the diagnosis of rhinovirus .
• Sheep brain is used for preparation of killed vaccine of rabies made by fixed
virus grown in the sheep brain & inactivated by phenol or beta propiolactone.
• Culture of Pox virus

42. Animal Uses
African green
monkey:
•Source of CV-1 cell line ( kidney fibroblast ) , vero cell line ( kidney
epithelia)
Resus Monkey:•Culture of pox virus
• Source of Rhesus monkey kidney cell line , Primary monkey kidney cell
line , HL-8 cell line.
• RBCs for heamagglutination of Measles virus and Adeno virus
Duck: •Its egg is used to grow Rabies virus
• Rabies duck embryo vaccine
• Its RBCs is used for viral hemagglutination of Influenza ,Flavi virus and
Toga virus .

43. Embryonated eggsEmbryonated eggs
Most useful form of living animal tissue for
Isolation of viruses
For titrating viruses (pock assay)
For large quantity cultivation in the production of viral
vaccines.
Viruses are inoculated mostly in chick embryo of 7-12
days old.

44. Advantage:Advantage:
Free from contamination of bacteria and many latent
viruses
Sterile and wide range of tissue and fluids available for
inoculation
Specific and nonspecific defenses are not involved in
embryonated egg
Widely used method for some viral vaccine production
like yellow fever(17 D strain) and rabies (flury strain).
Disadvantage:
Difficulty in inoculation in different sites
Different sites for different virus

46. Procedure:Procedure:
First egg shell is disinfected with iodine and spirit.
Penetration is done by small sterile drill.
After inoculation, opening is sealed with gelatine/parafin
and incubated at 36˚C for 2-3 days.
After incubation, egg is brokened and virus is isolated
from tissue of egg or virus growth is observed by
Death of embryo
Cell damage
Formation of pocks/lesions on egg membrane.

47. Candling
box

50. Egg holdersEgg holders

51. Flexible drill with shaftFlexible drill with shaft

52. Allantoic inoculation:Allantoic inoculation:

53. Chorioallantoic membrane Chorioallantoic membrane
inoculation:inoculation:
For inoculation in the ectodermal
layer of chorioallantoic membrane ,
an artificial air space has to be
created and the original air sac
obliterated. A small equilateral
triangle is cut out of the shell and a
hole is pierced into the blunt end of
the shell, perforating the air sac.
After lifting the triangluar piece, a
small opening is made in the shell
membrane and slight suction is
applied to the hole in the blunt end.
A new air space developes at the
top of horizontally placed egg and
the inoculum is dropped in.

55. ‘‘POCK’ LESIONS ON CAMPOCK’ LESIONS ON CAM

56. Amniotic cavity Amniotic cavity
inoculation:inoculation:
The translucent
membrane is pierced and
closed forceps are
inserted. The forceps are
opened and the amniotic
membrane is gripped and
pulled out. The
inoculation is made with
a fine needle syringe. This
methods involves more
risk to the embryo.

57. Yolk sac inoculation:Yolk sac inoculation:
Egg shell at the site of
air sac is removed and
then direct
inoculation in yolk sac
is possible.

58. Tissue cultureTissue culture
Important method of viral cultivation, demonstrated by Enders,
Weller and Robbins in 1949.
An additional advantage is that in contrast to most antigen and nucleic
acid detection methods, viral culture allows detection of multiple
viruses, not all of which may have been suspected at the time the
culture was ordered.
Types:
Organ culture
Explant culture
Cell culture

59. Organ culture:Organ culture:
Small bits of organs can be maintained in vitro for days
and weeks ,preserving their original architecture and
function.
Useful for the viruses which appear to be highly
specialized parasites of certain organs.
For example: tracheal rings for isolation of coronavirus.

60. Explant culture:
Fragments of minced tissue can be grown as ‘explant’
embedded in plasma clots.
They may be cultivated in suspension.
Example: Adenoid tissue explants cultures used for
isolation of adenovirus.

61. Cell culture:Cell culture:
It is a process by which cells are grown
under controlled conditions, outside of
their natural environment.
For this , cell lines can be obtained from
human, animal or mosquito.

62. Types of cell culture:Types of cell culture:
Based on their origin, chromosomal
characters and number of generation 3
types:
Primary cell culture:
Diploid cell strains
Continous cell lines

63. Primary cell cultures:
Normal cells taken from body and culture
Only limited growth in culture and can not be
maintained in serial culture
Example: Monkey kidney, Human embryonic kidney,
human amnion and chick embryo cell culture.

64. Diploid cell strains:
Can retain the original diploid chromosome number
and karyotype during serial subcultivation for limited
number of times.
After a specific no. of serial passage , they undergo
‘senescence’(gradual deterioration of functional capacity
or biological aging)
Example:
WI-Human embryonic lung cell strain
HL-8-Resus embryo cell strain

65. Continuous cell lines:
Cells usually derived from cancer cells that are capable
of continuous serial cultivation indefinitely.
Example:
HeLa: Human carcinoma of cervix cell line
Hep-2: Human epithelioma of larynx cell line
KB: Human carcinoma of nasopharyngeal cell line
McCoy: Human synovial carcinoma cell line
Detroit 6: Sternal marrow cell line
Chang C/I/L/K :Human conjunctival/intestine/liver/kidney cell lines
Vero: Vervet monkey kidney cell line
BHK-21: Baby hamster kidney cell line

66. Other types:Other types:
Type Shape after attachment
Epithelial cell line Flattened and polygonal
Fibroblast cell line Bipolar and elongated
Lymphoblast cell line Do not attach, remain in
suspension

67. Cell line can be adherent or floating.
Adherent cell line:
Adherent to surface like plastic/glass
Form a monolayer
When all surface is covered by cells, growth slows and
ceased so repeated passaging of cell lines is required
Passaging ratio is variable with different cell line.
Floating cell lines:
Remain free in the medium.
Also called suspension culture medium
Usually grow in magnetically rotated spinner.

68. Because no one cell culture type can support the growth
of all medically relevant viruses, virology laboratories
must maintain several different cell culture types.
The minimum requirements are :
Primary monkey kidney cell line: used for the isolation of
respiratory and enteroviruses
Human fibroblast line : Used for the isolation of cytomegalovirus
(CMV), varicella-zoster virus (VZV), and rhinoviruses.
A continuous human epithelial cell line such as HEp-2 :
required for the isolation of RSV.

69. The shell vial culture methodThe shell vial culture method
First developed for CMV ,
Dramatically decreases the time required for detection of viruses
in cell culture
 The method involves centrifugation of the specimen onto the cell
culture monolayer and incubation for 1–2 days, followed by
fluorescent antibody staining of the cell culture, regardless of
whether CPE is visible.
In addition to detection of CMV, shell vial cultures have also been
used to speed the detection of HSV, VZV, respiratory viruses, and
the enter viruses.

70. Recent advance in cell cultureRecent advance in cell culture
Use of micro carriers- 3 dimensional cell
culture is possible.

71. Difficulties with cell cultures:Difficulties with cell cultures:
Specific difficulties arise in the extraction of  cells from a "safe" tissue.
Available from NCCS (National centre of cell science, Pune) or globally
from ATCC( American type culture collection).
 Slow growth rates with doubling times in between 12 and 28 hours
Complex requirements of growth medium
Many cell lines grow in an adherent manner and a suitable surface for
attachment has to be provided for these cells to proliferate.

72. •Susceptible to bacterial contamination.
•Susceptible to toxic substances which may be present in the specimen.
•Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses,
parvovirus, papillomavirus
• Mammalian cells have originated from  the multi-cellular organisms, they still
carried the genetic program of inducing their own cell death; a process called
"apoptosis" or "programmed cell death". It can limit culture productivity in
biotechnological processes

73. Dulbecco's Modified Eagle Media (DMEM) Dulbecco's Modified Eagle Media (DMEM)
Basal medium consisting of amino acids , vitamins , gulcose , salts, 
and a pH indicator, which contains no growth promoting agents.
 Also called minimum essential medium.
 Therefore, it needs supplementation to be a "complete"
medium.
It is commonly supplemented with 5-10% Fetal Bovine Serum
(FBS).
DMEM used a sodium bicarbonate buffer system (3.7 g/L) and
therefore requires artificial levels of CO2 to maintain the required
pH. 7-10% CO2 is optimal but many researchers successfully use
it in 5% CO2. The potential problem with too low CO2 level is
that the pH may become too high. When exposed to ambient
levels of CO2, the sodium bicarbonate in the medium will cause
DMEM to become basic very rapidly.

74. Why to do subculture?Why to do subculture?
In order to keep the cells actively and healthy growing
This sub cultivation process involves breaking the bonds or cellular 'glue'
that attaches the cells to the substrate and to each other by using
proteolytic enzymes such as trypsin , dispase , or collagenase.
Sometimes, these enzymes or dissociating agents are combined with
divalent cation chelators such as EDTA which binds calcium and magnesium
ions.
Cells then reattach, begin to grow, divide and after a suitable incubation
period (depending on the initial inoculum size, cell lines and  growth
conditions), and again reach confluency( 70-80%) or saturation. At this
point, the sub cultivation cycle is repeated.
 

75. Cell Culture (Attached Cell)_HIGH.mp4

77. Detection of virus growth in Detection of virus growth in
cell culturecell culture
Cytopathic effect
Metabolic inhibition
Hemadsorption
Interference
Transformation
immunofluorescence

78. Neuro2a cells infected with street virus

79. CV
400x
400x
BHK 21 cells infected with rabies virus

80. Cytopathic effects:Cytopathic effects:
Structural changes that are caused by viral invasion in the host cell.
Infective virus causes lysis of host cell or cell dies without lysis due to
inability to reproduced.
Diagnostic uses of CPE:
Important in viral diagnosis, because they are characteristic features
Can be seen in unfixed, unstained cells under low power of optical
microscope
Usually appear 4-5 days in vitro with low multiplicity of infection, 1-2
days with high multiplicity of infection
Helpful in assessing the virus viability, viral-load, efficacy of new drug
in sample by doing plaque assay.

81. Different cytopathic effects:Different cytopathic effects:
Total destruction of host cell monolayer:
Most severe form of CPE
Whole cell monolayer shrinks rapidly, detached from glass surface within 3
days.
Seen in Enterovirus.
Subtotal destruction:
Less severe form
Detachment of some but not all cells
Seen with Toga ,Picorna and Paramyxoviruses

82. Focal destruction:
Localized attack of host cell monolayer in the initial stages
Focally occurs due to direct cell to cell transfer of virus rather than
diffusion through extracellular medium.
Initially host cell become enlarged, rounded and refractile and eventually
detached from surface.
Spreading of virus occurs concentrically, so that cell lifting off are
surrounded by enlarged, rounded cells which are surrounded by healthy
tissue.
Can observed only with fixation and staining of cell.
Seen in herpes, Pox virus
Syncystium formation:
Due to cell fusion
So enlarged cell with at least 4 nuclei are seen
Can be seen without staining
Seen in herpes virus

83. Swelling & clumping:
Host cell swells significantly
Once enlarged, cell clumps together in clusters and detached.
Seen in Adenovirus
Foamy degeneration/Vacuolization:
Formation of large and/ or numerous vacuoles in the cytoplasm
Seen in Rotavirus , Flavivirus, Paramyxovirus

84. Plaque assay:Plaque assay:
 The standard method used to determine virus
concentration in the term of infectious dose.
Determines the plaque forming units (pfu) in a viral
sample.
Done in petri dish/ multiwell plates.
A confluent monolayer of host cell is infected with
the virus at varying dilution and covered with a
semi-solid medium such as agar/ carboxy methyl
cellulose to prevent virus infection from spreading
indiscrimately.
When virus causes infection of cell, it will lysed and
spread the infection to adjacent cells.
Infected cell area will create a plaque which is an
area of infected cell surrounded by uninfected cells.
It can be seen visually or by optical microscope.
Usually takes 3-14 days depending upon virus being
analyzed.
Results are counted manually and after multiplying
with the dilution factor, it gives virus no. as pfu/ml.

85. Other assays:Other assays:
End point dilution assay:
TCID 50-Tissue culture infective dose 50
Use to measure the infectious virus titers
Used in clinical research application where lethal dose of virus must be
determined or if virus does not form plaques.
It will determine the virus required to kill 50% of infected hosts or to
produce a cytopathic effect in 50% of inoculated tissue culture.
Hemagglutination assay:
Dilution of influenza samples are incubated with 1% erythrocytes solution
for 1 hours and virus dilution at which HA take place first is visually
determined.
Takes only 1-2 hours only.


86. Antigen detection methodsAntigen detection methods
Particularly useful for viruses that grow slowly or are labile, making
recovery in culture difficult.
The most important targets are:
HSV and VZV in cutaneous specimens
CMV and hepatitis B virus (surface antigen) in blood specimens
NS 1 antigen for Dengue
P24 antigen detection for HIV
Rabies antigen detection in brain samples

87. Viruses such as the enteroviruses and the rhinoviruses
that have extensive antigenic heterogeneity and lack
cross-reacting antigens are not suitable for antigen-
detection techniques.
The advantages of antigen- detection techniques are
lack of requirement for viral viability in the specimen,
allowing greater flexibility in the handling and transport
of specimens.

88. Methods of antigen detection include:
Fluorescent antibody (FA) staining
Immunohistochemical staining
EIA
Of these, FA staining is the now most widely used in diagnostic
virology. FA staining methods continue to be improved through
the use of cytocentrifugation to prepare specimens and
simultaneous staining with multiple different antibodies labeled
with different fluorescent labels .

89. Immunohistochemical stain-brown Immunohistochemical stain-brown
coloured rabies antigencoloured rabies antigen

90. Antibody detection methods:Antibody detection methods:
Detection of rising titres of antibody between acute and convalescent
stages of infection, or the detection of IgM in primary infection.
•Immunofluroscence test
•Neutralisation test
•Complement fixation test (CFT)
•Heamagglutination inhibition tests
EIA (Enzyme immunoassays)
RIA (Radial immunoassays)

91. In Primary Infection
•4 fold or more increase in titre of IgG or total
antibody between acute and convalescent sera
•Presence of IgM
•Seroconversion
In Reinfection
• More increase in titre of IgG or total antibody
between acute and convalescent sera
•Absence or slight increase in IgM

92. MolecularMolecular
PCR
Conventional,
Real time,
Reverse transcriptase,
Nested,
Multiplex
Hot start
NASBA
Microarray

93. •Advantages:
•High sensitivity
•Rapid turnaround times
•Ability to detect noncultivable or fastidious viruses
•Detection of virus present in low concentration
•Best for detection of slow growing viruses
•Quantitative detection of virus is possible

94. Lab diagnosis of viral infections of Lab diagnosis of viral infections of
skin and mucous membraneskin and mucous membrane
Virus Methods
Herpes Simple Culture, fluorescent antibody
staining
Varicella zoster Culture, fluorescent antibody
staining
Enterovirus Culture
Human papilloma PCR , Histology, Cytology
Poxvirus Electron microscopy, Histology

95. Viral meningitis and encephalitisViral meningitis and encephalitis
Virus Methods
HSV PCR
VZV PCR
Enterovirus RT-PCR(preferred for throat, stool), Culture(CSF)
Arbovirus Serology(IgM) on CSF & serum
Rabies RT-PCR ( saliva, skin,CSF),FA staining(skin, brain),
Culture(saliva), serology(CSF & serum)
Mumps Serology (IgM), culture (CSF, saliva,urine)
Measles(SSPE) Serology
LCM Serology, Culture (CSF, blood0

96. Preferred test for diagnosis of Preferred test for diagnosis of
cytomegalovirus infectioncytomegalovirus infection
Diagnostic need Preferred test
Immune status Serology (IgG)
Congenital infection ,post partumCulture of urine
Congenital infection ,in uteroCulture or PCR analysis of amniotic
fluid(Ig M)
Mononucleosis in normal host Serology (IgM)
Systemic infection, Immunosuppressed
host, Initiation of presumptive therapy
Pp65 antigenemia assay, PCR analysis,
NASBA analysis
Monitoring response to therapyQuantitative PCR analysis
Retinitis PCR analysis of vitreous fluid
CNS disease PCR of CSF
GIT disease, pneumonitis Histology, immunohistochemistry

97. HIVHIV
Use Test
Routine diagnosis Immunochromatography,ElA, Western
blot
Diagnosis of acute infectionPlama HIV RNA analysis
Congenital infection HIV DNA PCR, Plasma HIV RNA analysis
Prognosis Plasma HIV RNA analysis
Response to therapy Plasma HIV RNA analysis
Blood donor screening EIA/ western blot, P24 antigen assay,
Plasma HIV RNA analysis
Resistance to antiretroviral drugsGenotypic or phenotypic susceptibility
assay

98. Miscellaneous viral infectionsMiscellaneous viral infections

99. Viral hepatitisViral hepatitis

100. SummarySummary
•Laboratory diagnosis of viral infections is
important in view of screening, detection,
epidemiology and research.
•Diagnostic methods for viral detection
include
–Microscopy
– Molecular methods
–Viral culture
–Serological methods

101. References: References:
Modern Uses of Electron Microscopy for Detection of
Viruses. Clinical microbiology reviews; Oct. 2009,P:552–
563 vol. 22, no. 4.
Lennette’s Laboratory Diagnosis of Viral Infections, Fourth
Edition, p.21-130.
Role of Cell Culture for Virus Detection in the Age of
Technology. Clinical microbiology reviews, Jan. 2007, P.
49–78 vol. 20, no.1.
Cell Culture and Electron Microscopy for Identifying
Viruses in Diseases of Unknown Cause. Emerging Infectious
Diseases , Vol. 19, No. 6, June 2013.
Transportation of viral sample. Clinical Microbiology
review,Apr.1990,p.120-131.

102. Thank you