2. Diagnostic techniques used in
the Virology Laboratory
1.Direct Staining for Antigen
2. Enzyme Immunoassay
3. Molecular Amplification
4. Viral Cell Culture
3. Detect Antigen in lesion
Direct Fluorescent antibody (DFA) stain
Collect cells from base of vesicular lesion
Stain with Fl antibody specific for HSV and/or VZV
Look for fluorescent cells using fluorescence microscope
Fluorescent cells = viral infected cells
More sensitive & specific method than Tzanck prep
(DFA 80% vs. Tzanck 50%)
Tzanck prep= Giemsa stain of lesion cells/examine
for multinucleated giant cells of Herpes virus
Tzanck
Tzanck DFA
4. Detection of Viral Antigens by EIA
Enzyme immunoassay –
Antigen/antibody complex formed – then
bound to a color producing substrate
Used most for detection of non-culturable
viruses – such as Rotavirus from stool
Detect Influenza A and B , & Respiratory
syncytial virus (RSV) from nasal/NP swab
Membrane EIA Liquid/well EIA
5. Molecular Amplification
Molecular Amplification (DNA or RNA)
Rapid/Sensitive/Specific for numerous viruses
Exceeds sensitivity of culture/replacing culture
Standard of practice for detecting respiratory viruses
Standard of practice for HSV and Enterovirus
detection from CSF
Culture <=20% PCR >=90%
Quantitative assays in transplantation - CMV
Hepatitis B and C detection and viral load
HIV viral load
Test of diagnosis not cure – can retain DNA/RNA
for 7 – 30 days after initial diagnosis
6. Viral Cell Culture
Inner wall coated with monolayer of cells
lines covered with liquid maintenance media
Three basic types of cell lines:
Primary cell lines – directly from animal organ
into culture tube (Rhesus monkey kidney-RMK)
Diploid cell lines– Can survive 20 – 50 passes
into new vials – human diploid fibroblast cells,
example: MRC-5-Microbiology Research Council 5
Continuous cell lines – can survive continuous
passage into new vials, usually of tumor
lineage, HEp-2 and HeLa
7. Viral Cell culture
Tubes/flasks read under microscope
for Cytopathic effect/ CPE
Appearance of cells in the monolayer
after being infected with a virus
Destruction is specific for each virus type
8. Spin Down Shell Vial
Virus Culture -
• Designed to speed up virus recovery
• Cells are on the round coverslip
• Specimen inoculated into vial
• Centrifuge vial to induce virus
invasion into cell monolayer
• Incubate @ 35*C, 24-72 hours
• Direct fluorescent antibody stain to
stain cells on coverslip – target early
• antigens for virus of interest
Cover slip
9. Specimen collection and
transport
Viral transport media (VTM) - Hanks
balanced salt solution with antibiotics,
Also known as Universal Transport Media
needed for the transport of lesions, mucous
membranes and throats to the laboratory
It is cell protective, protect the cell / protect the virus
Short term transport storage 4˚C
Long term transport(>72hours) storage-70˚C
VTM specimens filtered (45nm filter) to
eliminate bacteria in specimen prior to being
placed on cell monolayer
10. Which viruses will survive the trip
to the laboratory?
Most likely to survive - HSV
Intermediate
Adenovirus
Influenza A and B
Enterovirus
Least likely to survive
Respiratory Syncytial Virus (RSV)
Cytomegalovirus (CMV)
Varicella Zoster virus (VZV)
Amplification preferred for these viruses due to
survival issues
11. Which viruses grow the fastest in
conventional cell culture?
Fast (>=24 hours)
HSV
Intermediate (5 -7 days)
Adenovirus Enterovirus
Influenzae VZV
Slow (10 - 14 days)
RSV
Slowest (14 - 21 days)
CMV
Amplification methods are superior for slow
growers
13. Herpes Viruses
Double stranded DNA virus
Eight human Herpes viruses
Herpes simplex 1
Herpes simplex 2
Varicella Zoster
Epstein Barr
Cytomegalovirus
Human Herpes 6, 7, and 8
Latent infection with recurrent disease is the
hallmark of the Herpes viruses
Latency occurs within small numbers of specific kinds
of cells, the cell type is different for each Herpes virus
15. Herpes virus diagnosis
Herpes 1 & 2 do well in culture
Grow within 24-48 hrs in Human diploid fibroblast cells
(MRC-5) - Observe for characteristic CPE
Antigen detection by direct fluorescent staining
of cells obtained from vesicular lesions
Amplification methods available for detection
from lesions and bodily fluids
Cytology/Histology - intra nuclear inclusions,
multinucleated giant cells
Serology – More helpful to detect past infection
HSV1 and HSV2 can x-react in serology
16. Negative fibroblast cell
Culture -uninfected cells
HSV infected monolayer
Rounded cells throughout
the monolayer in cell culture
Multinucleated Giant Cells
of Herpes Simplex in tissue
histology
17. Varicella Zoster Virus
Transmission: close contact
Latency: dorsal root ganglia
Diseases:
Chickenpox (varicella)
Shingles (zoster – latent infection)
Chicken pox disease has decreased due to
effective vaccine program – most serious disease
occurs in immune suppressed or adult patients which
progresses to pneumonia and encephalitis
Histology – multi-nucleated giant cells like those of
Herpes simplex
Serology useful for immune status check
Amplification useful for disease diagnosis
18. Varicella-Zoster Diagnosis
In cell culture –
Limited # of Foci in
monolayer
Require 5- 7 days to
develop
Sandpaper look to the
Monolayer background with
scattered rounded cells -
diploid fibroblast monolayer
Younger wet vesicular lesions area
the best for culture and/or
molecular testing
19. Cytomegalovirus (CMV)
Transmitted by blood transfusion , vertical and horizontal
transmission to fetus, also by close contact
Latency: Macrophages
Disease: Infection asymptomatic in most individuals
Congenital – most common cause of TORCH
Perinatal
Immunocompromised – Primary disease most serious
Laboratory Diagnosis:
Cell culture CPE (Human diploid fibroblast)
PCR and quantitative PCR (best method)
Histopathology: Intranuclear and
intracytoplasmic inclusions
“Owl Eye” Inclusions
Treatment:ganciclovir, foscarnet, cidofovir
20. CMV pneumonia with
viral inclusions
CMV infected fibroblast
monolayer - Focal grape like
clusters of rounded cells
21. Epstein Barr virus (EBV)
Transmission - close contact, saliva
Latency - B lymphocytes
Diseases include:
Infectious mononucleosis
Lymphoreticular disease
Oral hairy leukoplakia
Burkitt’s lymphoma
Nasopharyngeal Carcinoma
1/3 Hodgkin’s lymphoma
Unable to grow in cell culture
Serology and PCR methods available for
diagnosis
EBV infection with
B cell transformatin
22. EBV Serodiagnosis using the
Heterophile Antibody
Heterophile antibodies (HA) react with
antigens phylogenetically unrelated to the
antigenic determinants against which they
were raised
HA secondary to EBV are detected by the
ability to react with horse or cattle rbcs
(theory of the Monospot test)
HA rise in the first 2 - 3 weeks of EBV
infection, then rapidly fall at @ 4 weeks
Cannot be used to diagnose children < 4
years of age
23. VCA = viral capsid antibody
EBNA = Epstein Barr nuclear antigen
EA = early antigen
24. Human Herpes virus
6, 7 & 8
HH6
Roseola [sixth disease]
6m-2yr high fever & rash
HH7
CMV like Disease
HH8
Kaposi’s sarcoma
Castleman’s disease
Onion skin of
Castleman disease
30. Parvovirus
DNA virus
Parvovirus B19
Erythema infectiosum (Fifth disease)
Cause fetal infection and stillbirths
Aplastic crisis in patients with chronic hemolytic
anemia and AIDS
Histology - virus infects
mitotically active erythroid
precursor cells in bone marrow
Molecular and Serology methods
for diagnosis
Slapped face appearance
of fifth disease
32. Papillomavirus
Diseases:
Pap smear
skin and anogenital warts,
benign head and neck tumors,
cervical and anal intraepithelial neoplasia and cancer
HPV types 16, 18, & 45 = 94% Cervical CA
HPV types 6 and 11 = 90% Genital warts
Pap Smear for detection
Hybrid capture DNA probe for detection and typing
PCR – FDA cleared platforms for detection/typing
Gardasil vaccine = To guard against HPV 6,11,16,18
33. Polyomavirus
JC virus [John Cunningham]
Cause of Progressive multifocal
leukoencephalopathy -
Encephalitis of immune suppressed
Destroys oligodendrocytes in brain
BK virus
Causes latent virus infection in kidney
Progression due to immune suppression
Hemorrhagic cystitis
Histology/PCR for diagnosis
Giant Glial Cells of JCV
35. Hepatitis B virus
Enveloped DNA – Hepadna virus
Hepatitis B clinical disease
90% acute
1% fulminant
9% chronic
Carrier state can lead to cirrhosis and
hepatic cell carcinoma
newer therapies – stops disease
progression
Vaccinate to prevent
36. Hepatitis B Serology
Surface Antigen Positive
Active Hepatitis B or Chronic Carrier
Do Hep B Quantitation
Do Hep e antigen – Chronic and “bad”
Core Antibody Positive
Immune due to prior infection,
acute infection or chronic carrier
Surface Antibody Positive
Immune due to prior infection or vaccine
37. Flaviviridae
RNA Viruses
Hepacivirus – Hepatitis C
Flavivirus – West Nile, Dengue,
and Yellow Fever
38. Hepatitis C virus
Spread parenterally - drug abuse, blood products or
organ transplants (prior to 1992), poorly sterilized
medical equipment, sexual (low risk)
Effects only humans and chimpanzees
Approx 3.2 mil persons in USA have chronic Hep C
Seven major genotypes (1-7)
Acute self limited disease that progresses to a
disease that mainly affects the liver
Infection persists in @ 75-85%/ no symptoms
5 - 20 % develop cirrhosis
1-5 % associated with hepatocellular CA
Require liver transplantation
39. Hepatitis C
Diagnosis:
Hepatitis C antibody test
If antibody positive do:
RNA qualitative or quantitative assay for
viral load
Requires Genotyping for proper therapy
Type 1 Hep C most common in USA
No vaccine – Antivirals currently in clinical
trials and/ or FDA cleared that can cure >=
85% of infected with Hepatitis C
40. Flaviviruses – Mosquito borne
Dengue – “breakbone fever”
Aedes mosquito / Asia and the Pacific
Fever, severe joint pain, rash
Small % progress to hemorragic fever
West Nile
Common across the US, Bird primary reservoir
Fever, Headache, Muscle weakness, 80%
asymptomatic. Small % progress to
encephalitis. Meningitis, flaccid paralysis
Mosquitoes – Aedes & Culex
Immunoassays for Antibody & PCR
Serum and CSF
41. Alpha virus –
Mosquito borne
Chikungunya virus – RNA virus
Mosquito borne – Aedes mosquito
Origin in Asia and African continents
Recent migration to the Caribbean and
SE USA with mosquito migration
Travel advisory to the Caribbean
Acute febrile illness with rash followed by
extreme joint pain, less fatalities than
Dengue / no hemorrhagic phase
Diagnosis – Serology(IgM, IgG) and PCR
42. Ebola Virus
>20 outbreaks since discovery in 1976
current outbreak Dec 2013 - West Africa
Prolonged due to area effected is high population
with limited medical facilities
Transmission direct contact with bodily fluids – fatality
rate 55%
Animal reservoir (?) fruit bats
Asymptomatic are not contagious
Fever, weakness, myalgias, headache, travel history
Consider malaria and typhoid
Susceptible to hospital disinfectants
Testing (EIA, PCR) at CDC – pos >= 4 days of illness
43. Coronovirus/SARS
Severe Acute Respiratory Syndrome (SARS)
Outbreak in China 2003 – spread to 29 countries
Incubation period of 2-10 days
2-7 days by dry cough and/or shortness of breath
Development of radiographically confirmed pneumonia
by day 7-10 of illness Lymphopenia in most cases
Laboratory testing for SARS-CoV available at state public
health laboratories. Available tests include antibody
testing enzyme immunoassay (EIA) and reverse
transcription polymerase chain reaction (RT-PCR) tests
for respiratory, blood, and stool specimens. In the
absence of person-to-person transmission of SARS-CoV,
the positive predictive value of a diagnostic test is
extremely low.
44. MERS CoV- Middle East
Respiratory Syndrome
Coronavirus
Isolated to Arabian peninsula (2012)
Close human to human contact can
spread infection – no outbreaks
2 unrelated cases in US from travel
Fever, rhinorrhea, cough, and malaise
followed by shortness of breath –
30% fatality rate
NP, Lower respiratory specimen and
serum for PCR at CDC
46. Enteroviruses
Diverse group of > 60 viruses – SS RNA
Infections occur most often in summer and fall
Polio virus - paralysis
Salk vaccine Inactive Polio Vaccine (IPV)**
Sabine vaccine Live Attenuated Vaccine (OPV)
Coxsackie A – Herpangina
Coxsackie B – Pericarditis/Myocarditis
Enterovirus – Aseptic meningitis in children,
hemorrhagic conjunctivitis
Echovirus – various infections, intestine
Rhinoviruses – common cold
Grow in cell culture (Diploid mixed cell – Primary
Monkey Kidney)
PCR superior for diagnosis of meningitis (CSF) and
more rapid and sensitive for all sites
47. CPE of Enterovirus
Teardrop and kite like cells in
Rhesus Monkey Kidney cell culture
48. Hepatitis A
Fecal – oral transmission
Can be cultured but not reliably
Usually – short incubation, abrupt onset, low mortality,
no carrier state
Travel
Diagnosis – serology, IgM positive in early infection
Vaccine available
50. Influenza A
Segmented RNA genome
Hemagglutinin and Neuraminidase glycoproteins spikes
on outside of viral capsid
Give Influenza A the H and N designations – such as H1N1
and H3N2
Antigenic drift - minor change in the amino acids of
either the H or N glycoprotein
Cross antibody protection will still exist so an
epidemic will not occur
Antigenic shift - genome re assortment with a “new”
virus created/usually from a bird or animal/ this could
create a potential pandemic
H5N1 = Avian Influenza
H1N1 = 2009 Influenza A
51. Influenzae A
Disease: fever, malaise …. death
Diagnosis
Cell culture obsolete [RMK]
Enzyme immunoassay (EIA) on paper membrane can
be used in outpatient setting – Rapid but low
sensitivity (60%) and can have specificity issues in
off season.
Amplification (PCR) gold standard for Influenza
detection
Treatment: Amantadine and Tamiflu (Oseltamivir)
Seasonal variation in susceptibility
Vaccinate to prevent
Influenza B
Milder form of Influenza like illness
Usually <=10% of cases /year
52. Paramyxoviruses – SS RNA
Measles
Parainfluenza 1,2,3,4
Mumps
Respiratory Syncytial Virus
Human Metapneumovirus
53. Measles
Measles
Measles syncytium
Fever, Rash, Dry Cough, Runny Nose,
Sore throat, inflamed eyes (photosensitive)
Can invade lung (see HE of Lung)
Respiratory spread - very contagious
Koplik’s spots – bluish discoloration inner
lining of the cheek
Subacute sclerosing panencephalitis [SSPE]
Rare chronic degenerative neurological disease
Persistent infection with mutated measles virus
due to lack of immune response
Diagnosis: Clinical symptoms and Serology
Vaccinate – MMR (Measles, Mumps, Rubella) vaccine
Treatment: Immune globulin, vitamin A
54. Parainfluenzae
Types 1,2,3, and 4
Person to person spread
Disease:
Upper respiratory tract infection in adults –
more serious in immune suppressed
Croup, bronchiolitis and pneumonia in
children
Heteroploid cell lines (Hep-2) for culture
PCR methods are the gold standard
Supportive therapy
55. Mumps
Person to person contact
Classic infection is Parotitis, but can
cause infections in other sites:
Testes/ovaries, Eye, Inner ear, CNS
Diagnosis: clinical symptoms,
serology available
Prevention: MMR vaccine
No specific therapy, supportive
56. Respiratory Syncytial Virus
Transmission:
Hand contact and respiratory droplets
Respiratory disease - from common cold to
pneumonia, bronchiolitis to croup, serious
disease in immune suppressed
Classic disease:
Young infant with bronchiolitis
Specimen: Naso-phayrngeal, nasal swab,
nasal lavage
Diagnosis: EIA, cell culture (heteroploid cell
lines), PCR is standard practice
Treatment: Supportive, ribavirin
57. Classic CPE = Syncytium formation
In heteroploid cell line
Respiratory syncytial virus CPE
Histology
58. Human Metapneumovirus
1st discovered in 2001 – community acquired
respiratory tract disease in the winter
Common in young children – but can be seen in all
age groups
@95% of cases in children <6 years of age
Upper respiratory tract disease
2nd only to RSV in the cause of bronchiolitis
Will not grow in cell culture
Amplification (PCR) for detection
Specimen: Nasal swab or NP
Treatment: Supportive
60. Rotavirus
Winter - spring season
6m-2 yrs of age,
Gastroenteritis with vomiting and fluid loss –
most common cause of severe diarrhea in
children
Fecal – oral spread
Major cause of death in 3rd world
Diagnosis – cannot grow in cell culture
Enzyme immunoassay, PCR
Vaccine available
62. Norovirus
Spread by contaminated food and water, feces
& vomitus – takes <=20 virus particles to
cause infection – so highly contagious
Tagged the “Cruise line virus” – numerous
reported food borne epidemics on land and sea
Leading cause of epidemic gastroenteritis –
more virulent GII.4 Sydney since spring 2012
Fluid loss from vomiting can be debilitating
Disease course usually limited, 24-48 hours
PCR for diagnosis
Cannot be grown in cell culture
64. Human Immunodeficiency virus
CD4 primary receptor to gain
entry for HIV into the lymphocyte
Reverse transcriptase enzyme
converts genomic RNA into DNA
Transmission - sexual, blood and blood product
exposure, perinatal
Non infectious complications:
Lymphoma, KS, Anal cell CA, non Hodgkins
Lymphoma
65. HIV Laboratory Diagnosis
Antibody EIA with Western Blot confirmation (old way)
Antibody test alone is NOT sufficient – all positive must
be confirmed with a western blot test
Western blot detects gp160/gp120 (envelope proteins),
p 24 (core), and p41(reverse trans)
Must have at least 2 solid bands on Western blot to
confirm as a positive result
New test - Antigen/antibody combination (4th generation)
immunoassay* that detects HIV-1 and HIV-2 antibodies and
HIV-1 p24 antigen to screen for established infection with HIV-
1 or HIV-2 and for acute infection
Positive patients on either test require additional testing:
HIV RNA/DNA quantitation >= 100 copies
Resistance Testing – report subtype
Most isolates in USA type B
Monitor CD4 counts for infection severity
66. HIV infectious complications
Non-compliant patients or newly diagnosed
Pneumocystis
Cryptococcus neoformans & Histoplasma
(disseminated)
TB/Mycobacterium avium complex
(disseminated)
Microsporidia and Cryptosporidium (stool)
Hepatitis B
Hepatitis C
STD’s – Syphilis, GC, Chlamydia
Syphilis rate high (mucosal contact)
68. Rubella
Known as the “Three day measles” – German measles
Congenital rubella – occurs in a developing fetus of a
pregnant women who has contracted Rubella, highest
% (50%) in the first trimester pregnancy
Deafness, eye abnormalities, congenital heart disease
Respirastory transmission
Diagnosis - Serology in combination with clinical
symptoms – Rash, low fever, cervical
lymphadenopathy
Live attenuated vaccine (MMR) to prevent
70. Hantavirus
USA outbreak in four corners (NM,AZ,CO,UT)
Indian reservation in 1993 brought attention to
this virus
Source - Urine and secretions of wild field mice
Deer mouse and cotton rat most implicated
Myalgia, headache, cough and respiratory
failure
Found in states west of the Mississippi River
Diagnosis by serology/ no therapy
72. Smallpox
Smallpox virus is also known as the Variola virus
Vaccinia virus is the strain used in Smallpox vaccine,
it is immunologically related to smallpox, Vaccinia can
cause disease in the immune suppressed, which
prevents vaccination of this population
Last case of Smallpox - Somalia in 1977
Disease begins as maculopapular rash and progresses
to vesicular rash - all lesions in same stage of
developemnt in body area – rash moves from central
body outward
Category A Bioterrorism agent (can maim or kill)
Requires BSL4 laboratory (self contained lab)
Any potential cases directly reported to public health
department – they will investigate and diagnose
73. Chickenpox vs Smallpox lesions
Chicken pox – Lesions in
different stage of development
Smallpox – all lesions same
stage of development
75. Rabies
Worldwide in animal populations
Bat and raccoons primary reservoir in US
Dogs in 3rd world countries
Post exposure shots PRIOR to the development of
symptoms prevent infection
Rabies is a neurologic disease – classic sympton is
salivation, due to paralysis of throat muscles
Detection of viral particles in the brain by Histologic
staining known as Negri bodies is diagnostic
Public health department should be contacted to assist
with diagnosis
76. Rabies virus particles
EM showing the bullet
shaped virus
Negri bodies –
Intracytoplasmic
brain biopsy specimen