This document provides an overview of diagnostic techniques used in virology laboratories, including direct antigen detection, rapid antigen detection assays, molecular detection methods like PCR, viral cell culture techniques, and specimen collection and transport. It discusses the diagnostic methods for specific virus families like Herpesviridae, Adenoviridae, Parvoviridae, Papovaviridae, Hepadnaviridae, Flaviviridae, Picornaviridae, and Orthomyxoviridae. Key points covered include the use of direct fluorescent antibody staining, enzyme immunoassays, viral culture characteristics and cytopathic effects, histopathology features, and serological assays for various virus identification.
3. Laboratory diagnosis
Direct antigen detection from lesions
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
Can provide a HSV and VZV diagnosis
More sensitive and specific than Tzanck prep
(DFA 80% vs. Tzanck 50%)
Tzanck prep= Giemsa stain/examine for
multinucleated giant cells of Herpes virus
Tzanck
4. Rapid detection of viral antigens
Enzyme immunoassay –
Antigen/antibody complex formed – then
combined with color forming compound
Detection of non-culturable viruses – such
as Rotavirus
Detection of Influenza A and B , and
Respiratory syncytial virus (RSV)
Membrane EIA
Liquid/well EIA
5. Molecular Detection
Amplification of DNA or RNA
Rapid results
Exceed sensitivity of culture
Standard of practice for detecting respiratory viruses
Standard of practice for HSV and Enterovirus
detection in CSF
Culture <=20%
PCR >=90%
CMV quantitative assays in transplantation
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
Viral cell culture
Inner wall coated with monolayer of cells
covered with liquid maintenance media
Primary cell lines – directly from animal
into tube (Rhesus monkey kidney-RMK)
Diploid cell lines– Can survive 20 – 50
passes into new vials – fibroblast cells
MRC-5-(Microbiology Research Council 5)
human diploid fibroblasts
Continuous cell lines – can survive
continuous passage into new vials, usually
of tumor lineage, HEp-2 and HeLa
7. Viral Cell culture
Cytopathic effect – CPE
Appearance of cell monolayer after being
infected with a virus
Specific for each virus type
8. Spin Down Shell Vial Method
Designed to speed up virus recovery
Cells are on the round coverslip
Specimen put into vial
Centrifugation to induce virus invasion
Incubate 24 - 72 hours
DFA stain cells on coverslip with early
antigen for virus of interest
Cover slip
9. Specimen collection and
transport
Viral transport media- Hanks balanced salt
solution with antibiotics, needed for the
transport of lesions, mucous membranes and
throats to the laboratory
I
10. Which viruses will survive the trip
to the laboratory?
Most likely viable - HSV
Intermediate
Adenovirus
Influenza A and B
Enterovirus
Least likely to survive
Respiratory Syncytial Virus (RSV)
Cytomegalovirus (CMV)
Varicella Zoster virus (VZV)
Amplification preferred due to survival issue
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
Molecular 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 for diagnosis
Cytology/Histology - intra nuclear inclusions,
multinucleated giant cells
Serology – Most helpful to detect past infection
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 +/- eliminated due to effective
vaccine program – most serious disease occurs in
immune suppressed or adult patient – can progress to
pneumonia and encephalitis
Histology –
Herpes simplex
multi-nucleated giant cells like those of
Serology useful for immune status check
Amplification useful for disease diagnosis
18. Varicella-Zoster Diagnosis
In cell culture –
Limited # of Foci
5- 7 days to develop
Sandpaper look to the
Background
Scattered rounded cells
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, close contact
Latency: Macrophages
Disease: Asymptomatic in most individuals infected
Congenital – most common cause
Perinatal
Immunocompromised – Primary disease most
serious
Diagnosis: Cell culture CPE (Human diploid fibroblast ,
PCR and quantitative PCR
Histopathology: Intranuclear and
intracytoplasmic inclusions
“Owl Eye” Inclusions
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 for diagnosis
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 in 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
26. Adenovirus
DNA - non enveloped/ icosahedral virus
Latent: lymphoid tissue
Transmission: Respiratory and fecal-oral route
Diseases:
Adenovirus type 14 – new virulent
respiratory strain / pneumonia
Pharyngitis (year round epidemics)
Gastroenteritis in children
Adenovirus types 40 & 41
Keratoconjuctivitis
Disseminated infection in transplant patients
Hemorrhagic cystitis in immune suppressed
27. Adenovirus
Round cells with
stranding
Diagnosis
Conventional cell culture (CPE)
2-5 days with round cells connected by
strands – Grows best in Heteroploid
continuous passage cell lines (HeLA, Hep-2)
Amplification
Histology - Intranuclear inclusions / smudge cells
Stool EIA for enteric infections
Antigen detection – staining respiratory cells by DFA
for Respiratory infections
PCR – has become the standard of practice
Supportive treatment – no specific viral therapy
30. Parvovirus
DNA virus
Parvovirus B19
Slapped face appearance
of fifth disease
erythema infectiosum (Fifth disease)
fetal infection and stillbirths
aplastic crisis in patients with chronic hemolytic
anemia and AIDS
Histology - virus effects
mitotically active erythroid
precursor cells in bone marrow
Molecular and Serology methods
for diagnosis
32. Pap smear
Papillomavirus
Diseases:
skin and anogenital warts,
benign head and neck tumors,
cervical and anal intraepithelial neoplasia and cancer
HPV types 16 and 18 = 70% Cervical CA
HPV types 6 and 11 = 90% Genital warts
Pap Smear for detection
Hybrid capture DNA probe for detection and typing
PCR – FDA approved platforms for detection/typing
Guardasil vaccine = To guard against HPV 6,11,16,18
33. Polyomavirus
Giant Glial Cells of JCV
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
35. Hepatitis B virus
Enveloped DNA – Hepadna virus
Virion called Dane particle
Surface Ag (HBsAg)- Australian Ag
Clinical Disease
90% acute
1% fulminant
9% chronic
Carrier state …….Hepatic cell carcinoma
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
38. Hepatitis C virus
Spread parenterally - drug abuse, blood products,
poorly sterilized medical equipment, sexual
Effects only humans and chimpanzees
Seven major genotypes
Acute self limited disease to start with progressive
disease that mainly affects the liver
Infection persists in 80%
20 - 30 % develop cirrhosis
Associated with hepatocellular CA
Require liver transplantation
39. Hepatitis C
Diagnosis:
Hep C antibody test
If antibody positive do:
RNA qualitative or quantitative assay for
viral load
Requires Genotyping for proper therapy
Type 1 most common
No vaccine – Antivirals currently in clinical trials
that can cure a large % of infected
40. Flaviviruses – Mosquito borne
St. Louis
Dengue – breakbone fever
Yellow fever
West Nile
Fever, Headache, Muscle weakness
Various species Mosquitoes
Serology / PCR
42. Enteroviruses
Diverse group of > 60 viruses
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)
44. 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
46. 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
47. Influenzae A
Disease: fever, malaise …. death
Diagnosis
Cell culture obsolete [RMK]
Enzyme immunoassay 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
Seasonal variation in susceptibility
Vaccinate to prevent
Influenza B
Milder form of Influenza like illness
Usually <=10% of cases /year
48. Paramyxoviruses – SS RNA
Measles
Parainfluenza 1,2,3,4
Mumps
Respiratory Syncytial Virus
Human Metapneumovirus
49. Measles
Measles
Fever, Rash, Dry Cough, Runny Nose,
Sore throat, inflamed eyes (photosensitive)
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
50. 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
51. Mumps
Person to person contact
Classic infection is Parotitis, but can
cause infections in other sites:
T
52. 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
53. Classic CPE = Syncytium formation
In heteroploid cell line
Respiratory syncytial virus CPE
Histology
54. 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
56. 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
58. 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
Disease course usually limited, 24-48 hours
PCR for diagnosis
Cannot be grown in cell culture
60. 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
61. HIV
Diagnosis
Antibody EIA with Western Blot confirmation
Antibody test alone is NOT sufficient
Western blot detects gp160/gp120 (envelope
proteins), p 24 (core), and p41(reverse trans)
Must have at least 2 bands on Western blot to
confirm the diagnosis of HIV
Positive patients 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
62. HIV infectious complications
Non-compliant patients or newly diagnosed
Pneumocystis
C. neoformans and Histoplasma
TB/Mycobacterium avium complex
Microsporidia and Cryptosporidium
Hepatitis B
Hepatitis C
STD’s – Syphilis, GC, Chlamydia
Syphilis rate high (mucosal contact)
64. Rubella
Respiratory transmission
Known as the “Three day measles” – German measles
Congenital rubella – occurs in a developing fetus of a
pregnant women who has contracted Rubella, highest
% in the first trimester pregnancy
Diagnosis - Serology in combination with clinical
symptoms – Rash, low fever, cervical
lymphadenopathy
Live attenuated vaccine (MMR) to prevent
66. 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
Myalgia, headache, cough and respiratory
failure
Found in states west of the Mississippi River
Diagnosis by serology/ no therapy
68. Smallpox
Smallpox virus is also known as the Variola virus
Vaccinia virus = vaccine strain used in Smallpox
vaccine, it is immunologically related to smallpox,
Vaccinia can cause disease in the immune
suppressed, which prevents vaccinated this population
Last case of Smallpox - Somalia in 1977
Disease begins as maculopapular rash and progresses
to vesicular rash - all lesions in same stage on a body
area – rash moves from central body outward
Potential agent of Bioterrorism
Any potential cases directly reported to public health
department – they will investigate and diagnose
69. Chickenpox vs Smallpox lesions
Chicken pox – Lesions of
different stage of development
Smallpox – all lesions same
stage of development
71. 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
Public health department should be contacted to assist
with diagnosis