1. The document discusses various methods for diagnosing viral infections, including direct fluorescent antibody staining, enzyme immunoassays, viral cell culture, and molecular amplification techniques like PCR.
2. Specific examples of viruses that can be diagnosed using these methods are provided, such as herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and adenovirus.
3. Details are provided on specimen collection and storage, as well as the cytopathic effects and characteristics of each virus in cell culture systems.
2. Specimens suspect for Class B viral pathogens can be processed in a laboratory
using BSL-2 precautions. Class B pathogens are ones that will not generally
cause fatal disease, such as Herpes simplex virus.
BSL-2 precautions include processing specimens inside a BSL2 hepa–filtered
biosafety cabinet, wearing a front closed laboratory gown and gloves
3. 1. Direct Staining Specimen for Antigen
2. Enzyme Immunoassay
3. Viral Cell Culture
4. Molecular Amplification
4. Direct Fluorescent antibody (DFA) stain for HSV1/2 & VZV
Collect cells from base of fresh vesicular lesion
Stain with antibody specific for HSV 1/2 and/or VZV
Look for fluorescent cells (virus infected) using fluorescence microscope
Sensitivity @ 80% compared to molecular assays
Tzanck prep
Cells from lesion stained with Giemsa stain /examine for multinucleated giant
cells consistent with Herpes virus family
Sensitivity @ 50% / Cannot differentiate HSV 1, HSV 2, or VZV
Tzanck DFA
5. • Enzyme immunoassay (EIA) –
Antigen in specimen forms a complex with test antibody, then a color
producing substrate is added and binds to Ag/Aby complex
Used for rapid point of care testing (20 min), moderate sensitivity (70%
compared to molecular assays) positive with high viral load, specificity
adequate during high prevalence viral season
Used most often for respiratory virus testing:
Influenza A /B, Respiratory syncytial virus (RSV), SARS-CoV-2
• Membrane lateral flow EIA Liquid/in-well EIA
6. Inner wall of container coated with monolayer of cells
grown in enriched liquid growth media
• Three types of cell lines used for culture:
Primary – obtained directly from animal or human organ, processed and
placed into culture vial , will only survive one subculture into new tube,
innate animal viruses can make culture reading difficult
Example: Rhesus monkey kidney-RMK
Diploid – semi continuous cell lines– Can survive 20 – 50 subcultures
Example: Human diploid fibroblast cells (MRC-5-Microbiology
Research Council 5)
Continuous cell lines – survive continuous passage (immortal)
Example: Tumor lineages such as HEp-2 and HeLa
7. Patient specimen inoculated onto cell monolayer, incubated for a
specified number of days, then read under light microscopy for
“Cytopathic effect” – the effect viral growth has on the cells
• The pattern of destruction of the cell monolayer is specific for each
virus type and the time in which destruction occurs is virus
specific.
• Sensitive but slow <= 21 days
8. Spin Down Shell Vial Culture
• Technique created to speed up viral cell culture
• Cell monolayer is prepared on a coverslip
• Specimen inoculated into vial with coverslip and growth
medium
• Centrifuge vial to rapidly induce virus invasion of cells
• Incubate @ 35*C for 24-72 hours
• Perform DFA stain to search for early virus antigens
Cover slip
9. Molecular Amplification of DNA or RNA (Qualitative)
• Rapid/Sensitive/Specific detection method for numerous viruses
• DNA viruses - direct amplification of genetic material
• RNA viruses (RT-PCR) – reverse transcriptase enzyme transcribes
complimentary copies of DNA using the RNA template
• More rapid and may exceed sensitivity of viral culture. Is the Standard of
Practice for detection of many viruses:
Viruses of respiratory, encephalitis, lesion, and diarrhea
• Test of diagnosis not cure – can continue to shed residual virus for 7 –
30 days after initial positive test
Quantitative molecular assays are useful for assessing severity of
infection, most used for CMV, Hepatitis B and C, and HIV
11. Reverse Transcriptase PCR (RT-PCR) is a
variation of the polymerase chain reaction that
amplifies target RNA.
Addition of reverse transcriptase (RT) enzyme
prior to PCR makes it possible to amplify and
detect RNA targets.
Reverse transcriptase enzyme transcribes the
template RNA and forms complementary DNA
(cDNA).
Single-stranded cDNA is converted into
double-stranded DNA using DNA polymerase.
These DNA molecules can now be used as
templates for a PCR reaction.
Reverse Transcriptase PCR (RT-PCR
12. Cycle threshold (Ct) is a semi-
quantitative value that can broadly categorize the
concentration of viral genetic material in a patient
sample following testing by PCR or RT PCR, it
tells approximately how much viral genetic
material is in the sample.
The cycle threshold (Ct) is the number of PCR
thermal cycles at which the fluorescent signal
exceeds that of the background and thus passes
the threshold for positivity
A low Ct indicates a high concentration of viral
genetic material, which is typically associated with
high risk of infectivity.
A high Ct indicates a low concentration of viral
genetic material which is typically associated with
a lower risk of infectivity.
13. Viral transport media (VTM) or Universal transport media
(UTM) - Hanks balanced salt solution protects the DNA &
RNA, antibiotics to prevent bacterial overgrowth
• A must for transport of swab collected specimens: lesions, mucous
membranes, nasopharyngeal & throat
• Collection for cell culture and molecular amplification assays
Storage of specimens prior to testing:
• Short term media storage at 4˚C
• Long term (>72hours) media storage at -70˚C
VTM/UTM is filtered (45nm filter) to eliminate
contaminating bacteria prior to cell culture inoculation
14.
15. Non-enveloped 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
Latency (hallmark of Herpes viruses) occurs
within low numbers of specific kinds of cells on
human tissue, the cell type is different for each
Herpes virus
16. Transmission: direct contact/secretions
Latency: dorsal root ganglia
Infection sites:
• Gingivostomatitis - common primary infection
• Herpes labialis
• Ocular
• Encephalitis
• Disseminated disease in immune suppressed
• Neonatal – If suspicious due to mother possibly
being HSV positive, skin surface viral surveillance
PCR assays should be performed
Therapy – Acyclovir and Valacyclovir
17. Viral Cell Culture
• Produce CPE within 24-48 hrs
• Human diploid fibroblast cells (MRC-5)
• Produces characteristic CPE
Negative
fibroblast
Cell line
HSV CPE consists of cell
rounding starting on the
edge of the monolayer.
Histology/Cytology – Observe for
multinucleated giant cells, cannot
differentiate HSV 1, 2, and VZV
Molecular Amplification -Standard
of practice for detecting HSV 1/2 from
lesions and CSF
Serology – used to screen for past
infection, not for acute diagnosis
18. Transmission: close contact
Latency: dorsal root ganglia
Diseases:
• Chickenpox (varicella)
More serious disease in adults than children.
Adults and immune suppressed possible
progression to pneumonia and/or encephalitis
• Shingles (zoster – latent form of VZV) lesions
usually appear over one isolated dermatome but
capable of dissemination
• Ramsay-Hunt syndrome – facial nerve infected
with facial paralysis (pictured)
19. Histology – multi-nucleated giant cells formed
Serology – 1º used for immune status check
Cell culture – growth in 5-7 days, fibroblasts
Sandpaper like appearance in cell monolayer
with scattering of rounded cells
Molecular Amplification (PCR) for disease
diagnosis has become standard of practice
Effective vaccine has lowered the incidence of
VZV in children and shingles in adults
20. Transmitted by blood transfusion , vertical and horizontal
transmission to fetus, close human contact, and sexual
contact
Latency: Macrophages
Infection:
• Initial infection asymptomatic in most and usually occurs
in the first decade of life
• Congenital infection– most common cause of TORCH
infection
• Perinatal acquisition of infection
21. Mononucleosis – fever, absence of swollen lymph nodes,
heterophile antibody test is negative (differs from EBV
mononucleosis)
Organ specific diseases can occur, primarily in the immune
suppressed host
• Gastrointestinal
• Hepatic
• Neurological
• Cardiovascular
• Primary infection is more serious than recurrent disease
22. Laboratory Diagnosis:
• Cell culture on Human diploid fibroblast cells
Cytopathic effect can take >=14 days
CPE described as grape like clustering of rounded cells
• Quantitative PCR useful to detect significant infection
Due to persistent CMV shedding from past infection
quantitative PCR best used to detect significant viral
loads associated with ongoing infection
• Histopathology – Infects epithelial cells,
macrophages and T lymphocytes. Intranuclear
and intracytoplasmic inclusions classic (Owl eye)
Treatment: Ganciclovir, Foscarnet, and Cidofovir
23. Transmission - close contact and saliva
CD21 cell receptor for B lymphocyte cell invasion
Latency in the B lymphocyte, undergoes proliferation
with active EBV infection
Diseases include
• Infectious mononucleosis
Heterophile antibody produced, the patient serum reacts with
horse and cattle red blood cells
• Lymphoreticular disorders
• Oral hairy leukoplakia
• Burkitt’s lymphoma
• Nasopharyngeal Carcinoma
• 1/3 of Hodgkin’s lymphoma cases
Will not grow in viral cell culture
Serology and molecular assays (PCR) for diagnosis
Examination of peripheral
WBC’s in EBV infection can
demonstrate enlarged reactive
T lymphocyte
24. VCA IgM VCA IgG EBNA-1 IgG
Negative Negative Negative No immunity
Positive Negative Negative Acute infection
Positive Positive Negative Acute infection
Negative Positive Positive Past infection
Negative Positive Negative Acute or past infection
Positive Positive Positive Late primary infection
Negative Negative Positive Past infection
VCA = viral capsid antibody
EBNA = EBV nuclear antigen
Serologic Diagnosis of EBV
Past
infection
25. HHV-6
Roseola [sixth disease] usual age 6m-2yr,
high fever and rash
Adult – encephalitis in the immune
compromised
Can undergo germ line integration and be a
source of false positive results in the normal
host when testing CSF, must be cautious in
test result interpretation
HHV-8
Kaposi’s sarcoma - Lesions in soft tissues. the
skin, lymph nodes, internal organs, and mucous
membranes. It often infects patients with immune
deficiencies, such as HIV/AIDS.
26. Castleman disease
Rare disorder that involves an overgrowth of cells in lymph
nodes and related tissue (lymphoproliferative disorder) There
are different forms with different findings and prognosis.
Primary effusion lymphoma
• Most often in HIV/AIDS patients
• Localized in body cavities without a tumor mass.
• Large cells , large round to irregular
nuclei and prominent nucleoli
Classic Lollipop
like lesion
28. DNA - non enveloped/ icosahedral virus
Latent: lymphoid tissue
Transmission: Respiratory and fecal-oral route
Diseases:
• Pharyngitis (year-round epidemics)
• Pneumonia
• Gastroenteritis in children
Adenovirus types 40 & 41 most common
• Kerato-conjunctivitis – usually bilateral red painful and
inflamed eyes for @ 2 weeks
• Disseminated infection in immunosuppressed –usually
pneumonia, @ 75% fatality/ no therapy
• Hemorrhagic cystitis in immune suppressed /
particularly in transplantation
29. Diagnosis
• Cell culture (CPE), <=5 days
• Molecular methods (PCR) superior
• Stool detection (40/41 strains) PCR & EIA
• Supportive treatment only – no specific viral therapy
• Histology - Intranuclear inclusions known as smudge cells
Basophilic and nuclear membranes become blurred
Round cells 2 – 5 days
with stranding – HeLA
or Hep-2 culture cells
30. Parvovirus
Canine parvovirus is a contagious virus mainly
affecting dogs. CPV is highly contagious and is
spread from dog to dog by direct or indirect
contact with their feces. Vaccines can prevent
this infection, but mortality can reach 91% in
untreated cases. This is not contagious to
humans.
31. Non-enveloped ss DNA virus infects humans
Infections:
• Erythema infectiosum (Fifth disease) – childhood infection with
headache, rash on cheeks, and cold-like symptoms
• Hydrops fetalis – infection in pregnant, can lead to miscarriage
• Chronic hemolytic anemia in patients with HIV/AIDS
• Arthritis and Arthralgia
• 20% do not have symptoms
Histology – In bone marrow, virus infects mitotically active
erythroid precursor cells, detected in bone marrow
Molecular and Serologic methods
confirm histology or establish diagnosis
Slapped face appearance
of fifth disease
33. Skin and anogenital warts
Benign head and neck tumors
Cervical and anal intraepithelial neoplasia and cancers
High risk HPV types 16, 18 cause @ 70% of cervical cancers.
Other high-risk types include: 31, 33, 45, 52, 58
Low risk HPV types 6 and 11 cause @ 90% genital warts
Diagnosis :
Pap Smear
Molecular methods: Detection and typing of HPV types
Guidelines suggest both PAP and molecular HPV testing for women 30 - 65
years of age / performed every 5 years
Vaccines - HPV 6,11,16,18,31,33,45, 52 and 58 for young females and males
Pap smear
34. • JC virus
Progressive multifocal leukoencephalopathy (PML)
Encephalitis in AIDS, cancer and immune suppression
Diagnosis:
Histology: Destruction of oligodendrocytes in brain with
formation of giant glial cells
• BK virus
Virus maintains latency in kidney causing nephropathy and a
hemorrhagic cystitis
Activates and progresses with immune suppression
Diagnosis:
Histology: Homogenous and purple intranuclear
inclusions, primarily in tubular epithelium
Giant Glial Cells of JCV
Demyelination is classic
finding on MRI in JCV
36. Enveloped/ double stranded DNA (an envelope aids survival to
pass between cells, but can be inactivated with detergents)
Acquisition from Hep B positive mother during birthing process
Spread by contact with blood and other body fluids
Spectrum of Hepatitis B symptomatology
• Acute phase - disease varies from subclinical hepatitis to icteric hepatitis
fulminant
• Chronic phase - chronic hepatitis, cirrhosis, and hepatocellular carcinoma
Vaccinate to prevent
Therapies effective in patients with minimal liver damage
Diagnosis
• Serology and Molecular assays
37. Surface Antigen Positive
• Patient has Active Hepatitis B or is a Chronic Carrier
Next perform Hep B Quantitation to assess viral load and
Perform Hep e antigen test – if positive, patient is a chronic carrier
of HBV. This carries a worse prognosis
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
Mosquito borne Flaviviridae:
Dengue
Zika
Yellow fever
West Nile
Tick borne Flaviviridae
Powassan fever
39. Single stranded RNA virus
Disease acquisition:
Parenteral transmission, drug abuse, sexual, blood products or
organ transplants (prior to 1992), poorly sterilized medical
equipment
Infection: humans and chimpanzees
Chronic Hep C common in US with estimated 3.2 mil
• Acute self-limited disease that progresses to a disease that mainly
affects the liver
• Seven genotypes (1-7), Type 1 virus most common in USA
• Infection persists in @ 75-85% without symptoms
• 5 - 20 % develop liver cirrhosis
• 1-5 % develop hepatocellular carcinoma
40. Diagnosis:
• Hepatitis C IgG antibody positive
• Must then perform:
RNA quantitative assay to assess viral load
Genotype of virus to aids in proper therapy
selection and duration
FibroScan to assess liver damage / therapy cannot
reverse cirrhosis
No vaccine available
Antivirals available capable of cure >= 85% of
patients Periportal mononuclear
inflammatory infiltrates and
mild interface activity.
41. • Dengue – “break bone fever”
• Vector: Aedes aegypti mosquito / tropical Asia and Caribbean
• Disease
Fever, rash, and severe joint pain
Small % progress to a hemorrhagic fever which can be fatal
Diagnosis: Serology / IgM for acute infection
Zika virus
• Vector: Aedes aegypti and A. albopictus mosquitoes
• Last outbreak 2016 in South America (Brazil) with spread to central America,
Caribbean and US (Miami)
• Milder disease than Dengue in most adults, with only fever and rash
• Neurologic tropism makes it very problematic during pregnancy
Microcephaly in fetuses borne to infected moms
Developmental issues can occur in young children infected with ZIKA
Guillain-Barre syndrome post infection sequelae in adults
• Diagnosis: Serum IgM / Molecular assays serum, urine, amniotic fluid and CSF
42. Yellow fever
Vector – Aedes aegypti mosquito
Outbreak Brazil 2018 /large numbers of infected mosquitoes
introduced into heavily populated Brazilian cities
Endemic in Africa, Central and South America
Most cases mild with 3-4 days fever, headache, chills, back pain,
fatigue, nausea, vomiting
• 15% experience liver damage (jaundice) and hemorrhagic issues (20 – 50%
fatality rate)
Diagnosis:
• Molecular testing for virus in serum
No specific anti-viral drugs for therapy
Vaccine – supplies life-long immunity
43. • West Nile
Vectors: Culex mosquito
Common across the US
Bird primary reservoir, humans and horses at risk
Disease
80% asymptomatic
20% fever, headache, muscle weakness
Small % progress to encephalitis, meningitis, flaccid paralysis
Serology and Molecular assays available for diagnosis
Molecular assays have low sensitivity due to narrow window of time when virus is present
in CSF, both serology (IgM) and molecular methods are needed for diagnosis
A positive WNV IgM with a clinical history and epidemiology usually diagnostic
NOTE: Cross reactions occur in serologic testing within the Flavivirus family:
Dengue, Zika and West Nile virus can present diagnostic problems/ Public health
laboratories can assist with molecular assays and neutralization testing.
44. Powassan (POW) virus
Vector: Ixodes ticks
Approximately 100 cases of POW virus disease were reported in the United
States over the past 10 years.
• Northeast and Great Lakes region of US
Fever, headache, vomiting, weakness, confusion, seizures, and memory
loss, can cause significant swelling in the brain
Long-term neurologic problems may occur.
There is no specific treatment
45. Chikungunya virus
Vector: Aedes mosquito including aegypti
Origin in Asia and African continents with recent migration to the
Caribbean and SE USA
Acute febrile illness with rash followed by joint pain, both disease and
location similar to Dengue, but no hemorrhagic phase
When screening a patient for ZIKA – need to rule out infection with Dengue
and Chikungunya, similar symptoms with very different sequelae.
Molecular tests if positive are diagnostic.
Serologic assays might require follow-up using viral neutralization assays
to assure specificity due to cross reactivity of the Flaviviruses.
46. >20 outbreaks since discovery in 1970’s/ ss RNA virus
• Animal reservoir : fruit bat
• Most recent outbreak in Uganda (2022) Sudan strain
• Major outbreak 2013 West Africa (DRC, Guinea) Zaire strain
Not contagious until symptoms develop which include, fever, weakness,
myalgia, and headache, as disease moves on there can be a “wet”
phase with copious vomiting and diarrhea and sometimes a
hemorrhagic phase with bleeding issues
• Transmission from direct contact with bodily fluids, patients are placed in
special pathogen containment units to handle unique patient care
biohazardous risks and waste disposal needs
• Biothreat Level A pathogen, testing in BSL-3 with BSL4 PPE
• Multifocal necrosis in liver, spleen, kidneys, testes and ovaries
• Fatality rate >=55%
Public health and special pathogen unit testing (PCR), detectable at >=
4 days of illness
Filoviridae:
48. Severe Acute Respiratory Syndrome 1 (SARS)
o Outbreak in China 2003 that spread to 29 countries / @ 80 cases in the US
o Bat coronavirus with civet cat identified as the intermediate host
o Dry cough and/or shortness of breath with pneumonia by day 7-10 of illness
o Laboratory testing available only in public health laboratories (CDC) Molecular
tests performed on NP, Throat, sputum, blood, and stool specimens.
Most coronaviruses cause cold like symptoms,
but a few took a very different path.
Middle East Respiratory Syndrome (MERS)
o Endemic in Arabian peninsula (1st reported 2012)
o Infection from direct contact with infected camels
o Close human to human contact can transmit infection but no outbreaks
o Fever, rhinorrhea, cough, malaise followed by shortness of breath
o 30% fatality rate from respiratory failure
49. SARS-CoV-2
• Current evidence: Bat coronavirus transmitted to human, ? intermediate host
• Spread primarily by respiratory droplet
• Influenza-like illness ranging from asymptomatic and mild symptoms to severe respiratory
illness with systemic complications & long-term neurologic complications
Most common presenting symptoms: Fever, dry cough, shortness of breath, sore throat,
nasal congestion, loss of sense of smell and taste, and diarrhea
Transmission, symptoms and severity can be COVID variant dependent and patient
specific, worse for obese, cardiac issues, diabetes, asthma, and immune suppression.
• Diagnosis:
Optimal specimen: Nasopharyngeal collected in universal transport medium (UTM)
Detection of viral RNA by molecular amplification assay is most sensitive and specific
Antibody detection (IgM and IgG) used to detect past COVID infection or response to vaccine
• Vaccine/ ongoing program for immunity and variant protection
50.
51. Diverse group of > 60 viruses – SS RNA
Seasonal viruses most often in summer and fall
• Polio virus/ poliomyelitis, CNS infection leads to flaccid paralysis
Salk Inactive Polio Vaccine (IPV) recommended
• Coxsackie A = vesicular oral lesion (Herpangina)
Hand, foot, and mouth disease
• Coxsackie B = Pericarditis and Myocarditis
• Enterovirus = (1) Parechovirus causes an aseptic meningitis in
children, with fever and rash, increase in cases in 2022
(2) EV-D68 meningitis with acute flaccid myelitis
• Echovirus = various infections, intestine
• Rhinoviruses = common cold
Grow in continuous cell lines in 5-7 days
• Teardrop or kite like CPE formed
Molecular assays superior for diagnosis
52. Fecal – oral transmission, contaminated food or person to person
Common traveler’s disease
Recent outbreaks in US homeless populations due to inadequate
sanitary facilities
Incubation 15 – 50 days with abrupt onset, low mortality, no carrier
state
80% infected develop symptoms of jaundice & elevated
aminotransferases
Diagnosis – serology with diagnostic IgM positive in early infection
Antibody (IgG) is protective and lasts for lifetime
Vaccination to prevent
53. Single stranded RNA virus
Hemagglutinin (H) and Neuraminidase (N) spikes on outside of the viral
capsid
• H and N provide virus strain typing information – H1N1, H3N2
Antigenic drift - minor change in the amino acids of either the H or N
glycoprotein, can occur yearly
Cross antibody protection exists that provides some protection
Antigenic shift - genome reassortment with a “new” virus
created/origin usually bird or animal (potential pandemic)
H5N1 = Avian Influenza strain
H1N1 = 2009 Influenza A pandemic strain
54. Disease: Acute onset of respiratory symptoms (nose, throat or lung) which can
progress to secondary bacterial lung infection
Usually, one H and N type dominates, most recently H3N2 and H1N1
Diagnosis
• Viral cell culture obsolete / no characteristic CPE formed
• Amplification (PCR) is gold standard for influenza diagnosis
Tests detect both Flu A and the H/N type, non-typable Flu A could be unique virus
Treatment: Tamiflu (Oseltamivir)
• Tamiflu has remained sensitive to evolving strains
Influenza B
• Milder form of respiratory symptoms compared to Flu A
• Usually <=10% of cases of total influenza cases most years
Vaccinate – Quadrivalent vaccine that contains 2 A types and 2 B types
55. Measles (Rubeola)
• Fever, Rash, Dry Cough, Runny Nose, Sore throat,
inflamed eyes (photosensitive), very contagious
• Koplik’s spot – small red spot with central bluish
discoloration- seen in the inner lining of the cheek
• Subacute sclerosing panencephalitis
Rare form of chronic progressive brain inflammation caused by
slow infection with certain defective strains of hypermutated
measles virus.
Diagnosis: Clinical symptoms plus molecular tests
performed on nasal or throat specimens
Histology for acute lung injury – multinucleated giant
inclusions with perinuclear halos
Vaccinate – MMR (Measles, Mumps, Rubella) vaccine
Treatment: Not specific, supportive
H and E stain/ lung
56. Types 1, 2, 3, and 4
Spread by respiratory droplets
Disease:
• Upper respiratory tract infection in adults and children with
fever, runny nose and cough
• Lower respiratory tract infection - Croup, bronchiolitis and
pneumonia more likely in children, elderly and immune
suppressed
Molecular methods are standard of practice for diagnosis
Supportive therapy only
No vaccine
57. Spread by respiratory droplets and contact
Infection of parotid and salivary glands, other sites
affected less commonly: testes/ovaries, Eye,
Inner ear, CNS
Diagnosis: clinical symptoms, serologic tests, and
molecular assays
Cell culture CPE = multinucleated cells formed
Histology – diffuse interstitial edema and an inflammatory
infiltrate composed of histiocytes, lymphocytes, and plasma
cells.
Vaccination: Measles/Mumps/Rubella (MMR)
No specific therapy, supportive
Mumps Orchitis
58. Respiratory disease - common cold to pneumonia, bronchiolitis to
croup (young infant) more serious in infants and immune suppressed
Transmission by contact and respiratory droplet
Best specimen for testing: Nasopharyngeal or nasal aspirate
Diagnosis: Cell culture in Hep-2 or HeLa cell lines (obsolete)
Molecular amplification testing standard of practice
Lung biopsy: Syncytium formation
Treatment: Ribavirin Vaccine: Clinical trials
Classic CPE =
Syncytium
formation(multinucleated
giant cell formation) in
HeLa cell line
Syncytium formation
In lung tissue
59. 1st discovered in 2001 – seasonal (winter) community acquired viral
respiratory tract disease
• 95% of cases in children <6 years of age but can be seen in the
elderly and immune suppressed
• Upper and lower respiratory tract disease
2nd only to RSV in the cause of bronchiolitis
Will not grow in viral cell culture
Molecular assays (PCR) for diagnosis
Treatment: Supportive, no specific anti-viral therapy available
No vaccine
60. Nonenveloped/double stranded RNA virus
Winter - spring seasonality
• Gastroenteritis with vomiting – common cause of severe diarrhea
in children 6m – 2 yr, can lead to dehydration
• Fecal – oral spread and can be transmitted by aerosols
• Resistant to detergents / hardy non-enveloped virus
Major cause of childhood death in developing countries
Diagnosis – unable to grow in cell culture
• Molecular amplification assays are standard of practice
• Vaccine (oral) has greatly decreased infection rate
Rota = Wheel
EM Pix
61. Non enveloped single stranded RNA virus
Spread by contaminated food and water, feces and vomitus, direct
contact with sick, and touching contaminated surfaces
<=20 virus particles in waste material can transmit infection making
Norovirus infection very contagious
Leading cause of epidemic gastroenteritis in all age groups,
particular problem in the elderly
• Vomiting and diarrhea
• Disease course usually lasts 24-48 hours
• No specific therapy / No vaccine
Diagnosis: Molecular amplification methods
• Will not grow in viral cell culture
62. Unique characteristics of HIV
• CD4 primary receptor site on lymphocyte for attachment and entry of
Retrovirus
• Reverse transcriptase enzyme converts genomic RNA into DNA
Transmission - sexual, blood and blood product exposure,
perinatal
Non-infectious complications of HIV/AIDS:
• Lymphoma, Kaposi’s sarcoma, anal cell carcinoma, and non-
Hodgkin’s lymphoma
Infectious complications:
• Pneumocystis, Cryptococcal meningitis, TB and Mycobacterium
avium complex, Microsporidia, Cryptosporidium, STD’s , Hepatitis B
and C
63. Antibody Enzyme immunoassay with Western Blot confirmation (old way)
Positive serology (EIA) test confirmed by Western blot
Western blot detects gp160/gp120 (envelope proteins), p 24 (core), and p41(reverse
transcriptase)
Must have positive serology plus at least 2 solid bands on Western blot to confirm as a
positive result for HIV diagnosis
Improved testing - Antigen/antibody combination (4th generation) immunoassay* that
detects IgG and IgM HIV-1 and HIV-2 antibodies plus HIV-1 p24 antigen.
Detects HIV infection earlier (at 2-4 weeks of infection) than previous methods (>=3 months)
and screens for both acute and established infection.
Patients testing positive require additional testing:
• Establish HIV-1 or HIV-2 virus infection
HIV viral load quantitation using quantitative PCR methods
Resistance gene testing – viral subtype knowledge optimizes therapy
Most isolates in US / type B
Monitor for low CD4 counts for HIV infection severity
65. Rubella, Three-day measles, or German measles
Relatively mild disease usually in children presenting with rash, low grade fever,
cervical lymphadenopathy
Respiratory transmission
Congenital rubella –
• Occurs in a developing fetus of a pregnant women who has contracted Rubella
• It was the neurotropic virus of the fetus prior to the occurrence of Zika
Infection caused deafness, eye abnormalities, congenital heart disease in the
newborn
Diagnosis: serology in combination with clinical symptoms
Live attenuated vaccine (MMR) to prevent
67. 1st outbreak occurred in 1993 on a farm in the four corner states
(NM,AZ,CO,UT), this led to disease description
Recent outbreak Yosemite National Park where mice worked their way
into insulation in the walls of cabins (2012)
Transmission
• Urine and secretions of wild deer mouse and cotton rat
• Transmission from pet rats to humans
Infected animals found in states west of the Mississippi River
Respiratory disease that can lead to respiratory failure, symptoms
usually begin with myalgia, headache, cough
Diagnosis: Serology
Supportive therapy
69. Variola virus – Smallpox, eradicated in 1977
Vaccinia virus - Smallpox vaccine strain,
immunologically related to smallpox
• Vaccinia can also cause limited disease after vaccine
administration in immune suppressed which would prevent
vaccination of this population
Smallpox begins as maculopapular rash on central
body moving outward and progressing to a vesicular
rash
BSL4 conditions are required for laboratory work,
Variola virus is Category A Biothreat agent
Possible infection reported to public health for case
investigation and molecular diagnostic testing
70. Historically, related to exposure to infected exotic animals,
endemic to Africa (DRC and Nigeria)
Worldwide human MPX outbreak, began May 2022 with
origin 2 large social gatherings in Europe
Symptoms: fatigue, fever, headache, aches and pains.
• Over the next week, Characteristic deep seated, well circumscribed
lesions develop (pictured), However, lesions can be variable in
appearance
Transmission by close contact
Diagnosis: Detect virus from lesions (PCR), must use
BSL-3 facility, or BSL-2 facility with BSL PPE precaution
to perform laboratory testing.
Antiviral therapy (Tecovirimat)
Smallpox vaccine can help to prevent (78% effective)
71. Appears as small flesh-colored papules on the skin
Commonly involved areas include groin and axillae but
disseminated disease may be seen in
immune compromised
Treatment dependent upon patient, location and extent of lesions
Histopathology shows a well
circumcised epithelial
proliferation with central
depression
73. Worldwide in animal populations
• Bat and raccoon primary reservoirs in US
• Dog reservoir in developing countries
Infections from bites and inhalation of animal aerosolized saliva, urine and feces
Post exposure rabies vaccine and rabies immunoglobulin PRIOR to the development of
symptoms prevent disease development
Classic disease symptom: excessive salivation due to paralysis of throat muscles
Diagnosis: Detection of viral particles (Negri bodies) in the brain and detection of
rabies genetic material testing saliva by molecular assays
Public health laboratories for assistance with diagnosis and therapy
Intracytoplasmic
Negri bodies
In brain biopsy
Bullet
shaped viral
particles
74. Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal
neurodegenerative disorder believed to be caused by an abnormal
cellular glycoprotein known as a prion protein.
Also known as transmissible spongiform encephalopathy (TSE) so
named due to the microscopic appearance of the infected brain
Spongiform changes observed in the gray matter on stained brain
biopsy. This is the most definitive test for diagnosis
Protein product 14-3-3 can be tested for in CSF
It is a biproduct of cell death in the brain
Not specific but helpful in ruling in/out diagnosis
Safety important to prevent transmission
• Standard Precautions
• Use disposable equipment when possible
Spongiform
change in
the
Gray matter