Margie Morgan, PhD, MT(ASCP),D(ABMM)

1.Direct Staining for Antigen2. Enzyme Immunoassay

3. Molecular Amplification4. Viral Cell Culture

Direct Fluorescent antibody (DFA) stain Collect cells from base of fresh vesicular lesion Stain with DFA specific for HSV and/or VZV

Look for fluorescent cells (virus infected) using fluorescence microscope

More sensitive & specific method than Tzanck prep (DFA 80% vs. Tzanck 50%)

Tzanck prep= Giemsa stained cells from lesion -/examine for multinucleated giant cells of Herpes

TzanckTzanck DFA

• Enzyme immunoassay (EIA) – Antigen/antibody complex formed – then

bound to a color producing substrate Used most often for:

Detection of non culturable viruses like Rotavirus Influenza A and B , & Respiratory syncytial virus

(RSV) from nasal/NP swab – point of care• Membrane lateral flow EIA Liquid/well

EIA

Molecular Amplification (for DNA or RNA)• Rapid/Sensitive/Specific for numerous viruses• Exceeds sensitivity of culture/ Gold standard for:

Respiratory viruses HSV and Enterovirus detection from CSF

Culture <=20% PCR >=90%• Tests of diagnosis not cure – can continue to shed

residual virus for 7 – 30 days after initial positive test

Molecular quantitative assays CMV - Quantitative assays in transplant patients Hepatitis B and C detection and viral load HIV viral load

• Inner tube wall of tube coated with monolayer

of cells in liquid growth media• Three types of cell lines:

Primary cell lines – direct from animal or human organ into culture tube , will only survive one subculture Rhesus monkey kidney-RMK

Diploid – semi continuous cell lines– Can survive 20 – 50 subcultures into new vials – Human diploid fibroblast cells, example: MRC-5-Microbiology

Research Council 5 Continuous cell lines – can survive continuous

passage into new vials, Tumor lineage, HEp-2 and HeLa

Patient specimens inoculated into cell culture tubes, incubated, then read under light microscopy for “Cytopathic effect” – the effect the virus has on the cell monolayer• The pattern of destruction of the cell

monolayer is specific for each virus type

Spin Down Shell Vial Culture – •A way to speed up viral cell culture•Cell monolayer is on a coverslip•Specimen inoculated into vial with coverslip•Centrifuge vial to induce virus invasion into cells•Incubate @ 35C, 24-72 hours•Direct fluorescent antibody stain of cells – target early virus antigens (those first formed )

Cover slip

Viral transport media (VTM) - Hanks balanced salt solution with antibiotics• Also known as Universal Transport Media (UTM)• Transport of lesions, mucous membranes and throats

– specimens collected with swab • 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 onto cell monolayer

Most likely - HSV Intermediate

• Adenovirus• Influenza A and B• Enterovirus

Least likely• Respiratory Syncytial Virus (RSV)• Cytomegalovirus (CMV)• Varicella Zoster virus (VZV)• Amplification preferred for these viruses due to

transport issues

Fast (@ 24-48 hours)• HSV

Intermediate (5 -7 days)• Adenovirus Enterovirus• Influenza VZV

Slow (10 - 14 days)• RSV

Slowest (10 - 21 days)• CMV

Amplification methods desirable for most – they can provide results the same day

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

Transmission: direct contact/secretions Latency: dorsal root ganglia Diseases

• Gingivostomatitis • Herpes labialis• Ocular• Encephalitis• Neonatal • Disseminated in immune suppressed

Therapy – Acyclovir, Valacyclovir

Herpes simplex 1 & 2 do well in culture• Produce CPE within 24-48 hrs • Human diploid fibroblast cells (MRC-5)Observe for

characteristic CPE

Negative fibroblastCell line

HSV CPE – cell rounding starting on the edge of the monolayer.

Cytology/Histology – multinucleated giant cell, intranuclear inclusions

Cannot differentiate from VZV

Amplification (PCR) Cell culture – HSV 1, HSV 2 Serology – more useful for proof of past infection

than for acute diagnosis, some cross reaction with 1 and 2

Transmission: close contact Latency: dorsal root ganglia Diseases:

• Chickenpox (varicella) • Shingles (zoster – latent infection)

Serious disease in immune suppressed or adult patients which progress to pneumonia or encephalitis

Ramsay-Hunt syndrome – facial nerve / facial paralysis Histology – multi-nucleated giant cells like those of

Herpes simplex Serology useful for immune status check Amplification useful for disease diagnosis Effective vaccine has lowered the incidence of VZV

in children

Varicella-Zoster Diagnosis

Cell culture at 5 – 7 daysLimited # of infected foci in monolayer

Sandpaper look to the monolayer background withscattered rounded cells –

Diploid fibroblast cells

Young wet vesicular lesions are best for culture and/or molecular testingLesions of zoster usually over one dermatome

Transmitted by blood transfusion , vertical and horizontal transmission to fetus, or close contact

Latency: Macrophages Disease: Infection is 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)

Due to persistent shedding it is best to do quantitative PCR to detect high viral loads, most consistent with ongoing infection

• Histopathology Treatment: ganciclovir, foscarnet, cidofovir

Cell culture -CMV infected fibroblast monolayer with grape like clusters of rounded cells

Histopathology – Intranuclear and Intracytoplasmic inclusions – knownas OWL EYE inclusions

Transmission - close contact, saliva Latency - B lymphocytes

• Cell receptor CD21 Diseases include:

• Infectious mononucleosis • Lymphoreticular disease• Oral hairy leukoplakia • Burkitt’s lymphoma• Nasopharyngeal Carcinoma• 1/3 Hodgkin’s lymphoma

Will not grow in cell culture Serology most used for diagnosis PCR techniques developed

EBV infection with B celltransformation

HA react with antigens phylogenetically unrelated to the antigenic determinants against which they were raised

Human HAs secondary to EBV are detected by the ability to react patient serum 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

VCA = viral capsid antibody EBNA = Epstein Barr nuclear antigenEA = early antigen

Anti-EBV antibodies Interpretation

VCA IgM VCA IgG EBNA-1 IgG

Negative Negative Negative No immunityPositive Negative Negative Acute infectionPositive Positive Negative Acute infectionNegative Positive Positive Past infectionNegative Positive Negative Acute or past infectionPositive Positive Positive Late primary infectionNegative Negative Positive Past infection

VCA = viral capsid antibody

Serologic Diagnosis of EBV

HH6 • Roseola [sixth disease]• 6m-2yr high fever & rash

HH8• Kaposi’s sarcoma • Castleman disease• Primary effusion lymphoma

Onion skin pattern of Castleman disease

DNA - non enveloped/ icosahedral virus Latent: lymphoid tissue Transmission: Respiratory and fecal-oral route Diseases:

• Adenovirus type 14 – virulent respiratory strain / pneumonia

• Pharyngitis (year round epidemics)• Gastroenteritis in children

Adenovirus types 40 & 41• Kerato-conjuctivitis – red eyes for @ 2 wks• Disseminated infection in transplant patients• Hemorrhagic cystitis in immune suppressed

Diagnosis• Cell culture (CPE)

CPE in 2-5 days with round cells connected by strands

Grows best in Heteroploid continuous passage cell lines (HeLA, Hep-2)

• Amplification (PCR)* superior for respiratory infection

• Histology - Intranuclear inclusions / smudge cells• Antigen detection –

staining respiratory cells by DFA for Respiratory infections

Stool antigen for 40/41 diarrhea strains• Supportive treatment – no specific viral therapy

Round cells withstranding

Adenovirus Smudge cells-(Membranes become blurred)and intranuclear inclusions

DNA virus Parvovirus B19

• Erythema infectiosum (Fifth disease) – headache rash and cold-like symptoms in the child

• In pregnant, infection in 1st trimester, hydrops fetalis leading to miscarriage

• Aplastic crisis in patients with chronic hemolytic anemia and AIDS

• Histology - virus infects mitotically active erythroid

precursor cells in bone marrow• Molecular and Serologic methods

to aid diagnosis

Slapped face appearanceof fifth disease

Infectious and oncogenic or potentially oncogenic DNA

viruses

Diseases: 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 of HPV Hybrid capture DNA probe for detection and typing PCR* – FDA cleared platforms for detection/typing, capable of detecting many HPV types

Three vaccines - 1°to guard against HPV 6,11,16,18

Pap smear

• JC virus [John Cunningham] Progressive multifocal leukoencephalopathy -

PML -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 to aid diagnosis

Giant Glial Cells of JCV

Enveloped DNA – Hepadna virus Hepatitis B clinical disease

• 90% acute• 1% fulminant• 9% chronic Carrier state in liver can lead to cirrhosis and hepatic cell carcinoma……. transplant

Antiviral therapies to prevent spread•Serology for diagnosis•Vaccinate to prevent

Surface Antigen Positive• Active Hepatitis B or Chronic Carrier, if detected

Do Hep B Quantitation Do Hep e antigen – if positive, Chronic carrier and 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

Hepacivirus – Hepatitis CFlavivirus – West Nile,

Dengue, Zika, and Yellow Fever

Spread parenteral - 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 US have chronic HepC Seven major genotypes (1-7)

• Acute self limited disease that progresses to a disease that mainly affects the liver• Type 1 virus most common in USA• Infection persists in @ 75-85%/ no symptoms• 5 - 20 % develop cirrhosis• 1-5 % associated with hepatocellular CA

liver transplantation

Diagnosis:• Hepatitis C antibody test

If Hep C antibody detected perform RNA quantitative assay for viral load Genotype of virus for proper therapy

selection/duration Assessment of liver disease - ? cirhhosis

No vaccine available Antivirals currently FDA cleared that can cure

>= 85% of patients infected with Hepatitis C

•Dengue – “breakbone fever”

Vector Aedes mosquito / Asia and the Pacific Fever, severe joint pain, rash Small % progress to hemorrhagic fever

•Diagnosis – Serology(IgM for acute infection) Zika virus•Vector: Aedes mosquito •Current outbreak began in South America (Brazil) and spread to central America, Caribbean and US (Miami)•Clinically a milder form of Dengue in most adults – but has a neurologic tropism

Microcephaly in fetuses borne to infected moms Potential developmental issues in infected children Guillain- Barre syndrome during ongoing Zika

infection•Diagnosis: Serum Antibody IgM and PCR serum, urine, amniotic fluid and CSF

• West Nile Vector Aedes and Culex mosquito Common across the US, Bird primary reservoir, horses also at risk Fever, Headache, Muscle weakness, 80%

asymptomatic. Small % progress to encephalitis. Meningitis, flaccid paralysis IgG) and PCR

Chikungunya virus Vector Aedes mosquito with 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/ RNA virus

When screening for ZIKA – need to rule out infection with Dengue and Chikungunya. Similar diseases with very different sequelae

>20 outbreaks since discovery in 1976 • Most recent Dec 2013 - West Africa• Prolonged outbreak due to area effected had high

population with limited medical resources Transmission direct contact with bodily fluids – fatality rate

55%• Animal reservoir (?) fruit bats

Asymptomatic are not contagious Fever, weakness, myalgia, headache, travel history

• Also consider malaria and typhoid in the differential Susceptible to hospital disinfectants Testing (EIA, PCR) at CDC – positive >= 4 days of illness RNA virus Level A agent of Bioterrorism

SARS - Severe Acute Respiratory Syndrome –Outbreak in China 2003 – spread to 29 countriesInitially dry cough and/or shortness of breath with development of pneumonia by day 7-10 of illness Lymphopenia in most casesLaboratory testing public health laboratories (CDC) -antibody testing enzyme immunoassay (EIA) and reverse transcription polymerase chain reaction (RT-PCR) tests for respiratory, blood, and stool specimens. These are RNA single strand viruses.

• MERS - Middle East Respiratory Syndrome• Isolated mostly to Arabian peninsula (2012)• Direct contact with infected camels• Close human to human contact can spread infection – no

outbreaks – 30% fatality rate• Fever, rhinorrhea, cough, malaise followed by shortness of breath

Diverse group of > 60 viruses – SS RNA• Infections occur most often in summer and fall• Polio virus - paralysis

Salk vaccine Inactive Polio Vaccine (IPV)** recommended Sabine vaccine Live Attenuated Vaccine (OPV)

• Coxsackie A – Herpangina – vesicular oral lesions• Coxsackie B – Pericarditis/Myocarditis• Enterovirus – Aseptic meningitis in children, hemorrhagic

conjunctivitis• Echovirus – various infections, intestine• Rhinoviruses – common cold

Grow in cell culture * 5-7 days (Diploid mixed cell culture – Primary Monkey Kidney)

PCR superior for diagnosis , more rapid and sensitive for all viruses

CPE of EnterovirusTeardrop and kite like cells inRhesus Monkey Kidney cell culture

Uninfected cells

Fecal – oral transmission, contaminated food or person to person

80% develop symptoms – jaundice & elevated aminotransferases

Usually – short incubation (15- 50 days), abrupt onset, low mortality, no carrier state

Diagnosis – serology, IgM positive in early infection to differ from other Hepatitis viruses

Antibody is protective and lasts for life Vaccine available

Influenza virus AInfluenza virus B

Hemagglutinin and Neuraminidase glycoproteins spikes on outside of viral capsid• Gives 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

Disease: fever, malaise …. Death from respiratory complications or secondary bacterial infection

Diagnosis• Cell culture obsolete [RMK]• Enzyme immunoassay (EIA) lateral flow membrane can

be used in point of care testing• Amplification (PCR) gold standard for detection

Treatment: Amantadine and Tamiflu (Oseltamivir)• Seasonal variation in susceptibility but Tamiflu usually

sensitive Influenza B

• Milder form of Influenza like illness• Usually <=10% of cases /year

Vaccinate – Trivalent vaccine -2 A viruses/1 B virus

MeaslesParainfluenza 1,2,3,4

MumpsRespiratory Syncytial VirusHuman Metapneumovirus

• Fever, Rash, Dry Cough, Runny Nose, Sore throat, inflamed eyes (photosensitive) Can invade lung

• Respiratory spread - very contagious• Koplik’s spots – bluish discoloration inner lining of

the cheek is pathognomonic• Subacute sclerosing panencephalitis [SSPE]

Rare chronic degenerative neurological disease Persistent infection with a mutated measles virus, due to

mutated virus there is total lack of an immune response• Diagnosis: Clinical symptoms and Serology • Vaccinate – MMR (Measles, Mumps, Rubella) vaccine• Treatment: Nothing specific, Immune globulin, vitamin A

Measles syncytium

H and E stain/ lung

Types 1,2,3, and 4 Person to person spread 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

Heteroploid - continuous cell lines (Hep-2) for culture – not suggested (slow and insensitive)

PCR** methods are gold standard Supportive therapy only available

Person to person contact Parotitis, but can also cause

infections in other sites: Testes/ovaries, Eye, Inner ear, CNS

Diagnosis: clinical symptoms and serologic tests

Prevention: MMR vaccine No specific therapy, supportive

Respiratory disease - common cold to pneumonia, bronchiolitis to croup, serious disease in infants and immune suppressed• Classic disease: Young infant with bronchiolitis

Transmission by contact and respiratory droplet Specimen: Nasophayrngeal, nasal swab, nasal

lavage Diagnosis: EIA (point of care), cell culture

(heteroploid, continuous cell lines), PCR is gold standard**, and lung biopsy

Treatment: Supportive, ribavirin Classic CPE = Syncytium formation heteroploid cell line

Syncytium formationIn lung tissue

1st discovered in 2001 – community acquired respiratory tract disease in the winter• @95% of cases in children <6 years of age• Upper respiratory tract disease• Lower 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

Rotavirus

Winter - spring seasonality• Gastroenteritis with vomiting and fluid loss –

most common cause of severe diarrhea in children 6m – 2 yrs

• Fecal – oral spread Major cause of childhood death / 3rd world

countries Diagnosis – cannot grow in cell culture

• Enzyme immunoassay, PCR Vaccine available

Rota = WheelEM Pix

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 sea aboard cruise liners

Leading cause of epidemic gastroenteritis – worldwide on land and sea• Fluid loss from vomiting can be debilitating

Disease course usually limited, 24-48 hours PCR for diagnosis

• Cannot be grown in cell culture

Human Immunodeficiency Virus

HIV

CD4 primary receptor site for entry of 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

Antibody EIA with Western Blot confirmation (old way) Positive tests must be confirmed with a Western blot test Western blot detects gp160/gp120 (envelope proteins), p 24

(core), and p41(reverse transcriptase) 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 IgG and IgM HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen to screen for established and acute infectionDetects infection earlier (@ 2- 4 weeks

Positive patients on either test require additional testing: HIV viral load quantitation >= 100 copies Resistance Testing – report subtype to optimize therapy Most isolates in USA type B

•Monitor CD4 counts for infection severity

Non-compliant patients or newly diagnosed• Pneumocystis – most common in US • Cryptococcus neoformans & Histoplasma

capsulatum (disseminated)• TB/Mycobacterium avium complex (disseminated)• Microsporidia and Cryptosporidium (Intestinal)• Hepatitis B• Hepatitis C• STD’s – Syphilis, GC, Chlamydia

Syphilis rate high (mucosal contact)

RNA VirusRubella

Known as the “Three day measles” or German measlesRash, low grade fever, cervical lymphadenopathyRespiratory transmissionCongenital rubella –

• occurs in a developing fetus of a pregnant women who has contracted Rubella, highest % (50%) in the first trimester pregnancy

• Prior to Zika it was the neurotropic virus of the fetus• Deafness, eye abnormalities, congenital heart disease

Diagnosis - Serology in combination with clinical symptomsLive attenuated vaccine (MMR) to prevent

Hantavirus

USA outbreak in the four corner states (NM,AZ,CO,UT) on an Indian reservation in 1993 brought attention to this virus

Source - Urine and secretions of wild field mice• Deer mouse (picture) and cotton rat

Myalgia, headache, cough and respiratory failure Found in states west of the Mississippi River Diagnosis by serology Supportive therapy

Smallpox virus (Variola virus)Vaccinia virus

Variola virus – agent of Smallpox Vaccinia virus - active constituent in the Smallpox

vaccine, it is immunologically related to smallpox, • Vaccinia can cause disease in the immune suppressed, which

prevents vaccination of this population • Eradication of smallpox occurred in 1977

Disease begins as maculopapular rash and progresses to vesicular rash - • all lesions in same stage of development in one body area –

rash moves from central body outward Category A Bioterrorism agent (can maim or kill) Requires BSL4 laboratory (self contained lab) Reported to public health department for investigation

Chicken pox – Lesions in different stage of development

Smallpox – all lesions same stage of development

Chickenpox vs Smallpox lesions

Rabies virus

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 symptom 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

Rabies virus particlesEM showing the bullet shaped virus

Negri bodies – Intracytoplasmicbrain biopsy specimen

Rare, degenerative fatal brain disorder Transmissible spongiform encephalopathies

(TSE) name established from the microscopic appearance of infected brain

Caused by type of protein - prion Confirmation by brain biopsy Safety – prevent transmission

• Universal Precautions• Use disposable equipment when possible

Spongiform change in the Gray matter