Margie Morgan, PhD, MT(ASCP),D(ABMM)microbeswithmorgan.com

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 Fl antibody specific for HSV and/or VZV

Look for fluorescent cells (virus infected) using fluorescence microscope

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

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

TzanckTzanck DFA

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

bound to a color producing substrate Used most often

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

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

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

Respiratory viruses HSV and Enterovirus detection from CSF

Culture <=20% PCR >=90% Tests of diagnosis not cure – can shed residual virus

for 7 – 30 days after initial positive test• CMV - Quantitative assays in transplantation• Hepatitis B and C detection and viral load• HIV viral load

• Inner tube wall coated with monolayer of cells

plus liquid growth media• Three types of cell lines:

Primary cell lines – direct from animal or human organ into culture tube subculture once 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 is specific for

each virus type

Spin Down Shell Vial Culture – •Speed up virus detection•Cell monolayer on coverslip/vial•Specimen inoculated into vial•Centrifuge vial to induce virus invasion into cells•Incubate @ 35C, 24-72 hours•Direct fluorescent antibody stain of coverslip – 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

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

Cytology/Histology – multinucleated giant cell, intranuclear inclusions

Cannot differentiate from VZV

Amplification (PCR) Serology – more useful for proof of past infection

than for acute diagnosis

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

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 incidence of VZV

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 testing

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

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 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

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 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 antibodyEBNA = 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 infection1Positive Positive Positive Late primary infectionNegative Negative Positive Past infection

Serologic Diagnosis of EBV

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

HH8• Kaposi’s sarcoma • Castleman’s 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 @ 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• Stool EIA for enteric infections• Antigen detection – staining respiratory cells by

DFA for Respiratory infections • Supportive treatment – no specific viral therapy

Round cells withstranding

Adenovirus Smudge cells-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 Serology 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 Three vaccines - 1°to guard against HPV 6,11,16,18

Pap smear

• JC virus [John Cunningham] 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 to aid diagnosis

Giant Glial Cells of JCV

Enveloped DNA – Hepadna virus Hepatitis B clinical disease

• 90% acute• 1% fulminant• 9% chronic Carrier state can lead to cirrhosis and hepatic cell carcinoma

Therapies under investigation•Serology for diagnosis•Vaccinate to prevent

Surface Antigen Positive• Active Hepatitis B or Chronic Carrier

Do Hep B Quantitation Do Hep e antigen – Chronic carrier and worse prognosis

Core Antibody Positive • Immune due to prior infection, acute infection or chronic carrierSurface Antibody Positive • Immune due to prior infection or vaccine

Hepacivirus – Hepatitis CFlavivirus – West Nile,

Dengue, and Yellow Fever

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 Hepatitis C 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• Can require 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 in clinical trials and/ or 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 hemorragic fever

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

•Diagnosis – Serology(IgM, IgG) and PCR

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

Zika virus• Vector Aedes mosquito • Common in South America (Brazil)• Clinically like a mild form of Dengue• **Microcephaly in fetuses born to infected mothers

Diagnosis • Immunoassays for Antibody & PCR (serum and CSF)

>20 outbreaks since discovery in 1976 • current outbreak Dec 2013 - West Africa• Prolonged due to area effected is high population with

limited medical reaources 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

SARS - Severe Acute Respiratory Syndrome –Outbreak in China 2003 – spread to 29 countriesInitially dry cough and/or shortness of breath 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.

• MERS - Middle East Respiratory Syndome• Isolated 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)** 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

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

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 Diagnosis

• Cell culture obsolete [RMK]• Enzyme immunoassay (EIA) on paper membrane can be

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

MeaslesParainfluenza 1,2,3,4

MumpsRespiratory Syncytial VirusHuman Metapneumovirus

Measles• 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

Measles syncytium

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 gold standard Supportive therapy only available

Person to person contact Classic infection is Parotitis, but can cause infections in other sites:

Testes/ovaries, Eye, Inner ear, CNS

Diagnosis: clinical symptoms and serology Prevention: MMR vaccine No specific therapy, supportive

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

Transmission by contact and respiratory droplet Specimen: Naso-phayrngeal, nasal swab, nasal lavage Diagnosis: EIA, cell culture (heteroploid cell lines), PCR is gold

standard, lung biopsy Treatment: Supportive, ribavirin Histology

Classic CPE = Syncytium formationIn heteroploid cell line

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

Rotavirus

Winter - spring seasonality• 6m-2 yrs of age most serious• Gastroenteritis with vomiting and fluid loss –

most common cause of severe diarrhea in children

• Fecal – oral spread Major cause of death in 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 – 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

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

Most isolates in USA type B•Monitor CD4 counts for infection severity

Non-compliant patients or newly diagnosed• Pneumocystis • 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” – 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

• Deafness, eye abnormalities, congenital heart disease

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

Hantavirus

USA outbreak in four corner states (NM,AZ,CO,UT) on 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 No 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 (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 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

Rabies virus particlesEM showing the bullet shaped virus

Negri bodies – Intracytoplasmicbrain biopsy specimen

Rare, degenerative fatal brain disorder Transmissable spongiform encephalopathies

(TSE) name established from the microscopic appearance of brain with infection

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

• Universal Precautions• Use disposable equipment when possible