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