Virology Update 2017

Mar. 7, 2017

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Virology Update 2017

  1. Margie Morgan, PhD, MT(ASCP), D(ABMM)
  2. 1.Direct Staining for Antigen 2. Enzyme Immunoassay 3. Molecular Amplification 4.Viral Cell Culture
  3.  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 Tzanck Tzanck DFA
  4. • 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
  5.  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
  6. • 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
  7.  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
  8. 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
  9.  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
  10.  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
  11.  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
  12.  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
  13.  Transmission: direct contact/secretions  Latency: dorsal root ganglia  Diseases • Gingivostomatitis • Herpes labialis • Ocular • Encephalitis • Neonatal • Disseminated in immune suppressed  Therapy – Acyclovir, Valacyclovir
  14.  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 fibroblast Cell line HSV CPE – cell rounding starting on the edge of the monolayer.
  15.  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
  16.  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
  17. Varicella-Zoster Diagnosis Cell culture at 5 – 7 days Limited # of infected foci in monolayer Sandpaper look to the monolayer background with scattered rounded cells – Diploid fibroblast cells Young wet vesicular lesions are best for culture and/or molecular testing Lesions of zoster usually over one dermatome
  18.  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
  19. Cell culture - CMV infected fibroblast monolayer with grape like clusters of rounded cells Histopathology – Intranuclear and Intracytoplasmic inclusions – known as OWL EYE inclusions
  20.  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 cell transformation
  21.  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
  22. VCA = viral capsid antibody EBNA = Epstein Barr nuclear antigen EA = early antigen
  23. Anti-EBV antibodies Interpretation 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 Serologic Diagnosis of EBV
  24.  HH6 • Roseola [sixth disease] • 6m-2yr high fever & rash  HH8 • Kaposi’s sarcoma • Castleman disease • Primary effusion lymphoma Onion skin pattern of Castleman disease
  25.  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
  26.  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 with stranding
  27. Adenovirus Smudge cells- (Membranes become blurred) and intranuclear inclusions
  28.  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 appearance of fifth disease
  29. Infectious and oncogenic or potentially oncogenic DNA viruses
  30.  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
  31. • 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
  32.  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
  33.  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
  34. Hepacivirus – Hepatitis C Flavivirus – West Nile, Dengue, Zika, andYellow Fever
  35.  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
  36.  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
  37. •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
  38. • 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
  39.  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
  40.  >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
  41. SARS - Severe Acute Respiratory Syndrome – Outbreak in China 2003 – spread to 29 countries Initially dry cough and/or shortness of breath with development of pneumonia by day 7-10 of illness Lymphopenia in most cases Laboratory 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
  42.  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
  43. CPE of Enterovirus Teardrop and kite like cells in Rhesus Monkey Kidney cell culture Uninfected cells
  44.  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
  45. Influenza virus A Influenza virus B
  46.  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
  47.  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
  48. Measles Parainfluenza 1,2,3,4 Mumps Respiratory Syncytial Virus Human Metapneumovirus
  49. • 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 Measlessyncytium H and E stain/ lung
  50.  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
  51.  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
  52.  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 formation In lung tissue
  53.  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
  54. Rotavirus
  55.  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 = Wheel EM Pix
  56. Norovirus
  57.  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
  58. Human Immunodeficiency Virus HIV
  59.  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
  60. 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 infection Detects 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
  61.  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)
  62. RNA Virus Rubella
  63. Known as the “Three day measles” or German measles Rash, low grade fever, cervical lymphadenopathy Respiratory transmission Congenital 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 symptoms Live attenuated vaccine (MMR) to prevent
  64. Hantavirus
  65.  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
  66. Smallpox virus (Variola virus) Vaccinia virus
  67.  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
  68. Chicken pox – Lesions in different stage of development Smallpox – all lesions same stage of development Chickenpox vs Smallpox lesions
  69. Rabies virus
  70.  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
  71. Rabies virus particles EM showing the bullet shaped virus Negri bodies – Intracytoplasmic brain biopsy specimen
  72.  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