Molecular Testing, Virology, HSV, HIV, HEP Flashcards
Desceibe nucleic acid testing
Molecular diagnostic
Relatively stable
Ubiquitous in nature (everythjng has it)
Purification processes are virtually identical irrespective of organism
Describe the steps of nucleic acid amplification tests
1- nucleic acid extraction
2- amplification
3- detection
4- interpretation of results
How js nucleic acid extracted
Lysis- release of nucleic acids (using heat,sonification, chemical or enzymatic
Purification - sequential wash steps eliminate contaminants
Recovery (elution)
What does PCR stand for
Polymerase chain reaction
What is PCR
Target site amplification Rapidly and exponentially amplify a particular DNA sequence Rapid Sensitive Specific
Describe the components of a pcr
Need a forward primer and a reverse primer
Template dna
Raw material
Dna polymerase (TAQ, thermostable)
Primers are small sefments of DNA complimentary to strands on template
How does PCR work?
Double stranded DNA separated, 94° heat applied, double strand turns to 2 single strands
Attach primers at lower heat (55°/anneal temp)
Anneal temo depends on primer
Get target site amplification, billions of copies from one double strand DNA
Find infection as long as nucleic acid is present
This reaction happens over and over
What is a virus
A package of genetic informstion protected by a protein shell for delivery into a host cell to be expressed and replicated
Where did the word virus come from
Greek meaninng poison
Initially described by edward jennner
What differentiates viruses from other micro organisms
Nucleic acid (one if either dna or rna) Lack of nuclear membrane and external cell wall Very small genomes, limited number of proteins Do not possess intracellular systems (obligate intracellular parasites) meaning they can not replicate on their own
What are bacteriophages
Viruses thst infect bacteria
How are viruses named or classified
- disease they are associated with
- cytopathology they cause
- site of isolation
- places or oeople that discovered them
- biochemical features
What is viral classification based upon
Size and shape Enveloped or naked (lipid envelop or no?) Nucleic acid composition Genome organization antigenic differences
Describe a virus structure
Capsomere: protein subunits of the capsid; smallest protein unit
Capsid: capsomeres assemble to form viral capsid - surrounds viral genome
Nucleocapsid: capsid + genomic nucleic acids (dna/rna); genetic material found within
Shape: capsid is usually symmetrical
Surface projections and glycoproteins; bind host receptors/allows entry
Envelope (sometimes)
What are the three different shapes of a virus
Icosehedral (cubic) MOST COMMON
Helical (influenza virus)
Complex (pox viruses);dont fit a shape
Describe the envelope
Lipid layer surrounding some bacteria; derived from host cell membrane
Does not protect cell more infact naked cells are more protected
Envelope makes it essier to kill bc it is lipid based
Envelope is less stable
Describe non enveloped viruses
Stable in environment
Resist desiccation, heat, detergents and acids
Transmitted easily on hands and fomites
Describe enveloped viruses
Labile in environment Must stay moist Damaged by drying, acid, detergents or heat (ex herpes viruses, hiv, hbv, influenza) Transmitted in droplets of body fluids Doesnt usually infect GI
How are RNA viruses organized
Single strand -> positive polarity or negative polarity (MRNA or not)
RNA double stranded -> one piece or segmented
Describe how DNA Virusss are organized
Single stranded or double stranded
How are viral infections acquired
Direct personal contact Airborne Parenteral (exposure to blood) Fomites Vectors Vertical transmission (mum to fetus) Enteral (food/waterborne)
What is the virus life cycle
Attachment Penetration Uncoating (expose genome) Transcription (dna/rna) Translation Genome replication Assembly of genome and protein material Release and infect other cells
Describe DNA/RNA Transcription
Dna can be directly transcribed
Negative RNA has to get MRNA to transcribe
Positive RNA directly transcribes
Describe stages of virus-host interaction
Entry to host Primary replication Spread Cell and tissue trophism Secondary replication Cell injury or persistance Host immune response (depending on virus)
What are consequences of virus-cell interaction
Viral proliferation and cell lysis
Latent infection (non replicating)
Persistent infection (ongoing replication)
Oncogenesis (cellular proliferation)
No apparent disease
How are viral infections disgnosed
Clinical features typical of infection
Laboratory diagnosis: serology or amplification, detection of antigens, antibody testing,virus isolation
What are prions
Proteinaceous infectious particles An infectious agent composed of protein Does not self replicate, induces existing proteins to take on the rogue form Very highly resistant Caused severe neurological diseases
What are some diseases caused by prions
Creutzfeldt-jacobs disease
Kuru
Animal forms: bovine spongiform encephalopathy (mad cow)
What herpesviridae affect humans?
Herpes simplex 1 & 2 Epstein barr Varicella zoster Cytomegalovirus Human herpes virus 6,7,8
Describe herpesviridae
Large ICOSAHERDREAL double stranded DNA viruses
Replicate in nucleus of cells
Enveloped w/ numerous glycoprotein spikes
Develop LATENT infections
Initial infection usually asymptomatic
Describe HSV 1/2
Infection of oral genital or ocular epithelium
Hsv- 1 more associated with oral and ocular infection
Hsv-2 frequently associated with genitsl infections
Describe herpes simplex virus
ENVELOPED ds-DNA virus with ICOSAHEDRAL CAPSID
Genital herpes = most common STI world wide
Describe clinical manifestations of primary infrction HSV type 1
Primary: Oral Genital Ocular Peripheral (dentists used to get this)
Describe clinical manifestations of HSV1 recurrent infection
Oral,ocular,genital,peripheral
Symptomatic
Asymptomatic shedding
What is herpes labialis
Cold sore
May be single or clustered
10-14 days
Treatment only necessary in immunocompromised patients
Describe HSV-2 infection
Not synonymous with genital herpes however:
Primarily infections of the genital tract
Neonates can be infected at delivery
Lesions do occur
Describe genital herpes
Many recognized first outbreaks may actually be reoccurrence
First episode tends to be more severe and longer in duration
Describe symptoms associated with genital Herpes
Fever, malaise, inguinal lymph node inflammation, headache, sometimes neck stiffness
Describe shedding for HSV 2
Reoccurrence more common
Asymptomatic shedding 28% of days
Shedding not completely suppressed by acyclovir
Describe shedding in HSV 1
Reoccurrence less frequent
Asymptomatic shedding does occur but less frequently
Describe neonatal herpes
In utero infection rare (does occur) intra partum (delivery) more likely
Primary infection in mum is highest risk
Asymptomatic shedding accounts for most transmission to infected neonates
Can be fatal if untreated
How is Herpes simplex diagnosed
Virus isolation
**PCR (nucleic acid amplification) especially with CSF to check for meningoencephalitis
Why is viral serology not a great diagnostic tool for Herpes simplex virus
70% have been exposed to HSV 1 so everyone is going to light up
How is HSV 1 & 2 treated?
Acyclovir: primary infections, prophylactic and immune compromised
Describe Epstein Barr Virus
Tranmission usually by saliva exchange
Virus has affinity for receptors on the surface of B cells
Viral infection results in local replication and a secondary viremia. There is proliferation of both b cells and T8 dubset of t cells
Describe the epidemiology of EBV infection
50% of 5 year olds have been infected
Children usually asymptomatic
Secon wave in teens more symptomatic
By 40 90-95% of adults have antibodies
What are clinical manifestations of EBV
Infectious mononucleosis (MONO): Illness lasts from 2-8 weeks, fatigue persists
What are symptoms of EBV
Tonsilar enlargement Pharyngeal redness Cervic adenopathy Large liver/spleen Skin rash esp. after ampicillin
How is EBV diagnosed
Serology more common
Lymphocytes & monocytes increased
Low neutrophils and low platelets
Treatment for EBV?
Usually supportive, treatment doesnt do much
Describe cytomegalovirus
Infections occur more in socioeconomic groups
2 peaks: childhood, adulthood
Most adults (70%) are sero positive
All infections resukt in viral latency
How is cytomegalovirus spread
Peri anal: intrauterine, cervix @ delivery, from breast milk
Saliva or close contact
Blood transfusion/organ transplant (RARELY)
During sexual inter course
Describe CMV infection in older hosts
Asymptomatic
Some get mononucleosis like syndrome but heterophile antibody does not develop
How is CMV diagnosed
Serology (determine immunity), molecular
Nucleicacid amplification
Virus isolation
Light microscopy
How is CMV treated/prevented
Usually no treatment required
Antiviral (gancyclovir) and immunoglobulin therapy usually used to prevent and treat immune compromised host
What is HIV
Retrovirus
What are characteristics of retroviruses
Enveloped positive strand RNA virus, encodes reverse transcriptase (reverse transcribes DNA)
Replicate through a DNA intermediate
DNA copy is integrated into host chromosome to become a cellular gene
Describe discovery of retrovirus
HIV-1 discovered 1981 by Gallo and Montagnier
HIV-2 discovered in west Africa
How is HIV transmitted
Blood
Semen
Vaginal fluids
What factors increase infectiousness of HIV
Primary infection - high viral load
Late stage - high viral load
Genital tract infections - mucosal breech and recruitment of inflammatory cells
Describe HIV transmission in mother to baby
Mother to baby 1 in 4 (25%) without treatment
4% with treatment
Describe replication of HIV
CD4 cells primary target
Mushroom shaped gp120 (protein on surface if HIV cell) interacts with CD4 surface molecule expressed on t helper lymphocytes and cells of the macrophage lineage
Reverse transcription after release
Copy of DNA produced
Integrsted into genome (integrase)
Each copy contains approx. 5 errors/mutations
How does HIV enter the cell?
HIV enters the cell by fusion eith the cellular envelope
Describe pathogenisis of HIV
HIV infection of CD4 lymphocytes results in cell death
CD4 primarily responsible for immunity
As CD4 cells are lost opportunistic infections and malignancies occur
Other cells survive and can store virus
What are CD4 cells
One of the most important immune cells
Primarily responsible for cell mediated immunity
What clinical syndromes can occur as a result of HIV
AIDS Toxoplasmosis Cryptococcal meningitis Pneumocystis pneumonia Aids related dementia Wasting syndrome Karposi sarcoma (end stage) Lymphoma Cervical cancer
What are aids defining illnesses
Toxoplasmosis
Cryptococcal menigitis
Pneumocystis pneumonia
How is HIV diagnosed
Serology (antibodies): WESTERN BLOT
Immunologic studies (CD4 counts)
Viral load testing (pcr quantification of RNA in plasma)
Resistance genotyping
Describe western blot
Serology test method
Less sensitive more specific
What is the biorad geenius
Differentiates HIV1 and HIV2
Rapid (~30min)
Removes subjectivity
What are the goals of HIV therapy?
Durable supression of viral load
Restoration/preservation of immune function (CD4 count)
Improvement of quality of life
Reduction of mortslity
Preservation of future trestment options (simple treatment)
How is HIV Treated
Anti retrovirals: nucleoside analogs, protease inhibitors fusion inhibitors integrase inhibitors
Usually Highly Active Anti Retroviral Therapy (HAART)
What is HAART
Highly Active Anti Retroviral Therapy
Describe risk of needle stick exposjre
Risk depends on stage of patients disease and how much blood
Describe post HIV needle stick steps
Confirm patient status
Document your status
Begin prophylaxis WITHIN 72 hours!!
What is chance of getting HIV from needle stick
0.3%
What are the 5 types of hepatitis
A: fecal oral transmission B: sexual fluids& blood to blood C: blood to blood D: travels with B; need B E: fecal oral transmission
Describe the hep A virus
A PICORNAVIRUS; single stranded, naked, icosahedral
Spread almost always by fecal oral spread; either person to person or food contamination
Very common in developing countries
Sporatic usually but can cause outbreaks
What is the incubation period for Hep A
2-4 weeks
Most cases are asymptomatic therefore jaundice only tip of the iceberg
Does Hep A produce a chronic carrier state?
Hep A does not produce a chronic carrier state and has no long term sequelae. Meaning you get Hep A then recover
How is Hep A diagnosed?
Disgnosis is confirmed by demonstrating the appearance of IgM antibodies
How is Hep A prevented?
Vaccine!! (Twinrex):
Usually for travellers, healthcare workers etc
Immune globulin (antibodies) post infection when you need instant protection
Describe Hep B
A double stranded HEPADNAVIRUS (DNA virus)
Patients who become carriers may develop a chronic active or persistent hepatitis
How is Hep B transmitted?
Parenteral, perinatal and sexually
How infectious is Hep B?
Very infectious!! 30% of needle stick injuries resukt in infection for non immune individuals
What is the rule of three?
Needle stick risk:
0.3% HIV
3% for Hep C
30% for Hep B
How common is Hep B
Very common
350,000,000 carriers
1,000,000,000 have serologic evidence of past infection
Describe Carrier state for Hep B
Chances of developing carrier state depend on age of infection
Younger (neonates) most likely to have carrier state
What are some consequences of chronic Hep C
No/minimal liver injury
Chronic active Hepatitis
Cirrhosis of liver (failure/hypertension)
Hepato-cellular carcinoma
How is Hep B treated and prevented
Post exposure prevention for needle stick = HBIG VACCINE
Vaccine: universal for kids
Treatment with lamivudine for chronic hepatitis
How is Hep B diagnosed
Detection of surface antigen in patients serum (HBsAg)
Detection of antibodies to hep B antigen: evidence of old infection or vaccine immunity
Liver biopsy
Describe Acute Hep B
Surface antigen increases and decreases as virus does
Develop antibody to surface antigen & anti HB
Describe Chronic Hepatitis B infection
Lots of surface antigens and anti HBE antibodies
No antibodies for surfave antigens unless patient is treated then you see SOME
If a patient js HBsAG positive
Anti HBsAG negative and ANTI HB core positive what is the likely interpretation of these results
They have a chronic infection
If a patient js HBsAG negative
Anti HBsAG positive (>10IU) and ANTI HB core negative what is the likely interpretation of these results
They have vaccine induced antibodies and are protected
If a patient js HBsAG negative
Anti HBsAG positive (>10 IU) and ANTI HB core positive what is the likely interpretation of these results
Previous natural infection and are protected from future infection
If a patient js HBsAG negative
Anti HBsAG positive (<10IU) and ANTI HB core negative what is the likely interpretation of these results
Vaccine induced antibodies but failed vaccine series
What does it mean if a patient has less than 10 (>10) International units (IU)
They failed their vaccine series
What does it mean if a patient is HBsAg positive
They have a chronic hep B infection
Describe Hep C
Small ENVELOPED RNA virus (flavivirus)
Usually parentally transmitted although less infectious than HBV (3% needle prick)
How does Hep C usually occur?
Usually occurs as sporadic disease in IV drug users, not as common sexual transmission, 75% mild or asymptomatic
What js the incubation period for Hep C
2-20 weeks
What are potential risks of Hep C infection
40-60% may get chronic liver disease, much higher than HEPB
20% may develop cirrhosis
Cancer may develop
How is Hep C diagnosed
Typically qualitative pcr (detects viral RNA)
recombinant immunoblot assay (RIBA)
How is Hep C treated and prevented?
No vaccine
Good public health measures
New hep C drugs >95% cure; largest breakthrough
Used to use interferon
Describe Hep D
Rare infection due to incomplete RNA virus that requires presence of Hep B
Found mostly in IVDUS
Concurrent infection may be more severe
Describe Hep E
A calivivirus (rna)
Almost never seen in north america
Fecal oral spread, lack of chronicity, low fatality rate
What is 16s rRNA
Essential gene common to all bacteria
Codes for small ribosomal units
Gene evolves at a very low rate so little mutation seen
How does 16s gene testing work
Perfectly conserved unchanging ateas of gene
Primers bind to conserved areas look for divergence in 16s rRNA
Only works on single bacteria type
Describe 16s rRNA gene sequencing
Molecular diagnosis
Pcr primers anneal to conserved regions
Any organism present will be amplified
Amplicon will contain several variable regions
What are some advantages of 16s rRNA gene sequencing
No need for viable organisms
Detection/identification of organisms in “partially” treated patients
Detection of slow growing or non growing organisms
What are some disadvantages of 16s rRNA gene sequencing
Only useful from normally sterile sites (monomicrobial)
Contamination significant problem
May take a week to obtain identification
Why do we use typing systems
To determine if a series of isolates obtained from epidemiologic cluster are clonally related
What are benefits of typing systems
Ease of interpretation Reproducibility Ease of performance Discriminatory power Cost
What are phenotypic systems
Bio typing
Antibiograms
Phage typing
Serotyping
Based on presence of metabolic or biologic activities
What are genotypic typing systems
Molecular
Substantial generic diversitt within microbial species
Evoluntionary divergence
What is pulsed field gel electrophoresis
System of electrical currents
What is whole genome sequencjng
Most pulsed field moved to whole genome sequencing
