Molecular Testing, Virology, HSV, HIV, HEP Flashcards
(118 cards)
1
Q
Desceibe nucleic acid testing
A
Molecular diagnostic
Relatively stable
Ubiquitous in nature (everythjng has it)
Purification processes are virtually identical irrespective of organism
2
Q
Describe the steps of nucleic acid amplification tests
A
1- nucleic acid extraction
2- amplification
3- detection
4- interpretation of results
3
Q
How js nucleic acid extracted
A
Lysis- release of nucleic acids (using heat,sonification, chemical or enzymatic
Purification - sequential wash steps eliminate contaminants
Recovery (elution)
4
Q
What does PCR stand for
A
Polymerase chain reaction
5
Q
What is PCR
A
Target site amplification Rapidly and exponentially amplify a particular DNA sequence Rapid Sensitive Specific
6
Q
Describe the components of a pcr
A
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
7
Q
How does PCR work?
A
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
8
Q
What is a virus
A
A package of genetic informstion protected by a protein shell for delivery into a host cell to be expressed and replicated
9
Q
Where did the word virus come from
A
Greek meaninng poison
Initially described by edward jennner
10
Q
What differentiates viruses from other micro organisms
A
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
11
Q
What are bacteriophages
A
Viruses thst infect bacteria
12
Q
How are viruses named or classified
A
- disease they are associated with
- cytopathology they cause
- site of isolation
- places or oeople that discovered them
- biochemical features
13
Q
What is viral classification based upon
A
Size and shape Enveloped or naked (lipid envelop or no?) Nucleic acid composition Genome organization antigenic differences
14
Q
Describe a virus structure
A
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)
15
Q
What are the three different shapes of a virus
A
Icosehedral (cubic) MOST COMMON
Helical (influenza virus)
Complex (pox viruses);dont fit a shape
16
Q
Describe the envelope
A
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
17
Q
Describe non enveloped viruses
A
Stable in environment
Resist desiccation, heat, detergents and acids
Transmitted easily on hands and fomites
18
Q
Describe enveloped viruses
A
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
19
Q
How are RNA viruses organized
A
Single strand -> positive polarity or negative polarity (MRNA or not)
RNA double stranded -> one piece or segmented
20
Q
Describe how DNA Virusss are organized
A
Single stranded or double stranded
21
Q
How are viral infections acquired
A
Direct personal contact Airborne Parenteral (exposure to blood) Fomites Vectors Vertical transmission (mum to fetus) Enteral (food/waterborne)
22
Q
What is the virus life cycle
A
Attachment Penetration Uncoating (expose genome) Transcription (dna/rna) Translation Genome replication Assembly of genome and protein material Release and infect other cells
23
Q
Describe DNA/RNA Transcription
A
Dna can be directly transcribed
Negative RNA has to get MRNA to transcribe
Positive RNA directly transcribes
24
Q
Describe stages of virus-host interaction
A
Entry to host Primary replication Spread Cell and tissue trophism Secondary replication Cell injury or persistance Host immune response (depending on virus)
25
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
26
How are viral infections disgnosed
Clinical features typical of infection
Laboratory diagnosis: serology or amplification, detection of antigens, antibody testing,virus isolation
27
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
```
28
What are some diseases caused by prions
Creutzfeldt-jacobs disease
Kuru
Animal forms: bovine spongiform encephalopathy (mad cow)
29
What herpesviridae affect humans?
```
Herpes simplex 1 & 2
Epstein barr
Varicella zoster
Cytomegalovirus
Human herpes virus 6,7,8
```
30
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
31
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
32
Describe herpes simplex virus
ENVELOPED ds-DNA virus with ICOSAHEDRAL CAPSID
| Genital herpes = most common STI world wide
33
Describe clinical manifestations of primary infrction HSV type 1
```
Primary:
Oral
Genital
Ocular
Peripheral (dentists used to get this)
```
34
Describe clinical manifestations of HSV1 recurrent infection
Oral,ocular,genital,peripheral
Symptomatic
Asymptomatic shedding
35
What is herpes labialis
Cold sore
May be single or clustered
10-14 days
Treatment only necessary in immunocompromised patients
36
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
37
Describe genital herpes
Many recognized first outbreaks may actually be reoccurrence
| First episode tends to be more severe and longer in duration
38
Describe symptoms associated with genital Herpes
Fever, malaise, inguinal lymph node inflammation, headache, sometimes neck stiffness
39
Describe shedding for HSV 2
Reoccurrence more common
Asymptomatic shedding 28% of days
Shedding not completely suppressed by acyclovir
40
Describe shedding in HSV 1
Reoccurrence less frequent
| Asymptomatic shedding does occur but less frequently
41
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
42
How is Herpes simplex diagnosed
Virus isolation
| **PCR (nucleic acid amplification) especially with CSF to check for meningoencephalitis
43
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
44
How is HSV 1 & 2 treated?
Acyclovir: primary infections, prophylactic and immune compromised
45
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
46
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
47
What are clinical manifestations of EBV
```
Infectious mononucleosis (MONO):
Illness lasts from 2-8 weeks, fatigue persists
```
48
What are symptoms of EBV
```
Tonsilar enlargement
Pharyngeal redness
Cervic adenopathy
Large liver/spleen
Skin rash esp. after ampicillin
```
49
How is EBV diagnosed
Serology more common
Lymphocytes & monocytes increased
Low neutrophils and low platelets
50
Treatment for EBV?
Usually supportive, treatment doesnt do much
51
Describe cytomegalovirus
Infections occur more in socioeconomic groups
2 peaks: childhood, adulthood
Most adults (70%) are sero positive
All infections resukt in viral latency
52
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
53
Describe CMV infection in older hosts
Asymptomatic
| Some get mononucleosis like syndrome but heterophile antibody does not develop
54
How is CMV diagnosed
Serology (determine immunity), molecular
Nucleicacid amplification
Virus isolation
Light microscopy
55
How is CMV treated/prevented
Usually no treatment required
| Antiviral (gancyclovir) and immunoglobulin therapy usually used to prevent and treat immune compromised host
56
What is HIV
Retrovirus
57
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
58
Describe discovery of retrovirus
HIV-1 discovered 1981 by Gallo and Montagnier
| HIV-2 discovered in west Africa
59
How is HIV transmitted
Blood
Semen
Vaginal fluids
60
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
61
Describe HIV transmission in mother to baby
Mother to baby 1 in 4 (25%) without treatment
| 4% with treatment
62
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
63
How does HIV enter the cell?
HIV enters the cell by fusion eith the cellular envelope
64
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
65
What are CD4 cells
One of the most important immune cells
| Primarily responsible for cell mediated immunity
66
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
```
67
What are aids defining illnesses
Toxoplasmosis
Cryptococcal menigitis
Pneumocystis pneumonia
68
How is HIV diagnosed
Serology (antibodies): WESTERN BLOT
Immunologic studies (CD4 counts)
Viral load testing (pcr quantification of RNA in plasma)
Resistance genotyping
69
Describe western blot
Serology test method
| Less sensitive more specific
70
What is the biorad geenius
Differentiates HIV1 and HIV2
Rapid (~30min)
Removes subjectivity
71
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)
72
How is HIV Treated
Anti retrovirals: nucleoside analogs, protease inhibitors fusion inhibitors integrase inhibitors
Usually Highly Active Anti Retroviral Therapy (HAART)
73
What is HAART
Highly Active Anti Retroviral Therapy
74
Describe risk of needle stick exposjre
Risk depends on stage of patients disease and how much blood
75
Describe post HIV needle stick steps
Confirm patient status
Document your status
Begin prophylaxis WITHIN 72 hours!!
76
What is chance of getting HIV from needle stick
0.3%
77
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
```
78
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
79
What is the incubation period for Hep A
2-4 weeks
| Most cases are asymptomatic therefore jaundice only tip of the iceberg
80
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
81
How is Hep A diagnosed?
Disgnosis is confirmed by demonstrating the appearance of IgM antibodies
82
How is Hep A prevented?
Vaccine!! (Twinrex):
Usually for travellers, healthcare workers etc
Immune globulin (antibodies) post infection when you need instant protection
83
Describe Hep B
A double stranded HEPADNAVIRUS (DNA virus)
| Patients who become carriers may develop a chronic active or persistent hepatitis
84
How is Hep B transmitted?
Parenteral, perinatal and sexually
85
How infectious is Hep B?
Very infectious!! 30% of needle stick injuries resukt in infection for non immune individuals
86
What is the rule of three?
Needle stick risk:
0.3% HIV
3% for Hep C
30% for Hep B
87
How common is Hep B
Very common
350,000,000 carriers
1,000,000,000 have serologic evidence of past infection
88
Describe Carrier state for Hep B
Chances of developing carrier state depend on age of infection
Younger (neonates) most likely to have carrier state
89
What are some consequences of chronic Hep C
No/minimal liver injury
Chronic active Hepatitis
Cirrhosis of liver (failure/hypertension)
Hepato-cellular carcinoma
90
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
91
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
92
Describe Acute Hep B
Surface antigen increases and decreases as virus does
| Develop antibody to surface antigen & anti HB
93
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
94
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
95
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
96
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
97
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
98
What does it mean if a patient has less than 10 (>10) International units (IU)
They failed their vaccine series
99
What does it mean if a patient is HBsAg positive
They have a chronic hep B infection
100
Describe Hep C
Small ENVELOPED RNA virus (flavivirus)
| Usually parentally transmitted although less infectious than HBV (3% needle prick)
101
How does Hep C usually occur?
Usually occurs as sporadic disease in IV drug users, not as common sexual transmission, 75% mild or asymptomatic
102
What js the incubation period for Hep C
2-20 weeks
103
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
104
How is Hep C diagnosed
Typically qualitative pcr (detects viral RNA)
| recombinant immunoblot assay (RIBA)
105
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
106
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
107
Describe Hep E
A calivivirus (rna)
Almost never seen in north america
Fecal oral spread, lack of chronicity, low fatality rate
108
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
109
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
110
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
111
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
112
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
113
Why do we use typing systems
To determine if a series of isolates obtained from epidemiologic cluster are clonally related
114
What are benefits of typing systems
```
Ease of interpretation
Reproducibility
Ease of performance
Discriminatory power
Cost
```
115
What are phenotypic systems
Bio typing
Antibiograms
Phage typing
Serotyping
Based on presence of metabolic or biologic activities
116
What are genotypic typing systems
Molecular
Substantial generic diversitt within microbial species
Evoluntionary divergence
117
What is pulsed field gel electrophoresis
System of electrical currents
118
What is whole genome sequencjng
Most pulsed field moved to whole genome sequencing
