Lecture 8: Molecular Applications to Infectious Diseases Flashcards
Applications of Molecular
Analyses to Infectious Diseases…
- Qualitative detection
- Quantitative detection
- Microbial “identity” testing
- Genotyping/drug resistance
What are the controls used for PCR?
- Positive control: positive template
- Negative control: negative template
- Amplification control: omnipresent template unrelated to target
- Reagent blank: no template present
Advantages of Molecular Detection of Resistance to
Antimicrobial Agents…
- Mutated genes are strong evidence of resistance
- Rapid detection without culturing
- Direct comparison of multiple isolates in epidemiological
investigations
A molecular marker for identification of bacterial species***
-this gene (1,500 bp) is large enough for informatics purposes
16S rRNA
presence in almost all bacteria, often existing as a multigene family, or operons;
16S rRNA
the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of _____________
time (evolution)
Overall, between 16S gene based and clinical identities, the study shows a
genus-level concordance rate of ___% and a species-level concordance rate of
____%.
96
87.5
The use of ______ gene sequencing in the clinical laboratory is becoming commonplace for identifying biochemically
unidentified bacteria or for providing reference identifications
for unusual strains
16S rRNA
What is pitfall with 16S rRNA gene sequencing?
- it has low phylogenetic power at the species level and poor discriminatory power for some genera
- For bacteria that are difficult to grow or identify
What is the “gold standard”
for proposed new species and for the definitive assignment of a strain with ambiguous properties to the correct taxonomic unit?
DNA-DNA hybridization
rapidly spreading outbreak of an infectious disease
Epidemic
a disease that sweeps across wide geographical areas
Pandemic
collection and analysis of environmental,
microbiological, and clinical data
Epidemiology
- An opportunistic, toxin-producing bacterial pathogen of the
gastrointestinal tract. - The most common cause of healthcare-associated diarrhea
Clostridum Difficile Infection
(CDI)
C difficile causes ___% to ___% of antibiotic-associated diarrhea and _____ % of pseudomembranous colitis cases.
25-30
> 95
Incidence has almost doubled since 1996, and the mortality rate is rising owing to emergence of a hypervirulent strain.
(027/NAP1/B1)
Clostridum Difficile Infection
Disease-causing C difficile strains produce 1 or both of 2 toxins, that are…
Toxin A is an enterotoxin and toxin B is a cytotoxin***
How do the C difficile toxins induce damage?
The toxins destroy cells and produce patches (colonized plaques) of inflammatory cells and decaying cellular debris inside the colon and
cause watery diarrhea
Ture or false:
other stains of C. diff produce neither toxin and are thought to colonize the colon without causing disease
true
What are the two different testing options for the detection of C difficile?
- An immunoassay to simultaneously assess for toxin and glutamate
dehydrogenase(GDH) as the presence of C. difficile antigen (sensitivity) - Nucleic acid amplification tests (NAAT) for C difficile toxin genes (test
code 16377).(Specificity)
Staphylococcal resistance to oxacillin/methicillin occurs when an isolate produces an altered penicillin-binding protein, PBP2a, which is encoded by the______ gene
mecA
-The variant penicillin-binding protein binds beta-lactams with lower avidity, results in resistance to this class of antimicrobial agents.
Strains that are oxacillin and methicillin resistant, historically termed methicillin-resistant S. aureus (MRSA), are resistant
to all __________.
ß-lactam agents
What is the antibiotic resistance gene?
mecA
What are tests for MRSA?
-Cefoxotin disk screen test
-Nucleic acid amplification tests, such as the polymerase chain
reaction (PCR (to detect mecA gene)
Cefoxotin disk screen test for MRSA:
- Latex agglutination test for _______
- or a plate containing 6 μg/ml of oxacillin in Mueller-Hinton agar supplemented with 4% NaCl as alternative methods of testing for MRSA.
PBP2a
_________ the most common gene that mediates oxacillin resistance in staphylococci
mecA
What is the downside of using PCR to detect MRSA?
mecA PCR tests will not detect novel resistance mechanisms such as mecC or
uncommon phenotypes such as borderline-resistant oxacillin resistance.
Aptima Combo 2 assay (Hologic) is used to detect what two diseases?
chlamydia trachomatis (CT) and neisseria gonorrhoeae (NG
What is the most sensitive nucleic acid amplification test available to detect CT and NG?
CT/NG test from Aptima Hologic
By targeting rRNA, Aptima Combo 2 enhances the sensitivity of amplification tests and virtually eliminates false negatives by
removing _____________.
amplification inhibitors.
What are other Molecular Detection Techniques for
Neisseria Gonorrhoeae?
-NASBA
-SDA (strand displacement amplification)
-LCR (Ligase Chain Reaction)
-Hybrid Capture II
combination DNA probe test that uses signal amplification to detect both C trachomatis and N gonorrhoeae in a single specimen
Hybrid Capture II (Digene Corporation, USA) test
reagents for RNA extraction and amplification, and a ruthenium-labelled probe against 16S rRNA for
electrochemiluminescent detection of amplicons.
This system has been evaluated for the detection of 16S rRNA of N gonorrhoeae in genital tract specimens
NASBA
- Solution hybridization, antibody capture assay
- Chemiluminescence detection of hybrid (DNA/RNA)
molecules
-DNA is denatured
-Hybridized to RNA probe
-Captured by bound anti DNA/RNA antibodies
Hybrid Capture Assay
Hybrid Capture Assay
for Human Papillomavirus uses hybridized _____ probe with target ______.
RNA, DNA
- Target DNA combines with specific RNA probes creating RNA:DNA hybrids
What needs to be done for hybrid capture assays?
release nucleic acids
- Clinical specimens are combined with a base solution which disrupts the virus or bacteria and releases target DNA.
How are capture hybrids assay performed?
RNA:DNA hybrids are captured onto a
microtiter well coated with capture antibodies specific for RNA:DNA hybrids
- Captured RNA:DNA hybrids are detected with multiple antibodies conjugated to alkaline phosphatase
Class I viruses…
ds DNA (use host polymerase)
Class II viruses…
ssDNA (use host polymerase)
Class III viruses…
ds RNA (Replicase)
Class IV viruses…
ssRNA “+” strand = sense strand (COVID19)
Class V viruses…
ssRNA “-” strand = noncoding strand
Class VI viruses…
retroviruses
What are the steps of virus life cycle?
-attachment (absorption)
-penetration
-uncoating and targeting
-gene expression and genome replication
-virion assembly/maturation
-release of new infectious virus
Viral life cycle:
penetration is ________ mediated
-phagocytosis
-direct membrane fusion
receptor
Naked viral genomes are more ________.
infectious
What makes + sense viruses more infectious?
- (+) = sense strand, makes protein
- Viral RNA serve as mRNA
- Make own replicase (for RNA synthesis) after entrance to cell
- Use its own RT to make v-DNA and its own integrase to integrated into host genome
- Use host RNA pol to make transcripts (of host and viral RNA)
- Use host cell E and v-Env gene products to produce proteins for packaging new viral particles
Retroviruses
HIV Structural Genes:
protein components of nucleo-protein core
gag
HIV Structural Genes:
reverse transcriptase; integrase; protease
pol***
What is used for early detection and screening of HIV?
gag
What is used for initiation and monitoring of Anti-Viral Therapy (ART) for HIV?
- CD4 counts
- Viral Load
What antibody/antigen testing is done for HIV diagnosis?
- Enzyme Immunoassays (EIAs)
- Rapid tests
- Western blot (WB)
Once infected with HIV, the virus begins to attack and destroy the _____ cells
of the person’s immune system. HIV uses the machinery of the ____ cells to multiply (produce more HIV virus) and spread throughout the body.
CD4
Why are CD4 counts done with HIV?
provide important information as to how healthy you are and whether the virus has progressed in your body, is the ART regime effective and what is the possible prognosis etc.
A CD4 count ranges from _________ cells/mm3 in healthy adults/adolescents
500–1,600
A CD4 count of fewer than _____ cells/mm3 is one of the qualifications for a diagnosis of stage 3 infection (AIDS).
200
Anti-Retroviral-Theray (ART) is recommended for everyone with HIV, but the urgency to start ART is greater in people with…
Low or rapidly falling CD4 counts
Typically, your HIV care provider will check your CD4 count every __ to ___ months when you are starting ART. Once your CD4 level rebounds and your viral load is suppressed, your provider may check your CD4 count less frequently.
3, 6
Viral Load:
Quantitative molecular assay measures amount of HIV in ____ milliliter blood
one
-Note: only 2% virus are present in blood
Other organ/tissue/cell also have virus
What is viral load testing used for?
Predict disease progression
Assist with deciding when to initiate anti-retroviral therapy
Monitors response to anti-retroviral therapy
What are the drawbacks of viral load testing?
-Expensive
-Labor-intensive
-Special facilities
What are the techniques used to measure HIV viral load?
-PCR (polymerase chain reaction)
-new ultrasensitive PCR
-bDNA (branched-chain DNA)
-NASBA
What is the most common test used for HIV viral load measurement?
A new ultrasensitive PCR test can measure down to 20 copies of HIV RNA.
This is the most common test used
Techniques in HIV Viral Load
Measurement:
creates a light signal whose brightness depends on the amount of viral RNA present.
bDNA (branched-chain DNA)
Techniques in HIV Viral Load
Measurement:
amplifies the viral RNA
that makes proteins, making HIV viral load easier to measure.
NASBA (nucleic acid sequence based amplification)
What is the most clinically used HIV viral load measurement technique used due to the simple requirements for instruments etc.?
NASBA (nucleic acid sequence based amplification)
Techniques in HIV Viral Load
Measurement:
What are the targets used?
gag 41, gag 24, polymerase, protease gene (in a conserved region)
Viral Load testing:
How is baseline determined?
Take 2 measurements 2-3 weeks apart to determine baseline.
Repeat every 3-6 months in conjunction with CD4 counts to monitor viral load ant T-cell count
Viral load testing:
Repeat ____ weeks after starting or changing antiretroviral therapy to
determine effect on viral load.
4-6
Viral load testing:
High: __________ to __________ copies (up to 1 million) per mL blood
100,000 to 500,000
Viral load testing:
- low: ________copies per mL of blood
- No detectable: _____ copies/ mL blood
20-500
<20
HIV Viral load testing:
> ______ copies/ml blood consider failure of anti-viral therapy
200***
PCR and qPCR Based Viral Testing can be used for…
- CMV
- HIV
- HIV Resistance
- HIV Genotyping
- HCV Viral Load
- HCV Genotyping
Second most common malignancy in women worldwide?
Cervical Cancer
-100% preventable by screening for pre-malignant disease
- Highly treatable if detected early
First solid, malignant tumor recognized as having a viral
origin
Cervical Cancer
What type of virus is Human papillomavirus (HPV)?
small, non-enveloped circular, double-
stranded DNA virus.
* viral genome is approximately 8-kb in length.
HPV:
The genome encodes for ___ early proteins responsible for virus replication and 2 late proteins, ____ and ___, which are the viral structural proteins
6
L1, L2
- Primary cause of cervical cancer.
- Over 70 site-specific types
HPV
___% of women greater than 35 years of age are persistent carrier of HPV
5-10
HPV E___ and E____ proteins inhibit p53 and pRB protein functions***
6, 7
- High clinical sensitivity for major carcinogenic HPV types
- High inter- and intra-test reproducibility
Digene’s Hybrid Capture® II
HPV DNA Test
Pros of Digene’s Hybrid Capture® II HPV DNA Test?
-simple and efficient procedure
-high inter- and intra- test reproducibility
* Objective, geographically standardized results
* Simple lab set-up and training
* Amenable to large testing volumes
Detection of HPV by hybrid capture
- RNA probe
- Denature ds viral DNA
- Capture RNA:DNA hybrids
onto a solid phase (in tube or
microplate format) - Detect amplified
chemiluminescent signal
HPV Viral Genotyping:
All viruses have demonstrated the potential to generate sequence _____________.
diversity
Types of HPV Sequence Diversity:
low risk types?
6, 11, 42, 43, 44
Types of HPV Sequence Diversity:
High risk types?
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
