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13 Cards in this Set
- Front
- Back
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Diagnosis of Infectious Diseases
Traditional techniques 1) Culture 2) Staining 3) Serology |
1) takes days to weeks, relies on being able to grow and ID a pathogen, some agents can't be grown in the lab or can't be ID'd once they are growing
2) relies on reaction of dyes with various pathogen components, most stains require >100,000 organisms per ml of specimen to detect microscopically - techniques like immunofluorescence can detect less than this but still fairly insensitive 3) relies in an immune response from the individual for the pathogen, usually takes weeks, may not occur (e.g. immunosuppressed individual) |
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Molecular methods
requires what? What used to the used? What is used now? |
knowledge of specific DNA or RNA sequences and a way to ID the sequences
Used to use Hybridization techniques (but relatively insensitive) Amplification techniques: allow 1 million fold multiplication of target sequence or specific detector molecule |
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Advantages of Molecular methods:
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- can detect very small # organisms, esp when target sequence is present in many copies in one orgamism
- theoretically fast (minutes to hours) - Can detect organisms that can't be cultured |
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Disadvantages of Molecular methods:
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- must have specific nucleic acid sequence
- must be able to interpret meaning of a positive test - must be able to exclude false positive tests (most often from amplicon contamination of the environment of the lab or promiscuous target) - practical issues and current technology limit turnaround time, may not allow theoretical speed of test (tests must be batched, may take days) |
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Molecular Techniques common in clinical use =
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PCR (polymerase chain reaction)
TMA (transcription mediated amplification) LCR (ligase chain reaction) bDNA (branched chain DNA amplification) |
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Molecular detection methods
Formats in use (2): |
Qualitative: use amplification to detect the smallest possible number of target molecules *Most sensitive, but may only be useful when ANY detection of the organism correlates with disease
Quanititative: (viral load) Generates a graded signal that correlates with a target #, *useful following therapy (e.g. HIV viral load) and distinguishing endogenous carriage from disease state (e.g. CMV viral load in immunosuppressed patients) |
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Agents detected by molecular techniques
Viral: |
Herpes (HSV, CMV, VZW, EBV, HHV6)
Enteroviruses (polio, echo, coxsackie A and B) Respiratory viruses (influenza A, parainfluenza, RSV, Adenovirus, Human Metapneumovirus) Hepatitis Viruses (HBV, HCV) HIV Polyoma viruses (JC and BK) |
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Agents detected by molecular techniques
Bacteria: |
Pertussis
Neisseria gonorrhea Chlamydia trachomatis Group A and B strep Staph Aureus Mycobacterium tuberculosis Mycoplasma pneumoniae Lyme Spirochetes (Borelia burgdorferi) Other |
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Agents detected by molecular techniques
Fungi: Protozoa: |
Histoplasma capsulatum
Coccidioides immitis Toxoplasma gondii Leishmania donovani |
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Example:
How do you diagnose Chlamydia and gonorrhea? Used Clinically for: Specimen types: |
TMA (transcription mediated amplification)
diagnosis of symptomatic STD screening asymptomatic populations endocervical swab urethral swab Urine test |
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Identification of microorganisms
techniques example |
hybridization in solution for identification of isolated colonies -> PCR and sequencing
case of endocarditis - ID unknown organism |
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Strain comparison for epidemiology:
techniques example |
See if organisms occurring in multiple individuals are of the same strain
PFGE (Pulse field gel electrophoresis) - Use rare-cutting restriction enzyme to make large fragments of bacterial DNA - Separate fragments in gel by programmed switching of electrical field orientation - Create pattern of 10-20 bands - Compare patterns of organisms for relatedness RFLP (restriction fragment length polypeptide) and other fragment ID schemes example: mysterious bacteremia in dialysis patients |
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Where can you look at gene sequences?
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NCBI (national center for biotechnology information) website, do a BlastN search
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