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13 Cards in this Set

  • Front
  • Back
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)
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
Advantages of Molecular methods:
- 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
Disadvantages of Molecular methods:
- 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)
Molecular Techniques common in clinical use =
PCR (polymerase chain reaction)
TMA (transcription mediated amplification)
LCR (ligase chain reaction)
bDNA (branched chain DNA amplification)
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)
Agents detected by molecular techniques

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)
Polyoma viruses (JC and BK)
Agents detected by molecular techniques

Neisseria gonorrhea
Chlamydia trachomatis
Group A and B strep
Staph Aureus
Mycobacterium tuberculosis
Mycoplasma pneumoniae
Lyme Spirochetes (Borelia burgdorferi)
Agents detected by molecular techniques


Histoplasma capsulatum
Coccidioides immitis

Toxoplasma gondii
Leishmania donovani

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
Identification of microorganisms


hybridization in solution for identification of isolated colonies -> PCR and sequencing

case of endocarditis - ID unknown organism
Strain comparison for epidemiology:


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
Where can you look at gene sequences?
NCBI (national center for biotechnology information) website, do a BlastN search