EPID 525 Lecture 3

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EPID 525 Lecture 3

• Introduction to Molecular Methods
• in Clinical Microbiology

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Specimen processing Target detection Post-amplif
ication detection Real-time detection Quality
assurance
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Specimen processing

• efficient target recovery
• maintain integrity of nucleic acid target
• removal of amplification inhibitors
• elimination of components affecting substrates
• sterilization of potentially hazardous organisms

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Specimen processing (cont.)
Nucleic acid extraction (total) - proteinase K
digestion - phenol-chloroform extraction -
precipitation with salts and ethanol - wash
pellet with cold ethanol - resuspend in sterile
water
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Specimen processing (cont.)
Nucleic acid extraction (RNA only) - treat with
guanidinium ITC RNA complexes with
guanidinium DNA and protein are removed -
precipitation with salts and ethanol - wash
pellet with cold ethanol - resuspend in sterile
RNase-free water RNazol, Tel-Test
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Specimen processing (cont.)
Heat treatment of specimens - specimen is added
to water or buffer - heating or boiling releases
NA - aliquot used as template for assay
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Specimen processing (cont.)
Silica particle methods - take advantage of NA
binding properties of silica - add digested
specimen to column - wash with buffer - elute
with water - can specifically elute RNA by
adding a DNase step Qiagen spin columns
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Specimen processing (cont.)
Tissue prior homogenation required CSF clean
specimen Blood (whole or fractionated) DO NOT
use heparin tubes HIV plasma (RNA), CMV whole
blood (DNA) Urine clean specimen Sputum
prior digestion and decontamination req. Feces
dirty specimen with inhibitors
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Automation
Specimen processing MagNA Pure (Roche) TECAN
(Roche) BioRobot (Qiagen) NucliSens
(BioMerieux)
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TECAN
MagNA Pure
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Step A The sample material, e.g. cells (up to
5x106), blood (up to 1000µl), or 10 mg of
homogenized tissue is placed into the wells of
the sample cartridge. Step B/C The
binding/lysis buffer and proteinase K is added to
the sample. Step D Magnetic glass particles are
added to the lysed sample. The DNA binds to the
surface of the particles. Step E The magnetic
glass particles with the adsorbed DNA are
recovered from the wells of the processing
cartridge by applying a magnet from the outside
of the pipette tips. Step F-H The DNA is washed
and eluted from the glass particles whereas the
glass particles are retained in the reaction tip
and discarded.
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Target detection
Target-directed probes - Gen-Probe for culture
confirmation - Digene Hybrid Capture for
HPV Target amplification - PCR for DNA
targets - Reverse transcriptase (RT)-PCR for
RNA - NASBA, TMA Signal amplification - Bayer
Branched DNA (bDNA) assays
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Target detection (cont.)
Target-directed probes - Synthetic genomic
sequences specific for an organism of
interest - Hybridize with DNA or RNA targets in
specimen (direct specimen or culture
isolate) - Probes are labeled with enzymes,
antigenic, chemiluminescent moieties, or
radioisotopes
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Digene Hybrid Capture Gen-Probe Culture ID
http//www.vysis.com/Hybridization_12956.asp
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Target detection (cont.)
Target amplification - Polymerase chain reaction
(PCR) of DNA targets - Primer-initiated
requires thermostable polymerase Denaturation
of ds DNA 94C Annealing of primers 50C or
higher Extension of primers 72C 1 min. each,
repeat for 25 to 40 cycles
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http//bric.postech.ac.kr/labinfo/n_protocol/servi
ce_view.php?nProtocolId961
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http//bric.postech.ac.kr/labinfo/n_protocol/servi
ce_view.php?nProtocolId961
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Target detection (cont.)
Target amplification - Reverse transcriptase
(RT)-PCR of RNA targets Conversion of RNA to
cDNA using RT Primer-initiated 42C, 1
hr. Random hexamers oligodT PCR
primers cDNA is used as template for PCR
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Target detection (cont.)
Target amplification - Nucleic acid
sequence-based amplification (NASBA) -
Transcription-mediated amplification
(TMA) Multiple RNA copies of targets
generated Use RT and/or T7 RNA
polymerase Detect product with labeled
probes Isothermal reaction
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Target detection (cont.)
Signal amplification - Branched DNA (bDNA) assay
(Bayer)
Target probes
Label probes
Preamplifiers
Substrate
Amplifiers
HIV-1
Hybridize target probes, preamplifiers and
amplifiers to microwell and HIV-1 RNA
Add lysis buffer to disrupt virus and release RNA
Hybridize label probes to amplifiers, add
dioxetane substrate, and measure chemiluminescence
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Post-amplification detection
Gel electrophoresis Colorimetric microtiter
plate Chemiluminescence Real-time fluorescence
detection
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http//bric.postech.ac.kr/labinfo/n_protocol/servi
ce_view.php?nProtocolId961
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Post-amplification detection (cont.)
Gel electrophoresis Agarose or polyacrylamide
gel with EtBr
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Post-amplification detection (cont.)
Colorimetric microtiter plate - product
captured by probe attached to plate -
enzyme-conjugated probes catalyze colorimetric
reaction - read on a spectrophotometer Chemilumi
nescence - conjugated capture probes catalyze
chemiluminescent reaction - read on a
luminometer
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Post-amplification detection (cont.)
Real-time fluorescence detection - dual-labeled
probe included in reaction reporter and
quencher - intact probe emits low
flourescence - Taq cleaves reporter from bound
probe - amount of reporter cleaved is
proportional to the amount of amplicon
produced Other detectors SYBR Green
dyes Molecular beacons
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Molecular beacons
SYBR green
FRET hybridization
Sunrise
TaqMan hydrolysis
Scorpion
ChemBioChem 2003, 4, 1120-1128
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Melting curve analysis
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Real-time quantitation
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Automation
Amplification and detection COBAS
(Roche) CT/NG, HCV, HBV, CMV COBAS TaqMan
48 LightCycler
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Quality assurance
RNase-free materials and environment - wear
gloves - DEPC-treated water Separation of work
areas and unidirectional flow - reagent prep -
specimen prep - amplification and detection
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Quality assurance (cont.)
Prevention of amplicon cross-contamination -
UNG - barrier tips, topical decontamination -
closed systems - blank wells or
tubes Processing controls - spiked
specimens Amplification controls - internal
controls - failure indicates inhibition
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Interpretation of results
Dead vs. live pathogens - molecular tests of
cure ex. persistent dead Chlamydia Presence of
nucleic acid vs. disease - results taken in
context of clinical presentation ex. HSV in
CSF Causal vs. casual - difference between
necessary and necessary and sufficient ex.
HPV and cervical cancer
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Applications of Molecular Methods in Clinical
Microbiology
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Laboratory detection of microbial pathogens
Culture (biological amplification) Growth Analys
is of macromolecular composition Analysis of
metabolic by-products Detection of Organisms
protein components (antigens) using
antibodies Patients immune response
(serology) Specific, characteristic nucleic acid
sequences (enzymatic amplification)
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Why Nucleic Acid Amplification Tests NAAT ?

• When conventional methods are
• 1. Too slow (e.g., mycobacteria, legionella)
• 2. Too insensitive (asymptomatic HIV, viral
  infection of central nervous system, etc.)
• 3. Too cumbersome (e.g., virus isolation)
• 4. Not available (unculturable agents HPV, HCV)

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The Promises of NAATs
Higher sensitivity and specificity Shorter
turn-around-time Overall reduction in patient
care costs
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Some organisms detected by PCR
M. tuberculosis Legionellae B. burgdorferii H.
influenzae B.pertussis N. meningitidis T.
pallidum H. pylori F. tularensis C. difficile E.
coli T. whipelii van, mec
HIV HTLV CMV HSV HHV VZV EBV Hepatides HPV Rubella
Influenza Rhino Env

• Adeno
• Rabies
• Parvo B19
• Arbo
• Yellow Fever
• Lassa
• JC/BK
• Candidae
• Cryptococcus
• Trypanosoma
• Toxoplasma
• Naegleria.

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Applications
Detection of uncultureables or slow
growers Organism ID via 16S rRNA
sequencing Prognostication by subtype
analysis Disease monitoring by
quantitation Genotypic approaches to resistance
testing Outbreak investigations
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Case 1 Patient S.M., 8 month old female Full
term delivery, normal weight gain 1 month PTA
(Dec, 02) Resp failure w/ presumed
pneumonia Sputum cx pos for H. influenzae and P.
aeruginosa Responded to medical and abtc
management in PICU Discharged home F/U at Pulm.
Clinic (Jan, 03) Noted resp. distress,
tachypnea, low oxygen sat CXR changes consistent
with viral pneumonia
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Current admission Labs WBC 6.4 K/µl (low
normal) ALC 1 K/µl (low) HCT 30.6 (low) Basic
metabolic panel WNL Cultures Respiratory P.
aeruginosa neg for fungi, AFB, viruses neg
for Chlamydia, B. pertussis Stool neg for
viruses Serum neg for Mycoplasma
BAL PCR for Pneumocystis
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What is going on?
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Immunodeficiency workup Sweat chloride
testing CF gene testing CH-50 analysis Ig
testing Oxidative burst testing T cell analysis
by flow
Normal
Negative
Normal
Slight dec. IgG IgM
Normal
CD4 117 (LLN 967) CD4CD8 0.3 (LLN 0.8)
Patient HIV by serology and qual. PCR Mother
HIV (previously unknown)
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Pneumocystis jiroveci (carinii) Previously
classified as a protozoa Currently classified as
a fungus - based on nucleic acid and biochemical
analysis Most healthy children exposed by age
4 Reactivation pneumonia in immunosuppressed -
Adults CD4 T cells lt200/µl - Pediatrics CD4
normal - low TMP-SMX prophylaxis reducing
incidence
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?GMS
? DFA
? Calcofluor
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PCR at UM Home-brew assay targeting gene
encoding Pneumocystis large subunit mitochondrial
rRNA Respiratory specimen digested and
concentrated Nucleic acid extracted using MagNA
Pure Amplify spiked specimen in
parallel Specimen tested at multiple dilutions
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Diluted patient specimens
Molecular size markers ?
Controls -
600 bp ?
Spiked
346 bp ?
Unspiked
600 bp ?
346 bp ?
Plan to convert to real-time platform
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Case 2 Patient P.N., 4 year old male no
significant past medical history Presents to ED
(Aug, 03) 2 week hx loose stools/diarrhea 1
week later, fever to 39.2C and respiratory
symptoms PCP ? right otitis media, Rx
antibiotics Fevers continued Irritability,
vomiting, nuchal rigidity for 2 days
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Clinical diagnosis?
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Meningitis Bacterial vs. Viral
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Lab values WBC 9300/µl, 60 lymphs Lumbar
puncture WBC 75/µl 72 neutrophils 8
lymphs 20 monos Protein 22 mg/dl
(normal) Glucose 60 mg/dl (normal)
Gram stain NOS, few PMNs
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Clinical course Blood, urine and CSF cultures
submitted Bacterial cultures no growth at 48
hrs. ? patient discharged, completed
10-day course of ceftriaxone 2 days post
discharge CSF viral culture
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Typical CSF findings in CNS infection
Menigitis Bacterial
Viral Encephalitis Cells/?l 500-10,000
10-500 0-1000 Neutrophils gt90
Early gt50 lt50 Late lt20 Glucose
(mg/dl) lt40 45-85
45-85 CSF/Serum glucose lt0.6 gt0.6
gt0.6 Protein (mg/dl) gt150
lt100 lt100
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Aseptic Meningitis
Aseptic no bacteria isolated from CSF Most
common cause of meningitis most often
viral Symptoms often indistinguishable from
bacterial meningitis Lymphocytic pleocytosis in
CSF Other causes bacteria, noninfectious
(post-surgery, drugs, lupus, cancer)
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Enteroviral meningitis
EVs cause 75-90 of diagnosed aseptic
cases Seasonality Peak during summer fall
Primarily affects children, young
adults Fecal-oral route of transmission Self
limited benign clinical course No treatment
available
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Enteroviruses (EVs)
Subgroup Serotype Poliovirus 1-3 Coxsackievir
uses A 1-22, 24 Coxsackieviruses
B 1-6 Echovirus 1-9, 11-27, 29-31 Numbered
EVs 68-71
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Enteroviral Meningitis Diagnosis
Culture 70 90 sensitive Variety of susceptible
cells TAT 5 - 10 days Serology Generally not
useful PCR Home brew assays on a variety of
platforms Some commercial detection kits
available TAT as low as 4 hrs.
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Utility of PCR in management of EV meningitis
Hamilton MS, et al Pediatr Infect Dis J
(1999) Reduces hospitalization time Reduces cost
of patient stay Robinson C, et al Pediatr
Infect Dis J (2002) When results available lt 24
hr Less antibiotic use 3000 less hospital
charges
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Critical factors Can specimen get to lab, be
tested, and result reported in lt 48 hrs. (i.e.
prior to bacterial culture results)?