EPID 525 Lecture 3
Introduction to Molecular Methods
in Clinical Microbiology
Specimen processing
Target detection
Post-amplification detection
“Real-time” detection
Quality assurance
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
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
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
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
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
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
Automation
Specimen processing
MagNA Pure (Roche)
TECAN (Roche)
BioRobot (Qiagen)
NucliSens (BioMerieux)
TECAN
MagNA Pure
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.
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
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
Digene Hybrid Capture
Gen-Probe Culture ID
http://www.vysis.com/Hybridization_12956.asp
Target detection (cont.)
Target amplification
- Polymerase chain reaction (PCR) of DNA targets
- Primer-initiated; requires thermostable polymerase
Denaturation of ds DNA
Annealing of primers
Extension of primers
94°C
50°C or higher
72°C
~1 min. each, repeat for 25 to 40 cycles
http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961
http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961
Target detection (cont.)
Target amplification
- Reverse transcriptase (RT)-PCR of RNA targets
Conversion of RNA to cDNA using RT
Primer-initiated; 42°C, 1 hr.
Random hexamers
oligodT
PCR primers
cDNA is used as template for PCR
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
Target detection (cont.)
Signal amplification
- Branched DNA (bDNA) assay (Bayer)
Target probes
Label
probes
Preamplifiers
Amplifiers
Substrate
O
O OCH
3
-2
OPO3
HIV-1
MICROWELL
Add lysis
buffer to
disrupt virus
and release
RNA
Hybridize target
probes, preamplifiers
and amplifiers to
microwell and HIV-1
RNA
MICROWELL
Hybridize label probes
to amplifiers, add
dioxetane substrate,
and measure
chemiluminescence
Post-amplification detection
Gel electrophoresis
Colorimetric microtiter plate
Chemiluminescence
Real-time fluorescence detection
http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961
Post-amplification detection (cont.)
Gel electrophoresis
Agarose or polyacrylamide gel with EtBr
Post-amplification detection (cont.)
Colorimetric microtiter plate
- product captured by probe attached to plate
- enzyme-conjugated probes catalyze
colorimetric reaction
- read on a spectrophotometer
Chemiluminescence
- conjugated capture probes catalyze
chemiluminescent reaction
- read on a luminometer
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
SYBR green
FRET hybridization
TaqMan hydrolysis
Molecular beacons
Sunrise
Scorpion
ChemBioChem 2003, 4, 1120-1128
Melting curve analysis
Real-time quantitation
Automation
Amplification and detection
COBAS (Roche)
CT/NG, HCV, HBV, CMV
COBAS TaqMan 48
LightCycler
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
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
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
Applications of Molecular Methods
in Clinical Microbiology
Laboratory detection of microbial pathogens
Culture (“biological amplification”)
Growth
Analysis of macromolecular composition
Analysis of metabolic by-products
Detection of:
Organism’s protein components (antigens) using
antibodies
Patient’s immune response (serology)
Specific, characteristic nucleic acid sequences
(“enzymatic amplification”)
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)
The Promises of NAATs
Higher sensitivity and specificity
Shorter turn-around-time
Overall reduction in patient care costs
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….
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
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
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
What is going on?
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)
CD4:CD8 0.3 (LLN 0.8)
Patient HIV+ by serology and qual. PCR
Mother HIV+ (previously unknown)
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 <200/µl
- Pediatrics: CD4+ normal - low
TMP-SMX prophylaxis reducing incidence
GMS
 DFA
 Calcofluor
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
Diluted patient specimens
Molecular size markers 
600 bp 
346 bp 
Controls
- +
Spiked
Unspiked
600 bp 
346 bp 
Plan to convert to real-time platform
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 39.2°C and respiratory symptoms
PCP  right otitis media, Rx antibiotics
Fevers continued
Irritability, vomiting, nuchal rigidity for 2 days
Clinical diagnosis?
Meningitis
Bacterial
vs.
Viral
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
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 +
Typical CSF findings in CNS infection
Menigitis
Cells/l
Bacterial
Viral
Encephalitis
500-10,000
10-500
0-1000
Neutrophils
>90%
Early >50%
Late <20%
<50%
Glucose (mg/dl)
<40
45-85
45-85
CSF/Serum glucose
<0.6
>0.6
>0.6
Protein (mg/dl)
>150
<100
<100
“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 (postsurgery, drugs, lupus, cancer)
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
Enteroviruses (EVs)
Subgroup
Serotype
Poliovirus
Coxsackieviruses A
Coxsackieviruses B
Echovirus
Numbered EVs
1-3
1-22, 24
1-6
1-9, 11-27, 29-31
68-71
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.
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 < 24 hr:
Less antibiotic use
~$3000 less hospital charges
Critical factors:
Can specimen get to lab, be tested, and
result reported in < 48 hrs. (i.e. prior to
bacterial culture results)?