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SOMC Overview and Future Directions
SOMC Grand Rounds
January 8, 2016
Timothy R. Cassity, Ph. D.
Microbiologist

The opinions expressed in this presentation
are those of the presenter. The material
presented here does not necessarily reflect
the views of Southern Ohio Medical Center.

The presenter has no financial interest in (i.e.
is not being paid by) any diagnostic testing
company or pharmaceutical company or
product that may be mentioned in this
presentation.

Due to the nature of the topic, some slides may
seem like an “infomercial.” I apologize in
advance!

Understand the advantages of molecular
methods over traditional diagnostic
microbiology tests.

Identify situations where
molecular methods are
not beneficial.

Understand problems
and limitations that
can occur with
molecular methods of
detecting etiologic
agents.
The level of molecular testing
offered at SOMC would not be
possible without the support of
Bridget Scott (Lab Administrative
Direct) and Dr. Vincent Randaisi
(Lab Medical Director) and the
SOMC Administration!
I would also like to express my
appreciation to the physicians
who have supported and
championed molecular
technology, and Dr. David Byers
and Dr. Elie Saab in particular.
I would like to acknowledge the
invaluable guidance of Dr. Darren
Adams in the design and
implementation of the targets
used by our OB/GYN providers.

What is “molecular diagnostic testing” in
infectious diseases?
 Detection of etiologic agents of infectious disease
by detecting a unique nucleic acid base sequence
for specific organisms.
 The “goal” of molecular testing is no different
from classical methods.

What is “molecular diagnostic testing” in
infectious diseases?
 Every living organism (and some that aren’t living)
are defined by the nucleotide base sequences on
their DNA or RNA.
 For our purposes, each unique DNA or RNA
sequence is loosely defined as a “target.”

What is PCR?
 Polymerase Chain Reaction
 It is the in vitro synthesis of strands of target DNA
base sequences directed by homologous primers.
 There are many other methods of “amplifying”
DNA, but I will use the term PCR to include all of
those.

What is PCR?
 There are many other methods of “amplifying”
DNA, but I will use the term PCR to include all of
those.
 At SOMC we also use:
▪
▪
▪
▪
▪
Strand Displacement Amplification
Loop-Mediated isothermal DNA amplification (LAMP)
Gold Nanoparticle-based Methods (Verigene)
RotorGene Q 5 channel in house developed PCR
Alere™ i isothermal nucleic acid amplification

What is DNA Amplification?
 It is the in vitro synthesis of strands of target DNA
base sequences directed by homologous primers
producing amplicons.

What is DNA Amplification?
 We do the same thing with a culture, but with a
culture we “amplify” by growing individual
bacterial cells to a colony that we can easily
detect visually.
 DNA amplification is a lot faster than growth!
Note: Product of an artistic tech
with too much time on her hands

What is multiplexing?
 Multiplexing is the detection of multiple gene
targets at the same time or on the same run.
▪ Respiratory Virus detects 17 targets simultaneously
▪ Blood culture detected 12-15 targets simultaneously
▪ HSV 1 & 2, Trichomonas vaginalis, Atopobium vaginae,
Gardnerella vaginalis, Candida albicans and Candida
glabrata are multiplexed.

What is RT-PCR?
 RT-PCR stands for “Reverse Transcriptase
Polymerase Chain Reaction
 It is used with RNA, such as with RNA-containing
viruses, like influenza, RSV, HCV
 Reverse transcriptase is first used to convert RNA
to DNA, then the DNA is amplified via
conventional PCR

What is “Real Time” PCR or molecular
detection?
 In “classical” PCR, amplification took place in a
thermocycler and detection in a separate
instrument.
 Real time combines amplification and detection
into one process.
 This is faster and less hands on than classical PCR

What is qPCR or qRT-PCR?
 The “q” refers to “quantitative”
 When we run a real time PCR, we generate a
cycling time. i.e. the number of cycles of
amplification it takes before the target is
detected.
 The number of cycles it takes to detect a target is
dependent on the number of targets present in
the original sample.

What is qPCR or qRT-PCR?
 By measuring the number of cycles on samples of
known concentration, a standard curve can be set
up.
 To determine the quantity of targets in the initial
sample, the number of cycles is compared to the
standard curve.
Magic?
Nay, Nay!

Steps to conventional real-time PCR
 Extraction
 Amplification and Detection
▪ Confirmation with DNA melt curve
 Data analysis
▪ Would not be possible without a computer. One of the
instruments we use takes 4,320 data readings a minute –
approximately 800,000 data points a run

Nucleic Acid Extraction
 Lyses cells and releases nucleic acids
 Proteases are incorporated to inactivate
nucleases and other enzymes
 Result is purified RNA and DNA

Amplification and Detection
 Done via thermocycling

To confirm ID (i.e. increase specificity) a DNA
melt temp curve is run

The Perfect Lab test would have 100%
sensitivity (No false negatives) and 100%
specificity (No false Positives)

Much more sensitive (i.e. few false negative
tests) than most conventional diagnostic
methods used with infectious agents.
 Example – only method acceptable for the
detection of HSV in CSF
 Analogy – if PCR could be used to detect sugar,
you could drop 2 sugar cubes into Lake Michigan
and detect it in Lake Michigan water.
Target
Organism
Molecular
Method
Conventional
Method
LoD
Molecular
LoD
Conventional
Influenza A & B
PCR
Viral Culture
10-50 PFU/ml
1,000 PFU/ml
Influenza A & B
Alere-i
Viral Culture
1,000 PFU/ml
1,000 PFU/ml
HSV-1 or 2
PCR
Viral Culture
5 - 15 PFU/ml
500-1,000 PFU/ml
4 cells/ml
1,000 cells/ml
Trichomonas
PCR
Wet Prep
CMV
PCR
Viral culture
100 PFU/ml
1,000 PFU/ml
N. gonorrhoeae Strand Disp
Culture
50 CFU/ml
100 CFU/ml
Cl. difficile
LAMP
Toxin Detect
30% more sens than toxin testing
B. pertussis
Nanopart
Culture
>70% more sens than culture
40% more sens than wet prep

If performed properly, specificity is also
excellent!
 Few false positive results, but some are still
possible
Target
Organism
Molecular
Method
Conventional
Method
% Specificity
Molecular
% Specificity
Conventional
Influenza A & B
PCR
Viral Culture
100%
100%
Influenza A & B
Alere-i
Viral Culture
97%
100%
HSV-1 or 2
PCR
Viral Culture
99%
100%
Trichomonas
PCR
Wet Prep
100%
90%
CMV
PCR
Viral culture
100%
100%
N. gonorrhoeae Strand Disp
Culture
98%
100%
Cl. difficile
LAMP
Toxin Detect
B. pertussis
Nanopart
Culture
95% (symptom)
95%
95%
100%

Specimens
 Except for GBS, which has an enrichment step,
molecular testing does not require viable
organisms.
 Can use viral swabs, ThinPrep, some dry swabs,
refrigerated CSF and sterile fluids
 Can be used in patients that have antibiotic
therapy before clinical sample collection.
 Specimen transport is not as important as with
conventional culture


Molecular methods are particularly well
adapted for diagnosis of infections caused by
fastidious and uncultivable bacterial species.
Beneficial for slow-growing organisms
Target
Organism
Molecular
Method
Conventional
Method
Min TAT
Molecular
Min TAT Conventional
Influenza A & B
PCR
Viral Culture
4 hours
1-2 days
Influenza A & B
Alere-i
Viral Culture
20 min
1-2 days
HSV-1 or 2
PCR
Viral Culture
4 hours
1 – 2 days
Trichomonas
PCR
Wet Prep
4 hours
20 min
CMV
PCR
Viral culture
4 hours
1 – 4 days
N. gonorrhoeae Strand Disp
Culture
3 hours
3 days
Cl. difficile
LAMP
Toxin Detect
1 hour
1 hour
B. pertussis
Nanopart
Culture
2 hours
5 days
RSV
Nanopart
Virus Culture
2 hours
2-4 days

In many cases, with blood cultures, rapid
detection of resistance genes influence initial
treatment.
 Example: E. coli with ESBL or CRE


Rapid results may permit discontinuance or
de-escalation of antibiotics earlier than
conventional culture.
Can be an important part of antibiotic
stewardship

Can decrease length of stay
 Several studies have shown positive blood
cultures identified by molecular detection average
a 1.2 day shorter length of stay

Too sensitive in some cases – can yield falsepositive results
 Example: C. difficile molecular detection
 Tests should be performed only on symptomatic
patients
 PCR results must be correlated with clinical
presentation and patient history

The biggest potential problem with PCRbased tests is amplicon contamination.
 Samples cannot be handled in the same area they
are amplified
 Instruments and the environment must be
“disinfected” after every use!


PCR tests cannot accurately differentiate
infection from commensalism or chronic
carriage
Not good for opportunistic pathogens and for
clinical specimens collected at nonsterile
sites.

Genetic Drift
 Genomes are not necessarily static
 If your target sequence changes in an organism
you can no longer detect it!
▪ Example – Staphylococcus aureus and mecA gene
cassette
▪ Some organisms such as Streptococcus sp. do not show
much genetic change. Others, like influenza viruses,
show genetic change more often


There are methods to detect some genes
responsible for resistance, such as mecA,
some ESBLs, and some CREs.
However, there are many more that we
cannot test, or don’t even know about.
 Therefore, for organisms that can have variable
susceptibility to antibiotics, culture and
conventional susceptibility testing is still
necessary.

Molecular-based diagnostics are much more
expensive than convention culture or antigen
testing!
 But for now, reimbursement is very good!
Laboratory Cost
Medicare
Reimbursement
Influenza A & B – Alere-i
$42
$96
HSV 1 & 2
$26
$96
Chlamydia/GC
$20
$90
Respiratory panel – complete panel
$110
$376
C. difficile
$29
$48
GBS
$33
$59
Atopobium/Gardnerella
$46
$96
Candida albicans/C. glabrata
$46
$96
Bordetella pertussis group
$42
$141
Test

Currently we have molecular tests for 58
infectious disease targets!
 15 Gram positive blood culture targets
 14 Gram negative blood culture targets
 18 Respiratory virus and pertussis targets
 10 STD and OB/GYN targets
 1 GI target (C. difficile), with 10 more under
development
Positive Blood Culture on the BacT/Alert
Subculture to plates and incubate
Prepare and stain Gram stain
Read and report Gram stain
Call provider & clinical
Pharmacist per protocol
Has patient been admitted to SOMC – not expired?
Yes
No
No
Do not perform molecular ID
Workup and Report from plates
Call provider & clinical Pharmacist per
protocol with Result
First POS Blood culture?
Yes
Perform molecular ID &
Workup and Report from plates
Organism
Gene Target
Staphylococcus aureus
S. aureus gyrB gene
Staphylococcus epidermidis
Staph epi hsp60 gene
Staphylococcus lugdunensis
Staph lugdunensis sodA gene
Streptococcus agalactiae
Strep agalactiae hsp60 gene
Streptococcus anginosus group
Strep anginosus gyrB gene
Streptococcus pneumoniae
Strep pneumoniae gyrB gene
Streptococcus pyogenes
Strep pyogenes hsp60 gene
Enterococcus faecalis
Enterococcus faecalis hsp60 gene
Enterococcus faecium
Enterococcus faecium hsp60 gene
Staphylococcus sp.
Staphylococcus sp tuf gene
Streptococcus spp.
Streptococcus tuf gene
Listeria sp.
Listeria sp. tuf gene
mecA
mecA gene
vanA
vanA gene
vanB
vanB gene
Bacterial Genera and Species
Acinetobacter spp.
Citrobacter spp.
Enterobacter spp.
Proteus spp.
Escherichia coli1
Klebsiella pneumoniae
Klebsiella oxytoca
Pseudomonas aeruginosa
Resistance Markers
CTX-M (blaCTX-M)
KPC (blaKPC)
NDM (blaNDM)
VIM (blaVIM)
IMP (blaIMP)
OXA (blaOXA)
Organism/Target
Method
Viable
Specimen
Needed?
Chlamydia trachomatis
Strand Displacement Amplification
No
Neisseria gonorrhoeae
Strand Displacement Amplification
No
Group B Streptococcus
Loop-Mediated Amplification
following Enrichment in LIM broth
YES
Herpes Simplex 1 & 2
Conventional real-time PCR
No
Trichomonas vaginalis
Conventional real-time PCR
No
Atopobium vaginae
Conventional real-time PCR
No
Candida albicans
Conventional real-time PCR
No
Gardnerella vaginalis
Conventional real-time PCR
No
Candida glabrata
Conventional real-time PCR
No


Typically Ordered by Groups, but can be
ordered individually if desired.
Groups include:
 Complete panel (12 targets)
 Influenza A & B (2 to 4 targets)
 RSV only (2 targets)
 Bordetella pertussis (3 targets)
 Can be customized per provider request

Influenza A & B – Rapid Method – Alere-i
 Two targets – influenza A & influenza B
 Done on a dry swab or viral swab in UTM
 Requires approximately 20 min to perform
 Unlike rapid antigen tests, it does not require PCR
or culture backup
 CLIA-waived, suitable for point of care

Influenza A & B – Verigene
 Done on a variety of specimens – swab in UTM or




other transport medium, BAL, Bronch Wash
Four targets
Gives H-type ID (ex. Influenza A H1)
Can be done in about 2.5 hours
>10X more sensitive than the Alere-i

Influenza A & B – Conventional RT-PCR
 Done on a variety of specimens – swab in UTM or
other transport medium, BAL, Bronch Wash
 Two targets – influenza A & influenza B
 Most sensitive method – LoD < 50 PFU
 Can be done in about 4.5 hours
 More labor-intensive – we do not do this much
anymore. Used only as backup or to resolve
questionable results.

RSV (only) – Done on the Verigene
 Done on a variety of specimens – swab in UTM or




other transport medium, BAL, Bronch Wash
Two targets (RSV A & RSV B)
Can be done in about 2.5 hours
More sensitive than rapid antigen tests
Can be done on adult patients as well as children

Common Respiratory Viruses
 Comprehensive Panel that includes:
▪ Influenza A & B, with influenza A H subtypes
▪ RSV A & RSV B
▪ Parainfluenza 1, 2, 3 & 4
▪ Human metapneumovirus
▪ Adenovirus
▪ Rhinovirus (it’s free so we throw it in for good measure)

Bordetella pertussis panel
 Include B. pertussis, B. holmesii, and B.
parapertussis/B. bronchiseptica
 Should be used only with symptomatic patients
 Used to diagnose whooping cough type illnesses

HSV and CMV
 These can also be ordered on respiratory tract
specimens.
 More relevant in immunosuppressed patients
than immunocompetent ones
 HSV-1 found in around 30-40% of specimens, but
most are not clinically significant
 These were routinely done with viral cultures

Currently, only C. difficile is offered
 Very sensitive - can detect asymptomatic carriage
 – must be performed only on symptomatic
patients
 30% more sensitive than toxin testing – only 1
specimen needed to rule out C. difficile

Molecular replacement for stool culture
under development – Spring 2016
 Stool culture TAT – 48 – 72 hours
 Molecular enteric pathogen TAT – 3 hours
 Expanded spectrum of etiologic agents

Disadvantages
 Stool culture for susceptibility testing will be
needed if Shigella sp. (and some Salmonella sp.)
are found.

Targets will include:
Bacteria
Toxins
Viruses
Campylobacter Group
Shiga Toxin 1 (stx1)
Norovirus
Salmonella spp.
Shiga Toxin 2 (stx2)
Rotavirus
Shigella spp.
Vibrio Group
Yersinia enterocolitica

Infectious Agents
 GI pathogens – estimated Spring, 2016
 Ureaplasma urealyticum
 HPV
 Mycoplasma genitalium (maybe)

Non-infectious targets
 BRAF Mutation Analysis
 EGFR (Epidermal Growth Factor Receptor)
 JAK 2 (Janus Kinase 2)
 KRAS mutation (Kirsten rat sarcoma viral oncogene homolog)