Geraldine S. Hall, PhD., D(ABMM), F(AAM)
Forestville, New York
June 16, 2015
 Describe the impact of existing and novel methods
available for the rapid detection of bacteria.
 Provide possibilities for the integration of rapid
methods in the laboratory as opportunities to improve
antimicrobial use and clinical and economic outcomes
 Explore the impact of existing and novel methods
possible for the rapid determination of antibiotic
susceptibility
 Identification
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Manual Biochemical Testing
Continuous monitoring BC machines
Screening of Urine Specimens
Automated (semi-automated) identification systems
o
o
o
o
o
o
Vitek (bioMerieux, Inc)
MicroScan ( Beckman Coulter)
BD Phoenix (Becton Dickinson)
VersaTrek (Thermofisher Scientific)
Biolog (Hayward, CA)
Other
 Often requires at least 24 hrs before results are available

Manual methods for Susceptibility testing
 Disk Diffusion
 E-Test
 MIC Trays

Automated (semi-automated) Methods for Susceptibility Testing

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Vitek
MicroScan
BD Phoenix
VersaTrek
Other
Requires isolated colonies; usually performed after the identification
is completed; typically requires at least 48 hrs

Better outcomes for the Patient
•
•
•
Lowered Mortality Rates
Use of appropriate, targeted antibiotics earlier
Less chance for more infections
 MRSA
 C. difficile

Lowered Costs for the Patient
•
•
•
Less time in the hospital
Fewer additional tests
? Fewer antibiotics, therefore fewer adverse reactions as a possibility
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Reduced Length of Stay (LOS) in the hospital
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Less development of antibiotic resistance

Identification and Susceptibility Testing for:
• Blood Cultures
• CSF and other sterile sites
• Pneumonia
• Wounds
• Urine Cultures
• Sexually Transmitted Diseases
• Other
 Doern, GV et al. 1994. JCM 32: 1757-62
 Semi-automated ID/AST systems vs. overnight
o
ID in 9.6 hr vs. 19.6 hr
o
AST in 11.3 Hr vs. 25.9 hr
 LOS was the same, but mortality was lower with rapid results, 8.8 vs
15.3%
 Fewer lab studies ordered
 Less time for appropriate antibiotics
 Barenfanger, JE et al. 1999. JCM 34: 208-9
 Evening shift verified VITEK results for ID and AST
 LOS was 10% with rapid vs 12% with conv.
 Mortality was 7.9% with rapid vs 9.6% with conv.
 $4,927 per patient with rapid vs. $6,677 with conv.
 Estimated saving $4 million per year
 Beekman, SE et al. 2003. JCM 41: 3119-25.
 917 blood cultures; most were SA, E. coli and CNS
• Collection of blood culture to Gram stain was 27.6 hr
 Continuous monitoring systems
• Time to notification significantly improved
–LOS for patient with BSI
–LOS after diagnosis of BSI
 Barenfanger, J et al. 2008. Am J Clin Path 130: 870-6.
 Staining and reporting of positive Blood Cultures
• <1 hr TAT: reported in 0.1 hr; 10% mortality
• > 1 hr TAT: reported in 3.3 hr; 19% mortality
1. MALDI-TOF
2. Sequencing
3. Direct Specimen Testing
• Probes
• NAAT
4. Other
Detection of Resistance markers
 Verogene
 Cepheid Xpert
 MRSA using GenOhm
 Other
Some questions as to whether genotypic
(molecular) detection will = phenotypic results
1.
Direct testing from a
positive BC:
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FISH probes
MALDI-TOF
Rapid MRSA testing
Verigene
Film Arrays
2. Direct testing from whole
blood:
 Out of U.S. presently:
• Light Cycler Septic FAST (Roche;
broad range PCR; IVD)
– Guido M et al. 2012. J Prev
Med Hyg 53: 104-8.
• SestiTest (Germany; homebrew)
Leitner E and Kessler HH. 2015. Meth
Mol Biol . 1287: 129-38
– Research in US
• T2 Candida
Neyda ND et al. 2013; DMID 77:
324-6.

Multi-faceted approach to optimize the use of antibiotics
while minimizing the development of resistance and other
adverse effects
Stewardship = acceptance for responsibility
for long-term management of something of
enormous value.

Good stewardship practices (GSP) is an active and dynamic
process of continuous improvement on antibiotic use — an
ethic with many steps of different sizes by all involved in
antibiotic use.

Prior term used: prudent antibiotic use
 MRSA, VRE, K. pneumoniae (CRE), P. aeruginosa (FQ-R),
A. baumannii (Amp/sulbactam-R)
 Resistant organisms: 7 d increase in LOS; even after confounding
variables were removed = 1 d less
 Resistant organisms: $70,000 increased charges; even after confounding
variables removed = $8000.
 Death rate was higher for HAI caused by VRE or KP (CRE) as
compared to CA-acquired; pneumonia and UTI were also cause of
higher death rates
 CA-VRE resulted in $69,000 higher charges than VSE
Nerdell et al. 2002. CID 55: 807-15
 Healthcare Infection Control Practices Advisor
Committee, in partnership with US DHHS, listed
Antimicrobial Stewardship as one of top 5 messages
for healthcare workers in 2009.
 MRSA infections were listed as 1 of the 6 categories
of healthcare associated infections for its 5 year
“National Prevention Target.”
Cost of SA bacteremia: $20,000 - $70,000/episode

Antimicrobial use guidelines/categorization
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Voluntary restriction policies
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Formularies
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Stop orders (test utilization)
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Education
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Regulation of access to antibiotics
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Dealing with COI issues
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Implementation of good Infection Control Practices
Prescott JF et al, 2014; Vet Microbiol 171: 273-8.
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Infectious Disease clinicians
Clinical Microbiologists
ID Pharmacists
Infection Control Practitioners
Patients
Industrial colleagues
o
o
Medical Device/Instruments
Pharmaceutical Industry
 Communicate best methods for specimen collection
 Use methods that will provide quickest and most accurate results for:

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Gram Stain
ID
Susceptibility
Serology/antigen testing
Direct specimen testing
 Communicate results as quickly as they are completed:
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By phone
By email
Electronically on lab information systems
All of above
 Mean decrease in survival of 7.6% for each hour
after onset of hypertension until effective
antibiotics are on board
 Use of inappropriate antibiotics within first 6 hrs
after recognition of septic shock is associated
with a 5-fold increase in mortality rate
 SO ------ answer will be “broad spectrum
antibiotics” which will further increase antibiotic
resistance
Kothari A et al. 2014. CID 59:72-8.
2014 Review
Direct Testing
from BC Bottle
Mortality
Benefit
Change in
LOS
Cost saving per
patient ($)
AS
Intervention
FISH probes
ND
2 d less
4005
YES
FISH probes
ND
2.2 d more
ND
NO
FISH probes*
ND
ND
1729
YES
FISH probes
16.8% vs 7.9%
2 d less**
19,441**
NO
GeneXpert
MRSA/SA
18% vs 26% **
6.2d less
21,387
YES
MALDI-TOF
5.6% vs
10.7%**
1.8d less
19,547 **
YES
MALDI-TOF
12.7% vs
20.3%
2.8 d less **
ND
YES
Verigene #
NO
21.7d less
60,729
YES
* = Yeast only; ** = difference was not statistically significant; # = enterococci
Kothari A, et al. 2014; CID 59:272-8

Xpert MRSA/SA compared to conventional methods at
OSU, employing partnership of Microbiology,
Epidemiology, Pharmacy, Infection Control and Infectious
Diseases:
o
o
o
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LOS: 6.2 d less with rapid methods
$21,387 less in patient costs
100% sensitivity and specificity as compared
to the conventional method
ID Pharmacist was essential
o
Bauer KA, et al. 2014. CID 51: 1074-80.

76 patients, mainly infected with P. aeruginosa—
19 died within 30 days; underlying conditions:
o
o
o
o
Coronary heart disease
Vasopressors
ICU diagnosis of sepsis/pneumonia
Received appropriate dose of antibiotics > 24 hr after BAL performed
(Kollef, KE et al. 2008; CHEST 134: 281-7)

E-test used directly on clinical specimens vs conventional AST testing
o
o
o
o
o
1.4 d vs. 4.2 d for results
Fewer days of fever
Fewer days of antibiotics before resolution of VAP
Lower antibiotic costs
Less C. difficile
(Bouza E et al. 2007; CID 44: 382-7.
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Imaging Techniques
• BACcel (Accelr8 or Accelerate ID/AST; RUO; Tucson, AZ)) : use in
positive Blood Culture Bottles

Fluorescence Activated Cell Sorting (FACS)
• Alone or in combination with microfluidics to distinguish cellular
size and shapes.
• Fluorescent stains can distinguish “dead” from “living “ cells

Microbial Cell Weighing by Vibrating Cantilevers
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Micro-calorimetry
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Rotating Magnets
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RNA Sequencing
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Testing in Microdroplets
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Bacteriophage Susceptibility
• Adaptations for TB testing in past
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OTHER
Van Belkum et al. Ann Lab Med 2013; 33:14-27
NO----also need:
 Better understanding of emergence and speed of
resistance
 Significant development of new antibiotics
 Sharing of global surveillance data
 Increase development of technical needs for our
technologists
• Large scale data management
• Bioinformatics
• Communications

Available urine screening methods with limited success/acceptance:
• Gram stain
• Dip sticks for nitrates and wbc’s
• Instruments that can enumerate microbes in broth rapidly

Reason for urine screen?
• Provide rapid indication that microbe count exceeds threshold (10 -10
• Reduce number of urines that need to be plated and read
4
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Save personnel time and money
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Use of appropriate antibiotics sooner
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Reduce use of antibiotics if screen not positive
5
UTI in a hospitalized patient is most often source of subsequent BSI
cfu/ml)
Operation Principle
Forward Laser Scattering combined with
Optical Density = sensitivity below
104 to above 1010 cfu/mL
Bacteria growth imaging:
27
UTI Screening Clinical Study Design
Rambam Healthcare Campus, a
1000-bed tertiary Israeli
hospital

BacterioScanTM 216R, two
CHROMagarTM plate cultures,
dipstick analysis and microscopy
had been performed
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2400 samples from a diverse
population, hospital wards and
collection methods were analyzed
Objective: quantitatively compare performance of various rapid
screening techniques with plate
culture
Chromagar plate 2
Chromagar plate 1
Dipstick
Clinitek Status
Vitek 2
Microscopy
10
10
Concentration change [cfu/ml]

10
10
10
10
10
BacterioscanTM 216R
9
8
7
6
5
4
3
0
50
100
150
Incubation time [min]
Urine Screening
with BacterioScan
(a) Exemplary growth curves for various Gram positive,
Gram negative bacteria and negative samples
(b) Additional output from BacterioScan 216R showing
Optical Density and mean size of the bacteria for two
coagulase negative Staphylococcus species.
216R Pathogens Detection Evaluation
100
• Statistical performance metrics of
BacterioScan 216R evaluated at various
incubation times
Sensitivity [%]
95
• ROC (Receiver Operating Characteristic)
curves for 30, 60, 90, 120 and 180 min
incubation are shown
90
85
30 min
• About 94% sensitivity with 78% specificity
obtained after 90 minutes of incubation
60 min
90 min
80
120 min
• Minor improvement in performance
observed with longer incubation periods.
180 min
75
0
10
20
100% - Specificity
30
40
Summary:
All Evaluated Rapid Techniques
Rapid Screening Test
Sensitivity
Specificity
NPV
PPV
Accuracy
Agar Plate
Culture
Microscopy
86%
94%
94%
86%
92%
86%
78%
96%
46%
79%
Dipstick BacterioScan 216R
37%
97%
83%
80%
83%
95%
80%
98%
62%
84%
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Use of a laser scatter instrument to measure changes in E. coli
(concentrations simulating a UTI) when exposed to varying concentrations
of ciprofloxacin
 Model 224R BacterioScan, St. Louis, MO)
 37 °C; measurements made over 5 hr.
 Starting concentration of E. coli of ~ 3 X 104 CFU/ml broth

Successful at measuring the growth rate with repeatability of +/- 10% and
3-min time resolution
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Ability to approximate a nominal MIC within 90 min
 MIC in instrument: between 0.004 and 0.008 μg/ml compared to CLSI QC
results for the E. coli of 0.004 to 0.015 μg/ml.
Marshall D et al. Poster at ASM Biodefense and Emerging Diseases Research Conference, Jan 2014.
1.
Donor blood was seeded with E. coli (1.5 X 105 cfu/ml) and 10 ml placed into
BACTEC BC bottles; incubated until machine detected a pos (+)
2.
4 aliquots removed from each of aerobic and anaerobic bottles:
•
•
•
•
1 tube not further processed
1 tube filtered
1 tube centrifuged
1 tube lysed
3. 2 μl of each sample was diluted (1000:1) and added to MHB with ciprofloxacin
and measurements made over 5 hr
Results:
Growth was detected and an accurate phenotypic MIC was obtained
within 3 hr after BC bottle was deemed positive.
Anbazhagan R et al. Poster submitted to ICAAC 2015.
(Beavers T and JG Wheeler. 2010. MLO 42: 20-22)

No longer passive
• Perhaps it is or should be interventional
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Specimen
• Reflex testing
• POC
• 24/7 testing
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Communications
• Active communication between clinicians and microbiologists
• Many different modes available
• DATA management and data systems must change
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Clinical and Public Health Impact
Costs vs benefits
• May cost more in lab but if LOS is decreased and cost to patient is decreased
and most importantly outcomes are better-----

The authors call this Collaborative Medicine
 The use of methods that reduce time to results have proven to be
better for the patient and for the medical community in general
•
•
•
•
Outcomes
LOS
Costs
Reduced antibiotic resistance development
 Rapid methods are now and will become more available
• One “shoe” will not fit all, but we all need to keep abreast of the
potential for these newer methods and find ones that fit our
circumstances
 Communication is key to using these rapid methods for obtaining
effective outcomes and achieving best antibiotic stewardship goals
• To Barbara Lapinskas and APUA for inviting me and
doing a wonderful job of planning & organizing
• You, the audience for listening and not sleeping (?)
• My family for their support