Transcript W12-1600-Dunn-ClinicalImpact

The Clinical Impact of
Real-Time Molecular Infectious
Disease Diagnostics
Jim Dunn, Ph.D., D(ABMM)
Cook Children’s Medical Center
Ft. Worth, TX
Molecular Microbiology
Fastest growing area in clinical
laboratory medicine
 Integral and necessary component of
many diagnostic laboratories
 Traditional methods being rapidly
displaced by molecular testing

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Clinical Value

Qualitative (pos/neg) nucleic acid tests are
especially valuable for the detection of
infectious agents that are:
Unculturable
 Present in extremely low quantities
 Fastidious or slow-growing
 Dangerous to amplify in culture
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Clinical Value

Quantitative (viral load) methods are
important for monitoring certain chronic
infections. These tests allow us to:
monitor therapy
 detect the development of drug resistance
 predict disease progression
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Real-Time PCR
Introduced in mid-1990’s
 Rapidly evolving field with numerous
technological advances
 Continuous fluorescence monitoring of
nucleic acid amplification within a closed
system.
 One tube amplification and detection

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Fluorescence Monitoring
Plateau:
Qualitative
end-point read
Exponential:
Quantitative
real-time read
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Real-Time PCR
Rapid assay development
 Simplified primer and probe design
 Simple and versatile to perform
 Pre-optimized universal master mixes
 Universal conditions for amplification
 Multiple chemistries available
 Choice of instrumentation
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What’s the impact on patient
management and outcomes?
Case #1
4 y.o. boy presents with 2-day history of
fever and headache
 Day of presentation began to complain of
neck pain
 Temp = 102.7oF
 Mild photophobia
 No rashes
 Intact neurologic exam

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Case #1
Complete Blood Count
- 9,300 cells/mm3
- 45% PMN, 40% lymph, 15 mono
 Cerebrospinal Fluid (CSF)
- WBC = 75 cells/mm3
- 72% PMN, 8% lymph, 20% mono
- protein = 22 mg/dl
- glucose = 60 mg/dl
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Case #1
CSF gram stain
mod WBC, no organisms
 I.V. ceftriaxone started
 Blood, CSF, urine bacterial
cultures obtained
 Enterovirus RT-PCR on CSF ordered
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Case #1

ANSWER
Blood, CSF, urine bacterial cultures = neg
Enterovirus RT-PCR = POSITIVE
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DIAGNOSIS: Viral Meningitis
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Aseptic Meningitis
Clinical and lab evidence of meningeal
inflammation not due to bacteria
 75,000 cases/year in US
 80 to 90% due to Enteroviruses
 Occur mainly in summer and fall
 Difficult to distinguish from bacterial
meningitis based on clinical features alone
 Enteroviral meningitis has good prognosis
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Enteroviruses
aseptic meningitis, myocarditis, flaccid
paralysis, neonatal sepsis-like disease,
encephalitis, febrile rash disease
 now probably >100 serotypes based on
capsid sequence analysis
 molecular diagnosis has replaced
traditional cell culture
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Enteroviruses

Comparison of RT-PCR vs. Viral Culture
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59 inpatient CSF samples tested
Result
RT-PCR
Culture
Pos
37
22
Neg
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37
Sensitivity of CSF viral culture = 60%
Culture time to detection = 3 – 5 days
RT-PCR time to detection = 3 – 4 hours
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Enteroviruses

Rapid diagnosis of enteroviral meningitis
by real time PCR impacts clinical
management:
Earlier hospital discharge
 Fewer additional diagnostic tests
 Decreased antibiotic usage
 Decreased overall health care costs
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Hospital-Acquired Infections
(HAIs)
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On an annual basis account for:
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~2 million infections
~100,000 deaths
$4-6 billion in health care costs

50–60% of the HAIs occurring in the USA each
year are caused by antibiotic-resistant bacteria
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High rate of antibiotic resistance increases
morbidity, mortality & costs associated with HAIs
Jones. Chest 2001;119:397S–404S
Weinstein. Emerg Infect Dis 1998;4:416–420
Vancomycin-Resistant
Enterococci (VRE)
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Since 1989, a rapid increase in the incidence of
infection and colonization with VRE has been
reported by U.S. hospitals
This poses important problems, including:
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Lack of available antimicrobial therapy for VRE
infections because most VRE are also resistant to
drugs previously used to treat such infections
Possibility that vancomycin-resistance genes present
in VRE can be transferred to other gram-positive
bacteria (e.g. Staphylococcus aureus )
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Vancomycin-Resistant
Enterococci (VRE)
E. faecium and E. faecalis that have
acquired genes vanA and/or vanB
 Most important reservoir for VRE is the
colonized gastrointestinal tracts of patients
 Transmission can occur:

Contaminated hands of healthcare workers
 Contamination of environment
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Vancomycin-Resistant
Enterococci
The Problem?
 Major nosocomial pathogen
Up to 6.3% of nosocomial enterococcal
bloodstream infections in pediatric hospitals
 28.5% of nosocomial enterococcal infections
in ICU patients (NNIS-2003)

Wisplinghoff, et al. Pediatr Infect Dis J 22:686, 2003.
NNIS. Am J Infect Control 32:470, 2004.
Vancomycin-Resistant
Enterococci
What Should Be Done?
 Active Surveillance (SHEA & CDC)

High Risk Patients/Locations:
Admission & Periodic (e.g. weekly)
VRE culture often requires ≥ 72 hrs.
 High Rate of False Negatives with Culture
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Muto, et al. Infect Control Hosp Epi 24:362, 2003.
CDC. MMWR 44:1, 1995.
Vancomycin-Resistant
Enterococci
Lab-Developed Taqman Real Time
Multiplex vanA/vanB PCR Assay
 Sens = 100%, Spec = 98%
 PPV = 91%, NPV = 100%
 Screening & Surveillance in Admitted
Oncology and Bone Marrow Transplant
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Pre-emptive isolation until VRE result known
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VRE by Real Time PCR
Greater sensitivity & More rapid results
 Rapid Detection → Infection Control
Measures
 Reduce Duration of Contact Isolation
 Excess costs associated with nosocomial
infections justify screening and preventive
infection control measures
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Cost-Effectiveness of VRE
Surveillance
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Attributable cost of surveillance vs.
cost of nosocomial infections
$1,000,000
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$761,320
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$800,000
$600,000
2-year period
Hosp #1
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$253,099
$400,000
$200,000
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Hosp #2
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$0
Hosp #1
No surveillance
Surveillance
Hosp #2
Muto, et al. Infect Control Hosp Epidemiol 23:429-435, 2002.
Cost-Effectiveness of VRE
Surveillance by Real Time PCR
University of Iowa Hospital
 Real Time PCR for VRE
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Average TAT = 1.3 days
(3.4 days for culture)
↓ length of stay by ~2 days for patients
discharged to long-term care facilities
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$205,000 annual savings
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Cost-Effectiveness of VRE
Surveillance by Real Time PCR
Rapid determination of VRE colonization
status prevented 2,348 isolation days/year
when compared to culture
 Annual savings = $87,600
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$187,200
$200,000
$150,000
$99,600
$100,000
$50,000
$0
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MPCR
Culture
Bordetella pertussis
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Bordetella pertussis
Endemic disease, occurs year-round,
epidemic cycles every 3 or 4 years
 Transmitted by large droplets
 Attack rates among close contacts as high
as 80 to 100%
 Waning immunity leads to susceptible
adolescents and adults
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Family members often source for infected
infants
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Bordetella pertussis
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Bordetella pertussis
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Diagnosis
Specimens
 NP swab or aspirate
 Throat & anterior nares swabs
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Lower rates of recovery
Ciliated respiratory epithelium not found in
pharynx
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Diagnosis
Find highest concentration of organism
during catarrhal stage and beginning of
paroxysmal stage
 Concentration of organism negatively
correlates with increasing age
conc. in infants
conc. adolescents/adults
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Diagnosis
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Culture: still “gold standard”
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DFA: low sens and variable spec
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Sens actually 15-60% compared to PCR
Special media/transport, long incubation
Always back-up with cx or PCR
Serology: not part of case definition
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Not standardized
Epidemiology/vaccine efficacy
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Real-Time PCR
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Very sensitive (~1 cfu/rxn)
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Don’t need viable organism
Good for mild, atypical cases, older patients
Results within hours
 Not standardized between labs
 Some labs multiplex with B. parapertussis
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Hospital-Acquired Pertussis
Among Newborns
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Cook Children’s
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6 infants admitted with pertussis w/in a few
days of each other
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Confirmed by real-time PCR w/in 24 hrs admit
4 infants in PICU
Investigation reveals all born at same local
hospital
 One HCW in newborn nursery with cough,
post-tussive emesis, dyspnea
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
PCR pos for B. pertussis
MMWR 57:600-603, 2008.
Timeline of Infants with Pertussis from a General Hospital Newborn Nursery
Nursery
Worker:
Prodrome?
§
** 07/10/2004
Infant # 1
*†
Infant # 2
*†
Infant # 3
*†
Infant # 4
Infant #6
*
§
PICU
§§
PICU
§§
PICU
§§
¶
§
PICU
§§
¶
§§
¶
§
*†
Infant # 8
§§
¶
§
†
¶
§
*†
Infant # 7
¶
§§
§
*†
Infant # 9
Infant # 10
§
*†
Infant # 5
¶
§
††7/17/2004
PICU
¶
§
*†
*†
¶
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 2
June
July
Aug
* Date born
† Exposure in nursery
§ Symptoms started
¶ Admission/Diagnosis Date
**Outbreak noted
†† HCW PCR +/Furlough
§§ Discharge Date
¶ 8/28 Out pt
§ unk ¶ 10/4
*†
Infant # 11
§§ 8/7
§ unk Out pt
Summary
HCW furloughed/treated
 Families of 110 infants born at local
hospital evaluated for cough illness
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18 with cough: PCR neg
2 additional PCR pos
Total of 11 infants with confirmed pertussis
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Attack rate ~10%
MMWR 57:600-603, 2008.
Cook Children’s Molecular Lab
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What’s So Cool About Real-Time PCR?
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Decreased Turnaround Times/High Throughput
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Closed system
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No additions made after specimen is added
Contamination control – No false positives
More Standardized
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Simultaneous amplification, detection, & data analysis
Pre-optimized master mixes, reproducible
Less expensive that traditional PCR
Increased Sensitivity
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Thanks