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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 - PowerPoint PPT Presentation
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The Clinical Impact of The Clinical Impact of Real-Time Molecular Infectious Real-Time Molecular Infectious
Disease DiagnosticsDisease Diagnostics
Jim Dunn, Ph.D., D(ABMM)Jim Dunn, Ph.D., D(ABMM)
Cook Children’s Medical CenterCook Children’s Medical Center
Ft. Worth, TXFt. Worth, TX
Molecular MicrobiologyMolecular Microbiology
Fastest growing area in clinical Fastest growing area in clinical
laboratory medicinelaboratory medicine Integral and necessary component of Integral and necessary component of
many diagnostic laboratoriesmany diagnostic laboratoriesTraditional methods being rapidly Traditional methods being rapidly
displaced by molecular testingdisplaced by molecular testing
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Clinical ValueClinical Value
QualitativeQualitative (pos/neg) nucleic acid tests are (pos/neg) nucleic acid tests are especially valuable for the detection of especially valuable for the detection of infectious agents that are:infectious agents that are:UnculturableUnculturablePresent in extremely low quantitiesPresent in extremely low quantitiesFastidious or slow-growingFastidious or slow-growingDangerous to amplify in cultureDangerous to amplify in culture
33
Clinical ValueClinical Value
QuantitativeQuantitative (viral load) methods are (viral load) methods are important for monitoring certain chronic important for monitoring certain chronic infections. These tests allow us to:infections. These tests allow us to:monitor therapymonitor therapydetect the development of drug resistancedetect the development of drug resistancepredict disease progressionpredict disease progression
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Real-Time PCRReal-Time PCR
Introduced in mid-1990’sIntroduced in mid-1990’sRapidly evolving field with numerous Rapidly evolving field with numerous
technological advancestechnological advancesContinuous fluorescence monitoring of Continuous fluorescence monitoring of
nucleic acid amplification within a closed nucleic acid amplification within a closed system.system.
One tube amplification and detectionOne tube amplification and detection
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Fluorescence Monitoring
Exponential:Quantitative real-time read
Plateau:Qualitative end-point read
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Real-Time PCRReal-Time PCR
Rapid assay developmentRapid assay developmentSimplified primer and probe designSimplified primer and probe designSimple and versatile to performSimple and versatile to performPre-optimized universal master mixesPre-optimized universal master mixesUniversal conditions for amplificationUniversal conditions for amplificationMultiple chemistries availableMultiple chemistries availableChoice of instrumentationChoice of instrumentation
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What’s the impact on patient What’s the impact on patient management and outcomes?management and outcomes?
Case #1Case #1
4 y.o. boy presents with 2-day history of 4 y.o. boy presents with 2-day history of fever and headachefever and headache
Day of presentation began to complain of Day of presentation began to complain of neck painneck pain
Temp = 102.7Temp = 102.7ooFFMild photophobiaMild photophobiaNo rashesNo rashes Intact neurologic examIntact neurologic exam
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Case #1Case #1
Complete Blood CountComplete Blood Count- 9,300 cells/mm- 9,300 cells/mm33
- 45% PMN, 40% lymph, 15 mono- 45% PMN, 40% lymph, 15 monoCerebrospinal Fluid (CSF)Cerebrospinal Fluid (CSF)
- WBC = 75 cells/mm- WBC = 75 cells/mm33
- 72% PMN, 8% lymph, 20% mono- 72% PMN, 8% lymph, 20% mono- protein = 22 mg/dl- protein = 22 mg/dl- glucose = 60 mg/dl- glucose = 60 mg/dl
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Case #1Case #1
CSF gram stainCSF gram stain
mod WBC, no organismsmod WBC, no organisms I.V. ceftriaxone startedI.V. ceftriaxone startedBlood, CSF, urine bacterialBlood, CSF, urine bacterial
cultures obtainedcultures obtainedEnterovirus RT-PCR on CSF orderedEnterovirus RT-PCR on CSF ordered
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Case #1Case #1
ANSWERANSWER
Blood, CSF, urine bacterial cultures = negBlood, CSF, urine bacterial cultures = neg
Enterovirus RT-PCR = Enterovirus RT-PCR = POSITIVEPOSITIVE
DIAGNOSIS: Viral MeningitisDIAGNOSIS: Viral Meningitis
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Aseptic MeningitisAseptic Meningitis
Clinical and lab evidence of meningeal Clinical and lab evidence of meningeal inflammation not due to bacteriainflammation not due to bacteria
75,000 cases/year in US75,000 cases/year in US80 to 90% due to Enteroviruses80 to 90% due to EnterovirusesOccur mainly in summer and fallOccur mainly in summer and fallDifficult to distinguish from bacterial Difficult to distinguish from bacterial
meningitis based on clinical features alonemeningitis based on clinical features aloneEnteroviral meningitis has good prognosisEnteroviral meningitis has good prognosis
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EnterovirusesEnteroviruses
aseptic meningitis, myocarditis, flaccid aseptic meningitis, myocarditis, flaccid paralysis, neonatal sepsis-like disease, paralysis, neonatal sepsis-like disease, encephalitis, febrile rash diseaseencephalitis, febrile rash disease
now probably >100 serotypes based on now probably >100 serotypes based on capsid sequence analysiscapsid sequence analysis
molecular diagnosis has replaced molecular diagnosis has replaced traditional cell culturetraditional cell culture
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EnterovirusesEnteroviruses
Comparison of RT-PCR vs. Viral CultureComparison of RT-PCR vs. Viral Culture 59 inpatient CSF samples tested 59 inpatient CSF samples tested
Sensitivity of CSF viral culture = 60%Sensitivity of CSF viral culture = 60% Culture time to detection = 3 – 5 daysCulture time to detection = 3 – 5 days RT-PCR time to detection = 3 – 4 hoursRT-PCR time to detection = 3 – 4 hours
ResultResult RT-PCRRT-PCR CultureCulture
PosPos 3737 2222
NegNeg 2222 3737
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EnterovirusesEnteroviruses
Rapid diagnosis of enteroviral meningitis Rapid diagnosis of enteroviral meningitis by real time PCR impacts clinical by real time PCR impacts clinical management:management:Earlier hospital dischargeEarlier hospital dischargeFewer additional diagnostic testsFewer additional diagnostic testsDecreased antibiotic usageDecreased antibiotic usageDecreased overall health care costsDecreased overall health care costs
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Hospital-Acquired Infections Hospital-Acquired Infections (HAIs)(HAIs)
On an annual basis account for:On an annual basis account for: ~2 million infections~2 million infections ~100,000 deaths~100,000 deaths $4-6 billion in health care costs$4-6 billion in health care costs
50–60% of the HAIs occurring in the USA each 50–60% of the HAIs occurring in the USA each year are caused by antibiotic-resistant bacteriayear are caused by antibiotic-resistant bacteria
High rate of antibiotic resistance increases High rate of antibiotic resistance increases morbidity, mortality & costs associated with HAIsmorbidity, mortality & costs associated with HAIs
Jones. Chest 2001;119:397S–404SWeinstein. Emerg Infect Dis 1998;4:416–420
Since 1989, a rapid increase in the incidence of Since 1989, a rapid increase in the incidence of infection and colonization with VRE has been infection and colonization with VRE has been reported by U.S. hospitalsreported by U.S. hospitals
This poses important problems, including:This poses important problems, including: Lack of available antimicrobial therapy for VRE Lack of available antimicrobial therapy for VRE
infections because most VRE are also resistant to infections because most VRE are also resistant to drugs previously used to treat such infectionsdrugs previously used to treat such infections
Possibility that vancomycin-resistance genes present Possibility that vancomycin-resistance genes present in VRE can be transferred to other gram-positive in VRE can be transferred to other gram-positive bacteria (e.g. bacteria (e.g. Staphylococcus aureusStaphylococcus aureus ) )
Vancomycin-Resistant Vancomycin-Resistant Enterococci (VRE) Enterococci (VRE)
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Vancomycin-Resistant Vancomycin-Resistant Enterococci (VRE)Enterococci (VRE)
E. faeciumE. faecium and and E. faecalisE. faecalis that have that have acquired genes acquired genes vanAvanA and/or and/or vanBvanB
Most important reservoir for VRE is the Most important reservoir for VRE is the colonized gastrointestinal tracts of patientscolonized gastrointestinal tracts of patients
Transmission can occur:Transmission can occur:Contaminated hands of healthcare workersContaminated hands of healthcare workersContamination of environmentContamination of environment
2020
Vancomycin-Resistant Vancomycin-Resistant EnterococciEnterococci
The Problem?The Problem?Major nosocomial pathogenMajor nosocomial pathogen
Up to 6.3% of nosocomial enterococcal Up to 6.3% of nosocomial enterococcal bloodstream infections in pediatric hospitalsbloodstream infections in pediatric hospitals
28.5% of nosocomial enterococcal infections 28.5% of nosocomial enterococcal infections in ICU patients (NNIS-2003)in ICU patients (NNIS-2003)
Wisplinghoff, et al. Pediatr Infect Dis J 22:686, 2003.Wisplinghoff, et al. Pediatr Infect Dis J 22:686, 2003.NNIS. Am J Infect Control 32:470, 2004. NNIS. Am J Infect Control 32:470, 2004.
Vancomycin-Resistant Vancomycin-Resistant EnterococciEnterococci
What Should Be Done?What Should Be Done?Active Surveillance (SHEA & CDC)Active Surveillance (SHEA & CDC)
High Risk Patients/Locations:High Risk Patients/Locations:
Admission & Periodic (e.g. weekly)Admission & Periodic (e.g. weekly)
VRE culture often requires VRE culture often requires ≥ 72 hrs.≥ 72 hrs.High Rate of False Negatives with CultureHigh Rate of False Negatives with Culture
Muto, et al. Infect Control Hosp Epi 24:362, 2003.Muto, et al. Infect Control Hosp Epi 24:362, 2003.CDC. MMWR 44:1, 1995. CDC. MMWR 44:1, 1995.
Vancomycin-Resistant Vancomycin-Resistant EnterococciEnterococci
Lab-Developed Taqman Real Time Lab-Developed Taqman Real Time Multiplex Multiplex vanAvanA//vanBvanB PCR Assay PCR Assay
Sens = 100%, Spec = 98%Sens = 100%, Spec = 98%PPV = 91%, NPV = 100%PPV = 91%, NPV = 100%Screening & Surveillance in Admitted Screening & Surveillance in Admitted
Oncology and Bone Marrow TransplantOncology and Bone Marrow Transplant Pre-emptive isolation until VRE result knownPre-emptive isolation until VRE result known
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VRE by Real Time PCRVRE by Real Time PCR
Greater sensitivity & More rapid resultsGreater sensitivity & More rapid resultsRapid Detection Rapid Detection → Infection Control → Infection Control
MeasuresMeasuresReduce Duration of Contact IsolationReduce Duration of Contact IsolationExcess costs associated with nosocomial Excess costs associated with nosocomial
infections justify screening and preventive infections justify screening and preventive infection control measuresinfection control measures
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Cost-Effectiveness of VRE Cost-Effectiveness of VRE SurveillanceSurveillance
Attributable cost of surveillance vs. Attributable cost of surveillance vs. cost of nosocomial infectionscost of nosocomial infections
$761,320
$253,099
$0
$200,000
$400,000
$600,000
$800,000
$1,000,000
Hosp #1 Hosp #2
Muto, et al. Infect Control Hosp Epidemiol 23:429-435, 2002.Muto, et al. Infect Control Hosp Epidemiol 23:429-435, 2002.
2-year period2-year period Hosp #1Hosp #1
No surveillanceNo surveillance
Hosp #2Hosp #2 SurveillanceSurveillance
Cost-Effectiveness of VRE Cost-Effectiveness of VRE Surveillance by Real Time PCRSurveillance by Real Time PCR
University of Iowa HospitalUniversity of Iowa HospitalReal Time PCR for VREReal Time PCR for VRE
Average TAT = 1.3 daysAverage TAT = 1.3 days
(3.4 days for culture)(3.4 days for culture)↓ ↓ length of stay by ~2 days for patients length of stay by ~2 days for patients
discharged to long-term care facilitiesdischarged to long-term care facilities$205,000 annual savings$205,000 annual savings
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Cost-Effectiveness of VRE Cost-Effectiveness of VRE Surveillance by Real Time PCRSurveillance by Real Time PCR
Rapid determination of VRE colonization Rapid determination of VRE colonization status prevented 2,348 isolation days/year status prevented 2,348 isolation days/year when compared to culturewhen compared to culture
Annual savings = $87,600Annual savings = $87,600
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Bordetella pertussisBordetella pertussis
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Bordetella pertussisBordetella pertussis
Endemic disease, occurs year-round, Endemic disease, occurs year-round, epidemic cycles every 3 or 4 yearsepidemic cycles every 3 or 4 years
Transmitted by large dropletsTransmitted by large dropletsAttack rates among close contacts as high Attack rates among close contacts as high
as 80 to 100%as 80 to 100%Waning immunity leads to susceptible Waning immunity leads to susceptible
adolescents and adultsadolescents and adultsFamily members often source for infected Family members often source for infected
infantsinfants2929
Bordetella pertussisBordetella pertussis
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Bordetella pertussisBordetella pertussis
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Diagnosis
SpecimensSpecimensNP swab or aspirateNP swab or aspirateThroat & anterior nares swabsThroat & anterior nares swabs
Lower rates of recoveryLower rates of recovery Ciliated respiratory epithelium not found in Ciliated respiratory epithelium not found in
pharynxpharynx
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Diagnosis
Find highest concentration of organism Find highest concentration of organism during catarrhal stage and beginning of during catarrhal stage and beginning of paroxysmal stageparoxysmal stage
Concentration of organism negatively Concentration of organism negatively correlates with increasing agecorrelates with increasing age
conc. in infantsconc. in infants
conc. adolescents/adultsconc. adolescents/adults
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Diagnosis
Culture: still “gold standard”Culture: still “gold standard” Sens actually 15-60% compared to PCRSens actually 15-60% compared to PCR Special media/transport, long incubationSpecial media/transport, long incubation
DFA: low sens and variable specDFA: low sens and variable spec Always back-up with cx or PCRAlways back-up with cx or PCR
Serology: not part of case definitionSerology: not part of case definition Not standardizedNot standardized Epidemiology/vaccine efficacyEpidemiology/vaccine efficacy
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Real-Time PCR
Very sensitive (~1 cfu/rxn)Very sensitive (~1 cfu/rxn) Don’t need viable organismDon’t need viable organism Good for mild, atypical cases, older patientsGood for mild, atypical cases, older patients
Results within hoursResults within hoursNot standardized between labsNot standardized between labsSome labs multiplex with Some labs multiplex with B. parapertussisB. parapertussis
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Hospital-Acquired Pertussis Among Newborns
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Cook Children’s
6 infants admitted with pertussis w/in a few 6 infants admitted with pertussis w/in a few days of each otherdays of each other Confirmed by real-time PCR w/in 24 hrs admitConfirmed by real-time PCR w/in 24 hrs admit 4 infants in PICU4 infants in PICU
Investigation reveals all born at same local Investigation reveals all born at same local hospitalhospital
One HCW in newborn nursery with cough, One HCW in newborn nursery with cough, post-tussive emesis, dyspneapost-tussive emesis, dyspnea PCR pos for PCR pos for B. pertussisB. pertussis
MMWR 57:600-603, 2008.MMWR 57:600-603, 2008.
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
Nursery Worker: § ** 07/10/2004 ††7/17/2004
Infant # 1 *† § ¶ §§PICU
Infant # 2 *† § ¶ §§PICU
Infant # 3 *† § ¶ §§PICU
Timeline of Infants with Pertussis from a General Hospital Newborn Nursery
Infant # 4 *† § ¶ §§
PICUInfant # 5 *† § ¶ §§
Infant # 7 *† § ¶ §§ PICUInfant # 8 *† § ¶ §§ 8/7
Infant # 9 *† § ¶ 8/28 Out ptInfant # 10 *†
§ unk ¶ 10/4Infant # 11 *† ¶ § unk Out pt
* Date born† Exposure in nursery§ Symptoms started¶ Admission/Diagnosis Date**Outbreak noted†† HCW PCR +/Furlough §§ Discharge Date
Prodrome?
Infant #6 * † § ¶ §§
Summary
HCW furloughed/treatedHCW furloughed/treatedFamilies of 110 infants born at local Families of 110 infants born at local
hospital evaluated for cough illnesshospital evaluated for cough illness 18 with cough: PCR neg18 with cough: PCR neg 2 additional PCR pos2 additional PCR pos
Total of 11 infants with confirmed pertussisTotal of 11 infants with confirmed pertussis Attack rate ~10%Attack rate ~10%
MMWR 57:600-603, 2008.MMWR 57:600-603, 2008.
Cook Children’s Molecular Lab
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What’s So Cool About Real-Time PCR?What’s So Cool About Real-Time PCR?
Decreased Turnaround Times/High ThroughputDecreased Turnaround Times/High Throughput Simultaneous amplification, detection, & data analysisSimultaneous amplification, detection, & data analysis
Closed systemClosed system No additions made after specimen is addedNo additions made after specimen is added Contamination control – No false positivesContamination control – No false positives
More StandardizedMore Standardized Pre-optimized master mixes, reproduciblePre-optimized master mixes, reproducible
Less expensive that traditional PCRLess expensive that traditional PCR Increased SensitivityIncreased Sensitivity
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