Presentation on theme: "Jim Dunn, Ph.D., D(ABMM) Cook Children’s Medical Center Ft. Worth, TX"— Presentation transcript:
1 Jim Dunn, Ph.D., D(ABMM) Cook Children’s Medical Center Ft. Worth, TX The Clinical Impact of Real-Time Molecular Infectious Disease DiagnosticsJim Dunn, Ph.D., D(ABMM)Cook Children’s Medical CenterFt. Worth, TX
2 Molecular Microbiology Fastest growing area in clinicallaboratory medicineIntegral and necessary component of many diagnostic laboratoriesTraditional methods being rapidly displaced by molecular testing
3 Clinical ValueQualitative (pos/neg) nucleic acid tests are especially valuable for the detection of infectious agents that are:UnculturablePresent in extremely low quantitiesFastidious or slow-growingDangerous to amplify in culture
4 Clinical ValueQuantitative (viral load) methods are important for monitoring certain chronic infections. These tests allow us to:monitor therapydetect the development of drug resistancepredict disease progression
5 Real-Time PCR Introduced in mid-1990’s Rapidly evolving field with numerous technological advancesContinuous fluorescence monitoring of nucleic acid amplification within a closed system.One tube amplification and detection
7 Real-Time PCR Rapid assay development Simplified primer and probe designSimple and versatile to performPre-optimized universal master mixesUniversal conditions for amplificationMultiple chemistries availableChoice of instrumentation
8 What’s the impact on patient management and outcomes?
9 Case #1 4 y.o. boy presents with 2-day history of fever and headache Day of presentation began to complain of neck painTemp = 102.7oFMild photophobiaNo rashesIntact neurologic exam
13 Aseptic MeningitisClinical and lab evidence of meningeal inflammation not due to bacteria75,000 cases/year in US80 to 90% due to EnterovirusesOccur mainly in summer and fallDifficult to distinguish from bacterial meningitis based on clinical features aloneEnteroviral meningitis has good prognosis
14 Enterovirusesaseptic meningitis, myocarditis, flaccid paralysis, neonatal sepsis-like disease, encephalitis, febrile rash diseasenow probably >100 serotypes based on capsid sequence analysismolecular diagnosis has replaced traditional cell culture
15 Enteroviruses Comparison of RT-PCR vs. Viral Culture 59 inpatient CSF samples testedSensitivity of CSF viral culture = 60%Culture time to detection = 3 – 5 daysRT-PCR time to detection = 3 – 4 hoursResultRT-PCRCulturePos3722Neg
16 EnterovirusesRapid diagnosis of enteroviral meningitis by real time PCR impacts clinical management:Earlier hospital dischargeFewer additional diagnostic testsDecreased antibiotic usageDecreased overall health care costs
18 Hospital-Acquired Infections (HAIs) On an annual basis account for:~2 million infections~100,000 deaths$4-6 billion in health care costs50–60% of the HAIs occurring in the USA each year are caused by antibiotic-resistant bacteriaHigh rate of antibiotic resistance increases morbidity, mortality & costs associated with HAIsJones. Chest 2001;119:397S–404SWeinstein. Emerg Infect Dis 1998;4:416–420
19 Vancomycin-Resistant Enterococci (VRE) Since 1989, a rapid increase in the incidence of infection and colonization with VRE has been reported by U.S. hospitalsThis poses important problems, including:Lack of available antimicrobial therapy for VRE infections because most VRE are also resistant to drugs previously used to treat such infectionsPossibility that vancomycin-resistance genes present in VRE can be transferred to other gram-positive bacteria (e.g. Staphylococcus aureus )
20 Vancomycin-Resistant Enterococci (VRE) E. faecium and E. faecalis that have acquired genes vanA and/or vanBMost important reservoir for VRE is the colonized gastrointestinal tracts of patientsTransmission can occur:Contaminated hands of healthcare workersContamination of environment
21 Vancomycin-Resistant Enterococci The Problem?Major nosocomial pathogenUp to 6.3% of nosocomial enterococcal bloodstream infections in pediatric hospitals28.5% 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.
22 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 CultureMuto, et al. Infect Control Hosp Epi 24:362, 2003.CDC. MMWR 44:1, 1995.
23 Vancomycin-Resistant Enterococci Lab-Developed Taqman Real Time Multiplex vanA/vanB PCR AssaySens = 100%, Spec = 98%PPV = 91%, NPV = 100%Screening & Surveillance in Admitted Oncology and Bone Marrow TransplantPre-emptive isolation until VRE result known
24 VRE by Real Time PCR Greater sensitivity & More rapid results Rapid Detection → Infection Control MeasuresReduce Duration of Contact IsolationExcess costs associated with nosocomial infections justify screening and preventive infection control measures
25 Cost-Effectiveness of VRE Surveillance Attributable cost of surveillance vs. cost of nosocomial infections2-year periodHosp #1No surveillanceHosp #2SurveillanceMuto, et al. Infect Control Hosp Epidemiol 23: , 2002.
26 Cost-Effectiveness of VRE Surveillance by Real Time PCR University of Iowa HospitalReal Time PCR for VREAverage TAT = 1.3 days(3.4 days for culture)↓ length of stay by ~2 days for patients discharged to long-term care facilities$205,000 annual savings
27 Cost-Effectiveness of VRE Surveillance by Real Time PCR Rapid determination of VRE colonization status prevented 2,348 isolation days/year when compared to cultureAnnual savings = $87,600
29 Bordetella pertussisEndemic disease, occurs year-round, epidemic cycles every 3 or 4 yearsTransmitted by large dropletsAttack rates among close contacts as high as 80 to 100%Waning immunity leads to susceptible adolescents and adultsFamily members often source for infected infants
32 Diagnosis Specimens NP swab or aspirate Throat & anterior nares swabs Lower rates of recoveryCiliated respiratory epithelium not found in pharynx
33 DiagnosisFind highest concentration of organism during catarrhal stage and beginning of paroxysmal stageConcentration of organism negatively correlates with increasing ageconc. in infantsconc. adolescents/adults
34 Diagnosis Culture: still “gold standard” Sens actually 15-60% compared to PCRSpecial media/transport, long incubationDFA: low sens and variable specAlways back-up with cx or PCRSerology: not part of case definitionNot standardizedEpidemiology/vaccine efficacy
35 Real-Time PCR Very sensitive (~1 cfu/rxn) Results within hours Don’t need viable organismGood for mild, atypical cases, older patientsResults within hoursNot standardized between labsSome labs multiplex with B. parapertussis
37 Cook Children’s6 infants admitted with pertussis w/in a few days of each otherConfirmed by real-time PCR w/in 24 hrs admit4 infants in PICUInvestigation reveals all born at same local hospitalOne HCW in newborn nursery with cough, post-tussive emesis, dyspneaPCR pos for B. pertussisMMWR 57: , 2008.
38 Timeline of Infants with Pertussis from a General Hospital Newborn Nursery Worker: § ** 07/10/ ††7/17/2004Prodrome?Infant # *† § ¶ §§ PICUInfant # *† § ¶ §§ PICUInfant # *† § ¶ §§ PICUInfant # *† § ¶ §§ PICUInfant # *† § ¶ §§Infant # * † § ¶ §§Infant # *† § ¶ §§ PICUInfant # *† § ¶ §§ 8/7Infant # *† § ¶ 8/28 Out ptInfant # *† § unk ¶ 10/4Infant # *† ¶ § unk Out ptJune July Aug* Date born† Exposure in nursery§ Symptoms started¶ Admission/Diagnosis Date**Outbreak noted†† HCW PCR +/Furlough§§ Discharge Date
39 Summary HCW furloughed/treated Families of 110 infants born at local hospital evaluated for cough illness18 with cough: PCR neg2 additional PCR posTotal of 11 infants with confirmed pertussisAttack rate ~10%MMWR 57: , 2008.
41 What’s So Cool About Real-Time PCR? Decreased Turnaround Times/High ThroughputSimultaneous amplification, detection, & data analysisClosed systemNo additions made after specimen is addedContamination control – No false positivesMore StandardizedPre-optimized master mixes, reproducibleLess expensive that traditional PCRIncreased Sensitivity