The Clinical Impact of Real-Time Molecular Infectious Disease Diagnostics

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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 Real-Time Molecular Infectious Disease DiagnosticsJim Dunn, Ph.D., D(ABMM)Cook Childrens Medical CenterFt. Worth, TX

  • Molecular MicrobiologyFastest growing area in clinical laboratory medicineIntegral and necessary component of many diagnostic laboratoriesTraditional methods being rapidly displaced by molecular testing

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  • 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*

  • 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*

  • Real-Time PCRIntroduced in mid-1990sRapidly evolving field with numerous technological advancesContinuous fluorescence monitoring of nucleic acid amplification within a closed system.One tube amplification and detection*

  • Fluorescence MonitoringExponential:Quantitative real-time read

    Plateau:Qualitative end-point read*

  • Real-Time PCRRapid assay developmentSimplified primer and probe designSimple and versatile to performPre-optimized universal master mixesUniversal conditions for amplificationMultiple chemistries availableChoice of instrumentation*

  • Whats the impact on patient management and outcomes?

  • Case #14 y.o. boy presents with 2-day history of fever and headacheDay of presentation began to complain of neck painTemp = 102.7oFMild photophobiaNo rashesIntact neurologic exam*

  • Case #1Complete Blood Count- 9,300 cells/mm3- 45% PMN, 40% lymph, 15 monoCerebrospinal Fluid (CSF)- WBC = 75 cells/mm3- 72% PMN, 8% lymph, 20% mono- protein = 22 mg/dl- glucose = 60 mg/dl*

  • Case #1CSF gram stainmod WBC, no organismsI.V. ceftriaxone startedBlood, CSF, urine bacterialcultures obtainedEnterovirus RT-PCR on CSF ordered*

  • Case #1ANSWER

    Blood, CSF, urine bacterial cultures = negEnterovirus RT-PCR = POSITIVEDIAGNOSIS: Viral Meningitis*

  • 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*

  • 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*

  • EnterovirusesComparison of RT-PCR vs. Viral Culture59 inpatient CSF samples tested

    Sensitivity of CSF viral culture = 60%Culture time to detection = 3 5 daysRT-PCR time to detection = 3 4 hours

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    ResultRT-PCRCulturePos3722Neg2237

  • EnterovirusesRapid diagnosis of enteroviral meningitis by real time PCR impacts clinical management:Earlier hospital dischargeFewer additional diagnostic testsDecreased antibiotic usageDecreased overall health care costs*

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  • Hospital-Acquired Infections (HAIs)On an annual basis account for:~2 million infections~100,000 deaths$4-6 billion in health care costs

    5060% of the HAIs occurring in the USA each year are caused by antibiotic-resistant bacteria

    High rate of antibiotic resistance increases morbidity, mortality & costs associated with HAIsJones. Chest 2001;119:397S404SWeinstein. Emerg Infect Dis 1998;4:416420

  • 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 )Vancomycin-Resistant Enterococci (VRE) *

  • 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*

  • Vancomycin-Resistant EnterococciThe 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.

  • Vancomycin-Resistant EnterococciWhat 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.

  • Vancomycin-Resistant EnterococciLab-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*

  • VRE by Real Time PCRGreater sensitivity & More rapid resultsRapid Detection Infection Control MeasuresReduce Duration of Contact IsolationExcess costs associated with nosocomial infections justify screening and preventive infection control measures*

  • Cost-Effectiveness of VRE SurveillanceAttributable cost of surveillance vs. cost of nosocomial infectionsMuto, et al. Infect Control Hosp Epidemiol 23:429-435, 2002.2-year periodHosp #1No surveillanceHosp #2Surveillance

  • Cost-Effectiveness of VRE Surveillance by Real Time PCRUniversity 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*

  • Cost-Effectiveness of VRE Surveillance by Real Time PCRRapid determination of VRE colonization status prevented 2,348 isolation days/year when compared to cultureAnnual savings = $87,600

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  • Bordetella pertussis*

  • 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*

  • Bordetella pertussis*

  • Bordetella pertussis*

  • DiagnosisSpecimensNP swab or aspirateThroat & anterior nares swabsLower rates of recoveryCiliated respiratory epithelium not found in pharynx*

  • DiagnosisFind highest concentration of organism during catarrhal stage and beginning of paroxysmal stageConcentration of organism negatively correlates with increasing ageconc. in infantsconc. adolescents/adults*

  • DiagnosisCulture: still gold standardSens 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

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  • Real-Time PCRVery sensitive (~1 cfu/rxn)Dont need viable organismGood for mild, atypical cases, older patientsResults within hoursNot standardized between labsSome labs multiplex with B. parapertussis*

  • Hospital-Acquired Pertussis Among Newborns*

  • Cook Childrens6 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 57:600-603, 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 AugNursery Worker: ** 07/10/2004 7/17/2004Infant # 1 * PICUInfant # 2 * PICUInfant # 3 * PICUTimeline of Infants with Pertussis from a General Hospital Newborn Nursery Infant # 4 * PICUInfant # 5 *

    Infant # 7 * PICUInfant # 8 * 8/7Infant # 9 * 8/28 Out ptInfant # 10 *