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The Clinical Impact of Real-Time Molecular Infectious Disease Diagnostics Jim Dunn, Ph.D., D(ABMM) Cook Childrens Medical Center Ft. Worth, TX.

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Presentation on theme: "The Clinical Impact of Real-Time Molecular Infectious Disease Diagnostics Jim Dunn, Ph.D., D(ABMM) Cook Childrens Medical Center Ft. Worth, TX."— Presentation transcript:

1 The Clinical Impact of Real-Time Molecular Infectious Disease Diagnostics Jim Dunn, Ph.D., D(ABMM) Cook Childrens Medical Center Ft. Worth, TX

2 Molecular Microbiology Fastest growing area in clinical Fastest growing area in clinical laboratory medicine Integral and necessary component of many diagnostic laboratories Integral and necessary component of many diagnostic laboratories Traditional methods being rapidly displaced by molecular testing Traditional methods being rapidly displaced by molecular testing 2

3 Clinical Value Qualitative (pos/neg) nucleic acid tests are especially valuable for the detection of infectious agents that are: Qualitative (pos/neg) nucleic acid tests are especially valuable for the detection of infectious agents that are: Unculturable Unculturable Present in extremely low quantities Present in extremely low quantities Fastidious or slow-growing Fastidious or slow-growing Dangerous to amplify in culture Dangerous to amplify in culture 3

4 Clinical Value Quantitative (viral load) methods are important for monitoring certain chronic infections. These tests allow us to: Quantitative (viral load) methods are important for monitoring certain chronic infections. These tests allow us to: monitor therapy monitor therapy detect the development of drug resistance detect the development of drug resistance predict disease progression predict disease progression 4

5 Real-Time PCR Introduced in mid-1990s Introduced in mid-1990s Rapidly evolving field with numerous technological advances Rapidly evolving field with numerous technological advances Continuous fluorescence monitoring of nucleic acid amplification within a closed system. Continuous fluorescence monitoring of nucleic acid amplification within a closed system. One tube amplification and detection One tube amplification and detection 5

6 Fluorescence Monitoring Exponential: Quantitative real-time read Plateau: Qualitative end-point read 6

7 Real-Time PCR Rapid assay development Rapid assay development Simplified primer and probe design Simplified primer and probe design Simple and versatile to perform Simple and versatile to perform Pre-optimized universal master mixes Pre-optimized universal master mixes Universal conditions for amplification Universal conditions for amplification Multiple chemistries available Multiple chemistries available Choice of instrumentation Choice of instrumentation 7

8 Whats the impact on patient management and outcomes?

9 Case #1 4 y.o. boy presents with 2-day history of fever and headache 4 y.o. boy presents with 2-day history of fever and headache Day of presentation began to complain of neck pain Day of presentation began to complain of neck pain Temp = o F Temp = o F Mild photophobia Mild photophobia No rashes No rashes Intact neurologic exam Intact neurologic exam 9

10 Case #1 Complete Blood Count Complete Blood Count - 9,300 cells/mm % PMN, 40% lymph, 15 mono Cerebrospinal Fluid (CSF) Cerebrospinal Fluid (CSF) - WBC = 75 cells/mm % PMN, 8% lymph, 20% mono - protein = 22 mg/dl - glucose = 60 mg/dl 10

11 Case #1 CSF gram stain CSF gram stain mod WBC, no organisms I.V. ceftriaxone started I.V. ceftriaxone started Blood, CSF, urine bacterial Blood, CSF, urine bacterial cultures obtained Enterovirus RT-PCR on CSF ordered Enterovirus RT-PCR on CSF ordered 11

12 Case #1 ANSWER ANSWER Blood, CSF, urine bacterial cultures = neg Enterovirus RT-PCR = POSITIVE DIAGNOSIS: Viral Meningitis DIAGNOSIS: Viral Meningitis 12

13 Aseptic Meningitis Clinical and lab evidence of meningeal inflammation not due to bacteria Clinical and lab evidence of meningeal inflammation not due to bacteria 75,000 cases/year in US 75,000 cases/year in US 80 to 90% due to Enteroviruses 80 to 90% due to Enteroviruses Occur mainly in summer and fall Occur mainly in summer and fall Difficult to distinguish from bacterial meningitis based on clinical features alone Difficult to distinguish from bacterial meningitis based on clinical features alone Enteroviral meningitis has good prognosis Enteroviral meningitis has good prognosis 13

14 Enteroviruses aseptic meningitis, myocarditis, flaccid paralysis, neonatal sepsis-like disease, encephalitis, febrile rash disease aseptic meningitis, myocarditis, flaccid paralysis, neonatal sepsis-like disease, encephalitis, febrile rash disease now probably >100 serotypes based on capsid sequence analysis now probably >100 serotypes based on capsid sequence analysis molecular diagnosis has replaced traditional cell culture molecular diagnosis has replaced traditional cell culture 14

15 Enteroviruses Comparison of RT-PCR vs. Viral Culture Comparison 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 days Culture time to detection = 3 – 5 days RT-PCR time to detection = 3 – 4 hours RT-PCR time to detection = 3 – 4 hours ResultRT-PCRCulture Pos3722 Neg

16 Enteroviruses Rapid diagnosis of enteroviral meningitis by real time PCR impacts clinical management: Rapid diagnosis of enteroviral meningitis by real time PCR impacts clinical management: Earlier hospital discharge Earlier hospital discharge Fewer additional diagnostic tests Fewer additional diagnostic tests Decreased antibiotic usage Decreased antibiotic usage Decreased overall health care costs Decreased overall health care costs 16

17 17

18 Hospital-Acquired Infections (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 year are caused by antibiotic-resistant bacteria 50–60% 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 HAIs 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

19 Since 1989, a rapid increase in the incidence of infection and colonization with VRE has been reported by U.S. hospitals 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: This 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 infections 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 ) Possibility that vancomycin-resistance genes present in VRE can be transferred to other gram-positive bacteria (e.g. Staphylococcus aureus ) Vancomycin-Resistant Enterococci (VRE) 19

20 Vancomycin-Resistant Enterococci (VRE) E. faecium and E. faecalis that have acquired genes vanA and/or vanB 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 Most important reservoir for VRE is the colonized gastrointestinal tracts of patients Transmission can occur: Transmission can occur: Contaminated hands of healthcare workers Contaminated hands of healthcare workers Contamination of environment Contamination of environment 20

21 Vancomycin-Resistant Enterococci The Problem? Major nosocomial pathogen Major nosocomial pathogen Up to 6.3% of nosocomial enterococcal bloodstream infections in pediatric hospitals Up to 6.3% of nosocomial enterococcal bloodstream infections in pediatric hospitals 28.5% of nosocomial enterococcal infections in ICU patients (NNIS-2003) 28.5% of nosocomial enterococcal infections in ICU patients (NNIS-2003) Wisplinghoff, et al. Pediatr Infect Dis J 22:686, NNIS. Am J Infect Control 32:470, 2004.

22 Vancomycin-Resistant Enterococci 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) VRE culture often requires 72 hrs. VRE culture often requires 72 hrs. High Rate of False Negatives with Culture High Rate of False Negatives with Culture Muto, et al. Infect Control Hosp Epi 24:362, CDC. MMWR 44:1, 1995.

23 Vancomycin-Resistant Enterococci Lab-Developed Taqman Real Time Multiplex vanA/vanB PCR Assay Lab-Developed Taqman Real Time Multiplex vanA/vanB PCR Assay Sens = 100%, Spec = 98% Sens = 100%, Spec = 98% PPV = 91%, NPV = 100% PPV = 91%, NPV = 100% Screening & Surveillance in Admitted Oncology and Bone Marrow Transplant Screening & Surveillance in Admitted Oncology and Bone Marrow Transplant Pre-emptive isolation until VRE result known Pre-emptive isolation until VRE result known 23

24 VRE by Real Time PCR Greater sensitivity & More rapid results Greater sensitivity & More rapid results Rapid Detection Infection Control Measures Rapid Detection Infection Control Measures Reduce Duration of Contact Isolation Reduce Duration of Contact Isolation Excess costs associated with nosocomial infections justify screening and preventive infection control measures Excess costs associated with nosocomial infections justify screening and preventive infection control measures 24

25 Cost-Effectiveness of VRE Surveillance Attributable cost of surveillance vs. cost of nosocomial infections Attributable cost of surveillance vs. cost of nosocomial infections Muto, et al. Infect Control Hosp Epidemiol 23: , year period 2-year period Hosp #1 Hosp #1 No surveillance No surveillance Hosp #2 Hosp #2 Surveillance Surveillance

26 Cost-Effectiveness of VRE Surveillance by Real Time PCR University of Iowa Hospital University of Iowa Hospital Real Time PCR for VRE Real Time PCR for VRE Average TAT = 1.3 days Average TAT = 1.3 days (3.4 days for culture) length of stay by ~2 days for patients discharged to long-term care facilities length of stay by ~2 days for patients discharged to long-term care facilities $205,000 annual savings $205,000 annual savings 26

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 culture Rapid determination of VRE colonization status prevented 2,348 isolation days/year when compared to culture Annual savings = $87,600 Annual savings = $87,600 27

28 Bordetella pertussis 28

29 Bordetella pertussis Endemic disease, occurs year-round, epidemic cycles every 3 or 4 years Endemic disease, occurs year-round, epidemic cycles every 3 or 4 years Transmitted by large droplets Transmitted by large droplets Attack rates among close contacts as high as 80 to 100% Attack rates among close contacts as high as 80 to 100% Waning immunity leads to susceptible adolescents and adults Waning immunity leads to susceptible adolescents and adults Family members often source for infected infants Family members often source for infected infants 29

30 Bordetella pertussis 30

31 Bordetella pertussis 31

32 Diagnosis Specimens NP swab or aspirate NP swab or aspirate Throat & anterior nares swabs Throat & anterior nares swabs Lower rates of recovery Lower rates of recovery Ciliated respiratory epithelium not found in pharynx Ciliated respiratory epithelium not found in pharynx 32

33 Diagnosis Find highest concentration of organism during catarrhal stage and beginning of paroxysmal stage Find highest concentration of organism during catarrhal stage and beginning of paroxysmal stage Concentration of organism negatively correlates with increasing age Concentration of organism negatively correlates with increasing age conc. in infants conc. adolescents/adults 33

34 Diagnosis Culture: still gold standard Culture: still gold standard Sens actually 15-60% compared to PCR Sens actually 15-60% compared to PCR Special media/transport, long incubation Special media/transport, long incubation DFA: low sens and variable spec DFA: low sens and variable spec Always back-up with cx or PCR Always back-up with cx or PCR Serology: not part of case definition Serology: not part of case definition Not standardized Not standardized Epidemiology/vaccine efficacy Epidemiology/vaccine efficacy 34

35 Real-Time PCR Very sensitive (~1 cfu/rxn) Very sensitive (~1 cfu/rxn) Dont need viable organism Dont need viable organism Good for mild, atypical cases, older patients Good for mild, atypical cases, older patients Results within hours Results within hours Not standardized between labs Not standardized between labs Some labs multiplex with B. parapertussis Some labs multiplex with B. parapertussis 35

36 Hospital-Acquired Pertussis Among Newborns 36

37 Cook Childrens 6 infants admitted with pertussis w/in a few days of each other 6 infants admitted with pertussis w/in a few days of each other Confirmed by real-time PCR w/in 24 hrs admit Confirmed by real-time PCR w/in 24 hrs admit 4 infants in PICU 4 infants in PICU Investigation reveals all born at same local hospital Investigation reveals all born at same local hospital One HCW in newborn nursery with cough, post-tussive emesis, dyspnea One HCW in newborn nursery with cough, post-tussive emesis, dyspnea PCR pos for B. pertussis PCR pos for B. pertussis MMWR 57: , 2008.

38 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 * § ¶ §§PICU Infant # 5 * § ¶ §§ Infant # 7 * § ¶ §§PICU Infant # 8 * § ¶ §§ 8/7 Infant # 9 * § ¶ 8/28 Out pt Infant # 10 * § unk ¶ 1 0/4 Infant # 11 * ¶ § unk Out pt * Date born Exposure in nursery § Symptoms started ¶ Admission/Diagnosis Date **Outbreak noted HCW PCR +/Furlough §§ Discharge Date Prodrome? Infant #6 * § ¶ §§

39 Summary HCW furloughed/treated HCW furloughed/treated Families of 110 infants born at local hospital evaluated for cough illness Families of 110 infants born at local hospital evaluated for cough illness 18 with cough: PCR neg 18 with cough: PCR neg 2 additional PCR pos 2 additional PCR pos Total of 11 infants with confirmed pertussis Total of 11 infants with confirmed pertussis Attack rate ~10% Attack rate ~10% MMWR 57: , 2008.

40 Cook Childrens Molecular Lab 40

41 Whats So Cool About Real-Time PCR? Decreased Turnaround Times/High Throughput Decreased Turnaround Times/High Throughput Simultaneous amplification, detection, & data analysis Simultaneous amplification, detection, & data analysis Closed system Closed system No additions made after specimen is added No additions made after specimen is added Contamination control – No false positives Contamination control – No false positives More Standardized More Standardized Pre-optimized master mixes, reproducible Pre-optimized master mixes, reproducible Less expensive that traditional PCR Less expensive that traditional PCR Increased Sensitivity Increased Sensitivity 41

42 Thanks


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