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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

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The clinical impact of real time molecular infectious disease diagnostics l.jpg

The Clinical Impact of Real-Time Molecular Infectious Disease Diagnostics

Jim Dunn, Ph.D., D(ABMM)

Cook Children’s Medical Center

Ft. Worth, TX


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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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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

  • 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

    • 59 inpatient CSF samples tested

    • 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)

  • On an annual basis account for:

    • ~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

  • 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


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Vancomycin-Resistant Enterococci (VRE)

  • 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:

    • 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.


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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

Muto, et al. Infect Control Hosp Epi 24:362, 2003.

CDC. MMWR 44:1, 1995.


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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

    • 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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Attributable cost of surveillance vs. cost of nosocomial infections

2-year period

Hosp #1

No surveillance

Hosp #2

Surveillance

Cost-Effectiveness of VRE Surveillance

Muto, et al. Infect Control Hosp Epidemiol 23:429-435, 2002.


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Cost-Effectiveness of VRE Surveillance by Real Time PCR infections

  • University of Iowa Hospital

  • Real Time PCR for VRE

    • Average 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


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Cost-Effectiveness of VRE Surveillance by Real Time PCR infections

  • Rapid determination of VRE colonization status prevented 2,348 isolation days/year when compared to culture

  • Annual savings = $87,600



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Bordetella pertussis infections

  • 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

    • Family members often source for infected infants




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Diagnosis infections

  • Specimens

    • NP swab or aspirate

    • Throat & anterior nares swabs

      • Lower rates of recovery

      • Ciliated respiratory epithelium not found in pharynx


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Diagnosis infections

  • 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 infections

  • Culture: still “gold standard”

    • Sens actually 15-60% compared to PCR

    • Special media/transport, long incubation

  • DFA: low sens and variable spec

    • Always back-up with cx or PCR

  • Serology: not part of case definition

    • Not standardized

    • Epidemiology/vaccine efficacy


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Real-Time PCR infections

  • Very sensitive (~1 cfu/rxn)

    • 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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Cook Children’s infections

  • 6 infants admitted with pertussis w/in a few days of each other

    • 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

    • PCR pos for B. pertussis

MMWR 57:600-603, 2008.


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Timeline of Infants with Pertussis from a General Hospital Newborn Nursery

Nursery

Worker: §** 07/10/2004 ††7/17/2004

Prodrome?

Infant # 1 *† § ¶ §§ PICU

Infant # 2 *† § ¶ §§ PICU

Infant # 3 *† § ¶ §§ PICU

Infant # 4 *† § ¶ §§ PICU

Infant # 5 *† § ¶ §§

Infant #6 * † § ¶ §§

Infant # 7 *† § ¶ §§ PICU

Infant # 8 *†§ ¶ §§ 8/7

Infant # 9 *† § ¶ 8/28Out pt

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

Infant # 10 *† §unk¶ 10/4

Infant # 11 *† ¶ §unk Out pt

* Date born

† Exposure in nursery

§Symptoms started

¶ Admission/Diagnosis Date

**Outbreak noted

†† HCW PCR +/Furlough

§§Discharge Date


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Summary Newborn Nursery

  • HCW furloughed/treated

  • Families of 110 infants born at local hospital evaluated for cough illness

    • 18 with cough: PCR neg

    • 2 additional PCR pos

  • Total of 11 infants with confirmed pertussis

    • Attack rate ~10%

MMWR 57:600-603, 2008.



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What’s So Cool About Real-Time PCR? Newborn Nursery

  • Decreased Turnaround Times/High Throughput

    • Simultaneous amplification, detection, & data analysis

  • Closed system

    • No additions made after specimen is added

    • Contamination control – No false positives

  • More Standardized

    • Pre-optimized master mixes, reproducible

  • Less expensive that traditional PCR

  • Increased Sensitivity


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Thanks Newborn Nursery


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