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

2. 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

3. 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

4. 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

5. 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

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

7. 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

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 pain
Temp = 102.7oF
Mild photophobia
No rashes
Intact neurologic exam

10. 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

11. 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

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

13. 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

14. 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

15. 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
Result
RT-PCR
Culture
Pos 37 22
Neg

16. 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

18. 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

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. 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 )

20. 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

21. 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.

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 Culture
Muto, 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 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

24. 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

25. Cost-Effectiveness of VRE Surveillance
Attributable cost of surveillance vs. cost of nosocomial infections
2-year period
Hosp #1
No surveillance
Hosp #2
Surveillance
Muto, et al. Infect Control Hosp Epidemiol 23: , 2002.

26. Cost-Effectiveness of VRE Surveillance by Real Time PCR
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

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
Annual savings = $87,600

28. Bordetella pertussis

29. Bordetella pertussis
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

30. Bordetella pertussis

31. Bordetella pertussis

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

33. Diagnosis
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

34. Diagnosis 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

35. Real-Time PCR Very sensitive (~1 cfu/rxn) Results within hours
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

36. Hospital-Acquired Pertussis Among Newborns

37. Cook Children’s
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: , 2008.

38. Timeline of Infants with Pertussis from a General Hospital Newborn Nursery
Worker: § ** 07/10/ ††7/17/2004
Prodrome?
Infant # *† § ¶ §§ PICU
Infant # *† § ¶ §§ PICU
Infant # *† § ¶ §§ PICU
Infant # *† § ¶ §§ PICU
Infant # *† § ¶ §§
Infant # * † § ¶ §§
Infant # *† § ¶ §§ PICU
Infant # *† § ¶ §§ 8/7
Infant # *† § ¶ 8/28 Out pt
Infant # *† § unk ¶ 10/4
Infant # *† ¶ § unk Out pt
June 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 illness
18 with cough: PCR neg
2 additional PCR pos
Total of 11 infants with confirmed pertussis
Attack rate ~10%
MMWR 57: , 2008.

40. Cook Children’s Molecular Lab

41. What’s So Cool About Real-Time PCR?
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

42. Thanks