1. Clinical context and utility of identifying respiratory virus coinfection using electrochemical detection
Derrick Chen MD, Sherilynn Vogel, Colleen Starkey,
Gary Procop MD, Belinda Yen-Lieberman PhD
Look at audience
We were interested in studying respiratory virus coinfections and the impact of multiplex testing on antibiotic stewardship

2. Respiratory Viral Infections
Respiratory tract infections among most common infectious diseases worldwide
Self-limiting to life-threatening illnesses
Most viral causes have no specific therapy
Respiratory tract infections among most common infectious diseases worldwide
Upper resp tract infxns frequent: children (6-9 infections/year), adults (2-4 infections/year) [Templeton 2007]
Lower RTI less frequent, but 1-13% patients hospitalized
Self-limiting to life-threatening illnesses
Particularly at extremes of age (younger and older patients), immunocompromised, cardiopulmonary diseases
Most have no specific therapy
Oseltamivir (Tamiflu, neuraminidase inh) – needs to be given <48 h of contact
?steroids for RSV (decr wheezing in bronchiolitis)
Palivizumab (Synagis) – RSV prophylaxis in high risk children (bronchopulmonary dysplasia, premature birth, congenital heart dz) 2
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3. Respiratory Viral Infections
Viral or bacterial?
Rapid detection guides clinical management
Infection control
Multiplex platforms
Viral or bacterial?
Clinical dx cannot reliably discriminate btw causative agents (viral vs bacterial) so need diagnostic tests [Templeton 2007]
Rapid detection guides clinical management
Limits unnecessary antibiotics, allows prescription of antivirals, reduces need for additional diagnostic studies and hospital procedures
Infection control
Isolation, droplet/contact precautions
Multiplex platforms [Popowitch et al. J Clin Microbiol 2013]
FilmArray RP (BioFire Diagnostics, Salt Lake City, UT)
Luminex xTAG RVPv1 (Luminex Molecular Diagnostics, Austin, TX)
Luminex xTAG RVP fast (Luminex Molecular Diagnostics, Austin, TX)
eSensor RVP (GenMark Dx, Carlsbad, CA) 3
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4. Materials and Methods Patient Selection Viral Test Procedure
Consecutive results from daily respiratory viral panel testing collected over 5-month period (Dec 2013-Apr 2014) were analyzed retrospectively
Medical records reviewed when positive for coinfection
Viral Test Procedure
Nucleic acid extraction using easyMAG (bioMerieux)
RT-PCR amplification on TC9700 thermal cycler (Applied Biosystems)
Exonuclease digestion results in single-stranded DNA
Add signal buffer containing ferrocene-labeled signal probes 4
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5. Electrochemical Detection
eSensor RVP on the eSensor XT-8 System
GenMark
Detection of 14 respiratory virus types and subtypes
1 target DNA is mixed with the signal probe solution, if the applicable target DNA is present, hybridization to the signal probes occurs immediately
2 the solution is pumped through the cartridge’s microfluidic chamber and the target DNA/signal probe complex completes the reaction with the pre-assembled capture probe
3 voltage is swept across each electrode and target DNA is analyzed by electrochemical detection 5
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6. Test results, and presence and rate of coinfection (N=2,073)
Viral targets
H1 (FA)
H3 (FA)
H1N1 (FA) FB
RSVA
RSVB
PIV1
PIV2
PIV3
HMPV
RHINO
ADENO B/E
ADENO C
Total
Percent of all performed tests (%)
Positive results (n) 1 11
113 14
156 76 21 8 88
240 38
777 37
Coinfection (n) 3 9 19 12 4 7 45 13 5
Rate of coinfection in positive tests (%) 27 16 34 15 6
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7. Results Patterns of Coinfection 31 (57%) males, 23 (43%) females
Viral targets
Cases
RHINO
RSVA 11
ADENO C 10
RSVB 9
HMPV 5
H1N1 (FA) 4
H3 (FA) 3
PIV1 2 1
PIV3
H1N1
Total 56
Patterns of Coinfection
31 (57%) males, 23 (43%) females
Range: 0 to 79 years-old
Mean: 12 yo, Median: 1 yo
10 (19%) immunocompromised
56 total tests that had coinfections
The 3 most common coinfections shown at the top of the table: rhino+RSVA, rhino+AdenoC, and rhino+RSVB 7
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8. Results Symptoms at presentation Possible exposures Cough (75%, n=42)
Fever (61%, n=34)
Rhinorrhea/congestion (52%, n=29)
Wheezing (23%, n=13)
Possible exposures
Household sick contacts (14%, n=8)
Daycare (11%, n=6) 8
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9. Results Empiric treatment Treatment after viral testing
Supportive (45%, n=25)
Antibiotics without oseltamivir (41%, n=23)
Antibiotics plus oseltamivir (9%, n=5)
Oseltamivir alone (5%, n=3)
Treatment after viral testing
Unchanged (80%, n=45)
Changed (20%, n=11)
Discontinuation of antibiotics (9%, n=5)
Addition of oseltamivir (5%, n=3)
Discontinuation of oseltamivir (4%, n=2)
Addition of ribavirin (2%, n=1)
Almost all patients improved with treatment, however
1 patient expired despite treatment (the one where Ribavirin was added)
Patient 24: 69F w rheumatoid arthritis on immunosuppressive therapy; presented with respiratory failure requiring intubation; (+)RSVB/(+)Rhino;unknown exposure; empiric: trimethoprim-sulfamethoxazole; definitive: trimethoprim-sulfamethoxazole, piperacillin-tazobactam, ribavirin; expired 9
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10. Conclusions
Detection of coinfection by multiplex respiratory viral panel test resulted in a change in treatment in 20% (n=11) of cases
Treatment was not modified in the majority of cases, possibly due to the desire to wait for bacterial culture results and to complete the full course of antibiotics
…including discontinuation of antibiotics and oseltamivir when not indicated, and addition of antivirals when appropriate 10
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12. Electrochemical Detection
Load PCR reactions into thermal cycler
eSensor RVP on the eSensor XT-8 System
RT-PCR
OPTIONAL HOLD POINT
Add 5uL of exonuclease to each sample
OPTIONAL HOLD POINT
Dispense sample and hybridization solution into cartridges
Load cartridges onto eSensor® XT-8 system
Generate report

13. Multiplex Platforms BioFire Diagnostics, Salt Lake City, UT
FilmArray RP
Luminex Molecular Diagnostics, Austin, TX
Luminex xTAG RVPv1
Luminex xTAG RVP fast
GenMark Dx, Carlsbad, CA
eSensor RVP
Popowitch et al. J Clin Microbiol 2013