Download C. albicans

Controls for
Blood Septicemia
Nucleic Acid Tests
Mark Manak
BBI Diagnostics, Inc.
A Division of SeraCare Life Sciences, Inc.
SoGAT XIX Meeting
Berne, Switzerland
June 15, 2006
Sepsis: Infection of Blood Stream
• Bacterial infection (Bacteremia); Fungal Infection
(Candidemia)
• Second leading cause of death among patients in non-coronary
intensive care units (ICU)
• Tenth leading cause of death overall in US
• Usually diagnosed by culture using specialized apparatus to
detect and identify any viable microorganisms.
• Limitations of Blood Culture
– Relatively insensitive and time consuming: 24 to 48 hrs to results
– >30% of septic patients are false negative by blood culture
Nucleic Acid Tests for Sepsis
Roche SeptiFastTM Test
• Highly specific – able to detect <100 organisms per ml blood
• Detects microorganisms that cause >90% of all blood stream
infections
• Real Time PCR on Light Cycler Platform
• 3 parallel reactions: gram positive, gram negative, and fungus
• Melting curve analysis of end product ensures proper sequence
• Rapid, automated results in less than 5 hours
ACCURUN 500
Design Specifications
• Manufactured from P. aeruginosa, S. aureus, and C. albicans
• Inactivated to reduce viability >6 logs.
• Diluted in negative human plasma
• Targeted at low concentration (about 300 CFU/ml )
Intended Use
• Estimate laboratory testing precision
• Detect errors in laboratory testing procedures.
• Controls for the entire workflow
Linearity of C. albicans in SeptiFast Test
cfu vs cp for c. albicans
28
cp
23
18
13
8
1
10
100
cfu
1,000
10,000
C. albicans
Quantification -Channel 640
Tm Calling – Channel 640
100 cfu/reaction
100 cfu/reaction
IC
NHP
NHP
P. aeruginosa
Tm Calling – Channel 670
Quantification -Channel 670
100 cfu/reaction
NHP
Excellent Reproducibility: Multiple Lots, Test Sites
S. aureus
P. aeriginosa
Test Site A Test Site B
cp
C. albicans
Test Site A Test Site B
Test Site A Test Site B
22
22
22
20
20
20
18
18
18
16
16
16
14
14
14
12
12
12
10
10
10
8
8
8
6
6
6
4
4
4
2
2
2
0
0
Lot Number
1
2
Mean:
SD:
%CV:
3
1
19.0
0.8
5%
2
3
Lot Number
1
2
3
1
2
Lot Number
20.1
0.6
4%
0
3
Lot Number
1
2
3
16.8
0.6
2%
1
2
3
Stability Studies
25
20
cp Value
Accelerated
Stability 37o C
S.aureus
P.aeruginosa
C.albicans
15
10
5
0
0
2
4
6
8
10
12
14
Days
25.0
20.0
Cp Value
Freeze-Thaw
Stability
15.0
S.aureus
P.aeruginosa
C.albicans
10.0
5.0
1
2
3
4
5
Number of Freeze Thaws
6
7
ACCURUN 500 Summary
Stable, low reactive independent quality control material
• Made from natural bacteria in plasma diluent to mimic clinical sample
• Controls for all test steps: extraction, amplification and detection
Well suited for the quality control of Nucleic Acid assays for
microorganisms in human blood.
• Gram positive, Gram negative and fungal controls showed amplification peaks
only in the respective channels for all lots
• Negative controls showed Internal Control peaks only
• Excellent Reproducibility between Control Lots, Test Runs, and Test Sites
Use of ACCURUN 500 will allow laboratories to detect
immediate testing errors and monitor the long term
performance of such assays.