Rapid Response Ebola Test
(RRET)
Technical Report
Overview: The Ebola Pandemic So Far…
Scary Stats:
• Africa
• Europe
• America
Country
Total Cases
Lab-confirmed
Cases
Total Deaths
Guinea
2283
2035
1412
Liberia
7719
2830
3177
Sierra Leone
7798
6317
1742
Mali
8
7
6
Senegal
1
1
0
Nigeria
20
19
8
Spain
1
1
0
United States
4
4
1
Total
17834
11214
6346
CDC – as at Dec. 9, 2014
2
Overview: Need for RRET
• Rapid diagnostics
 30mins T/A time
• Surveillance
 Ports of entry and
other health centres
3
RRET Features
•
Rapid genetic diagnostic test
•
Based on the Loop-mediated
isothermal amplification (LAMP)
technique
•
Single isothermal temperature
(630C)
– Water bath or heating block
sufficient
– No need for a thermal cycler
or PCR equipment
•
Visual detection
– Fluorescence at 240nm260nm
4
Comparison with gold standard - PCR
2mins
3mins – 1hr
Sample Collection
Urine
Blood
Sputum
Swab
RNA Extraction Phase
2mins
3mins – 1hr
(Kits from Quiagen, Illumina, etc.)
cDNA Synthesis
and Amplification
Minimum of 2hrs
30mins
RT-LAMP
RT-PCR
(RRET)
Gel
Electrophoresis
5
15 – 30mins
Gel
Electrophoresis
30mins
(Optional)
Fluorescence
Detection
1min
RRET Validation Data:
From current outbreak samples
S/N
Sample
ID
RTPCR
RRET
1
061195
NEG
NEG
2
061228
NEG
NEG
3
061215
NEG
NEG
4
5
6
7
8
061305
061306
061231
061307
061338
NEG
NEG
NEG
NEG
NEG
S/N
Sample
ID
RTPCR
RRET
9
061337
NEG
NEG
10
018
POS
POS
11
MAINL
AND
10
POS
POS
12
061232
POS
POS
13
061214
POS
NEG
14
019
POS
POS
15
PS
POS
POS
NEG
CLARIFICATIONS
•
RRET Primers
target only
– ZEBOV Strain
•
PCR Wobble
Primers target
– Filo
– Marburg
– ZEBOV
NEG
NEG
NEG
NEG
6
•
Positive Control
RefSeq
– PCR (GP Seq)
– RRET (Trailer Seq)
RRET Sensitivity/Specificity Values:
With respect to gold standard
SENSITIVITY
80%
SPECIFICITY
100%
POSITIVE PREDICTIVE
VALUE
100%
NEGATIVE PREDICTIVE
VALUE
90%
Sensitivity defined as probability that a test will be positive when disease is present.
Specificity defined as probability that a test will be negative when disease is absent.
Positive Predictive Value defined as probability that the disease is present when the test is positive.
Negative Predictive Value defined as probability that the disease is not present when test is negative.
7
Marketing and Deployment
•
Ports of entry
– Surveillance
•
Secondary & tertiary health centres
– General hospitals
– Teaching hospitals
– Specialist centres
•
Other diagnostic labs
– Universities/other educational institutions for student screening
– Private/public sector institutions for staff screening
•
Mobile diagnostics
– Sports screening (ANC, 2015 @E.Guinea)
– Supplies for volunteer clinicians at hot zones
RRET Accessories
needed @staging
area or mobile lab:
1.
Water bath or a
heating block.
2.
Adjustable UV lamp
or phototransilluminator
(wavelengths 240nm
– 370nm).
3.
Protective glass
shield or eye
googles.
4.
Hand gloves.
5.
Bio-harzard bin.