Jornadas de arbovirosis: dengue, chikungunya y Zika. Preparación y respuesta de los países del Cono Sur. Buenos Aires, Argentina, 4-5 noviembre 2015.

1. Update on Commercially Available Options of
Chikungunya Virus Diagnosis
Expert Consultation in Virology and Laboratory Diagnostics in the
Southern Cone
BW Johnson,1
CH Goodman,1
K Holloway,2
PM de Salazar,3
AM Valadere,3
M Drebot2
1
Diagnostic & Reference Laboratory, Arboviral Diseases Branch, Division of Vector-
Borne Diseases, Centers for Disease Control and Prevention (CDC), Fort Collins,
Colorado USA
2
Viral Zoonosis, Public Health Agency of Canada, Canadian Science Centre for Human
and Animal Health, National Microbiology Laboratory (NML), Winnipeg, Manitoba,
Canada
3
Caribbean Public Health Agency (CARPHA), Port-of-Spain, Trinidad and Tobago

2. Introduction
Since the introduction of Chikungunya virus (CHIKV) to the
Caribbean in 2013, public health laboratories throughout the
Americas have initiated diagnostic testing
Large CHIKV outbreaks and clinical similarity to other
arbovirus infections result in large number of samples
submitted for testing
Testing algorithms have been developed based on the high
titered viremia, immune response, and timing of specimen
collection
Expansion and endemicity of CHIKV likely and laboratories
will need to build and maintain high-volume diagnostic
testing capacity
Laboratories need commercial CHIKV diagnostic assays
that are validated and reliable

3. Specimen type and timing of collection determines test
Diagnostic test results for 35 travelers infected with Chikungunya virus,
2006*
*From: Lanciotti, et al. Chikungunya virus in US travelers returning from India, 2006. EID 2007

4. Specimen type and timing of collection determines test
Diagnostic test results for 35 travelers infected with Chikungunya virus, 2006*
*From: Lanciotti, et al. Chikungunya virus in US travelers returning
from India, 2006. EID 2007;13:764-767.

5. CDC Diagnostic Testing Algorithm for detection
of CHIKV infection
<Day 6 POI ≥Day 6 POI
qRT-PCR
POS
REPORT
NEG
IgM ELISA
POS NEG
REPORTREPORT
PRNT
*Meets Clinical Case Definition: Fever and arthralgia in a person returning from a
CHIK-endemic or epidemic region.
POI, post-onset of illness.
Serum* collected
From RS Lanciotti, unpublished

6. Results from subset of samples tested at
CDC from U.S. Virgin Islands (n=174)
Category Total # %
PCR NEG + SER NEG 46 26.4%
PCR POS + SER NEG 78 44.8%
PCR NEG + SER POS 31 17.8%
PCR POS + SER POS 5 2.9%
Equivocals 14 8%
From RS Lanciotti, unpublished

7. Days POI
Number of qRT-PCR Positive Samples
by Days Post-Onset of Illness
From RS Lanciotti, unpublished

8. CDC Diagnostic Testing Algorithm for detection
of CHIKV infection
<Day 6 POI ≥Day 6 POI
qRT-PCR
POS
REPORT
NEG
IgM ELISA
POS NEG
REPORTREPORT
PRNT
*Meets Clinical Case Definition: Fever and arthralgia in a person returning from a
CHIK-endemic or epidemic region.
POI, post-onset of illness.
Serum* collected
From RS Lanciotti, unpublished

9. CDC Arbovirus Diagnostic Laboratory:
Conclusions
 Approximately 50% of samples tested are acute and
for these qRT-PCR is the primary diagnostic test
 Few samples (2.9%) are qRT-PCR and IgM ELISA
positive
 Labs with only qRT-PCR OR MAC-ELISA capacity
should test samples at all days POI; negative results
interpreted as inconclusive

10. Samples tested at CDC in 2014
 1,452 Samples Received (46% <Day 6)
 660 Tested by qRT-PCR; 324 POS
 1120 Tested by IgM ELISA; 433 POS
 CHIKV infections reportable disease in USA starting
in 2015
 Some state public health labs have initiated CHIKV
testing
From RS Lanciotti, unpublished

11. Commercial sources for CHIKV
diagnostic testing

12. CHIKV qRT-PCR kits
• CHIKV RNA reference reagent (CHIKV-RR) US FDA – Anez et al. Vox
Sanguinis 2015
• Vircell (Spain): Lyophilized RNA (strain S27) rehydrated in 50 µl;
can be diluted 1:100 ≈ 5 ml – validated by B Russell, CDC, unpublished
• Zeptometrix (USA): NATtrol inactivated virus (strain India 2006)
1 ml pre-extraction - validated by B Russell, CDC, unpublished
 RealStar®
Chikungunya RT-PCR Kit (Altona Diagnostics GmbH
Hamburg, Germany) – M Panning et al. Performance of the RealStar Chikungunya virus
RT-PCR Kit. J Clin Microbiol 2009;47:3014–3016.
• genesig®
Chikungunya Non structural protein 2 standard (nsp2) RT-
PCR kit (Primerdesign Ltd, Southampton UK) - no validity information
CHIKV RNA PC

13. Two sets Chikungunya primers and probes (purchased from
commercial source)**
Lanciotti RS, et al. 2006. EID 2007;13:764-767
CHIKV 3855- 3957 (threshold of detection ≈1 RNA transcript)
Designed to Caribben isolates (Lanciotti unpublished)
CHIK 856 -962 (threshold of detection ≈1 RNA transcript)
RNA PC (strain India 2006)*
*Protocol with primer/probe sequences and RNA lysate PC available from CDC
upon request.
**All positive and equivocal samples are repeated with a second set of
primer/probes for confirmation. A positive result in any of the negative controls
invalidates the entire run. Failure of the positive control to generate a positive
result also invalidates the entire run.
CDC qRT-PCR protocol*

14. Commercial CHIKV IgM Detection Assays
Manufacturer Location Assay name and format
Reference
no.
No.
samples per
kit Price
Plate ELISA
IBL International Germany CHIK IgM micro-capture ELISA RE58841 91 $525
CTK Biotech USA/China RecombiLISA CHIK IgM Test E0315 91 unknown
Genway* Germany CHIKV IgM μ-capture ELISA 40-521-475066 91 $585
Abcam* Germany Anti-CHIKV IgM human ELISA kit ab177848 91 $475
SD Diagnostics Korea CHIKa IgM ELISA 16EK10 91 unknown
Euroimmun Germany Anti-CHIKV ELISA (IgM) EI293a-9601M 93 $341
Inbios USA CHIKjj Detect MAC-ELISA Research use only 92 NA
Rapid test
CTK Biotech USA On-site CHIK IgM Combo Rapid test R0066C 30 unknown
SD Diagnostics Korea SD BIOLINE Chikungunya IgM 46FK10 25 unknown
IFA
Euroimmun Germany Anti-CHIKV IIFT (IgM) Fl293a-1010 G/M 50 $250
.
*Novatec original equipment manufacturer (OEM).

15. Evaluations of CHIKV IgM detection assays:
Three Approaches
I: CDC – Evaluated 9 kits with a reference panel of well-
characterized sera
II: NML – Euroimmun ELISA evaluated with samples
submitted for diagnostic testing from travelers to the
Caribbean and tested with in-house assays
III: CARPHA – Euroimmun ELISA and IIFT, Inbios ELISA,
and Abcam ELISA evaluated with selected samples from
patients and controls from different CARPHA member
states, which had initially been tested at CDC

16. Manufacturer Assay name and format
No.
samples
per kit
Sample
volume
needed
Est. time
to test 20
samples
Storage
conditions
Microplate MAC-ELISA (n = 6)
Abcam Anti-CHIKV IgM human
ELISA
91 10 µl 4 h 2-8°C
CTK Biotech RecombiLISA CHIK IgM Test 91 10 µl 2 h 2-8°C
Euroimmun Anti-CHIKV ELISA (IgM) 93 2 µl 3.5 h 2-8°C
Genway CHIKV IgM μ-capture ELISA 91 10 µl 4 h 2-8°C
Inbios CHIKjj Detect MAC-ELISA
Research use only (RUO)
92 4 µl 3.5 h Most 2-8°C;
antigen
-20to-
80°C
SD Diagnostics CHIKa IgM ELISA 91 10 µl 2 h 2-8°C
Rapid test (n=2)
CTK Biotech On-site CHIK IgM Combo
Rapid test
30 30 µl 0.5 h 2-30°C
SD Diagnostics SD BIOLINE Chikungunya
IgM
25 50 µl 0.5 h 1-30°C
Indirect Immunofluorescence assay (n=1)
Euroimmun Anti-CHIKV IIFT (IgM) 50 ≈15 µl 3 h 2-8°C
Part I: CHKV IgM detection assays evaluated at CDC

17. •CHIKV Asian strain:
• 48 CDC CHKV IgM+ and PRNT+ sera from Yap (2013), the
Philippines, and Caribbean (2014) outbreaks
•CHIKV East/Central/South African (ECSA) strain:
• 4 CDC CHKV IgM+ and PRNT+ sera from Comoros (2005)
and travelers to India (2006)
•CHIKV IgM- sera
• 3 CDC Normal control reference serum
• 28 CDC CHKV IgM- sera from fever patients from
Thailand, Yap, and Caribbean
• 4 DENV IgM+ sera (CDC CHIK IgM-)
• CDC IgM Positive control serum for other alphaviruses
• CDC CHIK IgM- :Venezuelan equine encephalitis,
Eastern equine encephalitis
• CDC CHIK IgM+: O'nyong-nyong (ONN), Mayaro
(MAY)
CDC Sample panel for evaluation (n≈90)

18. CDC CHIKV IgM kit evaluation procedure
1. Each assay first tested with 10-20 sample subset consisting
of multiple positive controls (PCs), negative control (NC),
and selected CHKV IgM+ and IgM- samples
2. Kit PC, NC, and cutoff calibrators met validity criteria
3. One CDC PC used as in-house positive control (IHPC) for
each test run; normal serum used as in-house negative
control (IHNC)
4. If all CDC PCs positive in test, proceed to test complete
panel
5. If CDC PCs negative in test, retest. If PCs negative in 2nd
test,
test with alternate conditions suggested by manufacturer
(eg., change incubation time and temp, handwash vs
autowash) or contact manufacturer
6. Repeat testing with different lot # kits

19. Summary of performance of CHIKV IgM
detection assays evaluated at CDC
*CDC IHPC had negative result in test.
** CDC IHPC had positive result in test.
ND, not done as specificity and agreement could not be calculated.

20.  Euroimmun and Inbios MAC-ELISA kits show highest performance
• Two lots of Euroimmune and one lot of Inbios kits has been evaluated at CDC
• Cross-reactivity with ONNV and MAYV IgM similar to CDC test
• Inbios higher sensitivity compared to Euroimmun for low CHIKV IgM+ samples
•
 Abcam Anti-CHIKV IgM human ELISA kit lot-to-lot variability
• Novatec re-formatted test
• Kit PC (biotin-labeled IgM) replaced wih CHIKV IgM+ PC
• Enhanced QA/QC procedures implemented, including low positives
• Kit will need to be re-evaluated if further changes are made
 Genway kit CHIKV MAC-ELISA sensitivity low
• Same format as Abcam kit
• One lot tested had similar performance to “bad” Abcam lots
• CDC PCs negative in test kits
 SD and CTK CHK MAC-ELISAs sensitivity low
• CDC PCs negative in test kits
 SD and CTK RTs sensitivity low
• CDC PCs negative in lateral flow assays
 Euroimmun IIFA high performance
• Requires experienced technician to “read” slides
• Immunosorb sample dilution buffer with IgG depletion sold separately
Summary of CDC evaluations

21. Part II: In-House Validation of Euroimmun IgM
Chikungunya ELISA at NML

22. Diagnostic Algorithm, NML
22
Prior to 2014:
•HAI (in house produced SMB antigen)
•and/or commercial IFA (Euroimmun)
February 2014*:
•All samples tested by MAC-ELISA based on CDC developed methodology
(tissue culture derived antigen)
•All equivocal samples tested by real time RT-PCR
•All IgM+ confirmed by PRNT and/or real-time RT-PCR
April 2015:
•All samples tested by Euroimmun or in-house MAC-ELISA (depending on
volume)
•PRNT only on questionable results
•Convalescent samples only requested when acute sample EQ
•qRT-PCR when requested
*Most samples submitted with no date of illness onset or collection.

23. CARPHA is the reference laboratory for 24 member states
(English-speaking Caribbean islands, Haiti, Belize, Surinam, Guyana, Aruba,
Cuacao, Sint Martin, and BES islands)
In late 2013 CARPHA began to receive specimens from British
Virgin Islands and Dominica for CHIKV diagnostic testing
Sample aliquots were initially sent to CDC for testing until
CARPHA implemented testing
A subset of samples with results reported from CDC were
selected to evaluate the Euroimmun ELISA and IIFT, Inbios ELISA,
and Abcam ELISA kits at CARPHA
Part III: Field evaluations of Euroimmun and
Abcam CHIK MAC-ELISAs at CARPHA
Preventing disease
Promoting and protecting health

24. CARPHA Laboratory CHIKV testing results 2014
Preventing disease
Promoting and protecting health
Samples received from 19 CARPHA Member States
Total number of received samples 4449
Number of samples tested 3039 (68%)
Total number non-tested samples 1310 (32%)
Not fulfilling clin/epi criteria for CHIKV testing 1156 (88%)
Other reasons 164
(sample contamination, inappropriate sample)
Number of samples CHIKV positive 1626 (54%)

25. Preventing disease
Promoting and protecting health
CARPHA Laboratory CHIKV testing algorithm 2014
Serum Sample
≤ 8 days after onset of symptoms > 8 days
RT-PCR CHIKV*
Positive Negative
Confirmed Chikungunya
IgM ELISA CHIKV**
Dengue testing
(Serology/PCR)
consider others
≤ 4 days > 4 days
Probable Chikungunya
PositiveNegative
*CDC, updated 2014.
**Abcam (ab177848).

26. Summary of testing at CARPHA
• Concordance high between samples tested at CDC and
those tested at CARPHA with Euroimmun, Inbios, and
abcam kits
• Variance low between test runs
• Variance within run low, but sample ODs at later
positions < sample ODs positioned at beginning of plate
• The 2 abcam lots evaluated at CARPHA were not tested
at CDC
Preventing disease
Promoting and protecting health

27. • Inbios CHIKjj Detect MAC-ELISA and Euroimmun Anti-CHIKV ELISA (IgM)
kits had highest performance of all kits tested that are currently
available commercially
• Sensitivity, specificity, and concordance >95%
• Low variance in replicate testing
• Inbios higher sensitivity than Euroimmun for detecting low CHIKV IgM+
• Cross-reactivity with Mayaro and ONN IgM
• Abcam Anti-CHIKV IgM human ELISA kit had variable performance
• Sensitivity high with 2 lots tested at CARPHA
• Sensitivity high with 3 lots, low with 2 lots tested at CDC
• Sensitivity high with 3 lots of reformatted kit at CDC
• Sensitivity low with initial evaluations at NML; not evaluated further
Euroimmun IIFT kit had high performance but required more retesting
• Background fluorescence results in more EQs
• Requires experienced reader
• Requires IFA microscope
• Immunosorb IgG depletion sample dilution buffer sold separately
Conclusions of the three evaluations

28. • Manuscript of CHK IgM kit evaluations by CDC, NML, and CARPHA
in preparation
• Guidance to laboratories: Euroimmun ELISA and IIFT, Inbios RUO
ELISA, and Abcam reformatted ELISA performance highly correlated
with reference laboratories
• Labs should implement IHPC in every run as part of QA/QC, and also
to monitor assay validity
• CDC will continue to provide limited ELISA reagents (antigen,
conjugate, PC) through PAHO until kit evaluations are published
• CDC CHIKV molecular and serological proficiency testing program
initiated in 2015 to support state and international public health
laboratories; recommend that PAHO continue PT program
• Euroimmun working towards FDA approval; Inbios kit for sale
as RUO
Future directions

29. Thank you!