CDC: producción y distribución de reactivos: Chikungunya y Zika (Bárbara Johnson, CDC)

CDC produccion y distribucion de
reactivos: Chikungunya y Zika
Barbara W. Johnson
Centers for Disease Control and Prevention
Division of Vector-Borne Infectious Diseases
Arboviral Diseases Diagnostic and Reference Laboratory
Fort Collins, Colorado
3 Noviembre 2015

CDC Diagnostic Testing Algorithm for detection of
CHIKV, dengue, and Zika 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
qRT-PCR
sensitive ≤ 6
days, IgM
ELISA
sensitive ≥ 6
days

Acute
Specimen
IgM
ELISA
IgG
ELISA
PRNT
No
Interpretation
< 8 days
ID
Virus
No
Interpretation
No
Interpretation
Possible 2°
Infection
Consensus
RT-PCR
Real-Time
RT-PCR
Virus
Isolation
Nucleic acid
sequencing
ID
Virus
POS POS
RT-PCR or
IFA
ID
Virus
POS
ID
Virus
Interpretations of test results for a single acute specimen
Result confirmed
with 1 test
Result confirmed
with 2 tests

Acute
Specimen
IgM
ELISA
IgG
ELISA
PRNT
No
Interpretation
< 8 days
ID
Virus
No
Interpretation
No
Interpretation
Possible 2°
Infection
Consensus
RT-PCR
Real-Time
RT-PCR
Virus
Isolation
Nucleic acid
sequencing
ID
Virus
POS POS
RT-PCR or
IFA
ID
Virus
POS
ID
Virus
Interpretations of test results for a single acute specimen
Negative test
result does not
mean person is
not infected

III. Geographical distribution of Arboviruses
Before 1999
SLE

Zika
2001-2006
III. Geographical distribution of Arboviruses

Global distribution of West Nile virus -2006

CHIKV Clades
W. African
Central / East African
Asian
Locations from which CHIKV has been isolated from individuals
Countries with endemic CHIKV activity
Tropic of Cancer
Tropic of Capricorn
Equator 0
23.3
23.3
Geographical Distribution of IdentifiedGeographical Distribution of Identified
Chikungunya Virus IsolatesChikungunya Virus Isolates
CHIKV Clades
W. African
Asian
Tropic of Cancer
Tropic of Capricorn
Equator 0
23.3
23.3
CHIKV Clades
W. African
Asian
Tropic of Cancer
Tropic of Capricorn
Equator 0
23.3
23.3
Geographical Distribution of IdentifiedGeographical Distribution of Identified
Chikungunya Virus IsolatesChikungunya Virus Isolates 2004

From: http://www.cdc.gov/chikungunya/geo/index.html
Countries and territories where chikungunya cases
have been reported* (as of October 20, 2015)
*Does not include countries or territories where only imported cases have been
documented. This map is updated weekly if there are new countries or territories that report
local chikungunya virus transmission.

ZIKV is an emerging arthropod-borne virus (arbovirus) that was first isolated
from a Rhesus monkey in Uganda, in 1947. This arbovirus is related to DENV
and they have similar epidemiology and transmission cycle in urban
environments. Until recently, only sporadic human ZIKV infections were
reported. In 2007, ZIKV emerged outside of Asia and Africa for the first time and
caused an epidemic on Yap Island in the Federated States of Micronesia,2
which
was followed by a large epidemic in French Polynesia in 2013–14.3
Subsequently, ZIKV spread to several countries in Oceania The clinical
presentation of ZIKV infection is not specific (mild fever, rash, arthralgia, and
conjunctivitis) and can be confused with other diseases, especially dengue and
chikungunya. Prior to the French Polynesian epidemic, during which severe
neurological complications (Guillain-Barre syndrome) were confirmed, ZIKV was
believed to cause only mild diseases.
The history of ZIKV resembles that of CHIKV, an alphavirus.5
First described in
Africa in 1952, CHIKV emerged in Asia and caused major epidemics in India
and southeast Asia between the 1950s and 1980s, before it disappeared
epidemiologically. In 2004, CHIKV re-emerged in east Africa and spread to Asia
again before spreading worldwide. CHIKV, similar to DENV, now circulates in all
inhabited continents, evolving to a global public health problem in the past
decade.

Countries that have past or current evidence of
Zika virus transmission (as of October 2015)
http://www.cdc.gov/zika/geo/index.html

Distribution of Zika and chikungunya viruses before
2005 and their expansion worldwide and in Oceania
between 2005 and 2015
Musso et al. Zika virus: Following the path of dengue and chikungunya?
The Lancet 2015;386(9990): 243-244.

Dengue, chikungunya and Zika virus infection outbreaks
or new virus circulation, Pacific Region, January 2012–17
September 2014 (n=28)*
*From: Roth et al. Eurosurveillance 2015;20(30):1-8.

From: Roth et al. Eurosurveillance 2015;20(30):1-8.

IV. Strategies for diagnostics where
multiple arboviruses are co-circulating
1. What is the appropriate test?
• Consider all the factors
• Which test has highest sensitivity AND/OR specificity?
• If both criteria cannot be met, is sensitivity or specificity most
critical?
• Does test require a second confirmatory assay?
• How will co-circulation of other arboviruses affect test results?
2. Can all appropriate tests be done on samples?
• Need to prioritize tests if not sufficient volume to test all samples
• Need to test sequentially if all tests cannot be run at same time
3. Are paired specimens available?
• Is the specimen appropriate for the test?
• How is a negative result interpreted?

First priority test
CHIKV serology ok with appropriately time
sample; Zika and Dengue need differential
diagnostic testing
From CDC/PAHO testing guidelines 2015

CDC Diagnostic Testing Algorithm for detection of
CHIKV infection
qRT-PCR
POS
REPORT
NEG
IgM ELISA
POS NEG
REPORTREPORT
PRNT
Serum* collected
qRT-PCR
sensitive ≤ 6
days, IgM
ELISA not
cross-reactive

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.

CDC Diagnostic Testing Algorithm for detection
of DENV and ZIKV infection in US travelers
qRT-PCR
POS
REPORT
NEG
IgM ELISA
POS NEG
REPORTREPORT
PRNT
Serum* collected
qRT-PCR
sensitive ≤ 6
days, most US
patients 1°
flavivirus
infection

CDC Diagnostic Testing Algorithm for detection of DENV and ZIKV
infection in areas where multiple flaviviruses are co-circulating
qRT-PCR
POS
REPORT
NEG
IgM ELISA
POS NEG
REPORTREPORT
PRNT
Serum* collected
qRT-PCR
sensitive ≤ 6
days, IgM
ELISA cross-
reactive

Table 1. IgG and IgM testing with heterologous flaviviruses of patients infected with ZIKV, Yap State, Micronesia, 2007*
I g G IgM
Patient Days after onset ZIKV ZIKV DENV YFV JEV MVEV WNV
Primary flavivirus ZIKV
822a 5 1 .5 23.2 1 .3 1.4 1.7 1 .1 –
822b 10 1 .2 39.5 1 .2 1.0 2.4 1 .2 –
822c 24 3 .3 13.1 2 .7 0.63 1.8 1 .3 –
830a 2 1 .1 1 .3 4 .4 0.48 4.4 2 .9 –
830b 21 1 .8 16.3 1 .9 0.63 1.3 1 .6 –
849a 3 1 .5 4 .5 0 .9 2 0.95 1.2 0 . 6 6 –
849b 18 3 .0 18.2 2 .2 1.0 2.7 1 .5 –
862a 6 1 .9 25.4 1 .7 1.1 1.8 1 .0 –
862b 20 2 .6 15.4 2 1.1 2.3 1 .1 E q
Secondary flavivirus ZIKV (probable)
817a 1 5 .9 1 .4 1 .7 0.8 1.7 0 .7 –
817b 19 5 .7 8 .1 5 .1 2.1 1.7 1 .0 –
833a 1 3 .4 1 .7 3 .7 1.0 2.8 1 .3 –
833b 19 8 .2 3 .1 2 .3 0.9 2.5 1 .3 –
844a 2 3 .8 3 .8 6 .8 2.0 21.5 0 .7 –
844b 16 8 .5 12.7 1 4 .9 7.0 42.9 1 .6 –
955a 1 5 .0 1 .8 3 .7 1.0 3.4 2 .4 E q
955b 14 2 6 . 6 10.9 3 .4 0.8 1.7 4 .0 E q
968a 1 4 .0 1 .7 1 .3 0.6 1.2 1 .2 –
968b 3 1 2 . 3 20.4 2 .9 0.8 0.9 2 .0 –
839a 3 1 0.92 3 .4 0.7 2.7 2 .1 –
839b 20 4 .9 17.2 2 .2 2.1 1.9 1 .8 –
847a 5 0 .9 0.94 4 .1 4.1 2.3 1 .3 –
847b 8 1 4 . 1 21.5 1 .4 3.3 1.1 2 .6 –
*Ig, immunoglobulin; ZIKV, Zika virus; DENV, dengue virus type 1–4 mixture; YFV, yellow fever virus; JEV, Japanese encephalitis virus; MVEV, Murray
Valley encephalitis virus; WNV, West Nile virus; –, negative. Eq, result in equivocal range of the assay. IgG and IgM testing was conducted by ELISA
except for WNV, which was tested by microsphere assay; ELISA values are patient optical densities divided by negative control optical densities; <2,
negative; 2–3 equivocal; >3 positive.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 1233

Table 2. Neutralization testing with heterologous flaviviruses of patients infected with ZIKV, Yap State, Micronesia, 2007*
Patient
Days after
onset ZIKV DENV1 DENV2 DENV3
PRNT90 titer
DENV4 JEV YFV WNV SLEV MVEV
Primary flavivirus ZIKV
822a 5 320 <10 < 1 0 <10 <10 <10 <10 <10 <10 <10
822b 1 0 2,560 1 0 1 0 1 0 1 0 <10 <10 <10 <10 <10
822c 2 4 5,120 1 0 1 0 1 0 10 <10 <10 <10 <10 <10
830a 2 <10 <10 NT‡ N T NT NT N T N T N T NT
830b 2 1 2,560 <10 < 1 0 <10 <10 <10 <10 <10 <10 <10
849a 3 <10 <10 < 1 0 <10 <10 <10 <10 <10 <10 <10
849b 1 8 10,240 <10 < 1 0 <10 <10 <10 2 0 <10 <10 <10
862a 6 320 <10 < 1 0 <10 <10 <10 <10 <10 <10 <10
862b 2 0 2,560 1 0 1 0 <10 <10 <10 <10 <10 1 0 <10
Secondary flavivirus ZIKV (probable)
817a 1 8 0 8 0 1 6 0 320 160 <10 <10 <10 4 0 4 0
817b 1 9 10,240 2,560 20,480 5,120 5,120 20 320 160 1,280 640
833a 1 160 320 8 0 4 0 2 0 <10 <10 <10 <10 <10
833b 1 9 81,920 20,480 5,120 5,120 1,280 <10 <10 8 0 320 320
844a 2 2 0 1,280 6 4 0 320 160 <10 <10 5 2 0 2 0
844b 1 6 10,240 40,980 10,240 5,120 1,280 5 <10 160 640 640
955a 1 4 0 1,280 6 4 0 160 320 <10 <10 <10 2 0 2 0
955b 1 4 163,840 81,920 20,480 10,240 5,120 10 <10 640 2,560 1,280
968a 1 8 0 320 3 2 0 8 0 40 <10 <10 <10 4 0 2 0
968b 3 10,240 640 6 4 0 160 160 <10 <10 1 0 4 0 2 0
839a 3 <10 <10 1 0 <10 <10 <10 4 0 <10 <10 <10
839b 2 0 10,240 4 0 3 2 0 8 0 80 <10 640 4 0 8 0 8 0
847a 5 <10 <10 < 1 0 <10 <10 <10 640 <10 <10 <10
847b 8 2,560 4 0 3 2 0 160 40 <10 1,280 8 0 320 320
*PRNT90 titer, 90% plaque reduction neutralization test titer; ZIKV, Zika virus; DENV, dengue virus; JEV, Japanese encephalitis virus; YFV, yellow fever
virus; WNV, West Nile virus; SLEV, St. Louis encephalitis virus; MVEV, Murray Valley encephalitis virus; NT, not tested (sample depleted).

Commercial sources for CHIKV
diagnostic testing

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

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*

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).

Evaluations of CHIKV IgM detection assays
Part I: CDC – Evaluated 9 kits with a reference panel of well-
characterized sera
Part II: NML – Euroimmun ELISA evaluated with samples
submitted for diagnostic testing from travelers to the Caribbean
and tested with in-house assays
Part 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
Some kit evaluations have been published. However, a side-by-side
comparison of all the available kits with one common set of samples, and
analyses of kit performance with diagnostic samples submitted from
travelers and in an outbreak setting was needed.

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

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
Four CHKV IgM detection assays with performance
similar to CDC assays

Commercial sources for Zika virus
diagnostic testing

Zeptometrix: ZIKA VIRUS Infectious Culture Fluid (BSL-
2 virus) (Uganda76 prototype MR766)
Zeptometrix: ZIKA VIRUS Purified Viral Lysate
(inactivated) (Uganda76 prototype MR766)
CDC: Zika virus in Qiagen AVL lysis buffer (inactivated)
(Zika H/PF/2013; human, French Polynesia)
*CDC: Zika viral RNA (inactivated) on FTA filter paper
*Projected in 2016.
Zika virus RNA PC
Zika virus

Primer
Genome
position† Sequence (5′ 3′)→
Sensitivity
no. copies
Specificity
‡
ZIKV 835 835–857 TTGGTCATGATACTGCTGATTGC
ZIKV 911c 911–890 CCTTCCACAAAGTCCCTATTGC 100 ZIKV
ZIKV 860-
FAM
860–886 CGGCATACAGCATCAGGTGCATAGGAG
ZIKV 1086 1086–1102 CCGCTGCCCAACACAAG
ZIKV 1162c 1162–1139 CCACTAACGTTCTTTTGCAGACAT 25 ZIKV
ZIKV 1107-
FAM
1107–1137 AGCCTACCTTGACAAGCAGTCAGACACTCAA
*RT-PCR, reverse transcription–PCR; ZIKV, Zika virus.
†Based on ZIKV MR 766 GenBank accession no. AY632535.
‡ZIKV specificity indicates a positive result with ZIKV only and no reactivity with dengue virus-
1 (DENV-1), DENV-2, DENV-3, DENV-4, West Nile virus, St. Louis encephalitis virus, yellow
fever virus, Powassan virus, Semliki Forest virus, o’nyong-nyong virus, chikungunya virus,
and Spondweni virus.
Description and performance characteristics of Zika
virus real-time RT-PCR primer/probe sets*
From: Lanciotti et al. Emerg Infect Dis 208;14(8):1232-1239.

Commercial Zika IgM Detection Assays
No commercial validated assays available
CDC: not recommending Zika MAC-ELISA
testing in regions where multiple flaviviruses are
circulating; no plans to provide Zika MAC-ELISA
reagents to PAHO labs

Conclusions
•Multiple arboviruses co-circulate in many areas of the Americas
•Emerging/re-emerging viruses need to be considered in testing algorithm
•The similarity of clinical symptoms between arboviruses and to other
pathogens makes laboratory diagnosis essential
•Cross-reactive flavivirus epitopes can confound interpretation of serological
tests; not specific
•Detection of viral RNA is specific confirmatory test
•RNA detection assays may not be sensitive for arbovirus infections with
low, transient viremia or for samples collected after the acute phase of
illness
•Testing algorithms should consider all the factors for the geographical
region
•“No interpretation” sometimes the only accurate interpretation of results

Source: Mary Huang
Even though arbovirus diagnostics may be like
Sisyphus pushing the rock up the hill:
We do the best we
can with the tools we
have, while
continually exploring
improved techniques

National Center for Emerging and Zoonotic Infectious DiseasesNational Center for Emerging and Zoonotic Infectious Diseases
Division of Vector-borne Diseases, Bacterial Diseases BranchDivision of Vector-borne Diseases, Bacterial Diseases Branch
The findings and conclusions in this report
are those of the author(s) and do not
necessarily represent the official position of
the Centers for Disease Control and
Prevention
Thank
you!

DomainDomain EpitopeEpitope SpecificitySpecificity
IIII A1A1 GroupGroup
A2A2 SubcomplexSubcomplex
A3A3 TypeType
A4A4 TypeType
A5A5 SubgroupSubgroup
IIIIII B1B1 TypeType
B2B2 TypeType
B3B3 SubcomplexSubcomplex
B4B4 SubcomplexSubcomplex
II C1C1 SubcomplexSubcomplex
C2C2 TypeType
C3C3 TypeType
C4C4 TypeType
 SubtypeSubtype – reacts with some but– reacts with some but
not all JE strainsnot all JE strains
 TypeType – reacts with all JEs– reacts with all JEs
 SubcomplexSubcomplex – reacts with 2 or 3– reacts with 2 or 3
members of the JE complexmembers of the JE complex
 ComplexComplex – reacts with all– reacts with all
members in JE complexmembers in JE complex
 SubgroupSubgroup – reacts with 2 or more– reacts with 2 or more
flavivirus complexesflavivirus complexes
 GroupGroup – reacts with all flaviviruses– reacts with all flaviviruses
High
Low
Specificity
Flavivirus infections elicit immune responses to viral
epitopes producing:
Virus species-specific antibodies
 Flavivirus cross-reactive antibodies
(From J. Roehrig, personal communication)

The effect of flavivirus cross-reactivity on
IgM ELISA results
•Both IgM and IgG antibodies exhibit significant cross-reactivity
among the flaviviruses.
•IgM less cross-reactive than IgG
•Anti-DENV IgM may react with JE virus antigen, even though the
viruses are not in the same serocomplex, producing a positive result
•Closely related flaviviruses in the same serocomplex, such as JEV,
WNV, and St. Louis encephalitis virus, highly likely to cross-react in
the IgM ELISA and difficult to distinguish by IgM ELISA alone.

IgM ELISA: Add serum sample to plate coated with
anti-human IgM Mab.
Anti-human IgM Mab
IgM
pentamer

Wash away unbound IgG and any
extraneous substances: All IgM in sample
binds to anti-human IgM Mab
JE IgM
JE antigenJE antigen
Anti JE IgGAnti JE IgG
conjugated to HRPconjugated to HRP
JE specific IgMJE specific IgM Non JE IgMNon JE IgM
From D.
Featherstone,
WHO, personal
communication

Add JEV Antigen with HRP enzyme conjugate:
binds to JEV-specific IgM
JE IgM
JE specific IgMJE specific IgM Non JE IgMNon JE IgM
From D.
Featherstone,
WHO, personal
communication

Possibilities of flavivirus cross reactivity
both JEV and DENV IgM
False positive result
JE specific IgMJE specific IgM
Dengue IgMDengue IgM
From D.
Featherstone,
WHO, personal
communication

P/N: O.D. patient serum on viral
antigen/O.D. negative control
serum on viral antigen
 P/N > 3 = positive
 P/N < 2 = negative
 P/N 2-3 = equivocal
Ref = pos control serum
N = normal control serum
Test validity criteria: OD for the test
specimen must be ≥ twice the mean OD of
the test specimen reacted on normal
antigen. If this requirement is not met, non-
specific background is being generated, and
the result MUST be reported as
uninterpretable.
CDC flavivirus differential diagnostics: serology
JE antigen
Den antigen
Each lot of reagents must beEach lot of reagents must be
standardized for each assaystandardized for each assay
S1S1
S2
S3
S4
S5
S6
S7 Ref
N
Viral
Antigen
Normal
Antigen
S8
S1S1
S2
S3
S4
S5
S6
S7 Ref
NS8
First test: IgM antibody capture ELISA

Days IgM P/N IgG P/N PRNT
Sample post-onset WN JEV WN JEV WN JEV
Typical primary WN Infection
acute serum 8 12.75 4.00 1.37 2.04 1:80 1:20
conv. serum 31 11.35 4.21 6.38 5.76 1:1280 1:80
Secondary flavivirus infection?
acute serum 4 1.59 1.42 3.12 2.62 <1:10 <1:10
conv. serum 15 9.01 3.96 10.00 9.90 1:640 1:320
Interpretation of ELISA Results Interpretation of PRNT results
P/N: O.D. patient serum/O.D. negative
control serum reacted on viral antigen.
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
PRNT titer > 10 = positive antibody
PRNT titer ≥4-fold over
heterologous flavivirus = positive
for specific antibody
or
PRNT titer ≥ 4-fold between
acute and convelescent
7. Sensitivity and specificity of diagnostic tests: Example
ELISA and PRNT tests to diagnose WNV infection

acute serum 8 12.75 4.00 1.37 2.04 1:80 1:20
conv. serum 31 11.35 4.21 6.38 5.76 1:1280 1:80
acute serum 4 1.59 1.42 3.12 2.62 <1:10 <1:10
conv. serum 15 9.01 3.96 10.00 9.90 1:640 1:320
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
or
In primary WNV infection, CDC MAC-ELISA
sensitive but not specific

acute serum 8 12.75 4.00 1.37 2.04 1:80 1:20
conv. serum 31 11.35 4.21 6.38 5.76 1:1280 1:80
acute serum 4 1.59 1.42 3.12 2.62 <1:10 <1:10
conv. serum 15 9.01 3.96 10.00 9.90 1:640 1:320
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
or
In primary WNV infection, PRNT specific confirmatory test

acute serum 8 12.75 4.00 1.37 2.04 1:80 1:20
conv. serum 31 11.35 4.21 6.38 5.76 1:1280 1:80
acute serum 4 1.59 1.42 3.12 2.62 <1:10 <1:10
conv. serum 15 9.01 3.96 10.00 9.90 1:640 1:320
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
or
In secondary flavivirus infection, CDC MAC-ELISA
sensitive but not specific and PRNT not specific

CDC: producción y distribución de reactivos: Chikungunya y Zika (Bárbara Johnson, CDC)

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