Kanal Pengetahuan | Seminar Nasional : “Tantangan baru pengendalian penyakit arbovirus di Indonesia: Dengue, Chikungunya atau Zika”
State of the art
diagnosis technology
for arboviruses
Philippe Buchy (MD, PhD)
Director Scientific Affairs and Public
Health
GSK Vaccines Asia-Pacific
Singapore
Annual Scientific Meeting, Faculty of Medicine,
Universitas Gadjah Mada, Yogakarta
25th March 2017
Disclosure
§ I’m an employee of GlaxoSmithKline Vaccines Asia-Pacific
2
Alphaviruses - Flaviviruses
Asian
lineage
ZIKV
African
lineage
DENV
Mosquito-borne flaviviruses
Single-strand, + polarity, RNA viruses
WNV
YFV
Phylogenetic tree of flaviviruses 1
ZIKV: Zika virus; DENV: dengue virus; WNV: West Nile virus;
JEV: Japanese encephalitis virus; YKV: Yellow fever virus
1. Duong V, Dussart P & Buchy P, Int J Infect Dis 2017; 54:121-8.
Tick-borne
flaviviruses
JEV
Direct vs indirect methods
OPPORTUNITY
DIRECT METHODS
Virus
Isolation
Genome
detection
INDIRECT METHODS
Antigen
detection
Serology
IgM
Serology
IgG
CONFIDENCE
Adapted from Peeling RW, Artsob H, Pelegrino JL, Buchy P, Cardosa MJ , Devi S et al. Nat Rev Microbiol 2010; 8:S30-38
4
Markers for dengue diagnosis
Secondary IgG
NS1
Primary
IgG
IgM
Viremia
0
5 - 7
15
-
21
>60
Days
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
5
Advances in dengue diagnosis by decade
Decades
Viral isolation
1950-1960
Newborn mice
1970-1980
Mammalian cell lines
Mosquito inoculation
1980-1990
1990-2000
Mosquito cell lines + IFA
(monoclonal Abs)
2000-2011
Shell vial for virus isolation
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
6
Advances in dengue diagnosis by decade
Decades
Viral isolation
Serology
1950-1960
Newborn mice
HI + CF
1970-1980
Mammalian cell lines
Mosquito inoculation
1980-1990
1990-2000
Mosquito cell lines + IFA
(monoclonal Abs)
IgM MAC-ELISA
IgG ELISA
2000-2011
Shell vial for virus isolation
Commercial kits
(ELISA & rapid tests)
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
7
Advances in dengue diagnosis by decade
Decades
Viral isolation
Serology
1950-1960
Newborn mice
HI + CF*
1970-1980
Mammalian cell lines
Mosquito inoculation
Gene detection
1980-1990
1990-2000
2000-2011
Mosquito cell lines + IFA
(monoclonal Abs)
Shell vial for virus isolation
IgM MAC-ELISA
IgG ELISA
RT/PCR
Commercial kits
(ELISA & rapid tests)
Real-time RT/
PCR
NASBA/LAMP
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
8
Advances in dengue diagnosis by decade
Decades
Viral isolation
Serology
Gene detection
Ag detection
1950-1960
Newborn mice
HI + CF*
1970-1980
Mammalian cell lines
Mosquito inoculation
IgM MAC-ELISA
IgG ELISA
RT/PCR
Immunohistochemistry
for antigen detection
Commercial kits
(ELISA & rapid tests)
Real-time RT/
PCR
NASBA/LAMP
1980-1990
1990-2000
2000-2011
Mosquito cell lines + IFA
(monoclonal Abs)
Shell vial for virus isolation
NS1 antigen detection
Guzman MG, Buchy P, Enria D, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
9
Diagnostic methods, techniques and clinical samples
Diagnostic method
Technique
Clinical Sample
Time of sample
Virus isolation
Mosquito, cell culture
inoculation
NS1 detection
Antigen detection by
Immunohistochemistry
serum, plasma,
tissues
serum, plasma,
urine, CSF
tissues
Acute sample
Antigen detection
Genome detection
Serology
Conditions of
refrigeration
20-8 0 for one day
Acute sample
RT/PCR
serum, plasma,
whole blood, urine,
Acute sample
Real-time RT/PCR
Isothermal amplification
saliva tissues,
methods (LAMP/NASBA...) tissues in paraffin
serum, plasma
collected after
IgM detection
blood collected on
5-6 days of fever
filter paper
paired sera or
acute sera collected
IgG detection
plasma
in the first 5 days of
blood collected on fever; convalescent
filter paper
sera (15-21 days of
acute sample)
collected after
IgA detection
Serum, saliva
7 days of fever
-80 0 for longer
periods
20-8 0 for one or
two day s
-20 0 for longer
periods
Guzman MG, Buchy P, Enria D, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
10
Farrar JJ. CABI 2014.
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
Interpretation of dengue diagnostic tests
Higly suggestive
Confirmed
One of the following:
One of the following:
1. IgM + in a single serum sample
1. PCR/RT-PCR +
2. IgG + in a single serum sample with a titer ≥
1280
2. Virus culture +
3. IgM conversion in paired sera *
4. IgG seroconversion in paired sera or
fourfold IgG titer increase in paired sera *
Buchy P & Peeling R. Laboratory diagnosis and diagnostic tests. Dengue: Guidelines for diagnosis, treatment, prevention and control. WHO 2009.
11
Advantages and limitations of dengue diagnostic
tests
Indications
Diagnostic tests
Nucleic acid detection
Diagnosis of
acute dengue
infection
Advantages
Limitations
- Most sensitive and specific
- Possible to identify serotype
- Early appearance (pre-antibody),
so opportunity to impact on patient
management
- Potential false positive due to
contamination
- Expensive
- Needs expertise and expensive
laboratory equipment
- Not possible to differentiate
between primary and secondary
infection
- Need expertise and facility for cell
culture and fluorescent microscopy
- Takes more than 1 week
- Not possible to differentiate
between primary and secondary
infection
Isolation in cell culture and - Specific
identification using
- Possible to identify serotype by
immuno-fluorescence
using specific antibodies
Antigen detection
in clinical specimens
- Easy to perform
- Opportunity for early diagnosis
may impact on patient treatment
Serologic tests:
- Useful for confirmation of acute
IgM tests
infection
Seroconversion: 4-fold rise - Least expensive
in HI or ELISA IgG titers - Easy to perform
between acute and
- Can distinguish between primary
convalescent samples
and secondary infection
- Typically not as sensitive as virus
isolation or RNA detection
- May miss cases because IgM
levels may be low or undetectable
in some secondary infections
- Need two samples
- Delay in confirming diagnosis
Buchy P & Peeling R. Laboratory diagnosis and diagnostic tests. Dengue: Guidelines for diagnosis, treatment, prevention and control. WHO 2009.
12
Advantages and limitations of dengue diagnostic
tests
Indications
Surveillance and
outbreak
identification;
Monitor
effectiveness of
interventions
Diagnostic tests
Advantages
Limitations
IgM detection
- Identify probable dengue cases
- Easy to do for case detection in
sentinel laboratories
- May miss cases because IgM
levels may be low in some
secondary infections
Viral isolation and
RNA detection (+/NS1?)
- Confirm cases
- Identify serotypes
- Can only be performed in
reference labs
- Need acute samples
Buchy P & Peeling R. Laboratory diagnosis and diagnostic tests. Dengue: Guidelines for diagnosis, treatment, prevention and control. WHO 2009.
13
Zika
14
Viremia
Titers
§ In Nicaragua, mean ZIKV
viremia (4.8 log10 [6.3 X104])
lower to that of CHIKV (6.9 log10
[7.9 X 106]) and DENV (6.5 log10
[3.1 X 106])1
Viremia in Nicaraguan patients 1
§ Maximum viremia with ZIKV is
1000-fold lower than with DENV
and 100,000-fold lower than with
CHIKV 1
103
Mean:6.9
Mean: 6.5
X106
§ Viremia from 2.5 X
to 8
in asymptomatic blood donors 2
Mean:4.8
§ ZIKV viremia in pregnant women
higher than in non-pregnant
patients (5.0 log10 vs 3.7 log10,
p=0.006) 1
§ Viremia (and temperature)
higher among hospitalized cases
1
§ Filter paper (Cook Islands) 3
1. Waggoner JJ et al. Clin Infect Dis 2016; 63(12): 1584-1590
ending 30th March 2014
2. Aubry M. Transfusion 2016; 56:33-40. 3. WHO 2014. Pacific syndromic surveillance report. Week 13,
15
Viremia
French Polynesia patients
Mean viremia: 9.9 x 104 copies/ml
Musso D et al. J Med Virol 2016; doi: 10.1002/jmv.24735
16
Viremia
Duration
§ Duration usually less than 10 days 1
§ Testing recommended by WHO in whole
blood and serum from patients
presenting with onset of symptoms ≤ 7
days 2
Blood plasma ZIKV RNA kinetics during
primary infection of macaques 3
§ Up to 2 months in whole blood of a
Israeli patient 3, and up to 10 weeks in a
Finnish pregnant women returning from
South America (consequence of active
viral replication in fetus or placenta?) 4
§ In cynomolgus macaques inoculated
with 1x106 PFU by SC route, peak of
viremia reached by day 2 post-infection,
undetectable in 90% of animals by day
5, in 10% by day 7 (animal with highest
viremia) 5
1. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-524. 2. WHO. Laboratory testing for Zika virus infection. Interim guidance. 23 March 2016. http://
www.who.int/csr/resources/publications/zika/laboratory-testing/en/. 3. Lustig Y et al. Euro Surveill 2016; 21(26)pii=30269 4. Driggers RW et al. N Engl J Med 2016;
374:2142-51. 5 Osuna CE et al. Nat Med 2016; 22:1448-55.
17
Virus detection in other body fluids
§ ZIKV RNA is detected more often in
saliva 1 and urine 2 than in blood
§ Urine:
– Viral load ranging between 3.8 X 103 to 2.2
X 108 copies/ml 3
– Duration > 10 days (up to 20 days) 2
– Testing recommended by WHO in urine
from patients presenting with onset of
symptoms ≤ 7 days 4
ZIKV RNA detection in urine specimens of 70
American travellers 6
§ Saliva:
– Duration up to 8 days but median 3.0 days
+/- SD 1.5 days 1
– Of interest when blood is difficult to collect
(children, neonates) 3
§ Virus isolation: possible on both urine
and saliva 5
Proportion od samples positive according to the
number of days after onset of symptoms (N=182
patients) 1
1.. Musso D et al. J Clin Virol 2015; 68:53-55. 2. Gourinat AC et al. Emerg Infect Dis 2015; 21:84- 3. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-5249. 4. WHO.
Laboratory testing for Zika virus infection. Interim guidance. 23 March 2016. http://www.who.int/csr/resources/publications/zika/laboratory-testing/en 5. Bonaldo MC et al. PLoS Negl
Trop Dis 2016; 10(6): e0004816 6. Bingham AM et al. MMWR Morb Mortal Wkly Rep. 2016; 65:475-8.
18
Virus detection in other body fluids
§ Breast milk:
– Viral load ranging between 2.9 X 104 to 2 X 106 copies/
ml 1
– Infectious particles isolated 2
§ Semen:
– Viral load ranging between 1.1 X 107 to 2.9 X 107
copies/ml 1
– RNA detected up to 93 days after onset of symptoms 3
1. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-5249. 2. Dupont-Rouzeyrol M et al. Lancet 2016; 387:1051 3. Oliveira Souto I et al. Enferm Infecc Microbiol Clin 2016; doi:
10.1016/j.eimc.2016.10.009
Antibody response
§ Neutralizing antibody detection by PRNT is the « gold standard » for differentiation
between anti-Flavivirus antibodies as it is relatively specific in primary Flavivirus
infections 1
§ PRNT requires experienced technicians, is time-consuming, expensive, poses
potentially biosafety issues (live virus) 1
§ Anti-ZIKV neutralizing antibodies can be detected as early as 5 days after the onset of
fever. 2
§ IgM response is relatively ZIKV-specific during primary infections with only limited
cross-reactivity with other flaviviruses. During secondary Flavivirus infections, a high
degree of serologic cross-reactivity with other flaviviruses is observed. 1
§ IgM develop within few days after onset of illness (as early as 3 days) and can
generally be detected up to 3 months but in some cases they may persist for longer
periods which complicates accurate diagnostic testing 2
§ IgG antibodies are highly cross-reactive and develop within days after IgM (after day
10) and can be detected for months to years 2
§ The extensive cross-reactivity between antibodies trigered by different flaviviruses
(infection or vaccination) makes the interpretation of serological results very
complicate 2. A novel IgM and IgG ELISA based on recombinant ZIKV NS1
demonstrated excellent sensitivity & specificity. 3
1. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-5249. 2. Charrel RN et al. Bull World Health Organ 2016; 94(8):574-584. 3. Steinhagen K et al. Euro
Surveill 2016; 21(50):oii=30426.
20
Summary of laboratory diagnosis of Zika infections
Atif M et al. Infection 2016; 44(6):695-705
21
Chikungunya
22
Markers for Chikungunya diagnosis
IgG
IgM
Viremia
0
5 - 7
Adapted from Caglioti C et al. New Microbiol 2013; 36:211-27.
15
-
21
>60
Days
23
Diagnostic tests for CHIKV infection
Diagnostic
method
Sample types
Virus isolation (in
vivo or in vitro)
Serum, plasma,
whole blood, and
fresh or FFPE
tissues
ELISA or
immunochromatoSerum and CSF
graphic assay (ICA)
for antigen detection
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
Sensitivity
(%)
Variable
85 (serum)
80 (CSF)
Specificity
Advantages Disadvantages
(%)
100
Technical, laborious
Requires biosafety
Highly specific level 2/3
containment
May take 1-2 weeks
Commercial assays
89 (serum)
Early diagnosis not widely available
87 (CSF)
24
Diagnostic tests for CHIKV infection
Diagnostic
method
Sample types
RT-PCR
Real-time RT-PCR
Sensitivity Specificity
(%)
(%)
100
Serum and dried
blood spots
Isothermal
amplification
methods (RT-LAMP)
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
100
100
Up to 100
Up to 100
95.25
Advantages
Disadvantages
Highly sensitive and
specific
Rapid turnaround
time
Multiplex available
Expensive
reagents and
specialized
equipment
Multiplex available
Expensive
reagents and
specialized
equipment
Does not require
specialized
equipment (i.e.,
thermocyclers)
25
Diagnostic tests for CHIKV infection
Diagnostic
method
ELISA
Sample
types
Serum
CSF
Sensitivity
(%)
Specificity
(%)
Advantages
Disadvantages
Possible crossreactivity with other
Widely available
alphaviruses
IgM: 17 (serum);
Relatively cheaper and
48 (CSF)
Elevated IgM does
IgM: 95 (serum)
easier to perform
IgG: 45 (serum); IgG: 53 (serum)
not distinguish recent
Rapid bedside tests are
63 (CSF)
past infection from
available
acute infection
IFA
Serum
85–97
90–98
PRNT
Serum
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
Lack the ability to
quantify antibodies,
Sensitive and specific are subjective, and
Commercially available require special
equipment and
training
Very specific for
Requires the use of
alphaviruses; gold
live virus (requires
standard for
Biosafety level 3
confirmation of
containment)
serologic test results
26
Most recent developments
in arbovirus diagnostic
Image credit: https://www.shutterstock.com
27
28
Microsphere based immunoassays (MIAs)
www.thermofisher.com/luminex
29
30
31
Nucleic acid sequencing-based amplification (NASBA)
§ NASBA is an isothermal nucleic
acid amplification system, based
on transcription and reverse
transcription .
§ The method utilizes RTase, RNA
polymerase, RNaseH, a primer
containing T7 promoter (primer 1),
and a reverse primer (primer 2).
§ First, primer 1 anneals with target
sense RNA, and RTase extends
the complementary DNA (steps 1
and 2).
§ RNaseH recognizes the DNA–
RNA hybrids, and only digests the
RNA chain (step 3).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
32
Nucleic acid sequencing-based amplification (NASBA)
§ RTase uses its DNA polymerase activity to
generate double-stranded DNA with primer
2 (step 4).
§ The interaction of T7 RNA polymerase with
T7 promoter on the double-stranded DNA
generates antisense RNA (step 5).
§ Using this antisense RNA as a template,
RTase generates a DNA chain (step 6).
RNaseH digests the RNA (step 7), and
RTase generates double-stranded DNA
(step 8).
§ Finally, considerable quantities of antisense
RNA are generated as reaction products
(step 9).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
33
Loop-mediated isothermal amplification (LAMP)
§ Loop-mediated isothermal amplification
(LAMP) is similar to the PCR-based
method, but uses DNA amplification
under isothermal conditions
§ The system employs a DNA polymerase
and four primers, including two “looping
primers” and two “stripping primers”.
§ First, the looping primers anneal on the
F2 or B2 regions, and complementary
DNA chains are amplified (2)
§ Next, the stripping primers anneal on the
F3 or B3 regions, and complementary
DNA chains are amplified(3)
§ This leads to the release of the
complementary DNA chains generated
by the looping primers
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
34
Loop-mediated isothermal amplification (LAMP)
§ Finally, the single chain containing both
terminals makes stem-loops (step 4).
§ In the 5′ terminal, the double-stranded
F1 or B1 region works as a primer,
resulting in the generation of a doublestranded chain containing a stem-loop
(step 5).
§ The stem-loop area (F2 or B2 region) is
a single-stranded chain, and therefore
new looping primers can anneal on the
F2 or B2 region.
§ A new complementary chain is
generated from the looping primer (step
6), and strips the corresponding chain
(step 7 and 8).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
35
Loop-mediated isothermal amplification (LAMP)
§ The extensions from the F2 or B2 region,
and the extension formed by the looping
primer on the stem-loop area, occur
alternately (steps 9 and 10).
§ Finally, the long reaction products
containing tandem target sequence
repeats are generated (step 11).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
36
“The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time
PCR”…..
”The RT-LAMP method used in our study is specific, sensitive, and suitable for
further investigation as a useful alternative to the current methods used for clinical
diagnosis of DENV1-4, especially in hospitals and laboratories
that lack sophisticated diagnostic systems”.
37
Microarrays
§ DNA microarray is a
collection of
oligonucleotide,
probes, or DNA spots
immobilized on a solid
surface. The
technology is applied
to high-throughput and
simultaneous wide
genomic screening.
DNA microarray is
also used in medical
diagnosis and
surveillance of
infectious diseases
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
38
39
Mass Spectrometry
Ho Y-P & Rezddy P. Clinical Chemistry 2010; 56:525-536.
40
Mass Spectrometry
Ho Y-P & Rezddy P. Clinical Chemistry 2010; 56:525-536.
41
42
Thank You
Terimakasih
© 2016 GlaxoSmithKline. All rights reserved
diagnosis technology
for arboviruses
Philippe Buchy (MD, PhD)
Director Scientific Affairs and Public
Health
GSK Vaccines Asia-Pacific
Singapore
Annual Scientific Meeting, Faculty of Medicine,
Universitas Gadjah Mada, Yogakarta
25th March 2017
Disclosure
§ I’m an employee of GlaxoSmithKline Vaccines Asia-Pacific
2
Alphaviruses - Flaviviruses
Asian
lineage
ZIKV
African
lineage
DENV
Mosquito-borne flaviviruses
Single-strand, + polarity, RNA viruses
WNV
YFV
Phylogenetic tree of flaviviruses 1
ZIKV: Zika virus; DENV: dengue virus; WNV: West Nile virus;
JEV: Japanese encephalitis virus; YKV: Yellow fever virus
1. Duong V, Dussart P & Buchy P, Int J Infect Dis 2017; 54:121-8.
Tick-borne
flaviviruses
JEV
Direct vs indirect methods
OPPORTUNITY
DIRECT METHODS
Virus
Isolation
Genome
detection
INDIRECT METHODS
Antigen
detection
Serology
IgM
Serology
IgG
CONFIDENCE
Adapted from Peeling RW, Artsob H, Pelegrino JL, Buchy P, Cardosa MJ , Devi S et al. Nat Rev Microbiol 2010; 8:S30-38
4
Markers for dengue diagnosis
Secondary IgG
NS1
Primary
IgG
IgM
Viremia
0
5 - 7
15
-
21
>60
Days
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
5
Advances in dengue diagnosis by decade
Decades
Viral isolation
1950-1960
Newborn mice
1970-1980
Mammalian cell lines
Mosquito inoculation
1980-1990
1990-2000
Mosquito cell lines + IFA
(monoclonal Abs)
2000-2011
Shell vial for virus isolation
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
6
Advances in dengue diagnosis by decade
Decades
Viral isolation
Serology
1950-1960
Newborn mice
HI + CF
1970-1980
Mammalian cell lines
Mosquito inoculation
1980-1990
1990-2000
Mosquito cell lines + IFA
(monoclonal Abs)
IgM MAC-ELISA
IgG ELISA
2000-2011
Shell vial for virus isolation
Commercial kits
(ELISA & rapid tests)
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
7
Advances in dengue diagnosis by decade
Decades
Viral isolation
Serology
1950-1960
Newborn mice
HI + CF*
1970-1980
Mammalian cell lines
Mosquito inoculation
Gene detection
1980-1990
1990-2000
2000-2011
Mosquito cell lines + IFA
(monoclonal Abs)
Shell vial for virus isolation
IgM MAC-ELISA
IgG ELISA
RT/PCR
Commercial kits
(ELISA & rapid tests)
Real-time RT/
PCR
NASBA/LAMP
Guzman MG, Buchy P, EnriaD, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
8
Advances in dengue diagnosis by decade
Decades
Viral isolation
Serology
Gene detection
Ag detection
1950-1960
Newborn mice
HI + CF*
1970-1980
Mammalian cell lines
Mosquito inoculation
IgM MAC-ELISA
IgG ELISA
RT/PCR
Immunohistochemistry
for antigen detection
Commercial kits
(ELISA & rapid tests)
Real-time RT/
PCR
NASBA/LAMP
1980-1990
1990-2000
2000-2011
Mosquito cell lines + IFA
(monoclonal Abs)
Shell vial for virus isolation
NS1 antigen detection
Guzman MG, Buchy P, Enria D, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
Farrar JJ. CABI 2014.
9
Diagnostic methods, techniques and clinical samples
Diagnostic method
Technique
Clinical Sample
Time of sample
Virus isolation
Mosquito, cell culture
inoculation
NS1 detection
Antigen detection by
Immunohistochemistry
serum, plasma,
tissues
serum, plasma,
urine, CSF
tissues
Acute sample
Antigen detection
Genome detection
Serology
Conditions of
refrigeration
20-8 0 for one day
Acute sample
RT/PCR
serum, plasma,
whole blood, urine,
Acute sample
Real-time RT/PCR
Isothermal amplification
saliva tissues,
methods (LAMP/NASBA...) tissues in paraffin
serum, plasma
collected after
IgM detection
blood collected on
5-6 days of fever
filter paper
paired sera or
acute sera collected
IgG detection
plasma
in the first 5 days of
blood collected on fever; convalescent
filter paper
sera (15-21 days of
acute sample)
collected after
IgA detection
Serum, saliva
7 days of fever
-80 0 for longer
periods
20-8 0 for one or
two day s
-20 0 for longer
periods
Guzman MG, Buchy P, Enria D, Vazquez. S. Laboratory diagnosis of dengue. In: Dengue and dengue hemorrhagic fever, 2nd edition. Glubler DJ, Ooi EE, Vasudevan S,
10
Farrar JJ. CABI 2014.
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
Interpretation of dengue diagnostic tests
Higly suggestive
Confirmed
One of the following:
One of the following:
1. IgM + in a single serum sample
1. PCR/RT-PCR +
2. IgG + in a single serum sample with a titer ≥
1280
2. Virus culture +
3. IgM conversion in paired sera *
4. IgG seroconversion in paired sera or
fourfold IgG titer increase in paired sera *
Buchy P & Peeling R. Laboratory diagnosis and diagnostic tests. Dengue: Guidelines for diagnosis, treatment, prevention and control. WHO 2009.
11
Advantages and limitations of dengue diagnostic
tests
Indications
Diagnostic tests
Nucleic acid detection
Diagnosis of
acute dengue
infection
Advantages
Limitations
- Most sensitive and specific
- Possible to identify serotype
- Early appearance (pre-antibody),
so opportunity to impact on patient
management
- Potential false positive due to
contamination
- Expensive
- Needs expertise and expensive
laboratory equipment
- Not possible to differentiate
between primary and secondary
infection
- Need expertise and facility for cell
culture and fluorescent microscopy
- Takes more than 1 week
- Not possible to differentiate
between primary and secondary
infection
Isolation in cell culture and - Specific
identification using
- Possible to identify serotype by
immuno-fluorescence
using specific antibodies
Antigen detection
in clinical specimens
- Easy to perform
- Opportunity for early diagnosis
may impact on patient treatment
Serologic tests:
- Useful for confirmation of acute
IgM tests
infection
Seroconversion: 4-fold rise - Least expensive
in HI or ELISA IgG titers - Easy to perform
between acute and
- Can distinguish between primary
convalescent samples
and secondary infection
- Typically not as sensitive as virus
isolation or RNA detection
- May miss cases because IgM
levels may be low or undetectable
in some secondary infections
- Need two samples
- Delay in confirming diagnosis
Buchy P & Peeling R. Laboratory diagnosis and diagnostic tests. Dengue: Guidelines for diagnosis, treatment, prevention and control. WHO 2009.
12
Advantages and limitations of dengue diagnostic
tests
Indications
Surveillance and
outbreak
identification;
Monitor
effectiveness of
interventions
Diagnostic tests
Advantages
Limitations
IgM detection
- Identify probable dengue cases
- Easy to do for case detection in
sentinel laboratories
- May miss cases because IgM
levels may be low in some
secondary infections
Viral isolation and
RNA detection (+/NS1?)
- Confirm cases
- Identify serotypes
- Can only be performed in
reference labs
- Need acute samples
Buchy P & Peeling R. Laboratory diagnosis and diagnostic tests. Dengue: Guidelines for diagnosis, treatment, prevention and control. WHO 2009.
13
Zika
14
Viremia
Titers
§ In Nicaragua, mean ZIKV
viremia (4.8 log10 [6.3 X104])
lower to that of CHIKV (6.9 log10
[7.9 X 106]) and DENV (6.5 log10
[3.1 X 106])1
Viremia in Nicaraguan patients 1
§ Maximum viremia with ZIKV is
1000-fold lower than with DENV
and 100,000-fold lower than with
CHIKV 1
103
Mean:6.9
Mean: 6.5
X106
§ Viremia from 2.5 X
to 8
in asymptomatic blood donors 2
Mean:4.8
§ ZIKV viremia in pregnant women
higher than in non-pregnant
patients (5.0 log10 vs 3.7 log10,
p=0.006) 1
§ Viremia (and temperature)
higher among hospitalized cases
1
§ Filter paper (Cook Islands) 3
1. Waggoner JJ et al. Clin Infect Dis 2016; 63(12): 1584-1590
ending 30th March 2014
2. Aubry M. Transfusion 2016; 56:33-40. 3. WHO 2014. Pacific syndromic surveillance report. Week 13,
15
Viremia
French Polynesia patients
Mean viremia: 9.9 x 104 copies/ml
Musso D et al. J Med Virol 2016; doi: 10.1002/jmv.24735
16
Viremia
Duration
§ Duration usually less than 10 days 1
§ Testing recommended by WHO in whole
blood and serum from patients
presenting with onset of symptoms ≤ 7
days 2
Blood plasma ZIKV RNA kinetics during
primary infection of macaques 3
§ Up to 2 months in whole blood of a
Israeli patient 3, and up to 10 weeks in a
Finnish pregnant women returning from
South America (consequence of active
viral replication in fetus or placenta?) 4
§ In cynomolgus macaques inoculated
with 1x106 PFU by SC route, peak of
viremia reached by day 2 post-infection,
undetectable in 90% of animals by day
5, in 10% by day 7 (animal with highest
viremia) 5
1. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-524. 2. WHO. Laboratory testing for Zika virus infection. Interim guidance. 23 March 2016. http://
www.who.int/csr/resources/publications/zika/laboratory-testing/en/. 3. Lustig Y et al. Euro Surveill 2016; 21(26)pii=30269 4. Driggers RW et al. N Engl J Med 2016;
374:2142-51. 5 Osuna CE et al. Nat Med 2016; 22:1448-55.
17
Virus detection in other body fluids
§ ZIKV RNA is detected more often in
saliva 1 and urine 2 than in blood
§ Urine:
– Viral load ranging between 3.8 X 103 to 2.2
X 108 copies/ml 3
– Duration > 10 days (up to 20 days) 2
– Testing recommended by WHO in urine
from patients presenting with onset of
symptoms ≤ 7 days 4
ZIKV RNA detection in urine specimens of 70
American travellers 6
§ Saliva:
– Duration up to 8 days but median 3.0 days
+/- SD 1.5 days 1
– Of interest when blood is difficult to collect
(children, neonates) 3
§ Virus isolation: possible on both urine
and saliva 5
Proportion od samples positive according to the
number of days after onset of symptoms (N=182
patients) 1
1.. Musso D et al. J Clin Virol 2015; 68:53-55. 2. Gourinat AC et al. Emerg Infect Dis 2015; 21:84- 3. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-5249. 4. WHO.
Laboratory testing for Zika virus infection. Interim guidance. 23 March 2016. http://www.who.int/csr/resources/publications/zika/laboratory-testing/en 5. Bonaldo MC et al. PLoS Negl
Trop Dis 2016; 10(6): e0004816 6. Bingham AM et al. MMWR Morb Mortal Wkly Rep. 2016; 65:475-8.
18
Virus detection in other body fluids
§ Breast milk:
– Viral load ranging between 2.9 X 104 to 2 X 106 copies/
ml 1
– Infectious particles isolated 2
§ Semen:
– Viral load ranging between 1.1 X 107 to 2.9 X 107
copies/ml 1
– RNA detected up to 93 days after onset of symptoms 3
1. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-5249. 2. Dupont-Rouzeyrol M et al. Lancet 2016; 387:1051 3. Oliveira Souto I et al. Enferm Infecc Microbiol Clin 2016; doi:
10.1016/j.eimc.2016.10.009
Antibody response
§ Neutralizing antibody detection by PRNT is the « gold standard » for differentiation
between anti-Flavivirus antibodies as it is relatively specific in primary Flavivirus
infections 1
§ PRNT requires experienced technicians, is time-consuming, expensive, poses
potentially biosafety issues (live virus) 1
§ Anti-ZIKV neutralizing antibodies can be detected as early as 5 days after the onset of
fever. 2
§ IgM response is relatively ZIKV-specific during primary infections with only limited
cross-reactivity with other flaviviruses. During secondary Flavivirus infections, a high
degree of serologic cross-reactivity with other flaviviruses is observed. 1
§ IgM develop within few days after onset of illness (as early as 3 days) and can
generally be detected up to 3 months but in some cases they may persist for longer
periods which complicates accurate diagnostic testing 2
§ IgG antibodies are highly cross-reactive and develop within days after IgM (after day
10) and can be detected for months to years 2
§ The extensive cross-reactivity between antibodies trigered by different flaviviruses
(infection or vaccination) makes the interpretation of serological results very
complicate 2. A novel IgM and IgG ELISA based on recombinant ZIKV NS1
demonstrated excellent sensitivity & specificity. 3
1. Musso D & Gubler DJ. Clin Microbiol Rev 2016; 29:487-5249. 2. Charrel RN et al. Bull World Health Organ 2016; 94(8):574-584. 3. Steinhagen K et al. Euro
Surveill 2016; 21(50):oii=30426.
20
Summary of laboratory diagnosis of Zika infections
Atif M et al. Infection 2016; 44(6):695-705
21
Chikungunya
22
Markers for Chikungunya diagnosis
IgG
IgM
Viremia
0
5 - 7
Adapted from Caglioti C et al. New Microbiol 2013; 36:211-27.
15
-
21
>60
Days
23
Diagnostic tests for CHIKV infection
Diagnostic
method
Sample types
Virus isolation (in
vivo or in vitro)
Serum, plasma,
whole blood, and
fresh or FFPE
tissues
ELISA or
immunochromatoSerum and CSF
graphic assay (ICA)
for antigen detection
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
Sensitivity
(%)
Variable
85 (serum)
80 (CSF)
Specificity
Advantages Disadvantages
(%)
100
Technical, laborious
Requires biosafety
Highly specific level 2/3
containment
May take 1-2 weeks
Commercial assays
89 (serum)
Early diagnosis not widely available
87 (CSF)
24
Diagnostic tests for CHIKV infection
Diagnostic
method
Sample types
RT-PCR
Real-time RT-PCR
Sensitivity Specificity
(%)
(%)
100
Serum and dried
blood spots
Isothermal
amplification
methods (RT-LAMP)
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
100
100
Up to 100
Up to 100
95.25
Advantages
Disadvantages
Highly sensitive and
specific
Rapid turnaround
time
Multiplex available
Expensive
reagents and
specialized
equipment
Multiplex available
Expensive
reagents and
specialized
equipment
Does not require
specialized
equipment (i.e.,
thermocyclers)
25
Diagnostic tests for CHIKV infection
Diagnostic
method
ELISA
Sample
types
Serum
CSF
Sensitivity
(%)
Specificity
(%)
Advantages
Disadvantages
Possible crossreactivity with other
Widely available
alphaviruses
IgM: 17 (serum);
Relatively cheaper and
48 (CSF)
Elevated IgM does
IgM: 95 (serum)
easier to perform
IgG: 45 (serum); IgG: 53 (serum)
not distinguish recent
Rapid bedside tests are
63 (CSF)
past infection from
available
acute infection
IFA
Serum
85–97
90–98
PRNT
Serum
Mardekiean & Roberts. BioMed Res Int 2015;:834371.
Lack the ability to
quantify antibodies,
Sensitive and specific are subjective, and
Commercially available require special
equipment and
training
Very specific for
Requires the use of
alphaviruses; gold
live virus (requires
standard for
Biosafety level 3
confirmation of
containment)
serologic test results
26
Most recent developments
in arbovirus diagnostic
Image credit: https://www.shutterstock.com
27
28
Microsphere based immunoassays (MIAs)
www.thermofisher.com/luminex
29
30
31
Nucleic acid sequencing-based amplification (NASBA)
§ NASBA is an isothermal nucleic
acid amplification system, based
on transcription and reverse
transcription .
§ The method utilizes RTase, RNA
polymerase, RNaseH, a primer
containing T7 promoter (primer 1),
and a reverse primer (primer 2).
§ First, primer 1 anneals with target
sense RNA, and RTase extends
the complementary DNA (steps 1
and 2).
§ RNaseH recognizes the DNA–
RNA hybrids, and only digests the
RNA chain (step 3).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
32
Nucleic acid sequencing-based amplification (NASBA)
§ RTase uses its DNA polymerase activity to
generate double-stranded DNA with primer
2 (step 4).
§ The interaction of T7 RNA polymerase with
T7 promoter on the double-stranded DNA
generates antisense RNA (step 5).
§ Using this antisense RNA as a template,
RTase generates a DNA chain (step 6).
RNaseH digests the RNA (step 7), and
RTase generates double-stranded DNA
(step 8).
§ Finally, considerable quantities of antisense
RNA are generated as reaction products
(step 9).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
33
Loop-mediated isothermal amplification (LAMP)
§ Loop-mediated isothermal amplification
(LAMP) is similar to the PCR-based
method, but uses DNA amplification
under isothermal conditions
§ The system employs a DNA polymerase
and four primers, including two “looping
primers” and two “stripping primers”.
§ First, the looping primers anneal on the
F2 or B2 regions, and complementary
DNA chains are amplified (2)
§ Next, the stripping primers anneal on the
F3 or B3 regions, and complementary
DNA chains are amplified(3)
§ This leads to the release of the
complementary DNA chains generated
by the looping primers
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
34
Loop-mediated isothermal amplification (LAMP)
§ Finally, the single chain containing both
terminals makes stem-loops (step 4).
§ In the 5′ terminal, the double-stranded
F1 or B1 region works as a primer,
resulting in the generation of a doublestranded chain containing a stem-loop
(step 5).
§ The stem-loop area (F2 or B2 region) is
a single-stranded chain, and therefore
new looping primers can anneal on the
F2 or B2 region.
§ A new complementary chain is
generated from the looping primer (step
6), and strips the corresponding chain
(step 7 and 8).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
35
Loop-mediated isothermal amplification (LAMP)
§ The extensions from the F2 or B2 region,
and the extension formed by the looping
primer on the stem-loop area, occur
alternately (steps 9 and 10).
§ Finally, the long reaction products
containing tandem target sequence
repeats are generated (step 11).
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
36
“The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time
PCR”…..
”The RT-LAMP method used in our study is specific, sensitive, and suitable for
further investigation as a useful alternative to the current methods used for clinical
diagnosis of DENV1-4, especially in hospitals and laboratories
that lack sophisticated diagnostic systems”.
37
Microarrays
§ DNA microarray is a
collection of
oligonucleotide,
probes, or DNA spots
immobilized on a solid
surface. The
technology is applied
to high-throughput and
simultaneous wide
genomic screening.
DNA microarray is
also used in medical
diagnosis and
surveillance of
infectious diseases
Sakurai A & Shibasaki F. Viruses 2012; 4:1232-57.
38
39
Mass Spectrometry
Ho Y-P & Rezddy P. Clinical Chemistry 2010; 56:525-536.
40
Mass Spectrometry
Ho Y-P & Rezddy P. Clinical Chemistry 2010; 56:525-536.
41
42
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