Download آنفولانزا3

Role of the Laboratory in
Diagnosis of Influenza
during Seasonal Epidemics
Ali rezvani
PhD of molecular medicine
accurate diagnosis of infection
may thus be helpful not only for
treatment-related decision
making but also for prevention of
person-to-person transmission
in both hospitals and
communities.
Influenza Virus
Type of nuclear
material
Neuraminidase
Hemagglutinin
A/Moscow/21/99 (H3N2)
Virus
type
Geographic
origin
Strain
number
Year of
isolation
Virus
subtype
‫سروتایپ های انفوالنزا‬
‫بر اساس تولید آنتی بادی علیه پروتئین های غشایی‬
‫هماگلوتینن ‪HA‬و نوروآمینیداز ‪NA‬ویروس انفوالنزا‬
‫نوع ‪B ،A‬و ‪C‬به سروتیپ های مختلفی تقسیم میشود که‬
‫در این میان نوع ‪A‬دارای ‪ 16‬سروتیپ و نوع ‪B‬و‬
‫‪C‬تنها دارای یک نوع سروتایپ هستند‪ ،‬سروتیپ های نوع‬
‫‪A‬شامل ‪:‬‬
‫‪H1N1, H1N2, H2N2, H3N2, H5N1,‬‬
‫‪H7N2 ,H7N3, H9N2, H10N7 ,….‬‬
Specimen choice and collection
Specimen quality limits test quality
Pathogen detection depends on:
–Appropriate collection site.
–Proper timing of specimen collection.
–Effective and timely processing of
sufficient specimen
Specimen storage and transport
Keep specimens at 4ºC
If delay >24hrs, freeze at 70ºC or
below.
Avoid any storage at -20ºC: greater
loss in infectivity.
In general, diagnostic tests can be
grouped into 3 categories.:
(1) direct detection,
(2) indirect examination (virus
isolation)
(3) serology.
Direct Examination
1. Antigen Detection
immunofluorescence, ELISA etc.
2. Electron Microscopy
morphology of virus particles
immune electron microscopy
3. Light Microscopy
histological appearance
inclusion bodies
4. Viral Genome Detection
hybridization with specific
nucleic acid probes
polymerase chain reaction (PCR)
Indirect Examination
1. Cell Culture
cytopathic effect (CPE)
haemabsorption
immunofluorescence
2. Eggs
pocks on CAM
haemagglutination
inclusion bodies
3. Animals
disease or death
‫روش های تشخیص انفوالنزا‬
‫شماری از روش های آزمایشگاهی برای شناسایی و تشخیص آنفوالنزا‬
‫وجود دارد که از حساسیت و اختصاصیت های متفاوتی در‬
‫افتراق آنفوالنزا نوع ‪A‬از ‪B‬و ‪C‬و یا افتراق‬
‫زیرگونه های نوع ( ‪A‬همانند افتراق ‪H1N1‬فصلی‬
‫از ‪H1N1‬جدید ‪ ) 2009‬برخوردار هستند این روش ها شامل‬
‫‪:‬‬
‫‪ -1‬روش تشخیصی ‪RIDTs‬‬
‫‪2‬روش ایمنوفلورسانس مستقیم ‪DFAs‬‬‫‪3‬کشت ویروس ‪Viral Culture‬‬‫‪4-‬روش ملکولی ‪Real timeRT – PCR‬‬
‫‪RIDTS ( (RAPID INFLUENZA DIAGNOSTIC TEST):‬‬
‫این تست ها قابلیت ردیابی نوکلوپروتئین های ویروسی را دارند و از‬
‫جمله ویژگی های آنها اختصاصیت ‪ % 95‬و اعالم نتیجه در کمترین‬
‫مدت زمان است‪.‬‬
‫این تست ها را بر اساس قابلیت افتراقی شان به سه دسته زیر تقسیم می‬
‫کنند ‪:‬‬
‫‪ -1‬تست های که آنفوالنزا نوع ‪A‬را تشخیص می دهند‪.‬‬
‫‪ -2‬تست های که دو نوع ‪A‬و ‪B‬را از یکدیگر تشخیص و افتراق می‬
‫دهند‬
‫‪ -3‬تست های که آنفوالنزا نوع ‪A‬و ‪B‬را تشخیص می دهند اما قابلیت‬
‫افتراق این دو نوع را از یکدیگرندارند‪.‬‬
‫‪DFAS‬‬
‫) ‪(DIRECT IMMUNOFLUORESCENCE ASSAYS‬‬
‫این روش نیز مانند روش ‪RIDTs‬قابلیت‬
‫تشخیص آنتی ژن ویروس را دارند و از‬
‫جمله ویژگی آن اختصاصیت ‪ % 96‬و‬
‫قابلیت تشخیص و افتراق دو نوع ‪A‬و ‪B‬‬
‫در مدت زمان ‪ 2-4‬ساعت دارد ‪.‬‬
‫معایب این دو روش‪:‬‬
‫‪ .1‬حساسیت ‪RIDTs‬و ‪DFAs‬در ردیابی ‪H1N1‬جدید‬
‫در مقایسه با ‪RT – PCR‬بترتیب ( ‪ )% 10-70‬و ( ‪-93‬‬
‫‪ )% 47‬است‪.‬‬
‫‪ .2‬به دلیل ایجاد پاسخ های منفی کاذب نتایج منفی آنها قابل‬
‫اعتماد نیستند ‪.‬‬
‫‪ .3‬کلیه نتایج منفی و گاها" نتایج مثبت تست های‬
‫‪RIDTs‬و ‪DFAs‬بایستی با ‪RT – PCR‬تایید شوند‬
‫‪.‬‬
Cell Culture
Viruses are obligate intracellular organisms
– require living cells for virus isolation
Advantages:
– Relatively sensitive and specific
– Can detect many different viruses
– Provides a viral isolate for further
characterization (serotyping, genotyping,
susceptibility)
Cell culture –limitations
Certain viruses don’t grow or grow
slowly
Other techniques for detecting
viral infection more cost effective
Successful culture depends on
viability of virus in specimen
Serology
Detection of rising titres of antibody between acute and
convalescent stages of infection, or the detection of IgM in
primary infection.
Classical Techniques
Newer Techniques
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Complement fixation tests (CFT)
Haemagglutination inhibition tests
Immunofluorescence techniques (IF)
Neutralization tests
Counter-immunoelectrophoresis
Radioimmunoassay (RIA)
Enzyme linked immunosorbent assay (EIA)
Particle agglutination
Western Blot (WB)
RIBA, Line immunoassay
Molecular Techniques
Molecular techniques for the direct
detection of viral genomes in the
specimen will play an increasingly
important role in the clinical virology
laboratory in the 21st century.
Why Expanding Role for Diagnostic
molecular Lab
Results in increasing demand for
rapid methods, viral load testing,
antiviral susceptibility, genotyping.
Increased pool of immunocompromised
Increasing antiviral agents
Real Time PCR in Diagnosis
*Quantitatively measurment of Human
Immunodeficiency Virus (HIV).
*Detection of Thalassemia, hemophilia,Sickle
cell anemia & favism by real time PCR.
*Cystic fibrosis.
*Phenyl ketonuria.
*Use in forensic medicine.
*Noninvasive Prenatal Diagnosis by Analysis
of Fetal DNA in Maternal Plasma.
*Detection and Quantitation of Circulating
Plasmodium falciparum DNA.
* &…
‫‪RT – PCR REAL TIME‬‬
‫این روش که بر اساس تکثیر اسیدهای نوکلئیک قطعه ای‬
‫از ‪RNA‬ژنومی استوار است ‪ ،.‬از اختصاصیت و‬
‫حساسیت باالی برخورداراست و قابلیت‬
‫تشخیص و افتراق انواع آنفوالنزا و زیر‬
‫گونه های آن را از یکدیگر دارد ‪.‬‬
Why use a molecular test to diagnose an
infectious disease?
 Need
an accurate and timely
diagnosis
 Important for initiating the
proper treatment
 Important for preventing the
spread of a contagious disease
What are the advantages of using a
molecular test?
 High
sensitivity
 Can theoretically detect the
presence of a single organism
 High specificity
 Can detect specific genotypes
 Can determine drug resistance
 Can predict virulence
What are the advantages of using a
molecular test?
 Speed
 Quicker
than traditional culturing
for certain organisms
 Simplicity
 Some assays are now automated
Nucleic Acid Detection
Short length of viral genome makes
them ideal candidate for nucleic-acid
based diagnosis
PCR
–conventional PCR – agarose gel
detection of product
–Real-time PCR- products detected
using probes or intercalating dyes
within the reaction.
–Microarrays – chip or bead based.
Reverse transcription polymerase chain reaction (RTPCR)
• RT-PCR is a process whereby RNA is first
converted to complementary DNA (cDNA)
and a section of the genome is then
amplified through the use of primers that
bind specifically to this target area.
• amplification of small amounts of nucleic
acid which enables highly sensitive
detection of minute amounts of viral
genome.
PCR - A little goes a long way
Conventional PCR assay for subtyping influenza virus
•
•
•
•
Based on amplification
of HA target gene
using specific primers
Amplicon can then be
used for sequence
analysis for strain
identification
Requires gel analysis
about about 2 days
Controls
Specimens
HA1 HA3 Infl B
Taqman technology for real-time
PCR
Very sensitive, very
specific
Turn around time about
6-8 hours from sample
receipt
Current Influenza A Taqman Assay
From 10-2 to 10 -8
Cycle
97% Efficiency
Y-intercept 35.4
R2 .995
Influenza A Virus- real time assay
Flu A target
Flu B target
M1 NS1
PB1 PB2 PA HA NP NA M2 NS2
• 16 Antigenically
distinct HAs
– (H1-H16)
• 9 Antigenically
distinct NAs
– (N1-N9)
Factors Influencing Results of Molecular Assays
• Influenza viral shedding in the upper respiratory
tract generally
declines substantially after
4 days
• Patients with lower respiratory tract disease
may have prolonged
replication.
•
influenza viral
Factors Influencing Results of Molecular Assays
• Immunosuppressed patients and persons
receiving systemic corticosteroids can also
have prolonged influenza viral replication
• Molecular tests can detect influenza
viral RNA (positive results) for a longer
duration than other influenza testing
Factors Influencing Results of Molecular Assays
o
o
Time from illness onset to collection
of respiratory specimens for testing
specimens should ideally be collected as early
as possible (ideally
less than 4 days
after illness onset when influenza
viral shedding is highest)
Source of respiratory specimens

The best upper respiratory tract specimens to
detect influenza viral RNA by RT-PCR are
nasopharyngeal swabs, washes or
aspirates; other acceptable specimens
are a nasal and/or throat swab.

Respiratory specimens
• Nasal washes and nasopharyngeal
aspirates tend to be more sensitive than
pharyngeal swabs.
• NP wash or aspirate is generally
considered the ‘gold standard’ for
virus isolation.
Swabs
• swabs with a synthetic tip (e.g.,
polyester or Dacron®) and an
aluminum or plastic shaft.
•
• Swabs with cotton tips and wooden
shafts are not recommended.
Respiratory specimens
• in order to increase the potential for
virus detection, multiple respiratory
specimens from different sites
should be obtained from the same
patient on at least two consecutive
days.
Interpretation of Testing Results
• even with RT-PCR, false
negative results can occur due to
improper or poor clinical
specimen collection or from poor
handling of a specimen after
collection and before testing.
•
• A negative result can also occur by
testing a specimen that was collected
when the patient is no longer shedding
detectable influenza virus.
• False positive results, although rare,
can occur (e.g., due to lab
contamination or other factors).
Negative result
o
For hospitalized patients, especially
for patients with lower respiratory
tract disease, if no other etiology is
identified and influenza is still
clinically suspected, additional
specimens should be collected and
tested,.
Positive result
o
o
o
does not necessarily mean viable
virus is present.
a person who recently received
intranasal administration of live
attenuated influenza virus vaccine
(LAIV) may indicate detection of
vaccine virus.
.
Advantages of Molecular Assays

likelihood of a false
positive or false negative
result is low and therefore, the
The
interpretation of the result is less
impacted by the level of influenza
activity in the community