Ali rezvani PhD of molecular medicine

Role of the Laboratory in Diagnosis of Influenza

during Seasonal Epidemics

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

A/Moscow/21/99 (H3N2)

Neuraminidase

Hemagglutinin

Type of nuclearmaterial

Virustype

Geographicorigin

Strainnumber

Year of isolation

Virus subtype

انفوالنزا های سروتایپهای پروتئین علیه بادی آنتی تولید اساس بر

هماگلوتینن نوروآمینیداز HAغشایی NAونوع انفوالنزا Aویروس ، B وC سروتیپ به

میان این در که میشود تقسیم مختلفی هاینوع 16دارای Aنوع و تنها Cو Bسروتیپ

های سروتیپ هستند، سروتایپ نوع یک دارایشامل :Aنوع 1 1H N ,1 2H N ,2 2H N ,3 2H N ,5 1H N ,7 2H N, 7 3H N ,9 2H N ,10 7H N.…,

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

انفوالنزا تشخیص های روش

و شناسایی برای آزمایشگاهی های روش از شماری

که دارد وجود آنفوالنزا و تشخیص حساسیت ازافتراق در متفاوتی های اختصاصیت

نوع افتراق Cو Bاز Aآنفوالنزا یا ونوع های افتراق ) Aزیرگونه همانند

H1N1 از ( 2009جدید H1N1فصلیشامل : ها روش این هستند برخوردار

تشخیصی- 1 RIDTsروشمستقیم- 2 ایمنوفلورسانس DFAsروشویروس- 3 Viral Cultureکشتملکولی- 4 Real timeRT – PCRروش

RIDTS ( (RAPID INFLUENZA DIAGNOSTIC TEST):

را ویروسی های نوکلوپروتئین ردیابی قابلیت ها تست ایناختصاصیت آنها های ویژگی جمله از و و % 95دارند

. است زمان مدت کمترین در نتیجه اعالمسه به شان افتراقی قابلیت اساس بر را ها تست این

کنند : می تقسیم زیر دستهنوع- 1 آنفوالنزا که های . Aتست دهند می تشخیص رانوع- 2 دو که های و Bو Aتست تشخیص یکدیگر از را

دهند می افتراقنوع- 3 آنفوالنزا که های دهند Bو Aتست می تشخیص را

. یکدیگرندارند از را نوع دو این افتراق قابلیت اما

DFAS (DIRECT IMMUNOFLUORESCENCE ASSAYS )

روش مانند نیز روش RIDTsاینویروس ژن آنتی تشخیص قابلیت

آن ویژگی جمله از و دارند راقابلیت % 96اختصاصیت و

نوع دو افتراق و در Bو Aتشخیصزمان دارد .2-4مدت ساعت

روش دو این :معایب

ردیابی DFAsو RIDTsحساسیت. 1 درH1N1 با مقایسه در RT – PCRجدید

.47-93و %( ) 10-70بترتیب ) است %(

کاذب. 2 منفی های پاسخ ایجاد دلیل بهنیستند . اعتماد قابل آنها منفی نتایج

3 " مثبت. نتایج گاها و منفی نتایج کلیههای با DFAsو RIDTsتست – RTبایستی

PCR. شوند تایید

Cell CultureViruses 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. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Counter-immunoelectrophoresis 5. 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.* & …

Introduction Advantage disadvantages Real Time PCRProduct Detectionin medicineConclusion

Reference

RT – PCR REAL TIME

نوکلئیک اسیدهای تکثیر اساس بر که روش ایناز ای از . RNAقطعه ، است استوار ژنومی

باالی حساسیت و اختصاصیتتشخیص و برخورداراست قابلیت

گونه زیر و آنفوالنزا انواع افتراق ودارد . یکدیگر از را آن های

Why use a molecular test to diagnose an infectious disease?

Need an accurate and timely diagnosisImportant for initiating the

proper treatmentImportant for preventing the

spread of a contagious disease

What are the advantages of using a molecular test?

High sensitivityCan theoretically detect the

presence of a single organismHigh specificity

Can detect specific genotypesCan determine drug resistanceCan predict virulence

What are the advantages of using a molecular test?

SpeedQuicker than traditional culturing

for certain organismsSimplicity

Some assays are now automated

Nucleic Acid DetectionShort length of viral genome makes them ideal candidate for nucleic-acid based diagnosisPCR–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 (RT-PCR)

• 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 sub-typing 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

Specimens

Controls

HA1 HA3 Infl B

Taqman technology for real-time PCR

Very sensitive, very specific

Turn around time about 6-8 hours from sample receipt

Cycle

From 10-2 to 10 -8

Current Influenza A Taqman Assay

97% EfficiencyY-intercept 35.4R2 .995

• 16 Antigenically distinct HAs –(H1-H16)

• 9 Antigenically distinct NAs– (N1-N9)

Influenza A Virus- real time assay

PB1 PB2 PA HA NP NAM1M2

NS1NS2

Flu A target Flu B target

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 influenza viral replication.

•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

oTime from illness onset to collection of respiratory specimens for testing

ospecimens should ideally be collected as early as

possible (ideally less than 4 days after illness onset when influenza viral shedding is highest)

Factors Influencing Results of Molecular Assays

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.

•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.

•

Interpretation of Testing Results

•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

oFor 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

odoes not necessarily mean viable virus is present.

oa person who recently received intranasal administration of live attenuated influenza virus vaccine (LAIV) may indicate detection of vaccine virus.

o.

o

Advantages of Molecular Assays

The likelihood of a false positive or false negative result is low and therefore, the

interpretation of the result is less impacted by the level of influenza activity in the community