3. Ali rezvani PhD of molecular medicine
Role of the Laboratory in Diagnosis of Influenza during Seasonal Epidemics
Ali rezvani PhD of molecular medicine

4. 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.

6. Influenza Virus A/Moscow/21/99 (H3N2) Virus type Geographic origin
Neuraminidase
Hemagglutinin
Type of nuclear
material
Virus
type
Geographic
origin
Strain
number
Year of
isolation
subtype

7. سروتایپ های انفولانزا
بر اساس تولید آنتی بادی علیه پروتئین های غشایی هماگلوتینن HA و نوروآمینیداز NA ویروس انفولانزا نوع A، B وC به سروتیپ های مختلفی تقسیم میشود که در این میان نوع A دارای 16 سروتیپ و نوع B و Cتنها دارای یک نوع سروتایپ هستند، سروتیپ های نوع A شامل : H1N1, H1N2, H2N2, H3N2, H5N1, H7N2 ,H7N3, H9N2, H10N7 ,….

8. 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

9. 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.

10. In general, diagnostic tests can be grouped into 3 categories.:
(1) direct detection, (2) indirect examination (virus isolation) (3) serology.

11. 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)

12. Indirect Examination 1. Cell Culture cytopathic effect (CPE)
haemabsorption
immunofluorescence
2. Eggs pocks on CAM
haemagglutination
inclusion bodies
3. Animals disease or death

13. روش های تشخیص انفولانزا
شماری از روش های آزمایشگاهی برای شناسایی و تشخیص آنفولانزا وجود دارد که از حساسیت و اختصاصیت های متفاوتی در افتراق آنفولانزا نوع A از B وC و یا افتراق زیرگونه های نوع A (همانند افتراق H1N1 فصلی از H1N1 جدید 2009 ) برخوردار هستند این روش ها شامل : 1- روش تشخیصی RIDTs 2- روش ایمنوفلورسانس مستقیم DFAs 3- کشت ویروس Viral Culture 4- روش ملکولی Real timeRT – PCR

14. RIDTs ( (Rapid Influenza Diagnostic Test):
این تست ها قابلیت ردیابی نوکلوپروتئین های ویروسی را دارند و از جمله ویژگی های آنها اختصاصیت 95 % و اعلام نتیجه در کمترین مدت زمان است. این تست ها را بر اساس قابلیت افتراقی شان به سه دسته زیر تقسیم می کنند : 1- تست های که آنفولانزا نوع A را تشخیص می دهند. 2- تست های که دو نوع A وB را از یکدیگر تشخیص و افتراق می دهند 3- تست های که آنفولانزا نوع A وB را تشخیص می دهند اما قابلیت افتراق این دو نوع را از یکدیگرندارند.

15. DFAs (Direct immunofluorescence assays )
این روش نیز مانند روش RIDTs قابلیت تشخیص آنتی ژن ویروس را دارند و از جمله ویژگی آن اختصاصیت 96 % و قابلیت تشخیص و افتراق دو نوع A وB در مدت زمان 4-2 ساعت دارد .

16. معایب این دو روش:
1. حساسیت RIDTs و DFAs در ردیابی H1N1 جدید در مقایسه با RT – PCR بترتیب ( %) و ( %) است. 2. به دلیل ایجاد پاسخ های منفی کاذب نتایج منفی آنها قابل اعتماد نیستند . 3. کلیه نتایج منفی و گاها" نتایج مثبت تست های RIDTs و DFAs بایستی با RT – PCR تایید شوند .

17. 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)

18. 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

19. Serology
Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.

20. 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.

21. 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

22. Advantage disadvantages
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 PCR
Product Detection
in medicine
Conclusion
Reference

23. RT – PCR Real time
این روش که بر اساس تکثیر اسیدهای نوکلئیک قطعه ای از RNA ژنومی استوار است .، از اختصاصیت و حساسیت بالای برخورداراست و قابلیت تشخیص و افتراق انواع آنفولانزا و زیر گونه های آن را از یکدیگر دارد .

24. 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

25. 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

26. What are the advantages of using a molecular test?
Speed
Quicker than traditional culturing for certain organisms
Simplicity
Some assays are now automated

27. 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.

28. 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.

29. PCR - A little goes a long way

30. 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
Controls
Specimens
HA1 HA3 Infl B

31. Taqman technology for real-time PCR
Very sensitive, very specific
Turn around time about 6-8 hours from sample receipt

32. Current Influenza A Taqman Assay
Cycle
From 10-2 to 10 -8
97% Efficiency
Y-intercept 35.4
R2 .995

33. Influenza A Virus- real time assay
16 Antigenically distinct HAs
(H1-H16)
9 Antigenically distinct NAs
(N1-N9)
Flu A target
Flu B target M1 M2
NS1
NS2
PB1
PB2 PA HA NP NA

36. 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.

37. 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

38. Factors Influencing Results of Molecular Assays
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)

39. 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.

40. 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.

41. 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.

42. 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.

43. 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.

44. 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).

45. Negative result
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,.

46. does not necessarily mean viable virus is present.
Positive result
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. .

47. 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