اولین مورد این بیماری در سال 1981 در جوانان همجنس باز...
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Transcript of اولین مورد این بیماری در سال 1981 در جوانان همجنس باز...
در جوان�ان 1981اولین م�ورد این بیم�اری در س�ال جوان�ان و س�پ�س ب�از سانف�رانسیس�کو همجن�س س�ال در نیوی�ورک �مش�ا�هده �ش�د�. ب�ا�ز ه�مجن�س
وی�ر�وس �بیم�ا�ری �کش�ف و� �معل�وم ش�د ک�ه 1983 ی�ک� جامع�ه را تم�ام� �اف�ر�ادا�ین� �وی�روس� ق�ا�در اس�ت
گ�ردش آل�ود�ه ك�ن�د. �وي�روس �بایس�ت�ی بنح�وی �وارد همان�ن�د خ�ون �( س�ازد آ�ل�ود�ه ت�ا �ف�رد �را� �ش�و�د
(�.� در �ح�ال �حا�ض�ر ب�يم�اري �اي�دز Bوی�ر�وس ه�پ�اتی�ت� از �ب�یما�ر�یه�ا�ی م�حس�وب �مق�ار�بتیبع�ن�وان �یک�ی
آ�ل�و�دگ�ي �تغی�ی�ر ا�نت�ش�ار�ا�گ�ر چ�ه� �ا�لگ�و�ی � �م�ی� �ش�و�د. �L �از� را�ه اک�ثرا آل�و�ده �ت�ز�ری�قک�ر�ده. وي�رو�س م�واد
ان�تشا�ر �می یابد.�مع�تاد�ان تز�ریقی�ب�خصو�ص د�ر
ن�انومتر ب�زرگی دارد.120 ت�ا 100وی�روس رت�رو ویروس�ها و تحت وی�روس از خ�انواده
می باشد. Lenti Virideaخانواده وی�روس از ن�وعRNA ویروس�های ت�ک رش�ته
ای است. ب�ه مخص�وص پذیرن�ده طری�ق از وی�روس
آل�وده آن�را و متص�ل میزب�ان س�لول س�طح میکند.
، ش�د س�لول وارد وی�روس ژن�وم وق�تی میزب�ا�ن ت�لفی�ق ش�ده �س�لو�ل �ژن�ومب�او�ی�روس�
سل�ول �مذکور� آلوده �باقی میماند.همیشهو�
دو ن�وع ( از این وی�روس وج�ود دارد HIV-1, HIV-2 :)
HIV-1 ر�ا ب�ه �بیم�اری ن�ژاد س�فی�د در اروپ�ا و آمریک�ا�
�در آفریق�ای HIV-2 مب�تال می �س�ازد. ح�اد �و کش�ن�ده
را ب�ه ن�ژاد س�یا�هزی�ر ص�حرا آن�دمیک� اس�ت �و ب�یش�تر
مبت�ال می� سازد�.مزمن �غیر کشند�هبیم�اری � منش�اء وی�روس ک�امال روش�ن نیس�ت ولی ب�ه نظ�ر
می� رس�د ک�ه� وی�روس �حاص�ل تغ�ی�یر ژ�ن�تیکی ی�افتن باشد.SIV ویروس
مولک�ول وی�روس اص�لی می CD4 پذیرن�ده س�ل T) مثبت CD4باش�د و ل�ذا س�لول ه�ای
ماکرو�فاژه�ا( م�ونوس�یت/ اص�ل�ی ه�او �میزب�ا�ن ویروس هستند.
:پذیرن�ده ه�ای کمکی ب�رای این وی�روسCCR5 �ولی T روی� س�لول CXCR4 �هس�تند.CXCR4و
CCR5 ک�ه CKR5 ن�یز نامی�ده میش�و�د روی �س�لول� وجو�د دارد. و ما�کروفاژ/مونوسی�ت هاTهای
وی�روس ه�ایT فق�ط س�لول ه�ای تروپی�ک T و و T ��س��لو�لهای تروپی��ک Mویروس��ها�ی . را آلوده می کنندماکروفاژها
Syncytium تروپی�ک معم�والTوی�روس مرح�ل�ه در و� میکن�د در ای�دز ایج�اد
وی�ر�وس ش�ود�. می دی�ده Mبیم�ا�ر آل�ودگی �در ت�روپی�ک ب�دن اوای�ل در
و ش��ود� می� دی��ده ش��خص ��مبتال� �مون�وس�یت و�ماکر�وف�اژ ه�ا بیش�تر در� �
بص�و�رت �ذ�خیر�ه باقی� میم�ا�ند.ها
تغییر ماهیت ویروس
وق�تی وی�روس وارد گ�ردش خ�ون می ش�ود بایس�تیس�لول � مثبتCD4ب�ا )بعلت� کن�د� برخ�و�رد فع�ال
ز�ی�ادتر�ی �مولک�ول � تع�د�اد آل�ود�گی(� CD4دا�ش�تن �ت�ا� 400ص�و�ر�ت گ�یر�د.� �مع�م�وال� در �ح�الت� ط�بی�عی �از� ه�ر
سل�ول �فعا�ل� است �.یک خ�ون محیط�ی Tس�لول عالوه ب�راين وي�روس ب�ه س�لول ه�اي خ�اطره اي
تمايل زيادي دارد. رفت�ه غ�دد لنف�اوی بع�داز اینک�ه س�لول آل�وده ش�د ب�ه
و د�ر آ�نج�ا بعل�ت �وج�ود� س�ل�ول� ه�ای �فع�ال ب�ی�ش�تر ، لن�ف� ب�ه منج�ر� ش�ده� وس�ی�عتر آد�نوپ�ا�تی آل�و�دگی
.(PGL) و پایدار می شودعمومی
10050
500
200CD4+ T-cell counts
Viral burden
AIDS
شاخص پيشرفت بيماري
Time from Initial HIV-1 Infection to AIDSIn
cid
ence
AID
S
Years infected with HIV-1
0 10 15
Children mean 4.7 yrs Adults
mean 10.9 yrs
5
ELISA ب�ا این تس�ت آن�تی ب�ادی علی�ه آن�تی ژن ه�ای :ی�ا p41 g �س�طحی� p120 g ی�ا p160 g م�ی مش�خص
شود. آزم��ایش بعن��وان بالت(: )وس��ترن ایمون��وبالت
تائیدی درخواست می شود. تع�یین ب�رای محیطی: خ�ون در وی�روس شمارش
در�خواس�ت می شود. پیش آگهی بیم�اری �وض�عیت وPCR افرادیک�ه در عالئم : دی�ده هیچگون�ه بیم�اری
ش�ک ول�ی )بعلتدا�رد ب�ه �آل�و�دگی �وج�ود نم�یش�ود خط�ر(� پ�ر� رفت�ا�ر ی�ا� مف�رط این�, الغ�ری تس�ت �
درخواست می شود.
Objectives Discuss the diagnosis of HIV and available
tests Describe the approach to the diagnosis of
acute retroviral syndrome Debate the advantages and disadvantages
of early treatment of acute HIV infection Discuss the evidence for the possibility of
superinfection / reinfection and the implications for patient education and management
Anonymous vs Confidential Anonymous
Identifying information not provided Results not linked to identifying information Allows reporting of HIV infection without breaching
confidentiality Disadvantage: may not be able to locate clients for test
results Confidential
Clients linked to test result by identifying information Results remain confidential
Informed consent
Pre-Test Counseling Goal: reduce HIV acquisition and transmission Accurate and current information about HIV Obtain informed consent Transmission and acquisition HIV test info: risk, benefits, meaning of potential
test results Assessment of individuals risks and appropriate
risk reduction activities Capacity to comprehend HIV testing and consent
Post-Test Counseling Accurate and current information about HIV Local resources Risk reduction education Referrals for ongoing care and support Healthy living strategies Meaning of test results and state reporting
guidelines Mental health support / counseling
Diagnosis of HIV Infection Viral antibodies Viral antigens Viral RNA/DNA Culture
Lancet, 1996; 348: 176.
Enzyme Immunoassay
Enzyme-Linked Immunosorbent Assay(EIA, ELISA)
Primary HIV antibody screening test Serum plasma, dried blood spots, oral fluids,
urine HIV-1/2, HIV-1, HIV-2 High degree sensitivity and specificity Repeatedly reactive: confirmatory testing
Negative Antibody Test Results HIV negative Recent infection: too early for seroconversion CDC: follow-up testing at 6 weeks, 12 weeks,
6 months
Confirmation Process Non-negative screenings should be
confirmed Western Blot (WB) Immunofluorescent Antibody Assay (IFA)
Higher specificity than EIA Interpretation can be subjective
HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT
پروفايل سرمي بيمار ايدز
DIRECT
INDIRECT
PRE-TEST COUNSELING, INFORMED CONSENT,CONFIDENTIALITY.
Different Test for HIV
Challenges of HIV Testing
Sensitivity- Early diagnostic ( window period)
Specificity- Cross reactivity
Easy to perform, low cost
Detection of HIV-1 and HIV-2 and discrimination between the two viruses
One test can not fulfill these requirements Need to perform a combination of HIV tests for
screening and confirmation
Detection of antibodies Screening tests
Enzyme immunosorbent assays (EIAs) Simple/rapid immuno-diagnostics assays
Confirmatory or supplemental tests Western blot (WB)
Alternatives to confirmatory tests Repetitive EIA or rapid assays
Current HIV technologies
Evolution of HIV ELlSAs First generation
Ag : Purified lysates of HIV Poor sensitivity and specificity
Second generation Ag : HIV-recombinant proteins and/or peptides Detection of HIV-1 and HIV-2 Poor sensitivity, improved specificity
Third generation Ag : HIV-recombinant proteins and/ or peptides Detection of IgM and IgG, improved sensitivity Detection of HIV group O
Fourth generation Capacity to detect Ag (P24) and antibodies
Sources of Error for HIV EIA TestsDocumented Sources of False Negative
Results
Sources of Error for HIV EIA TestsDocumented Sources of False Negative
Results
AIDS Patients
Early Seroconverters
Operator Error
Equipment Error
AIDS Patients
Early Seroconverters
Operator Error
Equipment Error
FAIL TO ADD SERUM OR REAGENT TO THE CORRECT WELL
REAGENT DILUTED IN WRONG DILUVENT IN WRONG DILUTION
Operator Error
MULTIPLE PREGNANCY
MULTIPLE TRANSFUSION
AUTO IMMUNE DISORDER
CHRONIC HEPATITIS,
CHRONIC ALCOHOLIC
HBV VACCINATION
ANTIBODY TO POLYSTERENE
Source of False Positive Results
Trouble shooting EIATrouble shooting EIAKit integrity
Controls (kit and in-house)
Equipment
Cross contamination
Sample quality
Personnel training
Correct validation and interpretation of results
Kit integrity
Controls (kit and in-house)
Equipment
Cross contamination
Sample quality
Personnel training
Correct validation and interpretation of results
Kit integrityKit integrity
Was cold chain maintained during kit transport?
Has the kit expired?
Has the kit been stored properly in your lab?
Was cold chain maintained during kit transport?
Has the kit expired?
Has the kit been stored properly in your lab?
ControlsControls
Kit Controls should be run on each EIA plate.Indicates whether the kit components are functioning.Used to validate EIA run, calculate cut off
In-house controlsKit controls are designed to be quite robust and do not reflect subtle changes in testing.In-house controls should be calibrated to test as a low positive (above cut-off, below maximum)
Kit Controls should be run on each EIA plate.Indicates whether the kit components are functioning.Used to validate EIA run, calculate cut off
In-house controlsKit controls are designed to be quite robust and do not reflect subtle changes in testing.In-house controls should be calibrated to test as a low positive (above cut-off, below maximum)
Equipment : PipettesEquipment : Pipettes
Single and Multi channel Pipettes should be calibrated on a monthly basis.
This can be done using a balance.
Inaccurate pipetting
Single and Multi channel Pipettes should be calibrated on a monthly basis.
This can be done using a balance.
Inaccurate pipetting
Equipment : Microplate Washers Equipment : Microplate Washers DailyPrime the washer with wash solution before running sample plates
Set the washer to wash the recommended number of times (with correct volume)
Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head
Listen for changes in the sound the washer makes, this can indicate a vacuum leak
At the end of the day prime the washer with DI water
DailyPrime the washer with wash solution before running sample plates
Set the washer to wash the recommended number of times (with correct volume)
Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head
Listen for changes in the sound the washer makes, this can indicate a vacuum leak
At the end of the day prime the washer with DI water
Equipment : Micro-plate WashersEquipment : Micro-plate WashersWeekly
If a washer is not used during the week rinse it out with DI water to reduce microbial growth.
MonthlyRun a 10% solution of ethanol through the washer to disinfect. This can also be done if the washer exhibits signs of contamination (high background).Thoroughly rinse the washer after alcohol is used.
WeeklyIf a washer is not used during the week rinse it out with DI water to reduce microbial growth.
MonthlyRun a 10% solution of ethanol through the washer to disinfect. This can also be done if the washer exhibits signs of contamination (high background).Thoroughly rinse the washer after alcohol is used.
Equipment : Micro-plate ReaderEquipment : Micro-plate Reader
DailyEach time a reader is turned on it runs a self test, it will then report any errors.
WeeklyRun a control plate weekly. Variations in positive or negative specimens could be a sign of a bad diode or a spill on a diode.
DailyEach time a reader is turned on it runs a self test, it will then report any errors.
WeeklyRun a control plate weekly. Variations in positive or negative specimens could be a sign of a bad diode or a spill on a diode.
Cross contaminationCross contamination
Can be caused by:
Reusing pipette tips (contaminated with + plasma)
Splashes from one well to another
During removal of plate covers
Can be caused by:
Reusing pipette tips (contaminated with + plasma)
Splashes from one well to another
During removal of plate covers
Sample QualitySample QualityProperly collected (no haemolysis)
Transport conditions
Storage conditions
Number of freeze/thaw cycles
Age of sample
Properly collected (no haemolysis)
Transport conditions
Storage conditions
Number of freeze/thaw cycles
Age of sample
Validation and Interpretation of ResultsValidation and Interpretation of Results
Product inserts provide guidelines
Positive and Negative controls must fall within a certain range.
Controls are used to calculate a cut-off.
Samples below cut-off are negative, those above are positive
Product inserts provide guidelines
Positive and Negative controls must fall within a certain range.
Controls are used to calculate a cut-off.
Samples below cut-off are negative, those above are positive
Western Blot (Immunoblotting)Western Blot (Immunoblotting)
Solid-phase EIA with immobilized viral antigens to detect antibodies to specific HIV proteins.
Creating Western Blot StripsCreating Western Blot Strips
Diagram reproduced from Commercial Methods in Clinical Microbiology, ASM Press
HIV lysate proteins are separated by size using gel electrophoresis
Proteins are transferred (blotted) onto the surface of a membrane
Strips are incubated with patient serum and antihuman IgG conjugated with an enzyme (and chromagen)
The membrane iscut into strips
HIV Western Blot Banding PatternHIV Western Blot Banding Pattern
Image reproduced from Commercial Methods in Clinical Microbiology. 2000. ASM Press.
env gp160gp120gp 41
gag p55p18p24
pol p65p51p31
Interpretation of Results(General Consensus)
Interpretation of Results(General Consensus)
Negative: No bands present
Positive: 2 ENV band present (WHO Guidelines)
Indeterminate: Any bands present but do not meet criteria for
positive
Negative: No bands present
Positive: 2 ENV band present (WHO Guidelines)
Indeterminate: Any bands present but do not meet criteria for
positive
Western Blot BandingWestern Blot Banding
Image reproduced from Commercial Methods in Clinical Microbiology, 2000. ASM Press.
*
*
*
gp160/120
p66p55/51gp41
p32
p24
p17
HIV-1 Seroconversion – Western blotBBI - Panel C
HIV-1 Seroconversion – Western blotBBI - Panel C
When should WB be used? When should WB be used?
Western Blot assay should not be used as a screening test.
WB should be viewed as a supplemental test which can be used to confirm positive results obtained from EIA.
HOWEVER: Specificity is less than that of EIA A significant number of indeterminate blots are seen in low risk populations
Western Blot assay should not be used as a screening test.
WB should be viewed as a supplemental test which can be used to confirm positive results obtained from EIA.
HOWEVER: Specificity is less than that of EIA A significant number of indeterminate blots are seen in low risk populations
AdvantagesAdvantages
Specific interaction of antibody and antigen can be directly visualized.Specific interaction of antibody and antigen can be directly visualized.
Disadvantages
Technically demanding
Expensive
Subject to interpretation
Presence or absence of bands
Intensity of those bands
Western Blot
50