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