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UMass Amherst MICROBIO 160 - HIV testing

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MICROBIO 160 1st Edition Lecture 22 Outline of Last Lecture I. What are VirusesII. Characteristics of HIVIII. Types of HIVIV. Major Viral ComponentsV. HIV binding to CD4+ T-cellVI. Replication of DNAVII. HIV IntegraseVIII. HIV ProteaseIX. HIV PathogenesisX. How does HIV destroy CD4+ cellsXI. SyncytiaOutline of Current Lecture I. When should you have an HIV testII. Where can testing be found?III. Types of HIV testsIV. Types of Screening TestsV. How the HIV ELISA test worksVI. Rapid HIV testVII. HIV ProteinsVIII. Western BlotIX. Progression of HIV infectionCurrent LectureSpecific guidelines on when to have an HIV test:- Having 3+ sexual partners in the last 12 monthsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Have received a blood transfusion prior to 1985, or have a sexual partner who received atransfusion and later tested positive for HIV- If you’re not sure about one’s sexual partner’s risky behaviors- If you are a male who has had sex with another male (MSM—men who have sex with men)- Using street drugs by injection—sharing needles (you don’t know if it is sterile) - Having a sexually transmitted disease- Being a healthcare worker with direct exposure to blood on the jobWhere can I be tested?- Globally, HIV testing is provided at a number of locations including hospitals, health clinics, doctors or surgeries, specialist HIV/AIDS voluntary counseling and testing sites- Other places that you can potentially be tested in include in the home (industrialized nations), schools, workplaces, religious facilities, through mail order or online, mobile and outreach testing in the community Types of HIV Tests:1. Screening tests: highly sensitive—in a small percentage of cases the test result will be positive even if the person is not infected2. Confirmatory test: Western Blot (looks for proteins)3. Tests of HIV progressiona. HIV viral blood: How much virus you have in your systemb. CD4 cell count: How many CD4+ cells you have in your blood system HIV/tests What do theytest for?What is the window period?How long for the results?Reliability Commonly used inAntibody testsHIV antibodies3 months Between a few days and a few weeksHigh EuropeAntigen (p24)testP24 viral proteins11 days to one monthA few days toa weekHigh for primary infection, increasingly unreliable afterwardsUK—standard screening assayFourth generation HIV antibodies 11 days to 2-14 days High UK—recommendetests and p24 viral proteinsone month d at first-line assay USPCR/NAT test Genetic material belonging to HIV12 days Up to a week High Most developed countriesRapid test HIV antibodies3 months Within 20 minutesSatisfactory for uncomplicatedHIV infectionResource-poor settings How the HIV ELISA test works:- Testing of blood for HIV antibodies- Well is coated with HIV antigen- Patient’s serum is added o The ELISA TEST: a color change in the final step indicates that the patient has HIV antibodies in their bloodstreamo Anti-human antibodies will bind any human antibody in the well. Washing between steps removes unbound antibodies- Anti-human antibody with attached enzyme is addedo Anti-human antibodies will bind any human antibody in the well. Washing between steps removes unbound antibodies- Substrate to enzyme is added o If enzyme is present, substrate activates it=color changeFalse Negatives: happen if a patient has HIV, but there is no color change (Rare)- This is because there is not enough antibody in the sample- Old test kit=bad HIV antigenFalse Positives: happen if a patient does not have HIV, but there is a color change- This is because there is not enough washingRapid HIV Test:1. HIV antibodies from oral fluid are collected through the swab2. Once the device is inserted in to the test tube, the oral fluid mixes with the liquid and travels up the test stick3. If C-Line turns dark it confirms the test is working properly. If nom C-Line appears, the test is not working. a. If only C-Line appears, the test is negative4. HIV antibodies collecting at the T-Line indicate the test is positiveHIV Proteins: the confirmatory test—The Western Blot—looks for specific HIV proteins in a patient’s blood- Envelope proteins: the enV gene codes for polyprotein that is cut into gp120 and gp41- Pol proteins: the POL gene codes for a polyprotein that is cut into p64/53 and p34—together these make the reverse transcriptase- Gag and vpr proteins: help HIV infect a host cellWestern Blot and Specific HIV Proteins: run out the proteins in a gel so that they get separated by the size of the protein (smallest size moves the fastest so it will be at the bottom)- No bands present—negative- Bands at either p31 OR p24 AND bands present at either gp160 OR gp120—Positive- Bands present, but pattern does not meet criteria for positivity—indeterminate- Gp160: Viral envelope precursor (env)- Gp120: Viral envelope protein (Env) binds to CD4- P24: Viral core protein (gag)- P31: Reverse transcriptase (pol)Progression of HIV infection:- The column marked “PC” is an HIV-positive test result and the column marked “NC” is anHIV-negative test result- Column 3-10 are tests performed on a single person beginning with the day when the person was first exposed to HIV (Column 3, Day 0) to when the person had a fully detected HIV infection (Column 10, Day 30)o The number and quantity of expressed HIV proteins is increasing over time Effect of Viral load on HIV Detection: a person with HIV can have a viral load from less than 50 copies to over 1,000,000 copies of the virus per millimeter of blood (copies/mL) the HIV virus is undetectable when the HIV concentration in the blood is less than 400 to less than 50 copies of the virus/mL on test sensitivity (undetectable value varies)- High: for example, greater than or equal to 100,000 copies/mL- Low: for example, fewer than 10,000-30,000 copies/mL- Undetectable: for example, either less than 400 or less than 50 copies/mL, depending onthe test usedIf you get HIV it will eventually kill you no matter what medication you are taking—HIV Resistance and Mutations: resistance is the decreased ability of drugs to control HIV- Resistance affects treatment options (limits)- Genotype test: check for mutation in HIV that cause drug resistance (looking at genes and making a prediction)- Phenotype test: challenges virus with all HIV drugs (in a test tube) (actually checking)- Virtual


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UMass Amherst MICROBIO 160 - HIV testing

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