Laboratory diagnosis of AIDS notes by SANJU SAHpptx.pptx

AIDS (Acquired Immunodeficiency Syndrome) is the advanced stage of HIV infection, characterized by a severely weakened immune system. This makes individuals vulnerable to opportunistic infections and certain cancers. Although no cure exists, antiretroviral therapy (ART) can manage the disease, improve quality of life, and reduce transmission. Early diagnosis and treatment are crucial.

1. Laboratory diagnosis of AIDS Presented by - SANJU SAH St. Xavier’s College, Maitighar , Kathmandu MS.C. Microbiology Department

2. AIDS is a medical condition caused by HIV HIV infection can cause AIDS to develop. It is possible to contract HIV without developing AIDS Without treatment, HIV can progress and, eventually, it will develop into AIDS in the vast majority of cases AIDS is the syndrome, which may or may not appear in the advanced stage of HIV infection

3. HIV is found throughout all the tissues of the body but is transmitted through the body fluids of an infected person (semen, vaginal fluids, blood, and breast milk) HIV is a virus that attacks immune cells called CD-4 cells, which are a subset of T cells HIV is a retrovirus that infects the vital organs and cells of the human immune system Retroviruses are distinguished from all other RNA viruses in the presence of unusual enzyme, reverse transcriptase Converts ssRNA viral genome into ds viral DNA

4. The rate of virus progression varies widely between individuals and depends on many factors. These factors include the age of the individual, the body's ability to defend against HIV, access to healthcare, the presence of other infections, the individual's genetic inheritance, resistance to certain strains of HIV, and more.

5. Life cycle Binding or Attachment stage Fusion stage Reverse transcription Replication Integration Assembly Budding

6. 1. Attachment ,penetration and uncoating : Entry of HIV into cell is binding of virion gp120 envelope protein of CD4 protein on cell surface Binding of gp120 with chemokine receptors CXCR4 (T cell tropic ) and CCR5 (macrophage tropic strain ) 2.Fusion After binding to the CD4 cell, the HIV virus then fuses its envelope (which serves as the covering of the virus) with the cell membrane of the CD4 cell. This enables the Virus to gain entry into the CD4 cell. An HIV enzyme called reverse transcriptase converts the singlestranded HIV RNA to double-stranded HIV DNA.

7. 3.Reverse transcription stage of HIV Replication For a HIV virus to replicate, it must change from RNA ( RiboNucleic Acid) to DNA ( DeoxyriboNucleic Acid); for HIV to effect this change, it uses a protein (enzyme) called  Reverse Transcriptase Once the HIV DNA is produced, it can then enter the  Nucleus  of the CD4 cell now This stage is blocked by two classes of HIV drugs:  Nucleoside Reverse Transcriptase Inhibitors  and  Non-Nucleoside Reverse Transcriptase Inhibitors . 4.Integration stage of HIV Replication Cycle Inside the CD4 nucleus, HIV then uses an enzyme known as  Integrase   to integrate (or insert) its vital DNA into the DNA of the CD4 cell This stage is blocked using  Integrase Inhibitors  drugs.

8. 5.Replication stage of HIV life Cycle In the Nucleus of the CD4 cell, the HIV DNA combines with the CD4 DNA so that anytime the CD4 cell wants to produce, it uses the cells proteins to produce more  HIV long chains Proteins  thereby multiplying the HIV protein copies 6.Assembly stage of HIV Life Cycle Once the HIV long chains proteins are produced, they move out of the Nucleus to the surface of the CD4 membrane to assemble into immature noninfectious HIV. 7.Budding stage of HIV Life Cycle After moving to the surface of the CD4 membrane, the HIV releases another enzyme called  Protease  that helps to cleave or break up the long chains of HIV proteins into short chains of proteins These short chains of HIV proteins then combine to form the Mature HIV virus that is infectious and can then infect another new CD4 cell By this repeated replication and infection, it reduces the CD4 cells count in the body and the immunity of the HIV patient goes down   Protease Inhibitors  are the HIV Drugs that block this stage

9. Pathogenesis HIV infects CD4 helper T cells and kills them resulting in suppression of cell mediated immunity On entry the dendritic cells that line the mucosa of genital tract is affected After this the local CD4 helper T cell become infected HIV binds with CD4 receptor of helper T cells with gp 120 There is depletion of helper T lymphocytes HIV acts as “ superantigen ” and thus activates many helper T cells and leads to their demise There are different types of these co receptors for different cell types that HIV variants can use for infection of cells Two main chemokine receptors have been identified to play a major role in HIV entry, CCR5 and CXCR4 (or fusin ).

10. S tages Stage 1: Acute HIV infection Within  2 to 4 weeks  after infection with HIV, people may experience a flu-like illness, which may last for a few weeks When people have acute HIV infection, they have a large amount of virus in their blood and are very contagious But people with acute infection are often unaware that they’re infected because they may not feel sick right away or at all Stage 2: Clinical latency (HIV inactivity or dormancy) This period is sometimes called  asymptomatic HIV infection  or  chronic HIV infection During this phase, HIV is still active but reproduces at very low levels. People may not have any symptoms or get sick during this time For people who aren’t taking medicine to treat HIV, this period can last a decade

11. Stage 3: Acquired immunodeficiency syndrome (AIDS) AIDS is the most severe phase of HIV infection People with AIDS have such badly damaged immune systems that they get an increasing number of severe illnesses, called opportunistic illnesses Without treatment, people with AIDS typically survive about 3 years Common symptoms of AIDS include chills, fever, sweats, swollen lymph glands, weakness, and weight loss People are diagnosed with AIDS when their CD4 cell count drops below 200 cells/mm or if they develop certain opportunistic illnesses People with AIDS can have a high viral load and be very infectious

12. Symptoms The later symptoms of HIV infection are the result of infections caused by bacteria, viruses, fungi, and/or parasites. These conditions do not normally develop in individuals with healthy immune systems, which protect the body against infection. fever chills joint pain muscle aches sore throat sweats (particularly at night) enlarged glands a red rash tiredness weakness

13. Lab diagnosis T he diagnosis and monitoring of HIV infection is performed on blood specimens. Generally for serological tests (antigen and antibody detection) serum/plasma/whole blood is used, whereas for CD4 enumeration tests only whole blood collected in K2/K3 ethylene diamine tetra acetic acid (EDTA) evacuated tubes is used. For DNA/RNA PCR, Dried Blood Spots (DBS) or whole blood collected in K2/K3 EDTA is used. Blood Collection, Storage, and Transport for HIV Antibody Test

14. 1. Serology ( a) Antibody tests  - ELISA Most frequently used method for screening of blood samples for HIV antibody. The sensitivity and specificity of the presently available commercial systems approaches 100% but false positive and false negative reactions occur Other test systems available include passive particle agglutination, immunofluorescence , Western blots and RIPA bioassays. Western blots Regarded as the gold standard and seropositivity is diagnosed when antibodies against both the env and the gag proteins are detected The sensitivity of the test systems are currently being improved by the use of recombinant antigens. 

15. (b) Antigen tests  - HIV p24 antigen(core), gp41, gp120, gp 160(envelope protein) can be detected early in the course of HIV infection before the appearance of antibody. It is undetectable during the latent period (antigen-antibody complexes are present) but become detectable during the final stages of the infection It was argued that the routine use of antigen screening tests in the blood transfusion service may result in earlier cases of HIV infection being identified

16. 2. Virus isolation V irus isolation is accomplished by the co cultivation of the patient's lymphocytes with fresh peripheral blood cells of healthy donors or with suitable culture lines such as T-lymphomas. The presence of the virus can be confirmed by reverse transcriptase assays, serological tests, or by changes in growth pattern of the indicator cells However virus isolation is tedious and time consuming (weeks) and is successful in only 70 to 90% of cases. Therefore virus isolation is mainly used for the characterization of the virus.

17. 3. Demonstration of viral NA NAAT: Nucleic acid amplification test This can be accomplished by probes or by PCR techniques The latter may be useful because of its extremely high sensitivity These are sensitive tests for diagnosis of HIV infections They use polymerase chain reactions (PCRs) for the detecting various HIV structural genes (usually gag, pol and env ) PCRs are the test of choice in certain situations, such as early infant diagnosis and during window period. Therefore, up to the age of 18 months, the diagnosis of HIV infection can only be reliably made by DNA PCR.

18. 4.Rapid Anti-HIV Tests Several rapid tests have been developed using recombinant and/or synthetic antigens The most commonly employed rapid anti-HIV tests are based on the principle of Immunoconcentration /dot blot immunoassay (vertical flow), Immunochromatographic (lateral flow), particle agglutination (e.g., gelatine or latex), and Dipstick and Comb assay based on EIA Rapid tests are visual point of care tests that do not require any special equipment These tests are available in smaller test packs.

19. 5 . Prognostic Tests T he following may be useful as prognostic tests; (1) HIV antigen (2) Serial CD 4  counts (3) Neopterin (4) B 2 -microglobulin. (5) Viral load Of these tests, only serial CD4 counts and HIV viral load are still routinely used.  

20. a. HIV viral load   It appears that HIV viral load has the greatest prognostic value. HIV viral load in serum may be measured by assays which detect HIV-RNA e.g. RT-PCR, NASBA, or bDNA HIV viral load has now been established as having good prognostic value, and in monitoring response to antiviral chemotherapy Patients with a low viral load during the incubation period had a better prognosis than those with a high viral load. Patients whose viral load decreased significantly following the commencement of antiviral therapy had a better prognosis than those who did not respond

21. b. CD4 counts  - Despite the increasing use of HIV-RNA assays, measurement of CD4 still has important value in monitoring disease progression and response to antiviral chemotherapy whereas CD4 count gives an indication of the stage of disease. “The measurement of  HIV viral load tells us where the disease is going, whereas CD4 count tells us where the disease is at this moment”

22. Treatment Antiretroviral therapy (ART) : It is the use of HIV medicines to treat HIV infection. People on ART take a combination of HIV medicines (called an HIV regimen) every day. ART can’t cure HIV, but HIV medicines help people with HIV live longer, healthier lives ART also reduces the risk of HIV transmission. HIV medicines prevent HIV from multiplying (making copies of itself), which reduces the amount of HIV in the body. Having less HIV in the body gives the immune system a chance to recover. Even though there is still some HIV in the body, the immune system is strong enough to fight off infections and certain HIV-related cancers.

23. Nucleoside reverse transcriptase inhibitors ( Abacavir sulfate, Didanosine , Lamivudine , Stavudine , Zalcitabine , Zidovudine ) Nonnucleoside reverse transcriptase inhibitors ( Rit ( Efavirenz , Delaviridine , Nevirapine ) Protease inhibitors onavir , Nelfinavir , Saquinavir , Amprenavir , Indinavir,lopinavir )

24. HIV DRUG RESISTANCE Once a person has HIV, the virus begins to multiply (make copies of itself) in the body As HIV multiplies, it sometimes mutates (changes form) and produces variations of itself. Variations of HIV that develop while a person is taking HIV medicines can lead to drug-resistant strains of HIV. With drug resistance, HIV medicines that previously controlled the person’s HIV are not effective against the new, drug-resistant HIV In other words, the HIV medicines can’t prevent the drug-resistant HIV from multiplying  Drug resistance can cause HIV treatment to fail.

25. Drug-resistant HIV can spread from person to person. People initially infected with drug-resistant HIV have drug resistance to one or more HIV medicines even before they start taking HIV medicines. Medication adherence means taking HIV medicines every day and exactly as prescribed HIV medicines prevent HIV from multiplying. Skipping HIV medicines allows HIV to multiply, which increases the risk that the virus will mutate and produce drug-resistant HIV. As a result of drug resistance, one or more HIV medicines in a person’s HIV regimen may no longer be effective.

26. Cross resistance It happens when resistance to one HIV medicine causes resistance to other medicines in the same HIV drug class(HIV medicines are grouped into drug classes according to how they fight HIV .) As a result of cross resistance, a person’s HIV may be resistant even to HIV medicines that the person has never taken. Cross resistance limits the number of HIV medicines available to include in an HIV regimen.

27. Antiviral susceptibility assays Because of the increasing range of anti-HIV agents available, there is increasing pressure on the provision of antiviral susceptibility assays There are two types of antiviral susceptibility assays: phenotypic and genotypic assays Phenotypic assays define whether a particular strain of virus is sensitive or resistant to an antiviral agent by determining the concentration of the drug needed to inhibit the growth of virus in vitro. e.g. Plaque-reduction assay for HSV, plaque-reduction assay for HIV In Genotypic assays, mutations that are associated with resistance are assayed for by molecular biology methods such as PCR and LCR.  However, these assays are tedious and are not suitable for a routine diagnostic laboratory.