HIV infection (AIDS)

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HIV infection
Mode of transmission, pathogenesis, clinical manifestations, laboratory diagnosis, treatment, prevention, prognosis, scope of AIDS vaccine.

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HIV infection (AIDS)

  1. 1. AIDSAIDS Acquired Immuno deficiency syndrome or AIDS, is a collection of symptoms due to underlying infections and malignancies resulting from specific damage to immune system caused by human immunodeficiency virus (HIV). 04/19/14 2Biochemistry for medics
  2. 2. IncidenceIncidence • HIV (human immunodeficiency virus) infection has now spread to every country in the world. • Approximately 40 million people are currently living with HIV infection, and • An estimated 25 million have died from this disease. • In the United States, approximately 1 million people are currently infected. 04/19/14 3Biochemistry for medics
  3. 3. A Global view of HIV InfectionA Global view of HIV Infection 04/19/14 Biochemistry for medics 4
  4. 4. AIDSAIDS • The first indication of this new syndrome came in 1981 in homosexual drug addict males; • they had two things in common- Pneumocystis pneumonia and Kaposi’s sarcoma. • Both these are markers of collapsed immune system, • The affected patients appeared to have lost their immune competence, rendering them vulnerable to overwhelming and fatal infections with relatively avirulent micro-organisms, as well as to lymphoid and other malignancies. • This condition was given the name Acquired Immuno deficiency Syndrome (AIDS). 04/19/14 5Biochemistry for medics
  5. 5. AIDS MarkersAIDS Markers Pneumocystis pneumonia and Kaposi’s sarcoma are considered AIDS markers, since they reflect the underlying collapsed immune system04/19/14 Biochemistry for medics 6
  6. 6. Human Immunodeficiency VirusHuman Immunodeficiency Virus • In 1986, The International Committee on virus Nomenclature decided on the generic name of the causative virus as the Human Immunodeficiency Virus. • HIV, the etiological agent of AIDS, belongs to lentivirus subgroup of the retroviridae family. • This family of viruses is known for latency, persistent viremia, infection of the nervous system, and weak host immune responses. 04/19/14 7Biochemistry for medics
  7. 7. Human Immunodeficiency VirusHuman Immunodeficiency Virus • HIV has high affinity for CD4 T lymphocytes and monocytes. • HIV binds to CD4 cells and becomes internalized. The virus replicates itself by generating a DNA copy by reverse transcriptase. • Viral DNA becomes incorporated into the host DNA, enabling further replication. 04/19/14 8Biochemistry for medics
  8. 8. Structural Characteristics of HIVStructural Characteristics of HIV • HIV is a spherical enveloped virus of about 90- 120 nm in diameter • There is a lipoprotein envelop, which consists of lipids derived from the host cell membrane and glycoproteins which are viral coded. • The major virus coded envelop proteins are the projecting knob like spikes on the surface and the anchoring transmembrane pedicles. 04/19/14 9Biochemistry for medics
  9. 9. Structural Characteristics of HIVStructural Characteristics of HIV • The spikes, gp 120 constitute the major surface component of the virus which binds to the cell CD4 receptors on susceptible host cells. • These specific receptors, known as cluster of differentiation- CD4 are present on certain cells in the body, the cells possessing these receptors are called CD 4 + cells and these are - T helper cells, B lymphocytes ,macrophages, monocytes and dendritic cells. • Transmembrane pedicles gp 41 cause cell to cell fusion. 04/19/14 10Biochemistry for medics
  10. 10. Structural Characteristics of HIVStructural Characteristics of HIV A cross sectional schematic diagram of HIV virion, showing  lipid bilayer  in the form of viral envelop , Nucleocapsid core, which includes a layer of  a protein called p17 and an inner layer of a protein called p24.  04/19/14 11Biochemistry for medics
  11. 11. Structural Characteristics of HIVStructural Characteristics of HIV • Interior to the envelope is an outer icosahedral nuclear capsid shell and an inner cone-shaped core containing ribonucleoproteins. • The enzymes integrase p32, protease p10, reverse transcriptase p55/66 and 2 copies of single stranded genomic RNA are present inside the core. • (The proteins and glycoproteins are indicated by their mass expressed as kilo Daltons) 04/19/14 12Biochemistry for medics
  12. 12. Genome of HIVGenome of HIV There are two types of genes analyzed- a) Structural genes encode for products which participate in formation of functional structure of virus 1) gag gene • encodes for core and shell of virus. • The gene product is a precursor protein p55, which is cleaved into p17, p24 and p15. • The p24 antigen (major core antigen) can be detected in serum during the early stages of infection till the appearance of the antibodies. 04/19/14 13Biochemistry for medics
  13. 13. Genome of HIV (Structural genes)Genome of HIV (Structural genes) 2) pol gene • Encodes for the polymerase reverse transcriptase and other viral enzymes such as protease and integrase. • It is expressed as a precursor protein, which is cleaved in to components like p64 which has reverse transcriptase and RNAse activity: p51 which has only reverse transcriptase activity: p10 is a protease that cleaves gag precursor and p32 is an integrase. 04/19/14 14Biochemistry for medics
  14. 14. Genome of HIV (Structural genes)Genome of HIV (Structural genes) 3) env gene - determines the synthesis of envelop glycoprotein gp 160 which is cleaved into gp 120 and gp41 . Glycosylation occurs after cleavage. The antibodies to gp 120 are the first to appear after HIV infection and are present in circulation till the terminal stage of infection. 04/19/14 15Biochemistry for medics
  15. 15. b) Non structural and Regulatoryb) Non structural and Regulatory genesgenes 1) vif - (Viral infectivity factor gene) influences infectivity of viral particles. 2) vpr-stimulates promoter region of the virus 3) vpu (in HIV-1 ) and vpx (in HIV-2) enhance maturation and release of progeny virus from cells. Detection of the type specific sequences vpu and vpx is useful in distinguishing between infection by HIV type 1 and 2.04/19/14 16Biochemistry for medics
  16. 16. b) Non structural and Regulatoryb) Non structural and Regulatory genesgenes 4) tat –( trans activating gene) – (2 copies) having stimulatory effect on synthesis of all viral proteins . 5) rev - (Regulator of viral genes) –(2 copies )– required for expression of structural genes. 6) nef ( negative factor gene) down regulates viral replication. It may be responsible for the regulation of latent state of virus. 04/19/14 17Biochemistry for medics
  17. 17. b) Non structural and Regulatoryb) Non structural and Regulatory genesgenes 7) LTR - (long terminal repeat) sequences flanking on both sides giving promoter, enhancer and integration signals. Schematic representation of HIV genome 04/19/14 18Biochemistry for medics
  18. 18. Antigenic variations in HIVAntigenic variations in HIV • Based on molecular and antigenic differences, two types of HIV have been recognized. • The original isolates of HIV and the related strains present all over the world belong to HIV type 1. • The HIV strains, first isolated from West Africa, which react with HIV type I antiserum very weakly or not at all have been termed as HIV type 2. • It has 40 % genetic similarity and is more closely related to Simian immunodeficiency virus than to HIV-1. • It can cause AIDS but is less pathogenic and is less common. 04/19/14 19Biochemistry for medics
  19. 19. Antigenic variations in HIVAntigenic variations in HIV • HIV is a highly mutable virus and exhibits frequent antigenic variations as well as differences in other features such as nucleotide sequences, cell tropism, growth characteristics and cytopathology. • Not only are there differences between isolates of HIV from different races or persons but also between sequential isolates from the same person, and even between those obtained from different sites of the same person at the same time. • This great variability is believed to be due to error prone nature of reverse transcription. 04/19/14 20Biochemistry for medics
  20. 20. Antigenic variations in HIVAntigenic variations in HIV • HIV-1 strains have been classified in to at least ten subtypes based on sequence analysis of their gag and env genes. • These subtypes have been designated as A to J and constitute the Group M (For major), which cause the large majority of HIV-1 infections worldwide. • A few HIV-1 strains isolated from West Africa do not fall within the major group and have been designated as group O ( For Outlier). • Some later isolates of HIV-1 distinct from M and O groups have been called Group N (for new) 04/19/14 21Biochemistry for medics
  21. 21. Mode of transmissionMode of transmission • HIV is transmitted when the virus enters the body, usually by injecting infected cells or semen. • There are several possible ways in which the virus can enter. • Sexual contact- In 75 % cases , transmission is by sexual contact. o Most commonly, HIV infection is spread by having sex with an infected partner. 04/19/14 22Biochemistry for medics
  22. 22. Mode of transmissionMode of transmission o The virus can enter the body through the lining of the vagina, vulva, penis, rectum, or mouth during sex. o People who already have a sexually transmitted disease, such as syphilis, genital herpes, chlamydial infection, gonorrhea, or bacterial vaginitis, are more likely to acquire HIV infection during sex with an infected partner. 04/19/14 Biochemistry for medics 23
  23. 23. Mode of transmissionMode of transmission • Parenteral- In 15 % cases, it is by blood transfusion or blood product transfusion. o Sharing of unsterilized needles or syringes in drug addicts contaminated with blood from an infected person can spread virus. o HIV can be spread in health-care settings through accidental needle sticks or contact with contaminated fluids. o HIV can also spread through organ transplantation. o Donors are now tested for HIV to minimize this risk. 04/19/14 24Biochemistry for medics
  24. 24. Mode of transmissionMode of transmission • From mother to child o Women can transmit HIV to their babies during pregnancy or birth, when infected maternal cells enter the baby's circulation. o 30% of children born to infected mothers have the acquired infection unless virus is treated by antiviral drugs before pregnancy. o In nursing mothers transmission can occur through breast milk. 04/19/14 25Biochemistry for medics
  25. 25. Mode of transmissionMode of transmission 04/19/14 Biochemistry for medics 26
  26. 26. Mortality/Morbidity in HIV InfectionMortality/Morbidity in HIV Infection • The course of HIV infection is characterized primarily by latency. • Profound immune suppression eventually develops and the illness appears to be almost uniformly lethal. • Progression from HIV infection to AIDS occurs 8-10 years after infection without antiretroviral treatment. 04/19/14 27Biochemistry for medics
  27. 27. Mortality/Morbidity in HIVMortality/Morbidity in HIV InfectionInfection • Since the introduction of highly active antiretroviral therapy (HAART) and prophylaxis against opportunistic pathogens, death rates from AIDS have declined significantly. • An HIV-positive patient older than 50 years with a nearly undetectable viral load and a CD4 count more than 350 now has less than a 5% chance of dying or progressing to full blown AIDS within 3 years.04/19/14 Biochemistry for medics 28
  28. 28. Age for HIV InfectionAge for HIV Infection • Most AIDS cases occur in adults aged 25-49 years (70% of cases). • Adolescents and young adults (aged 13-24 y) represent 25% of new cases. • Young children represent fewer than 1% of AIDS cases in the United States. • Internationally, children younger than 15 years are estimated to account for close to 10% of all HIV cases. 04/19/14 29Biochemistry for medics
  29. 29. PathogenesisPathogenesis • Infection is transmitted when virus enters the blood or tissues of a person and comes in to contact with a suitable host cell, principally the CD4 lymphocytes. • The virus may infect any cell bearing the CD4 antigen on the surface. • Primarily these are the CD4 + helper T lymphocytes. 04/19/14 30Biochemistry for medics
  30. 30. PathogenesisPathogenesis • Some other immune cells possessing CD4 antigens are also susceptible to infection, like B lymphocytes, monocytes and macrophages including specialized macrophages such as Alveolar macrophages in the lungs and Langerhans cells in the dermis. • Glial cells and microglia cells are also susceptible. 04/19/14 Biochemistry for medics 31
  31. 31. Steps of viral entry in to the host cellSteps of viral entry in to the host cell • Attachment of virus into the host cell – Specific binding of the virus to the CD4 receptors is by the envelop glycoprotein gp120. • Cell to cell fusion – For infection to take place the cell fusion is essential. o This is brought about by the transmembrane glycoprotein gp 41. HIV-1 utilizes two major co-receptors along with CD4 to bind to, fuse with, and enter target cells; these co-receptors are CCR5 and CXCR4, which are also receptors for certain endogenous chemokines. 04/19/14 32Biochemistry for medics
  32. 32. Steps of viral entry in to the hostSteps of viral entry in to the host cellcell o Strains of HIV that utilize CCR5 as a co-receptor are referred to as macrophage tropic viruses (M –tropic viruses) o Strains of HIV that utilize CXCR4 are referred to as T - tropic viruses. o Many virus strains are dual tropic in that they utilize both CCR5 and CXCR4. 04/19/14 Biochemistry for medics 33
  33. 33. Steps of viral entry in to the host cellSteps of viral entry in to the host cell • Uncoating of the viral envelope and entry of nuclear capsid core into the cell o After fusion of virus with the host cell membrane, HIV genome is uncoated and internalized in to cell. o Viral RNA is released into the core cytoplasm 04/19/14 34Biochemistry for medics
  34. 34. Steps of viral entry in to the host cellSteps of viral entry in to the host cell • Viral transcription o viral reverse transcriptase mediates transcription of its RNA; o RNA-DNA hybrid is formed. o Original RNA strand is degraded by ribonuclease H, followed by o synthesis of second strand of DNA to yield double strand HIV DNA 04/19/14 35Biochemistry for medics
  35. 35. Steps of viral entry in to the host cellSteps of viral entry in to the host cell • Integration into the host DNA as provirus o The double stranded DNA is integrated in to the genome of the infected host cell through the action of the viral integrase enzyme, causing a latent infection. • Fate of provirus o From time to time, lytic infection is initiated releasing progeny virions, which infect other cells. o The long and variable incubation period of HIV is because of the latency. o In an infected individual the virus can be isolated from the blood, lymphocytes, cell free plasma, semen, cervical secretions, saliva, urine and breast milk 04/19/14 36Biochemistry for medics
  36. 36. Steps of viral entry in to the hostSteps of viral entry in to the host cellcell 04/19/14 Biochemistry for medics 37
  37. 37. Steps of viral exit from host cellSteps of viral exit from host cell • Transcription back into RNA o The viral DNA is transcribed into RNA and multiple copies of viral RNA are produced. o There are only nine genes in HIV RNA, and these code for the production of structural proteins, accessory proteins, and enzymes essential for the virus's replicative cycle. 04/19/14 38Biochemistry for medics
  38. 38. Steps of viral exit from host cellSteps of viral exit from host cell • Virion assembly - With the help of viral protease, the new virions are assembled into the polypeptide sequences needed for HIV virion formation and infectivity. • Cell lysis. The infected cell is made to burst open, presumably by the action of cellular proteins. 04/19/14 39Biochemistry for medics
  39. 39. 04/19/14 40Biochemistry for medics
  40. 40.   Causes of immune deficiencyCauses of immune deficiency • The primary pathogenic mechanism in HIV infection is the damage caused to the CD4+ T lymphocytes o The T4 cells decrease in numbers and the T4:T8 cell ratio is reversed. o The infected cells do not release cytokines. o This has a marked damping effect in the cell mediated immune response. o Though the major damage is to cellular immunity, the humoral mechanisms are also affected. AIDS patients are unable to respond to new antigens. o There is polyclonal activation of B lymphocytes leading to hypergammaglobulinemia. o These are non specific antibodies and are irrelevant to antigens. 04/19/14 41Biochemistry for medics
  41. 41. Causes of immune deficiencyCauses of immune deficiency • Monocyte, macrophage system is also affected apparently due to the lack of the activating factors by the T4 lymphocytes. • The activity of NK cells and Tc (T Cytotoxic) cells are also affected. • The clinical manifestations are due to failure of the immune responses. • This renders the patient susceptible to life threatening opportunistic infections and malignancies. 04/19/14 42Biochemistry for medics
  42. 42. Immune deficiency in HIV InfectionImmune deficiency in HIV Infection 04/19/14 43Biochemistry for medics
  43. 43. Clinical ManifestationsClinical Manifestations • AIDS is only the last stage in the wide spectrum of clinical features in HIV infection. • The center for disease control (USA) has classified the clinical course of HIV infection under various groups. • Acute HIV infection • Asymptomatic or Latent infection • Persistent generalized lymphadenopathy (PGL) • AIDS related complex • Full blown AIDS (Last stage) 04/19/14 44Biochemistry for medics
  44. 44. 1.  Acute HIV infection1.  Acute HIV infection • A flu-like illness with fever, sore throat, headache, tiredness, skin rashes and enlarged lymph nodes in the neck within  several days to weeks after exposure to virus. • These symptoms usually disappear of their  own within a few weeks.  • The test for HIV antibodies appears negative  while HIV antigenemia (p24 antigen) and viral  nucleic acids can be demonstrated at the  beginning of the phase.  04/19/14 45Biochemistry for medics
  45. 45. 1.  Acute HIV infection1.  Acute HIV infection • This phase is also  called window period or phase of Sero conversion. • Some patients do not develop symptoms after they first get infected with HIV. 04/19/14 Biochemistry for medics 46
  46. 46. 2. Asymptomatic or2. Asymptomatic or  Latent infectionLatent infection • All persons infected with HIV, pass through a phase  of symptomless infection (Clinical latency), which  may last up to several years.   • The progression of disease varies widely among  individuals.  • This state may last from a few months to more than  10 years.   • During this period, the virus continues to multiply  actively and infects and kills the cells of the immune  system.  • The virus destroys the CD4 cells that are the  primary infection fighters. 04/19/14 47Biochemistry for medics
  47. 47. 2. Asymptomatic or2. Asymptomatic or  Latent infectionLatent infection • The patients show positive antibody tests during this phase.  • Even though the person has no symptoms, he  or she is contagious and can pass HIV to others. • The median time between primary HIV  infection and development of AIDS has been  stated as approximately 10 years.  • About 5-10 % percent of the infected appear to  escape clinical AIDS for 15 years or more.  • They have been ‘long term survivors” or “long term non progressors”. 04/19/14 48Biochemistry for medics
  48. 48. 33.. Persistent generalized lymphadenopathy  Persistent generalized lymphadenopathy  (PGL)(PGL) • This has been defined by presence of  enlarged lymph nodes, at least I cm in  diameter, in two or more non contiguous  extra inguinal sites, that persist for at least  three months, in the absence of any current  illness or medication that may cause  lymphadenopathy. •  These are diagnostic of HIV when blood  tests are positive for antibodies. 04/19/14 49Biochemistry for medics
  49. 49. 4. AIDS related complex4. AIDS related complex • The patients present with weight loss – of  more than 10% of body weight, persistent  fever, diarrhea, generalized fatigue and  signs of other opportunistic infections may  be apparent.  • The opportunistic infections are oral  candidiasis, herpes zoster, salmonellosis or  Tuberculosis and hairy cell leucoplakia.  04/19/14 50Biochemistry for medics
  50. 50. Opportunistic infections in AIDSOpportunistic infections in AIDS 04/19/14 Biochemistry for medics 51
  51. 51. 4. AIDS related complex4. AIDS related complex • The patients are usually severely ill and many  of them progress to AIDS in few months.  • The CD4 cell count decreases steadily when the  count falls to 200, or less, clinical AIDS usually  sets in.  • For this reason the case definition by CDC  includes all HIV infected cases with CD4 + T  cell counts of 200 or less, irrespective of  clinical condition. 04/19/14 52Biochemistry for medics
  52. 52.   5.5.  Full blown AIDSFull blown AIDS • This is the end stage disease representing the  irreversible break down of immune defense  mechanisms.  • In addition to the opportunistic infections the  patient may develop primary CNS lymphomas  and progressive multifocal  leukoencephalopathy, dementia and other  neurological abnormalities.  • Kaposi sarcoma and Pneumocystis pneumonia  are almost always observed in a majority of  patients. 04/19/14 53Biochemistry for medics
  53. 53. Kaposi’s sarcomaKaposi’s sarcoma • Kaposi’s sarcoma- is an indolent, multifocal  non metastasizing mucosal or cutaneous tumor  probably of endothelial origin, represented in  the form of purple spots in the skin. 04/19/14 54Biochemistry for medics
  54. 54. Disease progression through different Disease progression through different  phases in HIV infected casesphases in HIV infected cases CD4 count comes down while the viral count  goes high with the passage of time 04/19/14 55Biochemistry for medics
  55. 55. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection 1) Non Specific Tests- The following tests help to establish  the immunodeficiency in HIV infection. a) Total Leukocyte and lymphocyte count- to  demonstrate leucopenia and lymphopenia. The  lymphocytic count is usually below 2000/mm3 b) T cell subset Assays- Absolute CD4+ cell count is less  than 200 /L.T4 T8 ratio is reversed.. The decrease in CD4  is the hall mark for AIDS. c) Platelet count-  shows Thrombocytopenia. d) IgA and Ig G levels are raised e) Diminished cell mediated Immunity as indicated by  skin tests f) Lymph node biopsy shows profound abnormalities. 04/19/14 56Biochemistry for medics
  56. 56. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection 2.Specific Tests for HIV infection- These  include demonstration of - • HIV antigen, • Antibodies, • Viral nucleic acids or other components and • Isolation of virus 04/19/14 57Biochemistry for medics
  57. 57. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection i) Detection of antigen o Following a contact, as by blood transfusion,  the viral antigen may be detectable in blood  after about 2 weeks.  o If the infecting dose is small, as following a  needle stick injury, the process may be  considerably delayed.  o The major core antigen p24 is the earliest  virus marker to appear in blood.  04/19/14 58Biochemistry for medics
  58. 58. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection • i) Detection of antigen (contd.) • Free p24 antigen disappears from circulation and  remains absent during the long asymptomatic phase  to reappear only when severe clinical disease sets  in. • The p24 Capture ELISA assay, which uses anti p24  antibody as the solid phase can be used for this.  • This test is positive in about 30% of the infected  persons.  • In the first few weeks after infection and in the  terminal phase, the test is uniformly positive.  04/19/14 59Biochemistry for medics
  59. 59. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection • Detection of antibodies o It takes 2-8 weeks to months for the  antibodies to appear in circulation o IgM antibodies appear first, to be followed  by IgG antibodies o Once antibodies appear they increase in  titer for the next several months o IgM antibodies disappear in 8-10weeks  while IgG antibodies remain through out. 04/19/14 60Biochemistry for medics
  60. 60. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection • Detection of antibodies (contd.) • There are two types of serological  tests- Screening tests and supplemental tests. i) Screening tests include- o  ELISA- ELISA is the most frequently used  method for screening of blood samples for  HIV antibody.  04/19/14 61Biochemistry for medics
  61. 61. Laboratory Diagnosis of HIV infectionLaboratory Diagnosis of HIV infection ELISA 1) First generation - whole viral lysates 2) Second generation - recombinant antigen 3) Third generation - synthetic peptide 4) Fourth generation - antigen + antibody  (Simultaneous detection of HIV antigen and  antibody) - HIV duo 04/19/14 62Biochemistry for medics
  62. 62. Laboratory Diagnosis of HIV Laboratory Diagnosis of HIV  infectioninfection Significance of ELISA • Antibody can be detected in a majority of  individuals within 6-12 weeks after infection using  the earlier generation of assays.  • But it can be detected within 3-4 weeks when  using the newer third generation ELISA.  • Due to their ability to detect p24 antigen, the  fourth-generation ELISA can be of value in  detecting early infection.  • The window period can be shortened to two  weeks using p24-antigen assay.04/19/14 63Biochemistry for medics
  63. 63. Laboratory Diagnosis of HIV Laboratory Diagnosis of HIV  infectioninfection • p24 Capture ELISA assay with HIV ELISA  is currently used for screening blood  donors. 04/19/14 Biochemistry for medics 64
  64. 64. Laboratory Diagnosis of HIV Laboratory Diagnosis of HIV  infectioninfection ii) Supplemental Tests a)Western Blot Test b) Indirect Immunoflorescence test       c)Radio ImmunoPrecipitaion Assay   iii) Rapid Tests a) Dot Blot assay b) Particle Agglutination tests c) HIV spot and comb test d)Fluorimetric micro particle technologies04/19/14 65Biochemistry for medics
  65. 65. Laboratory Diagnosis of HIV Laboratory Diagnosis of HIV  infectioninfection Western blotting •  Western blots are 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. 04/19/14 66Biochemistry for medics
  66. 66. Laboratory Diagnosis of HIVLaboratory Diagnosis of HIV infectioninfection 3. Demonstration of viral Nucleic acid • This can be accomplished by probes or by PCR techniques.  • The latter may be useful because of its extremely high  sensitivity.  • Cases of HIV are occasionally missed because individuals  can have negative antibody tests during the early stages of  infection.  • Also, a few people with long-term HIV infection may have  false negative antibody tests or may be chronic carriers  who are clinically asymptomatic. 04/19/14 67Biochemistry for medics
  67. 67. Laboratory Diagnosis of HIVLaboratory Diagnosis of HIV infectioninfection • PCR -In this the target HIV  RNA or proviral DNA is  amplified enzymatically in vitro by chemical reaction.  • It is an extremely sensitive  assay because a single copy  of proviral DNA can be  amplified.  • Qualitative PCR is useful for  diagnostic purposes. 04/19/14 68Biochemistry for medics
  68. 68. Laboratory Diagnosis of HIVLaboratory Diagnosis of HIV infectioninfection • Three different  techniques namely RT- PCR, nucleic acid  sequence based  amplification (NASBA)  and branched-DNA (b- DNA) assay have been  employed to develop  commercial kits. 04/19/14 Biochemistry for medics 69
  69. 69. Laboratory Diagnosis of HIVLaboratory Diagnosis of HIV infectioninfection 4. Virus isolation  • virus 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.  • 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. 04/19/14 70Biochemistry for medics
  70. 70. Laboratory Diagnosis of HIVLaboratory Diagnosis of HIV infectioninfection 5. Alternative to classical tests a) Oral fluid (saliva) HIV tests b) Urine tests 04/19/14 71Biochemistry for medics
  71. 71. Treatment of HIV InfectionTreatment of HIV Infection • All treatment for HIV infection and AIDS  today focuses on arresting the progression  of the disease within the body as measured  by T cell counts and tests for viral load. • There are two principal approaches to  treatment: immunotherapy and anti-HIV drug treatments 04/19/14 72Biochemistry for medics
  72. 72. Treatment of HIV InfectionTreatment of HIV Infection a)Immunotherapy •  Immunotherapy is transfusion based treatment designed  to replace lost immunoglobulins needed to fight HIV  infection (passive immunotherapy), to provide cellular  factors such as interleukins (IL-2) or to introduce selected  or altered immune cells to attack cells harboring the virus  (adoptive immunotherapy).  • The results of trials using this latter approach, however,  have been inconclusive, and no group has yet shown a  survival benefit. 04/19/14 73Biochemistry for medics
  73. 73. Treatment of HIV InfectionTreatment of HIV Infection b) Anti-HIV drug treatment  • Treatment with anti-HIV drugs attempts to  reduce viral load by blocking new infection  in the host cell.  • The drugs used target two major enzymes  of HIV which are needed for the infection  cycle: reverse transcriptase and protease. 04/19/14 74Biochemistry for medics
  74. 74. Treatment of HIV InfectionTreatment of HIV Infection I) Reverse transcriptase inhibitors   • Reverse transcriptase inhibitors act at the pre-integration  stage - before the viral RNA has been converted to DNA  and enters the host cell nucleus to integrate into the cell  chromosome.  • These drugs block the reverse transcription of viral RNA  into viral DNA.  • There are two types of reverse transcriptase inhibitors,  both of which accomplish the same objective: nucleoside  and nucleotide analogues, and non-nucleoside reverse  transcriptase inhibitors.  04/19/14 75Biochemistry for medics
  75. 75. Treatment of HIV InfectionTreatment of HIV Infection i) Nucleoside analogues • Constitute the most effective family of  antiretroviral drugs • Operate by mimicking nucleic acids normally  incorporated into viral DNA.  • They interfere with reverse transcriptase and thus  prevent infection.  • Nucleotide analogues have the same action but  are based around a different sugar.  04/19/14 76Biochemistry for medics
  76. 76. Treatment of HIV InfectionTreatment of HIV Infection ii) Non-nucleoside reverse transcriptase inhibitors  • Directly inhibit reverse transcription and, unlike  nucleoside analogues,  • Do not have to go through chemical changes in the  infected cell before beginning their action.  • The two main non-nucleoside reverse  transcriptase inhibitors are Nivirapine, and  Delavirdine. 04/19/14 77Biochemistry for medics
  77. 77. Treatment of HIV InfectionTreatment of HIV Infection 2) Protease inhibitors  • Protease inhibitors block HIV replication after  integration.  • These drugs inhibit the function of the protease  needed to process the ‘gag-pol’ polypeptide into  proteins.  • As a result, no infectious virus can be produced. • Since protease inhibitors act after integration, they  can obstruct infectious HIV production in both acutely  and chronically infected T cells and macrophages.  • Saquinavir, Ritonavir, Indinavir, and Nelfinavir are the  licensed protease inhibitors used in the clinical  practice04/19/14 78Biochemistry for medics
  78. 78. Treatment of HIV InfectionTreatment of HIV Infection 3) Combination treatment  • Since reverse transcriptase inhibitors and protease  inhibitors address different stages of viral replication,  using both families of drugs in combination has been  shown to be more effective than monotherapy in impeding  the spread of HIV in the body and reducing viral loads.  • This combination of two reverse transcriptase inhibitors,  one of which is a thymidine analogue, and a protease  inhibitor, blocks infection both before and after integration  and in both activated and resting T cells.  • This combinational therapy is termed as highly active anti- retroviral therapy (HAART). 04/19/14 79Biochemistry for medics
  79. 79. Treatment of HIV InfectionTreatment of HIV Infection 4) The future • In addition to the positive results shown by  combination therapy trials, a number of  developments may hold promise for the near  future.  • These include genetically engineered killer T cells  which attack HIV before it reproduces, and  research into genetically deactivating the CXCR4  and CCR5 T cell co-receptors, which are a path for  HIV entry into cells.  04/19/14 80Biochemistry for medics
  80. 80. Treatment of HIV InfectionTreatment of HIV Infection 04/19/14 81Biochemistry for medics
  81. 81. PreventionPrevention • The risk of contracting HIV increases with the  number of sexual partners.  • A change in the lifestyle can reduce the risk. • HIV-infected mothers are not recommended to  have children at present and pregnancy itself can  to be a risk factor for seropositive mothers.  • A recent clinical trial demonstrated the efficacy of  AZT in preventing transmission of HIV from the  mother to the fetus. 04/19/14 82Biochemistry for medics
  82. 82. PreventionPrevention • The spread of HIV through blood  transfusion had virtually been eliminated  since the introduction of blood donor  screening in many countries.  • Blood products such as factor VIII now  undergo routine treatment which appears  to inactivate any HIV present effectively. 04/19/14 83Biochemistry for medics
  83. 83. Development of vaccineDevelopment of vaccine Development of vaccine is fraught with several problems  unique to this virus. These include- 1) HIV can mutate rapidly, thus, it is not possible to design  antibodies against all antigens. 2) Antibody alone is not sufficient, cell mediated immunity  may also be necessary. 3) Virus enters the body not as free virions but also as  infected cells, in which the virus or the provirus is  protected against antibody or cell mediated lysis. 4) Virus readily establishes life long latent infection hiding  from antibodies. 04/19/14 84Biochemistry for medics
  84. 84. Approaches to an AIDS vaccineApproaches to an AIDS vaccine The main types of approaches to an AIDS vaccine are as  follows: • Live attenuated virus • Inactivated virus • Live recombinant viruses • Synthetic peptides • Recombinant DNA products (gp120, gp160) • Native envelope and/or core proteins • Anti-idiotypes antibodies • Passive immunization • To-date, the best hope lies in an inactivated vaccine 04/19/14 85Biochemistry for medics
  85. 85. Cause of death in HIV infectionCause of death in HIV infection • Despite progress in dealing directly with HIV, however, the  virus, by impairing the immune system, exposes the  infected person to a range of opportunistic viral, bacterial,  and parasitic infections and malignancies.  • It is these which are the actual cause of death in most  patients with AIDS and, notwithstanding the success of  anti-HIV drug treatment in reducing viral load, it is still  unclear whether HIV induced damage to the immune  system can be reversed.  • New strains of HIV undetectable by current screening  methods and resistant to the best antiretroviral drugs  currently available have also now been discovered.  04/19/14 86Biochemistry for medics
  86. 86. SummarySummary • Acquired immunodeficiency syndrome, is a disease caused  by Human Immuno deficiency Virus and is characterized  by marked Immunosuppression. • The mode of transmission is by sexual contact, blood  transfusions, needle prick injury or from mother to child. • About 5-10 % percent of the infected appear to escape  clinical AIDS for 15 years or more. They have been ‘long  term survivors” or “ long term non progressors”. • ELISA is the method most commonly employed for the  screening purpose and western Blotting is used for the  confirmation of the diagnosis. • Treatment is imparted simply to check the progression of  the disease, there is no cure of AIDS and there is no vaccine  available for prophylaxis of this disease.04/19/14 87Biochemistry for medics

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