Diagnostic Techniques In HIV

DIAGNOSIS
  • ELISA is the best screening test (Sensitivity > 99.9%)
  • Western blot is the confirmatory test (specificity when combined with ELISA > 99.9%)
  • Indeterminate results with Western blot: early HIV infection, HIV-2 infection, autoimmune disease, pregnancy, and recent tetanus toxoid administration
  • p24 is the earliest viral marker; p24 antigen capture assay (ELISA) is best for screening
  • The receptor for the virus is CD4 antigen, and therefore the virus may infect any cell bearing the CD4 antigen on the surface this is primarily the CD4 + (Helper) T Lymphocyte PCR is the most sensitive and specific in all stages and is the gold standard for diagnosis
  • HIV in newborns diagnosed by PCR (ELISA cannot be used for 18 months as IgG from mother is present)
  • HIV viral load - a measure of disease progression and response to antiviral medications. Best tests available for diagnosis of acute HIV infection (prior to seroconversion) Absolute CD4 cell count - Most widely used predictor of HIV progression
  • CD4 lymphocyte percentage - more reliable than the CD4 count.
  • Risk of progression to an AIDS opportunistic infection or malignancy is high with percentage < 14% in the absence of treatment
  • Chemokine co-receptor for HIV found on macrophage is CCR5
  • Virus isolation is by co cultivation of patient's lymphocytes with uninfected lymphocytes in the presence of IL-2
  • T cell anergy is commonly seen in HIV
  • Abnormal response of T cells to mitogens is a test used in HIV
  • HIV causes immune suppression, it causes hypergammaglobulinemia
HISTOPATHOLOGY OF HIV:
  • Florid reactive hyperplasia-may be: Collections of monocytoid B cells in sinuses
  1. Neutrophils
  2. Features of dermatopathic lymphadenopathy
  • Often reactive germinal centers show 'follicle lysis':i.e. invagination of mantle lymphocytes into germinal centers associated with:
  1. Disruption of centers ('moth-eaten appearance')
  2. Distinctive clustering of large follicular center cells resulting appearance termed explosive follicular hyperplasia
  • Occasional polykaryocytes:
  1. Warthin-Finkeldey cells
  2. May be multinucleated form of follicular dendritic cell
  • Electron microscopy:
  1. Sometimes prominent follicular dendritic cells exhibit alterations of their fine processes
  2. Interfollicular tissue may show prominent vascular proliferation: vague resemblance to Castleman's disease
  3. These areas and subcapsular region may reveal earliest signs of Kaposi's sarcoma
  4. Sometimes advanced lymphocyte depletion: may be abnormal (regressively transformed) germinal centers
WINDOW PERIOD
  • The time period between primary infection and antibody detection is called window period
  • Window period in HIV infection is 4-8 weeks
  • For -ve ELISA and western blot - window period is 22 days
  • It is associated with increased p24 antibody: basis for detection in window period.
  • For p24 detection(antigen) - 16 days
  • RNA PCR - 12 days
DISEASE MONITORING
  • CD4+ cell counts
  • HIV RNA - viral load
  • Beta-2-microglobulin and Neopterin(concentration increase with advancing disease)
  • DIAGNOSIS OF HIV IN INFANTS:
  • In adults HIV can be easily diagnosed by detecting IgG antibody to HIV (Elisa and western blot test). 
  • Average incubation period of AIDS is 10 years
  • But this method is not helpful in case of neonates because all neonates born to HIV infected mothers will have IgG antibody in their blood, regardless of infection status in them. These antibodies are passively transferred to the newborns from their mothers (IgG can cross placenta). 
  • These infants continue to test positive for IgG antibodies for upto 18 months.
  • At the end of 18 month they will lose these antibodies so uptil 18 month of age this method of diagnosis cannot be used.
  • The presence of IgA or IgM anti HIV in the infant's blood can indicate HIV infection, because these classes of antibodies do not cross the placenta. However detectable quantities of IgA antibodies are detected only after 3 months of life and detection of IgM antibodies is very unreliable (both insensitive and nonspecific). o So direct viral detection assays are used for the diagnoses of HIV in newborn.
  • These are
  1.  Detection of HIV DNA or RNA by PCR. 
  2. HIV p24 antigen
  3. HIV culture 
  4. Immune complex dissociated p24 antigen.
  5. These are very useful in young infants allowing a definitive diagnosis in most infected infants by 1 — 6 months of age. 
  6. Out of these method detection of viral DNA by PCR is the preferred method in developed countries.
CD4 COUNT & HIV ASSOCIATED DISEASES:
CD4 count 200- 500/μL CD4 count < 200/μL CD4 count < 100/μL
Tuberculosis Pneumocystis jeroveci Cryptococcal meningitis
Pneumococcal pneumonia Toxoplasmosis Primary CNS lymphoma
Herpes zoster, Herpes simplex Histoplasmosis Non Hodgkin lymphoma
Oropharyngeal candidiasis Cryptosporidiosis HIV associated dementia
Vaginal candidiasis Microsporidium PML
Oral Hairy leukoplakia Esophageal candidiasis Bartonella quintana
Kaposi sarcoma Extrapulmonary TB CMV colitis
Extraintestinal salmonellosis HIV associated Wasting CD4 count < 50/ pL
HIV associated ITP Peripheral neuropathy CMV retinitis
Lymphoid interstitial pneumonitis Nephropathy Disseminated MAC
Cervical intraepithelial neoplasia II,III Lymphoma
DIAGNOSIS OF AIDS ASSOCIATED DISEASES: 
  • Pneumonia in AIDS:
  1. Pneumocystis carinii is suspected as the causative organism
  2. The cysts, when stained with methenamine silver, have a characteristic cup or boat shape; the trophozoites are difficult to demonstrate without electron microscopy.
  3. It is also worth knowing that sputum samples are not nearly as effective as bronchial washes in demonstrating the organisms.
  • Penicillium marneffi:
  1. It is a dimorphic fungus
  2. It forms yeast like cells that are often intracellular resembling histoplasmosis.
  3. It causes systemic infections in AIDS patients in Asia
  • Candida tropicalis:
  1. All candida species can occur in yeast like form
  2. Bronchopulmonary candidiasis can occur as a rare complication of preexisting pulmonary or systemic disease. Crvptococcus neoformans
  3. Microscopy reveals capsulated yeasts
  4. It can cause lung infection in AIDS patient.
Tuberculosis:
  • In tuberculosis in an AIDS patient the chest X-ray looks like Miliary shadow
Exam Question

  • AIDS is associated with increased p24 antibody: basis for detection in window period.
  • Abnormal response of T cells to mitogens is a test used in HIV
  • HIV causes Hypergammaglobulinemia
  • Methenamine silver stain is used to detect cyst of Pneumocystis carinii in AIDS
  • Chemokine co-receptor for HIV found on macrophage is CCR5
  • During the Window period of patient with AIDS ELISA & western blot both are negative
  • Window period in HIV is from Infection to appearance of antibodies in serum
  • p24 antigen disappears from the blood after 6-8 weeks in HIV 
  • In HIV infected individual Gram stain of lung aspirate shows yeast like morphology that indicates Candida tropicalis, Cryptococcus neoformans or Pencillium marneffi infection Rapid progression of disease with full blown manifestation in AIDS occurs when T4 cell count falls below 200/microL
  • DNA-PCR, Viral culture, p24 antigen assay are used for diagnosis of HIV infection in a 2 month old child
  • According to CDC recommendations, HIV screening of pregnant women is Opt out testing
  • Warthin-Finkeldey cells, Marked follicular hyperplasia, Moth-eaten appearance are seen in lymph node biopsy of AIDS patients
  • Window period in HIV infection is 4-8 weeks
  • In tuberculosis in an AIDS patient the chest X-ray looks like Miliary shadow
  • Average incubation period of AIDS is 10 years
  • Best laboratory test to diagnose HIV infection Western blot
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