HUMAN IMMUNODEFICIENCY

VIRUS(HIV)

DR. SHRADDHA SIWAKOTI BPKIHS Dharan, Nepal

STRUCTURE OF HIV Virus• Spherical, enveloped virus,• 90-120nm.• Nucleocapsid-outer icosahedral Shell, inner cone shaped core• Genome diploid-composed 2 identical single-stranded positive sense RNA copies,Viral enzymes(RT,IT).

VIRAL GENES AND ANTIGENS

• Genome of HIV virus contains 3 structural genes(gag, pol & env).

• Other nonstructural and regulatory genes specific for virus.

• Product of these genes act as antigens.

• HIV shows 2 distinct antigenic types: HIV1 and HIV2.

• Envelope antigens of both types are different,

• core polypeptide shows some degree of cross-reactivity

STRUCTURAL GENES1. Gag gene• determines core and shell of virus.• Expressed as precursor protein p55 which is cleaved into 3 proteins• P15,p18 and p24.• p24 major core antigen- appears at early stage before antibody

detection.• Reappear late in course of infection; denotes exacerbation of illness.

STRUCTURAL GENES2. Env determines synthesis of envelope glycoprotein gp160;Gp120-forms surface spikesGp41-transmembrane anchoring protein

3. Pol gene codes for polymerase reverse transcriptase, protease, endonuclease.Expressed as precursor protein, cleaved into proteins p31,p51 and p66.

NONSTRUCTURAL GENES AND REGULATORY GENES

1. Tat (transactivating gene)-expression of all genes.

2. Nef (negative factor gene)- down regulating viral replication.

3. Rev (regulator of virus gene)-expression of structural proteins.

4. Vif (viral infectivity factor gene)-influencing infectivity of viral particles.

5. Vpu (HIV-1) and vpx (HIV-2)- enhancing maturation and release of progeny virus from cells.

6. Vpr stimulating promoter region of virus.

7. LTR(long terminal repeat) sequences

VIRAL REPLICATION

• Virus(gp120) binds to CD4 proteins on cell surface.

Gp120 also interacts with chemokine receptors(CXCR4 or CCR5).

• Gp41 mediates fusion of viral envelope with cell-membrane

• Entry of virus into cell and Uncoating

• Virion RNA dependent DNA polymerase transcribes

• Genome RNA into double stranded DNA.(provirus)

• Integrates with host cell DNA by viral integrase. LATENT INFECTION

Host cell RNA polymerase transcribes viral mRNA from proviral DNA

Viral mRNA encodes several proteins (gag, env, pol)

• Immature virions containing precursor polyproteins assembled in cytoplasm.

• Cleavage by viral protease occurs as immature virions buds from cell mem

• Production of mature infectious HIV

• Acquires lipoprotein envelope;

• lipids from host cell membrane, glycoproteins virus coded

PATHOGENESIS OF HIV INFECTION

• HIV primarily sexually transmitted pathogen; also IV drug abuse.

• HIV shows tropism for CD4 expressing cells;

• Helper T cells(primarily),macrophages(10-20%),B lymphocytes(5-10%)

• In genital tract, HIV infection begins in dendritic cells lining the mucosa)

• Followed by infection of local CD4+ helper T cells in genital tract

• Then viral replication occurs .

PATHOGENESIS OF HIV INFECTIONLATENT INFECTION

• Provirus can remain latent for long periods, though it influences host cell function.

• Long and variable incubation period is due to this latency.

• During latency, high level of viral replication occurs

• From time to time ,lytic infection is initiated releasing progeny virions which infect

other cells.

PATHOGENESIS OF HIV INFECTION• Killing of infected T cells and lytic release to infect new cells

• Leads to decrease in CD4 cells number, and T4: T8 cell ratio reversed.

• Viral infection also suppresses function of infected cell without causing structural

damage.

• Infected T4 cells do not appear to release normal amounts of IL-2,IFN-

gamma,other lymphokines.

• Marked damping effect on cell mediated immune response.

HOST IMMUNITY-CELLMEDIATED IMMUNUTY

• Development of cellular response produced against HIV proteins.

• HIV directly binds to CD4 receptor of T cells, depleting T cells.

• Causes Cell mediated T cell immunity gradually fail to

1. mount cytotoxic T cell response to virally infected cells

2.to process new foreign substance presented to immune system.

• Monocyte-macrophage function is also affected ,Due to lack of secretion of activating factors by T4 lymphocytes.

• Chemotaxis, antigen presentation and intracellular killing by monocytes/macrophages are diminished

HOST IMMUNITY-HUMORAL IMMUNUTY• Helper T cell activity is essential for optimal B cell function,• Particularly in responding thymus dependent antigens.• HIV infection leads to polyclonal activation of B lymphocytes• leading to hypergammaglobulinemia which is more of hinderance• Composed of useless Ig to irrelevant antigens and autoantibodies.• But development of neutralizing antibodies against HIV proteins is low

Natural history of HIV infectionStages include:• Primary infection, asymptomatic infection, early symptomatic infection

and advanced immunodeficiency with opportunistic complications,• Duration between primary infection and progression to clinical disease

averages 10yrs.• Untreated cases-death usually within 2yrs after onset of clinical

symptoms.• Following primary infection, there is 4-11days period between mucosal

infection and initial viremia.

Natural history of HIV infection• Viremia is detectable for about 8-12 weeks.• Virus widely disseminated throughout body and lymphoid organs

become seeded.• Significant drop in circulating CD4T cells at this early time.• Immune response to HIV occurs 1wk to 3mths after infection, plasma

viremia drops and level of CD4 cell rebound.• Immune response is unable to clear infection completely, HIV infected

cells persists in lymph nodes.

Natural history of HIV infection• Period of clinical latency may last as long as 10yrs-high level of

ongoing viral replication.• Half life of virus in plasma bout 6hrs,virus life cycle-2.6d.• Eventually patient will develop constitutional symptoms and clinical

apparent disease(opportunistic infections, neoplasm)• Higher levels of virus –advanced stage.

OPPURTUNISTIC INFECTIONs: AIDS-DEFINING DISEASES

BACTERIAL1. Extrapulmonary tuberculosis2. Disseminated nontuberculous mycobacterial infection3. Recurrent non-typhi salmonella septicemia

PARASITIC4. Chronic cryptosporidium, 5. Chronic isosporiasis6. Cerebral toxoplasmosis

OPPURTUNISTIC INFECTIONs: AIDS-DEFINING DISEASES

FUNGAL1. Esophageal candidiasis 5.Aspergillosis (not AIDS defining)2. Cryptococcal meningitis3. Pneumocystis jirovecii pneumonia4. Disseminated mycosis(coccidiomycosis, histoplasmosis)VIRAL5. Cytomegalovirus(retinitis or infection of other organs)6. Chronic mucocutaneous herpes simplex

Pneumocystis jirovecii pneumonia(PCP)

• Caused by Pneumocystis jirovecii.• Trimethoprim-sulfamethoxazole (TMP-SMX) : prophylactic agent .• Before use of PCP prophylaxis and ART, PCP occurred in 70% to 80% of

patients with AIDS.• 90% of PCP cases- (CD4 cell) counts <200 cells/mm3.• Cannot be cultured in vitro; Requires tissue culture/cell lines.• Diagnosis by induced sputum or Broncho alveolar lavage(BAL) .• Stains with wright-giemsa, methamine silver and direct

immunofluorescence.

Cluster of P. jirovecii stained with immunofluorescingantibody

Cyst wall stained with Silver stain

Cerebral toxoplasmosis

• Caused by the protozoan Toxoplasma gondii.• Primary infection-eating undercooked meat containing tissue cysts • or ingesting oocysts shed in cat feces .• Disease(Encephalitis): reactivation of latent tissue cysts.• Patients with CD4 counts <50 cells/µL are at greatest risk.• Diagnosis-seropositive for anti-toxoplasma (IgG) antibodies, CT, MRI.• seropositive patients with CD4 counts <100 cells/µL should receive

prophylaxis against TE.• (TMP-SMX), preferred regimen for (PCP) prophylaxis, is effective against TE.

Cryptosporidiosis • protozoan parasite Cryptosporidium parvum.• Infection occurs through ingestion of Cryptosporidium oocysts.• Common cause of chronic diarrhea in AIDS patients in developing

countries, with up to 74% of diarrheal stools.• CD4 <100 cells/µL—greatest risk for prolonged, severe, diarrhea or

extra intestinal cryptosporidiosis.• Diagnosis: microscopic identification of oocysts in stool or tissue with

acid-fast staining or direct immunofluorescence.

Modified ZN stained oocyst of cryptosporidiumImmunofluorescence of cryptosporidium oocyst

ISOSPORIASIS• protozoan parasite Isospora belli.• AIDS patient-extreme diarrhea that can lead to weakness, anorexia,

and weight loss.• Diagnosis-identification of oocyst through examining a stool sample

Modified ZN stained oocyst

Mycobacterium tuberculosis Infection • Tuberculosis (TB) infection occurs when susceptible person inhales

droplet nuclei containing Mycobacterium tuberculosis. • Immune response usually limits multiplication of tubercle bacilli

within 2 to 12 weeks after infection.• But viable bacilli persist for yrs -latent TB infection (LTBI). • Individuals with LTBI are asymptomatic and are not infectious.• TB disease (clinically active) can develop soon after exposure

(primary disease) or after reactivation of latent infection.

Mycobacterium tuberculosis Infection • with LTBI, risk of reactivation with TB disease increases after HIV

infection( 3 -12 times higher than general population)• TB disease can occur at any CD4 cell count, but risk increases with

progressive immunodeficiency.• ART results in prompt and marked decrease in incidence of TB disease• Diagnosis- chest xray, sputum smear and culture .

ZN stained AFB LJ media with growth

Disseminated nontuberculous mycobacterial infection

• M. avium.• Common agent causing mycobacterium avium complex(MAC)• inhalation, ingestion, or inoculation via respiratory or GI tract.• MAC disease occurs in patients with (CD4) cell counts <50 cells/mm3.• AIDS patients not on ART, MAC disease-disseminated, multi-organ infection.• Diagnosis: isolation of MAC from cultures of blood, lymph node,• bone marrow, or other normally sterile tissue or body fluids.

Numerous acid fast organisms growing within macrophages

ESOPHAGEAL CANDIDIASIS • Esophageal candidiasis are common in HIV-infected patients.• CD4T cell count <100 cells/mm3, • Causes painful swallowing.• Diagnosis: direct endoscopic visualization of lesions with histopathologic• demonstration of characteristic yeast forms in tissue • and confirmation by fungal culture.• Oropharyngeal candidiasis also common in HIV-infected patients but not

AIDS defining illness.CD4 Tcell <200 cells/mm3.

Esophageal candidiasisYeast infection in esophagus PAS stain

CRYPTOCOCCL MENINGITIS• Most HIV-associated cryptococcal infections are caused by

Cryptococcus neoformans.• HIV-infected patients, cryptococcosis commonly presents as • subacute meningitis or meningoencephalitis.• Most cases- CD4 T cell counts <100 cells/µL.• Diagnosed through culture, CSF microscopy, • or by cryptococcal antigen (CrAg) detection.

• India ink staining of CSF demonstrates encapsulated yeast in 60% to 80% of cases.

C. neoformans india ink prep

INVASIVE ASPERGILLOSIS• Aspergillus fumigatus most common• others; Aspergillus flavus, Aspergillus niger, • Invasive aspergillosis occurs in patients with advanced HIV infection

and was more common before ART.• CD4 T cell counts <100 cells/mm3, • History of other AIDS-defining opportunistic infections, and are not

receiving potent ART.

SEPTATE HYPHAE Aspergillus growth in SDA

Cytomegalovirus Disease

• DNA virus of Herpes virus family• Retinitis(most common), CD4 count <50 cells/mm3, • Colitis, Esophagitis, pneumonitis.• Most clinical disease occurs in previously infected with CMV

(seropositive) • represents either re-activation of latent infection or re-infection with

novel strain.

Chronic Mucocutaneous Herpes Simplex

• HSV infections are unrecognized clinically. • Oro labial herpes- most common manifestation of HSV-1 infection• untreated patients; 5 to 10 days. Lesions recur 1 to 12 times per year • Genital herpes- most common manifestation of HSV-2 infection• HSV-2 infection increases the risk of HIV acquisition 2-3fold,• CD4 ) T cell counts of <100 cells/µl• HSV DNA PCR, and viral culture diagnosis of mucocutaneous HSV.

LABORATORY DIAGNOSIS1. Serodiagnosis

A. Demonstration of antibodies(6-12 wks after infection)

I. Screening Tests II. Supplementary/Confirmatory tests.

B. Demonstration of viral antigen

2. Viral isolation

3. Polymerase chain reaction (PCR)

Screening Tests• Usually highly sensitive.• used for initial screening of serum samples for presence of HIV antibodies.1. ELISA • used for detection of HIV-1 and HIV-2 specific antibodies in serum .• High sensitive and specific.• Can also be used for antibodies detection in saliva.• Four types depending upon nature of antigen used • And detecting both antibodies and antigen in serum.

ELISA in HIV TYPE OF ELISA SOURCE OF ANTIGEN ANTIGEN/ANTIBODY

DETECTIONFirst-generation ELISA Cultured virus lysate Detection of antibodies

Second-generation ELISA Recombinant antigen Detection of antibodies

Third-generation ELISA Synthetic peptides Detection of antibodies

Fourth-generation ELISA Mixture of both Synthetic peptides And Recombinant glycopeptides.

Detection of both antigens and antibodies

Screening Tests 2. Rapid tests• Simple tests which can be performed in any laboratory• without requiring expensive instrument or skilled manpower.• Tests results can be read rapidly within 30mins of receipt of specimen

Supplementary/Confirmatory Tests• Used as confirmatory tests for detection of HIV antibodies.• Designed for higher specificity than screening tests,• Hence used as test of choice to verify results of screening tests.• Western blot, line immunoassay, indirect immunofluorescence.• Earlier western blot was used to confirm positive result of any

screening test.(cumbersome, expensive,not readily available)• Now-perform 2 different types of ELISA or an ELISA with any of rapid

tests.

WESTERN BLOT

WESTERN BLOT

• Test considered positive if it shows bands1. against at least 2 of 3 viral proteins,p24, gp41 and gp120/gp160.

• Equivocal/early infection-band at 1 site,p24 or gp120.• Negative-absence of any band.

WINDOW PERIOD• Time interval before an antibody appears in serum.• Varies 3-4 weeks.• Serum of patient tested during this period is negative for serum

antibodies but positive for viral antigens.

DEMONSTRTION OF VIRAL ANTIGENS• Can be detected during window period when antibodies can’t be

detected.• p24 antigen appears usually 16 days after infection.• Antibody sandwich ELISA using specific monoclonal antibodies to HIV p

24-used to detect p24 antigen.• P24 antigen become undetectable after HIV antibodies develop in blood.• Due to formation of immune complex by p24 with antibodies• But p24 appear late in course of infection-poor prognosis.

VIRAL ISOLATION• Not routinely used for diagnosis, Used for research purpose. • Virus mostly isolated from lymphocytes in peripheral blood,

COCULTIVATION• Cocultivating potentially infected and uninfected mononuclear cells to

facilitate replication of HIV.• Viral growth –presence of p24 antigen and HIV reverse transcriptase.• positive: 7-14days,upto 28d.• Useful for detection of high virus titre ,(early infection).

POLYMERASE CHAIN REACTION• As most sensitive and specific, has become gold standard for

diagnosis in all stages of infection.• Two forms1. DNA PCR-sensitive method for detection of HIV provirus2. RNA PCR (Reverse transcriptase polymerase chain reaction; RT-PCR)• can be used for diagnosis as well as level of viremia.(viral load)• Useful for early diagnosis of HIV infection in infants born to infected

mothers.

Sequence of appearance of laboratory markers for HIV-1 infection

MONITORING STATUS OF HIV INFECTION1. CD4+ T cell count• Reflects current immunological competence of patient.• HIV positive persons should have frequent CD4 Tcell counts.• If <500-indication of disease progression; <200-risk of serious infection.2. Measurement of HIV RNA• Marker for disease progression and are prognostic marker to monitor

effectiveness of anti-HIV therapies.3.B2 macroglobulin and neopterin ,measured in serum/urine.Concentration low in asymptomatic stage, rise with advancing disease.

NATIONAL GUIDELINESFOR VOLUNTARY HIV/AIDS COUNSELING AND TESTING

• Voluntary HIV/AIDS counseling, testing and referral (VCT) is a major strategy in HIV/AIDS prevention and care.• All testing must be accompanied by pre-and post- test counseling with

informed consent. • Client should be informed of result of testing only with post-test

counseling.• Test results must be kept confidential

VCT Process

PMTCT• Testing during pregnancy and for prevention of mother to child

transmission of HIV.• To be able to make informed decisions about safe infant feeding and

access to antiretroviral (ARV) therapy, pregnant women needs to know and understand her HIV status.• during pregnancy, during labor and delivery and after birth through

breastfeeding.• Pre- and post-test counseling sessions

HIV Test Algorithms For Nepal• After assessing current situation in country regarding technical and

logistic problems of ELISA machines and kits,

MINIMUM STANDARD• use of 2 or more rapid tests based on different test principles

(antigen) is recommended to be followed • at all levels of health care delivery system (hospitals, health centers,

clinics, etc.) in government, private and NGO settings.

National standard HIV testing strategy

Universal Precautions