training manual hiv
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TRAINING MANUAL
HUMAN IMMUNODEFICIENCY
VIRUS (This Manual is for internal circulation only)
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Introduction
Human immunodeficiency virus or HIV is a retrovirus that causes AcquiredIimmune Deficiency Syndrome (AIDS), a condition in humans in which the immune system begins to fail, leading to life-threatening opportunistic infections. HIV infection in humans is now pandemic. As of January 2006, the Joint United Nations Programme on HIV/AIDS (UNAIDS) and the World Health Organization (WHO) estimate that AIDS has killed
more than 25 million people since it was first recognized on December 1,
1981, making it one of the most destructive pandemics in recorded history. According to the latest figures published today in the UNAIDS/WHO 2006 AIDS Epidemic Update, an estimated 39.5 million people are living with HIV. There were 4.3 million new infections in 2006 with 2.8 million (65%) of these occurring in sub-Saharan Africa and important increases in Eastern Europe and Central Asia, where there are
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some indications that infection rates have risen by more than 50% since 2004. In 2006, 2.9 million people died of AIDS-related illnesses.
Origin and discovery
The AIDS epidemic was discovered in June 5, 1981, when the U.S. Centers for Disease Control and Prevention reported a cluster of Pneumocystis
carinii pneumonia in five homosexual men in Los Angeles.
In 1983, scientists led by Luc Montagnier at the Pasteur Institute in France first discovered the virus that causes AIDS. They called it lymphadenopathy-associated virus (LAV).
Two species of HIV infect humans: HIV-1 and HIV-2. HIV-1 is thought to have originated in southern Cameroon after jumping from wild chimpanzees (Pan troglodytes troglodytes) to humans during the twentieth century. HIV-2 may have originated from the Sooty Mangabey (Cercocebus atys), an Old World monkey of Guinea-Bissau, Gabon, and Cameroon.
HIV-1 is more virulent. It is easily transmitted and is the cause of the majority of HIV infections globally. HIV-2 is less transmittable and is largely confined to West Africa. HIV-1 is the virus that was initially discovered and termed LAV.
Transmission
Since the beginning of the pandemic, three main transmission routes for HIV have been identified:
• Sexual route. The majority of HIV infections are acquired through unprotected sexual relations. Sexual transmission can occur when infected sexual secretions of one partner come into contact with the rectal, genital or oral mucous membranes of another.
• Blood or blood product route. This transmission route can account for infections in intravenous drug users, hemophiliacs and recipients of blood transfusions (though most transfusions are checked for HIV in the developed world) and blood products. It is also of concern for persons receiving medical care in regions where there is prevalent substandard hygiene in the use of injection equipment, such as the reuse of needles in Third World countries. Health care workers such as nurses, laboratory workers, and doctors, have also been infected, although this occurs more rarely. People who give
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and receive tattoos, piercings and scarification procedures can also be at risk of infection.
• Mother-to-child transmission (MTCT). The transmission of the virus from the mother to the child can occur in utero during the last weeks of pregnancy and at childbirth. In the absence of treatment, the transmission rate between the mother and child is 25%. However, where drug treatment and Cesarian section are available, this can be reduced to 1%. Breast feeding also presents a risk of infection for the baby.
Structure and Genome
HIV is different in structure from other retroviruses. It is about 120 nm in diameter and roughly spherical.
It is composed of two copies of positive single-stranded RNA that codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein, p24. The single-stranded RNA is tightly bound to nucleocapsid proteins, p7 and enzymes needed for the development of the virion such as reverse transcriptase, proteases and integrase.
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A matrix composed of the viral protein p17 surrounds the capsid ensuring the integrity of the virion particle. This is, in turn, surrounded by the viral envelope which is composed of two layers of fatty molecules called phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell.
Embedded in the viral envelope are proteins from the host cell and about 70 copies of a complex HIV protein that protrudes through the surface of the virus particle. This protein, known as Env, consists of a cap made of three molecules called glycoprotein gp 120, and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope.
This glycoprotein complex enables the virus to attach to and fuse with target cells to initiate the infectious cycle. Both these surface proteins, especially gp120, have been considered as targets of future treatments or vaccines against HIV.
Of the nine genes that are encoded within the RNA genome, three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles.
Env, for example, codes for a protein called gp160 that is broken down by a viral enzyme to form gp120 and gp41.
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The six remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease.
Tropism
The term viral tropism refers to which cell types HIV infects. HIV can infect a variety of immune cells such as CD4+ T cells, macrophages, and microglial cells. HIV-1 entry to macrophages and CD4+ T cells is mediated through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells and also with chemokine coreceptors.
Replication cycle
Entry to the cell
HIV enters macrophages and CD4+ T cells by the adsorption of glycoproteins on its surface to receptors on the target cell followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell.
The interactions of the gp160 spike and both CD4 and a chemokine receptor on the cell surface. The gp160 spike contains binding domains for both CD4 and chemokine receptors
The first step in fusion involves the high-affinity attachment of the CD4 binding domains of gp120 to CD4. Once gp120 is bound with the CD4
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protein, the envelope complex allows for a more stable pronged attachment, which allows the N-terminal
fusion peptide gp41 to penetrate the cell membrane.
This is followed by a collapse of the extracellular portion of gp41 into a hairpin. This loop structure brings the virus and cell membranes close together, allowing fusion of the membranes and subsequent entry of the viral capsid.
Once HIV has bound to the target cell, the HIV RNA and various enzymes, including reverse transcriptase, integrase and protease, are injected into the cell.
HIV can infect dendritic cells and they are one of the first cells encountered by the virus during sexual transmission. They are currently thought to play an important role by transmitting HIV to T cells once the virus has been captured in the mucosa by DCs.
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Replication and transcription
Once the viral capsid enters the cell, an enzyme called reverse transcriptase liberates the single-stranded (+)RNA from the attached viral proteins and copies it into a complementary DNA. This process of reverse transcription is extremely error-prone and it is during this step that mutations may occur.
The reverse transcriptase then makes a complementary DNA strand to form a double-stranded viral DNA intermediate (vDNA). This vDNA is then transported into the cell nucleus. The integration of the viral DNA into the host cell's genome is carried out by another viral enzyme called integrase.
This integrated viral DNA may then lie dormant, in the latent stage of HIV infection. To actively produce the virus, certain cellular transcription factors need to be present, like the NF kappa B.
In this replication process, the integrated provirus is copied to mRNA which is then spliced into smaller pieces. These small pieces produce the regulatory proteins Tat (which encourages new virus production) and Rev. At this stage, the structural proteins Gag and Env are produced from the full-length mRNA. The full-length RNA is actually the virus genome; it binds to the Gag protein and is packaged into new virus particles.
HIV-1 and HIV-2 appear to package their RNA differently; HIV-1 will bind to any appropriate RNA whereas HIV-2 will preferentially bind to the mRNA which was used to create the Gag protein itself. This may mean that HIV-1 is better able to mutate (HIV-1 infection progresses to AIDS faster than HIV-2 infection and is responsible for the majority of global infections).
Assembly and release
The final step of the viral cycle, assembly of new HIV-1 virons, begins at the plasma membrane of the host cell. The Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by protease and processed into the two HIV envelope glycoproteins gp41 and gp120.
These are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. The Gag (p55) and Gag-Pol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell. Maturation either occurs in the forming bud or in the immature virion after it buds from the host cell.
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During maturation, HIV proteases cleave the polyproteins into individual functional HIV proteins and enzymes. The various structural components then assemble to produce a mature HIV virion. This cleavage step can be inhibited by protease inhibitors. The mature virus is then able to infect another cell.
Genetic variability
HIV differs from many other viruses as it has very high genetic variability. This diversity is a result of its fast replication cycle, with the generation of 109 to 1010 virions every day, coupled with a high mutation rate. This complex scenario leads to the generation of many variants of HIV in a single infected patient in the course of one day. This variability is compounded when a single cell is simultaneously infected by two or more different strains of HIV.
Three groups of HIV-1 have been identified on the basis of differences in Env: M, N, and O.
Group M is the most prevalent and is subdivided into eight subtypes (or clades), based on the whole genome, which are geographically distinct. The most prevalent are subtypes B (found mainly in North America and Europe), A and D (found mainly in Africa), and C (found mainly in Africa and Asia); these subtypes form branches in the phylogenetic tree representing the lineage of the M group of HIV-1.
Coinfection with distinct subtypes gives rise to circulating recombinant forms (CRFs). In 2000, the last year in which an analysis of global subtype prevalence was made, 47.2% of infections worldwide were of subtype C, 26.7% were of subtype A/CRF02_AG, 12.3% were of subtype B, 5.3% were of subtype D, 3.2% were of CRF_AE, and the remaining 5.3% were composed of other subtypes and CRFs. Most HIV-1 research is focused on subtype B; few laboratories focus on the other subtypes.
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The genetic sequence of HIV-2 is only partially homologous to HIV-1 and more closely resembles that of SIV than HIV-1.
The clinical course of infection
A generalized graph of the relationship between HIV copies (viral load) and CD4 counts over the average course of untreated HIV infection; any particular individual's disease course may vary considerably.
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CD4+ T cell count (cells per µL) HIV RNA copies per mL of plasma
WHO clinical staging system for HIV infection and related
Disease in adults.
Stage 1: The first stage of infection, the primary, or acute infection, is a period of rapid viral replication that immediately follows the individual's exposure to HIV leading to an abundance of virus in the peripheral blood with levels of HIV commonly approaching several million viruses per mL. This response is accompanied by a marked drop in the numbers of circulating CD4+ T cells. This acute viremia is associated in virtually all patients with the activation of CD8+ T cells, which kill HIV-infected cells, and subsequently with antibody production, or seroconversion. Symptoms
• Asymptomatic • Persistent generalized lymphadenopathy. Performance scale 1: asymptomatic, normal activity
Stage 2: (Also known as Clinical Latency Period): A strong immune defense reduces the number of viral particles in the blood stream, marking the start of the infection's clinical latency stage. Clinical latency can vary between two weeks and 2 years. During this early phase of infection, HIV is active within lymphoid organs, where large amounts of virus become trapped in the follicular dendritic cells (FDC) network. The surrounding tissues that are rich in CD4+ T cells may also become infected, and viral particles accumulate both in infected cells and as free virus. Following which the symptoms start to appear. Symptoms
• Weight loss < 10% of body weight. • Minor mucocutaneous manifestations (e.g. oral ulcerations, fungal nail infections) • Herpes zoster within the last 5 years. • Recurrent upper respiratory tract infections (e.g. bacterial sinusitis) Performance scale 2: symptomatic, normal activity
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Stage 3: When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and infections with a variety of opportunistic microbes appear. Common opportunistic infections and tumors, most of which are normally controlled by robust CD4+ T cell-mediated immunity then start to affect the patient. Symptoms
• Weight loss > 10% of body weight. • Unexplained chronic diarrhoea for more than 1 month. • Unexplained prolonged fever for more than 1 month. • Oral Candidiasis (thrush). • Oral hairy leukoplakia. • Pulmonary TB • Severe bacterial infections (pneumonia, pyomyositis). Performance scale 3: bedridden < 50% of the day during the last month.
Stage 4: In the final stages of AIDS, infection with cytomegalovirus (another herpes virus) or Mycobacterium avium complex is more prominent. Not all patients with AIDS get all these infections or tumors, and there are other tumors and infections that are less prominent but still significant. Symptoms • HIV-wasting syndrome, as defined by CDCa. • Pneumocystis carinii pneumonia. • Toxoplasmosis of the brain. • Cryptosporidiosis with diarrhoea, for more than 1 month. • Cryptococcosis, extrapulmonary. • Cytomegalovirus (CMV) disease of an organ other than liver, spleen, lymph nodes. • Herpes virus infection, mucocutaneous for more than 1month, or visceral any duration. • Progressive multifocal leukoencephalopathy (PML). • Any disseminated endemic fungal infection. • Candidiasis of the oesophagus, trachea, bronchi or lungs • Atypical mycobacteriosis, disseminated • Non-typhoid salmonella septicaemia. • Extrapulmonary TB. • Lymphoma.
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• Kaposi sarcoma • HIV encephalopathy, defined by CDCb. Performance scale 4: bedridden > 50% of the day during the last month a HIV wasting syndrome = weight loss > 10% of body weight, plus either unexplained diarrhoea for more than one month or chronic weakness and unexplained fever for more than one month. b HIV encephalopathy = clinical findings of disabling mental or motor dysfunction, interfering with activities of daily living, progressing over weeks and months, in the absence of a concurrent illness or condition other than HIV infection which could explain the findings.
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Diagnosis
Terminology
The window period is the time from infection until a test can detect any change. The average window period with antibody tests is 22 days. Antigen testing cuts the window period to approximately 16 days and NAT further reduces this period to 12 days.
Performance of medical tests is often described in terms of:
• Sensitivity: The percentage of the results that will be positive when HIV is present
• Specificity: The percentage of the results that will be negative when HIV not present.
All diagnostic tests have limitations, and sometimes their use may produce erroneous or questionable results.
• False positive results indicate that HIV is found to be present when, in fact, it is not.
• False negative results do not identify HIV that is present.
Nonspecific reactions, hypergammaglobulinemia, or the presence of antibodies directed to other infectious agents that may be antigenically similar to HIV can produce false positive results. Autoimmune diseases, such as systemic lupus erythematosus, can also cause false positive results.
Principle
HIV infection is usually diagnosed through detection of antibodies to the virus. Production of these antibodies usually begins 3–8 weeks after infection. Diagnosis of HIV infection is also possible through detection of the virus p24 antigen, nucleic acid based tests or culture.
HIV Antibody tests The most widely available way of identifying HIV-infected individuals is the detection of HIV antibodies in serum or plasma samples. There is a range of different principles that are employed for the detection of infection from the virus:
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• RAPIDS Rapid Antibody Tests are qualitative immunoassays intended for use as a point-of-care test to aid in the diagnosis of HIV infection. These tests should be used in conjunction with the clinical status, history, and risk factors of the person being tested. Some of the commonly used techniques for rapid tests are:- A: Immunochromatography: This is also known as the lateral flow technique. In this technique the sample (ag/ab) is complexed with a conjugate, this conjugate – ag/ab complex then flows through the immunochromatographic strip. Which is then detected with the help of antibodies immobilized on the test line. B. FLOW THROUGH TECHNOLOGY: HIV antigens are immobilized on a porous immunofiltration membrane. Sample and reagents pass through the membrane and are absorbed into the underlying absorbent. As the patient's sample passes through the membrane, HIV antibodies, if present, bind to the immobilized antigens. Conjugate binds to the Fc portion of the HIV antibodies to give distinct pinkish purple DOT(s) against a white background.
HIV TRI-DOT HIV TRDOT from J. Mitra is a CE certified, indigenously developed product, which based on the patented Flow Through technology. HIV TRIDOT designed using gp-41, C terminal of gp-120 & gp-36 representing the immunodominant regions of HIV-1 & HIV-2 envelope gene structure respectively.
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The device (an immunofiltration membrane) includes a "Built-in Quality Control DOT" which will develop colour during the test, thereby, confirming proper functioning of the device, reagents and correct procedural application. This CONTROL DOT is the "Built -in Quality Control."
The following are the Salient Features of HIV TRI –DOT:-
• This test is based on Flow through Technology having the unique washing steps same as that in Elisas, which ensure better results.
• It can detect all subtypes of HIV-1 & HIV-2 including HIV-1 subgroup C & O.
• Separate dots for HIV-1 & HIV-2. • Result in less than 3 minutes • Built in quality control • High Sensitivity & Specificity • Kit presentation- 10 T, 50 T, 100 T, 200 T. • Long shelf life. • Evaluations from reputed agencies like WHO, have found the kit to
be excellent in performance and easy to use, and also stated that the kit is able to detect the infection in about 1.7 days after the reference test.
PERFORMANCE CHARACTERISTICS
Sensitivity and Specificity studies were carried out on samples fresh as well as frozen from low risk as well as high-risk groups. Performance of the test with reference to sensitivity and specificity has been determined by NATIONAL HIV REFERENCE CENTRES of Govt. of India and WHO, Geneva using various testing panels. These evaluations indicate the following Sensitivity and Specificity:
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* Evaluation Reports of National HIV Reference Laboratories Govt. of India (AIIMS New Delhi, CMC Vellore, NICD New Delhi) ** As per evaluation of WHO; CH-1211, GENEVA, 27 SWITZERLAND, August 1999.
Other Evaluations:
• Evaluation from UNAIDS, WHO. Claiming the sensitivity to 100% and specificity to be 100%.
• Evaluation from JAIDS JOURNAL OF ACQUIRED IMMUNODEFICIENCY SYNDROMES, Claiming the sensitivity to 99.5% and specificity to be 99.9%.
• Evaluation from JCM, JOURNAL OF CLINICAL MICROBIOLOGY, Claiming the sensitivity to 99.5% and specificity to be 99.9% resp.
• Blood Bank, SMS Jaipur, Claims the test to be suitablefor use in blood bank and known to produce good results.
HIV COMB HIV EIA COMB is a CE certified product which has been specially researched, developed and designed using gp-41, C terminal of gp-120 & gp-36 representing the immunodominant regions of HIV-1 & HIV-2 envelope gene structure respectively. The comb (a polystyrene comb) includes a Built – in quality Control Dot, which will develop colour during the test, thereby, confirming proper functioning of the comb, reagents and correct procedural application.
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Other Evaluations:
• National AIDS Research Institute has found the specificity and sensitivity to be 100%, for HIV Comb.
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HIV EIA COMB This Test has been developed and designed using gp-41, C terminal of gp 120 & gp-36 representing the immunodominant regions of HIV-1 & HIV-2 envelope gene structure respectively. The comb (a polystyrene comb) includes a "Built -in Quality Control DOT" which will develop colour during the test, thereby, confirming proper functioning of the comb, reagents and correct procedural application. This test has been specially researched, developed and engineered using several thousands of serum/plasma specimens. The Sensitivity and Specificity has been extremely high in these samples of diverse origin. PRINCIPLE OF THE TEST
HIV EIA comb is an enzyme immunoassay (EIA) and employs the binding of enzyme with chromogenic substrate to visualize the immobilized immune complex. HIV antigens are immobilized as circular spot on the polystyrene comb. When incubated with a specimen containing HIV-1 and/or HIV-2 antibodies, these antibodies bind specifically to the immobilized antigens. The comb is washed to remove unbound antibodies. The comb is then placed in microwells containing enzyme conjugate (Alkaline phosphatase conjugated anti-human IgG). This conjugate will bind to antigen antibody complex present on the comb. Finally the comb is placed in microwells containing substrate and is incubated. The bound conjugate will react with substrate. The results are directly visualized by the presence of distinct grey-blue dot(s) on the surface of comb.
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PERFORMANCE CHARACTERISTICS
An elaborated study has been done on HIV EIA COMB to determine its performance as an anti-HIV screening test. The performance of the test with reference to sensitivity and specificity was evaluated in-house with fresh as well as frozen samples from low risk as well as high risk groups by using a panel containing 5165 nos. of known serum samples. The results of all the sera with a defined HIV status were fully comparable with these of HIV EIA COMB. The results of the in-house study done are as follows:
Sensitivity: 100% (161/161 Western Blot Positive Sera & Elisa positive sera) Specificity: 99.9% (5001/5004 EIA Negative Sera)
Other Evaluations:
National AIDS Research Institute has found the specificity and sensitivity to be 100%, for HIV EIA Comb.
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• ELISAS The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.
MICROLISA – HIV The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. MICROLISA-HIV
from J. Mitra is developed to detect anti-HIV ENV (envelope) antibodies to HIV-1 and / or HIV-2 with equal reactivity. It has been observed that the core protein of HIV-1 and HIV-2 show cross reactivity whereas envelope proteins are more type specific and moreover antibodies against the envelope gene products can be found in almost all infected people. Microlisa-HIV has been developed and designed to be extremely sensitive and specific using recombinant proteins (gp41, C terminus of gp120 and gp36) representing the immunodominant regions of HIV-1 & HIV-2 envelope gene structure respectively. PRINCIPLE OF THE TEST
Microlisa HIV test is an enzyme immunoassay based on Indirect ELISA. Recombinant proteins gp41, C terminus of gp 120, and gp 36 for HIV-1 and HIV-2 representing immunodominant epitopes are coated onto microtiter wells. Specimens and controls are added to the microtiter wells and incubated. Antibodies to HIV-1 and HIV-2 if present in the specimen, will bind to the specific antigens adsorbed onto the surface of the wells. The plate is then washed to remove unbound material. Horseradish peroxidase (HRP) conjugated antihuman IgG is added to each well. This conjugate will bind to HIV antigen-antibody complex present. Finally substrate solution containing chromogen and hydrogen peroxide is added to the wells and incubated. A blue colour will develop in proportion to the amount of HIV-1 and / or HIV-2 antibodies present in the specimen. The colour reaction is stopped by a stop solution. The enzyme substrate reaction is read by an EIA reader for absorbance at a wavelength of 450 nm. If the sample does not contain HIV-1 or HIV-2 antibodies then enzyme conjugate will not bind and the solution in the wells will be either colourless or only a faint background colour develops.
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PERFORMANCE CHARACTERISTICS Sensitivity and Specificity studies were carried out on samples fresh, as well as frozen, from low risk as well as high-risk groups. Performance of the test with reference to sensitivity and specificity has been determined by NATIONAL HIV REFERENCE CENTRES of Govt. of India and WHO Collaborating Centre, using various testing panels. These evaluations indicates the following Sensitivity and Specificity:
* As per evaluation report dated 20th Feb. 1998, of WHO Collaborating Centre, Instituutvoor Tropische Geneeskunde Nationalestraat 155-B 2000 Antwerpen, Balgium. ** Evaluation Reports of National HIV Reference Laboratories of Govt. of India (CMC Vellore). Drug Controller General (India), Directorate General of Health Services, Govt. of India, New Delhi. (Letter dated 8th May 1997). Seven HIV-O sera were included in reference serum panel. Microlisa-HIV has detected all the seven HIV-O positive samples as reactive thereby confirming its 100% Sensitivity & Specificity for HIV-O positive samples as well. A low performance and mixed panel from BBI (Boston Biomedica Inc.) were also tested with Microlisa - HIV were identical with the Western Blot data provided by BBI for the above mentioned low performance and mixed panel. Other Evaluations
• Indian Journal of Medical Microbiology, claims sensitivity and specificity of MICROLISA HIV to be 100% and 99.5%.
• Institute of Microbiology, Madras Medical College claimed the kits to be very satisfactory in performance.
• Department of Microbiology, Nizam’s Institute of Medical Sciences claimed the specificity, sensitivity and efficiency to be 100%.
• Western blot The specimen is reported as repeatedly reactive by the above tests are then tested by Confirmatory Test, such as Western Blot.
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HIV Western Blot
It is an in vitro qualitative immunoassay for the detection of antibodies to HIV-1 & HIV-2 in human serum / plasma. It is manufactured by J. Mitra & Co. Pvt. Ltd., based on lab research at CRI, Mumbai, under sponsorship from DBT (Department of Biotechnology) Govt. of India, New Delhi. It is intended to be used as a more specific & supplemental assay on samples found initially reactive using ELISA and other screening tests.
PRINCIPLE OF THE TEST
The HIV Western Blot is manufactured from HIV-1 cell line. The HIV-1 viral antigen is purified and then separated by SDS gel electrophoresis. SDS denatures viral components and yields proteins, which migrate in the gel according to their molecular weight to produce various bands. Low molecular weight components migrate faster and are found at the bottom of the gel, while high molecular weight proteins remain near the top. They are then transferred from SDS-PAGE gel on to nitrocellulose membrane, which is also impregnated with HIV-2 antigen and a control band. The membrane is cut and packaged as strips. To perform the assay, the strip is incubated with the patient serum/plasma diluted in a buffer. Antibodies to HIV- 1 & 2 if present, bind to viral antigens located on the strip. Unbound material is washed off and then the strip is incubated with anti-human IgG conjugated to alkaline phosphatase. After washing the unbound conjugate, substrate (BCIP/NBT) is added which results in the staining of bands. If antibodies to HIV-1 antigens are present in the sera three [two ENVELOPE with GAG (p24)] or more of the following bands will be seen: p24, p31, gp41, p51/p55, p66, gp120 & gpl60. If antibodies to HIV-2 antigens are present, HIV-2 band is also observed along with some of the other bands. If HIV specific antibodies are not present, the band pattern does not meet the required criteria.
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PERFORMANCE CHARACTERISTICS
1. Several known (ELISA positive sera /plasma samples as well as sera from known control subjects) and unknown sera /body fluids were tested on different batches of strips. A total of 476 samples from different hospitals at Mumbai were tested initially at the Virology Laboratory, Cancer Research Institute, Mumbai. These samples included mainly sera but also included milk from HIV positive mothers, CSF from HIV positive children with neurological complications as well as sera from healthy volunteers. However, finally 250 coded sera originating from five different national institutes (National Institute of Communicable Diseases, New Delhi; PGIMER, Chandigarh; Christian Medical College, Vellore; National AIDS Research Institute, Pune and National Institute of Cholera and Enteric Diseases, Calcutta) were sent by Dept of Biotechnology (DBT), Govt. of India, New Delhi. Results were compared with those obtained by the source institutes and the sensitivity & specificity were evaluated. The results showed 100%
sensitivity & 100% specificity. This compares excellently with the major commercially available kit performances. The specificity of HIV W. Blot was also checked with normal sera and sera with other viral infections. The results showed 100% Specificity. SPECIFICITY OF HIV Western BLOT WITH SERA OF NORMAL DONOR AND SERA WITH OTHER VIRAL INFECTIONS IS AS FOLLOWS:
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2. The performance of HIV W. Blot was also evaluated in clinical studies and compared to one licensed Western Blot test. The results are as follows:
The HIV Western Blot was positive in 74/74 of cases identified as positive by Licensed Western Blot test, demonstrating 100% sensitivity. The HIV Western
Blot also showed 100% specificity when compared with Licensed Western Blot test.
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Antigen tests
The p24 antigen test detects the presence of the p24 protein of HIV (also known as CA), a major core protein of the virus. Monoclonal antibodies specific to the p24 protein are mixed with the person's blood. Any p24 protein in the person's blood will stick to the monoclonal antibody and enzyme-linked antibody to the monoclonal antibodies to p24 causes a color change if p24 was present in the sample.
But as p24 dissapears is a short span, detection based on the p24antigen only is not that reliable.
Nucleic Acid Based Tests (NAT)
Nucleic-acid-based tests amplify and detect a 142-base target sequence located in a highly conserved region of the HIV gag gene. Blood screened with nucleic-acid-based tests, shortening the window period between infection and delectability of disease to about 12 days. Since these tests are relatively expensive, the blood is screened by first pooling some 10-20 samples and testing these together; if the pool tests positive, each sample is retested individually. In the RT-PCR test, viral RNA is extracted from the patient's plasma and is treated with reverse transcriptase so that the RNA of the virus is transcribed into DNA. The polymerase chain reaction (PCR) is then applied, using two primers thought to be unique to the virus's genome. After the PCR amplification process is complete, the resulting amplified segments bind to specific oligonucleotides bound to the vessel wall and are then made visible with a probe bound to an enzyme. The amount of virus in the sample can be quantified with sufficient accuracy to detect three-fold changes. In the branched DNA test, plasma is centrifugated to concentrate the virus, which is then opened to release its RNA. Special oligonucleotides are added which bind to viral RNA and to certain oligonucleotides bound to the wall of the vessel. In this way, viral RNA is fastened to the wall. Then new oligonucleotides are added which bind at several locations to this RNA; and other oligonucelotides, which bind at several locations to those oligonucleotides. This is done to amplify the signal. Finally, oligonucleotides that bind to the last set of oligonucleotides and that are bound to an enzyme are added; the enzyme action causes a color reaction, which
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allows quantification of the viral RNA in the original sample. Monitoring the effects of antiretroviral therapy by serial measurements of plasma HIV-1 RNA with this test has been validated for patients with viral loads greater than 25,000 copies per milliliter.
FFOOUUTTHH GGEENNEERRAATTIIOONN AANNTTIIGGEENN AANNDD AANNTTIIBBOODDYY TTEESSTTSS
HIV fourth generation test, is a recent development in HIV Diagnosis. This test brings together the advantages of both Antibody based tests and Antign based tests, and at the same time nullifies the limitations offered by both of them.
Specific antibody to HIV is synthesized soon after infection, although the precise time may depend on several factors, including both host and viral characteristics. Significantly, antibody may be present at low levels during early infection; however, these levels may be below the minimum concentration detectable by some assays. Antibody is detected in a majority of individuals within 6 to 12 weeks after infection with the earlier generations of assays, but antibody levels can be detected within 3 to 4 weeks after infection when the newer third-generation antigen sandwich assays are used. This window period can be
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shortened to about 2 weeks using p24 antigen assays or to 1 week with the implementation of nucleic acid detection assays. Although nucleic acid testing and viral culture are highly sensitive and specific methods to identify infection, respectively, these procedures are time-consuming, laborious, and expensive. Therefore the detection of p24 antigen is a simple and cost-effective technique to demonstrate viral components in blood, thereby verifying infection and/or identifying early infection, and offers the same performance advantages as antibody detection. 4th Generation HIV TRI-DOT + Ag
The HIV TRI-DOT + Ag test is a visual, rapid, sensitive and accurate
immunoassay for the differential detection of HIV-1 p24 antigen & HIV -1
and HIV-2 antibodies in Human Serum or Plasma.
Principle:
This test is based on the same Flow Through technology as HIV TRI-DOT; the
additional advantages offered by this test is the detection of p24 antigens
along with the differential detection of antibodies to HIV –1 and HIV – 2.
This thus reduces the window period to 4 - 9 days (According to Journal of
Clinical Microbiology), and is thus very useful for testing even early
infections where the seroconversion has not taken place.
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HIV TRI-DOT + Ag has been developed and designed using anti –p 24, gp
41, C terminal of gp 120 & gp 36 representing the immuno-dominant
regions of HIV –1 & HIV –2 envelope gene structure respectively. The
device (an immuno-filtration membrane) includes a “ Built – in quality
control Dot” which will develop colour during the test, thereby, confirming
proper functioning of the device, reagents and correct sequential
addition of reagents.
Salient Features:
• First of its kind 4th Generation rapid flow-through test for the
detection of HIV –1 p24 antigen and antibodies to HIV-1 and HIV-2.
• Significant reduction in Window Period.
• Unique washing steps for clear interpretation of results.
• Excellent Specificity and sensitivity.
• Results in less than 5 mins.
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• This test also provides for the indication regarding the phase of
infection the patient is in.
4th Generation HIV MICROLISA Ag + Ab
MICROLISA HIV (Ag & Ab) is developed to detect anti-HIV ENV (envelope)
antibodies to HIV-1 and / or HIV-2 with equal reactivity and Antigen to
HIV-1. Antigen can generally be detected in acute phase and during
symptomatic phase of AIDS and antibodies can detected throughout the
infection.
It has been observed that the core protein of HIV-1 and HIV-2 show cross
reactivity whereas envelope proteins are more type specific and
moreover antibodies against the envelope gene products can be found
in almost all infected people. Microlisa HIV (Ag & Ab) has been
developed and designed to be extremely sensitive and specific using
recombinant proteins (gp41, C terminus of gp120 , gp36 and HIV-1 P24
antibodies ) representing the immunodominant regions of HIV-1 & HIV-2
envelope gene structure respectively.
PRINCIPLE:
Microlisa HIV (Ag & Ab) test is an enzyme immunoassay based on
“Sandwich ELISA”. Recombinant proteins gp41, C terminus of gp 120, and
gp 36 for HIV-1 and HIV-2 representing immunodominant epitopes and
P24 antibodies are coated onto microtiter wells. Specimens and controls
are added to the microtiter wells followed by addition of enzyme
conjugate (HIV-1 & 2 antigen and HIV - 1 P24 antibodies linked with
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HRPO). A sandwich complex is formed in the well where in HIV-1 or HIV-2
antibodies or P24 antigen (from serum sample) is sandwiched between
the antigens & antigen HRPO and antibody & antibody HRPO conjugate.
The plate is then washed to remove unbound material. Finally substrate
solution containing chromogen and hydrogen peroxide is added to the
wells and incubated. A blue colour will develop in proportion to the
amount of HIV-1 and / or HIV-2 antibodies and /or HIV -1 antigen present
in the specimen. The colour reaction is stopped by a stop solution.
The enzyme substrate reaction is read by EIA reader for absorbance at a
wavelength of 450 nm. If the sample does not contain HIV-1 or HIV-2
antibodies then enzyme conjugate will not bind and the solution in the
wells will be either colour-less or only a faint background colour develops.
Salient Features:
• Significant reduction in window period.
• Enhanced specificity and sensitivity.
• Easy to use. Easy to adapt to an automated and semi automated
processors.
• Colour coded reagents to monitor procedural steps.
• Results in mere 90 mins
• Shelf like of 12 Months.
• Other tests used in HIV/AIDS treatment
The CD4 T-cell count is not an HIV test, but rather a procedure where the
number of CD4 T-cells in the blood is determined. A CD4 count does not
check for the presence of HIV. It is used monitor immune system function
in HIV-positive people. Declining CD4 T-cell counts are considered to be a
marker of progression of HIV infection. In HIV-positive people, AIDS is
officially diagnosed when the count drops below 200 cells/mL or when
certain opportunistic infections occur. This use of a CD4 count as an AIDS
criterion was introduced in 1992; the value of 200 was chosen because it
corresponded with an greatly increased likelihood of opportunistic
infection. Lower CD4 counts in people with AIDS are indicators that
prophylaxis against certain types of opportunistic infections should be
instituted.
Low CD4 T-cell counts are associated with a variety of conditions,
including many viral infections, bacterial infections, parasitic infections,
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sepsis, tuberculosis, coccidioidomycosis, burns, trauma, intravenous
injections of foreign proteins, malnutrition, over-exercising, pregnancy,
normal daily variation, psychological stress, and social isolation. This test is
also used occasionally to estimate immune system function for people
whose CD4 T cells are impaired for reasons other than HIV-infection, which
include several blood diseases, several genetic disorders, and the side
effects of many chemotherapy drugs.
Generally speaking, the lower the number of T cells, the lower the immune
system's function will be. Normal CD4 counts are between 500 and 1500
CD4+ T cells/microliter, and the counts may fluctuate in healthy people
depending on recent infection status, nutrition, exercise and other factors.
Women tend to have somewhat lower counts than men.
In addition to CD4 cell counts, CD4 or CD8 percentage is also diagnosed.
Percentages are usually more stable than counts over time. A normal CD4
cell percentage is about 30-60 percent, and a normal CD8 cell
percentage is about 20-50 percent. Sometimes doctors also look at the
CD4/CD8 ratio. Healthy HIV- people usually have at least 1-2 CD4 cells for
every CD8 cell. But HIV+ people may have many more CD8 cells than
CD4 cells.
Prevention
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Sexual contact or the sharing of needles
nearly always transmits HIV infection is
almost completely preventable.
Unfortunately, the measures required for
prevention—sexual abstinence or
condom use and access to clean
needles—are sometimes personally or
socially unpopular. Many people have
difficulty changing their addictive or
sexual behaviors, so they continue to
engage in behavior that puts them at
risk for HIV infection. Additionally, safe
sex practices are not foolproof:
condoms can leak or break.
Vaccines for preventing HIV infection or
slowing the progression of AIDS in
people who are already infected have
so far proved elusive. Research
continues, and several promising
vaccines are being tested.
Because HIV is not transmitted through
the air or by casual contact (such as
touching, holding, or dry kissing),
hospitals and clinics do not isolate HIV-
infected people unless they have another contagious infection. HIV-
contaminated surfaces can easily be cleaned and disinfected because
HIV is inactivated by heat and by common disinfectants such as
hydrogen peroxide and alcohol. People who are likely to come into
contact with blood or other body fluids at their job should wear protective
gear, including latex gloves, masks, and eye shields. These universal
precautions apply to body fluids from all people, not just those from
someone with HIV, for two reasons: people with HIV may not know that
they are infected, and other viruses can be transmitted by body fluids.
People who have been exposed to HIV from a blood splash, needlestick,
or sexual contact may reduce the chance of infection by taking a brief
course of anti-HIV drugs. These drugs must be started as soon as possible
after the exposure. Four weeks of preventive treatment with two or three
drugs is currently recommended. Because the risk of infection varies,
doctors and infected people make treatment decisions individually based
on the type of exposure.
Strategies for Preventing the Transmission of HIV
• Abstain from sexual activity
• Use a latex condom for each act of
intercourse with an infected partner or a
partner whose HIV status is unknown
(vaginal spermicides and sponges do not
protect against HIV infection)
• If engaging in oral sex, withdraw before
ejaculation; avoid brushing teeth for
several hours before and after oral sex
• Newly monogamous couples should get
tested for HIV and other sexually
transmitted diseases (STDs) before
engaging in unprotected sexual
intercourse
• Never share needles or syringes
• Wear rubber gloves (preferably latex)
when touching body fluids of a person
who might be infected with HIV
• If exposed to HIV by needlestick,
seek treatment to prevent infection
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Treatment
Three classes of drugs are available to treat HIV infection: nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse transcriptase
inhibitors, and protease inhibitors. Both types of reverse transcriptase
inhibitors work by interfering with the HIV enzyme reverse transcriptase,
which converts viral RNA into DNA. Protease inhibitors interfere with the
HIV enzyme protease, which is needed to activate certain proteins inside
newly produced viruses. Failure to activate these proteins results in
immature, defective HIV that does not infect new cells. None of these
drugs kill HIV; they prevent the virus from replicating. If replication is
sufficiently slowed, the destruction of CD4 cells by HIV is decreased
dramatically and CD4+ counts begin to rise. The result can be reversal of
much of the damage to the immune system caused by HIV.
HIV usually develops resistance to any of these drugs when they are used
alone. Resistance can develop after a few days to several months of use,
depending on the drug and the person. Therefore, treatment is most
effective when at least two or three of the drugs are given in
combination—usually one or two reverse transcriptase inhibitors plus a
protease inhibitor. This combination of drugs is sometimes referred to as a
"drug cocktail." Combinations of drugs are used for three reasons. First,
combinations are more powerful than single drugs in reducing levels of
HIV in the blood. Second, combinations help prevent the development of
drug resistance. Third, some HIV drugs (like ritonavir, Some Trade Names
NORVIR) boost the blood levels of other HIV drugs (including most
protease inhibitors) by slowing their removal from the body. Drug
combinations have delayed the onset of AIDS in HIV-infected people, thus
extending their lives.
Combinations of HIV drugs have both unpleasant and serious side effects.
Disturbances in the metabolism of fats appear to be caused primarily by
the protease inhibitors. Symptoms are the slow migration of body fat from
the face, arms, and legs to the abdomen ("protease paunch") and
sometimes to the breasts of women. Blood levels of cholesterol and
triglycerides, two forms of fat in the blood, are increased—probably
increasing the risk of future heart attacks and strokes.
Nucleoside reverse transcriptase inhibitors damage mitochondria, a
critical site of energy generation in human cells. Their side effects include
anemia, painful feet caused by nerve damage, and liver damage that
rarely progresses to liver failure. Individual drugs differ in their tendency to
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cause these problems. Careful monitoring and changes of drugs can
usually prevent serious problems.
Drug treatment is beneficial only when the drugs are taken on schedule.
Missed doses allow the virus to replicate and develop resistance. The goal
of combination therapy is to reduce the viral load so it is below
detectable levels. No treatments have proven able to eliminate the virus
from the body, although levels often fall below what can be measured; if
treatment is stopped, viral load increases and CD4+ counts begin to fall.
It is not yet clear for which infected people drug treatment should be
started, but people with low CD4+ counts or high viral loads require
treatment, even if they have no symptoms. Because of the many
significant and unpleasant side effects and because the drugs are very
expensive, it is not easy for people with HIV infection to take the drugs for
many years without fail. Because taking HIV drugs irregularly often leads to
drug resistance, doctors try to ensure that anyone prescribed these drugs
is both willing and able to adhere to the treatment schedule.
People with low CD4+ counts are routinely prescribed drugs to prevent
opportunistic infections.
Prognosis
Exposure to HIV does not always lead to infection, and some people who
have had repeated exposures over many years remain uninfected.
Moreover, many infected people have remained well for more than a
decade. Doctors do not fully understand why some people become ill so
much sooner than others, but a number of genetic factors appear to
influence both susceptibility to infection and progression to AIDS after
infection.
Of the people infected with HIV who do not receive drug treatment, each
year 1 to 2% develop AIDS for the first several years after infection. Every
year thereafter, about 5% of the people with untreated HIV infection
develop AIDS. Within 10 to 11 years of contracting HIV infection, half of
the people who have not received treatment develop AIDS. Eventually,
more than 95% of untreated infected people develop AIDS, and it is
possible that they all will if they live long enough, although a few people
have remained well for more than 15 years.
Early in the AIDS epidemic, many people with AIDS experienced a rapid
decline in their quality of life after first being hospitalized for the infection—
often spending much of their remaining time in the hospital. Most people
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died within 2 years of developing AIDS. However, current therapy has
changed AIDS into a more stable, manageable disease. Many people
have lived for years with AIDS, continuing to lead productive and active
lives. Nevertheless, illness from infections and the expense and side effects
of drugs may reduce quality of life. For people unable to tolerate or take
drugs consistently, the natural progression of the disease resumes.
Cure is not yet possible, although intensive research on a cure continues.
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