SPECIAL STUDY

SCREENING AND MONITARING OF HIV / AIDS INFECTED ADULT PATIENTS

NAME : THINES RAMALINGAM NIM SEM : 0802005189 : 5

FACILITATOR : dr. IGK. Darmada, SpKK EVALUATOR :

FACULTY OF MEDICINE, UDAYANA UNIVERSITY 2010 / 2011

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CONTENT

1.1INTRODUCTION
1.1 Definition Acquired immune deficiency syndrome or acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). On the other hand, HIV can also be addressed as any of various strains, serotypes, or clades of HIV1 and HIV-2 that infect and destroy helper T cells of the immune system causing the marked reduction in their numbers that is diagnostic of AIDS. This virus is called also AIDS virus human immunodeficiency virus. To add on, AIDS a disease of the human immune system that is characterized cytologically especially by reduction in the numbers of CD4-bearing helper T cells to 20 percent or less of normal thereby rendering the subject highly vulnerable to lifethreatening conditions (as Pneumocystis carinii pneumonia) and to some (as Kaposi's sarcoma) that become life-threatening and that is caused by infection with HIV commonly transmitted in infected blood especially during illicit intravenous drug use and in bodily secretions (as semen) during sexual intercourse.

1.2 Virology

HIV another type retroviruses. It is roughly spherical with a diameter of about 120 nm, around 60 times smaller than a red blood cell, yet large for a virus. 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
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as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein p17 surrounds the capsid ensuring the integrity of the virion particle. The viral envelope that is composed of two layers of fatty molecules are 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. The RNA genome consists of at least seven structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS) and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev) encoding 19 proteins. Three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles. For example, env codes for a protein called gp160 that is broken down by a viral enzyme to form gp120 and gp41. 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. The two Tat proteins (p16 and p14) are transcriptional transactivators for the LTR promoter acting by binding the TAR RNA element. The TAR may also be processed into microRNAs that regulate the apoptosis genes ERCC1 and IER3. The Rev protein (p19) is involved in shuttling RNAs from the nucleus and the cytoplasm by binding to the RRE RNA element. The Vif protein (p23) prevents the action of APOBEC3G (a cell protein that deaminates DNA:RNA hybrids and/or interferes with the Pol protein). The Vpr protein (p14) arrests cell division at G2/M. The Nef protein (p27) down-regulates CD4 (the major viral receptor), as well as the MHC class I and class II molecules.

Nef also interacts with SH3 domains. The Vpu protein (p16) influences the release of new virus particles from infected cells. The ends of each strand of HIV RNA contain an RNA sequence called the long terminal repeat (LTR). Regions in the LTR act as switches to control production of new viruses and can be triggered by proteins from either HIV or the host cell. The Psi element is involved in viral genome packaging and recognized by Gag and Rev proteins. The SLIP element (TTTTTT) is involved in the frameshift in the Gag-Pol reading frame required to make functional Pol.

1.3 Origin and Discovery Even thought, AIDS are now know to be a very common sexually transmitted disease, however, AIDS was only first reported June 5, 1981, when the U.S. Centers for Disease Control (CDC) recorded a cluster of Pneumocystis carinii pneumonia (now still classified as PCP but known to be caused by Pneumocystis jirovecii) in five homosexual men in Los Angeles. Earlier, this disease was not given any official name for the disease. often it is referred by way of the diseases that were associated with it, for example, lymphadenopathy, the disease after which the discoverers of HIV originally named the virus. They also used Kaposi's Sarcoma and Opportunistic Infections, the name by which a task force had been set up in 1981. Gay-related immune deficiency (GRID) is also another name rised for this syndrome. However, by September 1982 the CDC started using the name AIDS, and properly defined the illness. Talking about the discovery, in 1983, two separate research groups led by Robert Gallo and Luc Montagnier independently declared that a novel retrovirus may have been infecting AIDS
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patients, and published their findings in the same issue of the journal Science. Gallo claimed that a virus his group had isolated from an AIDS patient was strikingly similar in shape to other human T-lymphotropic viruses (HTLVs) his group had been the first to isolate. Gallo's group called their newly isolated virus HTLV-III. At the same time, Montagnier's group isolated a virus from a patient presenting lymphadenopathy and physical weakness, two classic symptoms of AIDS. Contradicting the report from Gallo's group, Montagnier and his colleagues showed that core proteins of this virus were immunologically different from those of HTLV-I. Montagnier's group named their isolated virus lymphadenopathy-associated virus (LAV). HIV was chosen as a compromise between the two claims (LAV and HTLV-III). However in 2008 half of Nobel Prize in Physiology or Medicine for the discovery of human immunodeficiency virus" was awarded to Luc Montagnier and his colleague Franoise Barr-Sinoussi.

2.0 EPIDEMIOLOGY

Globally, During 2009, some 2.6 million people became infected with HIV, including an estimated 370,000 children. Most of these children are babies born to women with HIV, who acquire the virus during pregnancy, labour or delivery, or through breast milk. Drugs are available to minimise the dangers of mother-to-child HIV transmission, but these are still often not reaching the places where they are most needed.The year also saw 1.8 million deaths from AIDS-related causes. The number of deaths probably peaked around 2004, and due to the expansion of antiretroviral therapy, declined by 19 percent between 2004 and 2009. By the end of 2009, the epidemic had left behind 16.6 million AIDS orphans, defined as those aged under 18 who have lost one or both parents to AIDS. Around half of people who acquire HIV become infected before they turn 25, and AIDS is the second most common cause of death among 20-24 year olds. Sub-Saharan Africa is by far the region most-affected by the AIDS epidemic. The region homes up to 68% of all people living with HIV. An estimated 1.8 million adults and children became infected with HIV during 2009 - contributing to a total of 22.5 million people living with HIV in the region. Adult HIV prevalence varies considerably across sub-Saharan Africa - from 0.2% in Madagascar to almost 26% in Swaziland. An estimated 1.3 million people died from AIDS7

related illnesses in 2009. Antiretroviral therapy has had a significant impact on the number of deaths from AIDS; in Southern Africa alone the scale-up of treatment contributed to an 18% decline in AIDS-related deaths between 2004 and 2009. The scale-up of PMTCT programmes has also contributed to a decline in the number of new HIV infections and AIDS-related deaths among children.Women are particularly affected by HIV in sub-Saharan Africa. Southern Africa accounts for around 40% of the global total of women living with HIV. In Asia, India account to an estimate of 4.9 million over the total HIV-infected population across the region. Other countries with large numbers of people living with HIV include China accounting an estimate of 740,000 people, Thailand (530,000 people) and Viet Nam (280,000 people). To add on, South & South East Asia are second worst affected; in 2007 this region contained an estimated 15% to 18% of all people living with AIDS, and an estimated 300,000 deaths from AIDS. An estimated 1.4 million people were living with HIV in Central and South America at the end of 2009. Around 92,000 people became newly infected with HIV in 2009, 7,000 less than 2001. Around 58,000 people died from AIDS-related illnesses in 2009 in the region.Adult HIV prevalence in most countries is below 1%. It is estimated that 2.3 million people are living with HIV in North America and Western and Central Europe, an increase from 1.8 million in 2001. In these two regions, a total of 35,000 people died from AIDS-related illnesses in 2009. The number of people living with HIV in Oceania almost doubled from 28,000 in 2001 to 57,000 in 2009. In recent years there has been a significant increase in new infections among gays in higher-income countries. In both North America and Western and Central Europe women account for less than a third of all people living with HIV. Two genetically different but related forms of HIV, called HIV-1 and HIV-2, have been isolated from patients with AIDS. HIV-1 is the virus that was initially discovered and termed both LAV and HTLV-III. It is more virulent, more infective, and is the cause of the majority of HIV infections globally. The lower infectivity of HIV-2 compared to HIV-1 implies that fewer of those exposed to HIV-2 will be infected per exposure. Because of its relatively poor capacity for transmission, HIV-2 is largely confined to West Africa. HIV-1 is the most common type associated with AIDS in the United States, Europe, and Central Africa, whereas HIV-2 causes a
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similar disease principally in West Africa and India. Although distinct, HIV-1 and HIV-2 share some antigens. Specific tests for HIV-2, however, are now available, and blood collected for transfusion is routinely screened for both HIV-1 and HIV-2 seropositivity. The ensuing discussion relates primarily to HIV-1 and diseases caused by it, but the information is generally applicable to HIV-2 as well.

3.0 RISK FACTORS

3.1 Sexual Transmission New HIV infections are among men who have sex with men and ~33% of new HIV infections are by heterosexual transmission, the most common mode of infection worldwide, particularly in developing countries. HIV2 has been demonstrated in seminal fluid both within infected mononuclear cells and in the cell-free state. The virus appears to concentrate in the seminal fluid, particularly in situations where there are increased numbers of lymphocytes and monocytes in the fluid, as in genital inflammatory states such as urethritis and epididymitis, con-ditions closely associated with other STDs6 (see below). The virus has also been demonstrated in cervical smears and vaginal fluid. There is a strong association of transmission of HIV with receptive anal intercourse, probably because only a thin, fragile rectal mucosal membrane separates the deposited semen from potentially susceptible cells in and beneath the mucosa and trauma may be associated with anal intercourse. Anal douching and sexual practices that traumatize the rectal mucosa also increase the likelihood of infection. It is likely that anal intercourse provides at least two modalities of infection: (1) direct inoculation into blood in cases of traumatic tears in the mucosa; and (2) infection of susceptible target cells, such as Langerhans cells, in the mucosal layer in the absence of trauma. Although the vaginal mucosa is several layers thicker than the rectal mucosa and less likely to be traumatized during intercourse, it is clear that the virus can be transmitted to either partner through vaginal intercourse. An history of STDs is most strongly associated with HIV transmission. In this regard, there is a close association between genital ulcerations and transmission, from the standpoints of both susceptibility to infection and infectivity. Infections with microorganisms such as Treponemapallidum and herpes simplex virus are important causes of genital ulcerations linked to transmission of HIV. In fact transmission was rare when the infected partner had a plasma level of <1500 copies of HIV RNA per milliliter. Furthermore, in a number of other studies, lack of circumcision has been strongly associated with a higher risk of HIV infection. This difference may be due to increased susceptibility of uncircumcised men to ulcerative STDs, as well as other factors such as microtrauma. In addition, the highly vascularized inner foreskin tissue contains a
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high density of Langerhans cells as well as increased numbers of CD4+ T cells, macrophages, and other cellular targets for HIV. Finally, the moist environment under the foreskin may promote the presence or persistence of microbial flora which, via inflammatory changes, may lead to even higher concentrations of target cells for HIV in the foreskin. In some studies the use of oral contraceptives was associated with an increase in incidence of HIV infection over and above that which might be expected by not using a condom for birth control. Oral sex is a much less efficient mode of transmission of HIV2 than is receptive anal intercourse. A number of studies have reported that the incidence of transmission of infection by oral sex among couples discordant for HIV was extremely low. However, there have been several reports of documented HIV transmission resulting solely from receptive fellatio and insertive cunnilingus. There are probably many more cases that go unreported because of the frequent practice of both oral sex and receptive anal intercourse by the same person. 3.2 Transmission By Blood Products HIV2 can be transmitted to individuals who receive HIV-tainted blood transfusions, blood products, or transplanted tissue as well as to IDUs7 who are exposed to HIV while sharing injection paraphernalia such as needles, syringes, the water in which drugs are mixed, or the cotton through which drugs are filtered. Parenteral transmission of HIV during injection drug use does not require intravenous puncture; subcutaneous ("skin popping") or intramuscular ("muscling") injections can transmit HIV as well, even though these behaviors are sometimes erroneously perceived as low-risk. Among IDUs, the risk of HIV infection increases with the duration of injection drug use; the frequency of needle sharing; the number of partners with whom paraphernalia are shared, particularly in the setting of "shooting galleries" where drugs are sold and large numbers of IDUs may share a limited number of "works"; comorbid psychiatric conditions such as antisocial personality disorder; the use of cocaine in injectable form or smoked as "crack"; and the use of injection drugs in a geographic location with a high prevalence of HIV infection, such as certain inner-city areas in the United States. It is estimated that 90 to 100% of individuals who were exposed to such HIV-contaminated products became infected. Transfusions of whole blood, packed red blood cells, platelets, leukocytes, and plasma are all capable of transmitting HIV infection.

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In addition to the above, individuals with hemophilia or other clotting disorders were infected with HIV2 by receipt of HIV-contaminated fresh-frozen plasma or concentrates of clotting factors. It is estimated that the risk of infection with HIV in the United States via transfused screened blood is approximately 1 in 725,000 to 1 in 835,000 donations. Prior to the screening of donors, a small number of cases of transmission of HIV2 via semen used in artificial insemination and tissues used in organ transplantation were well documented. At present, donors of such tissues are prescreened for HIV infection. 3.3 Perinatal transmission Maternal transmission to the fetus occurs most commonly in the perinatal period. Factor that is associated with higher rates of transmission is the presence of high maternal levels of plasma viremia. Low maternal CD4+ T cell counts have also been associated with higher rates of transmission; however, since low CD4+ T cell counts are often associated with high levels of plasma viremia, in one study using multivariate analysis including plasma viral load and CD4+ T cell count, only the level of plasma HIV RNA was significant. A prolonged interval between membrane rupture and delivery is another well-documented risk factor for transmission. Other conditions that are potential risk factors, but which have not been consistently demonstrated, are the presence of chorioamnionitis at delivery; STDs6 during pregnancy; hard drug use during pregnancy; cigarette smoking; preterm delivery; and obstetric procedures such as amniocentesis, amnioscopy, fetal scalp electrodes, and episeotomy. Vitamin A deficiency had been reported to be associated with higher transmission rates. With regard to levels of viremia, several studies indicate that the risk of transmission increases with the maternal plasma HIV RNA level. It has also been speculated that if the mother experiences acute primary infection during pregnancy, there is a higher rate of transmission to the fetus, owing to the high levels of viremia that occur during primary infection. The risk factors for mother-to-child transmission of HIV via breast-feeding are not fully understood. However, factors that increase the likelihood of transmission include detectable levels of HIV in breast milk, the presence of mastitis, low maternal CD4+ T cell counts, and maternal vitamin A deficiency. The risk of HIV infection via breast-feeding is highest in the early months of breast-feeding.

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3.4 Occupational Transmission Occupational risk of HIV2 transmission is greatly seen in health care workers and laboratory personnel and potentially others who work with HIV-containing materials, particularly when sharp objects are used. An increased risk for HIV infection following percutaneous exposures to HIV-infected blood is associated with exposures involving a relatively large quantity of blood, as in the case of a device visibly contaminated with the patient's blood, a procedure that involves a needle placed directly in a vein or artery, or a deep injury. Factors that might be associated with mucocutaneous transmission of HIV include exposure to an unusually large volume of blood, prolonged contact, and a potential portal of entry. In addition, the risk increases for exposures to blood from patients with advanced-stage disease, probably owing to the higher titer of HIV in the blood as well as to other factors, such as the presence of more virulent strains of virus. Despite these few cases, the risk of transmission from an infected health care worker to patients is extremely low; in fact, too low to be measured accurately.

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4.0 PATHOGENESIS OF HIV AND AIDS

This eight step mechanism starts with the binding of the virus tothe CD4+ T cell. Once HIV has entered the bloodstream, it attaches to the surface of a CD4+ T cell by binding to a CD4 receptor that has a high affinity for HIV. However, binding to the CD4 receptor is not sufficient for infection; the virus must also bind with other surface molecules (chemokine coreceptors) that bind the gp120 and gp41 envelope glycoproteins. This process is known as attachment. The second step allows for the internalization of the virus. After attachment, the viral envelope peptides fuse to the CD4+ T cell membrane. Fusion results in an uncoating of the virus, allowing the contents of the viral core (the two single strands of viral RNA and the reverse transcriptase, integrase, and protease enzymes) to enter the host cell. The chemokine coreceptors are critical components of the HIV infection process. It has recently been found that people with defective coreceptors are more resistant to developing HIV, despite repeated exposure. The third step consists of DNA synthesis. In order for the HIV to reproduce, it must change its RNA into DNA. It does this by using the reverse transcriptase enzyme. Reverse transcriptase makes a copy of the viral RNA, and then in reverse makes another mirror-image copy. The result is doublestranded DNA that carries instructions for viral replication. The fourth step is called integration. During integration, the new DNA enters the nucleus of the CD4+ T cell and, with the help of the enzyme integrase, is inserted into the cells original DNA. The fifth step involves transcription of the double-stranded viral DNA to form a single-stranded messenger RNA (mRNA) with the instructions for building new viruses. Transcription involves activation of the T cell and induction of host cell transcription factors. The sixth step includes translation of mRNA. During translation, ribosomal RNA (rRNA) uses the instructions in the mRNA to create a chain of proteins and enzymes called a polyprotein. These polyproteins contain the components needed for the next stages in the construction of new viruses. The seventh step is called cleavage. During cleavage, the protease enzyme cuts the polyprotein chain into the individual proteins that will make up the new viruses. Finally, during the eighth step, the proteins and viral RNA are assembled into new HIV viruses and released from the CD4+ T cell. HIV replication involves the killing of the CD4+ T cell and the release of copies of the HIV into the bloodstream. These viral particles, or virions, invade other CD4+ T cells, allowing the infection to progress. Every day, millions of infected

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CD4+ T cells are destroyed, releasing billions of viral particles into the bloodstream; but each day, nearly all the CD4+ T cells are replaced, and nearly all the viral particles are destroyed. The problem is that over years, the CD4+ cell count gradually decreases through this process, and the number of viruses detected in the blood of persons infected with HIV increases. Until the CD4+ cell count falls to a very low level, a person infected with HIV can remain asymptomatic, although active viral replication is still taking place and serologic tests can identify antibodies to HIV.19 These antibodies, unfortunately, do not convey protection against the virus. Although symptoms are not evident, the infection proceeds on a microbiologic level, including the invasion and selective destruction of CD4+ T cells. The continual decline of CD4+ T cells, which are pivotal cells in the immune response, strips the person with HIV of protection against common organisms and cancerous cells.

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5.0 DIAGNOSIS

5.1 Clinical manifestation

The clinical course of HIV varies from person to person. Most60% to 70%of those infected with HIV acquire AIDS 10 to 11 years after infection. These people are the typical progressors.21 Another 10% to 20% of those infected progress rapidly. They acquire AIDS in less than 5 years and are called rapid progressors. The final 5% to 15% are slow progressors, who do not progress to AIDS for more than 15 years. There is a subset of slow progressors, called longterm nonprogressors, who account for 1% of all HIV infections. These people have been infected for at least 8 years, are antiretroviral nave, have high CD4+ cell counts, and usually have very low viral loads (Fig. 22-4). A. Opportunistic Infections Opportunistic infections begin to occur as the immune system becomes severely compromised. The number of CD4+ T cells directly correlates with the risk for developing opportunistic infections. Once the CD4+ cell count drops below 200 cells/L, the risk for developing an opportunistic infection is 33% after 1 year and 58% after 2 years. Opportunistic infections involve common organisms that normally do not produce infection unless there is impaired immune function. Although a person with AIDS may live for many years after the first serious illness, as the immune system fails, these opportunistic illnesses become progressively more severe and difficult to treat. Most opportunistic infections can be categorized as bacterial, fungal, protozoal, or viral. Bacterial opportunistic infections include bacterial pneumonia, tuberculosis, salmonellosis, and Mycobacterium aviumintracellulare complex (MAC) infection. Fungal opportunistic infections include candidiasis, coccidioidomycosis, cryptococcosis, and

histoplasmosis. Protozoal opportunistic infections include cryptosporidiosis, isosporiasis, pneumocystiasis, and toxoplasmosis. Viral infections include cytomegalovirus (CMV), herpes, and progressive multifocal leukoencephalopathy (PML). B. Respiratory Manifestations The most common causes of respiratory disease in persons with HIV infection are PCP and pulmonary tuberculosis (TB). Other organisms that cause opportunistic pulmonary infections in persons with AIDS include CMV, MAC, Toxoplasma gondii, and Cryptococcus neoformans.

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Pneumonia also may occur because of more common bacterial pulmonary pathogens, including Streptococcus pneumoniae, Haemophilus influenzae, and Legionella pneumophila. Some persons may become infected with multiple organisms. Kaposis sarcoma also can occur in the lungs. C. Gastrointestinal Manifestations Diseases of the gastrointestinal tract are some of the most frequent complications of HIV and AIDS. Esophageal candidiasis (thrush), CMV infection, and herpes simplex virus infection are common opportunistic infections that cause esophagitis in people with HIV.30 Persons experiencing these infections usually complain of painful swallowing or retrosternal pain. The clinical presentation can range from asymptomatic to a complete inability to swallow and dehydration. Endoscopy or barium esophagography is required for definitive diagnosis. Diarrhea or gastroenteritis is a common complaint in persons with HIV. The most common protozoal infection that causes diarrhea is Cryptosporidium parvum. The clinical features of cryptosporidiosis can range from mild diarrhea to severe, watery diarrhea with a loss of up to several liters of water per day. The most severe form usually occurs in persons with a CD4+ cell count of less than 50 cells/L, and also can include malabsorption, electrolyte disturbances, dehydration, and weight loss. Other organisms that cause gastroenteritis and diarrhea are Salmonella, Shigella, and Giardia species, CMV, Clostridium difficile, Escherichia coli, and microsporida.30 These organisms are identified by examination of stool cultures or endoscopy. D. Nervous System Manifestations Human immunodeficiency virus infection, particularly in its late stages of severe immunocompromise, leaves the nervous system vulnerable to an array of neurologic disorders, including AIDS dementia complex (ADC), toxoplasmosis, and PML. These disorders can affect the peripheral or central nervous system (CNS) and contribute to the morbidity and mortality of persons with HIV. E. Cancers and Malignancies Persons with AIDS have a high incidence of certain malignancies, especially Kaposis sarcoma (KS), non-Hodgkins lymphoma, and noninvasive cervical carcinoma. It has been reported that KS or lymphoma is likely to develop in as many as 30% to 40% of people with HIV.35 The increased incidence of malignancies probably is a function of impaired cell-mediated immunity. F. Wasting Syndrome

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In 1997, wasting became an AIDS-defining illness. The syndrome is common in persons with HIV infection or AIDS. Wasting is characterized by involuntary weight loss of at least 10% of baseline body weight in the presence of diarrhea, more than two stools per day, or chronic weakness and a fever. This diagnosis is made when no other opportunistic infections or neoplasms can be identified as causing these symptoms. Factors that contribute to wasting are anorexia, metabolic abnormalities, endocrine dysfunction, malabsorption, and cytokine dysregulation. G. Metabolic and Morphologic Disorders A wide range of metabolic and morphologic disorders is associated with HIV infection, including lipodystrophy insulin and mitochondrial and disorders, glucose hypercholesterolemia, tolerance. Metabolic

hypertriglyceridemia,

resistance,

impaired

complications among people with HIV on HAART have been increasing during the past five years.41 Diabetes is now seen in 0.5% to 4.4% of people on HAART. Insulin resistance is seen in as many as 55% of patients receiving protease inhibitors (PIs). It is still not known why insulin resistance occurs in people with HIV. Lipid abnormalities such as increases in triglycerides and cholesterol are also becoming more common in persons with HIV. Most protease inhibitors (PIs) have been shown to increase LDL and triglycerides. Increased triglycerides can lead to pancreatitis.

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5.2 Laboratories Evaluation

The diagnosis of HIV2 infection depends upon the demonstration of antibodies to HIV and/or the direct detection of HIV or one of its components. As noted above, antibodies to HIV generally appear in the circulation 2 to 12 weeks following infection.

5.2.1 HIV Screening Assays

A. Enzyme-Linked Immunosorbent Assays/Enzyme Immunoassays ELISA is the most commonly used type of test to screen for HIV infection because of its relatively simple methodology, inherent high sensitivity, and suitability for testing large numbers of samples, particularly in blood testing centers. A common feature of all varieties of ELISA is the use of enzyme conjugates that bind to specific HIV antibody, and substrates/chromogens that produce color in a reaction catalyzed by the bound enzyme conjugate. The most popular ELISA involves an indirect method in which HIV antigen is attached to a well of a 96-well microtiter plate. Antibody in the sample is allowed to react with the antigencoated solid support, usually for 30 minutes at 37 C or 40 C. After a wash step to remove unbound serum components, addition of a conjugate (an antihuman immunoglobulin with a bound enzyme) binds to the specific antibody that is attached to the antigens on the solid phase. Following another wash, addition of an appropriate substrate results in color development that is detected by a spectrophotometer and is proportional to specific HIV antibody concentration in the sample. Optical density (OD) values are produced as the colored solution absorbs transmitted light, and provide an indication of the amount of color, which is proportional to the amount of antibody bound (ie, antibody concentration). A mathematical calculation, usually based on the OD of the negative controls multiplied by a factor, produces a cutoff value on which the OD of the sample is compared to determine the antibody status; samples with OD cutoff values >1.0 (in an indirect ELISA) are considered antibody reactive (positive). Several indirect ELISA tests incorporate polyvalent conjugates (anti-IgG and anti-IgM) and antigen-sandwich configurations in order to increase sensitivity for detecting early infection (during seroconversion). Alternate ELISA methodologies include a competitive format in which specific HIV antibody in the sample competes with an enzyme-bound antibody reagent for antigen sites on the
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solid phase. In this method, color development is inversely proportional to specific HIV antibody concentration. A more recent addition to ELISA technology is the antigen sandwich method in which an enzyme (alkaline phosphatase or horseradish peroxidase) is conjugated to an HIV antigen (similar to the immobilized antigen on the solid phase). The antibody in the sample is "sandwiched" between 2 antigen molecules, 1 immobilized on the solid phase and 1 containing the enzyme. Subsequently, the addition of substrate results in color development in proportion to antibody concentration. The antigen sandwich ELISA is considered the most sensitive screening method, given its ability to detect all isotypes of antibody (including IgM). One disadvantage of this method is the relatively large volume (150 L) of sample required, which may make repeat testing and testing of samples from infants difficult.

B. Antibody Testing Strategies for Identifying Early HIV Infection and Estimating Incidence: Sensitive/Less-Sensitive ("Detuned") Assays Laboratory-based strategies that can distinguish recently infected individuals from those with established infection have been devised. In these methods, the procedures of conventional ELISA or rapid assays have been modified to allow discrimination of antibody titer or antibody avidity. These modified assays have been called "detuned" assays or "sensitive/less-sensitive" (S/LS) assays. During acute HIV infection, prior to the appearance of antibody (window period or preseroconversion), HIV infection can be confirmed only by the demonstration of circulating p24 antigen, or by the presence of viral RNA or DNA. Although highly sensitive antibody assays exist to detect very low levels of HIV antibody in blood, the window period prior to appearance of antibody rarely can be shortened to less than 3 weeks. Once antibody has appeared, titers progressively increase during 3-5 months until levels peak, at which time they remain fairly constant throughout the remainder of infection. Also, antibodies during early infection usually are of low avidity, but avidity increases as infection progresses. Therefore, HIV infection can be divided into categories of recent or established infection, depending on the quantity of antibody present or their avidities. These parameters can be exploited as tools in order to estimate the relative time that HIV infection occurred. For example, if antibody titers or antibody avidity is low, it is likely that infection occurred within the past 4 months; conversely, high-titer or high20

avidity antibodies signal an established infection that has been present for longer than 4 months. Several epidemiological studies have used the S/LS testing strategy to predict incidence in San Francisco and in Rio de Janeiro, Brazil. The first S/LS strategy, then, is based on the principle that antibody titer increases with time and that recent infection can be assumed if test results become nonreactive following dilution of the individual's serum. In such a case, an initially reactive sample, when tested with the routine (sensitive), becomes nonreactive when diluted in a modified (less-sensitive) assay. Conversely, the serum of an individual with established HIV infection would remain reactive following dilution in the less-sensitive assay due to high levels of antibody. This strategy is used only on individuals who are confirmed positive using the Centers for Disease Control and Prevention (CDC) interpretive criteria via Western blot, as persons who are negative for antibody would not be candidates for determining the time of infection. This system, also known as the Serologic Testing Algorithm for Determining Recent HIV Seroconversion (STARHS), has been developed and adopted by the CDC. The initial assay system that was modified by dilution and validated using persons of known seroconversion or infection times was the FDA-licensed, firstgeneration Abbott HIV-1 Viral Lysate ELISA (3A11).(9) Specific modifications in the procedure of the 3A11 ELISA were made to 4 parameters in order to decrease the sensitivity (for the lesssensitive assay). To construct the less-sensitive test, the sample dilution was increased to 1:20,000, the sample incubation time was reduced to 30 minutes, the conjugate incubation time was reduced to 30 minutes, and the OD cutoff value was adjusted. In order to compare results obtained with the less-sensitive assay, OD readings for individual samples are standardized by calculating standardized OD (SOD) values based on the formula: SOD = (sample OD value negative control OD value)/positive control OD value). A cutoff SOD (0.75) has been determined statistically and nonreactive samples have an SOD less than the cutoff. When such a sample shows this reversion by the S/LS test, the time interval from seroconversion was calculated to be 129 days or about 4 months (95% confidence interval: 109-149 days). Several studies have validated the S/LS algorithm by analyzing individuals with known early infection as determined by clinical evaluation, recent seroconversion, high-risk behavior, and antigen and nucleic acid analyses. A limitation of the S/LS test strategy may be the detection of individuals with long-standing infection (0.4%) and late-stage AIDS (2%). Thus, CD4 cell counts and

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clinical information may be required to support results obtained by the S/LS test algorithm for maximum accuracy. The S/LS strategy is inexpensive, reproducible, and can give a fairly accurate estimate of the time of infection. Because the 3A11 ELISA no longer is available, other test kits (Vironostika, bioMrieux) have been substituted, and have been considered to be equivalent in performance. More recently, another quantitative ELISA method has been introduced, and reportedly performs effectively with samples from persons who are infected with non-B HIV clades. This assay, the BED assay (Calypte; Lake Oswego, OR), incorporates synthetic peptide antigens and can classify infections for clades B, E, C, and A/D. The second method to identify the time of infection for incidence estimation is based on antibody avidity and has been developed using a third-generation ELISA. This method is known as the Avidity Index Protocol. Avidity describes the collective interactions between antibodies and a multivalent antigen. Avidity measurements are used with a variety of infectious diseases to offer confirmatory evidence of acute infection, to distinguish reactivation from primary infections, and to permit diagnosis of acute infection from a single sample. An individual's differential binding or avidity index (AI) correlates with the estimated length of time from the initial infection by HIV. Thus, the strength of the interaction between antigen and the antibody present in early infection is weak because low-avidity HIV-1 antibody comprises the majority of antibodies found in early infection. The relative avidity of antibody is stronger in established infection and can be estimated serologically based on resistance of the antigen-antibody complex to chaotropic agents. Chaotropic agents are dissociating reagents such as urea (at concentration of 4, 6, and 8 M), potassium thiocyanate (KSCN; 1-3 M), magnesium chloride (2 and 4 M), diethylamine (0.025, 0.05, and 0.1 M), and guanidine HCl (3 and 6 M). The most widely recognized AI test is a recombinant viral lysate enzyme immunoassay (EIA) from Bio-Rad Laboratories (Hercules, CA), modified by the incorporation of a dissociation and wash step. The chaotropic agent that demonstrated the ability to dissociate low avidity HIV antibody molecules most effectively was 2.5 M KSCN. Procedurally, duplicate wells of a diluted sample are incubated with HIV antigen. Antibodies to HIV bind to the antigen, and following a wash step, a solution of dissociating reagent is added to one of the wells (test)
22

while wash solution is added to the other well (control). Results are interpreted based on a calculation of the AI from a percentage of the ratio of the OD of the KSCN-treated specimen to that of the nontreated control. Samples demonstrating an AI <80% are taken to represent early infection and are associated with the 3- to 4-month (120-day) time period following seroconversion. This method has been validated with samples from seroconversion panels and samples from individuals with clinically established HIV infection. Another development of a rapid S/LS method id by using the Uni-Gold HIV test, a 10minute, visually read, rapid test. This method, based on a dilution of serum for the LS mode, has shown excellent results in comparison with the Abbott 3A11 assay and when assessed using samples from individuals with known seroconversion dates. In addition, the obtained preliminary results using an HIV saliva test, SalivaCard (Trinity Biotech), that shows utility as an S/LS tool. More recently, the developed a simple and low-cost particle agglutination assay as an S/LS assay and shown it to be 97% accurate (unpublished observation). The advantage of rapid and simple S/LS assays is that they are portable and can be used to identify high-incidence populations in remote areas where ELISA instrumentation cannot be supported. Further, even in developed countries, they can be adapted easily for use in mobile testing centers to identify recently infected individuals so that they can be counseled appropriately to find contact persons within the past several months or to immediately direct individuals to appropriate treatment centers. Finally, the noninvasiveness of saliva-based rapid assays may increase testing participation. C. Third-Generation Assays for the Simultaneous Detection of HIV Antigen and Antibody Antibody can be detected in a majority of individuals within 6-12 weeks after infection using the earlier generations of assays, but may be detected within 3-4 weeks when using the newer thirdgeneration antigen sandwich assays. The window period can be shortened to about 2 weeks using p24 antigen assays or reduced to 1 week with the implementation of nucleic acid detection assays. Consequently, the window period between infection and detection of infection may be <2 weeks if a comprehensive testing approach is utilized. The detection of p24 antigen by ELISA is a simple cost-effective technique to demonstrate viral capsid (core) p24 protein in blood during

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acute infection due to the initial burst of virus replication after infection. In order to maximize the detection of all infected individuals, including those in early infection, antibody, antigen, and viral RNA tests should be used. However, viral RNA tests are expensive, time consuming, and unavailable in many laboratories. Laboratories that possess ELISA capability can increase the ability to detect most infections by testing for both HIV antibody and p24 antigen. During the late 1990s, assays in an ELISA format that have the capability to detect both HIV antibody and HIV p24 antigen simultaneously were developed, thereby eliminating the need to perform separate assays. D. Forth-Generation Assays for the Simultaneous Detection of HIV Antigen and Antibody The new generation of combination ELISAs that simultaneously detect both antigen and antibody has been developed and marketed, and offers advantages for decreasing the time, personnel, and costs necessary to perform each assay individually. These assays have demonstrated a high analytical sensitivity of detection that is most likely attributed to the combination of a third-generation format (antigen sandwich) for antibody detection and the ability to simultaneously detect HIV p24 antigen. To date, there are 8 commercial, combination antibody and antigen assays that have been developed and evaluated. These fourth-generation assays include the VIDAS HIV DUO Ultra (bioMrieux; Marcy l'Etoile, France), EnzymunTest-HIV-Combi (Boehringer; Mannheim, Germany), Vironostika HIV Uni-Form II Ag/AB (Organon Teknika; Boxtel, Netherlands), AxSYM-HIV Ag/AB (Abbott Laboratories; Abbott Park, IL), Enzygnost HIV Integral (Dade Behring; Marburg, Germany), Genescreen Plus HIV Ag-AB (Bio-Rad), and COBAS Core HIV Combi (Roche Diagnostics; Mannheim, Germany). The eighth assay is an 18-minute, double-antigen sandwich combination assay called the Elecsys-HIV Combi (Boehringer) that has been reported to have a specificity of 99.8% when challenged with a cohort of hospitalized patients. This rapid assay is based on electrochemiluminescence and is reported to reduce the window period by 5 days over antibody tests. A ninth, unidentified assay is a lineal immunoenzymatic assay evaluated to have a sensitivity of only 99.5% and a specificity of 94.8%.

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The benefits of testing for both antibody and antigen are justifiable due to the need to identify individuals with both established and early HIV infection not only for the blood donor population but also for some clinical applications. Early detection of infection via antigen testing promotes the prompt referral of infected individuals for the initiation of treatment, counseling, and prevention interventions to reduce the risk of transmission. Due to their ability to detect p24 antigen, the fourth-generation ELISAs will be of value in detecting early infection. These assays are highly applicable for the diagnosis of early and established HIV infection by hospital and private clinical laboratories and other laboratory settings. In these settings, individuals to be screened for infection are of higher risk groups than the blood donor population, and thus require the use of testing methodologies with high levels of analytical sensitivity to detect primary infection. Of significance, the high level of analytical and epidemiological sensitivity demonstrated by most of the fourth-generation assays with seroconversion and clade panels, as well as a variety of patient populations, makes them ideal for use in a variety of testing situations for the diagnosis of early and established infection. In routine laboratory settings, HIV-infected samples that are identified via antigen detection would not have been identified by the usual screening antibody assays, because antigen testing of patients is not performed commonly as a screening tool outside blood banks. The detection of early infection has been shown to be beneficial for the prompt initiation of appropriate antiretroviral therapy in a clinically relevant time frame. Additionally, early detection will help in the timely implementation of interventions such as the counseling of patients, prevention of transmission, and management of infection. E. Rapid Tests Rapid assays for detecting specific HIV antibody were developed in the late 1980s, and are defined as tests that can yield results in <30 minutes. These tests gained popularity in the early 1990s, and as technology became refined, proved to be as accurate as the ELISA when performed carefully by experienced personnel. Technical errors are common with these assays, however, because users become careless with these simple procedures. For example, pipettes are not always held in a vertical position as recommended, resulting in an incorrect delivery of

25

reagent volumes. In addition, many laboratory workers attempt to test multiple samples simultaneously, resulting in inaccuracies in the timing of steps. When performed correctly, rapid HIV assays are accurate and have wide utility in a number of testing situations. Application includes emergency rooms, physicians' offices, pointof-care testing, autopsy rooms, funeral homes, small blood banks, and situations involving stat HIV testing (where immediate treatment is recommended for exposures). Rapid HIV assays have proven particularly useful for testing pregnant women in labor who have not received prenatal care (ie, of unknown HIV status). It has been shown that the institution of antiretroviral therapy (eg, zidovudine) is effective in reducing transmission of HIV, and that this should be provided as early as possible to the mother and subsequently to the newborn. Rapid HIV testing of the mother who is near delivery allows treatment to be initiated prior to delivery if a positive serostatus is determined.(32) Importantly, these rapid assays are easy to perform and have utility in developing countries, where facilities may not be optimal, stable electricity may be unavailable, and formal education programs for laboratorians are absent. One class of rapid tests is the "dot blot" or "immunoblot"; they produce a wellcircumscribed colored dot on the solid phase surface if the test is positive. Most of these rapid assays now incorporate a built-in control to indicate that the test was performed correctly. This control is an antihuman immunoglobulin that binds any immunoglobulin in the sample and produces a separate indicator when all reagents are added appropriately. In addition, several varieties are available that include 2 "dots," which allows the differentiation of HIV-1 and HIV-2 infection. The procedures for the dot-blot assays are similar regardless of the exact format of the test. Most require drop-wise additions of reagents in the following sequence: buffer, sample, wash buffer, conjugate, wash buffer, substrate, and stop solution. Some assays substitute an IgG binding dye (protein A gold reagent) for the antiimmunoglobulin conjugate, thereby decreasing the procedure by a step. The newer 1-step rapid assays, also known as immunochromatographic assays, are convenient, self-contained tools for HIV serologic testing, consisting of a flat cartridge device, usually plastic or paper. Whole blood, oral fluid, or serum is placed at the tip of the device and allowed to diffuse along a strip that is impregnated with reagents (often protein A colloidal gold)
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that bind and permit visual detection of HIV antibodies; some use third-generation (antigen sandwich) technology. These tests can be completed in <10 minutes (some within 2 minutes), require little or no addition of reagents, and contain a built-in quality-control reagent to control for technical errors. Some tests can be stored at a wide range of temperatures (from 15 C to 30 C), and are transported easily. For example, one type (Determine; Abbott) comes in "cards" of 10 tests each, making it possible to carry 100 tests in a shirt pocket; the cards require no reagents, just addition of serum or plasma. The test can also be performed on whole blood, or blood collected via fingerstick (this requires 1 buffer addition). These types of rapid HIV tests are gaining in popularity because of their simplicity, ease of interpretation, and robustness.(33,34) In particular, the fingerstick collection method is taught easily to health care personnel in outreach situations or mobile vans. The use of fingerstick specimens also may prevent unnecessary collection and discarding of full units of donated blood (where blood is collected prior to testing at a remote laboratory and held until results become available). Another variety of lateral flow devices allows for the use of saliva, plasma, whole blood, or fingerstick specimens, thereby adding flexibility in sample type. Other rapid test formats include dipsticks, in which antigen is attached on the "teeth" of comblike devices; several of these rapid tests have the ability to differentiate HIV-1 and HIV-2. Disadvantages include a subjective interpretation, difficulty in reading if the laboratorian is color-blind, and a higher cost than that of the ELISA. Currently, 4 rapid HIV tests are approved for use in the United States. F. Simple Test This type of HIV test requires longer than 30 minutes for results, but consists of procedures that can be performed easily without instrumentation. Within this class of tests are agglutination assays in which antigen-coated particles (red blood cells, latex particles, or gelatin particles) are allowed to react with serum antibodies to form visible clumping (agglutination). If red blood cells are used, the technique is termed passive hemagglutination; with the use of latex particles, it is known as latex agglutination. In East Asia, an HIV gelatin particle agglutination test is popular, offering good sensitivity, low cost, and ease of performance. It incorporates a quality control system to detect nonspecific antibodies directed toward the gelatin particles themselves,
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and results can be obtained within 2 hours with minimal hands-on time. Although appropriate for use in facilities with limited testing capabilities, this test must be performed under temperaturecontrolled conditions.

5.2.2 HIV Confirmation Assays

A. Weatern Blot The most commonly used confirmatory test is the western blot (Fig. 173-23). This assay takes advantage of the fact that multiple HIV2 antigens of different, well-characterized molecular weights elicit the production of specific antibodies. These antigens can be separated on the basis of molecular weight, and antibodies to each component can be detected as discrete bands on the western blot. A negative western blot is one in which no bands are present at molecular weights corresponding to HIV gene products. In a patient with a positive or indeterminate EIA113 and a negative western blot, one can conclude with certainty that the EIA reactivity was a false positive. On the other hand, a western blot demonstrating antibodies to products of all three of the major genes of HIV (gag, pol, and env) is conclusive evidence of infection with HIV. Criteria established by the U.S. Food & Drug Administration (FDA) in 1993 for a positive western blot state that a result is considered positive if antibodies exist to two of the three HIV proteins: p24, gp41, and gp120/160. Using these criteria, ~10% of all blood donors deemed positive for HIV-1 infection lacked an antibody band to the pol gene product p31. Some 50% of these blood donors were subsequently found to be false positives. Thus, the absence of the p31 band should increase the suspicion that one may be dealing with a false-positive test result. In this setting it is prudent to obtain additional confirmation with an RNA-based test and/or a follow-up western blot. By definition, western blot patterns of reactivity that do not fall into the positive or negative categories are considered "indeterminate." There are two possible explanations for an indeterminate western blot result. The most likely explanation in a low-risk individual is that the patient being tested has antibodies that cross-react with one of the proteins of HIV. The most common patterns of cross-reactivity are antibodies that react with p24 and/or p55. The least likely explanation in this setting is that the individual is infected with HIV and is in the process of mounting a classic antibody response. In either instance, the western blot should be repeated

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in 1 month to determine whether or not the indeterminate pattern is a pattern in evolution. In addition, one may attempt to confirm a diagnosis of HIV infection with the p24 antigen capture assay or one of the tests for HIV RNA (discussed below). While the western blot is an excellent confirmatory test for HIV infection in patients with a positive or indeterminate EIA, it is a poor screening test. Among individuals with a negative EIA and PCR114 for HIV, 20 to 30% may show one or more bands on western blot. While these bands are usually faint and represent cross-reactivity, their presence creates a situation in which other diagnostic modalities [such as DNA PCR, RNA PCR, the (b)DNA assay, or p24 antigen capture] must be employed to ensure that the bands do not indicate early HIV infection. Most authorities consider it the gold standard for validation of HIV results. It is based on using an electrophoretic technique to separate HIV antigens derived from a lysate of virus grown in culture. This technique denatures the viral components, imparts a negative charge to the antigens, and separates them primarily on the basis of their molecular weights. The separation of antigens in the technique allows for the identification of specific antibodies to each of the viral antigens in a subsequent set of steps similar to the ELISA methodology. A purified HIV antigen mixture is layered onto a sodium dodecyl sulphate (SDS) polyacrylamide gel slab and then electrophoresed. The viral proteins (HIV antigens) migrate through the molecular pores of the gel at rates determined by electrical charge and molecular weight. The proteins with higher molecular weight migrate less and form bands closer to the starting point. The proteins on the gel are then transferred ("blotted") to nitrocellulose paper by another electrophoretic procedure. This paper is cut into thin strips, each with the full distribution of viral protein antigen bands. A single test strip is incubated with a 1:50 or 1:100 dilution of a test sample or a control and then washed and incubated with a labeled (tagged) antihuman globulin. At this point, the procedure is similar to any other indirect immunoassay. The label usually is an enzyme (horseradish peroxidase or alkaline phosphatase) that will react with a specific colorless substrate to produce an insoluble colored band on the strip wherever there is an antigen-antibody complex. Reaction with a positive serum sample produces a pattern of bands on the strip that is characteristic of HIV. Many of these bands have been identified as specific viral gene products. The HIV-1 viral antigens are separated as follows (from top to bottom): gp160, gp120, p66, p55, p51, gp41, p31,
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p24, p17, and p15 (Figure 1). The "gp" designation refers to glycoproteins; "p" indicates proteins. The numeric values (x100) indicate molecular weights. It is important to remember that nonviral proteins derived from the host cells in which the virus was grown also are present on the nitrocellulose strip. They can form bands in many places, but often are near the middle molecular weight (40,000 to 60,000) region. These nonviral protein bands may produce difficulty in interpretation of results by producing nonspecific reactions. The significance of an indeterminate Western blot result varies depending on the risk factors, clinical status of the patient, and the Western blot profile produced. For example, individuals with a history of high-risk behavior are more likely to be the ones who later seroconvert, because the chances of their being infected are high. In addition, some Western blot profiles are more suggestive of early infection (eg, p24, p31, and p55) than are others (eg, p17 only). Many initially indeterminate results that subsequently become negative or remain indeterminate probably are a result of nonspecific reactions, hypergammaglobulinemia, the presence of cross-reactive antibodies, infection by HIV2, or infection by an unknown, but related retrovirus. There have been a few reports where autoimmune diseases (eg, systemic lupus erythematosus) can cause false-positive HIV tests, including Western blot.(35) Also, it is known that some individuals with AIDS may lose reactivity to p24, and perhaps other antibodies, later in disease, so that even AIDS patients may have indeterminate Western blot results by some criteria. Ancillary tests, such as polymerase chain reaction (PCR) and viral culture may be helpful in resolving these indeterminate results if the diagnosis is in question.

B. Indirect Immunofluorescent Antibody Assay In this technique, cells (usually lymphocytes) are infected with HIV and are fixed to a microscope slide. Serum containing HIV antibodies is added and reacts with the intracellular HIV. The slide is washed and then allowed to react with antiimmunoglobulin antibodies with a covalently bound fluorescence label attached. The reaction is visualized using a fluorescent microscope. This technique has the advantage of sometimes providing definitive diagnosis of samples that have yielded indeterminate results by Western blot analysis. Disadvantages to its use include the requirement of an expensive microscope and a subjective interpretation, thus necessitating well-trained individuals.
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C. Line Immunoassay Another alternative to the classic Western blot and IFA confirmatory tests is the line immunoassay (LIA). In this assay, recombinant or synthetic peptide antigens are applied on a nitrocellulose strip, rather than electrophoresed as in the Western blot. This use of "artificial" antigens decreases the presence of contaminating substances derived from cell culture that can cause interference and sometimes false reactions. The use of LIA is popular in Europe, but these tests have not been licensed for use in the United States. A number of reports have verified that the accuracy is equivalent to the Western blot. 3.2.3 Morphology. The anatomic changes in the tissues (with the exception of lesions in the brain) are neither specific nor diagnostic. In general, the pathologic features of AIDS include those of widespread opportunistic infections, KS, and lymphoid tumors. Most of these lesions are discussed elsewhere because they also occur in patients who do not have HIV infection. To appreciate the distinctive nature of lesions in the central nervous system, we discuss them in the context of other disorders affecting the brain. Here we concentrate on changes in the lymphoid organs. Biopsy specimens from enlarged lymph nodes in the early stages of HIV infection reveal a marked follicular hyperplasia.[163] The enlarged follicles have irregular, sometimes serrated borders, and they are present not only in the cortex, but also in the medulla and may even extend outside the capsule. The mantle zones that surround the follicles are markedly attenuated, and hence the germinal centers seem to merge with the interfollicular area. These changes, affecting primarily the B-cell areas of the node, are the morphologic reflections of the polyclonal B-cell activation and hypergammaglobulinemia seen in patients with AIDS. In addition to B-cell expansion within germinal centers, activated monocytoid B cells are present within and around the sinusoids and trabecular blood vessels. Under the electron microscope and by in situ hybridization, HIV particles can be detected within the germinal centers. Here they seem to be concentrated on the villous processes of follicular dendritic cells, presumably trapped in the form of immune complexes. During the early phase of HIV infection, viral DNA can be found within the nuclei of CD4+ T cells located
31

predominantly in the follicular mantle zone. With disease progression, the frenzy of B-cell proliferation subsides and gives way to a pattern of severe follicular involution. The follicles are depleted of cells, and the organized network of follicular dendritic cells is disrupted. The germinal centers may even become hyalinized. During this advanced stage, viral burden in the nodes is reduced, in part because of the disruption of the follicular dendritic cells. These "burntout" lymph nodes are atrophic and small and may harbor numerous opportunistic pathogens. Because of profound immunosuppression, the inflammatory response to infections both in the lymph nodes and at extranodal sites may be sparse or atypical. For example, mycobacteria may not evoke granuloma formation because CD4+ cells are deficient. In the empty-looking lymph nodes and in other organs, the presence of infectious agents may not be readily apparent without the application of special stains. As might be expected, lymphoid depletion is not confined to the nodes; in later stages of AIDS, the spleen and thymus also appear to be "wastelands." 5.2.4 Radiology The imaging asseeement for HIV-AIDS are case disease specific and may help in making the diagnosis, however there are no specific imaging for this syndrome particularly. The preferable imagings are X-ray, MRI, CT-Scan and others.

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6.0

LABORATORY

MONITORING

OF

PATIENTS

WITH

HIV

INFECTION
The epidemic of HIV2 infection and AIDS1 has provided the clinician with new challenges for integrating clinical and laboratory data to effect optimal patient management. The close relationship between clinical manifestations of HIV infection and CD4+ T cell count has made measurement of the latter a routine part of the evaluation of HIV-infected individuals. Determinations of CD4+ T cell counts and measurements of the levels of HIV RNA in serum or plasma provide a powerful set of tools for determining prognosis and monitoring response to therapy. While the CD4+ T cell count provides information on the current immunologic status of the patient, the HIV RNA level predicts what will happen to the CD4+ T cell count in the near future, and hence provides an important piece of prognostic information.

6.1 CD4+ T Cell Counts The CD4+ T cell count is the laboratory test generally accepted as the best indicator of the immediate state of immunologic competence of the patient with HIV2 infection. This measurement, which is the product of the percent of CD4+ T cells (determined by flow cytometry) and the total lymphocyte count [determined by the white blood cell count (WBC) and the differential percent] has been shown to correlate very well with the level of immunologic competence. Patients with CD4+ T cell counts <200/uL are at high risk of infection with P. carinii, while patients with CD4+ T cell counts <50/uL are at high risk of infection with CMV124 and mycobacteria of the M. avium complex (MAC) (Fig. 173-26). Patients with HIV infection should have CD4+ T cell measurements performed at the time of diagnosis and every 3 to 6 months thereafter. More frequent measurements should be made if a declining trend is noted. According to most guidelines, a CD4 T cell count <350/uL is an indication for consideration of initiating antiretroviral therapy, and a decline in CD4+ T cell count of >25% is an indication for considering a change in therapy. Once the CD4+ T cell count is <200/uL, patients should be placed on a regimen for P. carinii prophylaxis, and once the count is <50/uL, primary prophylaxis for MAC infection is indicated. As with any laboratory measurement, one

33

may wish to obtain two determinations prior to any significant changes in patient management based upon CD4+ T cell count alone.

6.2 HIV RNA Determinations Facilitated by highly sensitive techniques for the precise quantitation of small amounts of nucleic acids, the measurement of serum or plasma levels of HIV2 RNA has become an essential component in the monitoring of patients with HIV infection. As discussed under diagnosis of HIV infection, the two most commonly used techniques are the RT-PCR125 assay and the bDNA assay. Both assays generate data in the form of number of copies of HIV RNA per milliliter of serum or plasma and, by employing a 1:10 concentration step with ultracentrifugation, can detect as few as 50 to 75 copies of HIV RNA per milliliter of plasma. Although earlier versions of the bDNA assay generated values that were ~50% of those of the RT-PCR assay, the more recent versions (version 3 or higher) provide numbers essentially identical to those of the RT-PCR test. While it is common practice to describe levels of HIV RNA below these cut-offs as "undetectable," this is a term that should be avoided as it is imprecise and leaves the false impression that the level of virus is 0. By utilizing more sensitive, nested PCR126 techniques and by studying tissue levels of virus as well as plasma levels, HIV RNA can be detected in virtually every patient with HIV infection. Measurements of changes in HIV RNA levels over time have been of great value in delineating the relationship between levels of virus and rates of disease progression, the rates of viral turnover, the relationship between immune system activation and viral replication, and the time to development of drug resistance. HIV RNA measurements are greatly influenced by the state of activation of the immune system and may fluctuate greatly in the setting of secondary infections or immunization. For these reasons, decisions based upon HIV RNA levels should never be made on a single determination. Measurements of plasma HIV RNA levels should be made at the time of HIV diagnosis and every 3 to 4 months thereafter in the untreated patient. In general, most guidelines suggest that therapy be considered in patients with >50,000 copies of HIV RNA per milliliter. Following the initiation of therapy or any change in therapy, plasma HIV RNA levels should be monitored approximately every 4 weeks until the effectiveness of the therapeutic regimen is determined by the development of a new steady-state level of HIV RNA. In most
34

instances of effective therapy this will be <50 copies per milliliter. This level of virus is generally achieved within 6 months of the initiation of effective treatment. During therapy, levels of HIV RNA should be monitored every 3 to 4 months to evaluate the continuing effectiveness of therapy.

6.3 HIV Resistance Testing The availability of multiple antiretroviral drugs as treatment options has generated a great deal of interest in the potential for measuring the sensitivity of an individual's HIV2 virus(es) to different antiretroviral agents. HIV resistance testing can be done through either genotypic or phenotypic measurements. In the genotypic assays, sequence analyses of the HIV genomes obtained from patients are compared to sequences of viruses with known antiretroviral resistance profiles. In the phenotypic assays, the in vivo growth of viral isolates obtained from the patient are compared to the growth of reference strains of the virus in the presence or absence of different antiretroviral drugs. A modification of this phenotypic approach utilizes a comparison of the enzymatic activities of the reverse transcriptase or protease genes obtained by molecular cloning of patients' isolates to the enzymatic activities of genes obtained from reference strains of HIV in the presence or absence of different drugs targeted to these genes. These tests are quite good in identifying those antiretroviral agents that have been utilized in the past in a given patient. In the hands of experts, resistance testing enhances the short-term ability to decrease viral load by ~0.5 log compared to changing drugs merely on the basis of drug history. While some have advocated the use of resistance testing in the selection of an intial treatment regimen, the value of resistance testing in this setting is unknown.

6.4 Other Tests A variety of other laboratory tests have been studied as potential markers of HIV2 disease activity. Among these are quantitative culture of replication-competent HIV from plasma, peripheral blood mononuclear cells, or resting CD4+ T cells; circulating levels of 2microglobulin, soluble IL127-2 receptor, IgA, acid-labile endogenous interferon, or TNF36-a; and the presence or absence of activation markers such as CD38 or HLA-DR on CD8+ T cells. While these measurements have value as markers of disease activity and help to increase our
35

understanding of the pathogenesis of HIV disease, they do not currently play a major role in the monitoring of patients with HIV infection

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