The HIV-1 virus (Fig. 25-1) is composed of a lipid membrane, structural proteins, and glycoproteins that protrude. The viral genome consists of three important structural components—pol, gag, and env. These gene components code for various products (Table 25-1). Long terminal redundancies (LTRs) border these three components. HIV-2 has a different envelope and slightly different core proteins.

Table 25-1

Viral Genome Components

Component	Product
pol	Produces DNA polymerase
pol	Produces endonuclease
gag	Codes for p24 and for proteins such as p17, p9, and p7
env	Codes for two glycoproteins, gp41 and gp120

Figure 25-1 The human immunodeficiency virus.
The envelope is made up of glycoproteins (gp) of 120 kDa and 41 kDa. The main core protein is p24. As an RNA virus, it relies on reverse transcriptase to produce complementary DNA for transcription and translation. (From Peakman M, Vergani D: Basic and clinical immunology, ed 2, London, 2009, Churchill Livingstone.)

Cells infected with HIV can be examined with an electron microscope. The virus may appear as buds of the cell membrane particles. The virion has a double-membrane envelope and an electron-dense laminar crescent or semicircular cores. An intermediate, less electron-dense layer lies between the envelope and core. In a mature, free extracellular virion, the core appears as a bar-shaped nucleoid structure in cross section. This structure appears circular and is frequently located eccentrically. It is composed of structural proteins and glycoproteins that occupy the core and envelope regions of the particle. The virion consists of knoblike structures composed of a protein called glycoprotein (gp) 120, which is anchored to another protein called gp41. Each knob includes three sets of these protein molecules. The core of the virus includes a major structural protein called p25 or p24 encoded for by the gag gene. After human exposure, these and other viral components may induce an antibody response important in serodiagnosis (Table 25-2).

Table 25-2

HIV Proteins of Serodiagnostic Importance

Virus	Protein	Location	Gene
HIV-1	gp41	Envelope (transmembrane protein)	env
	gp160/120	Envelope (external protein)	env
	p24	Core (major structural protein)	gag
HIV-2	gp34	Envelope (transmembrane protein)	env
	gp140	Envelope (external protein)	env
	p26	Core (major structural protein)	gag

Retroviruses contain a single, positive-stranded ribonucleic acid (RNA) with the genetic information of the virus and a special enzyme called reverse transcriptase in their core. Reverse transcriptase enables the virus to convert viral RNA into deoxyribonucleic acid (DNA). This reverses the normal process of transcription in which DNA is converted to RNA—thus, the term retrovirus.

The genomes of all known retroviruses are organized in a similar way. In the provirus, which is formed when complementary DNA (cDNA) synthesis is completed from the retroviral RNA template, viral core protein, envelope protein, and reverse transcriptase are encoded by the gag, env, and pol genes, respectively, whereas viral gene expression is regulated by tat, trs, sor, and 3′orf gene products. The gag gene encodes a polyprotein found at high levels in infected cells and is subsequently cleaved to form p17 and p24, both of which are associated with viral particles. The pol gene encodes for reverse transcriptase, endonuclease, and protease activities. The sor gene stands for small open-reading frame. The sor gene product is a protein that induces antibody production in the natural course of infection. The tat gene also represents a small open-reading frame; the protein product has not been identified to date.

The env gene encodes for a polyprotein that contains numerous glycosylation sites. The glycoprotein gp160 is found on infected cells but is deficient on viral particles; however, gp160 gives rise to two glycoproteins, gp120 and gp41, which are associated with the viral envelope. The encoding genes and gene products, or antigens, of the AIDS virus may induce an antibody response after human exposure (Table 25-3).

Table 25-3

Encoding Genes and Antigens of AIDS Virus

Encoding Gene	Antigen
gag	p55
gag	p24
gag	p17
pol	p66
pol	p51
sor	p24
env	gp160
env	gp120
env	gp41
3′ orf	p27

LTRs, which exist at each end of the proviral genome, play an important role in the control of viral gene expression and the integration of the provirus into the DNA of the hosts. Although a structural similarity exists between the genomes of HIV-1 and HIV-2 (HTLV-IV), the nucleotide sequence homology is limited. There is a nucleotide sequence homology of only 60% between the gag genes and 30% to 40% between the remainder of the genes of HIV-1 and HIV-2.

Viral Replication

The replication of HIV is complicated and involves several steps (Fig. 25-2). The HIV life cycle is that of a retrovirus (Box 25-1). Retroviruses are so named because they reverse the normal flow of genetic information. In body cells, the genetic material is DNA. When genes are expressed, DNA is first transcribed into messenger RNA (mRNA), which then serves as the template for the production of proteins. The genes of a retrovirus are encoded in RNA; before they can be expressed, the RNA must be converted into DNA. Only then are the viral genes transcribed and translated into proteins in the usual sequence.

Box 25-1 Summary of HIV-1 Life Cycle

1. The virus attaches to the CD4 membrane receptor and sheds its protein coat, exposing its RNA core.

2. Reverse transcriptase converts viral RNA into proviral DNA.

3. The proviral DNA is integrated into the genome (genetic complement of host cell).

4. New virus particles are produced as the result of normal cellular activities of transcription and translation. Once the viral genome is integrated into host cell DNA, the potential for viral production always exists and the viral infection of new cells can continue.

5. New particles bud from the cell membrane.

Figure 25-2 HIV replication cycle
Steps in the HIV replication cycle
1. Fusion of the HIV cell to the host cell surface; 2. HIV RNA, reverse transcriptase, integrase, and other viral proteins enter the host cell; 3. viral DNA is formed by reverse transcription; 4. viral DNA is transported across the nucleus and integrates into the host DNA; 5. new viral RNA is used as genomic RNA and to make viral proteins; 6. new viral RNA and proteins move to the cell surface and a new, immature, HIV virus forms; 7. the virus matures by protease releasing individual HIV proteins. (Courtesy National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.)

Target Cells

The infectious process begins when the gp120 protein on the viral envelope binds to the protein receptor, called CD4, located on the surface of a target cell. HIV-1 has a marked preference for the CD4+ subset of T lymphocytes (Fig. 25-3). In addition to T lymphocytes, macrophages, peripheral blood monocytes, and cells in the lymph nodes, skin, and other organs also express measurable amounts of CD4 and can be infected by HIV-1. About 5% of the B lymphocytes may express CD4 and may be susceptible to HIV-1 infection. Macrophages may play an important role in spreading HIV infection in the body, both to other cells and to the target organs of HIV. Monocyte-macrophages enable HIV-1 to enter the immune-protected domain of the central nervous system (CNS), including the brain and spinal cord.

Figure 25-3 Mechanisms of HIV entry into a cell.
In the model, depicted sequential conformational changes in gp120 and gp41 promote fusion of the HIV-1 and host cell membranes. The fusion peptide of activated gp41 contains hydrophobic amino acid residues that promote insertion into the host cell plasma membrane lipid bilayer. (Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2007, Saunders.)

Fusion of the virus to the membrane of a host cell enables the viral RNA and reverse transcriptase to invade the cytoplasm of the cell. However, CD4 receptors are not sufficient for HIV envelope fusion with the T4 cell membrane or for HIV penetration or entry into the interior of the cell. Chemokine coreceptors to CD4, which HIV uses to enter a host cell after binding to it, have been identified. Beta chemokine receptors are cell surface proteins that bind small peptides. They are classified into three groups, depending on the location of the amino acid cysteine (C) in the peptide. These receptors are identified by the individual chemokine(s) that bind(s) to them. In essence, the reference to a specific chemokine also identifies its receptor. The first example of a coreceptor was CXCKR-4 (FUSIN R-4). Other coreceptors include CCKR-2 (R-2), CCKR-3 (R-3), and CC-CKR-5 (R-5). Current research involves exploring ways to block or fill the chemokine receptors with a harmless molecule, thus blocking the binding site of the HIV on the host cell.

Although some cells do not produce detectable amounts of CD4, they contain low levels of mRNA encoding the CD4 protein, which indicates that they do produce some CD4. Because these cells can be infected by HIV in culture, the expression of only minimal CD4 or an alternate receptor molecule may be sufficient for HIV infection to occur. These cell types include certain brain cells, neuroglial cells, a variety of malignant brain tumor cells, and cells derived from bowel cancers. Cells of the gastrointestinal system do not produce appreciable amounts of CD4, although chromaffin cells sometimes appear to be infected by HIV in vivo.

Replication

Retroviruses carry a single, positive-stranded RNA and use reverse transcriptase to convert viral RNA into DNA. The life cycle of the HIV-1 virus consists of five phases (see Box 25-1):

1. The virus attaches and penetrates target cells (e.g., lymphocytes) that express the CD4 receptor. After penetration, the virus loses its protein coat, exposing the RNA core.

2. Reverse transcriptase converts viral RNA into proviral DNA.

3. The proviral DNA is integrated into the genome (genetic complement of the host cell).

4. New virus particles are produced as a result of normal cellular activities of transcription and translation.

5. These new particles bud from the cell membrane.

Once the viral genome is integrated into host cell DNA, the potential for viral production always exists and the viral infection of new cells can continue.

Immunologic activation of CD4+ cells latently infected with HIV induces the production of multiple viral particles, leading to cell death. The extensive destruction of cells leads to the gradual depletion of CD4+ lymphocytes. Progressive defects in the immune system include a severe B cell failure, defects in monocyte function, and defects in granulocyte function.

Epidemiology

HIV causes a chronic infection that leads to a progressive disease. Without treatment, most persons with HIV develop AIDS, which results in substantial morbidity and premature death.

• CD4+ T-lymphocyte count of <200 cells/µL or CD4+ T-lymphocyte percentage of total lymphocytes of <14 or documentation of an AIDS-defining condition (see Appendix A). Documentation of an AIDS-defining condition supersedes a CD4+ T-lymphocyte count of >200 cells/µL and a CD4+ T-lymphocyte percentage of total lymphocytes of >14. Definitive diagnostic methods for these conditions are available in Appendix C of the 1993 revised HIV classification system and the expanded AIDS case definition (2) and from the National Notifiable Diseases Surveillance System (available at http://www.cdc.gov/epo/dphsi/casedef/case_definitions.htm).

HIV Infection, Stage Unknown

• No information available on CD4+ T-lymphocyte count or percentage and no information available on AIDS-defining conditions. (Every effort should be made to report CD4+ T-lymphocyte counts or percentages and the presence of AIDS-defining conditions at the time of diagnosis.)

2008 Surveillance Case Definition for HIV Infection Among Children Aged <18 Months

The 2008 case definition of HIV infection among children aged <18 months replaces the definition published in 1999 and applies to all variants of HIV (e.g., HIV-1 or HIV-2). The 2008 definition is intended for public health surveillance only and not as a guide for clinical diagnosis.

Criteria for Definitive or Presumptive HIV Infection

A child aged <18 months is categorized for surveillance purposes as definitively or presumptively HIV infected if born to an HIV-infected mother and if the laboratory criterion or at least one of the other criteria is met.

Laboratory Criterion for Definitive HIV Infection

A child aged <18 months is categorized for surveillance purposes as definitively HIV infected if born to an HIV-infected mother and the following laboratory criterion is met.

• Positive results on two separate specimens (not including cord blood) from one or more of the following HIV virologic (non-antibody) tests:– HIV nucleic acid (DNA or RNA) detection– HIV p24 antigen test, including neutralization assay, for a child aged >1 month– HIV isolation (viral culture)

Laboratory Criterion for Presumptive HIV Infection

A child aged <18 months is categorized for surveillance purposes as presumptively HIV infected if (1) born to an HIV-infected mother, (2) the criterion for definitively HIV infected is not met, and (3) the following laboratory criterion is met.

• Positive results on one specimen (not including cord blood) from the listed HIV virologic tests (HIV nucleic acid detection test; HIV p24 antigen test, including neutralization assay, for a child aged >1 month; or HIV isolation [viral culture] for definitively HIV infected) and no subsequent negative results from HIV virologic or HIV antibody tests.

Other Criteria (for Cases that Do Not Meet Laboratory Criteria for Definitive or Presumptive HIV Infection)

• HIV infection diagnosed by a physician or qualified medical-care provider based on the laboratory criteria and documented in a medical record. Oral reports of prior laboratory test results are not acceptable.

• When test results regarding HIV infection status are not available, documentation of a condition that meets the criteria in the 1987 pediatric surveillance case definition for AIDS.

Criteria for Uninfected With HIV, Definitive or Presumptive

A child aged <18 months born to an HIV-infected mother is categorized for surveillance purposes as either definitively or presumptively uninfected with HIV if (1) the criteria for definitive or presumptive HIV infection are not met and (2) at least one of the laboratory criteria or other criteria are met.

Laboratory Criteria for Uninfected With HIV, Definitive

A child aged <18 months born to an HIV-infected mother is categorized for surveillance purposes as definitively uninfected with HIV if (1) the criteria for definitive or presumptive HIV infection are not met and (2) at least one of the laboratory criteria or other criteria are met.^¶

• At least two negative HIV DNA or RNA virologic tests from separate specimens, both of which were obtained at age ≥1 month and one of which was obtained at age ≥4 months.

• At least two negative HIV antibody tests from separate specimens obtained at age >6 months.

and

• No other laboratory or clinical evidence of HIV infection (i.e., no positive results from virologic tests [if tests were performed] and no current or previous AIDS-defining condition)

Laboratory Criteria for Uninfected With HIV, Presumptive

A child aged <18 months born to an HIV-infected mother is categorized for surveillance purposes as presumptively uninfected with HIV if (1) the criteria for definitively uninfected with HIV are not met and (2) at least one of the laboratory criteria are met.

• Two negative RNA or DNA virologic tests, from separate specimens, both of which were obtained at age ≥2 weeks and one of which was obtained at age ≥4 weeks.^#

• One negative RNA or a DNA virologic test from a specimen obtained at age ≥8 weeks.

• One negative HIV antibody test from a specimen obtained at age ≥6 months.

• One positive HIV virologic test followed by at least two negative tests from separate specimens, one of which is a virologic test from a specimen obtained at age ≥8 weeks or an HIV antibody test from a specimen obtained at age ≥6 months.

and

• No other laboratory or clinical evidence of HIV infection (i.e., no subsequent positive results from virologic tests if tests were performed, and no AIDS-defining condition for which no other underlying condition indicative of immunosuppression exists).

Other Criteria (for Cases that Do Not Meet Laboratory Criteria for Uninfected With HIV, Definitive or Presumptive)

• Determination of uninfected with HIV by a physician or qualified medical-care provider based on the laboratory criteria and who has noted the HIV diagnostic test results in the medical record. Oral reports of prior laboratory test results are not acceptable.

and

• No other laboratory or clinical evidence of HIV infection (i.e., no positive results from virologic tests [if tests were performed] and no AIDS-defining condition for which no other underlying condition indicative of immunosuppression exists).

Criteria for Indeterminate HIV Infection

A child aged <18 months born to an HIV-infected mother is categorized as having perinatal exposure with an indeterminate HIV infection status if the criteria for infected with HIV and uninfected with HIV are not met.

2008 Surveillance Case Definitions for HIV Infection and AIDS Among Children Aged 18 Months to <13 Years

The 2008 laboratory criteria for reportable HIV infection among persons aged 18 months to <13 years exclude confirmation of HIV infection through the diagnosis of AIDS-defining conditions alone. Laboratory-confirmed evidence of HIV infection is now required for all reported cases of HIV infection among children aged 18 months to <13 years.

Criteria for HIV Infection

Children aged 18 months to <13 years are categorized as HIV infected for surveillance purposes if at least one of laboratory criteria or the other criterion is met.^∗∗

Laboratory Criteria

• Positive result from a screening test for HIV antibody (e.g., reactive EIA), confirmed by a positive result from a supplemental test for HIV antibody (e.g., Western blot or indirect immunofluorescence assay).

• Positive result or a detectable quantity by any of the following HIV virologic (non-antibody) tests^††:– HIV nucleic acid (DNA or RNA) detection (e.g., PCR)– HIV p24 antigen test, including neutralization assay– HIV isolation (viral culture)

Other Criterion (for Cases that Do Not Meet Laboratory Criteria)

Criteria for AIDS

Children aged 18 months to <13 years are categorized for surveillance purposes as having AIDS if the criteria for HIV infection are met and at least one of the AIDS-defining conditions has been documented.

^∗Rapid tests are EIAs that do not have to be repeated but require a confirmatory test if reactive. Most conventional EIAs require a repeatedly reactive EIA that is confirmed by a positive result with a supplemental test for HIV antibody. Standard laboratory testing procedures should always be followed.

^†For HIV screening, HIV virologic (non-antibody) tests should not be used in lieu of approved HIV antibody screening tests. A negative result (i.e., undetectable or nonreactive) from an HIV virologic test (e.g., viral RNA nucleic acid test) does not rule out the diagnosis of HIV infection.

^‡Qualified medical-care providers might differ by jurisdiction and might include physicians, nurse practitioners, physician assistants, or nurse midwives.

^§An original or copy of the laboratory report is preferred; however, in the rare instance the laboratory report is not available, a description of the laboratory report results by a physician or qualified medical-care provider documented in the medical record is acceptable for surveillance purposes. Every effort should be made to obtain a copy of the laboratory report for documentation in the medical record.

HIV nucleic acid (DNA or RNA) detection tests are the virologic methods of choice for the diagnosis of exclusion of infection in children aged <18 months. Although HIV culture can be used, culture is less standardized and less sensitive than nucleic acid detection tests. The use of p24 antigen testing to exclude infection in children aged <18 months is not recommended because of poor sensitivity, especially in the presence of HIV antibody. Commercial tests for RNA and DNA detection have become widely available. Quantitative RNA tests have been approved by the Food and Drug Administration (FDA) for monitoring HIV infection, and qualitative RNA tests have been approved to aid diagnosis. The quantitative and qualitative RNA tests meet FDA standards for high analytic and clinical sensitivity and specificity (14-16). All available tests detect the subtypes of group M and strains of group O. HIV-2 can be diagnosed with HIV-2 DNA PCR. HIV RNA tests sometimes do not detect HIV-2 because the viral loads in some HIV-2–infected persons are below detectable levels. Because of the possibility of mutation or recombination involving the sequences detected by a particular test, occasionally, virus might not be detected in a specimen from an HIV-2 infected individual. If HIV-2 infection seems likely but results are negative, testing with a different assay might be advisable.

^¶Suspected cases of HIV infection among children aged <18 months who are born to a documented HIV-uninfected mother should be assessed on a case-by-case basis by the appropriate health care and public health specialists.

^#If specimens for both negative RNA or DNA virologic tests are obtained at age ≥4 weeks, specimens should be obtained on separate days.

^∗∗Children aged 18 months to <13 years with perinatal exposure to HIV are categorized as uninfected with HIV if the criteria for uninfected with HIV among children aged <18 months are met.

^††For HIV screening among children aged 18 months to <13 years infected through exposure other than perinatal exposure, HIV virologic (non-antibody) tests should not be used in lieu of approved HIV antibody screening tests. A negative result (i.e., undetectable or nonreactive) by an HIV virologic test (e.g., viral RNA nucleic acid test) does not rule out the diagnosis of HIV infection.

Adapted from Centers for Disease Control and Prevention: Revised Surveillance Case Definitions for HIV Infection Among Adults, Adolescents, and Children Aged <18 Months and for HIV Infection and AIDS Among Children Aged 18 Months to <13 Years—United States, 2008. MMWR 57(RR10);1-8, December 5, 2008.

Infectious Patterns

Acquired immunodeficiency syndrome is present worldwide. In some countries and regions (e.g., sub-Saharan Africa, Thailand, India), more than 90% of HIV-1 infections are acquired through heterosexual transmission, in contrast to 10% or less in the United States and Western Europe. HIV-1 and HIV-2 are distinct but related viruses, and both can cause AIDS.

HIV-1

HIV-1 is responsible for the main AIDS epidemic. By analyzing genome sequences of representative strains, HIV-1 has been divided into four groups: group M (for major), including at least nine subtypes, three sub-subtypes of A, and two sub-subtypes of F (A1, A2, A3, B, C, D, F1, F2, G, H, J, and K); group O (for outlier); group N (for non-M, non-O), and group P.

The progression of the natural history and immunopathogenesis of HIV-1 infection can be demonstrated in six discrete stages (Fig. 25-4). These stages are based on the sequential appearance in plasma of HIV-1 viral RNA, the gag p24 protein antigen, antibodies that bind to fixed viral proteins. No matter how HIV-1 was acquired, the timing of the appearance of viral and other markers of infection is generally uniform and follows an orderly pattern.

Figure 25-4 Typical course of HIV infection.
During the early period after primary infection, there is widespread dissemination of virus and a sharp decrease in the number of CD4 T cells in peripheral blood. An immune response to HIV ensues, with a decrease in detectable viremia followed by a prolonged period of clinical latency. The CD4 T cell count continues to decrease during the following years until it reaches a critical level below which there is a substantial risk of opportunistic diseases. (From Pantaleo G, Graziosi C, Fauci A: The immunopathogenesis of human immunodeficiency virus infection, N Engl J Med 328:327-335, 1993.)

HIV-2

HIV-2 is endemic in parts of West Africa. HIV-2 strains have been classified into at least five subtypes (A through E). Epidemiologic data have indicated that the prevalence of HIV-2 infections in the U.S. population is extremely low.

The primary mode of transmission of HIV-2 is via heterosexual contact. The period between infection and disease may be longer and milder for persons with HIV-2 than for those with HIV-1. HIV-2 appears to be less harmful (cytopathic) to the cells of the immune system and it reproduces more slowly than HIV-1. Compared with persons infected with HIV-1, those with HIV-2 are less infectious early in the disease course. As the disease advances, HIV-2 infectivity seems to increase compared with HIV-1, but the duration of this increased infectivity is shorter.

Modes of Transmission

The HIV virus has been isolated from blood, semen, vaginal secretions, saliva, tears, breast milk, cerebrospinal fluid (CSF), amniotic fluid, and urine. Only blood, semen, vaginal secretions, and breast milk have been implicated in the transmission of HIV to date. HIV has been found in saliva and tears in very low quantities from some AIDS patients. It is important to understand that finding a small amount of HIV in a body fluid does not necessarily mean that HIV can be transmitted by that body fluid. HIV has not been recovered from the sweat of HIV-infected persons. Contact with saliva, tears, or sweat has never been shown to result in transmission of HIV.

HIV can be transmitted as the virus itself or as a cell associated with HIV. The virus is held within leukocytes and carried in fluid (e.g., blood, semen) to the body of another person. Transmission of HIV is believed to be restricted to intimate contact with body fluids from an infected person; casual contact with infected persons has not been documented as a mode of transmission. The risk of HIV infection to children born to women with HIV is 20% to 30%. HIV-2 seems to be less transmissible from an infected woman to her fetus or newborn.

Viral transmission of HIV-1 can be cervicovaginal, penile, rectal, oral, percutaneous, intravenous, in utero or breastfeeding after birth. More than 80% of adults infected with HIV-1 became infected through the exposure of mucosal surface to the virus; most of the remaining 20% were infected by a percutaneous or IV route.

Health care workers have been infected with HIV after being stuck with needles containing HIV-infected blood or, less frequently, after infected blood enters a worker’s open cut or a mucous membrane (e.g., eyes, inside of nose). Viral transmission can result from contact with inanimate objects, such as work surfaces or equipment recently contaminated with infected blood or certain body fluids, if the virus is transferred to broken skin or mucous membranes by hand contact.

Signs and Symptoms

The CDC has a classification used to define stages of HIV-related illness (Fig. 25-5). Infection with HIV produces a chronic infection with symptoms that range from asymptomatic to the end-stage complications of AIDS.

Figure 25-5 CDC classification.
The CDC classification is used to define stages of HIV-related illness. (From Peakman M, Vergani D: Basic and clinical immunology, ed 2, London, 2009, Churchill Livingstone.)

Typically, patients in the early stages of HIV infection are completely asymptomatic or show mild chronic lymphadenopathy. The early phase may last from many months to many years after viral exposure. Although the course of HIV-1 infection may vary somewhat in individual patients, a common pattern of development has been recognized. The newly revised HIV classification system provides uniform and simple criteria for categorizing conditions (see Box 25-2).

During the early period after primary infection, widespread dissemination of the virus occurs, with a sharp decrease in the number of CD4+ T cells in peripheral blood. The early burst of virus in the blood, viremia, is often accompanied by flulike symptoms that can be so severe that the affected person may seek help at a hospital emergency department. An immune response to HIV develops, with a concurrent decrease in detectable viremia. It was previously believed that the human immune system could drive the AIDS virus into a latent period that kept it inactive for years. However, this concept has been replaced with the new vision of a virus that is furiously creating copies of itself throughout the disease course, even when the patient appears healthy. Even when HIV cannot be detected in the blood (viremia), it infects lymphatic tissues (in large quantities), including the tonsils and lymph nodes throughout the body. The absence of viremia generally lasts until the end stage of the disease.

This phase is followed by a prolonged period of clinical latency (range, 7 to 11 years; median, 10 years). During the period of clinical latency, the patient is usually asymptomatic. Differences in the infecting virus, host’s genetic makeup, and environmental factors (e.g., concomitant infection) have been suggested as causes of the variable duration of clinical latency in persons not receiving antiretroviral therapy. Treatment with inhibitors of viral reverse transcriptase (e.g., zidovudine [Retrovir]) and prophylaxis for pneumonia caused by Pneumocystis jiroveci (previously called P. carinii) have increased AIDS-free time in HIV-1–infected persons.

Opportunistic Infections

Since AIDS was first recognized in the early 1980s, remarkable progress has been made in improving the quality and duration of life for HIV-infected persons in the industrialized world. During the first decade of the epidemic, this progress resulted from improved recognition of opportunistic disease processes, improved therapy for acute and chronic complications, and introduction of chemoprophylaxis against key opportunistic pathogens. The second decade of the epidemic witnessed extraordinary progress in developing highly active antiretroviral therapy (HAART), as well as continuing progress in preventing and treating opportunistic infections. HAART has reduced the incidence of opportunistic infections and extended life. In addition, prophylaxis against specific opportunistic infections continues to provide survival benefits even among patients receiving HAART.

The absolute number of CD4+ T lymphocytes continues to diminish as the disease progresses. When the number of cells reaches a critically low level (<50 to 100 ×10⁹/L), the risk of opportunistic infection increases. The period of susceptibility to opportunistic processes continues to be accurately indicated by CD4+ T lymphocyte counts for patients receiving HAART.

The end stage of AIDS is characterized by the occurrence of neoplasms and opportunistic infections (Box 25-3). The most common opportunistic infections are P. jiroveci (P. carinii) (Fig. 25-6), cytomegalovirus (CMV), Mycobacterium avium-intracellulare, Cryptococcus, Toxoplasma, Mycobacterium tuberculosis, herpes simplex, and Legionella. Histoplasma capsulatum is being recognized with increasing frequency. The most frequent malignancy observed is an aggressive, invasive variant of Kaposi’s sarcoma, discovered in many cases on autopsy. Malignant B cell lymphomas are increasingly recognized in patients with or at high risk for AIDS.

Figure 25-6 *Pneumocystis jiroveci (P. carinii)* from tracheobronchial aspirate (methenamine silver). (From Markell EK, Voge M: Medical parasitology, ed 5, Philadelphia, 1981, Saunders.)

Kaposi’s Sarcoma

Kaposi’s sarcoma (KS) was first described in 1872 by the dermatologist Moritz Kaposi. Since then, until the AIDS epidemic, KS remained a rare tumor. Classic KS usually occurs in males. The tumor typically presents with one or more asymptomatic red, purple, or brown patches; plaque; or nodular skin lesions. The disease is often limited to single or multiple lesions, usually localized to one or both lower extremities, especially involving the ankle and soles. Classic KS most often has a relatively benign, indolent course for 10 to 15 years or longer, with slow enlargement of the original tumors and gradual development of additional lesions. Up to one third of patients with classic KS develop a second primary malignancy, usually non-Hodgkin’s lymphoma. An increased incidence of Hodgkin’s disease occurs in HIV-infected homosexual men.

Cryptosporidiosis

Cryptosporidiosis is a disease caused by the parasite Cryptosporidium parvum. As late as 1976, this parasite was not thought to cause disease in human beings. In 1993, more than 400,000 people in Milwaukee, Wisconsin, became ill after drinking water contaminated with the parasite. Cryptosporidiosis can be chronic and severe in immunocompromised persons. The watery diarrhea can be prolonged and debilitating, and may be fatal.

Persons at risk of severe cryptosporidiosis include AIDS patients, cancer or organ marrow transplant patients taking drugs that weaken the immune system, and persons born with genetically weakened immune systems.

Disease Progression

Although a large enough dose of the right strain of HIV-1 can cause AIDS on its own, cofactors can influence the progression of disease development. Debilitated patients, weakened by a preexisting medical condition before HIV-1 infection, may progress toward AIDS more quickly than others. Stimulation of the immune system in response to later infections can also hasten disease progression. Other pathogenic microorganisms, such as a herpesvirus called human B lymphotropic virus (human herpesvirus 6 [HHV-6]), can interact with HIV in a way that may increase the severity of HIV infection. HHV-6 is usually easily controlled by the immune system. If HIV compromises the immune system, however, HHV-6 may replicate more freely and become a health threat. The main host of HHV-6 is the B cell, but this virus can also infect CD4+ cells. If these T cells are simultaneously infected by HIV, HHV-6 can stimulate the virus, which further impairs the immune system and promotes disease progression.

The progressive decline of CD4+ cells leads to a general decline in immune function and is the primary factor in determining the clinical progression of AIDS. Plasma HIV-1 RNA is a strong, CD4+ T cell–independent predictor of a rapid progression to AIDS after HIV-1 seroconversion.

Infection with HIV is presently considered to lead to death. When the clinically apparent disease develops, untreated patients usually die within 2 years, some exposed or HIV-1–infected patients never develop AIDS. The current hope is that an AIDS-free generation is on the horizon because of prevention strategies, e.g., safe sex, and pre-exposure prophylaxis.

Although scientists have known since 1986 that CD8 T cells, when stimulated, could release molecules capable of suppressing HIV, the identity of these substances eluded researchers for more than a decade. Studies have suggested that three large proteins, identified as alpha-defensins 1, 2, and 3, could be major contributors to the CD8 antiviral factor that protects some patients against AIDS. In another study, scientists at the National Institutes of Health (NIH) have linked HIV resistance to a different molecule secreted by CD8 T cells, called perforin. More studies related to each category of molecules are needed before either of these theories is confirmed.

Another study at the National Institute of Allergy and Infectious Diseases has examined variations in a gene called RANTES (regulated on activation, normal T cells expressed, and presumably secreted) in HIV-infected and HIV-resistant individuals. This study searched for changes in a single nucleotide polymorphism (SNP). The results showed that one such SNP appears more often in HIV-positive than in HIV-negative persons. In addition, this particular alteration increases the activity of the RANTES gene and is associated with up to twice the risk of HIV infection. However, HIV-infected patients with this SNP take about 40% longer to develop AIDS.

Immunologic Manifestations

Cellular Abnormalities

The HIV-1 virus has a marked preference for the CD4+ subset of lymphocytes because the CD4 surface marker protein on these cells serves as a receptor site for the virus. Immunologic activation (e.g., participation in an immune response to HIV-1 or viruses in other cells) of CD4+ cells latently infected with HIV-1 induces the production of multiple viral particles, leading to cell death. The extensive destruction of T cells leads to the gradual depletion of the CD4+ lymphocytes. The major phenotypic cell populations affected by AIDS are CD4+ and CD8+ subsets of T lymphocytes. Normally, the CD4+/CD8+ ratio is 2:1 in heterosexuals and 1.5:1 in homosexuals. A reversal of these subsets is evident in, but not diagnostic of, AIDS. In patients with AIDS, the ratio is less than 0.5:1. It is important to note that this results from a marked decrease in the absolute number of circulating CD4+ cells, rather than from an absolute increase in suppressor or CD8+ cells. This abnormality exists in the lymph nodes and circulating T cells. A diminished CD4+/CD8+ ratio (altered lymphocyte subpopulation) can also be seen in individuals with other disorders, such as cutaneous T cell lymphoma, systemic lupus erythematosus (SLE), and acute viral infections. The ratio, however, reverts back to normal after recovery from a viral infection in non–AIDS patients.

A decreased lymphocyte proliferative response to soluble antigens and mitogens exists in AIDS. Functional testing reveals a diminished response to pokeweed mitogen. This disease also demonstrates defective natural killer (NK) cell activity.

Immune System Alterations

The HIV virus is fragile and, as the virus particle leaves its host cell, a molecule called gp120 frequently breaks off the outer coat of the virus. Glycoprotein 120 can bind to the CD4 molecules of uninfected cells and, when that complex is recognized by the immune system, these cells can be destroyed. The lysis of infected cells and gp120-bound uninfected cells leads to the gradual depletion of the CD4+ lymphocytes. Defects in immunity are related to this T cell depletion. Progressive defects in the immune system also include a severe B cell failure and defects in monocyte and granulocyte function.

Although HIV-1 destroys CD4+ cells directly and hampers the immune system, this process does not cause the severe immunodeficiency seen in AIDS. The severe deficiency can be explained only if the cells are also destroyed by other means. Several indirect mechanisms have been suggested. Infection by HIV can cause infected and uninfected cells to fuse into giant cells called syncytia, which are nonfunctional. Autoimmune responses, in which the immune system attacks the body’s own tissues, may also be at work. In addition, HIV-infected cells may send out protein signals that weaken or destroy other cells of the immune system. It is possible that the binding of HIV to a target cell triggers the release of the enzyme protease. Proteases digest proteins; if released in abnormal quantities, they might weaken lymphocytes and other cells and decrease cell survival. The decline in T cells and subsequent alteration of the immune mechanism are the underlying factors in the progression of HIV infection.

Serologic Markers

Detection of Core Antigen

After initial infection, the body mounts a vigorous immune response against the viremia (see Color Plate 10). The first signal of an immune response to HIV-1 infection is the appearance of acute-phase reactants, including α₁-antitrypsin and serum amyloid in plasma 3 to 5 days after transmission. This is followed by a steep rise in the HIV-1 viral load (ramp-up viremia) that coincides with a large burst of inflammatory cytokines led by interferon-α and interleukin-15 (IL 15) and by a burst of plasma microparticles derived from infected and activated CD4 T cells undergoing apoptosis.

Immunologic activities include the production of different types of antibodies against HIV. Some antibodies neutralize the virus, others prevent it from binding to cells, and others stimulate cytotoxic cells to attack HIV-infected cells.

The time and sequence vary for the appearance and disappearance of antibodies specific for the serologically important antigens of HIV-1 during the course of infection. A window period of seronegativity exists from the time of initial infection to 6 or 12 weeks or longer thereafter. Through an enzyme immunofluorescence assay (EIA) based on defined HIV-1 proteins produced by recombinant DNA methods, antibodies specific for gp41 are detectable for weeks or months before assays specific for p24. The appearance of antibodies specific for p24 has been shown to precede that of anti-gp41 in serum specimens undergoing Western blot analysis. This discrepancy in the sequence of antibody appearance is believed to be caused by the greater sensitivity of Western blot compared with viral lysate–based EIAs used for the detection of anti-p24. The gp41 antibodies persist throughout the course of infection. Antibodies specific for p24 not only rise to detectable levels after gp41, but also can disappear unpredictably and abruptly.

Increased production of core antigen is believed to be associated with a burst of viral replication and host cell lysis. The disappearance of antibody directed against p24 occurs concomitantly with an increase in the concentration of core antigen in the serum. This parallel activity may result from the sequestration of antibody in immune complexes; the sudden decrease in anti-p24 is considered to be a grave prognostic sign in HIV-1–infected patients.

Antibodies to HIV-1

Antibodies to HIV-1 appear after a lag period of about 6 weeks between the time of infection and a detectable antibody response. Because of this, some virus-positive, antibody-negative individuals would be missed by initial screening assays.

In addition to a positive HIV antibody test in 85% to 90% of patients, increased antibody titers to other viruses (e.g., cytomegalovirus [CMV], Epstein-Barr virus, hepatitis A or B, Toxoplasma gondii) and circulating immune (antigen-antibody) complexes can be found. Other ancillary findings include polyclonal hypergammaglobulinemia, elevated levels of interferon-α (IFN-α), α₁-thymosin, and β-microglobulin, and reduced levels of IL-1 or IL-2.

Specific intrathecal synthesis of HIV antibody should be assessed simultaneously with an assay for total CSF immunoglobulin M (IgM) and for the intrathecal synthesis of total immunoglobulin G, as well as IgG specific for an appropriate control organism (e.g., adenovirus). In progressive encephalopathy related to AIDS, an increase in HIV antibody may suggest intrathecal rather than extrathecal synthesis.

Diagnostic Evaluation and Monitoring

Infection with HIV is established by detecting antibodies to the virus, viral antigens, or viral RNA-DNA or by the gold standard, viral culture. The standard test is for antibody detection. Laboratory evaluation of asymptomatic HIV-infected patients consists of the assessment of cellular and humoral components.

Screening of blood donors and patients at risk is usually done by serologic methods. In patients who have developed the signs and symptoms of AIDS, assessment of T lymphocytes and viral load concentrations are important, along with the diagnosis and treatment of opportunistic infections.

Both leukopenia and lymphocytopenia can exist in the AIDS patient. Total leukocyte and absolute lymphocyte concentrations need to be periodically assessed. The common denominator of AIDS is a deficiency of a specific subset of thymus-derived (CD4+) lymphocytes. Enumeration of lymphocyte subsets is usually performed by flow cytometry (see Plate 11).

Additional testing includes viral load assay and resistance testing, an in vitro method to measure the resistance of HIV to antiretroviral agents. Resistance testing can aid in antiretroviral drug selection but has limitations.

Testing Methods

Testing assays for HIV (Table 25-4) are categorized into the following three main types:

Table 25-4

HIV Assays and Characteristics

Assay	Format	Target Molecule	Comments
Lymphocyte CD4 absolute count	Flow cytometry	CD4+
HIV antigen assay for serum and plasma	EIA	HIV p24 antigen
HIV types 1 and 2 (HIV-1, HIV-2) antibody detection in serum or plasma	EIA (first- and second-generation tests)	Recombinant HIV-1 env and gag and HIV-2 env proteins or Purified, inactivated HIV-1 virus propagated in T-lymphocyte culture	If reactive, confirm with molecular testing (Western blot).
Detection of HIV-1 groups M and O	EIA (third generation)	Purified, inactivated HIV-1 viral lysate proteins, envelope proteins, and HIV-1 group O transmembrane protein or Purified gp160 and p24 recombinant proteins from HIV-1, HIV-2 transmembrane gp36, and synthetic epitope of HIV-1 group O
Enzyme-linked fluorescence p24 or EIA p24	EIA (fourth generation)	HIV-1 gp160, p24 antigen, and peptides representing regions of gp41 from HIV-1 group O and gp36 from HIV-2 or HIV-1 antigens p31 and gp41, HIV-2 p36 recombinant protein, HIV-1 group O gp41, and anti-p24 monoclonal antibodies
HIV antibody detection in serum or plasma	Western blot	Purified and inactivated HIV-1 strain LAV grown in CEM cell line or Purified and inactivated HIV-1 propagated in H9/HTLV-IIIB T lymphocyte cell line
HIV viral load assays	PCR	Reverse-transcriptase PCR or Nucleic acid sequence–based amplification or Signal amplification, branched-chain DNA
Rapid testing	Rapid immunoassay	Uses recombinant proteins representing regions of HIV-1 envelope proteins or Uses synthetic HIV structural proteins
Molecular Testing
HIV-1 RNA	Quantitative real-time PCR		Aids in assessing viral response to antiretroviral treatment
HIV-1 RNA	Quantitative bDNA	Quantitative branched-chain DNA	Aids in assessing viral response to antiretroviral treatment
HIV-1 DNA	Qualitative PCR		Detects HIV-1 proviral DNA in infants <48 hr old; repeat testing at 1-2 mo and 3-6 mo of age
HIV-2 antibody		Qualitative enzyme immunoassay, qualitative immunoblot	Screen for HIV-2 infection in a patient with an epidemiologic link to Africa.
HIV-1 genotyping	Reverse transcription PCR/nucleic acid sequencing		Detect changes in the viral genome associated with drug resistance. Use in conjunction with CD4 measurement to monitor treatment efficacy.
HIV-2 antibody confirmation	Qualitative immunoblot		Confirm positive screening results.
HIV-1 antibody	Qualitative chemiluminescent immunoassay

EIA, Enzyme immunoassay; PCR, polymerase chain reaction.

Adapted from Zetola N, Klausner JD: HIV testing: an update, MLO Med Lab Obs 38:58–62, 2006.

1. Detection of HIV antibodies

2. Detection of antigens, particularly p24

3. Detection or quantification of viral nucleic acids

HIV-1 Antibodies

Detection of HIV antibodies by EIA was the first technology developed for HIV diagnosis in 1985. Diagnostic testing is classified as screening or confirmatory testing. Screening tests include traditional EIAs and newer methods. Confirmatory tests include Western blot (WB), indirect immunofluorescent antibody assay (IFA), and HIV RNA detection by nucleic acid amplification testing (NAAT).

Antibodies to HIV can be detected by EIA (specificity 99%, sensitivity 98%; Table 25-5) and confirmed by the immunoblot technique. Antibody testing by EIA remains the standard method for screening potential blood donors. Simultaneous testing for p24 antigenemia is considered unnecessary. Third-generation serologic assays have demonstrated that seroconversion typically occurs 3 to 12 weeks after infection, but significant delays can occur in some individuals.

Table 25-5

Causes of False-Positive and False-Negative HIV Enzyme Immunoassay Results

False-Positive Result	False-Negative Result
Positive RPR (syphilis serology) test	Laboratory glove starch
Hematologic malignant disorder	Window period before seroconversion
DNA viral infections	Immunosuppressive therapy
Autoimmune disorders	Malignancies
Alcoholic hepatitis	Bone marrow transplantation
Vaccinations (e.g., hepatitis B, influenza)	Kits that mainly detect antibodies to p24
Chronic renal failure
Renal transplantation

RPR, Rapid plasma reagin.

Adapted from Specialty Laboratories, Santa Monica, Calif.

HIV Antigen and Genome Testing

Enzyme Immunoassay: p24 Antigen

The enzyme immunoassay for the HIV-1 antigen detects primarily uncomplexed p24 antigen. This procedure is applicable to blood or CSF testing as evidence of an active infection and can be diagnostic before seroconversion, can predict a patient’s prognosis, and is useful for monitoring response to therapy. Disadvantages of the procedure include poor sensitivity, inability to detect in patients with a high titer of p24 antibody, and failure of the method to detect HIV-2 antigen. Antibodies to p24 antigen are a better predictive marker of progression than p24 antigen.

Polymerase Chain Reaction

The polymerase chain reaction (PCR) allows for the direct detection of HIV-1 by DNA amplification. This ultrasensitive PCR technique has revolutionized HIV-1 detection. In addition to confirmatory testing, DNA amplification can be used for the diagnosis of very early, postexposure HIV infection in the window period before production of antibodies.

The goal of direct detection of active virus in patient specimens by an ultrasensitive method is to detect less than 100 molecules of viral nucleic acid in the peripheral blood cells isolated from 1 mL of blood. This number is the assay target because as few as 1 in 10,000 lymphocytes express viral RNA in HIV-1–infected individuals. Therefore, of approximately 10⁶ lymphocytes/mL blood, about 100 contain viral nucleic acid, corresponding to 100 to 150 copies of HIV-1 DNA. The presence of HIV-1 DNA in lymphocytes of antibody-positive, asymptomatic patients can be used to confirm exposure to the virus. The presence of viral RNA might be a sensitive indicator of viral replication and possibly of further disease progression.

The basis of PCR is the amplification of minute amounts of viral nucleic acid in lymphocyte DNA. In HIV-1–infected cells, the DNA template is a provirus that exists as integrated or episomal DNA. After amplification, isotope or nonisotope methods can detect the amplified product. The most effective means of target amplification is PCR. A pair of specific oligomer primers initiates DNA synthesis in combination with heat-stable Taq I DNA polymerase. After this first round of primer extension, the material is heated to denature the product from its template and cooled to 37° C (98.6° F) to permit annealing of the primer molecules to the original template DNA and to the newly synthesized DNA fragments. Primer extension is then resumed. By repetition of these cycles of denaturation, annealing, and extension, the original DNA can be increased exponentially.

Viral RNA can also be specifically amplified with some additional steps. The gag region is probably the best choice of a sequence for amplification. Detection of viral RNA and DNA in clinical specimens might prove to be a better indicator of biologically active virus than DNA detection alone. The presence of both provirus and viral RNA transcriptase would be a strong indication of viral replication. Quantitation of HIV RNA in plasma is useful for determining free viral load, assessing the efficacy of antiviral therapy, and predicting progression and clinical outcome in AIDS patients.

Confirmatory Testing

Western Blot

Before an HIV result is considered positive, the results should be reproducible and confirmable by at least one additional test. Western blot (WB) analysis is currently the standard method for confirming HIV-1 seropositivity (Fig. 25-7).

The WB assay is based on the recognition of the major HIV proteins (p24, gp41, gp120/160) by fractionating them according to their weight by electrophoresis and then visualizing their binding with specific antibodies over nitrocellulose sheets. A positive result is indicated by the presence of any two of the following bands—p24, gp41, and gp120/160. If the test is positive for bands gp41 and/or p24 in conjunction with a positive EIA test result, it is regarded as a confirmatory test. A negative result demonstrates the absence of bands. Indeterminate results can be found in 10% to 20% of EIA-positive tests. In general, the presence of a band at p24, p31, or p55, although still classified as indeterminate, is more indicative of true infection as compared with other band patterns.

The WB appears to work best with samples that contain high levels of antibody. Antibody specificities against known viral components (generally, the core component p24 and envelope component gp41) are considered true-positive results, whereas antibodies specific against nonviral cellular contaminants are nonspecific, false-positive results.

The WB technique is time-consuming and expensive. It is also open to considerable interpretation and has many sources of error. Variables in the test include the following:

• The technical skill and experience of the technologist performing the procedure

• Characteristics of the technical methodology

• General sensitivity of the WB in detecting antibodies specific for various HIV-1 antigens (especially during the window period of seronegativity)

• Frequent lack of specificity because of contamination of the viral reference preparation by histocompatibility and other antigens that electrophoretically migrate with p24 and gp41

• Variation in band reactivity patterns in sera from an individual over the course of HIV-1 infection

Indeterminate test results account for 4% to 20% of WB assays with positive bands for HIV-1 proteins. Indeterminate WB results can be caused by the following:

• Serologic tests in the process of seroconversion; anti-p24 is usually the first antibody to appear.

• End-stage HIV infection, usually with loss of core antibody

• Cross-reacting nonspecific antibodies, as seen with collagen vascular disease, autoimmune diseases, lymphoma, liver disease, injection drug use, multiple sclerosis, parity, or recent immunization

• Infection with O strain or HIV-2

• Recipients of HIV vaccine

• Perinatally exposed infants who are seroconverting (losing maternal antibody)

• Technical or clerical error

In addition, nonspecific reactions producing indeterminate results in uninfected persons have occurred more frequently in pregnant women or mothers than in persons in other groups characterized by low HIV seroprevalence. The incidence of indeterminate WB results is relatively low. The immunofluorescence assay can be used to resolve an EIA-positive, WB-indeterminate sample.

The most important factor in evaluating indeterminate results is risk assessment. Patients in low-risk categories with indeterminate test results are almost never infected with HIV-1 or HIV-2. Repeat testing usually continues to show indeterminate results, and the cause of this pattern is seldom established. Follow-up serology testing at 3 months is recommended to verify the previous results. Patients with indeterminate tests who are in the process of seroconversion usually have positive WB test results within 1 month; repeat tests at 1, 2, and 6 months are generally advocated, with appropriate precautions to prevent viral transmission in the interim.

False-positive WB results, especially those with a majority of bands, are extremely uncommon.

Quantitative RNA Assay

Recently, the U.S. Food and Drug Administration (FDA) approved a quantitative RNA assay for the confirmation of HIV infection.

Immunofluorescence Assay

IFA can provide a definitive diagnosis in samples that test indeterminate with other confirmatory tests. IFA is used to locate the HIV-1 antigen in infected cells. Infected cells are treated with polyclonal or monoclonal antibody against p17 or p24. After being washed, the cells are incubated with fluorescein isothiocyanate or rhodamine conjugate as a secondary antibody and then washed, mounted, and examined using a fluorescence microscope. The limitations of this technique include the need for expensive equipment and the fact that fluorescence fades quickly.

Immunohistochemical Staining

In immunohistochemical (IHC) staining, infected cells are incubated with HIV-1 antibody. After incubation, the cells are treated with an enzyme-labeled secondary antibody (usually alkaline phosphatase or horseradish peroxidase) and an appropriate substrate is added. The cells are washed and examined using simple light microscopy. IHC staining has the advantages of IFA but is simple and inexpensive and does not require extensive expertise. Morphologic changes can also be observed.

Fourth-Generation Testing

Previously, most tests used in the diagnostic setting detected only HIV antibodies. Fourth-generation assays detect HIV-1 p24 antigen up to 20 days earlier than Western blot and 5 to 7 days earlier than third-generation enzyme immunoassays. Levels of p24 antigen increase early after initial infection. More specific or supplemental tests for HIV-1 and HIV-2 (e.g., NAAT), Western blot, or immunofluorescence, must be performed to verify the presence of HIV-1 p24 antigen or antibodies to HIV-1 or HIV-2.

Fourth-generation assays allow for the differentiation between acute infection (p24 only, no HIV-1 antibody) and established infections (both p24 antigen and HIV-1 antibody). The gold standard for acute infection screening is NAAT. HIV-1 RNA can identify HIV infection as early as 5 days after exposure.

In 2010, the FDA approved the first fourth-generation immunoassay that detects both antigen and antibodies to HIV (ARCHITECT HIV Ag/Ab Combo Assay, Abbott Laboratories, Abbott Park, Ill). This test is a chemiluminescent microparticle immunoassay. The ARCHITECT HIV Ag/Ab Combo Assay was the first diagnostic test approved by the FDA for use in children as young as 2 years of age and pregnant women.

Other fourth-generation assays (e.g., GS HIV Combo Ag/Ab EIA, Bio-Rad Laboratories, Hercules, Calif) use EIA methodology (see later, procedure). These methods simultaneously test for HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma.

Rapid Testing

Routine HIV testing of whole blood in the emergency department has been shown to find unidentified cases. Currently, six rapid point-of-care testing (POCT) assays have FDA approval, including the OraQuick ADVANCE Rapid HIV-1/2 Antibody Test (OraSure Technologies, Bethlehem, Pa; see later, procedure). OraQuick ADVANCE can screen oral fluid and whole blood. In 2007, Inverness (Waltham, Mass) acquired rights to market the Chembio (Medford, NY) rapid test for the detection of HIV-1 and HIV-2 antibodies in fingertip blood, whole blood, serum, or plasma. Tests use protein A colloidal gold, which allows for the visual detection of HIV antibodies, or second-generation EIA so-called sandwich technology, with HIV-1 gp41 and HIV-2 gp36 synthetic antigen.

A disadvantage of rapid tests includes lower sensitivity than third- and fourth-generation EIA assays. The sensitivity of the currently available rapid tests is similar to that of second-generation EIA assays.

Currently, most protocols recommend confirming any positive rapid tests with WB or EIA. Follow-up with WB or EIA should be done 4 weeks later if confirmatory test results are negative or indeterminate.

Tests for Therapeutic Monitoring

Viral Load Testing

Testing of the viral load should be performed as soon as patient treatment begins. Subsequent viral load testing can be used as a marker for HIV viremia and should be carried out every 3 to 6 months for patients undergoing treatment. New guidelines recommend testing of viral load every 2 to 8 weeks after the initiation of HAART to determine early response to therapy. Fully automated assays have a fast turn-around time. Fully automated assays and real-time PCR are noted to have a lower rate of false-negative results when compared with nucleic acid sequence-based amplification.

CD4 T Lymphocyte Testing

Monitoring of CD4 T lymphocytes measures immune function. This information guides the initiation of antiretroviral therapy (ART) and monitors a patient’s response to antiretroviral treatment. CD4 T lymphocyte counts are expressed as absolute counts or as a percentage of the total population of CD4/CD8 as a ratio. Flow cytometry, in conjunction with fluorescence-activated cell sorting (FACS; see Chapter 13) is considered to be the gold standard in CD4 T lymphocyte counting.

Prevention

Reducing Viral Transmission

The CDC estimates 1.2 million people in the United States (U.S.) are living with HIV infection. One in five (20%) of those people are unaware of their infection. Despite increases in the total number of people in the U.S. living with HIV infection in recent years. New infections constitute approximately 50,000 Americans becoming infected with HIV each year. The CDC has issued new testing recommendations, making HIV screening a routine part of medical care for all patients 13 to 64 years of age. CDC officials hope that these revised guidelines will increase early HIV diagnosis so that individuals can access treatment, know their health care status, and prevent transmission to others.

Health care personnel should assume that the blood and other body fluids from all patients are potentially infectious (Standard Precautions; see Chapter 6).

Vaccines

Types of potential HIV vaccines include the following:

• Live attenuated vaccines

• Subunit vaccines

• DNA vaccines

• Recombinant vector vaccines

To date, the results of clinical trials have been disappointing (Box 25-4). Live attenuated vaccines are not currently being developed for use in human beings because of safety concerns. The first AIDS vaccine using the subunit concept, AIDSVAX gp120 vaccine, failed to protect against HIV infection in an efficacy trial. Many of the current AIDS vaccines in development are DNA vaccines. DNA vaccines will not cause HIV infection because they do not contain all the genes of the live pathogen. Another common strategy in AIDS vaccine development is recombinant vector vaccines. These will not cause HIV infection because they contain copies of only one or several HIV genes, not all of them. It is hoped that the addition of a vector will allow the vaccine to be more effective in creating an immune response than a DNA vaccine used alone.

Box 25-4 Clinical Trials for a Candidate HIV-AIDS Vaccine

Phase I

Phase I trials are the first human tests of a candidate vaccine, generally conducted on small numbers (10 to 30) of healthy adult volunteers who are not at risk for the disease in question. The main goal is evaluation of safety and, to a lesser extent, analysis of the immune responses evoked by the vaccine and of different vaccine doses and immunization schedules. A phase I trial usually takes 8 to 12 months to complete.

Phase II

Phase II testing involves a larger number of volunteers (50 to 500), usually a mixture of low-risk people and higher risk individuals from the population in whom phase III (vaccine efficacy) trials will eventually be conducted. Phase II trials generate additional safety data as well as information for refining the dosage and immunization schedule. Although not set up to determine whether the vaccine actually works, phase II trials are sometimes large enough to yield preliminary indications of efficacy. These trials generally take 18 to 24 months, with the increase over phase I primarily resulting from the additional time required for screening and enrolling larger numbers of trial participants.

Phase III

Phase III trials are the definitive test of whether a vaccine is effective in preventing disease. Using thousands of volunteers from high-risk populations in geographic regions in which HIV is circulating, the incidence of HIV in vaccinated people is compared with that in people who receive a placebo. Successful demonstration of efficacy in a Phase III trial can then lead to an application for licensure of the vaccine.

Phase III trials of AIDS vaccines are generally expected to require a minimum of 3 years for enrollment, immunizations, and assessments of efficacy. Although there has been recent progress in increasing access to treatment and prevention programs, HIV continues to outpace the global response, with at least 80% of those in clinical need of antiretrovirals (ARVs) worldwide not receiving them. Furthermore, although a decline in national HIV prevalence has occurred—for example, in some Sub-Saharan African countries—these trends are not strong or widespread enough to have a major impact on the epidemics.

Adapted from International AIDS Vaccine Initiative: Vaccine science, 2007 (www.iavi.org).

Continued testing of vaccines is needed to determine whether they are more immunogenic in different doses, in different populations, and in combination with other candidate HIV vaccines (see Chapter 16).

More than 30 clinical trials have been in progress to develop a vaccine to prevent HIV (see www.iavi.org). The first generation of successful HIV vaccines likely will offer some protection but will not be entirely protective—no vaccine is 100% effective. Future generations of a preventive HIV vaccine will become increasingly more effective over time as scientific knowledge improves. However, even partially effective vaccines could make a difference in the following ways:

1. Protect some vaccinated individuals against HIV infection.

2. Reduce the probability that a vaccinated individual who later becomes infected will transmit the infection to others.

3. Slow the rate of progression to AIDS for those who later become infected with HIV.

An HIV vaccine, RV144, is being studied by the U.S Military HIV Research Program at the Walter Reed Army Institute of Research. This clinical trial has involved more than 16,000 adult volunteers in Thailand. In this study, the recipients of the vaccine had a 31% lower chance of becoming infected with HIV than those in the placebo group. Since the study results reported in 2009, more than 100 scientists from 25 institutions have been searching for molecular clues to explain why the vaccine showed a modest protective effect. An antibody formed by vaccinated individuals who made high levels of the antibody resulted in their being significantly less likely to become infected than those who did not. This particular binding antibody attaches to a part of the outer coat of the virus, the first and second variable regions, or V1V2, which may play an important role in HIV infection of human cells. The antibody belongs to the IgG family. Vaccinated study participants who built different antibodies to the vaccine appeared to have less protection from HIV. Further studies of this vaccine will determine whether the V1V2 antibody response is merely a marker of HIV exposure or of decreased susceptibility to HIV infection. The most recent studies of this vaccine have generated the hypothesis that V1V2 antibodies may have contributed to protection against HIV-1 infection, but high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.

An HIV vaccine could substantially alter the course of the AIDS pandemic and reduce the number of people newly infected, even if vaccine efficacy and population coverage levels are relatively low.