9: Human Immunodeficiency Virus

CHAPTER 9
Human Immunodeficiency Virus


Richard L. Hodinka


University of South Carolina School of Medicine, Greenville, and Greenville Health System, Greenville, SC, USA


9.1 Introduction


Currently, a number and variety of techniques and instrumentation are available for use in the diagnosis and management of patients infected with human immunodeficiency virus (HIV) (Table 9.1). The selection of which tests to perform depends greatly on the patient population and clinical scenario and the intended use of each individual assay (Table 9.2). Serological screening and confirmatory assays designed for antibody detection or a combination of antibodies and p24 antigen are most commonly used for the laboratory diagnosis of HIV infection. Tests that simultaneously measure antibody and p24 antigen, assays that detect p24 antigen alone, and molecular amplification assays for identification of viral DNA or RNA allow for the detection of acute HIV infection in newborn babies and other individuals at risk and aid in shortening the diagnostic “window-period” between infection and seroconversion. The additional use of quantitative nucleic acid-based assays provides invaluable information about the prognosis and response to therapy, while phenotypic, genotypic, and tropotypic tests are commonly used to detect antiretroviral drug resistance. The choice of whether to incorporate each of these methods into the clinical laboratory will depend on the patient population and clinical needs as well as the number of specimens to be tested; the cost, turnaround time, and ease of testing; and the resources and capabilities of the individual laboratory.


Table 9.1 Characteristics of available virological methods for the diagnosis and monitoring of HIV infection










































































Test Specimen required Cost Technical difficulty Turn-around (days) Diagnostic value
Screening immunoassays* Serum or plasma Low Low 1–2 High in patients > 15–18 months of age
Standard p24 antigen Serum or plasma Low Low 1–2 Low in all groups
ICD p24 antigen Serum or plasma Low Low 1–2 Moderate to high in patients > 1 month of age
Qualitative proviral DNA PCR PBMC Moderate Moderate 1–2 High in all groups
Qualitative RNA NAAT§ Plasma, serum Moderate Moderate 1–2 High in all groups
Viral culture PBMC High High 14–28 High in all groups
Quantitative RNA NAAT Plasma Moderate Moderate 1–2 High in all groups
Phenotypic drug susceptibility assays Patient’s viral isolate High High 14–42 High in all groups
Genotypic drug susceptibility assays Plasma High High 1–2 High in all groups

ICD, immune complex dissocation; PCR, polymerase chain reaction; NAAT, nucleic acid amplification test; PBMC, peripheral blood mononuclear cells.


* Screening immunoassays currently include antibody-only and antigen/antibody combination laboratory-based and rapid, less-sophisticated tests.


Individual p24 antigen-only assays are not routinely used in most clinical settings; used primarily in resource-poor countries and in research settings.


Qualitative proviral DNA PCR has limited commercial availability; assay also can be performed on whole blood, dried blood spots, plasma, other body fluids, and tissues.


§ Qualitative RNA NAATs are commercially available and FDA licensed, and have largely replaced qualitative proviral DNA tests.


Viral culture is primarily used in support of HIV research studies and is seldom needed today in a clinical setting; assay also can be performed on plasma, other body fluids, and tissues.


Table 9.2 Use of laboratory tests for HIV diagnosis and management


































Clinical situation Recommended test(s)
Blood donor screening Antibody screening immunoassay (EIA or CLA) and confirmatory test (WB or IFA), qualitative RNA NAAT
Routine screening/diagnosis of HIV infection Antibody-only and antigen/antibody combination EIA or CLA laboratory-based and rapid, less-sophisticated screening tests and confirmatory/differentiation assays
Acute HIV infection EIA or CLA antigen/antibody combination immunoassays, qualitative RNA NAAT
Infant (≤18 months of age) born to HIV-infected mother Qualitative proviral DNA PCR or qualitative RNA NAAT
Indeterminate HIV-1 WB Repeat HIV-1 antibody screening immunoassay and WB in ~2–3 weeks, perform HIV-2-specific antibody immunoassay and WB, qualitative RNA or proviral DNA NAAT
Indeterminate HIV-1/HIV-2 antibody differentiation assay Qualitative RNA NAAT
Prognosis Quantitative RNA NAAT
Response to therapy Quantitative RNA NAAT
Antiretroviral drug resistance Phenotypic, genotypic, tropotypic resistance assays

EIA, enzyme immunoassay; CLA, chemiluminescent assay; WB, Western blot; IFA, immunofluorescence assay; NAAT, nucleic acid amplification assay, PCR, polymerase chain reaction.


The emphasis of this chapter is on the many and varied technologies that are currently available commercially for the diagnosis and monitoring of HIV infections, and is not meant to duplicate other extensive reviews of HIV. Important characteristics of the assays are described, and the key advantages and disadvantages are presented. Due to competition among manufacturers and strict regulation worldwide, many excellent commercial diagnostic products have been developed that are well standardized and of high sensitivity and specificity. Most of the commercial assays are Conformité Européenne (CE) marked and are compliant with the European Union and have received regulatory approval for widespread clinical use in Europe, Asia, Japan, Canada, or Australia, while a smaller number have been registered in the United States and have been approved by the US Food and Drug Administration (FDA) for diagnostic use. The rapid pace of development of HIV-related tests has led to an ever changing market, with the introduction of many new assays and the discontinued use of others. A number of manufacturers and vendors are new to the field, while others have merged or left the market place. Consequently, some products have been transferred from one vendor to another, and many of the kit names have changed. Some products are also made by one company and distributed and sold under multiple brand names, making it more difficult to compile a comprehensive list. The information presented in this chapter about each commercial assay was obtained either from the published literature or from the individual manufacturers through their websites, written materials, and/or personal communications with company representatives. While every attempt has been made to include any and all manufacturers of HIV-related test products, users are advised that the amount of material is great and not every available test, device, product, or instrument may be included. Additional tests, devices, instruments, and products may be available elsewhere and the reader must thoroughly evaluate the availability and regulatory compliance of all products in their country or region. Individuals should contact the manufacturers listed in the Appendix of this manual for a more comprehensive description and the current list price and availability of a particular product.


9.2 Markers of HIV infection


HIV is classified into two major types, HIV type 1 (HIV-1) and type 2 (HIV-2). HIV-1 is responsible for the majority of HIV infections worldwide and is divided into four groups, designated M (major), O (outlier), N (non-O), and P. Group M is the most prevalent and is further subdivided into subtypes or clades designated A–D, F–H, J, and K. Coinfection with different subtypes of HIV-1 can give rise to circulating recombinant forms (CRFs).


Following infection with HIV-1, different HIV markers of diagnostic significance appear at various times in the blood of an infected individual, including viral RNA, p24 antigen, and circulating antibodies [16, 37, 99, 100, 122]. Measurable HIV-1 RNA is the first marker to be detected by using qualitative and/or quantitative nucleic acid amplification tests. This normally occurs within 10–11 days of an individual being infected and the levels of viral RNA rise rapidly and become markedly elevated over the next few weeks before decreasing to baseline levels that remain detectable over time. HIV-1 p24 antigen is the next marker to rise in an infected individual and can be detected within 4–10 days after the initial detection of HIV-1 RNA by using assays that detect p24 antigen alone or by using HIV antigen/antibody combination assays. The production of p24 antigen is transient and declines to low or undetectable levels with the appearance of antibodies to this protein. This is due to the formation of immune complexes that interfere with p24 antigen detection. Finally, HIV antibodies appear as the infection with HIV results in the induction of a humoral antibody response specific to viral proteins, with the production of immunoglobulin M (IgM), IgG, and IgA. The structural proteins of HIV-1 are the targets for the majority of the circulating antibodies directed against the virus. These include the envelope (env) proteins (surface glycoprotein [gp 120], transmembrane glycoprotein [gp 41], and their precursor glycoprotein [gp160]), polymerase (pol) proteins (reverse transcriptase [p65], endonuclease-integrase [p31], and protease [p10]), and core (gag) proteins (matrix protein [p18], internal capsid protein [p24], nucleocapsid protein [p7], and their precursor protein [p55]). Antibodies to HIV-1 are detectable in most persons within 4–12 weeks after infection and in virtually all patients within 6 months. Variable levels of immunoglobulin M (IgM) antibodies appear first, quickly reaching a peak and declining over the following weeks. HIV-specific IgM antibodies are detectable 3–5 days after the initial appearance of p24 antigen and 10–13 days after viral RNA is detected. About 1 week later, IgG antibody levels rise significantly, reach a plateau within a few months, and remain high for many years. The time at which IgG antibodies can be detected will vary depending upon the sensitivity of the serologic immunoassay used for screening and may be as short as 4–7 days and as long as 18–38 days or more after the initial detection of viral RNA. In some individuals, antibody to core proteins (p24 and/or p18) and sometimes polymerase proteins (p65 and/or p31) may drop significantly, and, in rare cases, even become undetectable with progression to HIV disease. Also, initiation of antiretroviral therapy prior to seroconversion can result in a delayed and decreased antibody response or a lack of a detectable antibody response to HIV infection. Elevated levels of HIV-specific serum IgA antibody can be found in infants as early as 3 weeks after birth and may persist for an extended time following acute infection.


9.3 HIV screening


Since 1989, tests for the detection of HIV-specific antibodies have been at the forefront for the diagnosis of HIV infection. They have been effectively used to determine if an individual has been exposed to HIV, to screen blood and plasma donations, and for epidemiological surveillance. During this time, the standard testing algorithm used in the United States and in many parts of the world involved a two-stage testing strategy. In this algorithm, HIV Infection is first identified by using an enzyme immunoassay (EIA), in which an enzyme and a substrate is used to produce a colorimetric signal to screen specimens for the detection of HIV-specific antibodies. If specimens are nonreactive for HIV antibodies when initially tested by EIA, no further testing is performed and the individual is considered to be uninfected. Specimens with initially reactive results by EIA are then repeated in duplicate using the same EIA. If one or both of the duplicate samples of the specimen are reactive by EIA, the final EIA result is considered positive. If the two repeat samples of the specimen are nonreactive, the specimen is considered negative for HIV antibodies by EIA. Repeatedly reactive specimens are then confirmed by Western blot or immunofluorescence assays (IFA) to assure the specificity of the EIA result. Initially, immunoassays and confirmatory tests were designed to detect antibodies only to HIV-1. In 1992, the Centers for Disease Control and Prevention (CDC) recommended specific screening for both HIV-1 and HIV-2 antibodies as the prevalence of HIV-2 increased worldwide, and also recommended the use of more specific confirmatory tests to verify the presence of antibodies against HIV-2 if the HIV-1 Western blot was negative or indeterminate and the clinical situation warranted testing for HIV-2. In 2004, it was recommended by the CDC that all specimens determined to be reactive by rapid HIV-specific antibody assays also be confirmed with either a Western blot or IFA specific for antibodies to HIV-1. For over 25 years, a determination of positive antibody reactivity by EIA followed by confirmation using one of the confirmatory tests remained the most convincing laboratory evidence for a diagnosis of HIV infection. However, there are shortcomings with this approach, including delays in turnaround time for HIV test results, lack of differentiation of HIV-1 from HIV-2 infection, and the inability to detect acute primary HIV infections.


More recently, there have been significant advances in HIV diagnostics that have led to the development of newer screening immunoassays with improved speed and performance and enhanced sensitivity and specificity, immunoassays that can distinguish and differentiate antibody responses to HIV-1 and HIV-2, and antigen- and nucleic-acid-based assays for earlier detection of HIV infection. In June of 2014, based on these changes in technology, the CDC and Association of Public Health Laboratories recommended a new algorithm for the diagnosis of HIV infection in adults and children > 24 months of age [19]. In the new algorithm, testing begins with a fourth-generation combination screening immunoassay that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen to test for established infections with either virus and acute infection with HIV-1. This is followed by a rapid antibody test that differentiates HIV-1 and HIV-2 antibodies for confirmation of specimens that are reactive on the antigen/antibody combination immunoassay. Specimens that are nonreactive or indeterminate on the rapid HIV-1/HIV-2 antibody differentiation immunoassay are then tested by an HIV-1-specific nucleic acid amplification assay for resolution of the results. It is also recommended that this same testing algorithm be used on serum or plasma specimens submitted for testing after a reactive preliminary result from any rapid HIV test and should be considered on specimens that are nonreactive by rapid HIV tests since laboratory-based antigen/antibody combination immunoassays detect HIV infection earlier than any of the rapid HIV tests, including commercially available rapid HIV-1/HIV-2 antigen/antibody combination test. The newly recommended algorithm offers distinct advantages over the previously used algorithm, including identification of acute HIV infection as much as 3–4 weeks earlier, more accurate diagnosis of HIV-2 infection, a decrease in indeterminate results and reporting times for most test results, and improved linkage to care and use of antiretroviral therapy [18, 19, 30, 99, 112, 122, 176]. Also, the HIV-1 Western blot and HIV-1 IFA are no longer part of the recommended algorithm.


Coincident with the changes in the testing algorithm, there have also been changes in the population for whom HIV testing is recommended. In 2006, the CDC recommended expansion from an approach of targeted testing of high-risk groups to a strategy that incorporates routine, universal screening of all patients aged 13–64 years [12]. The US Preventive Services Task Force (USPSTF) has since recommended that all adolescents and adults aged 15–65 years be screened for HIV infection and that screening be performed on all pregnant women, including those who present in labor who are untested and whose HIV status is unknown, and younger adolescents and older adults who are at increased risk for HIV infection [109].


9.4 Laboratory-based immunoassays


A wide selection of laboratory-based immunoassay kits that detect HIV-specific antibodies have been developed over the years and made available from a variety of commercial manufacturers (Table 9.3). The assays offer the distinct advantage of using highly standardized and stable immunoreagents that provide accurate and objective results. They normally require minimal training and equipment and are applicable to large numbers of specimens at a reasonable cost. The different test kits vary in their configurations, the number and type of antigens used to capture HIV-specific antibodies, the specimen sources that can be tested, and whether the assays are designed for detecting antibodies to either HIV-1 or HIV-2 or for the simultaneous detection of antibodies to both viruses or for the combined detection of both antibodies and antigens. Many of the newer immunoassays now use chemiluminescence, fluorescence, phosphorescence, and electrochemiluminescence measurements instead of the enzyme/substrate detection system used by the more traditional EIAs.


Table 9.3 Screening immunoassays for the detection of HIV antibodies and p24 antigen

c09-tbl001

CMIA, chemilumenescent microparticle immunoassay; EIA, enzyme immunoassay; SPR ELFA, solid-phase receptacle enzyme-linked fluorescent assay; CLIA, chemilumenescence immunoassay; ECLA, electrochemiluminescence assay; IgG, immunoglobulin G; Ab, antibody; Ag, antigen; IgM, immunoglobulin M; NF, none found.


Since being approved for diagnostic use in 1985, immunoassays for the detection of HIV-specific antibodies have evolved through four generations of change and improvement, with the source of antigen(s) used and the assay design being significant factors in determining the overall sensitivity and specificity of the tests (see reference [24] for a review). With each new generation of HIV immunoassay, the rate of false-positive results was decreased and the time to positivity was reduced, with a corresponding improvement in the detection of early infection.


The original first-generation kits used viral proteins prepared from lysates of HIV-1-infected human T-cell lines as the source of antigens that were bound to a solid phase for capturing HIV-specific antibodies from a patient’s specimen. The antigen–antibody complexes that form were then detected with the addition of an enzyme-labeled, anti-human antibody that binds to the complexes and reacts with a chromogenic substrate to produce a color change. The intensity of the color generated was measured in a spectrophotometer and compared with a set of positive and negative controls performed with each batch of specimens. Horseradish peroxidase and alkaline phosphatase were the most common enzyme labels, and the surfaces of microwell plates and polystyrene beads were used as the solid-phase carriers. With first-generation assays, specimens had to be significantly diluted to overcome cross-reactivity with cellular proteins that contaminated the prepared lysates.


Scientific and technological advances over the years led to the incorporation of recombinant antigens and synthetic peptides into second- and third-generation immunoassays to improve their sensitivity and specificity over traditional tests based on whole viral lysates. The assay format of second-generation assays, however, was similar to that for first-generation tests and, like first-generation assays, detected only IgG antibody to HIV-1. Third-generation assays underwent a more radical design change adding antigens to detect antibodies to HIV-1 and HIV-2 and using an immunometric antigen sandwich format in which antibodies in the specimen are sandwiched between HIV-specific antigens attached to the solid phase and to HIV antigens that are conjugated to indicator molecules and added to the reaction. This format has the distinct advantage of efficiently and simultaneously detecting IgG and IgM class antibodies to both virus types. Third-generation assays also have a greater sensitivity for detecting HIV antibodies in the early stages of infection, thereby shortening the time to seroconversion. The time to positivity for first- and second-generation assays has been estimated to be approximately 35–45 days and 25–35 days, respectively, following infection, while the time to positivity for third-generation assays is approximately 20–30 days [19, 24].


Over the years, the availability of commercial first- and second-generation laboratory-based HIV screening immunoassays in the United States and international markets has diminished to the point that few, if any, kits remain, while some third-generation assays continue to be sold.


More recently, a number of fourth-generation screening immunoassays have been developed and commercialized for the simultaneous detection of HIV-1 p24 antigen and antibodies to HIV-1 (including group O) and HIV-2 (Table 9.4). These combination assays use synthetic peptide or recombinant protein antigens in the same antigen–antibody–antigen sandwich format as third-generation assays to detect IgG and IgM antibodies to HIV-1 and HIV-2, and also include monoclonal antibodies to capture and detect HIV-1 p24 antigen. The format of these assays usually does not allow for the differentiation of antibody reactivity from antigen reactivity, although, more recently, several assays have been designed to discriminate between antibody and antigen detection. Fourth-generation assays provide an increase in sensitivity over tests that only detect antibody and allow a reduction in the window period between infection with HIV-1 and detectable seroconversion. The time to positivity for fourth-generation assays has been estimated to be 15–20 days following infection [19, 24].


Table 9.4 Rapid and/or simple immunoassays for the detection of HIV antibodies and p24 antigen

c09-tbl002

LF, lateral flow; FT, flow through; MPEIA, magnetic particle EIA; ID, immunodot; PA, particle agglutination; Ab, antibody; Ag, antigen; NF, none found.


In general, the different generations and formats of laboratory-based immunoassays used over the years to screen specimens have been considered to be sensitive and specific for the detection of antibodies to HIV. The predictive value of a positive result, however, can vary considerably and depends on the prevalence of HIV infection in the population being tested. Few false-positive results are observed when testing high-risk populations, while a high rate of false-positive results may occur in populations with low prevalence of HIV infection, such as blood donors. When using first-generation laboratory-based assays, biological false-positive reactions were mainly due to reactivity of antibodies to human leukocyte antigen (HLA) proteins expressed by the T-cell lines used to prepare the viral lysates. With second-, third-, and fourth-generation assays, the use of recombinant antigens or synthetic peptides significantly decreased the number of false-positive results but has not completely eliminated them. False-positives may be the result of passive immunoglobulin administration, receipt of an experimental HIV vaccine, recent exposure to certain vaccine preparations (e.g., influenza vaccine), or the patient having cross-reactive antibodies to contaminating bacterial or yeast proteins used in recombinant antigen-based immunoassays. Therefore, the immunoassay, although an excellent test for screening, should not be used as the sole test for making a diagnosis of HIV infection. A positive confirmation test is also necessary to exclude false-positive results.


A negative antibody screening immunoassay result normally means HIV infection is unlikely. However, false-negative results may occur and may be due to immunosuppressive therapy, replacement transfusion, severe hypogammaglobulinemia (B-cell dysfunction and defective antibody synthesis), genetic diversity of the virus itself, and testing too early or too late in the course of illness. Although it is possible that levels of HIV-specific antibodies may fall during advanced disease, they rarely drop below the detection limits of current immunoassays. Group O HIV-1 strains have been described that have evolved to the extent that antibodies to this group are not detected by some immunoassays on the market. Group O HIV-1 strains have been seen predominantly in Africa and Europe, although a small number of infections with this group have also been identified in the US. Immunoassay kits using viral lysates as the antigen source are more efficient at detecting this highly divergent group than kits that use recombinant antigens or synthetic peptides. Most current commercial immunoassays have been reformulated to contain specific antigens to group O in order to recognize infection with this group. Similarly, most commercial tests are now designed to detect antibodies to HIV-1 and HIV-2 to effectively screen for both virus types. Separate HIV-2 screening and supplemental tests are also commercially available and should be considered if it is suspected that HIV-2 infection is probable. Specific HIV-2 antibody testing should be considered for persons from West Africa, where the virus is endemic, individuals who have received blood transfusions from or have had sexual relations with someone from this region, and children of women at risk of infection or known to be infected with HIV-2.


Lastly, laboratory-based immunoassays have the greatest potential for automation, and a number of semi-automated and fully automated immunoassay analyzers are now commercially available for the performance of HIV serological assays. The majority of the automated immunoassay analyzers provide walk-away simplicity to perform assays from sample processing through interpretation of results (see Chapter 17).


9.5 Rapid, less-sophisticated immunoassays


Rapid (10–20 min) and less sophisticated immunoassays for the detection of HIV antibodies have also been developed by a number of manufacturers (Table 9.4). The tests offer the distinct advantages of lower costs and same-day results and are packaged as ready-to-use kits with all reagents and materials included. The kits are designed to be performed either in the laboratory or at the point of care (POC) where the specimen was collected using a single step or a few simple steps and self-contained, disposable devices. The performance of these rapid assays requires no specialized equipment and only limited technical expertise; some of the kits have the added advantage of possessing stabilized biochemicals that guarantee a long shelf life when stored at room temperature. Rapid HIV tests have the major disadvantages of low throughput and subjective reading of the test results (see references 10, 11, 134 for a review).


Currently, there are four basic formats for these rapid HIV tests, including membrane flow-through immunoconcentration devices, lateral-flow immunochromatographic strips, particle agglutination, and immunodot comb assays. In membrane flow-through devices, HIV antigens immobilized on a membrane capture and concentrate HIV-specific antibodies on the surface of the device as the specimen flows through and is absorbed into an absorbent pad. This is followed by the sequential addition of enzyme-labeled anti-human antibody and a colorless substrate. Enzymatic hydrolysis of the substrate leads to a colorimetric result that is read visually as a dot or line that forms on the membrane. Many of the membrane flow-through kits include procedural controls to verify the satisfactory performance of the assay. Lateral-flow immunochromatographic strips, or so-called dipsticks, are the most recent addition to the development of rapid and simple assays for HIV. The specimen is applied to an absorbent pad and migrates by capillary action along a solid-phase strip, where it combines with HIV antigens and detector reagents to produce a visual line on the strip when HIV-specific antibodies are present. A procedural control is normally included on the strip and is also indicated by a visible line. Immunodot comb assays use a solid plastic comb with teeth that are sensitized at several spots with different HIV antigens and control material. Patient specimens are placed in individual wells that accommodate single teeth of the comb. The comb is then transferred from specimen wells to reagent wells, and the teeth are saturated by the different solutions; spots that form at reactive positions on the individual teeth indicate positive results. Lastly, in particle agglutination assays, HIV-specific antibodies in a patient’s specimen will visually agglutinate or aggregate when cross-linked with particles (e.g., latex, red blood cells, or gelatin) that are coated and sensitized with HIV antigens.


Most of the described rapid tests use multiple recombinant and/or synthetic peptides to detect antibodies to HIV-1 and HIV-2, and some even include specific proteins to efficiently detect HIV-1 group O and the various subtypes of HIV-1 group M, and to differentiate antibody responses to either HIV-1 or HIV-2. Some rapid assays can detect both IgG and IgM responses, while most detect only IgG; many of the assays have procedural controls that ensure adequate test performance and control for specimen adequacy by the detection of human immunoglobulin molecules. Serum and plasma are the specimens of choice for the majority of the rapid devices, although many of the assays have also been adapted to allow the use of fingerstick or venipuncture whole blood or oral fluids. These assays have sensitivities and specificities similar to those of the more traditional second-generation laboratory-based antibody-only immunoassays when performed and read by properly trained personnel, although some assays perform better than others. Predictive values comparable to those of the standard combination of laboratory-based immunoassay and Western blot testing can be obtained using multi-test algorithms comprised of a combination of two or more rapid tests. Some countries outside of the United States now use these combinations of rapid tests as a less expensive and more rapid alternative to using laboratory-based immunoassays and Western blotting for blood screening, diagnostic testing, and epidemiological surveillance.


The rapid HIV tests are commonly used in developing countries, where resources and facilities may make it impractical or even impossible to perform the more technically demanding laboratory-based immunoassays that require time, sophisticated equipment, and performance in a clinical laboratory. The tests are intended for use in emergency departments, hospital clinics, sexually transmitted diseases clinics, family planning clinics, and HIV outreach programs. In these settings, rapid testing may provide same visit results to individuals seeking HIV testing, as many persons, including those infected with HIV, never return to receive their results following the delay in reporting that may occur when using traditional testing. The rapid availability of test results may assist in providing more timely essential medical and prevention services to these individuals. Rapid tests also may be useful in assessing the risk of HIV transmission following exposure to possibly HIV-contaminated materials or in screening pregnant women presenting for delivery with unknown HIV serostatus. It has been shown that antiretroviral therapy reduces occupational transmission of HIV after percutaneous exposures and reduces vertical transmission when used in the intra- or postpartum periods.


In 2012, the FDA approved the OraQuick In-Home HIV Test (Orasure Technologies, Inc., Bethlehem, PA) as the first rapid home-use, over-the-counter kit that is performed by the consumer and does not require sending a sample to a laboratory for analysis [70, 77, 96]. The test is a single-use immunoassay to detect antibodies to HIV-1 and HIV-2 in oral fluid specimens, and provides a test result in 20–40 min. The assay has the potential to identify large numbers of previously undiagnosed HIV infections and is targeted to people who otherwise would be reluctant to visit their physician or a healthcare facility to be tested. Positive test results using this test must be confirmed by follow-up laboratory-based testing and the test can be falsely negative until at least 3 months after infection with HIV. Prior to this test, the Home Access Express HIV-1 Test System (Home Access Health Corp., Hoffman Estates, IL) was FDA-approved in 1996 and was actually a home sample collection system in which dried blood spots were submitted by mail to a laboratory for HIV antibody screening and confirmation [39]. More recently in 2013, the FDA approved the first rapid POC test that detects both HIV-1/2 antibodies and HIV-1 p24 antigen (Determine HIV-1/2 Ag/Ab Combo Test, Alere, Waltham, MA). The test is a fourth-generation assay that is performed in three simple steps and takes 20 min to complete. The assay can be performed on fingerstick or venipuncture whole blood, serum, or plasma samples and can detect HIV infection earlier than first-, second-, or third-generation assays that detect only antibody to HIV. Similar to laboratory-based, fourth-generation combination assays, this test displays excellent performance for detecting established HIV infection, but appears to have a much lower sensitivity when compared to laboratory-based assays for detecting acute HIV infection [73, 100, 136]. At least two other rapid POC assays that detect both HIV-1/2 antibodies and HIV-1 p24 antigen have been developed and may be commercially available internationally (see Table 9.4).


9.6 Specimen matrices for HIV screening


Serum and plasma are the most widely used specimens in both the laboratory-based and rapid, less-sophisticated immunoassays. However, a number of these assays have been validated for use with other specimen types, including fingerstick and venipuncture whole blood, oral fluids, dried blood spots, cadaveric blood, urine, and, to a lesser extent, cerebrospinal fluid. The approval by the FDA of oral fluids [57, 97, 126], dried blood spots [7, 9, 71, 72, 79, 138], and urine [97, 98, 165] specimens for HIV antibody provides fast, convenient, and relatively noninvasive alternatives in specimen collection. Particular attention has been given to the value of oral fluids and urine specimens for the diagnosis of infection with HIV. Screening assays primarily intended for serum or plasma have been modified for use with oral fluid and urine specimens, and extremely sensitive assays specifically designed for these specimen types have also been developed and FDA approved. When such changes are made and assay protocols are optimized to accept oral-fluid or urine specimens, the sensitivities and specificities of these assays for the detection of HIV-specific antibodies are equal to, or only slightly less than, those when testing serum or plasma. Several commercial devices have also been developed specifically for the collection of oral mucosal transudate specimens, and the FDA has approved one of the devices (OraSure; OraSure Technologies) for diagnostic use. The devices provide a homogeneous specimen rich in plasma-derived IgG and IgM that is passively transferred to the mouth across the mucosa and through the gingival crevices (for a detailed description of the devices, see reference 57).


Testing oral fluids, urine, fingerstick whole blood, or dried blood spots for antibody to HIV has wide application in the management of patients and epidemiological surveillance, particularly under circumstances in which collecting serum or plasma is less practical. The collection of these specimens does not require laboratory personnel with special training: patients can easily obtain the sample themselves. HIV antibodies in urine, oral fluids, and dried blood spots are stable for extended times at room temperature, and specimens can be mailed or shipped without degradation. Collection of oral fluids or urine also increases compliance and alleviates the fear that patients may experience when having their blood drawn. It reduces the potential danger to the health professional through blood exposure and may benefit more challenging populations whose blood may be difficult to obtain, including children, hemophiliacs, obese people, and the elderly and infirm. The use of oral fluids or urine may permit improved access for the surveillance of intravenous drug users, homeless persons, sex industry workers, and persons in developing countries. Finally, collection of these specimens may afford a greater opportunity to screen for HIV antibodies in POC settings, physicians’ and dentists’ offices, public health institutions, and community outreach programs.


9.7 Confirmatory and supplemental tests


The Western blot assay has been the principle supplemental test used worldwide to confirm the specificity of positive results obtained from HIV screening immunoassays. Western blot assays are essentially solid-phase immunoassays that use immobilized viral antigens to detect antibodies to specific viral proteins. The major advantage of Western blot assays over the screening immunoassays is that the specific interaction of antibody and antigen can be directly visualized. These assays have the disadvantages, however, of being technically demanding, relatively expensive, and subject to interpretation. A number of Western blot assays have been commercially developed for detecting antibodies to either HIV-1 or HIV-2 (Table 9.5). Only a few have been FDA approved as confirmatory tests. Most Western blot tests are similar to first-generation immunoassays in that viral lysates are used as the source of HIV antigens. Several commercial manufacturers have developed immunoblot assays that utilize recombinant HIV proteins and/or synthetic peptides that are applied in separate lines or bands to nitrocellulose membranes. These assays can differentiate between HIV-1 and HIV-2 infection and are more sensitive and specific than conventional Western blot assays. In the United States, due to the low prevalence of HIV-2, Western blotting is usually performed for the confirmation of antibodies specific to HIV-1. HIV-2-specific Western blotting is not routinely done on all specimens that are positive by HIV screening immunoassays but is performed only when warranted by the clinical situation.


Table 9.5 Confirmatory/supplemental tests for the detection of HIV antibodies













































































































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    Manufacturer
    (alphabetical)
    Product name Assay type Sample Detection Antigens Selected references
    Alere ImmunoComb HIV 12 CombFirm ID Serum, plasma HIV-1/HIV-2 RP + SP NF
    Biokit S.A. Bioblot HIV 1 Plus WB Serum, plasma HIV-1, indicates suspected HIV-2 VL + SP NF
    Bionor Bionor HIV-1 & 2 Confirmatory Test MPEIA Serum, plasma, whole blood HIV-1/HIV-2 RP + SP 153
    Bio-Rad Genetic Systems HIV-1 Western Blot WB Serum, plasma, DBS HIV-1 VL 115
    Multispot HIV-1/HIV-2 Rapid Test FT Serum, plasma HIV-1/HIV-2 RP + SP 18, 123, 133
    Geenius HIV 1/2 Supplemental Assay LF Serum, plasma, or whole blood HIV-1/HIV-2 RP + SP 93, 108
    DiaSorin (MP Biomedicals) HIV-1 Blot 1.3 WB Serum, plasma HIV-1 VL NF
    HIV Blot 2.2 WB/LIA Serum, plasma HIV-1/HIV-2 VL + SP NF
    HIV-2 Blot 1.2 WB Serum, plasma HIV-2 VL NF
    Fujirebio (Innogenetics) Inno-Lia HIVI/II Score LIA Serum, plasma HIV-1/HIV-2 RP + SP 144, 145
    Maxim Biomedical, Inc. Cambridge Biotech HIV-1 Western Blot WB Serum, plasma, urine, oral fluids, DBS HIV-1 VL 67, 103, 164, 165
    OraSure Technologies OraSure HIV-1 Western Blot WB Oral fluids HIV-1 VL 44
    Sanochemia Pharmazeutika Fluorgonost HIV-1 IFA IFA Serum, plasma, DBS