Laboratory Testing for HIV Infection

Laboratory Testing for HIV Infection

Updated January, 2011

Joseph Nkeze, Niel Constantine, Richard Y. Zhao

History and Evolution of HIV Testing

Following the identification of the human immunodeficiency virus (HIV) in the United States in 1984, the first test for HIV was licensed by the Food and Drug Administration (FDA) in 1985. This was an enzyme immunoassay (EIA) and was used mainly to ensure the safety of blood supply. People were deterred from using the blood bank just for learning about their HIV status and opinions were divided about the implication of a positive test result (5). Counseling did not exist until 1987 when the United State Public Health Service (USPHS) issued guidelines making HIV testing and counseling a preventive strategy for those at high risk of infection. USPHS also recommended testing for those who were seeking treatment for other sexually transmitted diseases (STD) (30). The first Western blot (WB) blood test kit was also licensed in 1987 and the first rapid test that provides results in as short as ten minutes was licensed in 1992.

The recommendations for HIV testing were extended in 1993 to include in- and out-patients in the hospitals, especially those in acute-care settings and emergency departments. Hospitals with HIV prevalence rates or an AIDS diagnostic rate of >1% were encouraged to adopt voluntary counseling and testing for all patients aged 15-54 years (32). In 1994, these guidelines for counseling and testing those at high risk specified specific prevention goals and strategies for each person (client-centered counseling) (27). The first oral fluid test was also licensed in 1994 and the test was subsequently granted a Clinical Laboratory Improvement Amendments (CLIA) waiver in 2004. In 1995, Zidovudine (AZT) was administered to HIV infected pregnant women and resulted in a significant reduction of prenatal transmission. The USPHS then recommended that all pregnant women be counseled and encouraged to undergo voluntary testing for HIV (27, 43). In 1996, the first HIV viral load test was approved and the first home and urine collection kits for HIV testing were introduced. In 2001 the recommendations were modified and are now part of routine prenatal care; the high prevalence health care testing recommendations were extended to include clinical venues in both private and public sectors. HIV testing was targeted toward and was the basis for those in low risk settings (33, 35). The OralQuick Advance Rapid HIV antibody test that uses fingerstick blood was approved in 2002 and was granted a CLIA waiver in 2003 (42, 68). Also in 2003, the Centers for Disease Control and Prevention (CDC) introduced the initiative to advance HIV Prevention: New Strategies for a Changing Epidemic. The goal was to make HIV testing a routine part of medical care and also to further reduce prenatal transmission of HIV by the universal testing of all pregnant women and by using rapid tests during labor and delivery or postpartum if the mother was not screened prenatally (26). In March 2004, as result of a meeting with health consultants, a recommendation was made by the CDC to simplify the HIV screening process in order to make it more efficient, cost-efficient, and to urge frequent diagnostic testing for patients with symptoms. A final recommendation on HIV testing in the healthcare setting was published in 2006 (34). This recommendation came from a series of meetings with healthcare providers, HIV infected individuals, and researchers. It recommended routine HIV screening for all adults, aged 13–64, and repeated screening at least annually for those at high risk. Screening should also be voluntary (18, 34). Finally, in the last few years, efforts have been extended to identify the large percentage of infected persons who do not know that they are HIV infected by offering testing to all persons admitted to emergency rooms.

Techniques of HIV Testing

Antibody–Based Diagnostic Tests: Detection of antibodies produced in response to HIV infection is the basis of most HIV screening tests. Such antibodies are usually detectable within 1-3 months following infection (depending on which test is selected) (29, 35). Several varieties of tests exist and they differ on principle, format, and the type of specimen tested (whole blood, serum, plasma, oral fluid, fingerstick blood, urine), where the test is carried out [laboratory, point- of- care (POC) site, or home sample collection] and how quickly the results are available (conventional or rapid) (52). Antibody detection tests are easy to perform, have high sensitivity and specificity, and they are the choice of test technologies for the initial detection of most people who have HIV infection. They are, however, limited in their ability to detect acute infection; that is, during the window period before antibody is produced in an infected person. Antibody tests are generally classified as screening assays (initial tests) or confirmatory (supplemental tests) that have a higher specificity to rule out false positive screening test results.

FDA Approves Test to Detect Both HIV Antibodies and Antigen, 2010

Delaney KP, et al. Evaluation of the Performance Characteristics of 6 Rapid HIV Antibody Tests. Clin Infect Dis 2011;52(2):257-263.

Rapid HIV Antibody Test: Rapid HIV antibody tests have been widely used for years and most of them demonstrate sensitivities and specificities comparable to EIAs (15, 18, 83, 96, 134). They play an important role in HIV testing and access to testing in both clinical and non-clinical settings by overcoming some of the barriers of early diagnosis, thus improving linkage to care for those infected (78). They are especially useful in clinical settings such as public clinics and emergency rooms where rapid turn around time for results is important. They are also required in cases of occupational exposure to provide results on the source patient so that the injured person can be treated in a clinically relevant time frame (usually within 2 hours) (50). Most assays are in a ready-to-use kit format that include all necessary reagents and require no specialized equipment (18). Rapid HIV tests, similar to enzyme-linked immunosorbent assays (ELISAs), are screening tests and thus, the test results require confirmation if a reactive result is produced (78). Currently FDA-approved rapid test are listed in Table 1.

All rapid tests are interpreted visually. HIV antigens fixed to the test membrane or contained in the test wells will bind to HIV antibodies if present in the specimen, and a test kit colorimetric reagent binds to the anti-HIV antibody, or a labeled antigen creates an indicator that is visually detectable (78). A reactive result is interpreted as a preliminary positive and requires confirmation by a more specific assay such as WB or Immunofluorescent Assay (IFA) (15, 95). Performing an EIA as a confirmatory test is not required and if performed, the specimen must still proceed to WB or IFA testing regardless of the EIA result (31). A negative test result requires no further confirmatory testing. As with ELISAs, false negative results do occur in those acutely infected and also occur in some patients receiving antiretroviral (ARV) therapy with undetectable virus in whom antibody levels have waned below the sensitivity range of the test (33, 112).

The three most common formats of assays that can be used with whole blood are particle agglutination, immunoconcentration, and immunochromatography (18, 116, 124). Particle agglutination tests require 10 to 60 minutes after the specimen containing antibodies is mixed with latex particles coated with HIV antigen; cross-linkage occurs and agglutination occurs. Immunoconcentration (flow through) devices use a solid-phase capture technology which involves immobilization of HIV antigens on a porous membrane (Figure 1). The specimen flows through the membrane and is absorbed into an absorbent pad. A dot or line visibly forms on the membrane after addition of a conjugate or color producing reagent. This assay format usually requires several steps for the addition of specimen, wash buffers, conjugate (e.g., enzyme and substrate) or signal generating reagent (e.g., colloidal gold). They are performed in 5 to 15 minutes. Immunochromatographic (lateral flow) tests are the most recent development and incorporate both the antigen and signal reagent into the device. The specimen, followed by a buffer is applied to an absorbent pad where it binds to a conjugate (e.g., labeled antigen), and the complex migrates along a nitrocellulose strip and is captured by an immobilized antigen. A positive reaction results in a visual line on the membrane where the immobilized HIV antigen was applied. A procedural control line is usually applied to the strip beyond the HIV-antigen line. Two visual lines indicate a positive result and one only at the control indicates a negative result. Results are usually obtained in 20 minutes (18). Figure 1 shows an immunoconcentration-based test procedure of the Bio-Rad’s multispot HIV-1/HIV-2 assay (immunoconcentration); Figure 2 shows an Immunochromatographic-based test procedure of the OraQuick® Advance Rapid HIV Antibody Test.

Home Sample Collection for HIV Testing: These test kit are made to encourage patients to get tested in a more confidential and convenient manner. Only one home sample collection kit is currently approved by FDA: Home Access® HIV-1 (Home Access Health Corporation, Hoffman Estates, IL) (21). This is an over-the-counter kit but is also available by mail. The patient collects the sample of blood with a provided lancet, places it in the test card (dried blood spots) and mails it back to the company where it is tested. If the result is positive the patient is transferred to a counselor by phone. This assay utilizes an ELISA and IFA on the dried blood card. The sensitivity and specificity of this strategy is claimed to approach 100% (21, 69).

Confirmatory Tests

There are a number of tests that can be used to confirm HIV infection following a reactive initial screening test result.

EIAs: A second EIA or an EIA following a rapid test are the most commonly used method in developing countries to confirm infection (this is considered as an alternative confirmatory strategy to the routine screening test and WB or IFA testing algorithm) (46). Most EIAs have a sensitivity and specificity reported to be about 99% and the turn-around-time for the test is usually 24 hours (87, 122, 132). By using two EIAs or a rapid test/EIA in tandem, the predictive value of the combined result is near that of the routine testing algorithm. There have been improvements in EIA methodologies such as the use of recombinant antigens instead of whole viral lysates and also the use of double-antigen sandwich configurations (third-generation assays), all of which have lead to increased sensitivity and specificity (23, 80). Because of viral gene heterogeneity, HIV-2 is not always detected in HIV -1 ELISA tests but this problem has been resolved by the use of combined HIV-1/HIV-2 ELISA tests (72, 113). The addition of HIV-1 subtype O antigens to the ELISA has allowed improved detection of this subtype of HIV-1 (7). There are a number of reasons for false-positive and false negative ELISA results. False positive results can be due to technical error, cross reacting antibodies, and several medical conditions (122). Problems of cross reacting antibodies have been minimized with the use of synthetic or recombinant HIV peptides. False positive results may also occur in individuals participating in HIV vaccine trials (130). Similarly, false negative results could also occur for a variety of reasons. A nonreactive HIV ELISA result in a person at high risk of infection should always prompt consideration of the window period before seroconversion. Serological testing should be repeated several weeks after a suspected infection (122). Reactive HIV ELISAs should be confirmed by WB or IFA techniques. There are several EIAs using plasma or serum samples that have been approved by the FDA for use as screening tests for HIV-1 or HIV-2. The Vironostika HIV-1 Plus O Microelisa System (bioMerieux, Inc, Durham, NC, USA) is the test most recently approved for diagnostic non-blood donor screens for HIV-1 infection, while the Genetic Systems HIV-1/HIV-2 Plus O (Bio-Rad Laboratories Inc, Hercules, CA, USA) was approved to screen for HIV-2 infection in addition to infection with HIV-1 (59). Figure 3 shows an example of the third generation of EIA methodology.

Western Blot and IFA: The sensitivity and specificity of WB range between 96% and 100% in most cases (36). In the WB assay, individual proteins are separated according to their sizes by gel electrophoresis and transferred (blotted) onto a nitrocellulose paper. The reaction between antibodies and specific viral proteins can be determined after the addition of patient serum. Interpretation of the WB is based on the spectrum of bands that is visualized. Different groups have proposed different interpretation of the bands. The Association of State and Territorial Public Health Laboratory Directors and CDC have defined a positive HIV-1 WB as the presence of any two of the following bands: p24, gp41, or gp120-gp160 (28). If no band is present, the test is considered negative. If, however, a single band is present or a combination of bands that do not meet the criteria for a positive result, the test is termed indeterminate. As many as 10- 20% of WB results performed on sera with repeatedly reactive HIV-1 ELISA are interpreted as indeterminate, but depends on the population being tested (37). Accessing the person’s risk factor for HIV and repeating the WB test several weeks after is necessary to determine the significance of an indeterminate HIV-1 WB since this result may represent an infection. If a positive profile does not occur after 6 months, the patient is usually considered as not infected. The CDC guideline states that “a person whose Western blot test results continue to be consistently indeterminate for at least 6 months - in the absence of any known risk factors, clinical symptoms or other findings - may be considered to be negative for antibodies to HIV-1” (28). If a recent HIV-1 infection is suspected, additional tests such as measurement of p24 antigen or HIV-1 nucleic acid tests may be useful. False negative results may occur if the patient is of the rare HIV-1 serotype O and false positive results have been reported in patients with hyperbilirubinemia, Human Leukocyte Antigen (HLA) antibodies, other retroviruses, connective tissue disorders, and polyclonal antibodies (87, 90). Figure 4 shows how to interpret a WB test result.

The IFA provides an alternative confirmatory assay to Western blot. In this assay, serum or plasma samples are incubated with T cells that have been infected with HIV and that express HIV antigens intracellularly and with uninfected T cells as a control. If a specimen contains antibodies against HIV antigens, the antibodies should bind to the infected T cells but not the control uninfected cells. Bound antibodies are then detected with an anti-human antibody conjugated to a fluorescent molecule such as fluorescein isothiocynate. The fluorescent molecule emits light when exposed to ultraviolet light. The degree and pattern of fluorescence determines whether a sample is infected with HIV (142). The Fluorognost HIV-1 IFA (Waldheim Pharmazeutika GmbH, Vienna, Austria) is an FDA-approved confirmatory IFA for the detection of HIV-1 (59, 142). Currently, there are no FDA-approved confirmatory IFA assays for HIV-2 infection (59). Figure 5 illustrates appearance of an IFA positive test result.

Detection of Acute HIV Infection

Diagnosis and effective treatment of HIV infection during the acute phase of infection has several important advantages. The immune system could be better preserved, transmission to others minimized, and the spread of infection could be slowed (66). Detectable HIV-1 viral RNA or antigen (viremia) is thought to occur soon after infection and can be detected within 11 days post-infection. Quantification of HIV-1 by nucleic acid tests and HIV p24 antigen tests after this period has revealed very high levels of HIV-1 in plasma (during the first month after infection) (47, 77, 110, 120, 133). Because of the time lag until seroconversion, routine serological testing using antibody tests such as the ELISA and the WB assay may initially yield negative or indeterminate results and measurement of serum p24, HIV DNA in peripheral blood mononuclear cells (PBMCs) or viral RNA levels in plasma are useful during this period. Serum p24 antigen levels are high initially but wane after 1 to 2 weeks following their appearance because of the production anti-p24 antibodies (41, 48). The sensitivity of p24 assays can be up to 89% with a specificity of 100 % (47). Plasma HIV-1 RNA testing has a high sensitivity near 100% and a specificity of 97% (47, 91). However, about 1% of infected persons have been reported to have undetectable viral loads (without treatment) (127). Finally the third option for the diagnosis of acute HIV infection, particularly in persons with undetectable viral loads, is the use of PCR for HIV-1 DNA in plasma or PBMCs. HIV-1 viral DNA in PBMC is also particularly useful in the detection of HIV infection in newborns due to limited blood volumes.

HIV Disease Monitoring

Quantitative HIV Test: Active replicating HIV viruses can be inhibited or minimized by treating patient with antiretroviral drugs. Therefore, monitoring the viral load in patients on antiretroviral is crucial in patient care and management (2, 49). Rapid reduction in viral load (0.7-3 logs) is typically observed in drug naïve patients within few weeks after initiation of antiretroviral (ARV) therapy. Quantification of HIV viral RNA has many advantages, e.g., used by clinicians to measure the baseline of viral load before initiation of ARV therapy; evaluate the efficacy of initial AVR therapy; predict drug resistance; and also to estimate the potential to develop AIDS related opportunistic infections and diseases (2).

HIV Nucleic Acid-Based Assays: Nucleic acid-based assays especially quantification of HIV-1 viral RNA (viral load) in the plasma compartment have been very useful in monitoring HIV disease progression. Plasma HIV RNA is usually measured in HIV infected individuals at baseline and thereafter. The level of viral load measured at times about 1-2 months after infection is known as the “set point” and is positively correlated with the disease progression. Determination of viral load also provides vital information especially to those individuals who are under active ARV therapy. The minimal change in viral load considered to be statistically significant (2 standard deviation) is a threefold, or a 0.5 log10 copies/ml. Suppression of viral replication by ARV therapy to a level that is below the limits of detection (below 40-75 copies/ml depending on the specific assay used) is the main objective of ARV therapy (84, 103, 106, 135). Three basic types of techniques are currently used to amplify HIV RNA for detection and quantification of viral load: (1) gene target-based amplification technology such as polymerase chain reaction (PCR) or reverse transcription followed by PCR (RT-PCR). This type of method is designed to detect and amplify the target gene of interest; (2) signal-based amplification technique such as branched DNA (bDNA), which amplifies the signal rather than the gene target sequence; and (3) probe-based amplification techniques, e.g., ligase chain reaction (LCR) that relies on amplification of the probes that are homologous to a specific gene target (9, 81). The gene-based amplification technologies can be further divided into PCR-based or non-PCR based methods. The PCR-based method is an artificial DNA amplification method that is performed at various temperatures using a specific piece of equipment known as the thermocycler. In contrast, most of the non-PCR methods take advantage of the natural nucleic acid amplification processes (Table 2). For example, ligase chain reaction (LCR) mimics enzymatic ligation process; nucleic acid sequence based amplification (NASBA) mimics viral RNA reverse transcription and transcription; strand displacement assay (SDA) resembles the DNA excision repair process; and Qβ-replicase RNA amplification resembles bacteriophage replication. Another common feature of these non-PCR-based assays is that these assays can be carried out at constant temperature without thermocycling.

The RT-PCR–based assays convert HIV RNA into DNA using an enzyme called reverse transcriptase (RT). This is followed by PCR, which increases the copy number of DNA for detection. The resultant DNA is then detected with a nucleic acid probe specific for a HIV-1 nucleic acid sequence that has been attached to an enzyme. The enzyme-nucleic acid complex can react with another chemical and produce a color change, the intensity of which is used to quantify the DNA.

The HIV-1 Amplicor Monitor Assay and the HIV-1 AmpliPrep TaqMan assay (Roche Molecular Diagnostics, Pleasanton, CA, USA) are examples of target-based amplification assays. The difference between the HIV-1 Amplicor Monitor and AmpliPrep Taqman assays are that the Monitor assay is based on the conventional PCR method and the TaqMan is a real-time PCR-based method. The real-time PCR is much more accurate, reproducible with broader linear dynamic range than the conventional PCR method (54, 85). Of note, the amplified HIV DNA product can also be used to sequence HIV-1 RT and PR genes by using assays such as the ViroSeq HIV-1 Genotyping System with the 3100 or 3700 Genetic Analyzer (Celera Diagnostics, Alameda, CA, USA) (59). Genotypic information of HIV-1 RT and PR genes by sequencing can provide information on emergence of viral resistance to ARV therapies and determination of the HIV-1 subtypes that are infecting the patients (80). Other examples of target-based amplification assays include the Abbott m2000 real-time PCR assay (Des Plaines, IL) and the Nuclisens HIV RNA QT (Organon-Teknika, Boxtel, The Netherlands) (1, 81).

The bDNA assay, also known as the Versant TM HIV RNA kit or formally known as Quantiplex TM, is one example of the signal amplification-based assays. HIV RNA is captured by complementary oligonucleotides called capture probes that are bound to the bottom of a plate. The hybridization probes are used to simultaneously bind the captured HIV RNA. Unlike the target-based assays, detection of the captured viral RNA is achieved by linkage of oligonucleotides-containing multi-amplifiers to the hybridization probes.

In the probe-based assays, the probes that are homologous to the target sequences are amplified. One of these assays is LCR (16, 137). LCR is based on the principle that ligation of a DNA molecule is most efficient when the molecules are aligned in a head-to-tail fashion. Thus, typically two detection probes are specifically design to be complementary to a specific gene target sequence of interest. Once these probes are annealed to the gene target, addition of DNA ligase will join the two detecting molecules (12). Repetition of hybridization, ligation and denaturation will achieve probe-based amplification. The Abbott Laboratories (IL, USA) markets HIV-1 LCx, which is based on LCR technology. Table 2 summaries nucleic acid-based amplification methods.

The ultra sensitive versions of these assays in general have lower limits of detection of ≤50 copies of viral RNA per ml of plasma (131, 138). In addition to these popular assays, other approved methods include; hybrid capture system (Digene Co), transcription mediated amplification (TMA, Gen-probe Inc.) and a reverse transcriptase viral load method (Exa Vir Load, Cavidi Tech, Uppsala Sweden) are also available commercially (74).

Although the sensitivity and specificity of these diagnostic tests are high (99% and 98%, respectively), they should be used in conjunction with serologic assays (24). In addition, because most of these tests are designed to detect HIV-1 subtype B, they sometimes cannot detect other HIV strains and subtypes. Recently, new real-time PCR-based assays have been introduced (Abbott Real Time HIV-1; Roche Amplicor TaqMan ver. 2.0) that are capable of detecting all subtypes of HIV group M and group O.

HIV-1 proviral DNA can also be used as a means for HIV detection. The Roche HIV-1 Amplicor test, which is a qualitative PCR-based assay, can be used to detect HIV-1 DNA. This test is particularly useful in HIV diagnosis for pediatric patients. Currently, there are no commercially available assays to quantify proviral DNA, which could potentially be useful in evaluating the efficacy of ARV therapies especially when the HIV-1 viral RNA load is undetectable (40, 61, 62). A number of research-based assays have been described for quantification of HIV-1 proviral DNA but these assays are based on conventional PCR (13, 39, 79, 86). A limitation of some of those assays is that conventional PCR methods were used. As a result, the DNA copy numbers are calculated based on the final amplified gene products, which vary widely due to various affecting factors of PCR. Several real-time PCR Taqman-based protocols for quantification of HIV-1 proviral DNA have been reported (53, 139, 140), which provide a highly accurate and reproductive detection method for HIV-proviral DNA with a wide level of detection.

p24 Antigen Detection: p24 antigenemia can also be used to monitor disease progression. p24 antigen can typically be detected using EIA in serum. These assays measure free HIV p24 antigen and but are less sensitive than HIV RNA-based assays. Their specificity is high as long as a neutralization (confirmation) step is used (24). These methods are commonly used in developing countries because of the ease of performance and low cost as compared with the nucleic acid-based tests.

A real-time immuno-PCR (IPCR)-based assay, which uses HIV-p24 antigen as a marker for quantification of antigenemia, has been reported (8). The real-time IPCR assay combines the p24 ELISA with real-time PCR amplification of the signal DNA molecules that are attached to the detecting antibody of p24. With the IPCR, the first protein is captured in the same way as in a typical ELISA but the second antibody is conjugated with biotin that, through strong binding of biotin to streptavidin, links to biotinylated oligonucleotides. The protein level can then be quantified by amplifying the signal oligonucleotide linked to the antibody. The sensitivity level is in the range of femtogram (10-15g)/mL, which is about least 103 times more sensitive than a conventional ELISA assay (60). Figure 6 shows a schematic representation of the immuno- PCR assay in comparison with the conventional ELISA.

Viral Culture: Standard HIV-1 culture is typically used to verify HIV infection status using patient’s PBMCs. However viral culture can also be done using plasma, cerebrospinal fluid, saliva, semen, cervical specimens, or breast milk (87). The patient specimen is incubated with uninfected donor PBMC and interleaukin-2 that will activate and stimulate cell growth. The progeny HIV-1 virions from the supernatant can then be tested qualitatively or quantitatively with assays for RT or p24 antigen (65). Most assays become positive within 21 days. Sensitivity is reported to be greater than 97% with a specificity of 100 % (88, 89).

When compared with plasma HIV-1 viral load testing, culture methods for HIV-1 are more laborious, time-consuming and less sensitive. The sensitivity is also lower than proviral DNA PCR when used in diagnosing infants born to infected HIV-1 mothers (19, 22). Culture testing is thus mostly used in clinical trails and/or for research studies.

Quantification of CD4+ T-Cells: The course of HIV infection can also be monitored by measuring the CD4+ levels of the patient which serve as a major indicator of immunodeficiency. It provides information as to when to initiate therapy, the potential threat of opportunistic infections, and it is also a predictor of disease progression (46, 55, 105). There is an inverse correlation between the CD4 count and the viral load. Flow cytometry has been widely used but the tests are relatively expensive, require significant technical expertise and expensive instruments. Relatively less expensive and simple methods, however, do exist including the Cytospheres (Beckman Coulter) and Dynabeads (Dynal Biotech) (45, 46). 2.3. HIV testing with other sample types In addition to blood, plasma or serum, HIV-1 testing can also be performed on other biological matrices such as oral fluids, urine, vaginal secretions and cerebrospinal fluid. For most of these assays, specimen collection is easy and handling is safer for providers than the blood-based assays. It is also more comfortable for patients who are adverse to venipunture or who have poor venous access. Such tests are widely used in resource limited regions where laboratory support is less available (52).

Oral Fluid: Most oral fluid-based assays collect patient’s saliva having concentrated IgG antibodies for EIA and WB detection. Using the Orasure collection device (Epitope, Inc., Beaverton, OR), the oral fluid is collected by placing a cotton pad between the cheek and gum for about 2 to 5 minutes (also see Figure 2). A hypertonic solution in the pad will encourage transudation of oral mucosal transudate (the fluid portion from blood that moves through the capillaries at the tooth-gum margin) which is high in HIV-1 IgG. The pad is then transported to a laboratory in preservative where an EIA or WB tests can be performed. Orasure has a sensitivity of 98% to 100% and a specificity of 99% to 100 % (57, 58, 70). The OraQuick rapid test is described under the rapid test section (see Table 1).

Urine: Similar to the oral fluid tests, the urine-based test also relies on detection of anti-HIV antibodies. The sentinel HIV-1 Urine Enzyme Immunoassay (Calpyte Biomedical Corporation, Alameda, CA) is a rapid EIA with a sensitivity of 99% (136). A urine-based WB assay could be used to confirm urine EIA. However, a blood-based test is generally required to confirm a positive test result. This is due to the low specificity of the urine test (33).

Vaginal Secretion and Seminal Fluid: Antibodies to HIV-1 and HIV-2 can be detected in both cervicovaginal secretions and seminal fluids. At present, no commercially available assays using vaginal secretions have been approved by FDA. However, rapid HIV EIA tests using seminal fluids are available to detect HIV antibodies with excellent sensitivity. An example is the Abbott recombinant HIV-1/HIV-2 third generation immunoassay (11, 98). Since HIV-1 and HIV-2 IgG can be detected in seminal fluid, an EIA for detection of these antibodies could be useful in rape situations where serostatus can guide the need for post exposure prophylaxis (10).

Monitoring HIV Infection in Resource Limited Regions

HIV infection in resource limited areas remains a major cause of morbidity and mortality despite increasing availability of ARV therapies (73). Quantification of HIV-1 viral RNA loads in plasma is one of the most valuable clinical tools for initiation of therapy, evaluation of the efficacy of ARV, and predicting disease progression (3, 17, 107). Plasma viral load measurement however requires venous blood extraction, use of RNAase-free materials, freezers for storage, constant electricity supply to run the freezers, and also transport in a cold chain which make it difficult to manage in resource limited countries (3, 102). Spotting and drying whole blood on a filter (dried blood spots) collected by lancet or fingerstick have proven to be highly economical and an effective alternative method for sample collection and storage. For example, the sample is easy to obtain, collection volume is small, no blood separation is required, and the samples can be transported at room temperature; thus, there is no need for cold-chain transportation (6, 109). In addition, the use of dried blood spot (DBS) or dried plasma spot (DPS) samples in viral load determination appears to generate results that are less sensitive (approx. 2,000 copies/ml) than standard liquid plasma-based testing but they nevertheless provide useful information in most of the clinical situations. Importantly, it has been demonstrated that HIV-1 RNA in DBS and DPS samples is stable over time under different conditions of temperature and humidity (6, 64, 71, 92, 93, 102, 107, 129).

Detection of HIV-1 Non-B Subtypes

HIV-1 can be classified into group M (major), a rare group O (outlier) and a “new” group N (non-M, non-O) (67, 125). Recently, a new HIV-1 group, which is designated as group P, has been reported (121). Group M can be further divided into subtypes (A-D, F-H. J and K) and circulating recombinant forms (CRFs) (97, 125). The subtype B virus is at present the predominate subtype in the United States. Recent data however suggest increasing prevalence of HIV-1 non-B subtype in the USA. The clinical significance of emerging non-B viruses to HIV treatment and patient care is currently unknown. However, in the area of vaccine development, the extent of HIV diversity is an important consideration. Early candidate vaccines were developed based on the U.S. HIV-1 B subtype type. However, non-B subtypes should also be included in future research and development of an effective vaccine against HIV (44). This increase in HIV-1 non-B subtype is likely driven by global travel, immigration, commerce, tourism, and military deployment (104). For example, in a study conducted from blood donors throughout the US from 1997-2000, the prevalence of non-B subtypes was 2.3% (51). In a recent study conducted in the city of Baltimore and the State of Maryland, the non-B prevalence, which was determined using DNA sequencing analysis, was 13.2% in a Maryland suburb of the Washington, DC area (25). A similar high level of non-B HIV-1 subtypes in other part of the US has also been reported (20, 99) Therefore, new methods that are capable of detecting all of the non-B subtypes including those circulating recombinant forms should be developed and used. Failure to detect or accurately quantify non-B HIV infection has been documented in some of the antibody and RNA-based assays (4, 38, 56, 75, 115, 119, 128).

Individualized Molecular Genetic Testing for ARV Therapies

The genetic background of an individual has been shown to affect ARV therapies or prognosis of HIV progression. For example, gene copy number of CCL3L1, a ligand of the CCR5 chemokine co-receptor or the CCL3L1-CCR5 genotypes, has been shown to be associated with each individual’s susceptibility to HIV infection or with prognosis of HIV disease progression (76, 94). However, no commercial genetic test is currently available for such a test. Certain variations in human leucocytes antigen (HLA) haplotypes such as HLA-B*5701 has also been reported to associate with drug hypersensitivity to Abacavir (82, 101, 123). About 4-8% of HIV patients treated with Abacavir (Ziagen) experience hypersensitivity that is characterized by rash, fever, gastrointestinal symptoms and could be life threatening (118). Studies have shown that, screening for HLA-B*5701 before treatment with Abacavir and withholding Abacavir from persons who are positive for HLA-B*5701 appears to reduce the risk of an Abacavir hypersensitivity reaction (100, 126). Current guidelines of the USDHHS recommend that patients be tested for HLA-B*5701 before Abacavir is initiated and that patients with HLA-B*5701 should not be given Abacavir. If HLA-B*5701 screening is not available, Abacavir may be used, with appropriate counseling and monitoring. It should be noted that Abacavir is FDA approved for anti-HIV therapy because studies have shown an improvement of CD4 T-lymphocyte counts and HIV viral load on a 3-drug regimen of zidovudine (AZT), lamivudine (3TC), and Abacavir, in comparison with the 2-drug regimen of AZT and 3TC (63). GlaxoSmithKline (GSK), manufacturer of Abacavir, has developed an approved genetic test for HLA-B*5701 (108).

TABLES AND FIGURES

Table 1: FDA-approved Rapid HIV Antibody Screening Tests

Table 2: Nucleic Acid-Based Amplification Methods

Figure 1: A Flow Through Device Capable of Differentiating HIV-1 and HIV-2 (111)

Figure 2: OraQuick® Advance Rapid HIV Antibody Test procedure (114)

Figure 3: Principle of the 3^rd Generation EIA

Figure 4: Typical Western Blot Results (HIV blot 2.2. Genelabs)

Figure 5: Distinctive Apple-Green Intracellular Staining Pattern in an HIV-1 Positive Specimen Using the Fluorognost ^TM HIV-1 IFA (117).

Figure 6. Schematic Presentation of Immuno-PCR in Comparison with ELISA

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