New methods for the detection of HIV

Clin Lab Med 22 (2002) 573–592

New methods for the detection of HIV Joseph A. DeSimone, MD, Roger J. Pomerantz, MD, FACP* Division of Infectious Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA

Several strides have recently been made in the ability to detect the presence of both HIV-1 and HIV-2. Serologic testing has been greatly refined, vastly improving the sensitivity and specificity of such tests. More rapid serologic methods now exist, as do kits for performance of testing at home. HIV-1 antibody tests of nonserum samples, such as urine and oral fluids, have recently been approved by the Food and Drug Administration (FDA). HIV-1 antigen testing and HIV-1 culture techniques have become useful in certain clinical situations. Finally, nucleic acid–based tests, which can allow direct detection of both HIV-1 RNA and complementary DNA (cDNA), have played an important role in the detection of acute HIV-1 infection in adults and in the postpartum period of infants born to HIV-1–infected mothers. These tests and their clinical uses are discussed in this article. ELISA and Western blot testing Serologic tests for the presence of HIV-1 and HIV-2 using the enzymelinked immunosorbent assay (ELISA) and Western blot methods remain the primary methods for the detection of HIV infection. Both the sensitivity and specificity for most recent HIV ELISA tests are reported to be greater than 99% [1,2]. Detection of antibody is usually possible within 6 to 8 weeks after infection, and nearly all tests are positive within 6 months, although longer seroconversion periods have been reported [1,3]. Because of the window period in antibody development immediately after infection, serologic testing performed during this time may lead to a false-negative result. Also, because of passive transfer of maternal antibody, serologic diagnosis of an

* Corresponding author. 1020 Locust Street, Suite 329, Philadelphia, PA 19107–6799. E-mail address: [email protected] (R.J. Pomerantz). 0272-2712/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved. PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 1 3 - 6

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infant born to an HIV-infected mother is often not helpful. Detection of HIV infection in these situations is discussed in detail later. The ELISA is the usual serologic test used initially for screening and diagnosis of both HIV-1 and HIV-2. Recent improvements in ELISA methodologies, such as the use of recombinant antigens instead of whole viral lysate, and the use of double-antigen sandwich assays (frequently referred to as third-generation assays), have improved both the sensitivity and specificity of the HIV ELISA [4,5]. Similarly, difficulties with detection of HIV-2 by ELISA testing have been resolved. Although mostly limited to western Africa and South America, HIV-2 has been identified in some patients in North America. Because of gene heterogeneity, HIV-2 is not always detected in HIV-1 ELISA tests, although cross-reactivity can occur [6,7]. Combined HIV-1–HIV-2 ELISA tests have helped resolve this problem in many instances. A testing algorithm, using an HIV-2 Western blot confirmatory test, has been recommended by the Centers for Disease Control and Prevention (CDC) and FDA for use with the HIV-1–HIV-2 combination ELISA [8]. Finally, the addition of HIV-1 subtype O antigens to the ELISA has allowed improved detection of this uncommon subtype of HIV-1 [9]. There are a number of reasons for false-positive and false-negative ELISA results. False-positive ELISA results are often weakly or only moderately reactive, are frequently transient, and are usually nonreactive by Western blot testing [2]. Causes of false-positive HIV ELISA results include human and technical error, cross-reacting antibodies, and numerous medical conditions [2]. Many of the problems with cross-reacting antibodies have been eliminated since ELISA tests began using synthetic or recombinant HIV peptides, although cross-reaction may still occur. False-positive ELISA results may also occur in individuals participating in HIV vaccine trials [10]. False-negative HIV ELISA results can occur for a variety of reasons [2]. Importantly, a nonreactive HIV ELISA result in a person at high risk for HIV infection should always prompt consideration of the window period before seroconversion. Such patients should have repeat serologic testing several weeks later in this clinical situation. Repeatedly reactive HIV ELISAs require confirmatory testing, and the Western blot technique is the most widely used test in this setting. In this assay, individual HIV proteins are separated according to size by gel electrophoresis and transferred (blotted) onto nitrocellulose paper. After addition of the patient’s serum, the reactivity of antibodies to specific viral proteins can be determined. Interpretation of the Western blot is based on the spectrum of bands that is visualized. It should be noted, however, that different consensus groups have proposed alternative criteria for interpretation of the HIV Western blot. The Association of State and Territorial Public Health Laboratory Directors and CDC have defined a positive HIV-1 Western blot as the presence of any two of the following bands: p24, gp41, or gp120–gp160 [11]. These criteria for a positive Western blot have been accepted by most laboratories performing this test [2]. If no bands are present, the test is considered

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negative. If a single band is present, or a combination of bands is present that do not meet criteria for a positive result, the test is termed indeterminate. The sensitivity and specificity of the HIV-1 Western blot assay are both excellent, ranging between 96% and 100% in most cases [12]. Nevertheless, false-negative and false-positive results can occur. For example, because these assays do not contain certain proteins from HIV-1 serogroup O, conventional Western blot results can be falsely negative when this relatively rare serogroup is present [13]. False-positive HIV-1 Western blots have been reported in patients with hyperbilirubinemia; HLA antibodies; other human retroviruses (including HIV-2); connective tissue disorders; and polyclonal gammopathies [1]. The indeterminate HIV-1 Western blot result remains a source of confusion and anxiety for both physicians and patients alike. As many as 10% to 20% of Western blot results performed on sera that are repeatedly reactive by HIV-1 ELISA are interpreted as indeterminate [14]. This result may be caused by false positivity, as mentioned previously, or may represent true HIV-1 infection before complete seroconversion. Assessing the patient’s risk factors for HIV acquisition, and repeat Western blot testing in several weeks, are both necessary to determine the significance of an indeterminate HIV-1 Western blot. Because this result may represent recent HIV-1 infection before complete seroconversion, it is recommended that repeat Western blot testing occur over the next 6 months. If an evolution of reactive bands does not occur over time, such patients are usually not infected. Guidelines from the CDC state 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’’ [11]. If recent HIV-1 infection is suspected based on risk factors or clinical presentation, additional tests, such as those used in the diagnosis of acute HIV-1 infection (discussed later), also may be useful.

Rapid HIV-1 antibody tests One of the drawbacks of the previously mentioned routine antibody tests is that several days may elapse before the results are known or reported to the ordering physician. For this reason, a number of rapid HIV-1 antibody tests have been developed, in which results can be determined within a matter of minutes. Clinical settings in which rapid results may be important include public clinics and emergency rooms, where patients infrequently return for their HIV-1 antibody test results [15]. By determining HIV-1 status quickly, these patients can then be counseled and advised immediately regarding further testing and treatment. In addition, these rapid tests have been frequently used in situations that require immediate results, such as in the case of occupational exposure.

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Currently, only one FDA-approved rapid antibody test is commercially available in the United States (the Single Use Diagnostic System, Abbott Laboratories, Abbott Murex, Norcross, GA), although other rapid test kits are being evaluated for approval. These assays, performed on plasma samples, used rapid ELISA or latex agglutination techniques. Results are usually available within 10 minutes. In one large trial, the sensitivity of the Single Use Diagnostic System assay was 100%, and the specificity was 99.5% [16]. This resulted in a positive predictive value of 88% and a negative predictive value of 100%. Other studies of this assay and other commercially available rapid assays have found similar results [17,18]. Importantly, it has been noted that specificity can be lowered with improper specimen handling or if the test is performed on patients who have indeterminate Western blots, such as those patients who are acutely infected [16,19]. Because of the relatively low specificity, it is recommended that all positive results be confirmed with standard antibody testing. The low specificity and positive predictive value also imply that this test should not be used as a screening test of the general population. Home sample collection kits Home sample collection kits were designed to encourage patients to undergo HIV-1 testing in a more confidential and convenient manner. Currently there is a single home sample collection kit which is FDA-approved: Home Access (Home Access Health Corporation, Hoffman Estates, IL) [20]. This is an over-the-counter kit that also is available by mail order. The patient collects a sample of blood with a provided lancet, places the blood on a test card, and mails the card to the company. The Home Access test utilizes an ELISA and immunofluorescent assay (IFA) on the dried blood sample. The patient can call a toll-free number for the results, which are kept anonymous by means of a code number. If the test result is either positive or indeterminate, the caller is transferred to a counselor regarding the results. Sensitivity and specificity of this assay approaches 100% [20,21]. It should be noted that this kit differs from home-use HIV-1 test kits, which allow consumers to interpret their own HIV-1 test results at home, and which have not been approved by the FDA due to inaccurate results [22]. Non–serum-based HIV-1 antibody tests To make HIV-1 testing simpler and safer, several non–serum-based antibody tests have been developed. There are several advantages to testing for HIV-1 antibodies in samples, such as oral fluids, urine, and vaginal secretions. For example, specimen collection and handling are easier and safer for the health care provider. Also, patients who are averse to venipuncture, or who simply have poor venous access, may be more willing to undergo HIV-1 testing if venipuncture can be avoided. Finally, such tests may allow

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wider HIV-1 testing in developing countries, where laboratory support may be minimal. Orasure (Epitope, Inc., Beaverton, OR) is the only FDA-approved HIV1 antibody test that relies on oral fluid specimens. Specimens are collected by placing a cotton pad between the cheek and gum for 2 to 5 minutes. This pad contains a hypertonic solution to encourage transudation of oral mucosal transudate, a serous fluid that contains high amounts of HIV-1 IgG [23]. A health care provider then places the pad in a preservative for transport to an Orasure laboratory. An Orasure ELISA and Western blot can then be performed on the oral mucosal transudate. In several trials, when compared with standard testing, Orasure had a sensitivity of 98% to 100% and specificity of 99% to 100%. This corresponded to a positive predictive value of 100% and a negative predictive value of 99.97% [23–25]. Importantly, it has been shown that oral pathology does not affect the results of oral mucosal transudate testing [26]. The FDA also has recently approved an HIV-1 antibody test for urine specimens. The Sentinel HIV-1 Urine Enzyme Immunoassay (Calpyte Biomedical Corporation, Alameda, CA) is a rapid IgG capture enzyme immunoassay with a reported sensitivity of 98.73% [27]. A urine specimen HIV-1 Western blot is now available for confirmation of positive urine enzyme immunoassay results [27,28]. Further confirmation by blood sample is recommended for positive urine results because of the lower specificity of the urine Western blot as compared with the serum-based Western blot [22]. An ELISA for detection of HIV-1 and HIV-2 IgG in vaginal secretions, although not FDA-approved, may be useful in certain clinical situations. Because HIV-1 and HIV-2 IgG can be detected in seminal fluid, one potential use for vaginal secretion testing may be in a rape victim, where serostatus can guide the need for postexposure prophylaxis [29].

p24 antigen detection p24 is an HIV-1 core protein encoded by the gag gene. It is expressed shortly after acquisition of HIV-1, and antibody to p24 forms shortly thereafter [30,31]. p24 antigen can be detected in serum, plasma, and cerebrospinal fluid [32]. Assays to detect p24 antigen use an antigen capture with ELISA technique. The patient’s serum is added to a plate or beads coated with anti-p24 antibody, allowing the p24 antigen to be captured. An enzyme-linked anti–HIV-1 IgG is then bound to the complex, and is subsequently measured colorimetrically. Because antigen-antibody complexes can limit the level of detection of p24 antigen, a dissociation step to separate these immune complexes has been added to the assay. This dissociation step has increased the sensitivity of the assay significantly [33,34]. Although uncommon, false-positive p24 antigen results, presumably caused by crossreacting proteins, have occurred in uninfected patients [35].

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Before the advent of plasma HIV-1 viral RNA measurement, the p24 antigen assay was used as a prognostic tool. Levels of p24 antigen were found to reappear or increase in HIV-1–infected patients shortly before or during the development of AIDS [36–38]. Similarly, this assay was useful in measuring the antiretroviral effect of medical therapy [33,39,40]. Use of the p24 antigen assay in these roles has been mostly supplanted by quantitation of plasma HIV-1 RNA assays. Although not FDA-approved for use as a diagnostic test, p24 antigen testing has played a role in the diagnosis of acute HIV infection and in infants born to mothers who are HIV-infected (discussed later).

Cultures of HIV-1 The HIV-1 can be cultured from plasma, serum, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid, saliva, semen, cervical specimens, and breast milk [1]. Standardized culture methods usually use the patient’s plasma or PBMCs, which are incubated with uninfected donor PBMCs. Interleukin-2 is present to activate and stimulate cell growth. The culture supernatant, which then contains progeny HIV-1 virions, can be tested qualitatively or quantitatively for the presence of HIV-1 with assays for reverse transcriptase (RT) or p24 antigen. Most cultures from HIV-1– infected patients, excluding those on virally suppressive highly active antiretroviral therapy, become positive within 21 days [41]. Sensitivity of these culture methods in patients who are HIV-1 seropositive has been reported at greater than 97%, with a specificity of 100% [42,43]. In the age of plasma viral RNA testing, the use of HIV-1 culture in clinical management is minimal. The in vitro rate of replication when culturing HIV-1 has been shown to correlate with the patient’s clinical status and may serve as a means of measuring response to antiretroviral therapy [44–47]. When compared with plasma viral RNA testing, however, culture methods for HIV-1 are far more laborious, time-consuming, and less sensitive. Similarly, although culture may be useful in diagnosing infants born to women who are HIV-1–infected, the sensitivity of this test is far lower when compared with a diagnostic method, such as proviral DNA polymerase chain reaction (PCR) testing [48,49]. For these reasons, culture testing of HIV-1 has mainly been relegated to the research and clinical trial realm.

Nucleic acid–based tests On entering human cells, HIV-1 RNA is converted into a complementary strand of cDNA by RT. These linear DNA molecules then integrate into the host genome, becoming the proviral form of HIV-1. After translation and the production of new virions, release of HIV-1 RNA into the plasma

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occurs. Molecular tests are now available for the detection of cDNA and plasma viral RNA. Although primarily used as prognostic and therapeutic markers, these tests also have been used as diagnostics. The presence of proviral DNA can be detected by using the PCR. In the HIV-1 DNA PCR assay, PCR using oligonucleotide primers amplifies a segment of the highly conserved HIV-1 gag gene. This is followed by hybridization of an identifying DNA probe, with a subsequent qualitative enzymatic colorimetric assay. The sensitivity of this technique has been reported as greater than 95%, with a specificity of greater than 98% [50,51]. The DNA-PCR method for detecting the presence of HIV-1 is highly reliable when testing for HIV-1 subtype B, the most common subtype in North America. This method, however, is less reliable when infection with non– subtype B strains of HIV-1 is present [52–54]. Because of the somewhat high inaccuracy rate, this test has not been recommended as a routine screening measure [55]. Use of this method in diagnosing infants is discussed later. Although less commonly used as a diagnostic test, the quantitation of circulating virion-associated HIV-1 RNA in plasma, commonly referred to as the plasma viral load, has had an enormous impact on the management of HIV-1 infection. This measurement has allowed greater understanding of HIV-1 viral dynamics, and the continuous, high-level rate at which viral replication occurs [56,57]. The advent of the plasma viral load allowed investigators to understand that a basal level of high viremia is continuously present, regardless of the patient’s clinical stage [58,59]. Furthermore, knowledge of the extraordinary rate at which HIV-1 replication occurs has helped explain the process and development of antiretroviral-resistant quasispecies, and the reasons behind antiretroviral failure [57]. Perhaps most importantly, the advent of assays to quantitate plasma HIV-1 RNA in the early 1990s gave clinicians a powerful new prognostic tool. Several studies performed during the mid-1990s consistently confirmed that the risk of progression to AIDS and death from HIV-1 infection was directly related to the plasma viral load [60–63]. In addition, the plasma viral load was found also strongly to predict the decline of CD4þ T lymphocytes [64,65]. Subsequent studies revealed the superiority of the plasma HIV-1 RNA level over the CD4þ T-lymphocyte count in predicting disease progression, but importantly the combined measurement of both plasma HIV-1 RNA and the CD4þ T-lymphocyte count was found to be a better prognosticator of disease progression than any single test alone [64,66,67]. New, simpler, and more rapid techniques of quantitating plasma HIV-1 RNA have allowed the plasma viral load to become the most clinically useful and meaningful prognostic tool. Of equal importance has been the impact of plasma viral load on the use and understanding of antiretroviral therapy. With the advent of the plasma viral RNA assays, investigators had a reliable means of measuring both the short- and long-term impact of antiretroviral therapy [63,68–72]. Baseline plasma HIV-1 RNA levels are an integral component in determining when

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and if a patient should receive antiretroviral therapy. Also, viral load reduction has now become the standard measure for determining response to antiretroviral therapy when comparing efficacy of different antiretroviral medications in clinical trials. Indeed, it was the response in plasma viral load that allowed investigators to propose that dual or triple antiretroviral therapy is of greater benefit than monotherapy [73]. Finally, the plasma viral load has proved useful in predicting disease progression in the setting of antiretroviral therapeutic response, and the progressive change in this level has been shown to be even more predictive of disease progression than the pretherapy level [66,74]. The three methods of quantitating plasma HIV-1 RNA, which are commercially available, include the RT-PCR assay, the branched DNA (bDNA) assay, and the nucleic acid sequence-based amplification (NASBA) technique [75–81]. In the RT-PCR assay, HIV-1 RNA from the patient is converted to cDNA by adding RT. A well-preserved portion of the gag gene is amplified by PCR and hybridized to an enzyme-linked DNA probe. Simultaneously, a competitive RNA template, with a known standard copy number, is used in competitive titration. The ratio of detected signal is compared with the signal of the known standard, determining the amount of HIV-1 RNA in the patient’s plasma. The bDNA technique for measuring viral RNA differs in concept from the RT-PCR method. In this assay, plasma HIV-1 RNA is captured by probes on to a microplate. Multiple DNA probes are hybridized to specific pol (or gag) gene segments of the bound RNA, thereby amplifying the signal. Alkaline phosphatase is then added in the presence of a substrate to generate a chemiluminescent reaction. The chemical light units are then compared with a standard to determine the amount of RNA in the sample. The bDNA method is based on signal amplification rather than target amplification. The NASBA method of quantifying HIV-1 RNA, like the RT-PCR method, involves repetitive rounds of target amplification [75]. In this assay, RNA transcriptases are used to amplify specific HIV-1 RNA targets. Although similar to the RT-PCR method in principle, this method differs in that NASBA amplifies viral RNA as opposed to cDNA. Also, with the addition of a unique RNA extraction step, NASBA can measure HIV-1 RNA in samples other than plasma. Most commercially available viral load tests are effective for measuring HIV-1 subtype B only. HIV-2 RNA viral load tests are not commercially available. Similarly, these tests are frequently ineffective in measuring the HIV-1 non–subtype B strains (eg, A, C, D, or E), which are commonly found outside of North America [82]. Because multiple DNA probes are used in the bDNA method, this may be the best available method for measurement of non–subtype B strains of HIV-1 RNA [83]. Nevertheless, there is currently no FDA-approved viral load assay for non–subtype B HIV-1 RNA.

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These three assay methods seem to be equivalent in sensitivity and specificity, particularly when measuring high or moderately high levels of viremia [84,85]. Variability among the assays, however, may be more pronounced when evaluating low or undetectable viral loads [86]. Currently, only the RT-PCR viral load test has been approved by the FDA for determining prognosis and for monitoring the response to therapy. Also, absolute levels of nucleic acid can vary among the assays. For example, the levels of viremia when determined by RT-PCR are typically two times higher than when measured by bDNA [64]. For these reasons, direct comparisons of viral load should occur only if the same methodology is used, and the same assay should be used when serially testing viral loads. The issue regarding HIV-1 viral load differences in men compared with women is not yet resolved. Some studies have noted that women tend to have lower HIV-1 RNA levels than men at seroconversion and early in infection, although other studies revealed no such differences [87–91]. Even when viral load was found to be lower in women than men at seroconversion, the plasma HIV-1 RNA levels seemed to equalize with time [92]. More recent data suggest that because viral loads are lower at seroconversion in women, and because the rate of progression to AIDS is similar in both sexes, guidelines recommending when to initiate therapy should be different for men and women [93]. Until the issue of gender differences on plasma HIV-1 viral load is clarified, it is unlikely that different treatment recommendations for men and women will be made. A number of factors can lead to variation in viral load for an individual patient. This variation can be related to assay methodology or can be biologic in nature. For example, use of heparin as an anticoagulant can result in lower quantities of HIV-1 RNA than with use of ethylenediaminetetraacetic acid as the anticoagulant [64,94]. Similarly, time to processing of the specimen can alter levels of RNA [95]. In addition, plasma viral load can be transiently affected by a number of host or biologic factors. Vaccination against tetanus, the pneumococcus, and influenza have all been shown to cause a moderate, but transient, increase in plasma HIV-1 RNA levels [96–99]. The cellular activation that results from vaccination may explain the increased HIV-1 viral expression and replication. Several infectious disorders, such as tuberculosis, herpes simplex virus, and bacterial pneumonia, have also been shown to cause a transient increase in HIV-1 viral load [100–102]. In fact, a transient increase in viral load has been associated with the development of any of several opportunistic infections, such as Candida esophagitis or Pneumocystis carinii pneumonia [103]. Again, this may be related to cellular activation in response to infection. Transient changes in plasma viral load may occur for less obvious reasons. A decrease in plasma viral load had been demonstrated in ovulating women, particularly during the early follicular phase to the mid-luteal phase, perhaps as a result of hormonal regulation of lymphocytes or

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cytokines [104]. Also, missing even a few doses of antiretroviral therapy before measurement of viral load can result in an increased HIV-1 RNA level [67]. Given the variability in viral load, it is strongly recommended that decisions regarding therapy be based on at least two plasma viral RNA determinations separated over time [105,106]. Recommendations regarding use of plasma HIV-1 RNA level in guiding therapeutic decisions have been made by the Department of Health and Human Services and the International AIDS Society [107,108]. For patients who are naive to antiretroviral therapy, determination of baseline plasma viral load, in addition to the CD4þ T-lymphocyte count, is an essential component to these guidelines. The plasma HIV-1 RNA threshold values used in determining when to initiate therapy differ slightly among the two sets of recommendations. The guidelines by the Department of Health and Human Services also consider different threshold values based on which assay method for plasma viral load is used. Both sets of guidelines state that if combination antiretroviral therapy is instituted, the goal of therapy is to obtain and maintain a plasma viral load below the level of detection. The level of detection, however, depends on the sensitivity of the assay. Both the RT-PCR and bDNA methods for plasma HIV-1 RNA can detect viremia as low as 50 copies/mL. Currently, the guidelines suggest that a viral load less than 50 copies/mL is optimal, and there are some data to suggest that a more durable virologic response is obtained if such a viral load can be achieved [109–111]. It is expected that a 1- to 2-log reduction of plasma HIV-1 RNA should occur within 4 to 8 weeks after starting therapy, and that the plasma viral load should be undetectable within 16 to 24 weeks. One factor that can affect the time it takes for the viral load to become undetectable after beginning therapy is the level of viremia at baseline. A viral load that is greater than 100,000 copies/mL has been associated with a poorer chance of attaining an undetectable viral load in the setting of highly active antiretroviral therapy [107,112,113]. It has also been demonstrated that the less time it takes to obtain an undetectable viral load after initiating therapy, the more likely the viral load remains undetectable while on therapy [113]. The lower the nadir, and the less time it takes to achieve an undetectable viral load, the greater the chance of successful therapy. The optimal frequency for performing plasma viral load once therapy has begun is not clear. Current guidelines recommend performing plasma viral load within 1 month after initiating or changing therapy, monthly until the goal of therapy is reached, and every 2 to 3 months thereafter [107]. The guidelines also make recommendations regarding a change in antiretroviral therapy if viral rebound or failure to attain an undetectable viral load occurs. This issue is somewhat less clear, because the exact plasma viral load threshold for changing therapy is unknown, and other factors, such as immune status and adherence and side effects of medications, should be considered before altering therapy.

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The diagnosis of acute HIV-1 infection Because the development of antibodies to HIV-1 may not occur until weeks after exposure to the virus, routine serologic testing for HIV-1 using ELISA and Western blot assays may initially be negative or indeterminate, particularly during the first few days to weeks after infection. Diagnosis and treatment of HIV-1 infection shortly after exposure may be beneficial. Potential benefits of identifying and treating HIV-1 during the acute phase include preserving immune function, reducing the risk of transmission, and potentially altering the natural history and progression of disease [114]. Initial viremia is thought to occur approximately 4 to 11 days after mucosal exposure [115]. Quantitation of HIV-1 and p24 antigen during this period has revealed extraordinarily high levels of HIV-1 in the plasma [32, 59,116,117]. Control of viremia with antiretroviral therapy during acute infection may limit viral replication, enhance the cytotoxic T-lymphocyte response and T-helper lymphocyte response, and improve overall prognosis [118–123]. Making the diagnosis of acute HIV-1 infection, combined with early antiretroviral therapy, may be very beneficial. Establishing this diagnosis, however, may be difficult. Although a welldescribed mononucleosis-like syndrome occurs in at least 50% of patients with acute HIV-1 infection, these findings are diverse and nonspecific, and easily can be attributed to other viral illnesses. In fact, less than 10% of acute HIV-1 infection is diagnosed by clinicians [124]. These symptoms usually occur 2 to 6 weeks after exposure and last for approximately 1 to 2 weeks [125]. Because HIV-1 serologic tests become positive an average of at least 22 days after exposure, other methods of detection of HIV-1 infection are necessary [126]. Measurement of serum p24 antigen, HIV-1 DNA in plasma or PBMCs, or plasma HIV-1 RNA may be useful during this serologic window period. As mentioned previously, p24 antigen levels are often initially high, but may wane after 1 to 2 weeks [116,127]. One recent trial determined that the sensitivity of the p24 antigen assay during acute HIV-1 infection is approximately 89%, with a specificity of 100% [128]. A more sensitive method of diagnosing acute HIV-1 infection may be the plasma HIV-1 RNA level. Studies evaluating the use of this marker in diagnosing acute HIV-1 infection have found that the plasma viral load is nearly always greater than 50,000 copies/mL, and often greater than 100,000 copies/mL [119,128]. The sensitivity of this assay during acute HIV-1 infection was 100% in a recent study, with a specificity of 97% [128]. It should be noted, however, that false-positive plasma viral loads (usually with less than 5000 copies/mL) have occurred and can lead to misdiagnosis [68,129]. Although these tests are performed more easily, a third option for diagnosis of acute HIV-1 infection is the PCR for HIV-1 DNA in plasma or PBMCs. The sensitivity of this assay in this situation is not known. Finally, if a diagnosis of acute HIV-1 infection is made based on these tests, standard serologic testing should still be performed at a later date to confirm the diagnosis.

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Detection of HIV-1 infection in infants The diagnosis of HIV-1 infection in infants, particularly in neonates, can be challenging. Standard serologic tests, such as ELISA and Western blot, are not useful in early infancy because of the presence of transplacentally derived maternal antibody. This maternal antibody may be present in the child until the second year of life [130]. For this reason, the diagnosis of an HIV-1–infected infant usually relies on demonstrating the presence of viral antigens, viral RNA or DNA, or by culturing the virus itself. Early and accurate diagnosis of an infant born to an HIV-1–infected mother is important for a number of reasons, most important of which is deciding on the need for antiretroviral therapy and opportunistic infection prophylaxis for the infant. Measurement of p24 antigen in the blood of such infants has been evaluated as a diagnostic method. Sensitivity in this setting varies greatly with age, and is rather poor (20%) if the infant is younger than 1 month old [49,131,132]. Although p24 antigenemia has a relatively high specificity in this setting, false-positive results, particularly in neonates, have been reported when testing infants [133]. The rather poor sensitivity of p24 antigenemia testing has virtually eliminated its diagnostic role in infants born to HIV-1–infected mothers. The presence of p24 antigenemia at birth, however, has been associated with the development of early and severe HIV1–related disease [49]. The current recommendations for testing in this situation include either PCR testing for HIV-1 proviral DNA, or culture of the virus itself [134,135]. Testing is recommended at birth, at 1 to 2 months of age, and again at 3 to 6 months of age. Umbilical cord blood should not be used, to avoid maternal contamination. Any positive test result should be confirmed with repeat testing from a separate blood sample. Although considered the gold standard for diagnosing HIV-1 infection in infants, HIV-1 culture testing is problematic in that it is labor-intensive, requires a specialized laboratory, and may require 2 to 4 weeks to obtain a result. Furthermore, the sensitivity of this test varies with the age of the infant. For example, two studies using HIV-1 culture methods detected less than 50% of the truly HIV-1–infected infants when the test was performed at birth [49,136]. The sensitivity of this test improves greatly by 6 months of age, and the specificity of this test also is very high. Nevertheless, the poor sensitivity of HIV-1 culture during the neonatal period, and several technical issues, has resulted in relatively low use of this test during infancy. Currently, the most commonly used test in diagnosing infants born to HIV-1–infected mothers is PCR testing for proviral DNA. When compared with HIV-1 culture in detecting HIV-1 infection in infants, PCR testing for HIV-1 DNA is superior [48]. The timing of testing, however, can affect DNA PCR results in infants. The sensitivity of DNA PCR below the age of 3 months is not clear at this time. Several trials report a sensitivity of

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90% for DNA PCR after the age of 3 months, but as low as 50% at 1 month of age [48]. A recent meta-analysis evaluated 271 HIV-1–infected infants who were evaluated prospectively to determine age-specific estimates of the sensitivity of DNA PCR [137]. This analysis found a sensitivity of 38% if DNA PCR was performed on the day of birth or the day after birth, but an unexpectedly large increase in sensitivity to 93% by the second week of life. Although further trials may clarify the true accuracy of DNA PCR testing of the neonate, this test remains the current diagnostic method of choice in this setting. Measurement of HIV-1 plasma RNA in infants born to mothers who are HIV-1–infected may be superior to any of these tests. At least two trials evaluating plasma RNA levels have been conducted in this setting [138, 139]. These trials used the NASBA technique to measure plasma HIV-1 RNA, and compared this assay with HIV-1 DNA PCR testing. Both found that plasma RNA testing had an equal or better sensitivity than DNA PCR testing in infants. Importantly, the plasma RNA test had a much higher sensitivity than the DNA PCR test when comparing tests in infants less than 1 month of age [139]. It should be noted, however, that false-positive plasma RNA results, although uncommon, were reported in these studies. Although plasma RNA testing is not yet FDA-approved as a diagnostic test, its use in the setting of neonatal testing certainly holds promise. Numerous advances have been made recently in the ability to detect the presence of HIV-1 and HIV-2 infection. These assays have enabled quicker and more efficient diagnosis in the clinical setting, and have had an impact on therapy and survival. Nevertheless, some uncommon subtypes of HIV-1, and certain clinical scenarios, continue to be problematic from a detection standpoint. The diagnosis of HIV infection in these situations requires further study.

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