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Current Diagnostic Methods for Human Immunodeficiency Virus

Paper Type: Free Essay Subject: Health
Wordcount: 5443 words Published: 11th Aug 2017

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Detection of human immunodeficiency virus (HIV) infection is essential for diagnosis and monitoring of the infection. There are several different types of diagnostic tools available that are based on detection of HIV-specific antibodies, virus antigen, or nucleic acid. Sensitivities and specificities of assays utilized for HIV detection have improved. Newer HIV testing technologies such as third-generation enzyme immunoassay (EIA) which detect HIV-specific IgG and IgM antibodies, fourth-generation EIA which detect both anti-HIV antibodies and HIV p24 antigen, and nucleic acid-based tests (NATs) for HIV RNA, have significantly decreased the window period. This review study provides an overview of current technologies for the detection and monitoring of HIV infection and recent advances in the field of HIV diagnosis.

Keywords: HIV diagnosis; HIV antibody test; human immunodeficiency virus; Immunoassay; Polymerase chain reaction (PCR)


Diagnosis of HIV infection contributes to evaluating the progression of disease, monitoring the effectiveness of antiretroviral therapy (ART), and prevention and control of HIV/AIDS. The diagnosis of HIV is associated with decrease in risky behaviors, reduced HIV transmission, and improved survival linked to increased case detection, earlier care and treatment. HIV-negative persons can also protect themselves from HIV when making sexual decisions by engaging in safer sex behaviors and sometimes, taking pre-exposure prophylaxis (PrEP). Early diagnosis of HIV infection provides an opportunity for risk reduction counseling and preventing further transmission of the disease, while late diagnosis of HIV infection is detrimental to infected patients and to the public health, and is associated with an increased rate of morbidity, mortality, and healthcare costs.

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Since the start of the epidemic, it is estimated that 78 million people have become infected with HIV and 35 million people have died from AIDS-related illnesses. In 2015, 2.1 million people became newly infected, 36.7 million people were living with HIV and 1.1 million people died from AIDS-related illnesses. New HIV infections have fallen by 6% since 2010. Sub-Saharan Africa, which bears the heaviest burden of HIV/AIDS worldwide, accounts for 65% of all new HIV infections. Other regions significantly affected by HIV/AIDS include Asia and the Pacific, Latin America and the Caribbean, and Eastern Europe and Central Asia (Table 1) [9].

The present study aims to conduct a narrative review to summarize and discuss the current diagnostic methods for HIV and recent developments. We start with a brief overview of HIV infection, follow by a description on the development of virological and immunological markers following HIV infection. Thereafter, we introduce current algorithms for laboratory HIV testing with different kind of current diagnostics techniques including various generations of enzyme immunoassays, rapid or point-of-care tests, and qualitative/quantitative PCR assays.

Overview of HIV Infection:

HIV-1 causes chronic infection which is usually characterized by progressive immune deficiency, a long period of clinical latency, and appearance of opportunistic infections [1, 2]. Characteristics of HIV include infection and viral replication in T lymphocyte expressing CD4 antigen. Qualitative defects in CD4 cell response and a gradual decline in their numbers increase the risk of opportunistic infections like Pneumocystis carinii pneumonia, and neoplasms such as Kaposi’s sarcoma and lymphoma [3-5]. HIV infection can disrupt functions of blood monocytes, tissue macrophages, and B lymphocytes, and also increase the potential of encapsulated bacteria for developing infections [6, 7]. Direct invasion of CD4 cells in the peripheral and central nervous systems can cause meningitis, peripheral neuropathy, and dementia [8].

The prognosis is variable between people infected with HIV-1. In adults, the average time between HIV exposure to AIDS stage is 10-11 years, but a remarkable proportion of individuals (~20%) progresses rapidly to AIDS within 5 years after HIV exposure. On the other hand, it is estimated that 12% of infected individuals will remain free of AIDS for 20 years [10]. Prophylaxis and in particular antiretroviral therapy (ART) significantly enhanced the overall prognosis of HIV disease against opportunistic infections [11].

The most common route of HIV infection is sexual transmission at the genital mucosa via direct contact with infected body fluids, such as blood, semen, and vaginal secretions. Infection may also occur via inoculation of infected blood, transfusion of infected blood products, transplantation of infected tissues, from an infected mother to her infant during pregnancy, or by reuse of contaminated needles [12].

The probability of transmission after a single exposure with an uncontrolled HIV source has been estimated to be 1/150 with needle sharing, 1/300 with occupational percutaneous exposure, 1/300-1/1000 with receptive anal intercourse, 1/500-1/1250 with receptive vaginal intercourse, 1/1000-1/3000 with insertive vaginal intercourse, and 1/3000 with insertive anal intercourse. The average risk is 12-50% for congenital (mother-to-child) transmission, 12% for breast-feeding, 90% for a contaminated blood transfusion, and 0.1-1.0% for nosocomial transmission [13]. The risk of HIV transmission during early or acute HIV infection appears to be greater than during chronic infection (251). Available data suggest that the viral load is an important predictor of the risk of heterosexual transmission, and patients with levels less than 1500 copies of HIV-1 RNA per milliliter are at lower risk of HIV transmission, whereas the probability of transmission is increased dramatically with increasing viral loads (250).

Laboratory markers for HIV-1 infection:

Several immunological and virological blood markers can be monitored during the course of HIV infection. These markers appear highly consistent between different individuals in a chronological order and allows classification of HIV infection into distinct laboratory stages including eclipse period, seroconversion window period, acute HIV infection, and established HIV infection (Figure 1) [14, 15].

Shortly after exposure to HIV-1, no viral markers are consistently detectable in plasma, but low levels of HIV-1 RNA can be found intermittently [16]. This period is called the eclipse phase. About 10 days after infection, HIV-1 RNA becomes detectable by NAT in plasma and quantities rise to very high levels [17], which subsequently decline rapidly until reaching a set point, a stable level that may persist for years. This stable level of HIV RNA represents an equilibrium between HIV and host immune responses and is an important indicator of subsequent disease progression, and potential transmission of HIV. It has been shown that the higher HIV-1 RNA plasma level is associated with faster progression to AIDS [18]. The set point plasma HIV-1 RNA level can be a helpful clinical tool for determining the timing of initiation of antiretroviral therapy for HIV-infected patients. For instance, patients with high set point levels can be started on aggressive antiretroviral therapy and patients with low set point levels can be monitored without initiating therapy [19].

HIV-1 p24 antigen is expressed and quantities rise to levels that can be measured by fourth-generation immunoassays within 17 days after infection (typical range 13-28 days) [15, 20]. Due to high titers of p24 antigen present in the sera of acutely infected patients during the interval prior to seroconversion, p24 Ag assay can be utilized to diagnose the primary HIV-1 infection [21]. Nevertheless, detection of p24 antigen is transient because, as antibodies begin to develop, they bind to the p24 antigen and form immune complexes that interfere with p24 Ag assay [22, 23].

The time interval between infection with HIV and the first detection of antibodies is known as the serological window period. The detection of HIV-specific antibodies indicates the end of the window period and the individual is known as seropositive [24]. The length of the window period depends on the design and the sensitivity of the immunoassay. Expression of IgM antibodies can be detected by immunoassays within 10 to 13 days after the appearance of viral RNA, 3 to 5 days after detection of p24 antigen, and peak between the 4th and the 5th week [15, 20, 25, 26]. Thereafter, the emergence of IgG antibodies occurs at about 3-4 weeks after infection and persist throughout the course of HIV infection [21]. Nevertheless, the immune responses are highly dependent on the ability of the individual’s immune system to produce the antibodies. Approximately, 50% of patients within 3-4 weeks and about 100% of them within 6 months have detectable antibodies, although there are reports indicating that a small percentage of patients may require up to 6 months for the appearance of antibodies [27].

Laboratory HIV testing algorithms:

Since 1989, the diagnostic algorithm for HIV testing recommended by CDC and the Association of Public Health Laboratories (APHL) relied on the confirmation of a repeatedly reactive HIV immunoassay with the more specific HIV-1 antibody test, either the HIV-1 Western blot or HIV-1 indirect immunofluorescence assay (IFA). The Western blot was previously considered to be the gold standard for the diagnosis of HIV infection by Clinicians [29, 30]. It should be noted that both the Western blot and IFA are first-generation assays that detect only IgG antibodies against HIV proteins. Retrospective testing of specimens from high-risk individuals pointed that antibody testing alone may miss a significant percentage of HIV infections detectable by virologic tests such as HIV antigen and nucleic acid assays.

In 2013, the CDC and the APHL released new guidelines on HIV testing that have led to the earlier diagnosis of HIV infection when compared with the previous diagnostic algorithm. The new recommended algorithm starts with a fourth-generation HIV-1/2 Ag/Ab immunoassay to screen for HIV infection that detects both HIV-1/2 antibodies and the HIV-1 antigen. When the result of initial immunoassay is nonreactive, further testing is not required for samples. Instead, testing with an HIV-1/HIV-2 antibody differentiation test is needed when the sample is reactive on the screening fourth-generation assay. Reactive results with the initial fourth-generation assay and the HIV-1/HIV-2 antibody differentiation immunoassay should be considered as reactive for HIV-1 antibodies, HIV-2 antibodies, or HIV antibodies, undifferentiated. Reactive results with the initial fourth-generation assay and nonreactive or indeterminate on the HIV-1/HIV-2 antibody differentiation immunoassay should be tested with an FDA-approved HIV-1 NAT to differentiate early HIV infection from a false-positive screening result [28] (Figure 2).

HIV diagnostic tests:

Serological diagnostic assays:

Enzyme Immunoassays (EIA):Significant advances in the development of HIV immunoassays have been created since the discovery of HIV in 1983. Based on different design principles, HIV immunoassays are generally classified into generations. The earliest immunoassays (first-generation) are indirect EIAs that used coated viral lysate antigens derived from cell culture on a solid phase for antibody capture and an indirect format that detected antibody utilizing an enzyme-conjugated antihuman IgG [36]. Antibody can be detected within 8-10 weeks postinfection by first generation immunoassay. These assays have 99% sensitivity and 95-98% specificity for HIV infection. Second-generation immunoassays use synthetic peptide or recombinant protein antigens alone or in combination with viral lysates to bind HIV antibodies, and they use an indirect immunoassay format that employs conjugated antihuman IgG, which binds to IgG with high affinity, to detect IgG antibodies [37]. Utilizing recombinant antigens in the second-generation assays improves sensitivity for HIV-1, HIV-1 group O, and HIV-2, allowing earlier detection of IgG antibodies. The sensitivity and specificity of second-generation assays have been reported to be ˃99.5% and ˃99%, respectively. First and second generation immunoassays can only detect IgG antibody to HIV. The window period was decreased to 4 to 6 weeks postinfection by second-generation assays. Third generation immunoassays also utilize synthetic peptide or recombinant antigens to bind HIV antibodies, but in an immunometric antigen sandwich format; HIV antibodies in the specimen bind to HIV antigens on the assay substrate and to antigens conjugated to indicator molecules. This allows detection of both IgM and IgG antibodies which leads to increase in sensitivity and specificity of the test. Lower sample dilutions and the ability to detect IgM antibodies (which are expressed before IgG antibodies) further decrease the window period to 2-3 weeks postinfection [38]. The reported sensitivity and specificity of third-generation assays is ˃99.5%.

Combination or fourth-generation tests use synthetic peptide or recombinant protein antigens in the same antigen sandwich format as third-generation assays for the detection of IgM and IgG antibodies, and also monoclonal antibodies for the detection of p24 antigen [39]. Inclusion of p24 antigen capture allows the detection of HIV-1 infection before seroconversion and further decreases the window period. Most fourth-generation antigen/antibody immunoassays (termed “combo” assays”) do not distinguish antibody reactivity from antigen reactivity [39]. Recent published data has shown that the fourth-generation assay was able to establish HIV infection in more than 80% of patients who tested NAAT positive but either nonreactive or indeterminate by other tests like Western blot, first to third generation immunoassays, and rapid tests [40-42].

Delaney et al. found that the fourth-generation immunoassay are able to detect HIV infection 1-3 weeks earlier than the first, second, and third generation immunoassay which cannot detect p24 antigen. The results of their study revealed that the median duration of the eclipse period was 11.5 days and 99% of specimens from HIV-infected patients could be reactive with Ag/Ab combination tests within 45 days of exposure. Moreover, for detection of antibodies by the IgG/IgM-sensitive and other plasma screening assays, 50 days or longer were required and after 3 month of exposure, infection could be detected by all tests.

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Several studies have reported sensitivities of 100% for fourth-generation immunoassay, whereas other surveys reported transient sensitivities range from 62-89% when assessed against HIV RNA assays. This decreased sensitivity can be attributed to the presence of a second diagnostic window. This situation is rare but it can happen. Second diagnostic window period lies between the p24 antigen detection and the anti-HIV antibody detection, and is associated with reduction in the p24 antigen and antigen/antibody complexes levels, as well as a delay in HIV-specific antibody development which totally may affect the sensitivity of fourth-generation immunoassays. So, it is possible that many acute HIV infections have been missed using fourth-generation assays. Despite negative results from a fourth-generation immunoassay in high-risk populations with suspected acute HIV infection, it is needed to repeat the test on new blood samples obtained several days later, as well as testing for HIV antibody alone, p24 antigen or use of an HIV RNA assay.

In 2015, an improved version of immunoassay, BioPlex 2200 HIV Ag-Ab screening test system, received FDA approval in HIV screening which detects both HIV antibody and the HIV-1 p24 antigen by providing separate results for each analyte. This test also provides separate results for HIV-1 and HIV-2 antibodies, so there is no need for a HIV-1/2 differentiation assay for antibody reactive samples. It was reported that the sensitivity and specificity of BioPlex 2200 HIV Ag-Ab assay were 100 and 99.5%, respectively [43].

HIV Confirmatory Tests:Screening tests must be highly sensitive to produce few false-negative results, whereas confirmatory assays are characterized with high specificity to produce few false-positive results [44]. If the result of a screening test is repeatedly reactive, this has to be confirmed by (at least) one confirmatory assay. Western blot or indirect IFA traditionally have been employed as confirmatory assay due to their higher specificity. The probability that both ELISA and Western blot would give false-positive results is extremely low (<1/140,000) [45].

In IFA technique, HIV-infected and uninfected lymphocytes are fixed on a slide. The slide is incubated first with patient serum and then with a fluorescent-conjugated antihuman antibody. The reaction is visualized utilizing a fluorescent microscope [30]. Considerable skilled and well-trained individuals are needed, and indeterminate results may be yielded in patients with autoantibodies and other conditions. In addition to its use as an independent supplemental test, IFA can be employed to resolve indeterminate HIV-1 Western blots. An HIV-2-specific IFA has been described but it is not FDA approved [48].

Relying on the Western blot and IFA to viral lysate antigens and conjugated anti-human IgG, causes the detection of HIV- 1 to occur later in comparison with the most currently available conventional initial immunoassays and thus may yield false-negative or indeterminate results. Also due to cross-reactivity, the HIV-1 Western blot has been interpreted as positive for HIV-1 in 46 to 85% of specimens from HIV-2 infected patients [49-51].

Rapid or point-of-care tests: Rapid diagnostic tests (RDTs) are single-use qualitative immunoassays with short incubation times intended for use in point-of-care testing (POCT) when Clinical Laboratory Improvement Amendment (CLIA)-waived or laboratories need rapid turnaround. RDTs have been FDA-approved since 2002 and offer several advantages, such as a turnaround time of 30 minutes or less, room temperature storage of reagents, no need for equipment, quick access to results, and simplicity of testing. Due to the low cost of acquiring and maintaining instruments, the use of RDTs is particularly important in limited-resource settings. As a point-of-care test, persons can learn their test result during the same visit, and thus access effective treatment earlier, with significant benefits for long-term survival and quality of life. It should be noted that the majority of HIV RDTs are second-generation assays that detect only IgG antibodies against HIV-1, HIV-2 or HIV-1 only; so they have the same limitations as other second generation assays such as a longer window period when compared with the third- or fourth-generation assays. This limitation may lead to missed early infections or false-negative results.

Point-of-care tests use oral fluid, fingerstick whole blood, venous whole blood, serum, or plasma samples. Oral fluid is a complex mixture of saliva secreted by parotid and other salivary glands, gingival crevicular fluid from the gingival crevice and secretions from the mucous membranes. It contains mainly HIV-specific IgG antibodies in infected persons, although the concentration of IgG in Oral fluid is substantially lower than that in serum or plasma.

In 2004, FDA approved the Oraquick Advance rapid HIV-1/2 Antibody test (OraSure Technologies, Inc) as the first oral fluid based rapid HIV Test kit for detecting antibodies to HIV-1 and HIV-2 in oral fluid, fingerstick whole blood, venipuncture whole blood and plasma specimens [67]. The Chembio DPP HIV 1/2 Assay (Chembio Diagnostic Systems, Inc), as the second oral fluid based rapid HIV Test kit, was approved in 2012 by FDA for the rapid detection of HIV-1/2 antibodies in oral fluid and all blood matrices using Chembio’s patented dual path platform (DPP).

Results of recent studies showed that the Oraquick Advance rapid HIV-1/2 Antibody test may be associated with missing persons with early infection. Several surveys have been carried out on evaluation of diagnostic accuracy of the two oral fluid HIV rapid tests. In one study carried out in Mozambique between May and September 2009, evaluation of performance of two rapid oral fluid tests for HIV detection demonstrated the sensitivity and specificity of 99.8% for the OraQuick Advance Rapid HIV-1/2 test, similar to the manufacturer’s data reported to the U.S. FDA. In addition, the Chembio DPP test demonstrated a slightly higher sensitivity (100%) with same specificity (99.8%) than the OraQuick Advance Rapid HIV- 1/2 test. It was found that the sensitivity and specificity of OraQuick with oral fluid were lower than with whole blood or serum. Therefore, the Oraquick performed in whole blood had a sensitivity range of 91.9 to 96.2% with 100% specificity, whereas the sensitivity reduced to 86.6% with oral fluid. In another survey, Pai et al. performed a systematic review and meta-analysis comparing oral versus blood-based specimens in adults using the Oraquick Advance rapid HIV-1/2. Data analyses have shown a lower sensitivity and similar specificity than blood-based samples using Oraquick. The test displayed 98.0% sensitivity and 99.7% specificity with oral fluid versus 99.7% sensitivity and 99.8% specificity with blood-based specimens.

The Alere Determine HIV-1/2 Ag/Ab Combo (Alere Inc., Waltham, MA) is the first and only FDA-approved 4th generation point-of-care HIV rapid test for the detection of HIV-1 p24 antigen and HIV-1/2 antibodies in human serum, plasma, capillary (fingerstick) whole blood or venipuncture (venous) whole blood. A meta-analysis of published reports evaluating the diagnostic performance and accuracy of the Determine Combo test demonstrated a high pooled sensitivity of antibody component for HIV antibodies (97.3%), while the antigen component had very low sensitivity (12.3%) for p24 antigen. The new Alere HIV Combo test showed improved sensitivity (88%) and specificity (100%) for detection of p24 antigen. Another study by Faraoni et al., have shown that this assay have 100% specificity and positive predictive value with an overall sensitivity of 88.2%.

The Bio-Rad Multispot HIV-1/2 rapid test is a single use qualitative immunoassay for detecting and differentiating circulating antibodies to HIV-1/2 in serum and plasma. This test is suitable for use in multi-test algorithms designed for statistical validation of an HIV screening test result or as part of an HIV-1/HIV-2 diagnostic testing algorithm that includes differentiation of HIV-1/2 antibodies. Sensitivity of the Multispot antibody differentiation assay for established HIV infection is comparable to that of the HIV-1 Western blot, but it produces fewer indeterminate results and accurately detects HIV-2 antibodies, including those in specimens misclassified as HIV-1 by the HIV-1 Western blot [31-34].

In October 2014, FDA approved the Geenius HIV-1/HIV-2 Supplemental Assay (Bio-Rad Laboratories, Redmond, WA) as a single use test for the confirmation and differentiation of HIV-1/HIV-2 antibody in different types of sample such as whole blood, serum or plasma [35]. Although the Geenius is considered as the rapid test, the assay is FDA-approved only as a confirmatory test in the fourth-generation algorithm, not as a screening procedure. The reported sensitivity and specificity of Geenius HIV-1/HIV-2 were 100 and 96%, respectively.

In a most recent meta-analysis study, it was found that rapid tests had a pooled sensitivity of 94.5% in comparison with laboratory-based fourth-generation HIV antigen/antibody tests and a sensitivity of 93.7% in comparison with NATs. In studies carried out in high-income countries (which often included high-risk populations, like homosexuals), rapid tests had a pooled sensitivity of only 85.7% when compared with the fourth-generation antigen/antibody tests or NATs. The sensitivity of rapid tests was higher (97.7%) in low-income countries. This difference between high-income and low-income countries could be due to the higher proportion of acute HIV infections in populations tested in high-income countries. It is suggested that in high-income countries, HIV rapid tests should be utilized in combination with fourth generation EIA or NAAT tests, except in special circumstances.

Molecular diagnostic assays:

Qualitative tests for RNA and DNA of HIV:In September 2006, the Aptima HIV-1 qualitative RNA assay was the first RNA nucleic acid amplification test (NAAT) approved by the FDA for identification of acute HIV-1 infection and neonatal HIV-1 infection, and for confirmation of HIV-1 infection in an individual whose specimen is frequently reactive for HIV antibodies [53]. This assay utilizes transcription-mediated amplification to detect HIV-1 RNA in plasma. The test targets both the 5′-long terminal repeat (5′-LTR) and the pol gene of the HIV-1 genome, which allows detection of all HIV-1 group M, N, and O viruses. This assay was performed in three steps: target-specific capture of HIV-1 RNA from the clinical specimen, transcription mediated amplification, and detection using a hybridization protection assay, and takes about 4.5 hours to perform. The Aptima HIV-1 qualitative RNA assay has a limit of detection of 30 copies/ml of plasma with a specificity of 99.8% [70, 71].

Detection of HIV-1 DNA can also be utilized for HIV diagnosis in specific situations like during the acute phase of infection prior to appearance of antibody, in newborns of infected mothers, or in individuals with inhibited viral replication resulting from antiretroviral therapy or immunologic control. An advantage of proviral DNA assays is that they generally remain positive even in individuals receiving effective antiretroviral therapy and individuals that naturally suppress viral replication. Due to persistence of proviral DNA despite suppressive therapy, it can be employed to determine tropism status when HIV-1 RNA is undetectable [72]. Analysis of proviral DNA sequence plays an important role in determining tropism status for patients considering treatment with maraviroc. Although current US guidelines indicate that proviral testing can be utilized to determine tropism in patients with undetectable viral load, they note that the clinical utility of this approach has not yet been determined. More rarely, viral DNA may be the best target for exploring archived drug resistance [73, 74]. HIV-1 DNA qualitative assays use PCR to amplify conserved regions of the HIV-1 genome to detect proviral HIV-1 DNA in peripheral blood mononuclear cells (PBMCs). Only one qualitative PCR test (Roche Amplicor HIV DNA assay, version 1.5) for the HIV DNA, which is not approved by the FDA, is commercially available and its production may be stopped by the factory [75].

Quantitative RNA tests for the HIV virus: Viral load tests measure the amount of viral RNA in the plasma. Viral load tests are broadly used to monitor changes in plasma viremia during antiretroviral therapy, because they are useful for predicting time to progression to AIDS and for monitoring responses to therapy [76, 77]. The high level of viral RNA with negative or indeterminate antibody HIV test confirms acute HIV infection before seroconversion. Quantitative tests are used to establish the baseline for viral RNA and (along with CD4 + cell count) to determine when the treatment starts or is been postponed which may be helpful, due to the fact that the viral RNA has been associated with reduced CD4. It should be noted that the baseline for the viral RNA is not a strong indicator baseline, like the CD4 + cell count.

Viral RNA is suddenly reduced 2-4 weeks after starting or changing antiretroviral therapy effectively, and after this period, the decrease was slower. Patients who reached viral suppression (below the lower limit of detection of sensitive tests, typically 20- 40 copies per ml) revealed the most stable response to antiretroviral therapy and clinical prognosis. If no change is observed in the RNA of the virus, then it indicates that the treatment is ineffective or inconsistent with the patient [27].

Five commercial tests in the United States that can be employed to quantify HIV-1 RNA in plasma virus have been approved by FDA. These tests include RT-PCR (Roche Amplicor) (with detection limit of 400-750,000 copies per ml), bDNA Variant 0.3 (Bayer) (with detection limit of 75-500,000 copies per ml), Nucleic acid sequence-based amplification (NASBA) (with detection limit of 176 – 5.3 million copies per ml, depending on the volume), Real Time HIV-1 assay (Abbott) (with detection limit of 40 – 10 million copies per ml), and COBAS Ampliprep/ COBAS TaqMan HIV-1 Test (Roche) (with detection limit of 40 – 10 million copies per ml). Two of these tests which have been confirmed recently, the COBAS AmpliPrep/COBAS TaqMan HIV-1, version 2 (COBAS TaqMan), and the Abbott Molecular m2000 RealTime System (Abbott RealTime), utilize Real-Time RT PCR technology. Most labs in America use RT-PCR (Roche or Abbott) test which replaces the older test of (and less-accurate) bDNA. Each test can be used to diagnose acute HIV infection and monitor the treatment process, but the test should be used to follow up patients in the long run [78]. It should be noted that the earlier versions of commercial RT-PCR tests (Roche Monitor and Roche COBAS TaqMan HIV-1 Version 1), although still on the list of FDA-approved tests, are insensitive to some subtypes of HIV-1, notably A, F and CRF02_AG, leading to the underestimation of the true viral load. Sollis et al. conducted a systematic review of the performance of six commercially available HIV viral load assays on plasma samples and respective results are summarized in Table 7 [79].

Dried Blood Spot (DBS) Testing:

For many years, DBS specimens have been used for the diagnosis of infectious diseases like HIV. These specimens can be useful in testing of individuals who are unwilling to undergo phlebotomy or who have poor vascular access, mass screening of populations, and for seroprevalence studies. Moreover, the costs of DBS specimen collection and shipment is lower than plasma samples. Furthermore, completely dried DBS specimens are noninfectious and safe. DBS may be an interesting alternative to plasma for HIV antibody, p24 antigen, and HIV RNA testing. In comparison with plasma or serum, DBS requires a smaller volume of blood, and minimizes the need for cold-chain storage.

Collection of DBS samples starts with a finger, heel or toe prick and spotting whole blood directly onto specially manufactured absorbent filter paper, which is then left to dry at room temperature. Once dried, DBS can be stored without being frozen or refrigerated, and shipped to central laboratories for HIV virologic testing. Currently, there are four FDA-approved tests that used DBS samples, including Fluorognost HIV-1 IFA, GS HIV-1 Western Blot, Avioq HIV-1 Microelisa System, and Home Access HIV-1 Test System and all of them detect only anti-HIV-1 antibodies. Until now, there is no FDA-approved assay using DBS for nucleic acid or p24 antigen tests. The use of DBS sample as a source for diagnostic assay has become increasingly popular in recent years for HIV-1 drug resistance genotyping and viral load monitoring particularly in remote regions and resource limited settings.

Nevertheless, there are some disadvantages found by the DBS specimens, like the overestimation of viral load levels. It can be explained by the fact that a plasma viral load assay quantifies only cell free HIV RNA, while a DBS viral load assay also measures proviral DNA within the peripheral blood mononuclear cel


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