Virological diagnostics

Philip Cunningham: NSW State Reference Laboratory for HIV, St Vincent’s Hospital, Sydney NSW
 
The diagnosis of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) infection is usually made by the detection of circulating antibodies in blood. Antibodies are identified by the use of a screening test (standard test), usually an enzyme immunoassay (EIA) followed by definitive diagnosis using test strategies that include supplemental EIAs, nucleic acid tests and a Western Blot assay (reference test). Reference testing strategies and algorithms are designed to provide the greatest positive predictive value through a combination of tests. In some situations, such as pre-seroconversion (acute infection) or neonatal infection, detection of HIV antibodies may be unreliable. In these cases, diagnosis of infection may require tests that detect HIV directly – whether by quantitating plasma HIV RNA or HIV proteins (p24 antigen), or by detection of HIV DNA in blood mononuclear leucocytes. In 2005, screening tests became available that detect both HIV antibody and p24 antigen in a single test, which improved the detection of individuals presenting with acute HIV infection. Combined antibody and antigen tests are so called fourth generation tests and are widely used as screening (standard) tests to detect antibodies to both HIV-1 and HIV-2. HIV-2 is endemic in West Africa and in some countries with historical socioeconomic links with West Africa (e.g. France, Spain, Portugal, and former Portuguese colonies such as Brazil, Angola, Mozambique, and parts of India). HIV-2 is not prevalent in the Australasian Pacific region but has been reported particularly in individuals with links to HIV-2 endemic countries. HIV-2 is similar to HIV-1 in its structure, genomic organisation, route of transmission and ability to cause acquired immune deficiency syndrome (AIDS). Because the viruses are closely related there is significant serological cross-reactivity in antibody tests so the use of specific HIV-2 Western Blot assays and nucleic acid tests are used to confirm the diagnosis.

HIV antibody and antigen testing

HIV enzyme immunoassay (EIA) 

Because of its rapidity, sensitivity and low cost, the EIA is the standard screening tool for HIV infection[1]. Synthetic and native HIV antigens, fixed on a solid phase, are exposed to and bound by HIV antibodies in test  serum. These antibodies are then detected by a second antihuman antibody, with a sensitivity of more than 99.5%. Most commercially available EIAs are automated and allow for high throughput testing. For example, most laboratories in Australia use testing instrument platforms that enable continuous loading of specimens and with results being available within 1 hour. The EIA also detects antibodies against a broad range of HIV types and subtypes, and is continually updated to include newly described HIV-1 subtypes.
 
Test results arising from the screening (standard) HIV test are classified as reactive or non-reactive (negative) or occasionally invalid.  Reactive screening test results must undergo further testing to confirm whether the reactivity is true or false. Initially reactive screening results are generally not reported until the confirmatory tests are completed. Test results obtained from the patient sample are compared with a cut-off value which classifies a test as being reactive or negative. In most cases, if a sample is found to be negative by the screening (standard) test no further testing is performed and the result is issued. If acute HIV infection is suspected, other more sensitive tests such as p24 antigen or nucleic acid tests may be indicated.  A positive antibody test is usually observed within 3 to 6 weeks following infection. Reactivity on the fourth generation antibody and antigen combination test may be observed several days earlier than antibody-only tests (2-4 weeks).[2]
 
The weeks between HIV infection and seropositivity are termed the window period and are associated with high levels of circulating HIV RNA, and potentially more efficient HIV transmission. Direct detection of HIV nucleic acids by molecular amplification tests such as polymerase chain reaction (PCR) or serological detection of HIV p24 antigen is usually possible before the detection of HIV antibodies. These direct detection virological tests are indicated in suspected cases of primary infection or seroconversion illness or having a high-risk exposure in the previous 6 weeks and suspected of having acute HIV infection.
 
False-positive test results are rare and the specificity of most EIAs is above 99.8%. Factors  associated with false-positive EIA results are not well characterised and may include antibodies to human leukocyte antigen (HLA) class II antigens, autoantibodies and immune complexes, malaria, recent vaccination and acute viral infections, but also laboratory/technical  errors in testing procedure and specimen handling. Therefore, although a negative enzyme-linked immunosorbent assay (ELISA) result, repeated at 3 months, effectively rules out a diagnosis of HIV infection, an initially reactive EIA result is not indicative of infection unless confirmed by supplemental tests including a positive Western Blot assay or nucleic acid test.

HIV rapid tests

The use of HIV rapid tests has gained popularity since their availability in the late 1980s. Rapid HIV tests were first introduced in Australia in 2013 despite being widely available in other countries for many years. These devices are screening tests intended to be performed in settings near or at the side of the patient (hence the name point-of-care HIV tests) and have rapidly become a convenient and highly acceptable form of testing that may lead to increased uptake and frequency of testing when compared with conventional clinic- and laboratory-based testing[3]. Introduction of rapid HIV tests in some countries has been driven by the lack of laboratory infrastructure mainly in resource limited settings but more recently through a growing recognition of the public health benefits of promoting and improving access to individuals who may not otherwise engage in conventional testing pathways. Point-of-care HIV tests generally involve alternate sample types including finger prick capillary blood or oral fluids and provide results in 10 – 30 minutes by visual reading of reactivity to the HIV antigen and the internal control lines. In Australia, it is a requirement that all reactive HIV point-of-care tests be confirmed by conventional diagnostic laboratory tests.
 
The Australian regulator requires rapid HIV tests to have a clinical sensitivity of 99.5% for whole blood, serum or plasma and 99% for oral fluid tests (based on testing performed outside of the seroconversion window period for the device) and a clinical specificity of at least 99% for detection of HIVinfection.[4]
 
While many rapid tests for HIV demonstrate a high level of sensitivity and specificity, it is recognised that most have limitations when compared with conventional tests. The most common limitation is the prolonged window period when detecting the presence of HIV antibodies by simple lateral flow technology. It has also been recognised that operators of rapid HIV tests undergo comprehensive training in their operation, understand the limitations of the tests and use appropriate key messages and language in the delivery of test results.
 
Figure 1 a. Trinity Unigold HIV-1 antibody test

(NR: valid non-reactive test; R: valid reactive test)
 
Note two lines C: internal procedural control and T: test line detects presence of HIV antibodies
 
Alere Determine

Figure 1b. Alere Determine HIV-1 and HIV-2 antigen and antibody Ag/Ab combination test
 
This test presents three lines (control; Ag and Ab)

Test strip marked 10B is a valid reactive test showing control line, antibody (Ab) and weak reactivity to antigen (Ag) lines.

Test strip marked 11A is a valid non-reactive test showing a control line indicating the test is valid, but no reactivity is visible in the Ag or Ab test areas.
 
 
 
Source: NSW State Reference Laboratory for HIV, St Vincent’s Hospital Sydney Limited. 

HIV-1 Western Blot assay

The Western Blot assay involves detection of antibodies in patient sera that react with a number of different viral proteins. These viral proteins are separated into bands of distinct molecular weight using protein gel electrophoresis.  After transfer (blotting) to a solid membrane, proteins that bind HIV antibodies in test sera can be identified. Antibodies to different HIV proteins appear in a defined order. First, antibodies to the structural gag proteins, the precursor (p55), p24 and p18, appear. These antibodies are closely followed by antibodies to the envelope glycoproteins – the precursor gp160, the extracellular gp120, the transmembrane gp41 – and then the polymerase components p31, p51 and p66. Antibodies to the smaller regulatory and accessory HIV proteins encoded by Vpr, Vpu, Vif, Rev, Tat and Nef may also be seen.
 
The Western Blot test is classified as negative if there is no reaction of the patient’s serum with any HIV protein bands. An individual is classified as seropositive for HIV antibodies according to specific criteria. There are a number of criteria defined by different peak organisations (Centers for Disease Control and Prevention [CDC], World Health Organization [WHO], American Red Cross, National Serology Reference Laboratory of Australia, Paul Ehrlich Institute) which has led to confusion about classification of HIV seropositive samples. Generally, a positive Western Blot result is defined by the detection of antibodies to all of the three main groups of HIV proteins – envelope (gp160, gp120 or gp41), gag (p24, p55) and polymerase (p68 or p51). Samples that do not meet the criteria for seropositivity but demonstrate reactivity to some viral proteins are classified as indeterminate. In Australia, there are four groups of indeterminate Western Blots each corresponding to the likelihood of HIV infection with reactivity profiles containing antibodies to envelope glycoproteins being the most specific or most likely to be associated with true HIV infection.
 
In some countries including the USA, the HIV-1 Western Blot test is being replaced by nucleic acid-based tests. In Australia however, reference laboratories are experienced in interpretation of Western Blot results and many experts believe it is important to retain a native HIV viral protein assay in the testing strategy to safeguard against possible false-negative results arising from HIV genetic diversity if nucleic acid-based tests are used.  However, there is great interest in the pathology sector regarding emerging nucleic acid tests for HIV diagnosis.
 
In some countries including the USA, the HIV-1 Western Blot test is being replaced by nucleic acid-based tests. In Australia however, reference laboratories are experienced in interpretation of Western Blot results and many experts believe it is important to retain a native HIV viral protein assay in the testing strategy to safeguard against possible false-negative results arising from HIV genetic diversity if nucleic acid-based tests are used.  However, there is great interest in the pathology sector regarding emerging nucleic acid tests for HIV diagnosis.

Figure 2. Typical appearance of an evolving HIV-1 Western Blot in HIV infection (seroconversion)
 
 
Note: serial Western Blot test strips exposed to serum from the patient demonstrating the typical stepwise appearance of bands to the specific viral antigens over time.

Source: NSW State Reference Laboratory for HIV, St Vincent’s Hospital Sydney Limited.
 
An indeterminate Western Blot assay should be followed by repeat HIV serology tests after 4 to 6 weeks, to exclude an evolving HIV antibody response. During seroconversion (changing from seronegative to seropositive) the evolving pattern of viral protein bands is dynamic with additional bands to different viral proteins occurring in days to weeks following infection. The application of tests that directly detect the presence of viral nucleic acid or protein (p24) may be used, especially when the index of suspicion is high. A positive result in one or more such assays leads to a presumptive diagnosis of HIV infection, pending the development of a Western Blot assay which meets positive criteria for HIV antibodies. Negative results from one or more of these assays, and a non-evolving indeterminate Western Blot assay, exclude HIV infection and indicate that the initial reactivity on the screening test is non-specific or false. These results are frequently called biological false positives (BFP).
 
HIV-2 infection is rare in Australia and antibodies to HIV-2 proteins in most sera from people with HIV-2 infection will cross-react with HIV-1 proteins in Western Blot assays. Specific HIV-2 Western Blot assays can be used to confirm HIV-2 infection and distinguish it from HIV-1. However, the cross-reactivity between HIV-1 and HIV 2 is often significant making it difficult to conclusively differentiate the infections. There have been reported cases of misdiagnosis of HIV-2 in cases of HIV-1 infection due to this cross-reactivity.

HIV quantification

HIV RNA quantification (or viral load) is a critical tool in the management of HIV disease. Detection of HIV RNA can help provide a positive diagnosis of HIV infection in certain clinical situations, such as acute or neonatal infection, where standard serological testing is inappropriate or unclear. Quantification of HIV RNA levels allows and predicts the rate of HIV disease progression[5] [6] and is the major laboratory tool for monitoring response to antiretroviral therapy.

Nucleic acid quantification

Different technologies are used to quantitate HIV RNA but all have now achieved a sensitivity of 20-50 copies of HIV RNA per millilitre (mL) of plasma. In acute HIV infection, plasma HIV RNA levels may be used to assist in the confirmation of acute or primary infection before the appearance of HIV antibodies detected by an EIA or Western Blot assay. Most true-positive results will show very high levels (105 to 107 copies/mL) of circulating HIV RNA, consistent with uncontrolled primary viraemia. False-positive results occur in less than 10% of cases; these usually involve low RNA levels (less than 104 copies/mL) and are usually not reproducible[7] [8]. Most manufacturers of HIV viral load tests do not make claims that support the use of these tests for diagnosis of HIV infection. However, an increasing number of products introduced to the market can be used for both monitoring and diagnostic purposes.

Real time PCR assay

Conventional nucleic acid-based quantification methods are being replaced in some laboratories with one of a number of real time PCR assays that have become available. The Roche COBASTM TaqManTM HIV-1 test combines automated sample preparation for HIV-1 RNA purification using the AmpliPrep, and real-time PCR amplification and detection using the COBAS AmpliPrep TaqManTMsystem. Similar to the Roche COBASTM Amplicor test, this assay targets dual conserved regions in HIV-1 and uses fluorescently-labelled probes to detect the amplified products in real time. The advantages are a wider dynamic range and greater sensitivity[9] [10]. Earlier versions of HIV RNA quantification viral load assays using single target amplification test systems were prone to variability in the levels of HIV-1 RNA reported caused by genetic diversity of HIV-1 and mutations and mismatches in the primer and probe target areas. The introduction of dual targets in highly conserved regions of the HIV-1 genome has reduced this variability and allows for greater equivalence in reported RNA results from patients with HIV infection with different subtypes.
 
Table 1. Comparison of three commercial HIV-1 viral load methods
 
HIV Viral Load Method Assay Target/s Measuring Range (cp/mL)
Roche gag and LTR 20 - 1 X 107
Abbot Integrase 40 - 1 X 107
Qiagen LTR 34 - 4.5X107
 
Source: NSW State Reference Laboratory for HIV, St Vincent’s Hospital Sydney Limited.

Table 2. Genomic structure of HIV-1
 
 
The Abbott RealTime HIV-1 assay, for use on the m2000 system, uses a unique partially double-stranded probe that targets the HIV-1 pol. The probe strands are labelled with a fluorophore (reporter) at the 5’ end, and a quencher moiety at the 3’ end of the shorter, complementary strand. In the presence of target pol sequences, the reporter probe preferentially binds, and upon release of the shorter quencher probe, fluoresces. Assay performance characteristics from Abbott Laboratories show that the assay has a 5-log10 linear range, assay specificity of 100% (n=259), 95% probability of detecting samples with a viral load of 25 copies/mL, and recognition of subtype panels from group M (A-H), group O, and group N.[11] [12] [13] [14] [15] [16]

HIV DNA PCR

The HIV DNA assay detects both unintegrated and integrated forms of HIV DNA present in circulating peripheral blood mononuclear cells. There are a number of newly emerging qualitative assays able to detect both HIV DNA and RNA (total nucleic acid) for supplementary reference testing and diagnostic testing strategies. The main clinical use of these assays is in the qualitative detection of HIV in situations where serological testing is inappropriate (e.g. pre-seroconversion, or neonatal infection where results may be confounded by the presence of maternal antibodies) or where serological testing has been disputed or is inconclusive (e.g. indeterminate Western Blot assay result). The HIV DNA PCR test is highly sensitive (greater than 99%, detecting one copy of HIV DNA per 10,000 to 100,000 cells) and specific (98%), but has not replaced serology either as a screening test or as a diagnostic test in isolation.  HIV DNA testing is the preferred test for early infant diagnosis when neonates are born to HIV seropositive mothers.
 
HIV DNA PCR and HIV RNA reverse transcriptase (RT)-PCR techniques were commonly used in laboratory research however they have made a rapid transition to routine clinical practice. Amplification may be performed on various genomic regions of interest, and the products used in such analyses as HIV subtype determination, phylogenetic analysis, detection of genetic mutations, and prediction of viral tropism and drug resistance.[17] [18]

HIV antigen testing

Assays are available to directly detect the major structural core protein HIV-1 p24 (designated p24 because the molecular weight of the protein is 24 kilodaltons). While initially examined for utility as an assay to monitor viral load[19] [20], it is no longer considered to be appropriate for viral load testing due to a lack of sensitivity[21] [22]. The p24 assay is widely used by reference laboratories in Australia, but is not commercially available in the USA and as such does not appear in recommended CDC testing algorithms. HIV-1 p24 antigen is particularly useful in diagnosis of acute HIV infection as a supplemental test when antibody tests may be negative, inconclusive or indeterminate. While nucleic acid tests offer superior sensitivity and specificity for the earliest detection of HIV infection, dedicated specimens are required for nucleic acid tests while HIV-p24 can still be performed on the primary serum sample submitted for antibody screening. The presence of HIV-1 p24 in newly identified sera or in sera which exhibit negative or inconclusive HIV-1 antibody tests should be confirmed by neutralisation with anti-p24 antisera.
 
In 2005, the so-called fourth generation HIV screening tests were developed. These tests detect HIV-1 p24 antigen and both HIV-1 and HIV-2 antibodies in a single test. They have been rapidly adopted globally as preferred screening tests for HIV infection and allow for the routine detection of recently acquired infections where antibody-only tests may be unreliable.

HIV tissue culture

Tissue culture allows HIV to be expanded and propagated in vitro by its culture with donor peripheral blood mononuclear cells in the presence of stimulatory factors such as interleukin 2 (IL-2). Coculturing of patient peripheral blood mononuclear cells C, or cells from other body compartments suspected to be infected with HIV, with stimulated donor peripheral blood mononuclear cells is an alternative method for the detection of very low levels of virus. Culture supernatants are assayed for the presence of p24 or reverse transcriptase, which generally appear over 1 to 2 weeks. The efficiency of the process falls dramatically with lower viral load, although it can be improved by using techniques such as the removal of inhibitory CD8 cells from the patient and donor peripheral blood mononuclear cells, stimulation with phytohaemagglutinin or IL-2. The tissue culture technique is expensive and time-consuming and must be performed only within a laboratory with appropriately certified biocontainment. For viral detection and isolation in clinical situations, tissue culture has now been replaced by the methods outlined above and is never performed as a routine test. However, it remains a mainstay of laboratory practice, where it is used for the maintenance and analysis of viral strains.

Drug resistance testing

HIV shows a high rate of genomic evolution due to the error- prone nature of RT (which introduces random sequence changes into newly produced viral RNA)[23],  the high rate of viral production, and the rapid turnover of productively infected cells{ref}Perelson AS, Neumann AU, Markowitz M, Leonard JM, Ho DD. HIV-1 dynamics in vivo: virion clearance rate, infected cell life- span, and viral generation time. Science 1996;271:1582-6.{/ref}. For these reasons HIV strains rapidly develop resistance to antiretroviral monotherapy through the acquisition of mutations in RT, envelope or protease genes that confer resistance of enzymatic function to the effects of the drug on the enzyme. The principle behind combination antiretroviral therapy (cART) is to achieve a low level of viral replication and thus a reduced opportunity for the introduction of advantageous mutations into newly produced viruses. Strains that have mutated to become resistant to one therapeutic agent may also have abnormally lowered replicative capacity (fitness), making the acquisition of mutations to further agents less likely.

The resistance profile of a viral strain can be estimated by examination of the nucleic acid sequence of the target genes for evidence of known resistance mutations, or directly assessed by tissue culture of the virus (full length or cloned into a common viral backbone) in the presence of a panel of antiretroviral drugs. Treatment may then be altered in the knowledge that a patient is carrying a virus resistant to one or more agents. Studies have demonstrated improved virological outcomes in patients whose therapeutic choices have been guided by the use of resistance testing[24] [25] [26] [27] [28] [29] [30] and the use of resistance testing is regarded as standard of care in Australia, western Europe and the USA[31] [32].
 
There are a number of commercially available methods for determining the resistance profile of an HIV isolate. The most common is genotypic resistance testing, which is the only assay routinely available in Australia. and is supported by the Medicare Benefits Schedule (MBS). Samples submitted for genotypic antiretroviral resistance testing should be accompanied by a request for a plasma HIV RNA level and a treatment history to aid in the interpretation of test results.

Antiretroviral drug resistance genotyping

Assessment of the HIV genotype (i.e. the nucleic acid sequence) involves the isolation of viral RNA followed by the RT-PCR amplification of portions of the target genes. These amplification products are then sequenced using standardised automated DNA sequencing procedures. Ultracentrifugation of a plasma sample with a low viral load (1000- 2000 copies/mL) will pellet free virus for RNA extraction and sequencing. Test sequences are compared with a wild-type control reference strain (i.e. no prior exposure to antiretroviral agents) for annotating differences in the predicted amino acid sequences. Mutations that have been associated with resistance to antiretroviral therapy are then identified by comparison with a database of known codon (genetic code) mutations. Resistance mutations are usually reported as primary (generally associated with phenotypically detectable drug resistance) or secondary (mutations generated after the primary mutation, and which may confer varying levels of resistance). The nomenclature of mutations is the wild-type amino acid (using the 20 letter amino acid code)/the amino acid position in the protein/the mutant amino acid e.g. for a change from the wild- type methionine residue at position 46 of protease to isoleucine is denoted M46I. Mutations that involve the insertion of amino acids follow similar nomenclature with the additional amino acids following the residue position. Tables 3 and 4 show HIV resistance mutations to the classes of antiretroviral agents.
 
Caution must be exercised in the interpretation and use of resistance genotyping data. Two major factors contribute to the complexity of this process: the contribution of minor species and mutational complexity. It is important to realise that genotypic resistance testing gives a snapshot of the dominant viral forms circulating in the plasma at the time of testing. Viral variants with different resistance profiles may circulate at low levels or be present latently in proviral DNA. These may rapidly re-appear and become dominant once the selective pressure of antiretroviral therapy changes. Only minor species that are circulating at substantial levels (above about 20%) may be detected and reported. For this reason, genotypic resistance data must always be interpreted in the light of a thorough history of treatment and previous resistance testing.

Mutations are associated with drug resistance on the basis of in vitro or in vivo data. These data are not always clear and the publication of new evidence means the assignment of mutation to phenotype is being continuously updated. The substantial sequence variability between HIV strains means that it can be difficult to distinguish naturally occurring viral variants from selected mutations that confer an evolutionary advantage particularly if this advantage is small or secondary to the existence of a primary mutation. When in doubt, consultation with the laboratory performing the assay is strongly indicated.
 
Table 3. HIV antiretroviral therapy resistance mutations: nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) and non-nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) mutations
Major HIV-1
Source: adapted from Standford University HIV Drug Resistance Database. For most current details of interpretation, please refer to http://hivdb.stanford.edu
 
Table 4. HIV antiretroviral therapy resistance mutations: protease inhibitor and integrase inhibitor Mutations
Source: adapted from Standford University HIV Drug Resistance Database. For most current details of interpretation, please refer to http://hivdb.stanford.edu 

Resistance phenotyping

Phenotypic drug resistance is considered the gold standard test for drug susceptibility and directly measures the competency of a virus isolate to grow in various concentrations of antiretroviral drugs. Phenotypic drug resistance assays are not routinely performed due to availability, lengthy turn-around time for results and cost. Nucleic acid sequences from the various target genes of HIV are extracted from patient plasma HIV RNA and are inserted into the backbone of a laboratory clone of HIV or used to generate pseudotyped viruses that express the patient-derived HIV genes of interest. Replication competence of the pseudotyped viruses at increasing drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference wild-type HIV strain. The antiretroviral drug concentration that inhibits viral replication by 50% is calculated, and the ratio of the IC50 (inhibitory concentration) of test and reference viruses is reported as the fold increase in resistance. The addition of phenotypic to genotypic testing is generally preferred for persons with known or suspected complex drug-resistance mutation patterns, particularly to protease inhibitors (PIs)[33]

Genotypic determination of HIV-1 CCR5/CXCR4 tropism

HIV-1 uses one of two coreceptors for entry into cells: CCR5 and CXCR4. A relatively new class of antiretrovirals, the CCR5 inhibitors, block the CCR5 receptor, but not the CXCR4 coreceptor, reducing viral entry, replication and therefore plasma viral load. CCR5 inhibitors, of which maraviroc is the only one licensed, are only effective if a patient’s HIV uses CCR5 as its coreceptor.[34] [35] If any proportion uses CXCR4, there will be no meaningful response to maraviroc. Therefore, the patient must be documented as having HIV that only uses CCR5 within the 3 months before the commencement of maraviroc.
Viral coreceptor usage is determined primarily by sequencing the section of the envelope gene encoding the V3 loop of the HIV-1 gp120 viral envelope protein. Previously, viral tropism has been determined by a phenotypic assay (Trofile®) which was expensive, required a significant blood volume and had a lengthy turn-around time. More recently, genotypic assays that use viral RNA from plasma or viral DNA from white blood cells have been developed and validated. These assays work like a genotypic resistance assay. The viral RNA and DNA is extracted, amplified and sequenced. This sequence is then examined by the Geno2Pheno (G2P) algorithm.

The G2P algorithm provides a statistical estimate of the likelihood that the virus binds to CXCR4. This process generates a false-positive rate, which is reported along with the tropism result. The lower the false-positive rate the more likely the patient’s HIV uses CXCR4. The higher the false-positive rate, the more likely the virus uses CCR5. Currently, the consensus position is to use a false-positive rate of 20% as the break-point for determining whether a virus binds CCR5 or CXCR4. As even a minor proportion of HIV using CXCR4 will compromise the activity of any CCR5 inhibitor such as maraviroc, the test is performed in triplicate and the result with the lowest false-positive rate determines the result reported. That is, for a virus to be reported as CCR5-using each of the three results must have an false-positive rate above 20. If any result is below 20, then the virus is reported as being CXCR4-using.
The plasma viral RNA G2P assay has undergone the most rigorous validation and is the test of choice for any viraemic patient. The pro-viral DNA G2P assay should be used only in patients who have viral loads of 2000 copies/mL or above because there is substantially less clinical experience with this test and the results generated are more variable.
Ethylenediaminetetraacetic acid (EDTA) anticoagulant treated whole blood is required. If a RNA-based tropism is ordered, a viral load must be ordered on the same sample, as the test only works in patients with viral loads of 2000 copies/mL or above, and will not be performed when the viral load is lower. As the DNA test is more variable it will not be performed as a default. The DNA test should be specifically requested.

The turn-around time for results is approximately 2 weeks, similar to that for genotypic resistance assays. The report will give the overall result as either:

  • Analysis of sequences of V3 loop of the HIV-1 envelope gene does not detect the presence of a substantial sub population of CXCR4 using virus. CCR5 inhibitors are likely to be effective
     or
  • Analysis of sequences of V3 loop of the HIV-1 envelope gene suggests the presence of CXCR4 using virus. CCR5 inhibitors are unlikely to be effective

The report will include the G2P results for the triplicate assays, including the false-positive rate and the interpretation of each of the triplicates performed.

Once a CXCR4-using virus has been detected, the test should not be ordered again. It is not appropriate to start a CCR5 inhibitor in a patient with a history of a CXCR4-using virus. Therefore, as the virus only evolves from CCR5 to CXCR4 using, once a virus has been labelled CXCR4-using, it will always be CXCR4-using, and a CCR5 inhibitor is not recommended. To start maraviroc a patient must have carriage of a CCR5-using virus within 3 months of the start date.

HLA B5701* allele genotyping for assessing the risk of abacavir hypersensitivity reaction.

Abacavir hypersensitivity reaction (AHSR) affects 4-8% of patients with HIV-1 infection within the first 6 weeks of starting abacavir. It is usually characterised by fever, rash, abdominal pains and lethargy. Symptoms related to abacavir hypersensitivity reaction deteriorate with continued therapy and improve within 72 hours of abacavir discontinuation. Rechallenging with abacavir after a hypersensitivity reaction usually results in recurrence of symptoms within hours and with the potential to be fatal.[36] 

GlaxoSmithKline and Western Australia investigators have independently found a strong association between a rare HLA type and the risk of developing abacavir hypersensitivity. Martin et al. [37]  showed a strong association between carriage of the 57.1 ancestral haplotype of major histocompatibility complex (MHC) genes and abacavir hypersensitivity in HIV patients using recombinant haplotype mapping. They found the occurrence of the HLA-B*5701 allele and a haplotypic variant of the Hsp70-Hom allele represented a highly predictive susceptibility marker for abacavir hypersensitivity. Genetic tests involving HLA-B5701 alone or in combination with the Hsp70-Hom M493T variant reduced the prevalence of definite abacavir hypersensitivity in the population predominantly of European origin from 8% to 0.4%. When using HLA-B*5701 alone, an estimated 1.6% of the tested population would be inappropriately denied access to abacavir, however, testing for the presence of both HLA-B*5701 and the Hsp70-Hom M493T variant would reduce this percentage to 0.4%. On the basis of many such studies it is recommended that HLA-B*5701 screening occur before commencement of abacavir-containing regimens and that HLA-B*5701–positive patients should not be prescribed abacavir.
 
A retrospective study by GlaxoSmithKline of patients with abacavir hypersensitivity reaction also found a strong association between the hypersensitivity reaction and HLA-B57. They identified a strong association between a point mutation (SNP) in the tumour necrosis factor-∝ (TNF-∝) gene and abacavir hypersensitivity reaction. However, this association is thought to be a secondary rather than a primary association as the HLA-B57 and TNF-∝ SNP genes are linked.
 
Molecular assays have been developed to identify HLA-B*5701 allele associated with abacavir hypersensitivity reaction. The HLA typing for HLA-B*5701 allele can be determined using a combination of sequence specific primers (SSP). This assay allows rapid HLA-B*5701 low resolution PCR typing using a primer mix containing four specific primer that can be used to discriminate between HLA-B*5701 and the related B57 subtypes HLA-B*5702, HLA-B*5703, HLA-B*5704, and potentially HLA-B*5705, HLA-B*5706, HLA-B*5709.
 
The test involves a number of key sequential steps: genomic DNA extraction from primary clinical specimens; PCR amplification containing a housekeeping gene, HLA-B57 gene and HLA-B5701 allele; agarose gel electrophoresis and sequencing if indicated; and interpretation of results.
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