Test
Method
Target
Specimen volume a
Range
Amplicor HIV-1 Monitor® version 1.5 (Roche Diagnostics Indianapolis, IN)
RT-PCR
HIV-1 gag gene
Standard
200 μl
400–750,000 copies/mL
Ultrasensitive
500 μl
50–100,000 copies/mL
COBAS® Amplicor HIV-1 Monitor version 1.5
(Roche Diagnostics, Indianapolis, IN)
RT-PCR
HIV-1 gag gene
Standard
200 μl
400–750,000 copies/mL
Ultrasensitive
500 μl
50–100,000 copies/mL
COBAS® AmpliPrep/COBAS® Amplicor HIV-1 Monitor version 1.5
RT-PCR
HIV-1 gag gene
Standard
250 μl
500–1,000,000 copies/mL
Ultrasensitive
750 μl
50–100,000 copies/mL
Versant® HIV-1 RNA 3.0 (bDNA) (Siemens Healthcare Diagnostics)
Branched DNA
HIV-1 pol gene
1 mL
75–500,000 copies/mL
COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test
(Roche Diagnostics, Indianapolis, IN)
RT-qPCR
v 1.0
HIV-1 gag gene
1 mL
48–10,000,000 copies/mL
v 2.0
HIV-1 gag gene and LTR
1 mL
20–10,000,000 copies/mL
RealTime Taqman® HIV-1 (Abbott Molecular)
RT-qPCR
HIV-1 integrase gene
1 mL
40–10,000,000 copies/mL
Increasing genetic diversity of HIV-1 isolates from individuals in the USA has been reported, primarily from those who have immigrated from Africa and Asia [22, 23], so detection of non-B subtypes is taking on increased importance. The RT-qPCR tests were intentionally designed to detect not only non-B subtypes but also many CRFs. The COBAS Taqman version 1 test measures all subtypes of group M and N viruses and many CRFs [24], while the recently approved version 2 test has improved quantification of CRFs and of Group O virus. The Abbott RealTime test quantifies all group M, N, and O viruses, and CRFs [25, 26]. Ongoing international surveillance of HIV-1 isolates ensures that these assays maintain their ability to detect evolving viral genetic diversity, which occurs because of the high recombination activity within the HIV-1 subtypes and CRFs [27].
Proper collection and processing of blood samples are essential to ensure accurate assessment of viral load levels; the key is to minimize RNA degradation. For conventional and RT-qPCR assays, EDTA is the preferred anticoagulant for blood collection, and the plasma must be separated from blood cells within 4–6 h of collection, as delays in processing may lead to a falsely decreased viral load value. Plasma specimens can be stored at 4 °C for several days without significant degradation of RNA, and HIV-1 RNA has been shown to remain stable after three cycles of freezing (−70 °C) and thawing [28]. For long term storage, plasma samples should be frozen at or below −70 °C [29]. Vacutainer PPTs may be acceptable for the collection of blood specimens for viral load testing, but this specimen type must be validated by the individual laboratory. The PPTs contain a gel barrier which, after centrifugation, physically separates plasma from the cellular components [30]. Whole blood collected in PPTs can be held at room temperature for as long as 6 h after collection and shipped as plasma (in the original tube) at ambient temperature or on wet or dry ice without affecting the HIV-1 viral load [31]. Freezing PPTs (after separating the plasma) prior to testing can give higher viral load values compared to those obtained when the plasma is separated and stored at 4 °C which is thought to be due to the lysis of cells releasing proviral DNA as well as virions adherent to platelets [32–36]. For this reason freezing specimens in PPTs in situ is not recommended, although Fernandes et al. showed that it could be done when using the Abbott platform, and this is likely due to the additional centrifugation prior to testing [37]. The PPTs provide a closed sample collection system, which is a safe, convenient, and practical approach to shipping specimens collected at sites remote from the laboratory.
Although measuring viral load in plasma is the standard of care in clinical practice, these tests have been adapted for use with other specimens, most notably serum, dried blood spots, cerebrospinal fluid (CSF), seminal fluid or semen, and cervical secretions. When serum specimens are used, the viral load is decreased approximately 50 % compared to plasma [38]. Both whole blood and plasma dried spots can be used for viral load testing; in fact, viral load levels from dried plasma spots are equivalent to those obtained from fresh frozen plasma specimens [39]. Similarly, HIV-1 RNA from dried whole blood spots, corrected for hematocrit (number of spot RNA copies per milliliter of blood)/([100-hematocrit]/100), yields viral load results comparable to those obtained from plasma [40]. HIV-1 RNA in dried plasma spots remains stable for up to 16 days when stored at 4 °C or ambient temperature [41]. RNA from dried blood spots has been shown to be stable up to 1 year at room temperature or cooler [42]. Viral load levels in CSF have been used in the evaluation of patients with AIDS dementia, and if virus is quantified, ART that penetrates into the CSF compartment is chosen for treatment [43].
Drug Resistance Assays
Antiretroviral resistance can be detected using either genotypic or phenotypic assays. HIV-1 genotypic assays identify mutations or changes in the nucleotide sequence known to confer decreased susceptibility to antiretroviral drugs. Phenotypic resistance testing, on the other hand, refers to a viral trait or behavior resulting from the expression of a specific genotype. HIV-1 phenotypic assays measure viral replication in the presence of antiretroviral drugs. Results of phenotypic assays are typically reported as the inhibitory concentration of a drug that reduces in vitro HIV-1 replication by 50 % (IC50). The IC50 is usually reported as the fold change in IC50 relative to a wild-type strain. A “virtual phenotype” is a test where the results of the genotypic assay are entered into a database containing matching genotypic and phenotypic results from thousands of clinical specimens, and the closest matching phenotypic results are averaged and reported as the virtual phenotype. Studies have demonstrated that the virtual phenotype is equivalent to conventional phenotyping for clinical decision making regarding changes in drug therapy. The virtual phenotype, while useful clinically, is not longer available [44, 45].
Two US FDA-cleared genotypic tests are available that include reagents for sequencing and software programs to assist with sequence alignment and interpretation (Trugene HIV-1 Genotyping Kit and OpenGene DNA Sequencing System, Siemens Healthcare Diagnostics, Tarrytown, NY; ViroSeq HIV-1 Genotyping System, Abbott Molecular, Des Plaines, IL) (Table 45.2). Though the Trugene HIV-1 assay has been widely used and much of our understanding of the clinical utility of HIV genotypic testing has come from studies with this test, effective December 2014 Trugene HIV-1 test is no longer available. HIV-1 RNA is extracted followed by reverse transcription and PCR amplification of the entire PR gene and most of the RT gene, which are sequenced using automated dideoxynucleotide terminator cycle sequencing. The PR and RT gene sequences are analyzed by comparison to a reference sequence (wild type HIV-1 strain) to identify mutations. For patients who have been exposed to INSTI, the HIV-1 integrase genotype (HIV-1 Integrase Inhibitor Resistance by Sequencing; GeneSeq® for Integrase Inhibitors Assay) can be performed by reference laboratories, since the typical genotypic tests only detect mutations in the PR and RT genes not the integrase gene where the INSTI resistance mutations reside.
Table 45.2
Available assays for resistance testing
Assay | Method | Comments |
|---|---|---|
Trugene® HIV-1 Genotyping Kit and Open Gene DNA Sequencing System (Siemens Healthcare Diagnostics)* | Genotypic | US FDA-cleared; Detects protease and reverse transcriptase mutations |
Viroseq® HIV-1 Genotyping System (Abbott Molecular) | Genotypic | US FDA-cleared; Detects protease and reverse transcriptase mutations |
GeneSeq® for Reverse Transcriptase and Protease Inhibitors Assay (Monogram Biosciences) | Genotypic | Detects protease and reverse transcriptase mutations |
GenoSure® MG (Monogram Biosciences) | Genotypic | Detects protease and reverse transcriptase mutations |
HIV-1 Genotype, RT and Protease Genes (Quest Diagnostics Nichols Institute) | Genotypic | Detects protease and reverse transcriptase mutations |
GeneSeq® for Integrase Inhibitors Assay (Monogram Biosciences) | Genotypic | Detects integrase mutations |
HIV-1 Integrase Inhibitor Resistance by Sequencing (ARUP, Salt Lake City, UT) | Genotypic | Detects integrase mutations |
PhenoSense®GT (Monogram Biosciences) | Combined genotypic and Phenotypic | Detects protease and reverse transcriptase mutations |
PhenoSense™ HIV (for Reverse Transcriptase and Protease Inhibitors) (Monogram Biosciences) | Phenotypic | Detects protease and reverse transcriptase mutations |
PhenoSense™ for Entry Inhibitor Susceptibility (Monogram Biosciences) | Phenotypic | Measures susceptibility to entry inhibitors (Fuzeon®) |
PhenoSense™ Integrase (Monogram Biosciences) | Phenotypic | Measures susceptibility to integrase inhibitors |
Trofile™ Co-receptor Tropism (Monogram Biosciences) | Tropism | Used prior to initiating therapy with maraviroc |
Phenotypic assays measure the ability of HIV-1 to replicate in the presence of various concentrations of an antiretroviral drug using high-throughput automated assays based on recombinant DNA technology. The single commercially available assay, PhenoSense (Monogram Biosciences, South San Francisco, CA) (Table 45.2) amplifies the PR and RT genes using RT-PCR and the amplified product is inserted into a modified HIV-1 vector which lacks RT and PR genes and has a luciferase reporter gene inserted into the viral envelope gene. Viral replication, in the presence of various drugs, is measured by quantification of luciferase expression [46] and results are reported as fold-change in IC50 compared to a wild-type control. Increases in IC50 of greater than 2.5-fold can be reliably detected by this assay.
Prior to initiating therapy with a CCR5 inhibitor (maraviroc), the patient’s virus must be assessed for use of the CCR5 as a co-receptor, as viruses that use CXCR4 as a co-receptor will not be susceptible to this drug. The commercially available Trofile assay (Monogram Biosciences, South San Francisco, CA) generates pseudoviruses using full-length env genes amplified from the patient’s virus. Co-receptor tropism is then determined by measuring the ability of the pseudoviral population to infect CD4+ U87 cells that express either CXCR4 or CCR5. Depending on which cells are infected, the patient’s virus is then designated X4-tropic, R5-tropic, or dual-tropic [47]. Patients are candidates for a CCR5 inhibitor if their virus is solely CCR5-tropic. Patients with CCR5-tropic virus that are treated with maraviroc may develop resistance to the drug due to either (1) mutations that allow the virus to adapt and use CXCR4 co-receptors or (2) structural changes in the envelope of a R5-tropic virus that prevent the drug from being effective [48, 49].
The currently available range of resistance testing (genotype and phenotype) provides clinicians with tools to better assess how to tailor the ART regimen, as well as determine whether drugs that are considered “resistant” by genotype may be usable in a salvage regimen after analysis by phenotypic testing.
Little data directly address specimen collection and processing for HIV-1 resistance testing which are very sensitive to RNA degradation because the methods require the amplification of a large portion of the 9 kilobase viral genome (1,200–1,600 base pairs). Current recommendations for resistance testing are to follow guidelines established for HIV-1 RNA viral load testing regarding collection, processing, and storage of specimens. Considering the cost of these assays and the variability of viral load measurements near the limit of quantification, resistance testing is not recommended until the viral load in the plasma is >1,000 copies/ml. Both the Trugene and ViroSeq assays successfully genotype non-B subtypes of HIV-1 [50].
Interpretation
Qualitative HIV-1 RNA Assays
CDC guidelines recommend use of HIV-1 RNA testing for the diagnosis of HIV infection when the screening result (by EIA) is not confirmed by the HIV-1/2 discriminatory test. This raises important issues regarding the implementation of the algorithm, as laboratory directors will need to determine whether to use plasma or serum samples, and assess if the same sample used for screening/discriminatory testing should be used for RNA testing. Testing for RNA after the serologic tests raises concerns of cross contamination between samples, as serologic testing is not routinely performed with the same precautions to prevent carryover of RNA between samples, as is needed for molecular testing. An assessment of the specimen integrity, including contamination prevention and storage conditions, following serologic testing is needed to assess the specimen adequacy for RNA testing. Removing an aliquot of plasma/serum prior to serologic testing for RNA testing, would reduce the risk of cross contamination and assure proper specimen storage, but this would be very labor intensive, particularly for laboratories with a low positivity rate, and risk misidentification. Asking for a second specimen to be collected for RNA testing requires an additional visit for the patient and may decrease the likelihood that testing is done, plus increases the turnaround time for final results. Consideration of these issues and discussions with healthcare providers prior to implementation of the testing algorithm for HIV-1 diagnosis should increase the likelihood of a successful adoption of the algorithm.
Viral Load Assays
HIV-1 RNA viral load assays have become the standard of care for monitoring response to ART. In order to effectively use HIV-1 viral load assays in clinical practice, the changes in viral load that represent a clinically important change in viral replication must be defined. This requires knowledge of both viral biology and assay performance. The available HIV-1 viral load assays have an intra-assay variability of 0.12–0.2 log10 on repeated testing of individual samples [38, 51]. Biologically, HIV-1 RNA levels are fairly stable in individuals who are not receiving ART; the biological variation is approximately 0.3 log10 [52]. Therefore, changes in HIV-1 RNA levels must exceed 0.5 log10 (three-fold) to represent biologically relevant changes in viral replication. For all the viral load assays, the intra-assay variability is even greater near the lower limit of quantification, so for HIV-1 RNA values less than 3 log10 (1,000 copies/ml), small changes in viral load should not be overinterpreted. Reporting viral load levels as log10-transformed data may assist in preventing clinicians from over-interpreting small changes in viral load.
Several clinical illnesses, including herpes simplex virus infections, acute infections, and opportunistic infections, as well as vaccinations for influenza, tetanus, or pneumonococcal infection, can lead to transient increases in HIV-1 RNA levels [53–55]. For some individuals these increases in viral load may be quite dramatic, even greater than 1 log10 change; however, HIV-1 RNA levels usually return to baseline within a month of the acute event. For this reason viral load measurements should be avoided during acute illness or within a month of vaccination.
False-positive results can occur with HIV-1 viral load assays and are attributed to carryover contamination with amplicons, limitations in assay chemistry, or cross-contamination of specimens during specimen processing. Carryover contamination has been essentially eliminated with the automated RT-qPCR tests (TaqMan and RealTime) since the test reaction is not open to the air after the amplification step. An advantage of the Versant bDNA assay is that carryover contamination does not occur with this signal amplification method. However, the Versant bDNA assay chemistry involves complex hybridization of nucleic acid probes which can result in nonspecific hybridization leading to false-positive results. The assay has a specificity of approximately 98 % when testing specimens from HIV-1-negative individuals [56]. Most of the false-positive samples have viral load values of less than 2,000 copies/ml. Contamination with HIV-1 RNA during specimen processing can lead to false-positive results with any of the assays, although this risk is reduced with the automated extraction systems used in the RT-qPCR tests.
Drug Resistance Assays
Interpretation of genotypic resistance testing is very complex and requires a detailed understanding of the genetics of resistance. For many drugs, the mutations associated with resistance have been well characterized when used as monotherapy. However, when drugs are used in combination, as is the standard of care for HIV-1-infected individuals, interactions may occur, which may increase or decrease individual drug efficacy. These interactions, although they are complex, must be understood to accurately interpret genotypic results. A current and comprehensive discussion of the specific mutations associated with each antiretroviral drug and the interactions of mutations is available from a variety of sources, including Los Alamos National Laboratory HIV Databases (http://hiv-web.lanl.gov), International AIDS Society-USA (http://www.iasusa.org), and Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu).
The proper interpretation of genotypic drug resistance assays involves (1) identification of resistance mutations and (2) interpretation of how these mutations alter viral susceptibility to specific antiretroviral drugs. While establishing appropriate quality control guidelines for the technical aspect of an assay is common practice, the complex interpretation of HIV-1 genotyping assays represents a challenge for molecular pathologists. Since interpretation of genotypic assays is so complex and critical to patient care, FDA-cleared assays provide software programs that assist in base calling, sequence alignment, and identification of the resistance mutations by comparing the sequence to a wild-type HIV-1 sequence.
After identifying the resistance mutations, a “rules-based” software program is used to interpret the implications of the various mutations for response to different drugs. For example, with the OpenGene system (Siemens Healthcare Diagnostics Inc, Tarrytown, NY), the manufacturer provides regular interpretation software updates which are cleared by the US FDA prior to release. In addition to listing the mutations identified in the RT and PR genes, an interpretive report is provided that lists each drug and provides a designation of either “no evidence of resistance,” “possible resistance,” “resistance,” or “insufficient evidence.” A similar approach is used in the ViroSeq assay (Abbott Molecular), though the interpretation of all mutations may not be identical for both systems. These rules-based interpretation systems are essential for providing clinicians with results in a user friendly format that is easily understood and clinically useful without the need for an extensive knowledge of the genetics of HIV-1 resistance. Clinically, resistance results must be interpreted in the context of the treatment history of each patient, which usually means avoiding drugs previously used to treat the patient when possible.
Mutations in HIV-1 are reported with a specific nomenclature in which amino acids are reported using single letter abbreviations. The wild-type amino acid encoded by the nucleotide triplet is followed by the location of the mutation (codon number) and then the mutant amino acid. For example, K103N indicates that the lysine (wild type) at codon 103 is replaced by an asparagine (mutant). Genotyping reports include a list of the mutations identified as well as the effect of the mutations on antiretroviral drug susceptibility (Table 45.3).
Table 45.3
Example of a genotypic resistance report
Resistance associated RT mutations: A62V, K65R, A98G, L100I, V179D, M184V | |
|---|---|
Nucleoside and nucleotide RT inhibitors | Resistance interpretation |
Abacavir (ABC) | Resistance |
Didanosine (ddI) | Possible resistance |
Zidovudine (AZT) | No evidence of resistance |
Lamivudine (3TC)/emtricitabine (FTC)
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