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III. Monitoring Tests
Once a patient has been diagnosed as being HIV-infected, several tests are required to evaluate, stage, and monitor the clinical progression of disease.
For HIV/AIDS staging considerations, please note that the New York State Department of Health AIDS Institute follows the Centers for Disease Control and Prevention (CDC) definition of HIV/AIDS and list of AIDS-defining conditions.
A. Viral Load Assays
Clinicians should repeat viral load tests that are inconsistent with the clinical presentation before management decisions are made. (AIII)
The most important goal of ART is to durably suppress viral load to below the limits of quantitation of an assay that measures <20 (or <50) copies/mL.
Several different HIV viral load tests have been developed, and five are currently approved for use in the United States:
- Amplicor HIV-1 Monitor test (Roche), also known as the PCR test
- Versant/Quantiplex HIV-1 RNA, or bDNA (Chiron/Siemens)
- NucliSens HIV-1 QT, or NASBA (bioMérieux)
- Abbott RealTime HIV-1
- COBAS AmpliPrep/COBAS TaqMan HIV-1 Test
Viral load assays quantify the amount of HIV-1 RNA circulating in the blood of an infected individual. Total quantification includes cell-free virus, virus in infected cells in all compartments of the body, and integrated provirus. However, the easiest measurement of viral load is that of cell-free virus in an individual's plasma. Because differences exist in the absolute copy number generated by different viral load assays, the same assay should be used to follow an individual's viral load. For a specific assay, the biologic variability of viral load is 2-fold.
Viral load tests are also approved for monitoring ART by tracking viral suppression, and to detecting treatment failure. Successful ART should decrease viral load 1.5 to 2 logs (30- to 100-fold) within 6 weeks, with the viral load decreasing below the limit of detection within 4 to 6 months.7
Newer assays have a greater dynamic range than either the standard or ultrasensitive assay (e.g., 40 to 10,000,000 copies/mL). Standard assays have a lower limit of detection of 400 copies/mL, whereas ultrasensitive assays may detect viral loads as low as 5 to 50 copies/mL. Cohort studies strongly suggest that patients with viral loads <50 copies/mL have more sustained viral suppression than patients with viral loads between 50 and 400 copies/mL. Assays that can detect <50 copies/mL are therefore more useful than standard viral load tests in predicting prolonged viral suppression and are recommended for monitoring patients who are receiving ART.
Brief viral rebounds, known as "blips," can result in viral load levels of 50 to 500 copies/mL in patients with previously undetectable viral loads (<50 copies/mL). Acute concurrent illness and/or recent vaccination may cause this transient rise; however, studies have suggested that low-level transient viremia represents random biologic and statistical variation or false elevations of viral load resulting from laboratory processing.8,9 "Blips" are not often associated with the development of resistance mutations or virologic failure and do not usually require a change in ART.9 Re-testing should be performed within 4 weeks to differentiate low-level transient viremia from virologic failure. ART should not be changed based on a single viral load elevation. For more information, refer to Antiretroviral Therapy: Section VI. A. Virologic and Immunologic Monitoring.
1. Roche Amplicor HIV-1 Monitor and Roche Amplicor HIV-1 Monitor Ultrasensitive (RT-PCR)
The Roche Amplicor HIV-1 Monitor Version 1.5 (see Appendix E) and Roche Amplicor HIV-1 Monitor Ultrasensitive Version 1.0 (RT-PCR) are approved by the FDA for the quantitation of HIV-1 RNA in plasma and are reported as copies/mL. This procedure is similar to the DNA PCR assay. For patients with good viral suppression, the ultrasensitive test is the preferred method for quantifying plasma HIV-1 RNA. HIV-1 RNA is isolated from the plasma; then a complementary strand of DNA (cDNA) is transcribed from the target RNA using RT. The cDNA is amplified using very specific oligonucleotide primers. Quantification of the RNA is achieved by hybridizing the amplified DNA to specific probes, followed by a colorimetric detection assay (see Appendix A for further description of this procedure).
2. Branched Chain DNA (bDNA)
The Versant HIV-1 RNA 3.0 assay is approved by the FDA for quantitation of HIV-1 RNA in plasma, and results are reported as units/mL. It uses the bDNA technology to measure viral load (see Appendix F). The bDNA assay consists of a series of hybridization procedures followed by an enzyme substrate reaction (see Appendix A for further description). In this assay, HIV-1 present in the patient's blood is disrupted to release the viral RNA. The RNA is captured by a set of capture probes (bound by solid phase), and a set of target probes hybridizes both the viral RNA and the preamplifier probes. The amplifier probe hybridizes to the pre-amplifier probe, forming a branched DNA (bDNA) complex. The bound bDNA is incubated with an enzyme and then with a chemiluminescence substrate.
3. Nucleic Acid Sequence-Based Assay (NASBA)
The NucliSens HIV-1 QT assay (bioMérieux) is a nucleic acid sequence-based assay that has been approved by the FDA for the quantitation of HIV-1 RNA in plasma. Results are reported as units/mL. In this viral load test, the HIV-1 is lysed and HIV-1 RNA is extracted and bound to silica beads (see Appendix G). Nucleic acid amplification then occurs using specific primers derived from the gag region of the genome. The amplified RNA is hybridized to capture probes attached to magnetic beads, and the nucleic acid is detected by measuring electrochemiluminescence. The isolation technique used in this assay allows diverse sample types (plasma, cerebrospinal fluid, lymph tissue, genital secretions, and cells) to be used as the source of viral nucleic acid; however, the FDA-approved assay, NucliSens HIV-1 QT, has only been validated for use with plasma.
4. Other Viral Load Tests
An inexpensive HIV viral load assay has been developed to measure viral RT activity (ExaVir Load Version 2; Cavidi AB, Uppsala, Sweden). The test is performed mostly manually and was designed primarily for resource-limited settings. The assay has a lower limit of detection of 400 copies/mL. It is not approved by the FDA for clinical use in the United States. Another investigational viral load assay is the real-time immuno-polymerase chain reaction (IPCR), which combines ELISA and PCR methods for quantification of HIV-1 p24 antigen detection.10
B. Lymphocyte Analysis
Clinicians should measure CD4 cell counts at the time of diagnosis of HIV infection and at differing follow-up intervals based on the individual patient's level and length of viral suppression if receiving ART and based on absolute CD4 level for those not receiving ART (see Antiretroviral Therapy: Section VI. A. Virologic and Immunologic Monitoring. (BIII)
Treatment decisions should not be made solely on the basis of a single CD4 cell measurement obtained at a single point in time. Treatment decisions should be made only after two successive measurements have been obtained. (AIII)
CD4 cell counts should not be used for diagnosis of HIV infection.
Historically, CD4 cell count has been used to establish a threshold for initiating ART. However, current guidelines in New York State recommend that all HIV-infected patients be evaluated for ART, regardless of CD4 cell count. For patients who may not be ready to initiate ART, CD4 cell count can be used to guide discussions between patient and provider regarding the urgency of initiating ART.
Low CD4 cell counts are seen in a number of disease processes and should therefore not be used for diagnosis of HIV. However, the CD4 cell count is important for determining the staging of HIV disease and the need for prophylaxis against opportunistic pathogens (see Primary Care Approach to the HIV-Infected Patient: Section VI. B. Opportunistic Infection Prophylaxis). CD4 cell counts <200 cells/mm3 (or <14% of total lymphocytes) meet the national surveillance case definition for AIDS.
Clinicians are unlikely to use CD4 counts to guide clinical decision-making in practice for virologically suppressed patients once their CD4 count remains above 200 cells/mm3. However, for persons infected with HIV-2 or HIV-1 variants that cannot be accurately quantitated using viral load assays, CD4 count remains the most effective monitoring tool for progression of disease. See Human Immunodeficiency Virus Type-2.
Absolute CD4 cell counts are calculated values that may fluctuate widely. The calculation is made by multiplying the total white blood cell count (in thousands) by the percentage of total lymphocytes and then by the percentage of CD4 lymphocytes. Therefore, any change in one of these three parameters will cause the absolute CD4 count to vary. As a result, CD4 percentage is a direct measurement and more reliable. Fluctuations in the absolute CD4 cell count in the setting of a stable CD4 percentage can assure both the patient and the clinician that immunologic stability is present.
Factors influencing lymphocyte subsets include sex, age, race, drugs (zidovudine, cephalosporins, cancer chemotherapy, nicotine, and corticosteroids), anti-lymphocyte antibodies, and splenectomy. Differences in reagents and equipment both within a laboratory and between laboratories may further contribute to variations in CD4 cell counts.
C. Drug Resistance Tests
Clinicians should perform resistance testing under the following circumstances:
- At baseline, regardless of whether ART is being initiated (genotypic testing) (AIII)
- In ART-naïve patients before initiation of ART (genotypic testing) (AII)
- In patients experiencing treatment failure or incomplete viral suppression while receiving ART (genotypic and/or phenotypic testing) (AII)
Resistance testing should be performed promptly in cases of virologic failure or incomplete viral suppression. Resistance testing should be performed while patients are still receiving therapy or have been off therapy for no more than 1 year. (AII)
Clinicians should consult with an expert to interpret the results of resistance assays because such results are often complex (the New York State AIDS Institute's Clinical Education Initiative line is available for phone consultation). (AIII)
The replication mechanism of HIV makes it prone to mutations (i.e., changes in its genetic sequence). Most currently available antiretroviral drugs are targeted to inhibit the activity of two specific viral proteins, the protease and RT. Many mutations have been identified in these proteins that alter the ability of one or more antiretroviral drugs to inhibit the viral protein, making the virus resistant to the drug(s). Antiretroviral drugs that inhibit fusion and viral entry and integration are now available; however, resistance mutations for these agents have also been identified.
In numerous studies, the emergence of drug resistance mutations has been associated with virologic failure during ART. Two general methodologies are used to determine drug resistance for HIV: genotyping and phenotyping. The clinical benefits of using resistance tests to guide treatment decisions for patients at various stages of infection and treatment are discussed in Antiretroviral Therapy: Section VI. Monitoring of Patients Receiving ART. In New York State, third-party reimbursement programs, including Medicaid, the New York State AIDS Drug Assistance Program (ADAP), and private insurers, often limit resistance testing to no more than three tests per year (within 12 months following date of first use), regardless of the type of resistance test that is performed.
New resistance mutations and the emerging clinical significance of these mutations frequently change. Several resources are available for more information on drug resistance and resistance testing. These include:
A genotypic assay provides an indirect measure of drug resistance because it is based on detection of the mutations known to be associated with resistance. Genotypic testing involves determining the sequence of the genomic region where resistance mutations occur, typically the coding region for the protein inhibited by the drug. This is best achieved by direct sequencing methods. Two direct sequencing-based methods have been approved by the FDA: the TRUGENE HIV-1 Genotyping assay (Siemens) and the ViroSeq HIV-1 Genotyping System (Celera Diagnostics). In addition, laboratory-developed ("in-house") genotyping assays are available through several commercial laboratories. Advances in genotyping assays continue to evolve. Testing for resistance to integrase strand transfer inhibitors and fusion inhibitors is now available and should be considered when resistance to these classes of drugs is a concern, such as when transmission of resistant virus is suspected or when a patient fails a regimen that includes one of these drugs.
In genotyping assays, the HIV-1 RNA is isolated from a plasma specimen and reverse transcribed to produce cDNA. Specific regions of the HIV genome are amplified by PCR and sequenced. This sequence is then compared with that of a drug-sensitive (wild-type) strain of HIV, and differences (mutations) present in the specimen sequence are noted. Computer software is generally used to perform this comparison and to predict whether resistance to specific drugs is likely to result from the particular combination of mutations detected in the virus. For most genotypic assays, this prediction is based on a set of rules derived from clinical observations, laboratory studies, and the advice of experts in the field. The actual prediction of resistance may vary from laboratory to laboratory for some combinations of mutations, depending on the interpretation algorithm used to define the rules. Currently available genotypic assays require a minimum viral load in the range of 500 to 2000 copies/mL, depending on the assay, and generally require 2 weeks or less for results.
One method of genotypic testing, the "virtual phenotype type" (VIRCO, vircoTYPE), uses a variation of the standard rules-based method of interpreting genotypic test results. The patient's mutation profile is analyzed using a comprehensive database consisting of correlated genotypic (sequence) and phenotypic (drug susceptibility) data. A patient's genotype is entered into the database, and viruses with similar genotypes to those of the patient's virus are identified. The drug susceptibility results (IC50 and fold change, see Section 2: Phenotyping) of these matching viruses are used to calculate the probable degree of drug susceptibility of the patient's virus. The report provides the mutations detected by sequencing, the predictive phenotype results, and the number of matches on which the prediction was based. A minimum number of matches to the patient's virus must be present in the database in order to obtain an interpretation. The advantages of this type of virtual phenotypic testing are that the results are available more rapidly and the interpretation is similar to that of a conventional phenotypic assay. However, a disadvantage is that the actual viral phenotype may be different from the result because of limitations of the database.
A phenotypic assay provides a direct measure of drug resistance. The currently available phenotypic assays use recombinant DNA methods to measure the ability of a patient's virus to grow in the presence of a drug. Therefore, results from a phenotypic test include the net effect of any and all resistance mutations.
In the phenotypic assay, HIV RNA is isolated from plasma and converted into cDNA, and the relevant region is amplified by PCR. This amplified material is inserted into a recombinant virus system whereby the susceptibility to different drugs can be tested. The result from the phenotypic assay is an IC50 value that defines the concentration of the drug required to reduce growth of the virus by 50%. The IC50 of the patient's virus is compared to the IC50 of a drug-sensitive (wild-type) reference virus, and the fold change is defined. If the IC50 value of a person's virus is greater than that of the reference virus for a particular drug, it indicates that the person's virus has decreased sensitivity to the drug. The relative fold change helps determine whether the drug should still be part of the therapy regimen or whether it should be removed entirely. Two companies, Tibotec-Virco (Antivirogram) and Monogram-Biosciences (PhenoSense), offer phenotypic resistance testing through many clinical laboratories. Phenotypic assays have a minimum viral load requirement of 500 to 1000 copies/mL and generally require 3 to 5 weeks for results. Phenotypic assays are more technically complex, labor-intensive, and expensive than genotypic assays; however, they may provide a more accurate indication of drug susceptibility, particularly when a patient's virus presents a complex mutation profile.
3. Co-Receptor Tropism Assay
Co-receptor tropism analysis determines the type of cellular co-receptor (either CCR5 or CXCR4) that an HIV-infected individual's dominant viral population uses to gain access to host cells. The majority of acutely or recently infected individuals, including perinatally infected children, have a CCR5-utilizing virus. The drugs that target the CCR5 co-receptor, such as maraviroc, will likely be effective in these patients (see New Antiretroviral Drugs).
Because the CCR5-tropic virus predominates early in HIV infection, whereas CXCR4-tropic virus is often present in late-stage disease, the CCR5 variant may be preferentially transmitted compared to CXCR4 variants. In chronically HIV-infected individuals, a population of mixed CCR5- and CXCR4-tropic viruses, as well as viruses with both tropisms, is also commonly encountered. Such viral populations are often referred to as dual/mixed or D/M HIV.
In the United States, co-receptor tropism testing is most often performed using phenotypic testing. However, the low cost of genotypic assays, which predict tropism with a variety of available algorithms that analyze V3 sequencing,11,12 may increase their availability in the future.
Although phenotypic testing can determine a viral population containing both tropisms, it is not sufficiently sensitive to differentiate between mixed and dual tropism. The Trofile (Monogram Biosciences) co-receptor tropism assay permits phenotypic identification of CCR5, CXCR4 co-receptor, or dual/mixed-tropic (CXCR4/CCR5-utilizing) HIV-1 before the initiation of co-receptor antagonists.
Another commercially available recombinant phenotypic assay for assessing HIV chemokine co-receptor tropism is the Phenoscript assay (Eurofins VIRalliance). In this assay, a 900-bp portion containing the patient's V1-V3 envelope virus is amplified and inserted into a HIV-1 vector lacking the corresponding V1-V3 section. The fully complemented HIV-1 is then able to produce virus that can be used to infect cell lines with either CCR5 or CXCR4 on their surfaces with a colorimetric readout. The results are reported in a similar manner as the Trofile (i.e., CCR5-trophic, CXCR4-trophic, or dual/mixed tropic). This assay has not been validated in a clinical trial setting or against the Trofile assay.
A third assay, the HIV-1 Coreceptor Tropism (Quest Diagnostics), is a DNA-based method that uses population sequencing of the HIV envelope V3 loop combined with heteroduplex tracking to detect the presence of CXCR4-tropic HIV-1.13
Resistance to the new class of CCR5 co-receptor antagonists develops by two unrelated mechanisms. First, the patient's viral population shifts its co-receptor usage (i.e., uses CXCR4 exclusively or uses both CCR5 and CXCR4 receptors to gain entry into the cell). The current assays are not sufficiently sensitive to discriminate between mixed- or dual-tropic populations. The second method by which resistance to a CCR5 receptor antagonist may develop is by the virus mutating and binding to the CCR5 receptor with the drug antagonist still in place. This second method can be discerned by a flattening of the IC90 curves in a phenotypic assay or potentially by genotypic analysis. Analysis by phenotypic assay is the preferred method for this purpose because genotypic data are more complex.
4. Other Drug Resistance Tests
Replicative capacity information is often provided as an adjunct to phenotypic or combination genotypic-phenotypic resistance assays. Currently, the test is configured by inserting patient-derived RT and protease sequences into a modified retroviral vector containing a luciferase indicator gene that allows quantification of viral replication. After normalizing the output of the assay on the basis of the viral inoculum, the ability of the vector to replicate is measured in the absence of an ARV drug. The relative replicative capacity of the virus from the source patient is calculated as the ratio of the luciferase activity from vectors containing patient-derived sequences to the luciferase activity from vectors containing wild-type sequences. A ratio of less than 1 reflects a reduced replicative capacity as compared with that of the wild-type control. The full clinical value of this adjunctive information remains under investigation.
D. Human Leukocyte Antigen Testing
Clinicians should perform HLA-B*5701 testing before initiating abacavir-based therapy. (AI)
Individuals with human leukocyte antigen (HLA)-B*5701, HLA-DR7, and HLA-DQ3 have an ostensible genetic predisposition to development of abacavir hypersensitivity. HLA-B*5701 testing is the most thoroughly documented and may be useful for addressing concerns about treatment with abacavir.14,15 See Antiretroviral Therapy. Unlike virus-specific tests (HIV genotype, phenotype, co-receptor tropism assays), HLA genotyping is necessary only once during an individual's lifetime, because it will not change over time.
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