Antiviral drug resistance testing:
See Human Immunodeficiency Virus Type 1 (HIV-1) Genotype (Molecular Assay) in Chapter
17
, Infectious Disease Assays.
Numerous studies have demonstrated improved patient outcome when therapy is guided by results of antiviral resistance testing, especially for initial treatment in areas with a high circulation of resistant viruses and for patients who fail a therapeutic regimen. HIV-1 reverse transcriptase, protease inhibitors, and substrate analogs are the most common types of antiviral agents used for treatment of HIV-1 infection and are most commonly reported in antiviral drug resistance testing. Development of drugs targeting other essential steps in HIV-1 infection, like fusion and integrase functions, as well as relevant resistance testing, is ongoing.
Resistance to antiviral agents can be determined by phenotypic or genotypic methods. Both methods require the amplification of informative sequences from HIV-1 RNA isolated from the patient plasma.
In genotypic assays, mutations in genes whose products are the targets of specific antiviral agents are detected. Most commonly, these mutations are detected by sequencing amplicons. Resistance interpretations for specific drugs or drug classes are maintained in frequently updated databases.
For phenotypic assays, the target sequences of a “reagent” HIV-1 virus are replaced by the genes amplified from HIV-1 RNA from the patient’s plasma. The recombinant virus is used to infect cell cultures in the presence of different antiviral agents, with results interpreted on the basis of the drug’s ability, or not, to prevent infection of the cell line. An advantage of phenotypic assays is that they do not depend on knowledge of specific mutations for interpretation of drug efficacy and they are efficient in detecting how multiple mutations in the patient’s HIV-1 RNA interact in terms of inhibiting or enhancing the activity of an antiviral agent.
Both methods are limited in their ability to yield results when the patient’s viral load is low (<1,000 copies/mL), primarily due to technical limitations of the laboratory processing. Resistant “quasispecies” may emerge due to selective pressure during antiviral therapy. Neither genotypic nor phenotypic assays are efficient in detecting relevant resistance in these quasispecies until they contribute more than approximately 30% of the HIV-1 RNA in the patient’s plasma.
Diagnostic Challenges
HIV viral load tests have a low rate of false-positive results, virtually all with levels <10,000 copies/mL. If viral load testing is performed in patients who are negative by HIV antibody testing (e.g., to evaluate possible infection during the “window phase” or for patients with positive HIV antigen in a fourth-generation screening test, but negative WB), positive results must be confirmed by subsequent antibody testing before an unequivocal diagnosis is established.
Placental transfer of HIV-1 IgG from an infected mother to her fetus complicates the diagnosis of HIV infection in the infant after delivery. In infants at risk for HIV-1 infection, viral culture or molecular diagnostic tests are recommended for diagnosis. Sequential testing at 48 hours after birth, at age 1–2 months, and at 3–6 months has been recommended. Testing for plasma HIV-1 RNA is reported to provide the greatest sensitivity for HIV-1 infection in the neonate. Positive results must be confirmed with subsequent testing. Detection of p24 antigen may be an alternative to HIV culture or molecular diagnostic testing, especially in areas where this testing is not immediately available, although this test is less sensitive and less specific compared to the other virologic assays. An ultrasensitive method for p24 antigen detection using dried blood spots has been described (see Knuchel et al. in Suggested Readings). Infants with negative virologic studies should be assessed serologically. Two negative HIV-1 serology tests, performed at least 1 month apart after 6 months of age, essentially excludes a diagnosis of HIV-1 infection in the infant. Specific testing may be needed for diagnosis of HIV-2 and non–M HIV-1 infections.
Other Considerations
Greater severity and persistence of symptoms during primary infection and severe depression of CD4
+
T lymphocytes beyond 2–3 months are associated more rapid progression to severe immunosuppression and AIDS.
The HIV-1 viral load at baseline is the best predictor of severity and progression early in disease; the CD4
+
T-lymphocyte count is the best predictor of progression in late disease.
