laboratory of clinical virology assays & services

HIV-1 Genotypic Resistance Testing

Resistance to antiretroviral drugs occurs through specific mutations in the target viral gene and is an important factor which determines treatment outcome in HIV-infected patients. The ARI-UCSF Laboratory of Clinical Virology (LCV) uses the TRUGENE™ Genotyping assay (Bayer HealthCare), which provides the specific mutations that contribute to resistance and the drug susceptibility interpretation for the HIV-1 reverse transcriptase and protease genes. Synonymous mutations, polymorphisms, and unexpected mutations at resistance sites are also determined. The assay is FDA-approved for diagnostic purposes. One ml of EDTA, ACD, or PPT blood plasma is required. In addition, the laboratory performs genotypic resistance testing on HIV RNA from CSF, seminal fluid, vaginal fluids, and cellular DNA.

HIV-1 Viral Load, Plasma, or CSF

For plasma HIV-1 viral load analyses, the LCV performs the AMPLICOR HIV-1 MONITOR version 1.5 assay using the COBAS or manual detection platforms. This assay is based on PCR-based amplification of the target signal in HIV-1 gag and is specific for HIV-1 group M, subtypes A–G. Determinations using the Standard extraction procedure (range 400–750,000 copies/ml, 0.5 ml requirement) and the Ultrasensitive extraction procedure (range 50–100,000 copies/ml, 0.2 ml requirement) use an FDA-approved procedure. The laboratory has also validated a modification which extends the lower limit of detection to 2.5 copies/ml (Ultra-Low procedure), requiring 2.0 ml clarified blood plasma or CSF (Havlir et al., 2001). The assay utilizes both internal quantitation standards for each sample and external standards for each run to determine the nominal value of RNA in the sample. The assays using the Ultrasensitive and Standard extraction procedures are FDA-approved for blood plasma and are specific for group M viruses, subtypes A–G. Blood plasma collected from EDTA, PPT, and ACD collection tubes is compatible with this assay.

HIV-1 Viral Load, Seminal Fluid, Vaginal Fluid

Viral load analyses from seminal fluid and vaginal secretions and washes are particularly challenging using PCR-based assays due to interfering substances, limited sample volume, and potentially low viral concentration. The laboratory has adopted a three-step procedure to purify viral RNA from these sources to yield intact, highly purified RNA with quantitative recovery for accurate PCR-based viral load and sequencing assays. Viral load assays on genital fluids are performed using the AMPLICOR HIV-1 MONITOR version 1.5 assay, with addition of the internal quantitation standard prior to the modified viral RNA extraction procedure to ensure quantitative recovery and to indicate the presence of interfering substances.

High Resolution Sequence-Based HLA Typing

The HLA class I and II cell surface glycoproteins are central in determining the specificity and breadth of the immune response against viruses and pathogens. Although critical for recognizing an essentially unlimited array of antigenic peptides, their degree of genetic polymorphism provides a challenge for HLA typing. Among the molecular typing methods available, high resolution sequence-based typing (SBT) allows the greatest degree of specificity, the most accurate level of allele typing, and assignment of novel alleles. In addition, various PCR amplification strategies followed by nucleotide sequencing allow superior resolution of cis-trans linkage for ambiguous typing results.

HLA class I and class II SBT is performed using the AlleleSEQR Sequencing-Based Typing kits from Atria Genetics (distributed by Abbott Laboratories). The pre-formulated sequencing mixes use dye-terminator cycle sequencing chemistry and provide complete sequence of the most polymorphic regions in both directions for accurate assessment of heterozygotes, deletions, and insertions. For class I typing, locus-specific amplicons are generated and followed by sequencing of exons 2, 3, and 4 (for HLA-A and -B) or exons 2 and 3 (for HLA-C). To address common ambiguous typing results when cis-linkages of heterozygotes at the HLA-B locus cannot be resolved using locus-specific primers, group-specific amplification primers will be used (AlleleSEQR-B GSA kits). For class II typing (DR, DQ, or DP), the beta chain exon 2 is amplified and sequenced in tandem with additional sequencing primers to resolve common potential cis-trans ambiguities. All sequencing reactions will be analyzed using a 16-capillary array on a 3100 Automated Sequence Analyzer (Applied Biosystems, Foster City, CA).

Allele assignment is a critical component of sequence-based typing. Assign SBT™ (Conexio Genomics, Perth, Australia) will be used for sequence analysis, editing, alignment, and allele assignment. This software package contains state-of-the-art algorithms for base calling and scoring, allows analysis of multiple loci in a single project, enables resolution of heterozygous ambiguities, and includes detection of insertions and deletions. Assign SBT™ is linked to the IMGT/HAL database sequence library with regular updates to ensure inclusion of novel alleles.

Miscellaneous Sequencing

Please contact the Research Director for inquiries on viral and host gene sequencing.

Qualitative PBMC Macroculture

This culture method is intended to produce high titer HIV-1 stocks from infected peripheral blood mononuclear cells (PBMC) from viremic individuals. A co-culture of patient PBMC and uninfected PHA-stimulated PBMC is maintained under ideal conditions in vitro, allowing viral replication. Most PBMC co-cultures will yield detectable HIV-1 gag p24 antigen. Success rates will vary with patient treatment status, viral load, condition of input patient PBMC (freshly prepared or frozen), and, potentially, host genetic factors of the HIV-1–negative host donor cells. Cultures are monitored every 3-4 days for viral growth by HIV-1 gag p24 antigen detection.

Qualitative PBMC Ultrasensitive Macroculture

This culture method is intended to produce HIV-1 stocks from infected peripheral blood mononuclear cells (PBMC) from individuals with undetectable (50 copies/ml) or low plasma viral load. A co-culture of patient PBMC and uninfected PHA-stimulated PBMC is maintained under ideal conditions in vitro, allowing viral replication. PBMC from the HIV-positive donor undergo CD8 T cell depletion by magnetic bead separation and are stimulated by immobilized anti-CD3, anti-CD28. PBMC from HIV-negative donors are similarly depleted of CD8T cells before activating with PHA. Activated patient and negative donor cells are co-cultured in flasks coated with anti-CD3 antibodies for continued stimulation. Fresh CD8-depleted PHA blasts are added weekly to propagate emerging virus. Success rates will vary with patient treatment status, viral load, condition of input patient PBMC (freshly prepared or frozen), and, potentially, host genetic factors of the HIV-1–negative host donor cells. Cultures are monitored every 3-4 days for viral growth by HIV-1 gag p24 antigen detection.

Quantitative PBMC Ultrasensitive Culture

This culture method is intended to determine the titer of replication competent cells from an individual with low or undetectable (<50 copies/ml) plasma viremia. Viral titers are expressed as infectious units per million (IUPM) cells. Multiple co-cultures of serially diluted HIV-1–infected PBMC with a constant number of uninfected cells from pooled HIV-1–negative donors are maintained under ideal conditions in vitro, allowing viral replication. PBMC from the HIV-positive donor undergo CD8 T cell depletion by magnetic bead separation and are stimulated by immobilized anti-CD3, anti-CD28. PBMC from HIV-negative donors are similarly depleted of CD8T cells before activating with PHA. Activated patient and negative donor cells are co-cultured in multiple flasks coated with anti-CD3 antibodies for continued stimulation. Fresh CD8-depleted PHA blasts are added weekly to propagate emerging virus. Success rates will vary with patient treatment status, viral load, condition of input patient PBMC (freshly prepared or frozen), and, potentially, host genetic factors of the HIV-1–negative host donor cells. Cultures are monitored every 3-4 days for viral growth by HIV-1 gag p24 antigen detection.

Batch HIV-1 p24 ELISA

The laboratory uses the NEN HIV-1 p24 ELISA (Enzyme-Linked Immunoabsorbant Assay) for detecting and quantifying virus production in vitro. The NEN HIV-1 p24 ELISA utilizes a mouse monoclonal specific for HIV-1 p24 antigen which is coated on microtiter plates. Neutralized tissue culture supernatant is added to the coated well, and nonspecific antibody binding sites are blocked to reduce background signal. P24 molecules bind to the immobilized antibody and are detected following adequate washing by adding biotinylated human polyclonal antibody. The wells are further washed, and the polyclonal antibody is detected by adding streptavidin-horseradish peroxidase conjugate. In the final step, the substrate OPD is added, turning the positive wells yellow. The absorbance is measured spectrophotometrically, and the intensity of the signal is directly proportional to the concentration of immobilized antigen. The concentration is directly measured against a p24 antigen standard curve.

PBMC Processing

Peripheral Blood Mononuclear Cells are separated from neutrophils, granulocytes, and red blood cells in whole blood by density gradient centrifugation using Ficoll-hypaque (density 1.077). Following separation and extensive washing, cells are counted and aliquoted as desired in cryopreservation media (for viable recovery) or as dry pellets (for nucleic acid extraction).

PBMC Processing and 2nd Type Aliquoting

This processing charge applies to an additional mode of aliquoting and storage (for example, processing and storage as viable cryopreservation in DMSO and as dry PCR pellets).