Analysis of HIV-1 reverse transcriptase and protease sequences in paired plasma and lymphoid tissue specimens from HIV-1 infected individuals.

OBJECTIVES: To determine how representative the genotype of HIV-1 circulating in plasma is of the genotype of the virus present in lymphoid tissue. METHODS: Paired plasma and tonsillar tissue samples were prospectively obtained from patients with various levels of plasma HIV-1 RNA who were receiving combination antiretroviral therapy. HIV-1 reverse transcriptase and protease sequences were amplified from plasma and lymphoid tissue specimens by nested polymerase chain reaction and analyzed using an automated sequencing system. Results were compared with consensus HIV-1 sequences to determine whether drug-resistance mutations were present in the regions analyzed. RESULTS: HIV-1 protease sequences were compared in 11 plasma/tissue pairs obtained from eight patients; HIV reverse transcriptase sequences were compared in 12 plasma/tissue pairs obtained from nine patients. Sequence homology between plasma and tissue RNA, tissue RNA and DNA, and plasma and tissue DNA ranged from 97% to 100%. Few discrepancies were found when the percentage of mutant sequences at resistance codons was compared among paired samples. In most instances, tissue RNA or plasma contained a higher percentage of mutant sequences than did tissue DNA. CONCLUSION: The genotype of plasma HIV-1 is similar to the genotype of the virus in lymphoid tissue. Resistance studies using plasma samples should provide accurate information regarding the genotype of HIV-1 in lymphoid tissues.

Quantitative image analysis of HIV-1 infection in lymphoid tissue.

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Outpatient biopsies of the palatine tonsil: access to lymphoid tissue for assessment of human immunodeficiency virus RNA titers.

OBJECTIVES: Our objective was to assess the feasibility of using tonsillar lymphoid biopsy specimens obtained on an outpatient basis to quantitate a patient's lymphoid human immunodeficiency virus (HIV) RNA titers. DESIGN: A pilot cohort study was performed. PATIENTS: We evaluated ten HIV-seropositive patients who ranged in age from 26 to 48 years and had CD4+ cell counts ranging from 110 to 833 at enrollment. MAIN OUTCOME MEASURES: The main outcome measures were tolerance and safety of outpatient tonsil biopsies and quantitation of HIV RNA titers in tonsillar lymphoid biopsy specimens, plasma, and peripheral blood mononuclear cells determined by a new method of HIV RNA signal amplification with branched DNA probes. RESULTS: Outpatient tonsil biopsies were well tolerated and were performed without complications. Nine of 10 tonsil biopsies from the HIV-seropositive patients examined were positive for significant concentrations of HIV RNA, ranging from 106 to 101 HIV RNA equivalents per gram of tissue. All of the HIV RNA-positive tonsillar lymphoid specimens had HIV RNA titers that were 101 to 104 times greater than those recovered from plasma (per milliliter) of the same patient obtained at the time of biopsy. CONCLUSIONS: Sufficient tonsillar tissue can be obtained in an outpatient clinic setting to quantitate lymphoid HIV titers by the new branched-DNA signal amplification method with relative ease and without complication. The biopsy method described here affords ready access to the lymphoreticular system, which may help to advance our understanding of the pathogenesis of myriad immune diseases without the need for excisional node biopsies.

Directions in HIV/AIDS research. A Minnesota perspective.

Clinical studies involving HIV therapeutics are becoming ever more challenging because of the changing epidemiology of HIV infection, the rapid evolution of standard clinical practice, and a more difficult economic environment for research. Recent studies relating to HIV pathogenesis have provided novel insights into the interactions between HIV, the immune system, and antiretroviral therapies. Current dogma now suggests that the interaction of HIV and the immune system is an incredibly dynamic process. HIV is an intimidating pathogen capable of replicating quickly and mutating very rapidly in response to antiretroviral therapy. An important question being studied by Minnesota-based investigators is how well plasma analysis reflects HIV-immune system interactions in the lymphoid tissue where most virus and CD4+ (T-helper) cells are located. Most of the HIV clinical studies in Minnesota are conducted under the auspices of the AIDS Clinical Trials Group (ACTG) or the AIDS Research Consortium of the Twin Cities (ARCTiC). Much expertise is available in Minnesota addressing a wide range of topics, from epidemiology, to basic virology, to prevention. To optimize patient access to clinical studies, we recommend that physicians discuss available clinical studies with an HIV investigator soon after first seeing a new HIV-infected patient.