The Infectious Diseases Research Laboratory

The Tulane Infectious Diseases Retrovirology Laboratory

The Infectious Diseases Research Laboratory was established in 1987 as a component of the original Tulane-LSU AIDS Clinical Trials Unit (ACTU). As one of several such university-based research retrovirology laboratories throughout the country, the Laboratory is an active participant in a number of treatment protocols for children and adults with HIV infection and its complications under the auspices of the Division of AIDS in the National Institute of Allergy and Infectious Diseases and the National Institutes of Health, Bethesda, MD. The Laboratory also participates in AIDS treatment protocols sponsored by pharmaceutical companies.

Shanker Japa, Ph.D. supervises two full-time laboratory research technicians and performs the following tasks: review of laboratory safety practices and techniques, review and interpretation of laboratory data, maintainance of equipment and inventory, and training of graduate students and research personnel in the use of technical equipment.

The laboratory has two primary functions:

Specimens to be sent to specialized laboratories elsewhere must be initially processed on-site before shipping. The laboratory is also a repository for plasma and other samples held for later study.

The special expertise of the Laboratory with studies related to HIV is available to all interested scientists and physicians in the State of Louisiana and as such has been utilized on a number of occasions in the past.

Automated DNA Sequencing for HIV Genotypic Drug Resistance

The highly polymorphic human immunodeficiency virus has tremendous potential for mutation in both the relatively conserved reverse transcriptase and protease gene regions, as well as within the hypervariable regions of the envelope gene. Even with rapid evolution of genetic mutations and multiple amino acid substitutions, the reverse transcriptase enzyme maintains its functional integrity. Therefore, drug resistant strains of virus appear to have near-equal survival advantage as wild type virus, and this has resulted in a rapid increase in HIV drug-resistant strains.

The use of direct sequencing of the enzyme-encoding regions of HIV (i.e. protease and polymerase) to assess for drug resistance has become recognized as an important means of designing appropriate treatment for patients. Sequencing has the advantage over drug susceptibility testing for several reasons: (1) in vitro susceptibility testing (phenotypic analysis) requires growth of the virus in high level biosafety facilities, (2) Phenotypic analysis is cumbersome and technically demanding, (3) Inter- and intra-assay variability is a barrier to standardized application of the test, and (4) the costs are prohibitive in many health care settings, particularly in the public health sector.

For these reasons, the Tulane Infectious Diseases laboratory acquired in late 1999, a state-of-the-art system to perform HIV sequencing analysis. The rapid DNA sequencer integrates with an Intel-based computer system to analyze DNA sequences in a matter of 35 minutes. Approximately 400 base pairs corresponding to the enzyme-encoding regions of the virus are sequenced with excellent fidelity using small, precast gels with a 50 micron width. The computer-generated image of the sequence is then automatically base-called, and the sequence data is compared to a customized HIV database which incorporates most known drug-encoding resistance mutations. In an optimized workflow, a single sequencing unit is capable of running at least 8 gels in a regular workday. A computer generated printout of the patient’s drug resistance result is obtained, which also includes unique polymorphisms of the entire 400 base pairs.