A number of factors, especially the prospect of antiviral chemotherapy, have made the need for rapid, sensitive, specific and practical assays for microbial diagnosis increasingly apparent. Progress has been limited in developing rapid viral diagnostic methods largely because of the limited amount of viral analyte molecules (antigen and nucleic acid) in clinical material. The conceptual breakthrough of amplifying an analyte molecule with the polymerase chain reaction has provided new promise for the development of needed assays. The principal investigator and his colleagues have been investigating gene amplification methods to detect viral nucleic acid in clinical material utilizing HIV- I as the prototype agent. Most recently they have been able to detect the sequential appearance of the 4 nucleotide mutations in the reverse transcriptase gene that account for AZT resistance in serial lymphocyte samples from patients receiving AZT therapy. An alternative method for sequence amplification has been developed that in an isothermal (37 degrees C), single cycle reaction that can generate 10(6) copies in 45 minutes. A bead based sandwich capture assay has also been developed that permits reproducible detection and quantitation of the products of this new amplification scheme. This assay is also amenable to nonisotopic detection. A device and method for the simultaneous assay for multiple agents in a single specimen has also been developed. In this proposal methods will be optimized and then applied to four sets of target organisms:herpesviruses, respiratory viruses, genital pathogens and ophthalmologic pathogens. The principal investigator has extensive experience in the clinical diagnosis of antibodies, antigens, and nucleic acids. Large inventories of well characterized clinical specimens for each of the relevant agents are available for these investigations. A systematic, sequential approach will be pursued to fulfill the following aims:
AIM 1 : Identify nucleic acid sequences from target organisms that will provide organism specificity while maintaining broad reactivity with all strains of that organism.
AIM 2 : Apply the specific amplification and detection methods to clinical materials known to contain the target agent (and controls) to verify sensitivity, specificity, reproduceability and when possible quantitation.
AIM 3 : Once assays for individual agents in a panel for the sets of target organisms have been developed, apply the plastic chamber or derivative technologies to develop a rapid diagnostic method for a clinical syndrome (respiratory infection, genital lesions, keratoconjunctivitis).
|Richman, D D (1996) Antiretroviral drug resistance: mechanisms, pathogenesis, clinical significance. Adv Exp Med Biol 394:383-95|
|Kozal, M J; Shah, N; Shen, N et al. (1996) Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays. Nat Med 2:753-9|
|Havlir, D V; Eastman, S; Gamst, A et al. (1996) Nevirapine-resistant human immunodeficiency virus: kinetics of replication and estimated prevalence in untreated patients. J Virol 70:7894-9|
|Pandori, M W; Fitch, N J; Craig, H M et al. (1996) Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein. J Virol 70:4283-90|
|Havlir, D; McLaughlin, M M; Richman, D D (1995) A pilot study to evaluate the development of resistance to nevirapine in asymptomatic human immunodeficiency virus-infected patients with CD4 cell counts of > 500/mm3: AIDS Clinical Trials Group Protocol 208. J Infect Dis 172:1379-83|
|Corbeil, J; Richman, D D (1995) Productive infection and subsequent interaction of CD4-gp120 at the cellular membrane is required for HIV-induced apoptosis of CD4+ T cells. J Gen Virol 76 ( Pt 3):681-90|
|Xie, H; Voronkov, M; Liotta, D C et al. (1995) Phosphatidyl-2',3'-dideoxy-3'-thiacytidine: synthesis and antiviral activity in hepatitis B-and HIV-1-infected cells. Antiviral Res 28:113-20|
|Corbeil, J; Richman, D D (1995) The role of surface CD4 in HIV-induced apoptosis. Adv Exp Med Biol 374:91-9|
|Richman, D D (1995) Drug resistance in relation to pathogenesis. AIDS 9 Suppl A:S49-53|
|Richman, D D; Havlir, D (1995) Early versus delayed treatment of HIV infection. Zidovudine should be given before symptoms develop. Drugs 49 Suppl 1:9-16;discussion 38-40|
Showing the most recent 10 out of 36 publications