Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV.

Activities of daily living capabilities and values of long-term-care facility residents.

OBJECTIVE: The Minimum Data Set for Nursing Home Resident Assessment and Care Screening was used to compare staff-report and self-report of residents' capabilities in eight activities of daily living (ADLs) in one long-term-care facility (LTCF). METHOD: The relative values residents placed on independence in each of the eight ADLs were compared with their self-reported capabilities in those ADLs. Subjects were 30 LTCF residents ranging in age from 45 to 96 years. RESULTS: Residents perceived themselves to be significantly more capable than did staff members for dressing (p < .05), toileting (p < .01). locomotion (p < .05), and personal hygiene (p < .001). For five of the ADLs, residents tended to report high capability in the ALDs they valued most. CONCLUSION: These findings support the need to include resident self-assessment in treatment planning, because staff members' and residents' perceptions of ADL capabilities may differ.

Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction.

We demonstrate the utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity. One of the limitations of the polymerase chain reaction (PCR) is the co-amplification of non-specific products caused by TaqPol activity on low stringency templates present in the initial cycle of PCR. We have used anti-TaqPol antibodies as thermolabile switches that inhibit TaqPol activity at low temperatures (20-40 degrees C) and release fully active TaqPol when they are inactivated by elevated temperatures in the PCR thermal cycling (70-98 degrees C). Several in a set of high affinity anti-TaqPol monoclonal antibodies fully inhibited TaqPol activity at 37 degrees C. The capacity for inhibition was ablated by incubation at temperatures high enough to denature antibodies but not sufficiently high to significantly reduce TaqPol activity. In a PCR model system, preincubation of TaqPol with these antibodies yielded PCR product consisting entirely of the intended product and the absence or significant reduction of non-specific products and primer dimers. In evaluation of clinical samples such antibody triggering yielded defined PCR product and higher sensitivity because of the absence of non-specific products.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction.

An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.