Development and validation of a polymerase chain reaction method for the precise quantitation of HIV-1 DNA in blood cells from subjects undergoing a 1-year immunotherapeutic treatment.

OBJECTIVE: To validate a quantitative polymerase chain reaction method developed to measure HIV-1 DNA in peripheral blood mononuclear cells. DESIGN: The assay was used to measure HIV-1 DNA in 15 consecutive blood samples taken from subjects enrolled in a multicenter, randomly allocated, double-blind, placebo-controlled trial using an HIV-1 immunogen. The assay was validated following the United States Pharmacopeia guidelines. The analytical parameters assessed were sensitivity, specificity, linearity and precision. METHODS: The quantitative analysis was obtained by (1) co-amplifying HIV-1 DNA targets with an endogenous control (globin); (2) extrapolating the target values using HIV-1 and globin standard curves; and (3) normalizing the HIV-1 copy numbers to the globin copy numbers (genomic DNA load). RESULTS: With United States Pharmacopeia assay validation methodology, the HIV-1 DNA polymerase chain reaction assay proved to be sensitive, specific, linear and precise and the evaluation of the relative difference between two consecutive blood samples was reproducible. The intra-assay variability, which examines the reproducibility of replicates, was determined using a conservative assessment (tolerance intervals). We established that an increase of 60% or more in the number of DNA copies or a decrease of 38% or more was significantly greater than the variation due to random or experimental error and therefore attributed this variability to a significant change in the HIV-1 DNA copy number. CONCLUSION: We developed and validated a polymerase chain reaction method for the precise quantitation of HIV-1 DNA in peripheral blood mononuclear cells. This assay was able to detect changes in viral loads in HIV-1-infected asymptomatic subjects enrolled in a double-blind placebo-controlled trial using an HIV-1 immunogen.

A flexible information management system. Better information management means increased productivity.

While the finite details and program listings have been purposely excluded, the system described in the preceding text provides the basis for a relatively inexpensive equipment management system. The Biomedical Engineering Department at Rochester General Hospital has utilized this system to increase productivity levels by nearly 15% since implementation in early 1990. With a seven-member technical staff this increase in productivity translated into the addition of approximately 2,100 man-hours in 1991, the equivalent of one extra person. The department was able to utilize these hours to expand into new areas of responsibility and reduce the average service cost per item by 58%. As previously indicated, the key to the system presented is flexibility. There is no hard and fast rule for this system, just a simple, common sense guideline: continuously review, revise, and develop the program. While adherence to this process provides a solid basis for any system whether computerized or manual, it is intended for use only as a "tool," not as a substitute for clinical engineering knowledge and instincts. The ease and speed with which studies are formulated facilitate the large-scale data dissection process so often associated with equipment management programs. Correspondingly, the ability to modify database structures and applications provides the means for expansion and refinement of the system on an ongoing basis.

Comparison of three methods of estimating the parameters of the Naka-Rushton equation.

The Naka-Rushton equation empirically describes the amplitude R of the dark-adapted electroretinogram b-wave, as a function of stimulus luminance L, as R/Rmax = Ln/(Ln + Kn). Estimating the three parameters Rmax, n, and K of this function from electroretinogram data is of both experimental and clinical interest. Several different approaches have been developed to accomplish this analysis, but these approaches may derive different estimates of the three parameters. To examine this possibility, we compared the results of three methods of fitting the Naka-Rushton equation to data sets obtained from 30 normal subjects. Two methods were nonlinear curve-fitting programs; the third method involved fitting a regression line to transformed data. The results indicate that solutions provided by these methods have consistent differences, which may be an important consideration when comparing results reported in studies that used different curve-fitting methods.

Toxicity and pharmacokinetics of ciprofloxacin.

Ciprofloxacin is a fluorinated quinolone antibiotic with a broad spectrum of activity against both gram positive and gram negative organisms. Previous studies have indicated that oral or parenteral preparations of ciprofloxacin resulted in therapeutic concentrations in the aqueous humor but intravitreal levels were found to be only marginally higher than the MIC90 for gram positive cocci. Toxicity was evaluated following intravitreal doses of ciprofloxacin injected into the mid-vitreous cavity of one eye of pigmented rabbits. As a control, an identical volume of normal saline was injected into the fellow eye. Four doses of ciprofloxacin were studied, 100 micrograms, 250 micrograms, 500 micrograms and 1000 micrograms. Electroretinograms (ERGs) were recorded prior to injection and 1, 7 and 14 days after injection. These were analyzed as b-wave amplitude ratios between treated and untreated eyes. On ophthalmoscopic examination focal areas of retinitis were observed following injection of both 500 micrograms and 1000 micrograms of ciprofloxacin but not at 250 micrograms. Additionally, ERG amplitude ratios were significantly reduced following the 1000 micrograms dose. At the 100 or 250 micrograms ciprofloxacin dose histological sections are comparable with control eyes and appear normal; ERG ratios were unchanged from the baseline level and careful indirect ophthalmoscopic examination revealed no alterations. Pharmacokinetic analysis after single intravitreal injection of 250 micrograms of ciprofloxacin (N = 3 rabbits/dose) disclosed vitreous fluid levels (microgram/ml) of 146 at 0 hr, 77 at 4 hr, 32 at 8 hr, 0.49 at 24 hr and 0.24 at 48 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and slow changes in the human cone electroretinogram during light and dark adaptation.

Changes in the response characteristics of the human cone electroretinogram (ERG) during light and dark adaptation were studied in two visually normal subjects. Cone ERG responses were isolated under all adaptation conditions through the use of 31 Hz flicker. To determine the time-course of changes in the cone ERG during adaptation, responses to stimuli of constant luminance were measured repeatedly during 15 min of exposure to an adapting field of 2.0 log cd/m2 and during 30 min of dark adaptation following adapting field termination. In addition, luminance-response functions were obtained before and immediately after adapting field onset, as well as before and immediately after adapting field termination. The results indicate that the human cone ERG is influenced by two major processes. One process has a relatively rapid time-course and serves to reposition the luminance-response function along the luminance axis following changes in ambient light levels. The second process, which has a slow time-course, scales response amplitudes during light and dark adaptation by the same proportion at all stimulus luminances. The results provide a framework for predicting the manner in which the cone ERG will change with alterations in the state of retinal adaptation.

'On' response defect in paraneoplastic night blindness with cutaneous malignant melanoma.

Response properties of rod and cone systems were assessed in a patient with an acquired form of night blindness associated with a metastatic cutaneous malignant melanoma. The night blindness, a sensation of shimmering lights, and selective reductions in the amplitudes of both rod and cone electroretinographic (ERG) b-waves were present before and after chemotherapy, confirming that this disorder was a paraneoplastic consequence of the melanoma rather than a response to chemotherapy. During ERG testing with flashes of extended duration, the cone b-wave abnormality was found to be a predominant loss of the cone ERG "on" response with relative preservation of the "off" response, similar to that observed in patients with congenital stationary night blindness. An impairment in signal transmission specific for retinal "on" pathways may be a primary defect in both of these forms of night blindness.

Comparison of the human electro-oculographic response to green and near-ultraviolet stimuli.

We recorded the electro-oculogram from 27 normal subjects by means of green and near-ultraviolet (UVA) stimulation. After a 40-minute dark-adaptation period, baseline responses were recorded. In response to the green stimulus, the electro-oculogram increased from this level by an average (+/- standard error of the mean) of 49.5% +/- 4.0%. Although the predicted scotopic effectiveness of the ultraviolet stimulus was more than 3 log units below that of the green stimulus, the near-ultraviolet-induced electro-oculogram increased to an average of 21.9% +/- 3.0% above baseline. This response cannot be due to lens fluorescence to the near-ultraviolet stimulus, since two aphakic subjects had electro-oculographic responses of 32% and 76% above baseline to near-ultraviolet stimuli. Neither the green nor the ultraviolet electro-oculogram changed significantly with age. These large responses to near-ultraviolet stimulation demonstrate the need for standardizing light sources for electro-oculographic testing because the degree of near-ultraviolet irradiance varies considerably according to their design characteristics.

Longitudinal electroretinogram study in a patient with metallosis oculi.

Serial electroretinograms were obtained from a patient with visual loss secondary to an intraocular metallic foreign body. Six days after the injury, the acute-phase electroretinogram was larger in the affected left eye. During the following months, the electroretinogram amplitude declined steadily in the affected eye to values well below normal. At 9 months, electroretinogram amplitudes were approximately 50% compared with those in the normal eye. Clinical signs confirmed the need for surgery, and the foreign body was removed. Visual acuity in both eyes remained essentially normal, and electroretinogram amplitudes stabilized over subsequent months. Approximately 8 months after surgery, dark-adaptometry testing showed a 1-log unit elevation of absolute threshold in the affected eye, and Rmax in the affected eye was 42% of that in the uninjured eye.

The isolated perfused frog eye: a useful preparation for the investigation of drug effects on retinal function.

The isolated frog eye perfused through its ophthalmic artery with a modified oxygenated Sickel's media responds to light stimuli of different wavelengths in a manner similar to that observed in the frog eye in vivo for periods of 9 hr and longer. Short- and long-duration field potentials from the globe (ERG) and optic nerve (ONP) can be recorded simultaneously. Drugs perfused through the eye can be shown to modify components of the ERG and ONP and to alter the flow rate of the perfusion media through the eye.

The use of disposable cautery to scrape corneal ulcers.

We used a handheld disposable cautery for the diagnostic scraping of corneal ulcers. Sterility of the tip is obtained by activating the unit until the tip reaches a red-hot color and then allowing it to cool. The unit has a sturdy, well-defined tip, and is self-sterilizing, reusable, readily available, and simple to use.

Positive after-image, PAI: early erasure by saccadic eye movement or Jendrassik manoeuvre.

Positive after-images (PAIs) evoked by low intensity stroboscopic flash are erased by voluntary large amplitude saccadic eye movements. The duration of the PAI is shortened by a Jendrassik manoeuvre. The results suggest that muscle spindle afferent impulses conducted centrally are involved in the erasure phenomenon. The duration of the PAI is modifiable by drugs. The social implications of PAI are considered briefly.