Prototype trial design for rapid dose selection of antiretroviral drugs: an example using emtricitabine (Coviracil).

Antiretroviral monotherapy for initial drug characterization risks the selection of resistant virus, yet monotherapy is the only setting where many fundamental properties of a new drug can be reliably determined. Using data on viral replication kinetics and dynamics, we designed an accelerated (14 day) open-label study of single agent emtricitabine (formerly known as FTC)--a nucleoside reverse transcriptase inhibitor--to select a dosing regimen for further therapeutic study. Five regimens (25 mg bd, 100 mg od, 200 mg od, 100 mg bd and 200 mg bd) were evaluated in HIV-1-infected subjects over a 14 day dosing period to determine the optimal dose and pharmacokinetics. Serial blood samples for virological, pharmacokinetic and intracellular FTC-triphosphate measurements were drawn frequently. A dose-response relationship for the antiviral activity of emtricitabine was established, with total daily doses of 200 mg or more producing the greatest median HIV-1 viral load suppression: 1.72-1.92 log10. Based on virological outcomes, dose-response analysis and intracellular triphosphate levels, a once-daily dose of 200 mg was selected for further long-term clinical study. Adverse events possibly related to emtricitabine were unremarkable. The antiviral activity of emtricitabine correlated well with intracellular FTC-triphosphate concentrations. This study design is a safe, useful tool for early dose selection for drugs with potent antiretroviral activity and linear pharmacokinetics.

Cytomegalovirus infectivity in whole blood following leukocyte reduction by filtration.

Cytomegalovirus (CMV) may be transmitted by transfusion of whole blood and cellular components processed according to standard processing procedures. A need exists to develop new procedures to remove CMV and other leukocyte-borne viruses from donor blood. Ten patients (AIDS/bone marrow transplants) who were CMV antigenemic (virus subsequently confirmed by isolation), donated 50 mL of venous blood within 24 to 72 hours of the initial antigen detection. Twenty-five-milliliter aliquots of each specimen were passed through Purecell Neo Neonatal Leukocyte Reduction Filters (Pall, East Hills, NY). The remaining 25-mL nonfiltered aliquots, as well as the blood filtrates, were subjected to infectivity endpoint determinations. The Purecell Neo filter effected a 3 to 4 log10 leukocyte reduction. CMV input titers ranged from less than 10 to 7.3 x 10(1) median tissue culture infectious dose (TCID50) per milliliter. CMV was not isolated from any postfiltration effluent (i.e., leukocytes, erythrocytes, or plasma). CMV DNA was not detected by nested polymerase chain reaction in 8 of 10 postfiltrate blood specimens. The Purecell Neo filter was efficacious in eliminating or significantly reducing viral (CMV) load in venous blood.

Evaluation of the PrimeCapture CMV DNA detection plate system for detection of cytomegalovirus in clinical specimens.

With the availability of anticytomegalovirus (CMV) therapeutic agents, rapid detection of CMV is important in the care and management of the immunosuppressed patient. The PrimeCapture CMV DNA Detection Plate System (PC-PCR) was evaluated for the detection of CMV in blood and cerebrospinal fluid (CSF). The resolution of discordant results was performed by consensus testing utilizing a combination of conventional cell culture (TC-CPE), the CMV-antigenemia (CMV-Ag) assay, one or more in-house CMV nested PCR assays, and/or patient evaluation and follow-up. Of 51 blood specimens from 34 patients, 23 (45%) were identified as true positives. PC-PCR was significantly more sensitive than the CMV-Ag assay, TC-CPE, or a combination of both tests. The sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) for PC-PCR, the CMV-Ag assay, TC-CPE, and a combination of CMV-Ag and TC-CPE were 78, 75, 72, 81%; 46, 100, 100, 70%; 39, 100, 100, 67%; and 58, 100, 100, 73%, respectively. CMV was not detected or isolated in CSF, resulting in a combined PC-PCR sensitivity, specificity, PPV, and NPV of 77, 90, 68, and 93%, respectively. Among those laboratorians considering the incorporation of molecular CMV diagnostics into their clinical microbiology or virology laboratories, the CMV PC-PCR offers a relatively simple-to-perform and sensitive assay system.

Stavudine resistance: an update on susceptibility following prolonged therapy.

The current report summarizes the available published and unpublished data from several investigators on resistance in clinical isolates following prolonged stavudine therapy. Results suggest that stavudine resistance is both modest in degree and infrequent in appearance. Phenotypic evaluation of 61 patients on stavudine therapy showed only modest changes in drug sensitivity following up to 29 months of treatment. The post-treatment isolates from 15 patients exhibited an increase in EC50 value > fourfold (level above variability of assay) when compared with the corresponding pretreatment isolates. However, the vast majority (11) of these pretreatment isolates either had unexpectedly low EC50 levels and/or had post-treatment isolates that lacked any amino acid changes within their reverse transcriptase (RT) gene to account for the observed change in sensitivity. Of the four remaining isolates, two appeared to have a multi-resistant phenotype to several nucleoside analogues and two had no detectable RT amino acid changes to account for the observed change in stavudine sensitivity. To date, clinical HIV-1 isolates displaying stavudine-specific resistance have yet to be reported. Furthermore, full or partial RT sequence analysis of 194 post-treatment isolates failed to identify any consistent amino acid changes. The strain-specific V75T mutation reported to confer stavudine resistance to the HXB2 HIV-1 strain in vitro, was found in only six isolates and did not correlate with stavudine resistance. This low incidence of stavudine resistance is in striking contrast to that observed with other nucleoside analogues and further supports the use of stavudine in first-line combination therapy for HIV patients.

Application of a standardized cytomegalovirus antigenemia assay in the management of patients with AIDS.

One-hundred twenty AIDS patients, tested by an optimized cytomegalovirus antigenemia (CMV-Ag) assay, were followed over a 6-month period in an attempt to correlate viral (CMV) load with severe CMV organ-specific disease. Among patients available for follow-up, CMV organ-specific disease was present in seven of eight (88%) with pp65 antigen levels > or = 50 cells per 4 x 10(5) polymorphonuclear leukocytes. One-hundred six patients of 107 patients with levels < 50 pp65 reactive cells did not develop CMV organ-specific disease within our 6-month follow-up period. The CMV-Ag assay, as standardized in our clinical setting, served as a marker and predictor of CMV organ-specific disease in our AIDS patient population.

A fifth human cytomegalovirus glycoprotein B genotype.

Genetic variation in glycoprotein B (gB) may play a role in human cytomegalovirus (HCMV) pathogenesis. Using restriction endonuclease digestion and DNA sequencing, a unique gB genotype was identified in eight HCMV strains isolated from five patients with the acquired immune deficiency syndrome. Nucleic acid homology to the four previously described gB genotypes ranged from 79 to 91% for the two major variable regions of gB. Studies of the role of gB in HCMV pathogenesis should recognize the existence of live gB genotypes.

A comparative trial of zidovudine administered every four versus every twelve hours for the treatment of advanced HIV disease.

Zidovudine is approved for administration in doses given every 4 hours. Less frequent dosing has been used in many clinical trials, but the toxicity and efficacy of such regimens have not been formally compared with the approved regimen. In this multicenter, randomized, double-blind, controlled trial, the safety, tolerance and efficacy of 600 mg of zidovudine given daily in two or six divided doses were compared. Three hundred and twenty patients with a CD4 lymphocyte count < 250 cells/mm3 (mean, 104 cells/mm3) or a prior AIDS-defining illness were treated with zidovudine 100 mg every 4 hours (regimen A) or 300 mg every 12 hours (regimen B). Eighty-eight patients (56%) and 94 patients (58%), assigned to regimens A and B, respectively, completed the planned 48 weeks of treatment. Serious anemia (hemoglobin < or = 7.5 g/dl) occurred in 13% and 7% of patients treated with regimens A and B, respectively (difference, 6%, 95% confidence interval [CI], 2, 12%; p = .13). The mean duration of treatment and the frequency of neutropenia and symptomatic complaints including nausea and headache were similar in the two treatment groups. The number of patients experiencing a new opportunistic infection (18% versus 20% for regimens A and B, respectively), and the number of deaths (five in each group) did not differ significantly between groups. The effect of treatment on CD4 lymphocyte counts and HIV p24 antigenemia also was similar for both regimens. Zidovudine given at the more convenient dose of 300 mg twice daily has similar safety, and tolerance and appears to have similar efficacy to the currently approved regimen. Use of this regimen should help simplify the treatment of HIV disease.

Significance of leukocyte concentration in the performance of the quantitative cytomegalovirus (CMV) antigenemia assay.

BACKGROUND: The quantitative cytomegalovirus-antigenemia (CMV-Ag) assay is an important technology in the regimen of tests utilized in the care and management of acquired immunodeficiency syndrome (AIDS) and other immunocompromised patient groups. Performance of this assay is contingent upon the appropriate processing of the polymorphonuclear leukocyte (PMNL) compartment of the peripheral blood. However, a cell input standard in the performance of the CMV-Ag assay, has not been established. Interpretive differences between laboratories utilizing the CMV-Ag assay may reflect this lack of test uniformity. OBJECTIVES: To determine the effect of different PMNL concentrations on the quantitation of CMV in peripheral blood. The leukocyte concentration resulting in optimal rates of viral detection, will be compared with the shell vial assay-indirect immunofluorescent assay (SVA-IFA), and conventional tube culture (TC-CPE). STUDY DESIGN: A total of 74 freshly collected blood specimens were tested by the CMV-Ag assay, using cytospin preparations consisting of 2 x 10(5), 4 x 10(5) and in preliminary experiments, 8 x 10(5) PMNLs/slide. Data obtained from these studies were compared to SVA-IFA and TC-CPE. Viral load was monitored among 11 symptomatic patients through sequential testing of these patients at the start of ganciclovir (GCV), foscarnet (PFA), or combination drug therapy. RESULTS: Among 74 blood specimens tested by the CMV-Ag assay, cytospin preparations consisting of 4 x 10(5) compared with 2 x 10(5) PMNLs/slide, affected a mean positive cell increase of 215% (P = 0.03). PMNL slide preparations consisting 8 x 10(5) cells produced background levels which prevented accurate reading of slides. The CMV-Ag assay was more sensitive than the SVA-IFA, but equivalent to TC-CPE. Among 11 patients started on drug therapy, viral load was markedly reduced in 8 within 2-3 weeks; three patients (2 deceased within 3 weeks after receiving therapy), showed no decrease in viral load. One patient was identified as harboring a PFA resistant strain. CONCLUSIONS: A PMNL concentration of 4 x 10(5) cells facilitated the reading of CMV-Ag assay slide preparations. The modified CMV-Ag assay furthermore, is applicable in the monitoring of viral load for the tracking of susceptible or resistant CMV strains.

Antiviral susceptibility testing-flow cytometric analysis (AST-FCA) for the detection of cytomegalovirus drug resistance.

Antiviral susceptibility testing-flow cytometric analysis (AST-FCA), an application of flow cytometry in conjunction with cell culture, was developed to identify susceptibility of cytomegalovirus (CMV) isolates to the antiviral drugs ganciclovir (GCV) and foscarnet (PFA). The viral isolates used in this study were obtained from peripheral blood of AIDS patients. Among GCV-susceptible strains, the mean 50% inhibitory concentration (IC50) using AST-FCA was 18 microns (SI50 = 1.4). Among GCV resistant strains, the mean IC50 was 47 microns (SI50 = 3.6). For PFA, the mean IC50 was 80 microns (SI50 = 1.0) for susceptible strains. IC50s for strains resistant to PFA, showed no significant reduction of infectivity at the highest drug concentration (i.e., 200 microns PFA) tested. Additional analyses confirmed the accuracy of AST-FCA by parallel testing using plaque reduction assay. AST-FCA is a novel, nonisotopic, and relatively simple to perform laboratory procedure for the identification of CMV drug-resistant strains.

Antiretroviral agents. Current usage.

Recent advances in the understanding of HIV pathogenesis, the development of a new potent class of antiretroviral agents, and new data on the effectiveness of older, less potent agents when used in combination are resulting in a new era of more effective therapies for HIV. Three classes of antiretroviral agents are approved for clinical use: nucleoside and nonnucleoside RT inhibitors and protease inhibitors. These classes of drugs are limited in duration of effectiveness owing to the emergence of resistance. Problems with the potency of therapy and resistance can be overcome to some extent by using these agents in combination. The most promising regimen to emerge thus far appears to be the combination of zidovudine, lamuvidine, and a potent protease inhibitor such as indinavir. Multiple additional combination regimens are now in clinical trials, and data on other promising regimens with potent antiretroviral activity may soon appear. Short-term results obtained with such combination regimens are impressive and represent a considerable advance over older approaches to the treatment of HIV infection. Data on the long-term clinical and virologic effectiveness of new combination regimens are needed, however, before the true impact of these treatments on the prognosis for HIV-infected patients can be assessed.

Effect of foscarnet on quantities of cytomegalovirus and human immunodeficiency virus in blood of persons with AIDS.

Four intravenous dosages of foscarnet given for 10 days were compared with no therapy in persons with AIDS who had asymptomatic cytomegalovirus (CMV) viremia. CMV viremia was quantitated by endpoint cell dilution microcultures, pp65 antigenemia assay, and measurement of CMV DNA in peripheral blood leukocytes by a quantitative-competitive PCR. Human immunodeficiency virus type 1 (HIV-1) viremia was quantitated by endpoint cell dilution microculture, serum p24 antigen assay, and PCR for HIV-1 RNA in plasma. Twenty-seven subjects who had received a median of 22 months of nucleoside antiretroviral therapy were enrolled. Twenty-two subjects received foscarnet, which was well tolerated and decreased the CMV burden, as reflected by all three indicator assays. During the 10 days of dosing, the level of CMV viremia, as measured by 50 percent tissue culture infective doses, decreased from 117.5 to 12.7 (P = 0.001), the amount of CMV DNA decreased from 20,328 copies to 622 copies per 150,000 leukocytes (P = 0.02), and the level of CMV pp65 antigenemia decreased from 14.9 to 1.6 positive peripheral blood mononuclear cells per 50,000 leukocytes (P = 0.008). A significant pharmacodynamic relationship was found between the peak foscarnet concentration and a decrease in the level of CMV antigenemia (P < 0.05). Foscarnet had no effect on quantitative HIV-1 microcultures during the 10 days of treatment, but the HIV-1 p24 antigen level in serum decreased significantly, from 454 to 305 pg/ml (P = 0.01). Also, a significant pharmacodynamic relationship was seen between plasma HIV-1 RNA concentrations and both peak foscarnet concentration (P < 0.01) and the area under the foscarnet time-concentration curve (P < 0.05). Reductions in the levels of CMV and HIV-1 viremia correlated quantitatively with systemic exposure to foscarnet, whereas control subjects actually experienced an increase in CMV and HIV-1 burdens. The dual antiviral activity of foscarnet shown in this trial encourages investigation of its use in combination with other antiretroviral therapies for persons with AIDS.

Early clinical markers and CD4 percentage in subjects with human immunodeficiency virus infection.

In a clinical trial involving asymptomatic, HIV-seropositive subjects treated with either the HIV-1 immunogen (an inactivated, gp120-depleted HIV-1 virus in incomplete Freund's adjuvant) or an adjuvant control, we examined the relationship between changes in the percentage of CD4 cells over time and early clinical markers of HIV disease progression. Subjects who had an early clinical event were more likely to have a greater decline in the percentage of CD4 cells than those subjects who did not have a clinical event (p = 0.054). The greatest decline in CD4 percentage occurred within 10 weeks prior to a clinical event (mean 11% decrease from baseline). Subjects from the quartile with the greatest decline in CD4 percentage had a fivefold greater risk of having a clinical event than subjects from the quartile with the second largest decline (p = 0.045). These results demonstrate a relationship between changes in the percentage of CD4 cells and early clinical events. Further validation of this association may be useful in clinical monitoring and in evaluating therapies to treat HIV infection.

Reverse transcriptase genotype and antiretroviral susceptibility of human immunodeficiency virus isolates from patients with advanced disease treated with didanosine: correlation with virologic response and survival.

To identify correlates of virologic response and survival, the reverse transcriptase (RT) genotype and in vitro antiviral susceptibility of human immunodeficiency virus (HIV) isolates from 20 patients treated with didanosine were studied. Patients had advanced HIV disease and were intolerant to or had failed zidovudine. Neither RT genotype nor antiviral susceptibility testing, as determined by a peripheral blood mononuclear cell-based assay, correlated with a virologic response to didanosine, as determined previously by quantitative serum culture. Only one (8%) of 12 isolates obtained after 6-12 months of treatment showed mutation at codon 74 conferring didanosine resistance. Reversions were seen in three of five patients with pre-treatment zidovudine resistance mutations at codons 70, but in none of eight with mutations at codon 215. Pretreatment isolates encoding mutations at RT codon 215 or encoding codon 123 asp were associated with both significantly greater CD4 lymphocyte depletion and shorter survival. In this cohort of patients with advanced HIV disease, neither rapid emergence of didanosine resistance nor rapid reversion of zidovudine resistance was observed. To better understand the relationship between virologic response and in vitro susceptibility to didanosine, more precise tools may be needed.

Cytomegalovirus glycoprotein B groups associated with retinitis in AIDS.

To determine if cytomegalovirus (CMV) retinitis occurs more frequently in patients infected with certain strains CMV isolates from the blood of 44 patients with advanced human immunodeficiency virus disease were grouped by the DNA sequence or the restriction endonuclease digest pattern of a portion of the glycoprotein B (gB) gene. Forty-two patients (95%) were followed clinically until the development of CMV retinitis or death. Fourteen (78%; 95% confidence interval, 7%-39%) of 26 with isolates belonging to other gB groups developed CMV retinitis (P = .002). Viremia caused by gB group 2 CMV strains is associated with higher risk of CMV retinitis than viremia due to other CMV gB groups. The association of CMV gB gene with retinitis suggests this gene, or one linked to it, is a virulence factor for CMV strains causing infection in AIDS patients.

Seroepidemiology of cytomegalovirus in patients with advanced HIV disease: influence on disease expression and survival.

To determine the prevalence and effect of cytomegalovirus (CMV) co-infection on clinical outcome, the seroepidemiology of CMV was examined in 196 demographically diverse patients with advanced HIV disease. Thirty-six (18.4%) were seronegative for CMV; 31 of these 36 (86.1%) were both non-black and non-homosexual. Invasive CMV disease developed in 41 of 160 (25.6%) seropositive patients and 0 of 36 (0%) seronegative patients (p = 0.00015). Among seropositive patients, the frequency of CMV disease varied markedly according to risk group for acquisition of HIV infection. CMV disease occurred in 26 of 73 (35.6%) homosexual men and 11 of 33 (33.3%) heterosexuals, but only 2 of 47 (4.3%) injection drug users. Sexual exposure as the only risk factor for the acquisition of HIV was a highly significant independent risk factor for invasive CMV when other covariables were considered in a proportional hazards model (risk ratio 5.4, p = 0.0019). The cumulative proportion of all seropositive patients developing CMV disease after 3 years was 31%. CMV serologic status had no effect on occurrence of AIDS-related illnesses other than CMV disease and no effect on survival. Risk for the development of CMV disease varies substantially among different groups of patients with advanced HIV disease and can be assessed using serologic and demographic criteria. The results of this study may be used to influence clinical management and help target prophylactic interventions for CMV disease to high-risk individuals.

Cell culture-PCR technique for detection of infectious cytomegalovirus in peripheral blood.

We determined the efficacy of PCR for the direct detection of infectious cytomegalovirus (CMV) in specimen-inoculated MRC-5 tube cultures (cell culture-PCR [CC-PCR]). Parallel testing was performed by a shell vial assay-indirect immunofluorescence assay (SVA-IFA) and isolation (conventional MRC tube cultures [TC-CPE]. The sensitivity, specificity, and positive and negative predictive values for CC-PCR, SVA-IFA, and TC-CPE were 81, 99, 94, and 94%, 86, 100, 100, and 96%, and 77, 100, 100, and 93%, respectively. Future application of CC-PCR may be directed toward its use as a screening tool for cytomegalovirus genotypic variants.

Serious Pseudomonas aeruginosa infection in AIDS.

During a 7-year period, 32 patients with Pseudomonas aeruginosa infection were identified on an HIV treatment service at a university-affiliated teaching hospital. The number of cases increased from 2 in 1986 to 13 in 1992. Affected patients had evidence of advanced HIV infection. In those treated with antiretroviral therapy, 96% of infections occurred > 1 year after initial presentation with HIV disease. Eighteen cases of pneumonia and 14 nonpulmonary (central venous access device, soft tissue, middle ear-mastoid, corneal, and peritoneal) infections were seen. Comparison with matched controls identified use of a central venous access device and administration of aerosolized pentamidine, corticosteroids, or ganciclovir as risk factors for infection (odds ratios, 5.3, 6.5, 15.0, and 9.0, respectively; p = 0.004, 0.007, 0.02, and 0.02, respectively). Seventy-five percent of cases had community onset, but time since last hospital discharge was significantly shorter in study patients than in controls (mean difference, -85 days; 95% confidence interval, -24 to -146; p = 0.01). Among evaluable cases, outcome was fatal (survival < or = 30 days) in 2 of 16 (13%) patients in whom initial antibiotic therapy was appropriate and 8 of 14 (57%) patients in whom initial therapy was not appropriate (p = 0.016). Ten recurrent infections were seen in 8 of 21 patients who survived the initial infection. Median survival after onset of infection was only 80 days. Pseudomonas aeruginosa infection is an increasingly frequent, severe complication of advanced HIV disease. Several treatment and prevention strategies used in the management of advanced HIV disease are associated with an increased risk of infection.

Improved immunogenicity of recombinant vaccinia virus-anchored gp120 lacking gp41.

To produce a vaccine against human immunodeficiency virus-1 with improved immunogenicity, the transmembrane and cytoplasmic tail regions of human immunodeficiency virus-1 were replaced with those of the Vesicular Stomatitis Virus glycoprotein, and cloned into vaccinia virus. This recombinant vaccinia virus, vvE13, was compared to one expressing full length envelope gp160, vvE1. Env products of both were located on the cell surface. Antibody response, lymphocyte proliferation and cytotoxicity were better with vvE13 than with vvE1 inoculated mice.