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Type 1 diabetes, which affects 1.25 million American children and adults, and more than 20 million people around the world, is a challenging chronic disease caused by the body's inability to make insulin. Among its most severe forms is brittle diabetes. People with brittle diabetes frequently experience large swings in blood sugar that can quickly move from too high to too low or vice versa. Severely low blood sugar, called hypoglycemia, can cause sudden and unexpected seizures, coma, heart attacks, and even death. Insulin is the main treatment for this common disease. But it isn't a cure. A type of cell transplant that comes close to a cure for some people with type 1 diabetes, a technique pioneered and tested in the United States, is now available in many countries but is still deemed an experimental procedure in the U.S., making it almost impossible to get. More than a decade ago, the United Kingdom's National Health Service approved islet cell transplantation for type 1 diabetes - an approval based on an extensive review of the evidence generated by clinical trials conducted in the United States. Our federal dollars supported that research, and this treatment ought to be available to U.S. citizens. Islet cell transplantation is not a panacea for all forms of type 1 diabetes. And transplantation of any organ, including islet cells, requires the use of anti-rejection drugs that can have a range of adverse side effects. Nevertheless, individuals with severe brittle diabetes who are fully informed of the risks and benefits should have the ability to access this lifesaving treatment option. We fully understand the FDA's efforts to rein in companies marketing unapproved stem cell products that have little or no evidence to support their use and that may put patients at risk. Yet the FDA should stay equally focused on its commitment to approving evidence-based transformative treatments for devastating diseases and conditions, including brittle diabetes.

Protease inhibitor (PI) therapy for patients infected with the human immunodeficiency virus has been associated with lipid disorders and insulin resistance. We compared the incidence of myocardial infarction (MI) among participants receiving treatment with PIs with or without nucleoside reverse transcriptase inhibitors (nRTIs) to nRTI therapy alone in 30 phase II/III double-blind, randomized studies conducted before 1999 for the first 4 PI drugs. In most trials included in this analysis, participants could receive combination therapy with a PI plus nRTIs in open-label extensions after the blinded phase concluded. Person-years (PY) of follow-up were calculated from treatment initiation to the diagnosis of MI, or to the end of the randomized phases for nRTI-only therapy or to the conclusion of the studies for PI-containing regimens. Separate analyses were conducted for the randomized and the randomized-plus-extension phases. Among 10,986 participants, 7951 (72%) received PI drugs at some point for an average duration of 12 months. There were 10 MIs (1.31/1000 PY) in the randomized phases and 19 MIs (1.63/1000 PY) in the randomized-plus-extension phases. The overall stratified relative risk of MI for PI-containing (1.82 MI/1000 PY) versus nRTI-only (1.05 MI/1000 PY) regimens of 1.69 was not significantly increased (95% confidence interval [CI], 0.54 to 7.48). The absolute difference in MI risk was +0.77 (95% CI, -0.71 to +2.26) MIs/1000 PY. Compared with NRTI-only therapy, patients receiving PI-containing regimens for an average of 1 year did not have significantly more MIs, but the upper bound of the 95% CI indicates there may be up to 2.3 additional MIs per 1000 PY. Although studies with a longer duration of PI therapy are in progress to assess whether a later increase in MI incidence occurs, our analysis did not demonstrate a dramatic increase in MI risk during the first year of PI therapy.

Background: Lopinavir is a newly developed inhibitor of human immunodeficiency virus (HIV) protease that, when formulated with ritonavir, yields mean trough plasma lopinavir concentrations that are at least 75 times as high as that needed to inhibit replication of wild-type HIV by 50 percent., Methods: We conducted a double-blind trial in which 653 HIV-infected adults who had not received antiretroviral therapy for more than 14 days were randomly assigned to receive either lopinavir-ritonavir (400 mg of lopinavir plus 100 mg of ritonavir twice daily) with nelfinavir placebo or nelfinavir (750 mg three times daily) with lopinavir-ritonavir placebo. All patients also received open-label stavudine and lamivudine. The primary efficacy end points were the presence of fewer than 400 HIV RNA copies per milliliter of plasma at week 24 and the time to the loss of virologic response through week 48., Results: At week 48, greater proportions of patients treated with lopinavir-ritonavir than of patients treated with nelfinavir had fewer than 400 copies of HIV RNA per milliliter (75 percent vs. 63 percent, P<0.001) and fewer than 50 copies per milliliter (67 percent vs. 52 percent, P<0.001). The time to the loss of virologic response was greater in the lopinavir-ritonavir group than in the nelfinavir group (hazard ratio, 2.0; 95 percent confidence interval, 1.5 to 2.7; P<0.001). The estimated proportion of patients with a persistent virologic response through week 48 was 84 percent for patients receiving lopinavir-ritonavir and 66 percent for those receiving nelfinavir. Both regimens were well tolerated, with the rate of discontinuation related to the study drugs at 3.4 percent among patients receiving lopinavir-ritonavir and 3.7 percent among patients receiving nelfinavir. Among patients with more than 400 copies of HIV RNA per milliliter at some point from week 24 through week 48, resistance mutations in HIV protease were demonstrated in viral isolates from 25 of 76 nelfinavir-treated patients (33 percent) and none of 37 patients treated with lopinavir-ritonavir (P<0.001)., Conclusions: For the initial treatment of HIV-infected adults, a combination regimen that includes lopinavir-ritonavir is well tolerated and has antiviral activity superior to that of a nelfinavir-containing regimen.

The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with reduced susceptibility. Mutations at positions 82, 54, 10, 63, 71, and 84 were most closely associated with relatively modest (4- and 10-fold) changes in phenotype, while the K20M/R and F53L mutations, in conjunction with multiple other mutations, were associated with >20- and >40-fold-reduced susceptibility, respectively. The median 50% inhibitory concentrations (IC(50)) of lopinavir against isolates with 0 to 3, 4 or 5, 6 or 7, and 8 to 10 of the above 11 mutations were 0.8-, 2.7-, 13.5-, and 44.0-fold higher, respectively, than the IC(50) against wild-type HIV. On average, the IC(50) of lopinavir increased by 1.74-fold per mutation in isolates containing three or more mutations. Each of the 16 viruses that displayed a >20-fold change in susceptibility contained mutations at residues 10, 54, 63, and 82 and/or 84, along with a median of three mutations at residues 20, 24, 46, 53, 71, and 90. The number of protease mutations from the 11 identified in these analyses (the lopinavir mutation score) may be useful for the interpretation of HIV genotypic resistance testing with respect to lopinavir-ritonavir (Kaletra) regimens and may provide insight into the genetic barrier to resistance to lopinavir-ritonavir in both antiretroviral therapy-naive and protease inhibitor-experienced patients.

Background: Changes in body fat distribution are an adverse effect of therapy with HIV protease inhibitors (PI). It has been suggested that nucleoside analogue reverse transcriptase inhibitors (NRTI) may also contribute to this so-called lipodystrophy syndrome, but the relative contribution of the two drug classes is unclear as they are usually administered concomitantly., Method: The occurrence of lipodystrophy, as reported by physicians using no standardized criteria, was followed in patients randomly assigned to treatment with either a PI alone or a PI combined with an NRTI. The patients were part of a multicenter, open-label, randomized comparison of ritonavir (RTV)/saquinavir (SQV) with or without the addition of stavudine (d4T) in HIV-1-infected patients without prior PI and d4T experience (the Prometheus study)., Results: Lipodystrophy was reported in 29 of 175 (17%) patients during 96 weeks of follow up. Overall, it was reported significantly more frequently in patients who were randomized to RTV/SQV/d4T (22/88; 25%), than in patients randomized to RTV/SQV alone (7/87; 8%) (P = 0.003). When the analysis was limited to patients without any prior antiretroviral experience, lipodystrophy likewise was significantly more frequent in patients randomized to RTV/SQV/d4T (12/50; 24%) than in those randomized to RTV/SQV (2/44; 5%) (P = 0.008)., Conclusion: This randomized clinical trial, in spite of not having been blinded, supports a contributory role of NRTI in the development of antiretroviral therapy-associated lipodystrophy. The low incidence of lipodystrophy in patients with no or limited NRTI exposure supports further evaluation of NRTI-sparing regimens as alternatives to current antiretroviral regimens.

Objective: To evaluate the safety and antiviral activity of different dose levels of the HIV protease inhibitor ABT-378 combined with low-dose ritonavir, plus stavudine and lamivudine in antiretroviral-naive individuals., Design: Prospective, randomized, double-blind, multicenter., Methods: Eligible patients with plasma HIV-1 RNA > 5000 copies/ml received ABT-378 200 or 400 mg with ritonavir 100 mg every 12 h; after 3 weeks stavudine 40 mg and lamivudine 150 mg every 12 h were added (group I, n = 32). A second group initiated treatment with ABT-378 400 mg and ritonavir 100 or 200 mg plus stavudine and lamivudine every 12 h (group II, n = 68)., Results: Mean baseline HIV-1 RNA was 4.9 log10 copies/ml in both groups and CD4 cell count was 398 x 10(6)/l and 310 x 10(6)/l in Groups I and II respectively. In the intent-to-treat (ITT; missing value = failure) analysis at 48 weeks, HIV-1 RNA was < 400 copies/ml for 91% (< 50 copies/ml, 75%) and 82% (< 50 copies/ml, 79%) of patients in groups I and II respectively. Mean steady-state ABT-378 trough concentrations exceeded the wild-type HIV-1 EC50 (effective concentration to inhibit 50%) by 50-100-fold. The most common adverse events were abnormal stools, diarrhea and nausea. No patient discontinued before 48 weeks because of treatment-related toxicity or virologic rebound., Conclusions: ABT-378 is a potent, well-tolerated protease inhibitor. The activity and durable suppression of HIV-1 observed in this study is probably attributable to the observed tolerability profile and the achievement of high ABT-378 plasma concentrations.

Objective: To evaluate the effect of treatment with ritonavir (RTV)/saquinavir (SQV)/6 stavudine (D4T) or RTV/SQV alone, with treatment intensification if needed, in protease inhibitor- and D4T-naïve HIV-1-infected individuals., Design: Multicentre, open-label, randomized controlled trial. Two-hundred and eight patients were randomized to receive treatment with RTV 400 mg/SQV 400 mg twice daily or RTV 400 mg/SQV 400 mg/D4T 40 mg twice daily. Intensification of study medication with reverse transcriptase inhibitors was permitted if serum HIV-RNA remained > 400 copies/ml after 12 weeks of treatment. Follow-up of this study was 48 weeks., Results: In a strict intention-to-treat analysis, counting all dropouts as virological failures, 63% [95% confidence interval (CI), 54-73%] of subjects in the RTV/SQV group (n = 104) reached a serum HIV-RNA < 400 copies/ml at week 48, as compared with 69% (95% CI, 60-78%) in the RTV/SQV/D4T group (n = 104; P = 0.379). In the on-treatment analysis these percentages were 88 and 91% respectively. Thirty-one patients intensified their study medication according to the protocol (28 in the RTV/SQV group, three in the RTV/SQV/D4T group). Thirty out of 31 (97%) patients had a serum HIV-RNA < 400 copies/ml at their last follow-up visit. Ten per cent of patients discontinued study medication due to adverse events., Conclusion: The concept of starting with a simple, potent regimen, that could be intensified if necessary, showed good virological results after 48 weeks in this study, comparable to starting with more drugs from the beginning. Longer follow-up is needed to determine the long-term efficacy of this treatment strategy.

Objectives: Although the treatment of pregnant women and their infants with zidovudine (ZDV) has been remarkably effective in preventing the perinatal transmission of human HIV-1, many potentially preventable infections still occur. To examine whether the risk of perinatal infection is increased among women who carry ZDV-resistant HIV-1, the role of genotypic ZDV resistance in perinatal transmission was evaluated., Methods: The reverse transcriptase (RT) region of clinical isolates from culture supernatants of 142 HIV-1-infected women enrolled in the Women and Infants Transmission Study (WITS), who had been treated with ZDV during pregnancy was sequenced. Results from genotypic sequencing were linked to demographic, laboratory, and obstetrical databases, and the magnitude of association of having consensus drug-resistant HIV-1 RT mutations with transmission was estimated., Results: Twenty-five per cent (34/142) of maternal isolates had at least one ZDV-associated resistance mutation. A lower CD4 cell percentage and count (P= 0.0001) and higher plasma HIV-1 RNA (P=0.006) were associated with having any ZDV resistance mutation at delivery. Having any RT resistance mutation [odds ratio (OR): 5.16; 95% confidence interval (CI): 1.40, 18.97; P=0 0.01], duration of ruptured membranes [OR: 1.13 (1.02, 1.26) per 4 h duration; P= 0.02], and total lymphocyte count [OR: 1.06 (1.01, 1.10) per 50 cells higher level; P=0.009] were independently associated with transmission in multivariate analysis., Conclusion: Maternal ZDV resistant virus was predictive of transmission, independent of viral load, in these mothers with moderately advanced HIV-1 disease, many of whom had been treated with ZDV before pregnancy.

Objective: To evaluate the safety and antiretroviral activity of ritonavir (Norvir) and saquinavir (Invirase) combination therapy in patients with HIV infection., Design: A multicenter, randomized, open-label clinical trial., Setting: Seven HIV research units in the USA and Canada., Patients: A group of 141 adults with HIV infection, CD4 T lymphocyte counts of 100-500 x 10(6) cells/l, whether treated previously or not with reverse transcriptase inhibitor therapy, but without previous HIV protease inhibitor drug therapy., Interventions: After discontinuation of prior therapy for 2 weeks, group I patients were randomized to receive either combination (A) ritonavir 400 mg and saquinavir 400 mg twice daily or (B) ritonavir 600 mg and saquinavir 400 mg twice daily. After an initial safety assessment of group I patients, group II patients were randomized to receive either (C) ritonavir 400 mg and saquinavir 400 mg three times daily or (D) ritonavir 600 mg and saquinavir 600 mg twice daily. Investigators were allowed to add up to two reverse transcriptase inhibitors (including at least one with which the patient had not been previously treated) to a patient's regimen after week 12 for failure to achieve or maintain an HIV RNA level < or = 200 copies/ml documented on two consecutive occasions., Measurements: Plasma HIV RNA levels and CD4+ T-lymphocyte counts were measured at baseline, every 2 weeks for 2 months, and monthly thereafter. Safety was assessed through the reporting of adverse events, physical examinations, and the monitoring of routine laboratory tests., Results: The 48 weeks of study treatment was completed by 75% (106/141) of the patients. Over 80% of the patients on treatment at week 48 had an HIV RNA level < or = 200 copies/ml. In addition, intent-to-treat and on-treatment analyses revealed comparable results. Suppression of plasma HIV RNA levels was similar for all treatment arms (mean areas under the curve minus baseline through 48 weeks were-1.9, -2.0, -1.6, -1.8 log10 copies/ml in ritonavir-saquinavir 400-400 mg twice daily, 600-400 mg twice daily, 400-400 mg three times daily, and 600-600 mg twice daily, respectively). Median CD4 T-lymphocyte count rose by 128 x 10(6) cells/l from baseline, with an interquartile range (IQR) of 82-221 x 10(6) cells/l. The most common adverse events were diarrhea, circumoral paresthesia, asthenia, and nausea. Reversible elevation of serum transaminases (> 5 x upper limit of normal) occurred in 10% (14/141) of the patients enrolled in this study and was associated with baseline abnormalities in liver function tests, baseline hepatitis B surface antigen positivity, or hepatitis C antibody positivity (relative risk, 5.0; 95% confidence interval 1.5-16.9). Most moderate or severe elevations in liver function tests occurred in patients treated with ritonavir-saquinavir 600-600 mg twice daily., Conclusions: Ritonavir 400 mg combined with saquinavir 400 mg twice daily with the selective addition of reverse transcriptase inhibitors was the best-tolerated regimen of four dose-ranging regimens and was equally as active as the higher dose combinations in HIV-positive patients without previous protease inhibitor treatment.

The chronicity, high mutation rates, and high circulating titers of HIV during the 'stable' phase of infection make rapid evolution of resistance mutations a key predictor of antiretroviral efficacy. Recent advances in measurement of viral RNA titers, turnover dynamics and the in vivo spectrum of resistance mutations allow realistic in vivo estimates of important kinetic parameters of within-patient evolution of viral resistance. First-order estimates of the frequency of viral genotypes necessary for resistance to many antiretroviral combination regimens indicate that many such genotypes pre-exist in patients prior to initiation of therapy. The combinatorial nature of observed multiply-resistant genotypes, however, along with current estimates of total-body viral load and viral turnover dynamics, imply a strikingly sharp transition associated with the change from two-drug to three-drug antiretroviral regimens: pre-existing resistance being near-certain in the first instance but highly unlikely in the second. This abrupt change, a 'combinatorial ledge', carries with it a number of important implications for the understanding and control of HIV infection and other potential targets of antiviral therapy.

Objective: To examine the patterns of vertical transmission of zidovudine (ZDV) resistance mutations., Design: HIV-1 reverse transcriptase codons 10-250 were sequenced from 24 pairs of ZDV-exposed women and their HIV-infected infants as part of the Women and Infants Transmission Study., Methods: Viral RNA was extracted from tissue culture supernatants and sequenced using fluorescent dye-primer chemistry and an automated sequencer., Results: For 17 of these pairs, maternal and infant sequences were identical to one another and lacking known ZDV resistance mutations. The remaining seven maternal sequences contained known mutations associated with ZDV resistance at reverse transcriptase codons 70, 210, 215 and 219. In each case where the maternal HIV isolate showed a pure mutant species, the infant sequence was identical. When the maternal sequence showed the presence of a sequence mixture at codon 70 or 219, the infant's virus showed only wild-type sequence even when the ZDV-resistant mutant was quantitatively dominant in the mother. The single maternal HIV isolate showing mixed sequence at codon positions 210 and 215 transmitted an unmixed mutant to the infant at both positions. When maternal mixtures were present at sites not associated with ZDV resistance, only the dominant species appeared in the infant., Conclusions: When maternal HIV isolates contained mixed wild-type and ZDV-resistant subpopulations, only a single component of the mixture could be detected in the infected infants. Resistance mutants without the codon 215 mutation were not transmitted from mixtures, even when the mutants formed the majority of circulating maternal virus. In perinatal HIV transmission, specific ZDV-resistant HIV genotypes circulating in the maternal virus pool may influence whether infection in the infant will be established by a wild-type or ZDV-resistant HIV strain.

The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 microM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, 50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.

The pharmacokinetic interaction between indinavir and ritonavir was evaluated in five groups of healthy adult volunteers to explore the potential for twice-daily (b.i.d.) dosing of this combination. All subjects received 800 mg of indinavir every 8 h (q8h) on day 2. In addition, subjects in group I received one dose of 800 mg of indinavir on day 1 and 800 mg of indinavir q8h on day 17. Subjects in Groups II and IV each received one dose of 600 mg of indinavir on days 1 and 17, and subjects in groups III and V each received one dose of 400 mg of indinavir on days 1 and 17. During days 3 to 17, ritonavir placebo or ritonavir at 200, 300, 300, or 400 mg q12h was given to groups I, II, III, IV, and V, respectively. Ritonavir at steady state probably inhibited the cytochrome P-450 3A metabolism of indinavir and substantially increased plasma indinavir concentrations, with the area under the plasma concentration-time curve (AUC) increasing up to 475% and the peak concentration in serum (Cmax) increasing up to 110%. The Cmax/trough concentration ratio decreased from 50 in standard q8h regimens to less than 14 when indinavir was administered with ritonavir. For a constant indinavir dose, an increase in the ritonavir dose yielded similar indinavir AUCs, Cmaxs, and concentrations at 12 h (C12s). For a constant ritonavir dose, an increase in the indinavir dose resulted in approximately proportional increases in the indinavir AUC, less than proportional increases in Cmax, and slightly more than proportional increases in C12. Ritonavir reduced between-subject variability in the indinavir AUC and trough concentrations and did not affect indinavir renal clearance. With the altered pharmacokinetic profile, indinavir likely could be given as a b.i.d. combination regimen with ritonavir. This could potentially improve patient compliance and thereby reduce treatment failures.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization. Structural elements of RT that determine processivity are poorly understood. The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity. To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the beta3-beta4 hairpin of HIV-1NL4-3 RT. The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs. When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme. Homology modeling the three-dimensional structures of wild type HIV-1NL4-3 RT and its finger insertion mutants revealed that the extended loop between the beta3 and beta4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to approximately 12 A. Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer. Our data suggest that the beta3-beta4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.

Objective: To determine markers that are associated with the durability of virologic response to therapy with HIV protease inhibitors in HIV-infected individuals., Design: This study encompassed two retrospective analyses of the duration of virologic response to protease inhibitor therapy. The first analysis included 29 patients receiving either monotherapy or combination therapy with the protease inhibitor ritonavir whose plasma HIV RNA levels rebounded from the point of greatest decline with mutations associated with resistance to ritonavir. The second analysis included a cohort of 102 patients who initially responded to randomized treatment with either monotherapy with ritonavir or combination therapy with ritonavir and zidovudine., Methods: Durability of response was defined as the time from the initiation of therapy to the point at which plasma HIV RNA displayed a sustained increase of at least 0.6 log10 copies/ml from the nadir value. In the first analysis, durability of response was analyzed with respect to baseline HIV RNA, HIV RNA at the nadir, and the drop in HIV RNA from baseline to the nadir. In the second analysis, time to rebound was examined using Kaplan-Meier analysis, stratifying by either baseline HIV RNA or HIV RNA at the nadir., Results: In both analyses, the durability of response was not highly associated with either baseline RNA or the magnitude of RNA decline from baseline. Instead, a strong relationship was observed between the durability of response and the nadir plasma HIV-1 RNA value (P < 0.01). The nadir in viral load was generally reached after 12 weeks of randomized therapy., Conclusions: Viral RNA determinations at intermediate timepoints may be prognostic of impending virologic failure of protease inhibitor therapy. Therapeutic strategies that allow intensification of initial antiretroviral regimens in the subset of patients with incomplete virological response before the emergence of high level resistance should be investigated.

The associations of CD4 cell count, plasma human immunodeficiency virus (HIV) type 1 RNA, infectious HIV titer in peripheral blood mononuclear cells, immune complex-disrupted (ICD) p24 antigen, and MT-2 assays with measures of disease progression after drug treatment were assessed in a subset of patients enrolled in AIDS Clinical Trials Group Study 175. Baseline plasma RNA levels and changes in RNA values at weeks 8 or 56 were more important predictors of disease progression than were baseline or changes in CD4 cell counts. Each 10-fold lower HIV RNA concentration at baseline and each 10-fold decrease in HIV RNA between baseline and week 8 was associated with increases of 49-61 CD4 cells/mm3 at weeks 56 and 104. In multivariate analyses, neither baseline values nor changes in infectious HIV titer nor ICD p24 antigen concentrations were associated with long-term changes in CD4 cell count. Plasma HIV-1 RNA appears to be the best predictor of long-term CD4 cell count responses and disease progression.

Virologic measurements are increasingly used to evaluate prognosis and treatment responses in human immunodeficiency virus (HIV) type 1 infection. Markers of HIV-1 replication, including infectious HIV-1 titer from peripheral blood mononuclear cells, serum HIV-1 p24 antigen, plasma HIV-1 RNA, CD4 cell numbers, and viral syncytium-inducing (SI) phenotype, were determined in 391 virology substudy participants in AIDS Clinical Trials Group study 175. The subjects had 200-500 CD4 cells/mm3. All markers of viral replication significantly correlated with one another and were inversely related to CD4 cell number. Disease progression to an AIDS-defining event or death or loss of >50% of CD4 cells was associated with infectious HIV-1 titer (P < .001), HIV-1 RNA (P < .001), and HIV-1 p24 antigen (P = .007). In multivariate proportional hazards models, p24 antigen was never significant when HIV-1 RNA level was included. In a model containing infectious HIV-1 titer (P = .038), HIV-1 RNA (P < .001), SI phenotype (P < .001), and CD4 cell number (P = .18), only the virologic parameters remained significantly associated with progression.

Background: Inhibitors of HIV-1 protease produce a rapid decrease in plasma HIV-1 RNA, with concomitant increases in CD4 T-helper lymphocyte counts. The main side-effects of the protease inhibitors currently in use include gastrointestinal disturbances, paraesthesias, hyperbilirubinaemia, and nephrolithiasis. The increasing use of these agents in patients with advanced HIV-1 infection and CD4 counts of less than 50 cells/microL may be associated with unforeseen adverse effects not observed in earlier studies of patients with higher CD4 counts., Methods: Five HIV-infected patients with baseline CD4 lymphocyte counts of less than 50 cells/mL were admitted to the Beth Israel Deaconess Medical Center (Boston, MA, USA) with high fever (> 39 degrees C), leucocytosis, and evidence of lymph-node enlargement within 1-3 weeks of starting indinavir therapy. Informed consent was obtained for studies that entailed CD4 lymphocyte counts, immunophenotyping, isolator blood cultures, and radiological scans. Biopsy samples of cervical, paratracheal, or mesenteric lymph nodes were taken for culture and pathology in four patients., Findings: Lymph-node biopsy samples showed that focal lymphadenitis after initiation of indinavir resulted from unsuspected local or disseminated Mycobacterium avium complex (MAC) infection. The prominent inflammatory response to previously subclinical MAC infection was associated with leucocytosis in all patients and with an increase in the absolute lymphocyte counts in four patients. Three patients with follow-up CD4 counts showed two-fold to 19-fold increases after 1-3 weeks of indinavir therapy. Immunophenotyping after therapy in two patients showed that more than 90% of the CD4 cells were of the memory phenotype., Interpretation: The initiation of indinavir therapy in patients with CD4 counts of less than 50 cells/mL and subclinical MAC infection may be associated with a severe illness, consisting of fever (> 39 degrees C), leucocytosis, and lymphadenitis (cervical, thoracic, or abdominal). The intense inflammatory reactions that make admission to hospital necessary may be secondary to significant numbers of functionally competent immune cells becoming available to respond to a heavy mycobacterial burden. Prophylaxis or screening for subclinical MAC infection, or both, should therefore be done before the beginning of protease-inhibitor therapy in patients with advanced HIV infection.

Zidovudine susceptibility was assessed for 525 clinical human immunodeficiency virus type 1 isolates, before and after reducing the number of replicates and zidovudine concentrations in the standardized consensus peripheral blood mononuclear cell culture assay. We conclude that omitting the 0.001 microM concentration and using duplicate rather than triplicate wells are valid and cost-effective modifications of this expensive assay.

A phase-I study was conducted to examine the safety, pharmacokinetics, and activity of combination 2',3'-dideoxyinosine (ddI) and ribavirin against human immunodeficiency virus type 1 (HIV-1)-positive individuals with CD4+ cell counts of < or = 500/microliter. Nineteen patients were enrolled into the study in which ddI monotherapy (200 mg p.o.b.i.d.) was administered for the first 4 weeks, followed by the coadministration of ribavirin (600 mg p.o.q.d.) and ddI (200 mg p.o.b.i.d.) for 8 or 20 additional weeks. The combination regimen was safe and well tolerated. Three patients did not complete 12 weeks of the study because of adverse events or voluntary withdrawal. The pharmacokinetic studies performed at weeks 4, 6, and 12 on specimens collected from the 15 individuals who completed 12 weeks of therapy revealed no pharmacokinetic interaction between ddI and ribavirin. A significant decline from baseline in HIV-1 titer as measured by quantitative HIV-1 culture was detected both during the ddI-monotherapy phase (week 4, p < 0.001) and during the combination-therapy ddI + ribavirin phase (week 12, p < 0.001); the median drop observed was 0.90 log10 at week 4 and 0.92 log10 at week 12. While the addition of ribavirin did not result in further reductions in viremia in the following weeks on study treatment, 13 (81%) of the 16 patients had at least a -0.5 log10 change in viral titer at week 12. The median decline in plasma viral RNA was 0.68 log10 at week 4(p < 0.001) and 0.67 log10 at week 12 (p = 0.005). CD4+ cell counts increased above baseline significantly during the ddI-monotherapy phase of the study (p = 0.0038). The median increase was +26 cells/mm3 at week 4 and +11 cells/mm3 at week 12; for patients who remained on treatment through 24 weeks, the median CD4+ cell count increase was +10 cells/mm3. The L74V ddI resistance-conferring HIV-I reverse-transcriptase mutation emerged in 53% of the patients. Patients with non-syncytium-inducing HIV variants demonstrated greater responses to treatment with larger decreases in virus load and greater increases in CD4+ cell count. Our results reveal that the combination of ddI and ribavirin in HIV-positive patients is safe, well tolerated, without adverse pharmacologic interaction, and associated with significant and sustained declines in virus load over 12 weeks of therapy.

Background: Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test., Objectives: We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form of the drug on the enzyme activity in a quantitative, cell-free RT assay., Study Design: Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC50 (50% inhibitory concentration) values were determined by application of the median effect equation., Results: Assays from nine post-nevirapine therapy isolates gave IC50 values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2',3'-dideoxyinosine (ddI), but not between the ZDV-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.

Background: We studied measures of human immunodeficiency virus (HIV) replication, the viral phenotype, and immune function (CD4 cell counts) and the relation of changes in these indicators to clinical outcomes in a subgroup of patients in a controlled trial of early antiretroviral treatment for HIV, the AIDS Clinical Trials Group Study 175., Methods: The 391 subjects, each of whom entered the study with a single screening CD4 cell count of 200 to 500 per cubic millimeter, were randomly assigned to receive zidovudine alone, didanosine alone, zidovudine plus didanosine, or zidovudine plus zalcitabine. Plasma concentrations of HIV RNA were assessed in 366 subjects, and viral isolates from 332 subjects were assayed for the presence of the syncytium-inducing phenotype., Results: After eight weeks, the mean (+/-SE) decrease from base line in the concentration of HIV RNA, expressed as the change in the base 10 log of the number of copies per milliliter, was 0.26+/-0.06 for patients treated with zidovudine alone, 0.65+/-0.07 for didanosine alone, 0.93+/-0.10 for zidovudine plus didanosine, and 0.89+/-0.06 for zidovudine plus zalcitabine (P<0.001 for each of the pairwise comparisons with zidovudine alone). Multivariate proportional-hazards models showed that higher base-line concentrations of plasma HIV RNA, less suppression of plasma HIV RNA by treatment, and the presence of the syncytium-inducing phenotype were significantly associated with an increased risk of progression to the acquired immunodeficiency syndrome and death. After adjustment for these measures of viral replication and for the viral phenotype, CD4 cell counts were not significant predictors of clinical outcome., Conclusions: Both the risk of the progression of HIV disease and the efficacy of antiretroviral therapy are strongly associated with the plasma level of HIV RNA and with the viral phenotype. The changes in the plasma concentration of HIV RNA predict the changes in CD4 cell counts and survival after treatment with reverse-transcriptase inhibitors.

The association of plasma human immunodeficiency virus type 1 (HIV-1) RNA level at study entry and over time with clinical progression was evaluated in 187 patients from AIDS Clinical Trials Group protocol 116A who had little or no prior zidovudine treatment. Three-fold-higher HIV-1 RNA levels at study entry and 3-fold increases by week 8 were associated with progression (relative hazard [RH], 1.67; 95% confidence limits [CL], 1.20, 2.32; and RH, 1.45; CL, 1.02, 2.05, respectively). Having 3-fold-higher CD4 cell count at entry was independently associated with a 52% reduction in risk for progression (adjusted RH, 0.48; CL, 0.33, 0.70). When stratified by length of prior zidovudine therapy, RNA level was predictive in drug-naive patients (adjusted RH, 1.87; CL, 1.23, 2.85) but not predictive in patients with up to 16 weeks of prior therapy (adjusted RH, 1.11; CL, 0.70, 1.76). Analysis suggests that the acquisition of mutations at HIV-1 reverse transcriptase codons 215 and 74 is associated with subsequent increases in HIV-1 RNA level (relative risk, 7.00; CL, 0.86, 56.90).

Human immunodeficiency virus (HIV)-1 RNA level in plasma was evaluated as a surrogate marker for disease progression in a clinical trial of advanced HIV-1 infection. Baseline HIV-1 RNA level was an independent predictor of disease progression (relative hazard [RH] for each doubling of HIV-1 RNA level, 1.26; 95% confidence interval [CI], 1.03-1.54; P = .02), after adjusting for the week 4 change in HIV-1 RNA level, baseline CD4 cell count, syncytium-inducing phenotype, clinical status at study entry, and therapy randomization. A 50% reduction in HIV-1 RNA level was associated with a 27% decrease in the adjusted risk of disease progression during the study (RH, 0.73; 95% CI, 0.52-1.02; P = .07). The partial validation of HIV-1 RNA as a predictor for clinical end points has implications for the use of HIV-1 RNA in clinical trials and practice.

Sequence-specific PCR was used in six laboratories and a ligase detection reaction was used in one laboratory to detect the zidovudine-resistance mutation at codon 215 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase DNA. The genotypes of 27 different clinical samples, including cultured HIV-1 isolates, peripheral blood mononuclear cells, and plasma, were correctly identified by 140 of 154 (91%) assays. The sensitivity for detecting a mutation was 96% for HIV-1 reverse transcriptase DNA clone mixtures containing 30% mutant DNA and 62% for mixtures containing 6% mutant DNA.

Zidovudine resistance mutations at reverse transcriptase codons 215 or 41 were found in two-thirds of human immunodeficiency virus type 1 (HIV-1) isolates obtained at baseline from patients enrolled in an AIDS Clinical Trials Group (ACTG) protocol that compared didanosine with continued zidovudine in patients with > or = 16 weeks of previous zidovudine therapy (ACTG 116B/117). The combined presence of mutations at both codons 215 and 41 conferred an increased risk for progression (relative hazard, 1.82; 95% confidence interval [CI], 1.02-3.26) and an increased risk for death (RH, 5.42; 95% CI, 1.92-15.30) in analyses that controlled for other factors predictive of progression. However, the benefit of switching to didanosine compared with continued zidovudine therapy was independent of the presence of these mutations. Although this information is not helpful in determining when to alter therapy, detection of zidovudine resistance mutations provides prognostic information in patients with advanced HIV disease.

Objective: To evaluate the association between resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine and clinical progression., Design: Retrospective analysis of specimens from patients in the AIDS Clinical Trials Group (ACTG) protocol 116B/117, a randomized comparison of didanosine with continued zidovudine therapy in patients with advanced HIV-1 disease who had received 16 weeks or more of previous zidovudine therapy., Setting: Participating ACTG virology laboratories., Patients: 187 patients with baseline HIV-1 isolates., Measurements: Zidovudine susceptibility testing and assays for syncytium-inducing phenotype were done on baseline HIV-1 isolates. Relative hazards for clinical progression or death associated with baseline clinical, virologic, and immunologic factors were determined from Cox proportional hazards regression models., Results: Compared with other patients, 15% (26 of 170) with isolates showing high-level zidovudine resistance (50% inhibitory zidovudine concentration > or = 1.0 microM) had 1.74 times the risk for progressing to a new AIDS-defining event or death (95% CI, 1.00 to 3.03) and 2.78 times the risk for death (CI, 1.21 to 6.39) in analyses that controlled for baseline CD4+ T-lymphocyte count, syncytium-inducing HIV-1 phenotype, disease stage, and randomized treatment assignment. The clinical benefit of didanosine was not limited to patients with highly zidovudine-resistant baseline HIV-1 isolates., Conclusions: High-level resistance of HIV-1 to zidovudine predicted more rapid clinical progression and death when adjusted for other factors. However, patients with advanced HIV-1 disease may benefit from a change in monotherapy from zidovudine to didanosine whether high-level HIV-1 resistance to zidovudine is present or absent, and laboratory assessment of zidovudine resistance is not necessary for deciding when to switch monotherapy from zidovudine to didanosine.

Background: Existing phenotypic tests of antiretroviral susceptibility in clinical isolates of human immunodeficiency virus (HIV) are expensive and slow, and require passage of virus in cell culture with the possible consequence of selecting variants., Objectives: We sought to develop a rapid 14-day assay for zidovudine susceptibility of cell-associated HIV performed directly in patient blood samples., Study Design: Twenty-three tests were performed prospectively in 21 children, and the results were compared with those of the AIDS Clinical Trials Group/Department of Defense consensus drug susceptibility assay (DSA) as well as certain clinical parameters., Results: Five strains from ZDV-naive children were sensitive by the rapid test. Three were tested by DSA, and all were sensitive. Six strains from children who had received >/=24 months of ZDV were resistant by the rapid assay. Four of these strains were tested by the DSA, and all were shown resistant. The viral strains from children who received <24 months of therapy or who had switched from ZDV to other antiviral therapy exhibited variable sensitivity by both tests. Changes in CD4 cells in the subsequent 6 months, as well as weight gain during this time were both correlated to the results of the rapid test. The syncytium-inducing capacity of the virus strains was analyzed similarly., Conclusions: The rapid intracellular virus susceptibility assay is a test of drug sensitivity performed on HIV growing in cells obtained directly from an infected patient. The test has a two-week turn-around time and, in this preliminary report, gives results which correlate with both time on zidovudine and also subsequent CD4 cell changes.

A standardized assay in 96-well microtiter plates for syncytium-inducing (SI) human immunodeficiency virus type 1 phenotype detection using MT-2 cells has been developed. SI variants were found in 67% of the patients with advanced human immunodeficiency virus disease. The occurrence of the SI phenotype increased with lower CD4+ counts. There was no association between p24 antigenemia and the SI phenotype.

Human immunodeficiency virus type 1 (HIV-1) isolates resistant to zidovudine (ZDV) have previously been demonstrated to exhibit in vitro cross-resistance to other similar dideoxynucleoside agents which contain a 3'-azido group. However, cross-resistance to didanosine (ddI) or dideoxycytidine (ddC) has been less well documented. ZDV, ddI, and ddC susceptibility data have been collected from clinical HIV-1 isolates obtained by five clinical centers and their respective retrovirology laboratories. All subjects were treated only with ZDV. Clinical HIV-1 isolates were isolated, amplified, and assayed for drug susceptibility in standardized cultures of phytohemagglutinin-stimulated donor peripheral blood mononuclear cells obtained from healthy seronegative donors. All five cohorts showed a correlation between decreased in vitro susceptibility to ZDV and decreased susceptibility to ddI and ddC. For each 10-fold decrease in ZDV susceptibility, an average corresponding decrease of 2.2-fold in ddI susceptibility was observed (129 isolates studied; P < 0.001, Fisher's test of combined significance). Similarly, susceptibility to ddC decreased 2.0-fold for each 10-fold decrease in ZDV susceptibility (82 isolates studied; P < 0.001, Fisher's test of combined significance). These data indicate that a correlation exists between HIV-1 susceptibilities to ZDV and ddI or ddC for clinical HIV-1 isolates.

A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 1 (HIV-1) isolates has been developed for use in clinical trials. The protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seronegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV-1 stock. The virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC. A standardized inoculum of 1,000 50% tissue culture infective doses per 10(6) cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 microM. The ZDV 50% inhibitory concentrations (IC50) for reference ZDV-susceptible and ZDV-resistant HIV-1 isolates ranged from 0.002 to 0.113 microM and from 0.15 to > 5.0 microM, respectively. Use of this consensus protocol reduced interlaboratory variability for ZDV IC50 determinations with reference HIV-1 isolates. Among eight laboratories, the coefficient of variation ranged from 0.85 to 1.25 with different PBMC protocols and was reduced to 0.39 to 0.98 with the standardized assay. Among the clinical HIV-1 isolates assayed by the standardized drug susceptibility assay, the median ZDV IC50 increased gradually with more ZDV therapy. This protocol provides an efficient and reproducible means to assess the in vitro susceptibility to antiretroviral agents of virtually all clinical HIV-1 isolates.

A quantitative rapid assay to detect resistant clinical human immunodeficiency virus type 1 (HIV-1) strains remains an important medical goal. A system incorporating a quantitative RNA.RNA hybridization assay that measures the amount of intracellular HIV-1-specific RNA has been employed to detect the level of inhibition by nucleoside analogues in sensitive and resistant HIV-1 strains. The RNA.RNA hybridization assay readily distinguished previously published zidovudine (ZDV; 3'-azido-3'-deoxythymidine)-resistant isolates from ZDV-sensitive isolates of HIV-1. The 50% inhibitory concentration (IC50) of ZDV for HTLV-IIIB and sensitive clinical HIV-1 isolates is between 0.01 and 0.04 microM. HIV-1 strains from three patients on long-term ZDV therapy displayed a greater than 20-fold increase in the ZDV IC50 compared to sensitive strains. The drug sensitivity system was confirmed by showing that mutations in the HIV reverse transcriptase gene from a ZDV-resistant isolate resulted in four amino acid changes (Leu-125----Trp, Ile-142----Val, Thr-215----Tyr, and Pro-294----Thr) including one change (Thr-215----Tyr) that has been previously reported to be associated with resistance. One clinical HIV strain with high-level ZDV resistance displayed a 5-fold increase in 2',3'-dideoxyinosine IC50 compared to that of HTLV-IIIB. A drug sensitivity assay employing RNA.RNA hybridization may be useful for extensive screening of HIV isolates from patients enrolled in clinical trials and permit the correlation of in vitro resistance with clinical outcome.

Certain anticonvulsant drugs, especially phenytoin and phenobarbital, interfere with vitamin K metabolism as indicated by a raised serum osteocalcin level. This finding may be of importance in the pathogenesis of side effects of these medications.