Comparative effectiveness of ledipasvir/sofosbuvir ± ribavirin vs. ombitasvir/paritaprevir/ritonavir + dasabuvir ± ribavirin in 6961 genotype 1 patients treated in routine medical practice.

BACKGROUND: Real-world data are needed to inform hepatitis C virus (HCV) treatment decisions. AIM: To assess the comparative effectiveness of ledipasvir/sofosbuvir ± ribavirin (LDV/SOF ± RBV) vs. ombitasvir/paritaprevir/ritonavir + dasabuvir (OPrD) ± RBV in genotype 1 HCV patients treated in routine medical practice. METHODS: Observational intent-to-treat cohort of genotype 1 patients initiating 8 or 12 weeks of LDV/SOF ± RBV or 12 weeks of OPrD ± RBV. Sustained virological response (SVR) required RNA below the limit of quantification at least 10 weeks after end of treatment. RESULTS: 6961 patients initiated LDV/SOF (N = 4478), LDV/SOF + RBV (N = 1269), OPrD (N = 297), and OPrD + RBV (N = 917) at 126 facilities. Intention-to-treat SVR rates were 91.4% (3813/4170) for LDV/SOF, 90.0% (1098/1220) for LDV/SOF + RBV, 95.1% (269/283) for OPrD and 85.8% (746/869) for OPrD + RBV. SVR rates in those completing 8 weeks of LDV/SOF were 91.7% (1223/1333) and 12 weeks of LDV/SOF 94.6% (2475/2615), LDV/SOF + RBV 92.2% (1033/1120), OPrD 98.0% (248/253) and OPrD + RBV 95.5% (705/738). Significant predictors of SVR were African American race (OR 0.71, 95%CI 0.59-0.86, P < 0.001), body mass index (BMI) > 30 kg/m(2) (OR 0.73, 95% CI 0.60-0.89, P = 0.002), FIB4 > 3.25 (OR 0.60, 95% CI 0.49-0.72, P < 0.001), OPrD + RBV compared to LDV/SOF (OR 0.60, 95% CI 0.48-0.76, P < 0.001) and subtype 1b (OR 1.38, 95% CI 1.11-1.71, P = 0.003). For those completing 12 weeks, FIB-4 > 3.25 and high BMI remained significant predictors. CONCLUSIONS: In this robust real-world cohort, SVR rates were similar to clinical trials. FIB-4 > 3.25 and high BMI were significant negative predictors of SVR. Reduced odds of SVR in African Americans and with OPrD + RBV likely arose from excess early discontinuation as these factors were no longer significant, when limited to patients completing a 12-week course.

Effectiveness of sofosbuvir-based regimens in genotype 1 and 2 hepatitis C virus infection in 4026 U.S. Veterans.

BACKGROUND: Real-world effectiveness data are needed to inform hepatitis C virus (HCV) treatment decisions. AIM: To assess sustained virological response (SVR) of sofosbuvir (SOF)-based regimens in routine medical practice. METHODS: Observational, intent-to-treat cohort analysis of genotype 1 and 2 HCV-infected veterans initiating SOF-based regimens with recommended treatment duration of 12 weeks. RESULTS: Four thousand and twenty-six veterans with genotype 1 (N = 3203) and genotype 2 (N = 823) comprise the cohort. SVR rates for genotype 1 were 66.8% for SOF + peginterferon + ribavirin (RBV), 75.3% for SOF + simeprevir (SIM), 74.1% for SOF + SIM + RBV and for genotype 2 were 79.0% for SOF + RBV. Genotype 1 patients were less likely to achieve SVR with BMI ≥30 (OR 0.64, 95% CI 0.49-0.84, P < 0.001), a history of decompensated liver disease (OR 0.51, 95% CI 0.36-0.71, P < 0.001), treatment experience (OR 0.58, 95% CI 0.48-0.71, P < 0.001), APRI >2 (OR 0.44, 95% CI 0.36-0.55, P < 0.001) and with SOF + PEG + RBV compared with SOF + SIM (OR 0.50, 95% CI 0.40-0.62, P < 0.001). Age, sex, race/ethnicity, diabetes and genotype subtype did not predict SVR. Odds of achieving SVR with SOF + SIM + RBV did not differ compared with SOF + SIM (OR 1.03, 95% CI 0.75-1.44, P = 0.86). Genotype 2 patients were less likely to achieve SVR with prior treatment experience (OR 0.55, 95% CI 0.35-0.88, P = 0.009) and APRI >2 (OR 0.39, 95% CI 0.25-0.62, P < 0.001). CONCLUSIONS: In this real-world cohort, SVR rates were lower than in clinical trials. Genotype 1 and 2 HCV-infected patients with advanced liver disease by APRI >2 or FIB-4 > 3.25 were significantly less likely to achieve SVR. For genotype 1, a SOF + SIM ± RBV regimen was associated with a higher likelihood of SVR.

Comparative effectiveness of the hepatitis C virus protease inhibitors boceprevir and telaprevir in a large U.S. cohort.

BACKGROUND: Limited data exist on the effectiveness of boceprevir and telaprevir in routine practice. AIM: To assess the comparative effectiveness of boceprevir and telaprevir regimens. METHODS: In this observational, intent-to-treat cohort analysis of hepatitis C genotype 1-infected veterans initiated on peginterferon/ribavirin and boceprevir (n = 661) or telaprevir (n = 198), we determined sustained virological response (SVR), treatment discontinuation rates and adverse haematological events. Inverse probability-of-treatment weighting (IPTW) was used to estimate the effect of one drug over the other, with matched pairs and unweighted logistic regression on the entire cohort for comparison. RESULTS: Of 835 veterans, SVR occurred in 50% and 52% receiving boceprevir- and telaprevir-based treatment, respectively (P = 0.72). No significant differences occurred among subgroups: cirrhotics (37% vs. 39%, P = 0.94), null responders (23% vs. 18%, P = 0.81), partial responders (39% vs. 58%, P = 0.15) and relapsers (60% vs. 77%, P = 0.11). Early discontinuation rates for boceprevir and telaprevir, respectively, were 31% and 28% by week 24 (P = 0.46) and 54% and 45% by 48 weeks (in those completing at least 28 weeks) (P = 0.14). Choice of telaprevir over boceprevir was significantly associated with SVR in multivariate models (IPTW OR: 1.57, 95% CI: 1.10-2.25, P = 0.01; matched-pairs OR: 1.91, 95% CI: 1.23-3.00, P = 0.004; unweighted OR: 1.50 95% CI: 1.05-2.14, P = 0.02). Rates of haematological adverse events in boceprevir- and telaprevir-treated patients were as follows: anaemia 59% vs. 51%, P = 0.30, thrombocytopenia 41% vs. 48%, P = 0.26, neutropenia 41% vs. 27%, P = 0.04. CONCLUSIONS: Sustained virological response was more likely with telaprevir-based regimens compared with boceprevir-based regimens in routine medical practice, after accounting for patient differences. Early discontinuation and haematological events, however, were similar.

Trends in uptake of recently approved antiretrovirals within a national healthcare system.

OBJECTIVE: The aim of the study was to describe Veterans Healthcare Administration (VHA) system-wide uptake of three HIV protease inhibitors: atazanavir, darunavir and tipranavir. METHODS: This retrospective cohort study evaluated VHA uptake of three target antiretrovirals and lopinavir/ritonavir in each complete 90-day quarter since approval to December 2007 using VHA HIV Clinical Case Registry data. We assessed uptake using number of new prescriptions, number of providers and facilities prescribing target agents, provider type, clinic type, facility size and location within four US regions. RESULTS: Overall, 6551 HIV-infected veterans received target antiretrovirals. Uptake was generally greatest within the first year after Food and Drug Administration (FDA) approval, and then slightly declined and plateaued. Geographically, early adoption of new antiretroviral drugs tended to occur in the Western USA, as evidenced by comparison of uptake patterns of new antiretrovirals to use of all antiretroviral agents. A small percentage of prescribers of all antiretrovirals were responsible for new prescriptions for target medications, particularly for darunavir and tipranavir. Providers at almost 50% of VHA facilities were prescribing these agents within the first year. CONCLUSIONS: Uptake of new antiretrovirals in the VHA generally reflected overall prescribing of all antiretrovirals, suggesting a lack of VHA impediments to new antiretrovirals in the healthcare system. Some regional variation in uptake among the targeted antiretrovirals occurred over time but tended to resolve after the first several months. Providers responsible for early prescribing of the target medications were limited to a fraction of providers who tended to be physicians who practised in infectious disease (ID) clinics at medium-sized facilities.

Pretreatment assessment and predictors of hepatitis C virus treatment in US veterans coinfected with HIV and hepatitis C virus.

The US Department of Veterans Affairs (VA) cares for many human immunodeficiency virus/hepatitis C virus (HIV/HCV)-coinfected patients. VA treatment recommendations indicate that all HIV/HCV-coinfected patients undergo evaluation for HCV treatment and list pretreatment assessment tests. We compared clinical practice with these recommendations. We identified 377 HIV/HCV-coinfected veterans who began HCV therapy with pegylated interferon and ribavirin and 4135 HIV/HCV-coinfected veterans who did not but were in VA care at the same facilities during the same period. We compared laboratory and clinical characteristics of the two groups and estimated multivariate logistic regression models of receipt of HCV treatment. Overall, patients had high rates of receipt of tests necessary for HCV pretreatment assessment. Patients starting HCV treatment had higher alanine aminotransferase (ALT), lower creatinine, higher CD4 counts and lower HIV viral loads than patients not starting HCV treatment. In the multivariate model, positive predictors of starting HCV treatment included being non-Hispanic whites, having higher ALTs, lower creatinines, higher HCV viral loads, higher CD4 counts, undetectable HIV viral loads and receiving HIV antiretrovirals. A history of chronic mental illness and a history of hard drug use were negative predictors. Most HIV/HCV-coinfected patients received the necessary HCV pretreatment assessments, although rates of screening for hepatitis A and B immunity can be improved. Having well-controlled HIV disease is by far the most important modifiable factor affecting the receipt of HCV treatment. More research is needed to determine if the observed racial differences in starting HCV treatment reflect biological differences, provider behaviour or patient preference.

Developmental toxicity of mixtures: the water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture.

The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA) and bromochloro (BCA) acetic acid are developmentally toxic in mouse whole embryo culture. Human exposure to these contaminants in drinking water would involve simultaneous exposure to all three HAs. This study explores the question of developmental toxicity interactions between these compounds. Gestational day (GD) 9.5 rat embryos were exposed to various concentrations of the three HAs (singly or in combination) for 48 h and then evaluated for dysmorphology. The embryonic effects from exposure to the single compounds and mixtures were evaluated using developmental score (DEVSC) as the parameter of comparison. Concentrations of individual compounds and mixtures were chosen (based on a dose-additivity model) which were predicted to produce scores 10 or 20% lower than control levels. Evaluations were performed on all possible combinations of the three HAs. The HAs were dysmorphogenic and resulted in primarily rotation and heart defects and to a lesser extent prosencephalic, visceral arch, and eye defects. The percent anomalies in the rat were comparable to those previously published for the mouse at comparable toxicant concentration. There was a low incidence of neural tube defects in the rat following exposure to the HAs. The rat neural tube appeared less sensitive to the HAs than did the mouse resulting in a higher rate of neural tube dysmorphology in the mouse. Following exposures to BCA and DBA, alone and in combination, there was a significant incidence of delayed embryonic caudal development with apparent normal development anterior to the second visceral arch. The developmental scores for embryos exposed to combinations of the three compounds, when compared to scores for embryos exposed to the single compounds, indicated that the dose-additivity model adequately predicted the observed toxicity and that the developmental toxicity of these water disinfection by-products appears to be additive in whole embryo culture (WEC).

The Immunology Case Registry.

The Veterans Health Administration (VA) is the nation's largest public integrated health care system and the largest single provider of health care to HIV-positive patients in the United States. First established in 1992, the Immunology Case Registry (ICR) is a national-level administrative database originally designed to monitor health care service utilization. With its interface to the VA electronic medical record, the ICR provides an opportunity to monitor and improve the quality of clinical care for HIV-positive patients receiving VA care. To realize this potential, key data element enhancements and quality review systems are being put into place. A brief description of the population included in the ICR is provided, along with current data limitations and planned improvements for data collection. The ICR, now managed by a national quality management center, is being updated and systematically improved for local clinical use. As local data capture improves, the ICR will prove to be an invaluable resource for assessing the quality of HIV care in the VA system and patient outcomes from that care.

A pilot trial of indinavir, ritonavir, didanosine, and lamivudine in a once-daily four-drug regimen for HIV infection.

OBJECTIVE: To evaluate the tolerance, pharmacokinetics, and virologic and immunologic outcomes of once-daily indinavir, ritonavir, didanosine, and lamivudine in HIV-seropositive individuals. DESIGN: Open-label 24-week pilot study. PATIENTS: Ten HIV-seropositive subjects who were either antiretroviral-naive or minimally experienced with short-term single-or dual-nucleoside therapy provided informed consent and were enrolled. All subjects received didanosine (400 mg) 30 to 60 minutes before a meal followed by indinavir (1200 mg), ritonavir (400 mg), and lamivudine (300 mg) concurrent with the aforementioned meal. METHODS: Safety laboratory tests, including a complete blood cell count and amylase, lipase, liver transaminase, and nonfasting lipid monitoring as well as plasma HIV viral load and CD4+ lymphocyte count, were carried out at monthly intervals. Genotyping was performed at baseline. Pharmacokinetic studies for indinavir and ritonavir were performed at week 8. RESULTS: Nine of 10 subjects completed 24 weeks of therapy. No subject demonstrated primary protease inhibitor mutations at baseline. Toxicities experienced by subjects were typically mild and consistent with those commonly reported for each of the medications, including two cases of hematuria. By week 24, median nonfasting cholesterol and triglyceride levels increased by 49% and 108%, respectively. Median baseline plasma HIV viral load and CD4+ lymphocyte count were 29,292 (4.47 log10) copies/ml and 224 cells/mm3, respectively. Eight of 10 subjects had a plasma HIV viral load of <50 copies/ml by week 12. The 2 subjects with a detectable HIV viral load reached <50 copies/ml by week 28. Median CD4+ lymphocyte counts increased by 193 cells/mm3 at week 24. Indinavir and ritonavir plasma concentrations remained above respective inhibitory and effective concentrations (IC95 and EC50) (uncorrected for protein binding) throughout the 24-hour dosing interval for 6 of 10 and 8 of 10 subjects, respectively. CONCLUSIONS: Our pilot study demonstrates excellent virologic suppression despite low minimum protease inhibitor concentrations during a dosing interval in some patients and is supportive of further study.

Tolerance and pharmacokinetic interactions of rifabutin and azithromycin.

This multicenter study evaluated the tolerance and potential pharmacokinetic interactions between azithromycin and rifabutin in volunteers with or without human immunodeficiency virus infection. Daily dosing with the combination of azithromycin and rifabutin was poorly tolerated, primarily because of gastrointestinal symptoms and neutropenia. No significant pharmacokinetic interactions were found between these drugs.

Decreased medical expenditures for care of HIV-seropositive patients. The impact of highly active antiretroviral therapy at a US Veterans Affairs Medical Center.

OBJECTIVE: To identify any changes in expenditures and in morbidity and mortality with the progression of treatment of the HIV-seropositive population from monotherapy with a nucleoside reverse transcriptase inhibitor (NRTI) [1993] through dual NRTI therapy (1995) to highly active antiretroviral therapy (HAART) [1997]. DESIGN AND SETTING: This study retrospectively compared 3 separate years of the total expenditures encountered in the management of HIV-seropositive individuals seen at a US Veterans Affairs Medical Center. INTERVENTIONS: Utilising a computerised hospital database, we identified those patients with HIV-related International Classification of Diseases, version 9 (ICD-9) codes and collected all healthcare-related expenditure data. The 3 eras selected for comparison were controlled for similar utilisation of prophylaxis against opportunistic infections, access to investigational antivirals, consistency between primary care providers and distribution of new anti-HIV therapies relative to that era. Cost data for inpatient and outpatient activities (visits and admissions) were derived from actual expenditures. Major categories were then compared, including total inpatient/outpatient expenditures and utilisation, laboratory and prescription costs, and morbidity and mortality rates. MAIN OUTCOME MEASURES AND RESULTS: The 3 periods had similar patient populations, with 86, 86 and 82% of patients in 1993, 1995 and 1997, respectively, having some degree of immunosuppression (defined as CD4+ lymphocyte counts < 500 cells/mm3). Morbidity and mortality were not changed by the addition of dual NRTI therapy. HAART therapy produced 60 and 70% declines in relative mortality when compared with the single and dual NRTI eras. Dual NRTI or HAART therapy decreased overall expenditures as compared with NRTI monotherapy. HIV-related outpatient resource utilisation other than pharmacy and laboratory costs fell by 25 and 59% in 1997 as compared with 1993 and 1995, respectively. The greatest fall in resource utilisation was for inpatient bed-days of care, where the average cost per patient fell by $US2782 between 1993 and 1997. Pharmacy and laboratory expenditures increased by $US1825 and $US231 per patient from 1993 to 1997, respectively. Overall, the impact of HAART was a decrease of $US1193 in the average total cost per patient from 1993 to 1997. CONCLUSIONS: The introduction of HAART provided a positive outcome on patient morbidity and mortality and on medical centre expenditures. The end result was a cost shift of expenditures from inpatient utilisation to outpatient pharmacy and laboratory costs. This information is important for patients and providers, who need to make clinical decisions on lifelong therapies, and for healthcare financial planners, who need to predict inpatient and outpatient healthcare utilisation during an era of limited healthcare dollars.

Tolerance and pharmacokinetic interactions of rifabutin and clarithromycin in human immunodeficiency virus-infected volunteers.

This study evaluated the tolerance and potential pharmacokinetic interactions between clarithromycin (500 mg every 12 h) and rifabutin (300 mg daily) in clinically stable human immunodeficiency virus-infected volunteers with CD4 counts of <200 cells/mm3. Thirty-four subjects were randomized equally to either regimen A or regimen B. On days 1 to 14, subjects assigned to regimen A received clarithromycin and subjects assigned to regimen B received rifabutin, and then both groups received both drugs on days 15 to 42. Of the 14 regimen A and the 15 regimen B subjects who started combination therapy, 1 subject in each group prematurely discontinued therapy due to toxicity, but 19 of 29 subjects reported nausea, vomiting, and/or diarrhea. Pharmacokinetic analysis included data for 11 regimen A and 14 regimen B subjects. Steady-state pharmacokinetic parameters for single-agent therapy (day 14) and combination therapy (day 42) were compared. Regimen A resulted in a mean decrease of 44% (P = 0.003) in the clarithromycin area under the plasma concentration-time curve (AUC), while there was a mean increase of 57% (P = 0.004) in the AUC of the clarithromycin metabolite 14-OH-clarithromycin. Regimen B resulted in a mean increase of 99% (P = 0.001) in the rifabutin AUC and a mean increase of 375% (P < 0.001) in the AUC of the rifabutin metabolite 25-O-desacetyl-rifabutin. The usefulness of this combination for prophylaxis of Mycobacterium avium infections is limited by frequent gastrointestinal adverse events. Coadministration of clarithromycin and rifabutin results in significant bidirectional pharmacokinetic interactions. The resulting increase in rifabutin levels may explain the increased frequency of uveitis observed with concomitant use of these drugs.

The impact of active herpes simplex virus infection on human immunodeficiency virus load.

The effect of a concurrent herpes simplex virus (HSV) infection on human immunodeficiency virus type 1 (HIV-1) load was evaluated. Sixteen subjects were identified with an active HSV infection and had pre-outbreak, acute-phase, and post-outbreak plasma (n = 16) and peripheral blood mononuclear cell (PBMC) (n = 8) samples for evaluation. All subjects were treated for an acute HSV outbreak with acyclovir for 10 days, followed by chronic prophylaxis. HIV-1 plasma RNA levels were determined by branched DNA, and intracellular HIV gag mRNA copy numbers were determined by quantitative reverse transcriptase-polymerase chain reaction ELISA. Plasma virus load increased a median of 3.4-fold during the acute outbreak (range, 0- to 10-fold; P = .002), while post-outbreak levels (30-45 days after the appearance of lesions) remained above pre-outbreak, baseline levels in some subjects. Intracellular HIV gag mRNA increased during the outbreak as well. Thus, an acute HSV episode can result in increased HIV transcription and plasma virus load.

Evaluation of Chiron HIV-1/HIV-2 recombinant immunoblot assay.

In a study to determine the reliability, sensitivity, and specificity of the Chiron RIBA HIV-1/HIV-2 Strip Immunoblot Assay (RIBA HIV-1/2 SIA) for confirmation of human immunodeficiency virus type 1 (HIV-1) and HIV-2 antibodies, 1,263 serum samples from various populations in the United States, Caribbean, Africa, India, and Thailand were evaluated by RIBA HIV-1/2 SIA, and the results were compared with those obtained by an HIV-1 Western blot (immunoblot) assay. All sera were tested by HIV enzyme immunoassay, RIBA HIV-1/2 SIA, and Western blotting. Samples with discrepant results were further tested by an HIV-1 and/or HIV-2 immunofluorescent-antibody assay and HIV-1 p24 antigen assay. The RIBA HIV-1/2 SIA detected all 17 HIV-1 and HIV-2 dually reactive serum samples, all 215 HIV-2-positive serum samples, and 480 of 481 HIV-1-positive serum samples for a sensitivity of 99.8%. Of 548 negative samples, 523 were RIBA HIV-1/2 SIA negative, for a specificity of 95.4%, with 22 (4%) samples interpreted as indeterminate and 3 (0.6%) interpreted as falsely positive. Western blotting detected 391 of 548 negative samples (specificity, 71.4%), with 152 (27.7%) samples interpreted as indeterminate and 5 (0.9%) interpreted as falsely positive. In conclusion, the RIBA HIV-1/2 SIA had a sensitivity comparable to that of Western blotting and could discriminate HIV-1 from HIV-2 in one blot, providing a cost advantage. Because of its high degree of specificity, the RIBA HIV-1/2 SIA further reduced the number of indeterminate results found by Western blotting, providing a more accurate means of assessing seronegative individuals.

Human immunodeficiency virus reverse transcriptase codon 215 mutations diminish virologic response to didanosine-zidovudine therapy in subjects with non-syncytium-inducing phenotype.

Eight zidovudine-experienced subjects received zidovudine and didanosine for 30 weeks followed by 30 weeks of didanosine monotherapy. At study entry, plasma from 4 subjects had human immunodeficiency virus RNA pol T215Y/F mutant and 4 had codon 215 wild type. All 8 subjects had non-syncytium-inducing virus phenotype. Sustained 10-fold decreases in plasma RNA levels were seen only in subjects who initially had 215 wild type RNA, despite the development of a T215Y/F mutation during combination therapy. Virologic and immunologic benefits were maintained in this group with didanosine monotherapy. No subject developed a pol L74V codon mutation. Significant differences in plasma virus load and CD4 cell responses were seen in this zidovudine-didanosine combination pilot study relative to codon 215 genotype.

Nonisotopic hybridization assay for determination of relative amounts of genotypic human immunodeficiency virus type 1 zidovudine resistance.

A nonisotopic hybridization assay for human immunodeficiency virus genotypic zidovudine resistance determination is described. Biotinylated PCR product was hybridized with enzyme-labeled probes for wild-type or resistant mutant sequences and detected colorimetrically or chemiluminescently in a microplate format. Changes in mutant-to-wild-type ratios allow for the monitoring of longitudinal patient samples.

Determination of human immunodeficiency virus RNA in plasma and cellular viral DNA genotypic zidovudine resistance and viral load during zidovudine-didanosine combination therapy.

Eleven human immunodeficiency virus (HIV)-infected subjects on long-term zidovudine (ZDV) therapy had didanosine (ddI) added to their antiretroviral regimen. HIV RNA in plasma was quantitated by branched-DNA signal amplification assay. Peripheral blood mononuclear cell (PBMC) HIV viral DNA was quantitated by PCR. The relative amounts of wild-type (WT) sequence, ddI resistance-associated codon changes (reverse transcriptase [RT] gene codon 65 K-->R [RT K65R], RT 174V, RT I135K/T/V, and RT M184I/V), and ZDV resistance-associated codon change (RT T215Y/F) from HIV RNA in plasma and RT T215Y/F from PBMC viral DNA were determined by differential hybridization of PCR products from 10 of 11 subjects. All subjects had evidence of RT T215Y/F mutation in both RNA in plasma and PBMC DNA at baseline. Subjects with a mixture of WT and RT T215Y/F HIV RNA in plasma at baseline demonstrated a decline in RNA levels in plasma after the addition of ddI. However, after 6 months of ZDV-ddI therapy, WT HIV RNA in plasma was undetectable in all subjects who had demonstrated a mixture at baseline. Subjects with only RT T215Y/F RNA present in plasma at baseline remained so and demonstrated no decline in RNA levels in plasma. In all subjects, no significant changes in PBMC DNA viral load and RT T215Y/F or WT levels were seen. HIV RNA in plasma demonstrated a significantly higher RT T215Y/F mutant/WT ratio than that of PBMC viral DNA, both at baseline and after ZDV-ddI combination therapy in all subjects. No subjects developed mutations associated with ddI resistance at codons 65, 74, 135, and 184 during this study. This study suggests that determination of relative amounts of RT T215Y/F and WT species from HIV RNA in plasma at baseline may be predictive of virologic response during ZDV-ddI combination therapy.

Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes.

This study compared the levels of human immunodeficiency virus (HIV) virion RNA in plasma from whole blood collected in VACUTAINER CPT (cell preparation tube), VACUTAINER PPT (plasma preparation tube), VACUTAINER SST (serum separation tube), and standard VACUTAINER tubes with sodium heparin, acid citrate dextrose, sodium citrate, and potassium EDTA used as anticoagulants. Quantitative plasma HIV RNA levels were measured by branched-DNA signal amplification. Blood from all tubes was either processed within 1 to 3 h after collection or stored at room temperature or at 4 degrees C for analysis at 6 to 8 and 30 h postdraw. Immediately separated plasma from sodium citrate CPT tubes held at 4 degrees C maintained better stability of HIV RNA equivalents than whole blood held at room temperature or 4 degrees C. The highest number of HIV RNA equivalents was seen with EDTA VACUTAINER tubes. HIV RNA equivalents in all types of plasma were significantly higher than in SST tubes. Although a decline in HIV RNA equivalents was seen in all collection devices after 30 h, a significantly greater decline in plasma HIV RNA equivalents occurred in acid citrate dextrose VACUTAINER tubes than in citrate CPT, PPT, and standard EDTA VACUTAINER tubes. In order to minimize the variability of quantitative HIV RNA test results, our data suggest that samples collected for a particular assay should be processed at the same time postdraw using a particular tube type throughout a given study.

Stabilities of quantitative plasma culture for human immunodeficiency virus, RNA, and p24 antigen from samples collected in VACUTAINER CPT and standard VACUTAINER tubes.

We evaluated the stability of human immunodeficiency virus (HIV) load markers from blood samples collected in VACUTAINER CPT or standard VACUTAINER brand tubes using sodium heparin or sodium citrate as anticoagulants. Quantitative plasma culture and p24 antigen concentrations were determined, and HIV RNA levels in plasma were measured by both reverse transcription-PCR-enzyme-linked immunosorbent assay (RT-PCR-ELISA) and branched DNA methods. All tubes were stored at room temperature for analysis at 2, 24, 48, and 72 h after the blood samples were drawn. No difference was seen between tube types with respect to the HIV titer in plasma or the positivity rate for all samples that demonstrated a fall in titer over time. Unbound p24 antigen levels in plasma decreased during the initial 48-h period in both tube types. Immune complex-dissociated p24 antigen levels decreased in CPT tubes but not in standard VACUTAINER tubes. The HIV RNA copy number in plasma measured by RT-PCR-ELISA was stable in most subjects and was significantly higher in CPT tubes than in standard VACUTAINER tubes at 24 and 72 h after the blood samples were drawn. The branched DNA probe assay detected a significant decline in HIV RNA equivalent in plasma over 72 h in both collection tubes, the decline being more dramatic in the standard VACUTAINER tube than the CPT tube. Overall, interday variability suggests that samples collected for a particular assay should be processed at the same time after blood is drawn and that a particular tube type be used throughout a given study.