Dried-plasma transport using a novel matrix and collection system for human immunodeficiency virus and hepatitis C virus virologic testing.

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.

Magnetic silica extraction for low-viremia human immunodeficiency virus type 1 genotyping.

Nucleic acid extraction and human immunodeficiency virus type 1 (HIV-1) genotyping using the NucliSens miniMAG platform and the TruGene HIV-1 genotyping kit gave HIV-1 sequence data from HIV-1-negative plasma spiked with 100 copies/ml reference HIV-1 RNA and from low-viremia clinical samples (<500 copies/ml) without the need for ultracentrifugation or nested second-round PCR.

Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1.

We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification (NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant recombinant viruses and all group M HIV-1 subtypes.

Markers of immune activation and viral load in HIV-associated sensory neuropathy.

BACKGROUND: HIV infection is associated with a painful distal sensory polyneuropathy (DSP) that can severely limit the quality of life of affected subjects. The pathogenesis of DSP is unknown, although both HIV proteins and products of immune activation triggered by HIV infection have been implicated. OBJECTIVE: To assess the association between baseline markers of immune activation and HIV RNA levels (viral load) and time to symptomatic DSP (SDSP). METHODS: A cohort of 376 subjects, most receiving highly active antiretroviral therapy (HAART), were followed semiannually for up to 48 months. Blood and CSF levels of HIV viral load, monocyte chemotactic protein-1, macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase-2, and tumor necrosis factor-alpha were measured in addition to CD4 lymphocyte cell count. RESULTS: In subjects without SDSP at baseline (62.5% of the cohort), among the virologic and immunologic markers, only baseline CSF M-CSF levels were associated with time to SDSP (hazard ratio = 2.97, p = 0.05). The Kaplan-Meier estimate of the 1-year incidence of SDSP was 21%, a 15% decrease from that observed in the Dana cohort, a pre-HAART cohort enrolled with the same inclusion/exclusion criteria. CONCLUSION: Highly active retroviral therapy (HAART) has changed the natural history of HIV-associated symptomatic distal sensory polyneuropathy (SDSP), which may explain, in contrast with studies from the pre-HAART era, the lack of association between SDSP and baseline HIV viral load and CD4 cell count.

Evaluation of HIV RNA and markers of immune activation as predictors of HIV-associated dementia.

OBJECTIVE: To evaluate whether baseline levels of plasma and CSF HIV RNA, tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-2 (MMP-2), or macrophage colony stimulating factor (M-CSF) are predictors of incident HIV-associated dementia (HIVD) in a cohort with advanced HIV infection. METHODS: A total of 203 nondemented subjects with CD4 lymphocyte counts less than 200/muL, or <300/microL but with cognitive impairment, underwent semiannual neurologic, cognitive, functional, and laboratory assessments. HIVD and minor cognitive motor disorder (MCMD) were defined using American Academy of Neurology criteria. The cumulative incidence of HIVD was estimated using Kaplan-Meier curves. Cox proportional hazards regression models were used to examine the associations between biologic variables and time to HIVD, adjusting for age, sex, years of education, duration of HIV infection, type of antiretroviral use, premorbid IQ score, and presence of MCMD. RESULTS: After a median follow-up time of 20.7 months, 74 (36%) subjects reached the HIVD endpoint. The dementia was mild in 70% of cases. The cumulative incidence of HIVD was 20% at 1 year and 33% at 2 years. Highly active antiretroviral therapy (HAART) was used by 73% of subjects at baseline. A plasma HIV RNA level was undetectable in 23% of subjects and a CSF HIV RNA level was undetectable in 48% of subjects. In adjusted analyses, neither plasma nor CSF HIV RNA levels (log10) were associated with time to HIVD; log10 levels of plasma TNFalpha (HR 3.07, p = 0.03) and CSF MCP-1 (HR = 3.36, p = 0.06) tended to be associated with time to HIVD. CONCLUSION: The lack of association between baseline plasma and CSF HIV RNA levels and incident dementia suggests highly active antiretroviral therapy may be affecting CNS viral dynamics, leading to lower HIV RNA levels, and therefore weakening the utility of baseline HIV RNA levels as predictors of HIV-associated dementia.

HIV in the brain: RNA levels and patterns of zidovudine resistance.

OBJECTIVE: To examine the association between HIV RNA levels, patterns of antiretroviral resistance, and neurologic status. METHODS: Autopsy samples from 13 HIV-infected subjects were examined for HIV-1 viral RNA (vRNA), and viral reverse transcriptase (RT) genotype was determined. All subjects had been clinically characterized using standard instruments before death. RESULTS: The median HIV-1 vRNA level in brain samples from subjects with moderate dementia was 7.79 log(10) copies/g (range 5.56 to 9.75 log(10) copies/g) compared with 5.44 log(10) copies/g (range 3.51 to 9.32 log(10) copies/g) for mildly demented subjects and 4.87 log(10) copies/g (3.51 to 6.86 log(10) copies/g) for those obtained from nondemented individuals. There were differences between subjects with moderate dementia and nondemented subjects (p = 0.0002) and between subjects with moderate and mild dementia (p = 0.0128). No significant differences among the groups were observed for vRNA levels in peripheral tissues. Some demented subjects had relatively low levels of HIV-1 vRNA, and paradoxically some nondemented subjects had high vRNA brain levels. Little subject effect in vRNA was noted in peripheral regions, but high regional variation in vRNA was noted within the brain. Patterns of the major zidovudine (ZDV) RT mutations in brain and peripheral tissues were concordant in most subjects. Subjects with longer duration of exposure to ZDV tended to have lower brain vRNA levels and a greater number of RT mutations than those with limited to no exposure. CONCLUSIONS: The presence and severity of HIV dementia correlates with the levels of productive HIV replication within the brain. Other pathophysiologic events (including macrophage activation) probably also contribute to neurologic dysfunction.

Substitution of a commercially available, RNA extraction procedure in an HIV-1 genotyping system improves sensitivity and allows reduced sample volume.

The use is described of a commercially available, silica-based extraction procedure for HIV-1 RNA that can be substituted for the extraction procedure supplied with a commercially available, PCR-based genotyping kit for HIV-1. The advantages of using this alternative, commercially available extraction procedure include the following: (1) reduced safety concerns, due to inactivation of virus in the initial step of the extraction procedure; (2) enhanced sensitivity, allowing the use of plasma with HIV-1 RNA levels of less than 2000 copies/ml; (3) improved yield, allowing a 60% reduction in plasma volume; and (4) convenience and improved reproducibility, with a single extraction providing RNA suitable for PCR-based sequencing and for quantitation of HIV-1 RNA levels.

HIV-1 reverse transcriptase sequence in plasma and cerebrospinal fluid of patients with AIDS dementia complex treated with Abacavir.

OBJECTIVE: To assess HIV-1 RNA levels and the relationship between HIV-1 reverse transcriptase (RT) genotype from plasma and cerebrospinal fluid (CSF) during treatment with abacavir (Ziagen, ABC) or placebo in combination with stable background therapy (SBG) in subjects with AIDS dementia complex (ADC) (study CNA3001). DESIGN: One-hundred and five HIV-1 infected adults with ADC were randomized to receive either ABC (600 mg twice daily) or ABC-matched placebo (twice daily) in addition to SBG for 12 weeks. METHODS: Plasma and CSF were collected for population sequencing at baseline and week 12 (CSF optional). Sequences were analyzed for mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTI). RESULTS: Sixty out of sixty-seven subjects with baseline plasma HIV-RT sequence data harbored virus with > or = 1 NRTI-associated mutations; 50 out of 67 had the M184V mutation. At week 12, more subjects in the ABC group had plasma HIV-1 RNA < or = 400 copies/ml than the SBG group (46% versus 13%, P = 0.002). Non-response to ABC was associated with multiple baseline zidovudine (ZDV)/stavudine (d4T)-associated mutations. Baseline RT mutation patterns differed in 14 out of 21 (67%) paired samples from plasma and CSF. Four subjects experienced > 1 log10 copies/ml reductions in CSF HIV-1 RNA, two in the absence of reductions in plasma HIV-1 RNA and two with undetectable plasma HIV-1 RNA at baseline. CONCLUSIONS: Substantial decreases in plasma and CSF HIV-1 RNA following addition of ABC were not precluded by baseline HIV-1 NRTI-associated mutations, including the M184V mutation, but non-responders commonly harbored multiple ZDV/d4T-associated mutations. HIV-1 RNA responses and RT genotype appear to be discordant between CSF and plasma in some subjects.

The efficacy of potent anti-retroviral drug combinations tested in a murine model of HIV-1 encephalitis.

Development of anti-retroviral regimens with enhanced efficacy against brain HIV-1 is essential if viral eradication is to be achieved. To address this, a severe combined immune deficiency mouse model of HIV-1 encephalitis was used to assay the effect of protease-containing and protease-sparing drug regimens on viral replication in brain macrophages. Here, HIV-1-infected human monocyte-derived macrophages (MDM) are inoculated into basal ganglia, causing a multinucleated giant cell encephalitis reminiscent of human disease. Drugs were administered at the time of MDM inoculation and continued until sacrifice. Immunohistochemical tests evaluated ongoing viral replication, glial immunity, and neuronal survival. Treatment with ddI/d4T decreased the numbers of infected cells by 75%, while ddI/d4T/amprenavir or ZDV/3TC/ABC diminished infection by 98%. Triple drug regimens decreased astrogliosis by > or = 25%. This small-animal model may be used to screen drug regimens that affect ongoing HIV-1 replication within its brain sanctuary.

Evaluation of a second-generation nucleic acid sequence-based amplification assay for quantification of HIV type 1 RNA and the use of ultrasensitive protocol adaptations.

Accurate assessment of plasma HIV RNA levels at low concentrations is clinically important. We evaluated a second-generation quantitative HIV RNA assay (NucliSens HIV-1 QT), and three simple adaptations of the NucliSens standard protocol to lower the lower cutoff level. The assays were evaluated in constructed panels with known HIV RNA concentrations and in clinical samples. Results were compared with those obtained with the first generation (NASBA HIV-1 QT) and with two other commercially available assays: the Amplicor HIV Monitor test and the Quantiplex assay. In a constructed panel, results obtained by NASBA QT were on average 0.13 log(10) copies/ml (SD 0.15) higher than those of NucliSens. The NucliSens assay could quantify HIV RNA in at least 50% of the samples down to 518 (2.71 log(10)) copies/ml and NASBA QT to 5.80 x 10(3) (3.76 log(10)) copies/ml). Both assays correlated well with the known input (R NucliSens = 0.99; R NASBA QT = 0.996), but results were more variable at lower input levels. With the three different ultrasensitive NucliSens adaptations, HIV RNA could be quantified in at least 50% of the samples down to 100 (2.00 log(10)), 46 (1.66 log(10)), and 10 (1.00 log(10)) copies/ml, respectively. In patient samples, Amplicor results were on average 0.11 (SD 0.20) log(10) copies/ml above, NucliSens 0.02 (SD 0.29) copies/ml above, and Quantiplex 0.13 (SD 0.19) copies/ml below the mean of the three assay results per sample. The variation remained the same over the range of RNA levels with all three assays. The NucliSens assay can quantify HIV RNA at lower levels than the NASBA QT and is comparable to other commercially available assays. The lower cutoff of the NucliSens can be lowered down to 10 copies/ml.

Evaluation of antiretroviral drug efficacy for HIV-1 encephalitis in SCID mice.

OBJECTIVES: To compare the efficacy of the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), and stavudine (d4T) to inhibit viral replication in brain macrophages. A severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE) was used to monitor spreading viral infection in the CNS. BACKGROUND: The development of antiretroviral therapies with CNS efficacy against neuroinvasive virus is important if eradication of HIV-1 can be achieved within critical "hidden reservoirs." METHODS: HIV-1-infected human monocyte-derived macrophages (MDMs) (after a single round of viral replication) were inoculated into the caudate and putamen of SCID mice. This resulted in the spreading of viral infection with a concomitant multinucleated giant cell encephalitis (astrogliosis, microglial activation, and neuronal injury). NRTIs were administered to animals at the time of intracerebral MDM inoculations and continued until the time of sacrifice. Antiretroviral effects were assessed by viral load and percentages of infected MDMs. RESULTS: In brains of SCID mice with HIVE, abacavir and lamivudine reduced HIV-1 p24 antigen-positive cells by 80% and 95%, respectively, whereas both decreased viral load by approximately 1 log. Zidovudine, didanosine, and stavudine showed variable effects. CONCLUSION: Abacavir and lamivudine showed significant antiretroviral activity in SCID mice with HIVE when compared with other NRTIs. The extrapolation of these results to humans with HIV-1 dementia awaits future investigations.

Presence of mutation conferring resistance to lamivudine in plasma and cerebrospinal fluid of HIV-1-infected patients.

To explore the relation between levels of HIV-1 in the cerebrospinal fluid (CSF) and plasma, simultaneously obtained samples from twelve patients who were receiving or had received lamivudine were examined. HIV-1 RNA levels were measured by nucleic acid sequence-based amplification procedure (NASBA). HIV-1 pol gene was amplified and sequenced. Median plasma and CSF HIV-1 RNA levels were 4.98 log and 2.93 log respectively. In total, 5 patients had CSF levels <100 copies/ml. A significant correlation between plasma and CSF HIV-1 RNA levels was found; 3 patients with disproportionately elevated CSF HIV-1 RNA levels had clinical evidence of central nervous system disease. Genotypic analysis was available for 9 plasma/CSF pairs. In 4, the lamivudine (3TC)-resistance mutation M184V was found in both CSF and plasma. Discordance was found in 2 pairs. In both, M184V was found in plasma but not CSF. These data suggest that CSF and plasma levels of HIV-1 may be correlated.

Characterization of viral dynamics in human immunodeficiency virus type 1-infected patients treated with combination antiretroviral therapy: relationships to host factors, cellular restoration, and virologic end points.

Biphasic plasma viral decays were modeled in 48 patients treated with ritonavir, zidovudine, and lamivudine. Estimated first- and second-phase decay rates were d1 as 0.47/day and d2 as 0.04/day. Interpatient differences in both decay rates were significant. The d1 was directly correlated with baseline CD4+, CD4+CD28+, and CD8+CD28+ T lymphocyte counts (P<.05) and inversely correlated with baseline virus load (P=.044) and the magnitude of CD4+ and CD8+ T lymphocyte recovery (P<.01). The d2 was directly correlated with baseline percentage of CD8+ T lymphocytes (P=.023), the CD8+CD38+ cell number (P=.024), and the level of IgG that binds to human immunodeficiency virus (HIV) type 1 gp120 (P=.02). Viral decay rates were not predictive of treatment failure or durability of viral suppression. These exploratory findings are consistent with a model in which immunologic factors contribute to elimination of HIV-infected cells and suggest a dynamic interplay between regulation of HIV expression and lymphocyte activation and recovery.

Beta-chemokines MCP-1 and RANTES are selectively increased in cerebrospinal fluid of patients with human immunodeficiency virus-associated dementia.

Human immunodeficiency virus-associated dementia (HAD) is associated with increased numbers of activated central nervous system (CNS) macrophages. Chemokines, which regulate infiltration of macrophages, were measured in the cerebrospinal fluid (CSF) of human immunodeficiency virus (HIV)-negative and HIV-positive individuals with and without neurological disease. Monocyte chemotactic protein (MCP)-1 and RANTES (but not MCP-3), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, or interleukin-8 (IL-8) was higher in HAD. MCP-1 correlated with CSF viral load and severity of dementia, and it increased over time in patients who developed dementia.

Evaluation of surrogate markers and clinical outcomes in two-year follow-up of eighty-six human immunodeficiency virus-infected pediatric patients.

OBJECTIVES: To evaluate the prognostic value of surrogate markers (HIV RNA copy number, CD4 counts and CDC clinical and immunologic categories) in HIV-infected children through a 2-year period. METHODS: Eighty-six HIV-infected children followed by the Duke Pediatric HIV Clinic in the fall of 1994 were evaluated for plasma HIV RNA concentration (viral load), CD4 lymphocyte percentage, age, antiretroviral treatment status and CDC clinical and immunologic categories. Follow-up evaluations were performed for approximately 2 years, and the time to progression to a new CDC category C diagnosis or death was noted. RESULTS: Of 86 children 22 had progression to new Category C diagnosis or death. Seven children died, 17 had a new Category C diagnosis and 2 had both. Among children who progressed, the median CD4 percentage at entry was 3% (absolute count, 75 cells/mm3), whereas children who had no disease progression entered with a median of 29% (868 cells/mm3). The overall median viral load at study entry was 4.58 log10 copies/ml (38,019 copies/ml, with a range of 1.7 to 6.78 logs). Children who had no disease progression had a median log copy number of 4.43, whereas 5.18 was the median for children whose disease progressed. Log copy number declined over time in children < 3 years of age, whereas it remained fairly consistent for children 3 years or older. Progression rates were determined by entry plasma HIV RNA concentration quartiles [quartile boundaries < 4.18, 4.58, > 5.08 log RNA copy/ml (< 15,136, 38,019 and > 120,226 copies/ml, respectively)]. Progression rates by quartile were 0 of 21, 4 of 22, 5 of 21 and 13 of 22. Kaplan-Meier survival curves defined by CD4% less than or greater than 15 and log RNA less than or greater than 5.0 (100,000) revealed that patients with CD4% less than 15 and plasma HIV RNA concentration > 5 log10 copies/ml did least well: 11 of 12 (92%) had a progression event at a median of 179 days. Patients with a high CD4 percentage and high viral load, or a low CD4 percentage and low viral load did similarly; 5 of 14 (36%) and 4 of 12 (33%) had progression events, respectively. Patients with high CD4 percentage and low viral load did best: only 2 of 48 (4%) had a progression event. CONCLUSIONS: The two most significant prognostic indicators of disease progression were the initial CD4 percentage and the plasma HIV RNA concentration, and a combination of CD4 percentage and virus load best predicted which children had progression events. Progression was less common in children who had < 100,000 HIV RNA copies/ml initially (6 of 60 vs. 16 of 26; P < 0.001; relative risk 0.16). Therefore it seems reasonable that in a child for whom complete suppression is not possible, a threshold of 100,000 (5 log10 copies/ml) can be used to mandate a change in therapy.

Relationship between human immunodeficiency virus-associated dementia and viral load in cerebrospinal fluid and brain.

Cerebrospinal fluid (CSF) human immunodeficiency virus (HIV) RNA levels were measured with the Nucleic Acid Sequence-Based Amplification (NASBA) assay to determine the relationship with neurological status; 37 subjects with HIV dementia (HIV-D) were compared with 77 with HIV with minor neurological signs (HIV-MCMD) and 93 neurologically normal HIV-seropositive individuals (HIV-NML). The NASBA assay had a lower limit of detection of 100 copies per milliliter. Mean CSF log HIV RNA levels were significantly higher in those with dementia after adjusting for CD4 count and were correlated with dementia severity. Plasma levels did not distinguish comparably immunosuppressed subjects with or without dementia. CSF and plasma RNA levels were significantly intercorrelated for subjects with CD4 counts <200/mm3 and also correlated inversely with CSF beta2-microglobulin. CSF RNA levels were independent of CSF pleocytosis or antiretroviral exposure. Brain RNA levels were consistently higher than CSF but correlated with CSF values for dementia subjects. The NASBA assay can be used reliably to determine HIV RNA levels in CSF, brain, and plasma samples. CSF HIV RNA may be a surrogate marker for brain infection, based on the observed correlation with brain levels. The association between plasma HIV RNA and CSF levels of HIV and beta2-microglobulin suggests that both viral load and CNS immune activation are important determinants of neurological disease.

Selective inhibition of topoisomerases from Pneumocystis carinii compared with that of topoisomerases from mammalian cells.

Type I and II topoisomerase activities were partially purified from Pneumocystis carinii. The catalytic (strand-passing) activities of both enzymes were selectively inhibited by members of a series of dicationic-substituted bis-benzimidazoles compared with those of topoisomerases of mammalian (calf thymus) origin. The most active inhibitors of the parasite enzymes were also highly effective in an in vivo animal model of P. carinii pneumonia. Selected dicationic-substituted bis-benzimidazoles also strongly inhibited the induction of the topoisomerase I- and II-mediated cleavable complex, suggesting that the biologically active DNA minor groove-binding molecules inhibit the enzyme-DNA binding step of the topoisomerase reaction sequence. The apparent selectivities for the parasite enzymes and the low levels of toxicity to mammalian cells for the biologically active bis-benzimidazoles suggest that these compounds hold promise as effective therapeutic agents in the treatment of a life-threatening AIDS-related disease, P. carinii pneumonia.