[show abstract][hide abstract] ABSTRACT: 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.
Journal of clinical microbiology 04/2009; 47(5):1491-6. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several markers of immune activation have been identified as potential prognostic markers for human immunodeficiency virus (HIV)-associated morbidity and mortality, but the results from studies are conflicting.
To evaluate whether neurocognitive status and baseline levels of plasma and cerebrospinal fluid tumor necrosis factor alpha (TNF-alpha), macrophage chemoattractant protein 1 (MCP-1), matrix metalloproteinase 2 (MMP-2), or macrophage colony-stimulating factor (M-CSF) are associated with time to death in a cohort with advanced HIV infection.
Enrollees in the Northeast AIDS Dementia Study.
Three hundred twenty-nine subjects who were positive for HIV-1 and had a CD4 cell count of less than 200/microL (or <300/microL but with cognitive impairment at baseline) were assessed for CD4 cell count, neurocognitive status, pertinent demographic and clinical variables, and plasma and cerebrospinal fluid HIV RNA, TNF-alpha, MCP-1, MMP-2, and M-CSF levels.
Cox proportional hazards regression models were used to examine the associations between the variables of interest (using time-dependent covariates, where applicable) and time to death, adjusting for possible confounders.
There were 50 deaths in the cohort after a median of 25.2 months of follow-up. The cumulative incidences of death were 7% at 1 year and 16% at 2 years. In Cox proportional hazards regression analyses adjusting for demographic, clinical, and immunological variables, HIV-associated dementia (hazard rate, 6.10; P = .001) was significantly associated with time to death; (log) plasma MCP-1 level (hazard rate, 3.38; P = .08) trended toward significance.
In patients with advanced HIV infection, HIV-associated dementia is an independent predictor of time to death.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Clinical Microbiology 02/2007; 45(2):572-4. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Clinical Microbiology 07/2006; 44(6):2280-2. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
To assess the association between baseline markers of immune activation and HIV RNA levels (viral load) and time to symptomatic DSP (SDSP).
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: Before the introduction of combination antiretroviral therapy (CART), neurological disease correlated with cerebrospinal fluid (CSF) levels of human immunodeficiency virus (HIV) RNA.
To investigate the relationships among HIV RNA levels, immune activation markers, and neurological status in patients receiving CART.
Multicenter cohort study.
Academic neurology departments.
A total of 371 patients unselected for neurological complaints and with CD4 cell counts less than 200/microL or with cognitive symptoms and CD4 cell counts less than 300/microL were enrolled into the Northeastern AIDS Dementia cohort in 1998-2002. Diagnoses of HIV-associated dementia (HIV-D) and minor cognitive-motor disorder (MCMD) were obtained with a computerized algorithm. Plasma and CSF levels of HIV RNA, monocyte chemotactic protein 1, macrophage colony-stimulating factor, and tumor necrosis factor alpha were quantified.
The mean +/- SD age was 41.5 +/- 7.2 years, and the mean +/- SD educational level was 12.3 +/- 2.2 years. Seventy percent of the cohort was black, and 30% were women. The mean +/- SD CD4 cell count was 136.8 +/- 87.9/microL, and CART was used in 71%. Twenty-nine percent of the patients were unimpaired (n = 106), 36% had MCMD (n = 133), and 35% had HIV-D (n = 128). Mean log(10) CSF HIV RNA copies per milliliter was 2.6 +/- 0.8, with no differences among the neurological groups, even after adjustments for baseline CD4 cell counts and antiretroviral therapy. Cerebrospinal fluid HIV RNA was undetectable in 47% of unimpaired, 46% of MCMD, and 43% of HIV-D patients (P = .91). Plasma levels of monocyte chemotactic protein type 1 and tumor necrosis factor alpha correlated weakly with HIV RNA levels but did not distinguish those with neurological deficits.
In contrast to observations in individuals not treated with CART, we found no relationship between CSF markers and neurological status in this CART-using cohort with advanced HIV/AIDS. This was not explicable by demographic differences or plasma virological control. CART may substantially attenuate the degree of central nervous system HIV infection and immune activation, and in CART users, CSF HIV RNA and immune activation markers may fail to discriminate milder degrees of HIV-D and MCMD.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: To examine the association between HIV RNA levels, patterns of antiretroviral resistance, and neurologic status.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Virological Methods 08/2001; 96(1):1-4. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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).
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.
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).
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
AIDS Research and Human Retroviruses 11/2000; 16(15):1507-17. · 2.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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."
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
Annals of Neurology 12/1998; 44(5):831-5. · 11.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Annals of Neurology 12/1997; 42(5):689-98. · 11.19 Impact Factor