Ultrasensitive Reverse Transcription-PCR Assay for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This “ultrasensitive” method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.

The measurement of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels has become an important tool for identifying individuals likely to benefit from antiretroviral therapy (12, 15, 16, 21, 26, 29) as well as monitoring patients on therapy (5, 6, 9, 12, 18, 20, 23) and is now regarded as standard medical practice for managing the treatment of HIV-1-infected individuals (1–4, 22, 25, 28). Recently, the use of combination therapy resulted in rapid and potent antiretroviral and immunological effects which lead to sharp declines in the plasma HIV-1 RNA concentration, frequently to an undetectable level (6, 18, 23). A more sensitive method with a lower detection limit for plasma HIV-1 RNA is therefore required.The AMPLICOR HIV-1 MONITOR Test, an in vitro nucleic acid amplification test for the quantitation of HIV-1 RNA in plasma, is intended to be used as an indicator of disease prognosis in conjunction with other laboratory markers and clinical presentation and as an aid in assessing the efficacy of antiretroviral therapy. The lower limit of quantitation of the AMPLICOR HIV-1 MONITOR Test is 400 RNA copies/ml of plasma (24). We introduce here a modified specimen preparation procedure (17, 27) that enhances the sensitivity of the standard MONITOR test. Increased sensitivity is obtained by increasing the input plasma volume by a factor of 2.5, performing high-speed centrifugation to concentrate the virus particles from the plasma, and reducing the final resuspension volume for the recovered nucleic acid by a factor of 4. If centrifugation yields 100% recovery of virus, this modified, ultrasensitive procedure should result in a 10-fold increase in the analytical sensitivity of the AMPLICOR HIV-1 MONITOR Test. We evaluated the sensitivity, specificity, linear range, reproducibility, and precision of the ultrasensitive test. We also analyzed the correlation between RNA concentrations measured by the ultrasensitive and the standard HIV-1 MONITOR Tests.     

Overestimation of Human Immunodeficiency Virus Type 1 Load Caused by the Presence of Cells in Plasma from Plasma Preparation Tubes

The human immunodeficiency virus type 1 (HIV-1) load is an important marker of disease progression and treatment efficacy in patients with HIV-1 infection. In recent years, an increase in the number of samples with detectable HIV-1 RNA has been reported among patients with previously suppressed viral loads, affecting clinical patient care and leading to repeat measurements of viral load and drug resistance. This rise seems to have coincided with the increased use of plasma preparation tubes (PPTs) for sample collection, and we have aimed to explain why PPTs might yield elevated HIV-1 RNA levels. The impacts of different sample-processing procedures on HIV-1 RNA levels were compared retrospectively. Prospectively, the presence of different cells and cell-associated HIV-1 nucleic acids in paired plasma samples from PPTs centrifuged before (PPT1) and after (PPT2) transportation to the laboratory was compared. A retrospective analysis of 4,049 patient samples with <1,000 HIV-1 RNA copies/ml showed elevated HIV-1 RNA levels in plasma from PPT1 compared with the levels from PPT2 and standard EDTA-containing tubes. Prospective data revealed cell-associated HIV-1 nucleic acids and abundant blood cells in plasma from PPT1 but not from the corresponding PPT2. The levels of HIV-1 RNA correlated with the lymphocyte counts in plasma in PPT1. Cells could be removed by the recentrifugation of PPT1 before analysis. In conclusion, the transportation of PPTs after centrifugation may render cells in the plasma fraction containing cell-associated HIV-1 nucleic acids that contribute significantly to the HIV-1 RNA copy numbers in patients with low viral loads.Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma is an essential tool in the clinical management of patients with HIV-1 infection and is used to monitor HIV-1 disease progression and the response to antiretroviral therapy (ART) (9, 12, 14, 15). It is also an important end point in most HIV-1 treatment clinical trials.Although intermittent low-level viremia is often reported among patients receiving seemingly effective ART (4, 13, 16, 23), the unexpected detection of HIV-1 RNA might have a clinical impact and lead to repeat quantification of HIV-1 RNA and testing for drug resistance. In recent years, several HIV-1 clinics and laboratories have reported an increased number of cases of detectable HIV-1 RNA in patients with previous suppressed viral loads, raising concerns about drug resistance and virologic failure (8, 17, 20). At the Oslo University Hospital, Ullevål, we have also experienced an unusual increase in the proportion of plasma samples with HIV-1 RNA levels in the range of 40 to 1,000 copies/ml. This seems to have coincided with a change in the routines for plasma sample collection, and here we present data providing an explanation for this phenomenon.It is recommended that blood samples for HIV-1 RNA quantification be collected in tubes with EDTA as an anticoagulant (3, 12). Standard EDTA-containing tubes require transfer of the plasma to a secondary tube within 6 h after sample collection to reduce the risk of RNA degradation (3). This is often inconvenient in a patient clinic, and as an alternative, plasma preparation tubes (PPTs; Becton Dickinson [BD], Franklin Lakes, NJ) are increasingly often used. Upon centrifugation of the PPTs, a gel barrier separates the plasma from most of the cellular elements, theoretically allowing transportation of the sample in the primary tube.The collection of samples in PPTs from patients receiving effective ART is reported to yield increased levels of HIV-1 RNA compared with the levels in plasma collected in standard EDTA-containing tubes (5, 6, 8, 17, 18, 20, 21). However, the reason for this discrepancy has remained unclear. Our data confirm the previous findings that PPTs generate significantly higher HIV-1 RNA levels in samples from patients with low-level viremia. Additionally, we have shown that the source of this overestimation is cell-associated HIV-1 nucleic acids. These findings necessitate a reconsideration of low-level viral load results in plasma obtained from PPTs not handled with care after centrifugation. Furthermore, we present a simple procedure that will circumvent the problem.     

Persistent Hijacking of Brain Proteasomes in HIV-Associated Dementia

Immunoproteasome induction sustains class 1 antigen presentation and immunological vigilance against HIV-1 in the brain. Investigation of HIV-1-associated alterations in brain protein turnover by the ubiquitin-proteasome system was performed by (1) determining proteasome subunit changes associated with persistent brain inflammation due to HIV-1; (2) determining whether these changes are related to HIV-1 neurocognitive disturbances, encephalitis, and viral loads; and (3) localizing proteasome subunits in brain cells and synapses. On the basis of neurocognitive performance, virological, and immunological measurements obtained within 6 months before death, 153 autopsy cases were selected. Semiquantitative immunoblot analysis performed in the dorsolateral prefrontal cortex revealed up to threefold induction of immunoproteasome subunits LMP7 and PA28α in HIV-1-infected subjects and was strongly related to diagnoses of neuropsychological impairment and HIV encephalitis. Low performance on neurocognitive tests specific for dorsolateral prefrontal cortex functioning domains was selectively correlated with immunoproteasome induction. Immunohistochemistry and laser confocal microscopy were then used to localize immunoproteasome subunits to glial and neuronal elements including perikarya, dystrophic axons, and synapses. In addition, HIV loads in brain tissue, cerebrospinal fluid, and blood plasma were robustly correlated to immunoproteasome levels. This persistent “hijacking” of the proteasome by HIV-1-mediated inflammatory response and immunoproteasome induction in the brain is hypothesized to impede turnover of folded proteins in brain cells. This would disrupt neuronal and synaptic protein dynamics, contributing to HIV-1 neurocognitive disturbances.People infected with HIV-1 are vulnerable to syndromes of neurocognitive impairment at a relatively young age, including HIV-associated dementia (HAD) and mild cognitive and motor disturbance (MCMD). Highly active antiretroviral therapy suppresses HIV-1 replication, prevents dementia, and prolongs survival, but does not eradicate HIV-1 infection.¹ Inflammation is the putative driving force behind MCMD and HAD.^2,3 HIV-1 enters the central nervous system (CNS) via infected macrophages and triggers inflammatory changes including the release of cytokines, neurotoxins, and toxic viral proteins. HIV-1 produces inflammatory changes neuropathologically that are known as HIV encephalitis (HIVE).⁴ HIVE and HAD are correlated with each other, which supports a proinflammatory mechanism for the pathophysiology of dementia in many, but not all cases.⁵Inflammation has an influence on protein turnover through the ubiquitin proteasome system (UPS).^6,7,8 The proteasome is a multicatalytic proteinase that is the main route of cellular protein degradation and turnover.⁹ Inflammatory mediators including interferon-γ (IFN-γ) and tumor necrosis factor α modify expression of proteasome subunits to promote the synthesis of the immunoproteasome complex (IPS).^{6,7,8,10,11,12,13,14,15} This causes switching from the synthesis of “standard” constitutive proteasome complexes (CPS), which process folded proteins through the UPS, to IPS complexes, which are specialized for processing unfolded polypeptides for class 1 antigen presentation in viral defense.^10,15 The “borrowing” of the UPS by IPS induction is not pathological to cells because it subsides quickly after an infected host eradicates the pathogen.⁷ Eradication of HIV-1 in the CNS, however, is not achieved and a vigilant immune defense must be maintained.^15,16,17 This persistent inflammatory drive in HIV/AIDS could exert a potentially harmful slowing of protein turnover through the UPS. That in turn could have a profound influence in the CNS because impairment of protein turnover interferes with synaptic function and impairs learning and memory formation.^18,19 A persistent slowing of protein turnover via the UPS probably leads to accumulation of misfolded ubiquitinylated proteins in pathological aging, which is a hallmark neuropathological change in neurodegenerative diseases.^{20,21,22,23,24,25,26,27} An increase in ubiquitin-protein conjugates was reported in HIV/AIDS brains that was associated with inflammation and altered synaptic protein content.²⁸ Here we report that HIV-1 infection exerts a strong influence on brain UPS that is associated with neurocognitive impairment and neuropathological changes.     

Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.