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Introduction: HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C. Methods: Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability. Results: The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2–6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region. Conclusions: We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions. [ABSTRACT FROM AUTHOR]

We report the selection of enfuvirtide-resistant human immunodeficiency virus type 1 in cerebrospinal fluid, resulting in subsequent loss of viral suppression in the plasma. This case report emphasizes the potential danger of low-level penetration of entry inhibitors into the central nervous system. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To investigate the mechanism explaining the persistence of human immunodeficiency virus (HIV) type 1 variants with multiple protease inhibitor (PI)-resistance mutations in the absence of PI therapy. METHODS: Longitudinal genotypic analyses were performed on sequential samples obtained from 2 HIV-1-infected patients who had stopped PI therapy for 4 years. Replication capacity (RC) was determined using recombinant viruses. Subsequently, the effect that changing individual protease mutations back to wild type has on RC was analyzed. RESULTS: We observed prolonged persistence (up to 4 years) of viruses with multiple protease mutations after PI therapy was stopped, despite the fact that the RC of the viruses was severely reduced. Forcing the virus to evolve toward wild type by changing individual protease mutations to wild type was unsuccessful, because all variants displayed a decreased RC in comparison with that of their predecessors. CONCLUSIONS: We propose compensatory fixation as a mechanism for the in vivo persistence of variants with multiple PI-resistance mutations in the absence of PI therapy. Viruses with multiple PI mutations have (partially) compensated for the initial loss in RC. Therefore, reversion of a single mutation causes a (further) reduction in RC and, as a consequence, the route to wild type is blocked. Copyright © 2007 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

BACKGROUND: Airway infection with influenza virus induces local expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which has been shown to enhance inflammatory mediator responses in vitro. Because secondary pneumococcal infections occurring shortly after recovery from influenza are associated with enhanced inflammatory responses, we hypothesized that IDO activity contributes to the enhanced response to bacterial challenges in mice previously infected with influenza virus. METHODS: On day 14 after influenza virus infection (with strain A/PR/8/34), C57Bl/6 mice were intranasally inoculated with 1 x 10(4) colony-forming units of S. pneumoniae (serotype 3). Matrix-driven delivery pellets that contained 70 mg of the IDO inhibitor 1-methyl-DL-tryptophan (MeTrp) released over a period of 7 days were subcutaneously implanted 48 h before pneumococcal infection. RESULTS: MeTrp treatment resulted in a 20-fold reduction in pneumococcal outgrowth 48 h after bacterial inoculation. Remarkably, pulmonary levels of interleukin-10 and tumor necrosis factor-alpha were significantly reduced in mice treated with MeTrp. CONCLUSIONS: Our data suggest that IDO expression during influenza virus infection alters the inflammatory response and facilitates the outgrowth of pneumococci during secondary bacterial pneumonia. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

During summer and fall, enterovirus infections are responsible for a considerable proportion of hospitalizations of young infants. We prospectively studied the incidence of enterovirus infections via real-time polymerase chain reaction (PCR) in blood, feces, and cerebrospinal fluid samples from infants ⩽ 60 days old who had received a clinical diagnosis of sepsis. Forty-five patients were included: 19 were admitted to the pediatric wards of 2 general hospitals, and 26 had been hospitalized since birth in the neonatal intensive care unit (NICU) of a tertiary care hospital. None of the NICU patients developed enteroviral disease. In contrast, an enterovirus was detected in 11 (58%) of the patients admitted to the 2 general hospitals, 10 of whom (53%) showed evidence of systemic infection. Enterovirus infections are an important cause of sepsis in infants admitted to the hospital. Real-time PCR in serum was a rapid and sensitive method for diagnosis of enterovirus infection. [ABSTRACT FROM AUTHOR]

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA. [ABSTRACT FROM AUTHOR]