Plasma RNA Load Research Articles

SARS-CoV-2 RNAemia as a reliable predictor of long-term mortality among older adults hospitalized in pulmonary intermediate care units: a prospective cohort study

BackgroundSARS-CoV-2 viremia is associated with disease severity and high risk for in-hospital mortality. However, the impact of SARS-CoV-2 viremia on long-term outcomes in hospitalized patients with COVID-19 is poorly understood.MethodsWe conducted a prospective cohort study and recruited a group of older adult patients with COVID-19 admitted to pulmonary intermediate care units of Peking University Third Hospital during December 2022 and January 2023. The plasma level of SARS-CoV-2 RNA was determined by a standardized RT-PCR technique, and SARS-CoV-2 RNAemia was defined as a plasma viral load ≥ 50 copies/ml. In-hospital and follow-up (180-day) outcome data were collected.ResultsA total of 101 patients with an average of 80.4 years were recruited, and 63.4% of them were severe or very severe cases. Twenty-eight patients (27.7%) had SARS-CoV-2 RNAemia, with a median viral RNA load of 422.1 [261.3, 1085.6] copies/ml. Patients with SARS-CoV-2 RNAemia were more likely to develop critical cases and had a higher incidence of sepsis. Accordingly, they had a higher 180-day mortality (57.1% vs. 19.7%, P < 0.001), as well as in-hospital mortality (50.0% vs. 13.7%, P < 0.001), independent of age, disease severity, sepsis, lymphocyte count and C-Reactive protein. In addition, the risk for 180-day mortality increased with the SARS-CoV-2 RNA load in plasma. Plasma cytokines, including IL-6, IL-8 and IL-10, were higher in patients with SARS-CoV-2 RNAemia.ConclusionsOur study indicates that SARS-CoV-2 RNAemia serves as a useful biomarker for predicting mortality, especially long-term mortality, in older adult patients hospitalized in pulmonary intermediate care units.Trial registrationChinese Clinical Trial Registry website (No. ChiCTR2300067434).

Effect of viral storm in patients admitted to intensive care units with severe COVID-19 in Spain: a multicentre, prospective, cohort study

The contribution of the virus to the pathogenesis of severe COVID-19 is still unclear. We aimed to evaluate associations between viral RNA load in plasma and host response, complications, and deaths in critically ill patients with COVID-19. We did a prospective cohort study across 23 hospitals in Spain. We included patients aged 18 years or older with laboratory-confirmed SARS-CoV-2 infection who were admitted to an intensive care unit between March 16, 2020, and Feb 27, 2021. RNA of the SARS-CoV-2 nucleocapsid region 1 (N1) was quantified in plasma samples collected from patients in the first 48 h following admission, using digital PCR. Patients were grouped on the basis of N1 quantity: VIR-N1-Zero (<1 N1 copies per mL), VIR-N1-Low (1-2747 N1 copies per mL), and VIR-N1-Storm (>2747 N1 copies per mL). The primary outcome was all-cause death within 90 days after admission. We evaluated odds ratios (ORs) for the primary outcome between groups using a logistic regression analysis. 1068 patients met the inclusion criteria, of whom 117 had insufficient plasma samples and 115 had key information missing. 836 patients were included in the analysis, of whom 403 (48%) were in the VIR-N1-Low group, 283 (34%) were in the VIR-N1-Storm group, and 150 (18%) were in the VIR-N1-Zero group. Overall, patients in the VIR-N1-Storm group had the most severe disease: 266 (94%) of 283 patients received invasive mechanical ventilation (IMV), 116 (41%) developed acute kidney injury, 180 (65%) had secondary infections, and 148 (52%) died within 90 days. Patients in the VIR-N1-Zero group had the least severe disease: 81 (54%) of 150 received IMV, 34 (23%) developed acute kidney injury, 47 (32%) had secondary infections, and 26 (17%) died within 90 days (OR for death 0·30, 95% CI 0·16-0·55; p<0·0001, compared with the VIR-N1-Storm group). 106 (26%) of 403 patients in the VIR-N1-Low group died within 90 days (OR for death 0·39, 95% CI 0·26-0·57; p<0·0001, compared with the VIR-N1-Storm group). The presence of a so-called viral storm is associated with increased all-cause death in patients admitted to the intensive care unit with severe COVID-19. Preventing this viral storm could help to reduce poor outcomes. Viral storm could be an enrichment marker for treatment with antivirals or purification devices to remove viral components from the blood. Instituto de Salud Carlos III, Canadian Institutes of Health Research, Li Ka-Shing Foundation, Research Nova Scotia, and European Society of Clinical Microbiology and Infectious Diseases. For the Spanish translation of the abstract see Supplementary Materials section.

Low anti‐SARS‐CoV‐2 S antibody levels predict increased mortality and dissemination of viral components in the blood of critical COVID‐19 patients

BackgroundAnti‐SARS‐CoV‐2 S antibodies prevent viral replication. Critically ill COVID‐19 patients show viral material in plasma, associated with a dysregulated host response. If these antibodies influence survival and viral dissemination in ICU‐COVID patients is unknown.Patients/MethodsWe studied the impact of anti‐SARS‐CoV‐2 S antibodies levels on survival, viral RNA‐load in plasma, and N‐antigenaemia in 92 COVID‐19 patients over ICU admission.ResultsFrequency of N‐antigenaemia was >2.5‐fold higher in absence of antibodies. Antibodies correlated inversely with viral RNA‐load in plasma, representing a protective factor against mortality (adjusted HR [CI 95%], p): (S IgM [AUC ≥ 60]: 0.44 [0.22; 0.88], 0.020); (S IgG [AUC ≥ 237]: 0.31 [0.16; 0.61], <0.001). Viral RNA‐load in plasma and N‐antigenaemia predicted increased mortality: (N1‐viral load [≥2.156 copies/ml]: 2.25 [1.16; 4.36], 0.016); (N‐antigenaemia: 2.45 [1.27; 4.69], 0.007).ConclusionsLow anti‐SARS‐CoV‐2 S antibody levels predict mortality in critical COVID‐19. Our findings support that these antibodies contribute to prevent systemic dissemination of SARS‐CoV‐2.

Multiscale model of hepatitis C virus dynamics in plasma and liver following combination therapy.

This work explores the application of a physiologically structured population (PSP) framework in modeling hepatitis C virus (HCV) kinetics. To do so, a model was developed for the viral RNA load in plasma and liver as observed in 15 patients treated with a combination therapy of pegylated interferon, ribavirin, and telaprevir. By including both intracellular and extracellular processes of the HCV lifecycle, the model provided a description of the treatment effect on the intracellular HCV lifecycle. Combining PSP models with a nonlinear mixed effects approach in a single model permits a natural inclusion of the direct‐acting antiviral effect on intracellular processes, which can then be integrated with the viral kinetics within the host while accounting for the interindividual variability between patients. This should allow an exploration of the treatment effect within the entire chronic HCV‐infected population.

Koala retrovirus (KoRV) subtypes and their impact on captive koala (Phascolarctos cinereus) health.

Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the impact of infection with multiple subtypes is not well understood. Accordingly, in this study, we surveyed a representative sample from a Japanese zoo population to determine the infection status for three KoRV subtypes (KoRV-A, B, and C) and to investigate the proviral and RNA load profiles in animals with single- and multiple-subtype infections, using peripheral blood mononuclear cells (PBMCs) and plasma. Six koalas were evaluated in the study; all were infected with KoRV-A, and two koalas were coinfected with non-A subtypes (KoRV-B and/or KoRV-C). The highest KoRV total RNA and viral loads in PBMCs and plasma were found in a koala infected with multiple subtypes (KoRV-A, -B and -C). The other koala infected with multiple subtypes (KoRV-A and B) showed the highest proviral PBMC load but the lowest RNA copy number in PBMC and plasma. PBMCs from this animal were cultured for further investigation, and KoRV RNA was detected in the cells and culture supernatant after 7 and/or 14 days. The koalas harboring multiple subtypes had a higher white blood cell count than those harboring only KoRV-A and were judged to be leukemic, and they subsequently died due to lymphoma. Accordingly, we conclude that coinfection with multiple KoRV subtypes may be linked to more-severe disease. In a sequence alignment, the detected KoRV-A env gene showed 100% sequence identity to the reference gene, whereas the KoRV-B and -C env genes varied from their reference sequences.

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19

BackgroundCOVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response.MethodsA total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients.ResultsThe frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183–12.968], 0.025), viral RNA load (N1) (1.962 [1.244–3.096], 0.004); viral RNA load (N2) (2.229 [1.382–3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia).ConclusionsSARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.

Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo.

Combined effect of Vacc-4x, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial

Immune priming before reversal of latency might be a component of a functional HIV cure. To assess this concept, we assessed if therapeutic HIV immunisation followed by latency reversal would affect measures of viral transcription, plasma viraemia, and reservoir size in patients with HIV on suppressive antiretroviral therapy. In this single-arm, phase 1B/2A trial, we recruited adults treated at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark (aged ≥18 years) with successfully treated HIV-1 with plasma RNA loads of less than 50 copies per mL for the previous year and CD4 counts of at least 500 cells per μL. Exclusion criteria included CD4 counts of less than 200 cells per μL within the past 2 years, active hepatitis B or C infections, and clinically significant cardiac disease, including QTc prolongation. Participants received six therapeutic intradermal HIV-1 immunisations with 12 mg/mL Vacc-4x and 0·6 mg/mL rhuGM-CSF over 12 weeks (at 0 weeks, 1 week, 2 weeks, 3 weeks, 11 weeks, and 12 weeks) before receiving 5 mg/m(2) intravenous romidepsin once a week for 3 weeks. This procedure was followed by analytical treatment interruption. Coprimary outcomes were changes in copies of HIV-1 DNA (total and integrated) per million CD4 T cells and infectious units per million (IUPM) resting memory CD4 T cells established by viral outgrowth, assessed in all patients receiving at least one dose of active treatment with assessable data. We assessed total HIV-1 DNA at screening, before romidepsin treatment, and 6 weeks after romidepsin treatment. We assessed integrated viral DNA at baseline, before romidepsin treatment, and 8 weeks after romidepsin treatment. We assessed IUPM at screening, 2 weeks before romidepsin treatment, and 6 weeks after romidepsin treatment. This trial is registered at ClinicalTrials.gov, number NCT02092116. Between May 19, 2014, and Oct 8, 2014, we enrolled 20 individuals, of whom 17 completed all Vacc-4x and rhuGM-CSF administrations and romidepsin infusions. 16 of 17 had assessable total HIV-1 DNA, 15 of 17 had assessable integrated HIV-1 DNA, and six of 17 had assessable IUPM at baseline and at one or more timepoints after study treatment. Total HIV-1 DNA declined from screening to 6 weeks after romidepsin treatment (mean reduction 39·7%, 95% CI -59·7 to -11·5; p=0·012). The decrease in integrated HIV-1 DNA from baseline to 8 weeks after romidepsin treatment was not significant (19·2%, -38·6 to 6·3; p=0·123). Among the six assessable participants, the mean reduction in IUPM from screening to 6 weeks after romidepsin treatment was 38·0% (95% CI -67·0 to -8·0; p=0·019). Of 141 adverse events, 134 (95%) were grade 1 and seven (5%) were grade 2-3. This in-vivo combinatorial approach provides the first evidence for the feasibility of a combined shock and kill strategy, but also emphasises that further optimisation of this strategy is needed to achieve a sizeable effect on the latent reservoir that will translate into clinically measurable benefits for people living with HIV-1. Bionor Pharma, the Research Council of Norway, and SkatteFUNN.

Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome.

Why is Breast-feeding a risk factor for HIV-1 transmission despite HIV-1 infected mothers receiving combination ART (cART)?

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) is an important source of HIV-1 infection in infants, and can occur at any point, including in utero, at the time of delivery, and via breastfeeding. In the absence of intervention, such as the use of antiretroviral therapy (ART), MTCT of HIV-1 during pregnancy or delivery can affect approximately 15–25% of infants, with a further 5–20% of infants becoming infected postnatally during breastfeeding. The use of maternal ART while breastfeeding reduces the MTCT risk from 35% without any intervention to less than 5%. Although a significant improvement, 5% of infants continue to be infected.i Maternal factors that contribute to this continued risk of transmission despite taking ART include the method of feeding, breast health, RNA and DNA viral load in breast milk and plasma, and maternal nutrition. Infantfactors such as the disruption in the integrity of the gastrointestinal mucosal surfaces also contribute to an increased rate of MTCT.Aims: This literature review aimed to investigate why breastfeeding is still a risk factor despite HIV-1-infected mothers being on effective combination ART (cART)? This review will explore the associated factors that contribute to the increased risk of HIV-1 transmission to the infant via breastfeeding.

Koala retrovirus in free-ranging populations—prevalence

The prevalence of koala retrovirus (KoRV) provirus (DNA) and the average number of proviral insertions per cell vary in different free-ranging koala (Phascolarctos cinereus) populations across Australia. Populations in the northern states of Queensland and New South Wales have 100% proviral prevalence and mean proviral copy number of 140-165 per cell. In contrast, the proviral prevalence in the southern states of Victoria and South Australia differs among populations, with a mean prevalence in these states' mainland populations of 73% and 38%, respectively and with the prevalence on southern island populations ranging from 0-50%. The proviral load in southern populations is comparatively low, with some populations having an average of less than 1 proviral copy per cell. The KoRV RNA load in plasma shows a similar discordance between northern and southern populations, with consistently high loads in northern koalas (103 to 1010 RNA copies per ml plasma), and loads ranging from 0 to 102 copies per ml in southern KoRV provirus-positive koalas. The variation in KoRV proviral prevalence and the disparity in proviral and viral loads between northern and southern koalas may reflect different types of infection in the two populations (endogenous versus exogenous). Alternatively, it is possible that KoRV has been present for a longer time period in northern populations resulting in differences in the host-virus relationship.

Disrupted cerebral metabolite levels and lower nadir CD4+ counts are linked to brain volume deficits in 210 HIV-infected patients on stable treatmentpatients on stable treatment

Cognitive impairment and brain injury are common in people with HIV/AIDS, even when viral replication is effectively suppressed with combined antiretroviral therapies (cART). Metabolic and structural abnormalities may promote cognitive decline, but we know little about how these measures relate in people on stable cART. Here we used tensor-based morphometry (TBM) to reveal the 3D profile of regional brain volume variations in 210 HIV + patients scanned with whole-brain MRI at 1.5 T (mean age: 48.6 ± 8.4 years; all receiving cART). We identified brain regions where the degree of atrophy was related to HIV clinical measures and cerebral metabolite levels assessed with magnetic resonance spectroscopy (MRS). Regional brain volume reduction was linked to lower nadir CD4 + count, with a 1–2% white matter volume reduction for each 25-point reduction in nadir CD4 +. Even so, brain volume measured by TBM showed no detectable association with current CD4 + count, AIDS Dementia Complex (ADC) stage, HIV RNA load in plasma or cerebrospinal fluid (CSF), duration of HIV infection, antiretroviral CNS penetration-effectiveness (CPE) scores, or years on cART, after controlling for demographic factors, and for multiple comparisons. Elevated glutamate and glutamine (Glx) and lower N-acetylaspartate (NAA) in the frontal white matter, basal ganglia, and mid frontal cortex — were associated with lower white matter, putamen and thalamus volumes, and ventricular and CSF space expansion. Reductions in brain volumes in the setting of chronic and stable disease are strongly linked to a history of immunosuppression, suggesting that delays in initiating cART may result in imminent and irreversible brain damage.

Hantaan Virus RNA Load in Patients Having Hemorrhagic Fever With Renal Syndrome: Correlation With Disease Severity

To investigate the role of viral load in the pathogenesis of hemorrhagic fever with renal syndrome, the Hantaan virus RNA load in plasma from 101 patients was quantified, and the relationships between viral load and disease course, severity, and level of specific humoral immunity were analyzed. The viral load, detectable in 79 patients, ranged from 3.43 to 7.33 log10 copies/mL of plasma. In the early stage of disease, patients in severe/critical group were found to have higher viral loads than those in the mild/moderate group (5.90 vs 5.03 log10 copies/mL; P = .001), suggesting an association between Hantaan virus load and disease severity.

Using HIV viral load to guide treatment-for-prevention interventions

To provide evidence that HIV-1 RNA load can guide treatment-for-prevention interventions to mitigate the HIV epidemic. Some HIV-infected individuals maintain increased levels of HIV-1 RNA load after acute infection for an extended period of time, and can disproportionately contribute to the spread of HIV in the community. The recent HIV Prevention Trials Network 052 study has demonstrated 96% efficacy for initiation of early antiretroviral treatment (ART) in HIV-1 serodiscordant couples. The level of HIV-1 RNA load in plasma is the major biological predictor of virus transmission. HIV-infected individuals who maintain increased levels of HIV-1 RNA load, extended high viremics, can transmit virus at higher rates. Combinatorial ART decreases HIV replication, thus reducing rates of virus transmission. Identifying high viremics and placing them on ART seems an attractive strategy that has the potential to achieve both individual benefits by lowering risk for early onset of clinical AIDS and public health benefits by reducing HIV transmission. A key logistical challenge is to screen for high viremics among HIV-positive individuals. Efficacy of the modified treatment-for-prevention approach focused on high viremics is being evaluated in ongoing and upcoming clinical trials. If efficacious, such an approach could be used widely to mitigate and control the HIV epidemic.

Advances in HIV laboratory testing

Laboratory tests are readily available for the diagnosis of HIV infection. These are based on the detection of HIV-specific antibodies and HIV p24 antigen in combination screening assays, followed by confirmation by Western blot. Managing established HIV infection, including the use of antiretroviral drugs, has been facilitated by the use of nucleic acid tests that measure HIV RNA load in plasma or detect mutations associated with drug resistance. Quality assurance programs ensure high-level performance of HIV assays.

Comparative Evaluation of the ExaVir Load Version 3 Reverse Transcriptase Assay for Measurement of Human Immunodeficiency Virus Type 1 Plasma Load

In resource-limited settings, the virological monitoring of antiretroviral therapy is limited by high cost and the lack of infrastructure. The Cavidi ExaVir Load assay employs a simple and inexpensive enzyme-linked immunosorbent assay format to measure human immunodeficiency virus (HIV) reverse transcriptase activity, which correlates with plasma RNA load. The version 3 assay has been described as having improved precision and sensitivity. There are limited data on its performance relative to those of current real-time assays. Our objective was to compare HIV type 1 (HIV-1) RNA load measurement in plasma by ExaVir Load version 3 (designated ExaVir), Abbott M2000sp/M2000rt RealTime HIV-1 assay (designated RealTime), and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 version 1 assay (designated TaqMan). Plasma from 119 patients (34 with subtype B infection, 85 with non-subtype B infection [A-H, CRF01, CRF02, CRF06, CRF12, CRF14, and complex]; 48 subjects were treatment experienced, 71 were naive) and serial dilutions of the second international standard (IS) were tested. Assay relationship and agreement were determined by linear regression, correlation analysis, and the Bland-Altman method. The ExaVir assay quantified 77/83 (92.8%) samples with viral loads of >2.3 log10 copies/ml by the molecular assays. Results were linearly associated and strongly correlated with RealTime and TaqMan measurements (R of 0.94 and 0.92, respectively) for both subtype B (R of 0.97 and 0.95, respectively) and non-subtype B (R of 0.93 and 0.91, respectively) samples. Mean differences were 0.28 and 0.18 log10 copies/ml in favor of the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples were outside the 95% level of agreement. ExaVir underquantified the IS by a mean of 0.2 (range, 0.0 to 0.5) log10 copies/ml. The ExaVir assay showed excellent concordance with real-time molecular assays, offering a suitable option for virological monitoring in settings with limited infrastructure.

Correlation between Human Immunodeficiency Virus Type 1 (HIV-1) RNA Measurements Obtained with Dried Blood Spots and Those Obtained with Plasma by Use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV Load Tests

The plasma human immunodeficiency virus (HIV) RNA load is used in the clinical routine for the monitoring of HIV infection and the patient's response to antiretroviral therapy. Other body fluids or dried blood spots (DBS) can be used, however, to assess the level of viremia. The use of DBS may be especially helpful for the monitoring of HIV-infected patients in resource-poor settings, where access to adequate laboratory facilities is often difficult. However, the correlation between the HIV RNA levels in plasma and those in DBSs has not been well established. Paired plasma and DBS samples obtained from HIV type 1 (HIV-1)-infected patients were tested for HIV RNA copy numbers by using two different commercial assays, the Nuclisens EasyQ HIV-1 (version 1.1) test (the Nuclisens test; Biomerieux) and the m2000rt RealTime HIV test (the m2000rt test; Abbott). Nucleic acid extraction was performed manually by using either the Nuclisens isolation kit (which uses the Boom methodology) or the m2000rt sample preparation kit (an iron particle-based method). A total of 103 paired plasma and DBS samples were tested. Viral load results were obtained for 97 (94.2%) samples with the Nuclisens isolation kit and 81 (78.6%) samples with the m2000rt kit. The overall correlation between the RNA loads in plasma and DBS was good, although better results were obtained by the Nuclisens test (R(2) = 0.87, P < 0.001) than by the m2000rt test (R(2) = 0.70, P < 0.001). While the specificities were excellent and similar for both the Nuclisens and the m2000rt tests (97.1% and 100%, respectively), the sensitivity was greater by the Nuclisens test than by the m2000rt test (75.8% and 56.6%, respectively). Overall, the viral loads in DBS tended to be lower than those in plasma, with mean differences of 0.3 log unit (standard deviation, 0.5 log unit) and 0.76 log unit (standard deviation, 0.8 log unit) for the Nuclisens and the m2000rt tests, respectively. The levels of agreement between the measurements in plasma and DBS were assessed by using the Bland-Altman plot for each assay. The Nuclisens test gave results within its defined limits (-0.65 to 1.26) for 95.9% of the samples, while the m2000rt test gave results within its limits (-0.83 to 2.33) for 100% of the samples. In summary, the HIV-1 load can accurately be quantified by testing DBS by either the Nuclisens or the m2000rt test, although the Nuclisens test may outperform the m2000rt test when nucleic acids are extracted manually.

Factors Associated with HIV-1 Proviral DNA Loads in Patients with Undetectable Plasma RNA Load

To evaluate factors associated with human immunodeficiency virus type 1 (HIV-1) proviral DNA load, we conducted a cross-sectional study of 36 chronically HIV-1-infected individuals with undetectable plasma viral RNA. We used real-time polymerase chain reaction to determine the number of HIV-1 proviral DNA copies per 106 peripheral blood mononuclear cells. The mean level of plasma viral RNA when the CD4+ T cell count was above 500 cells/µL without highly active antiretroviral therapy (HAART) was significantly associated with proviral DNA load at the time of undetectable plasma HIV RNA with HAART. Strategies to reduce the level of plasma viral RNA when patients' CD4+ T cell counts are above 500 cells/µL without HAART could help reduce HIV-1 proviral DNA load.

Correlation Between HIV-1 RNA Load in Blood and Seminal Plasma Depending on Antiretroviral Treatment Status, Regimen and Penetration of Semen by Antiretroviral Drugs&amp;#167;

To assess the correlation between HIV-1 RNA load in blood and semen by antiretroviral therapy status and the relative penetration of antiretroviral drugs in seminal plasma. We performed a cross-sectional cohort study of 119 HIV-1 subjects divided into three groups according to treatment status. Blood and semen were collected concurrently. Seminal viral load determined by NucliSens HIV-1 QT PCR (BioMerieux). Viral suppression over time was assessed in a second semen sample collected from 10 treated subjects. Antiretroviral plasma concentrations were measured by high performance liquid chromatography and recovery experiments were performed on semen samples to validate quantitation in this matrix. All subjects taking non nucleoside reverse transcriptase inhibitors (n = 36, mean treatment 33 months +/- 14) or protease inhibitors (n = 45, mean treatment 31 months +/- 25) had blood viral load < 50 copies/mL and seminal viral load < 250 copies/mL. In untreated subjects (n = 38), blood and semen viral loads were positively correlated (Spearman's rho = 0.489, p = 0.002). Blood and semen nevirapine concentrations were positively correlated (r(2) = 0.795, p = 0.005) and therapeutic concentrations achieved in both compartments. Lopinavir and atazanavir also penetrated semen but efavirenz did not. We find that there is compartmentalisation of HIV-1 within the male genital tract and propose that new infections may originate from untreated men and that suppressive antiretroviral regimens may reduce the risk of sexual transmission.

Highly Sensitive Methods Based on Seminested Real-Time Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 Unspliced and Multiply Spliced RNA and Proviral DNA

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.