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Background: Dried blood spots (DBS) have been used as alternative specimens to plasma to increase access to HIV viral load (VL) monitoring and early infant diagnosis (EID) in remote settings. We systematically reviewed evidence on the performance of DBS compared to plasma for VL monitoring and EID., Methods and Findings: Thirteen peer reviewed HIV VL publications and five HIV EID papers were included. Depending on the technology and the viral load distribution in the study population, the percentage of DBS samples that are within 0.5 log of VL in plasma ranged from 52-100%. Because the input sample volume is much smaller in a blood spot, there is a risk of false negatives with DBS. Sensitivity of DBS VL was found to be 78-100% compared to plasma at VL below 1000 copies/ml, but this increased to 100% at a threshold of 5000 copies/ml. Unlike a plasma VL test which measures only cell free HIV RNA, a DBS VL also measures proviral DNA as well as cell-associated RNA, potentially leading to false positive results when using DBS. The systematic review showed that specificity was close to 100% at DBS VL above 5000 copies/ml, and this threshold would be the most reliable for predicting true virologic failure using DBS. For early infant diagnosis, DBS has a sensitivity of 100% compared to fresh whole blood or plasma in all studies., Conclusions: Although limited data are available for EID, DBS offer a highly sensitive and specific sampling strategy to make viral load monitoring and early infant diagnosis more accessible in remote settings. A standardized approach for sampling, storing, and processing DBS samples would be essential to allow successful implementation., Trial Registration: PROSPERO Registration #: CRD42013003621.

Increased life expectancy due to improved efficacy of cART has uncovered an increased risk of age-related morbidities in HIV+ individuals and catalyzed significant research into mechanisms driving these diseases. HIV infection increases the risk of non-communicable diseases common in the aged, including cardiovascular disease, neurocognitive decline, non-AIDS malignancies, osteoporosis, and frailty. These observations suggest that HIV accelerates immunological ageing, and there are many immunological similarities with the aged, including shortened telomeres, accumulation of senescent T cells and altered monocyte phenotype/function. However, the most critical similarity between HIV+ individuals and the elderly, which most likely underpins the heightened risk of non-communicable diseases, is chronic inflammation and associated immune activation. Here, we review the similarities between HIV+ individuals and the aged regarding the pathogenesis of inflammatory diseases, the current evidence for mechanisms driving these processes and discuss current and potential therapeutic strategies for addressing inflammatory co-morbidity in HIV+ infection.

Background: Viral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring., Methods and Findings: A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2-26% and 9-70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0-5.1%) and 5.44% (range 1.17-30.00%) across the range of VL counts (2log10-7log10)., Conclusions: This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same technology platform to ensure appropriate interpretation of changes in VL. Prospero registration # CD42013003603.

Chronic HIV infection is associated with increased risk of cardiovascular disease (CVD), including in patients with virological suppression. Persistent innate immune activation may contribute to the development of CVD via activation of monocytes in these patients. We investigated whether changes in monocyte phenotype predict subclinical atherosclerosis in virologically suppressed HIV-positive individuals with low cardiovascular risk. We enroled 51 virologically suppressed HIV-positive individuals not receiving protease inhibitors or statins and 49 age-matched uninfected controls in this study. Carotid artery intima-media thickness (cIMT) was used as a surrogate marker for CVD, and traditional risk factors, including Framingham risk scores, were recorded. Markers of monocyte activation (CD14, CD16, CCR2, CX3CR1, CD38, HLA-DR and CD11b) were measured in whole-blood samples by flow cytometry. Associations were assessed using univariate and multivariate median regressions. Median cIMT was similar between HIV-positive and HIV-negative participants (P=0.3), although HIV-positive patients had significantly higher Framingham risk score (P=0.009) and systemic inflammation. Expression of two monocyte markers, CD11b and CX3CR1, independently predicted carotid artery thickness in HIV-positive individuals after controlling for Framingham risk score (P=0.025 and 0.015, respectively). These markers were not predictive of carotid artery thickening in controls. Our study indicates that monocyte surface markers may serve as novel predictors of CVD in HIV-positive individuals and is consistent with an important role for monocyte activation in the progression of HIV-related cardiovascular pathology.

Objectives: Glucose metabolism plays a fundamental role in supporting the growth, proliferation and effector functions of T cells. We investigated the impact of HIV infection on key processes that regulate glucose uptake and metabolism in primary CD4 and CD8 T cells., Design and Methods: Thirty-eight HIV-infected treatment-naive, 35 HIV+/combination antiretroviral therapy, seven HIV+ long-term nonprogressors and 25 HIV control individuals were studied. Basal markers of glycolysis [e.g. glucose transporter-1 (Glut1) expression, glucose uptake, intracellular glucose-6-phosphate, and L-lactate] were measured in T cells. The cellular markers of immune activation, CD38 and HLA-DR, were measured by flow cytometry., Results: The surface expression of the Glut1 is up-regulated in CD4 T cells in HIV-infected patients compared with uninfected controls. The percentage of circulating CD4Glut1 T cells was significantly increased in HIV-infected patients and was not restored to normal levels following combination antiretroviral therapy. Basal markers of glycolysis were significantly higher in CD4Glut1 T cells compared to CD4Glut1 T cells. The proportion of CD4Glut1 T cells correlated positively with the expression of the cellular activation marker, HLA-DR, on total CD4 T cells, but inversely with the absolute CD4 T-cell count irrespective of HIV treatment status., Conclusion: Our data suggest that Glut1 is a potentially novel and functional marker of CD4 T-cell activation during HIV infection. In addition, Glut1 expression on CD4 T cells may be exploited as a prognostic marker for CD4 T-cell loss during HIV disease progression.

Combined antiretroviral therapy (cART) extends the lifespan and the quality of life for HIV-infected persons but does not completely eliminate chronic immune activation and inflammation. The low level of chronic immune activation persisting during cART-treated HIV infection is associated with the development of diseases which usually occur in the elderly. Although T-cell activation has been extensively examined in the context of cART-treated HIV infection, monocyte activation is only beginning to be recognized as an important source of inflammation in this context. Here we examine markers and sources of monocyte activation during cART-treated HIV infection and discuss the role of monocytes during cardiovascular disease, HIV-associated neurocognitive disorder, and innate immune aging.

African-born Australians are a recognised "priority population" in Australia's Sixth National HIV/AIDS Strategy. We compared exposure location and route for African-born people living with HIV (PLHIV) in Victoria, Australia, with HIV-1 pol subtype from drug resistance assays and geographical origin suggested by phylogenetic analysis of env gene. Twenty adult HIV positive African-born Victorian residents were recruited via treating doctors. HIV exposure details were obtained from interviews and case notes. Viral RNA was extracted from participant stored plasma or whole blood. The env V3 region was sequenced and compared to globally representative reference HIV-1 sequences in the Los Alamos National Library HIV Database. Twelve participants reported exposure via heterosexual sex and two via iatrogenic blood exposures; four were men having sex with men (MSM); two were exposed via unknown routes. Eight participants reported exposure in their countries of birth, seven in Australia, three in other countries and two in unknown locations. Genotype results (pol) were available for ten participants. HIV env amplification was successful in eighteen cases. HIV-1 subtype was identified in all participants: eight both pol and env; ten env alone and two pol alone. Twelve were subtype C, four subtype B, three subtype A and one subtype CRF02_AG. Reported exposure location was consistent with the phylogenetic clustering of env sequences. African Australians are members of multiple transnational social and sexual networks influencing their exposure to HIV. Phylogenetic analysis may complement traditional surveillance to discern patterns of HIV exposure, providing focus for HIV prevention programs in mobile populations.

Tetraspanins are a family of membrane-organizing proteins that mediate diverse functions. Little is known of their expression or function in myeloid cells. Here, expression of CD9, CD53, CD63, and CD81, tetraspanins that have been implicated in HIV-1 pathogenesis, were characterized in normal monocyte subsets, in MDM, and in HIV-1-infected donors. We show that tetraspanins are expressed differentially by monocyte subsets, with higher CD9 and CD63 and lower CD53 and CD81 levels on CD14++CD16- monocytes compared with CD14++CD16+ and CD14+CD16++ subsets. Maturation of monocytes resulted in increased CD9 expression and apparent relocation of CD63 and CD53 from surface to intracellular membranes. Expression was modulated by cytokines, and CD9 was a marker of anti-inflammatory and CD53 a marker of proinflammatory MDM. Tetraspanin expression on monocyte subsets from HIV-1-infected donors receiving antiretroviral therapy was unchanged compared with that in uninfected donors. However, CD53 expression was inversely correlated with viral load in HIV-1-infected donors not on therapy. This study is the first to comprehensively characterize tetraspanin expression on monocyte subsets and macrophages in health and during HIV-1 infection. It demonstrates regulation of tetraspanin expression by cytokines, and CD53 expression as a novel correlate of a proinflammatory phenotype. This paper characterizes tetraspanins in myeloid cells and shows that tetraspanins are expressed differentially in monocyte subsets and are modified in inflammatory conditions.

Background: Human immunodeficiency virus (HIV)-infected patients on combination active antiretroviral therapy (cART) are at increased risk of age-related complications. We hypothesized that nucleos(t)ide reverse transcriptase inhibitors (NRTI) may contribute to accelerated aging in HIV-infected individuals on cART via inhibition of telomerase activity., Methods: Telomerase activity and telomere length (TL) were measured by quantitative polymerase chain reaction in vitro in activated peripheral blood mononuclear cells (PBMCs) cultured with NRTI and ex vivo in PBMCs from uninfected patients exposed to NRTI and from HIV-infected patients on NRTI-containing cART., Results: Lamivudine, abacavir, zidovudine, emtricitabine, and tenofovir significantly inhibited telomerase activity in activated PBMCs in vitro. Tenofovir was the most potent inhibitor of telomerase activity and caused greatest shortening of TL in vitro at the therapeutic concentration of 0.3 μM. PBMCs from HIV-infected patients receiving NRTI-containing cART (n = 39) had significantly lower telomerase activity than HIV-uninfected patients (n = 47; P = .011) and HIV-infected patients receiving non-NRTI-containing cART (n = 11; P < .001). TL was significantly inversely associated with age (P = .009) and the total duration on any NRTI (P = .01)., Conclusions: NRTIs and, specifically tenofovir at therapeutic concentrations, inhibit telomerase activity leading to accelerated shortening of TL in activated PBMCs. The relationship between NRTI, reduced telomerase activity, and accelerated aging requires further investigation in HIV-infected individuals on cART.

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.

Background: Aging is associated with immune dysfunction and the related development of conditions with an inflammatory pathogenesis. Some of these immune changes are also observed in HIV infection, but the interaction between immune changes with aging and HIV infection are unknown. Whilst sex differences in innate immunity are recognized, little research into innate immune aging has been performed on women., Methods: This cross-sectional study of HIV positive and negative women used whole blood flow cytometric analysis to characterize monocyte and CD8(+) T cell subsets. Plasma markers of innate immune activation were measured using standard ELISA-based assays., Results: HIV positive women exhibited elevated plasma levels of the innate immune activation markers CXCL10 (p<0.001), soluble CD163 (sCD163, p = 0.001), sCD14 (p = 0.022), neopterin (p = 0.029) and an increased proportion of CD16(+) monocytes (p = 0.009) compared to uninfected controls. Levels of the innate immune aging biomarkers sCD163 and the proportion of CD16(+) monocytes were equivalent to those observed in HIV negative women aged 14.5 and 10.6 years older, respectively. CXCL10 increased with age at an accelerated rate in HIV positive women (p = 0.002) suggesting a synergistic effect between HIV and aging on innate immune activation. Multivariable modeling indicated that age-related increases in innate immune biomarkers CXCL10 and sCD163 are independent of senescent changes in CD8(+) T lymphocytes., Conclusions: Quantifying the impact of HIV on immune aging reveals that HIV infection in women confers the equivalent of a 10-14 year increase in the levels of innate immune aging markers. These changes may contribute to the increased risk of inflammatory age-related diseases in HIV positive women.

Background: The HIV epidemic in Indonesia remains concentrated in vulnerable populations, namely injecting drug users (IDUs), commercial sex workers (CSWs) and men who have sex with men (MSM). We aimed to determine the HIV-1 subtypes present in Indonesia and to establish the extent of the viral overlap between individuals with different risk factors., Methods: Venous blood samples were collected from HIV-positive individuals primarily from sexually transmissible infection clinics and drug rehabilitation centres in Bali and Jakarta, and applied to filter paper. A polymerase chain reaction-based assay designed to amplify a 330-bp region of the HIV-1 envelope was used to determine HIV-1 subtype result and to perform phylogenetic analysis., Results: Of the 175 individuals recruited to the study, a subtype result was obtained for 108 (62%). Four subtypes were found to exist in the population, CRF01&#95;AE (n=96, 88.9%), B (n=10, 9.3%), C (n=1, 0.9%) and G (n=1, 0.9%). Of these 108 individuals, 65 (60%) were IDUs, and the remaining 40% were CSWs, MSM, transgender individuals, people with multiple sexual partners or those with no obvious risk factor. CRF01&#95;AE was found to be more common among IDUs with 100% of individuals infected with this subtype. Subtype B was more common among MSM and CSWs (P=<0.001). Phylogenetic analysis revealed a lack of viral segregation between risk groups., Conclusions: In Indonesia, CRF01&#95;AE continues to dominate the HIV epidemic, although HIV subtype B is responsible for a significant number of sexually acquired infections.

Foam cells are a pathological feature present at all stages of atherosclerosis. Foam cells develop from monocytes that enter the nascent atheroma and subsequently ingest modified low density lipoproteins (LDL). The regulation of this process has previously been studied in vitro using cultured macrophage fed modified LDL. We used our existing in vitro model of transendothelial migration (TEM) to study this process in a more physiologically relevant setting. In our model, monocytes undergo TEM across a primary endothelial monolayer into an underlying three-dimensional collagen matrix in the presence of 20% human serum. Foam cells were detected by Oil Red O staining for intracellular lipid droplets. We demonstrate that sub-endothelial monocytes can develop into foam cells within 48 h of TEM across TNF-α activated endothelium, in the absence of additional lipids. Our data indicate a role for both monocyte-endothelial interactions and soluble factors in the regulation of foam cell development, including oxidation of LDL in situ from lipid present in culture medium following TNF-α stimulation of the endothelial cells. Our study provides a simple model for investigating foam cell development in vitro that mimics cell migration in vivo, and demonstrates the critical role of inflammation in regulating early atherogenic events., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chronic inflammation in older individuals is thought to contribute to inflammatory, age-related diseases. Human monocytes are comprised of three subsets (classical, intermediate and nonclassical subsets), and despite being critical regulators of inflammation, the effect of age on the functionality of monocyte subsets remains to be fully defined. In a cross-sectional study involving 91 healthy male (aged 20-84 years, median 52.4) and 55 female (aged 20-82 years, median 48.3) individuals, we found age was associated with an increased proportion of intermediate and nonclassical monocytes (P = 0.002 and 0.04, respectively) and altered phenotype of specific monocyte subsets (e.g. increased expression of CD11b and decreased expression of CD38, CD62L and CD115). Plasma levels of the innate immune activation markers CXCL10, neopterin (P < 0.001 for both) and sCD163 (P = 0.003) were significantly increased with age. Whilst similar age-related changes were observed in both sexes, monocytes from women were phenotypically different to men [e.g. lower proportion of nonclassical monocytes (P = 0.002) and higher CD115 and CD62L but lower CD38 expression] and women exhibited higher levels of CXCL10 (P = 0.012) and sCD163 (P < 0.001) but lower sCD14 levels (P < 0.001). Monocytes from older individuals exhibit impaired phagocytosis (P < 0.05) but contain shortened telomeres (P < 0.001) and significantly higher intracellular levels of TNF both at baseline and following TLR4 stimulation (P < 0.05 for both), suggesting a dysregulation of monocyte function in the aged. These data show that aging is associated with chronic innate immune activation and significant changes in monocyte function, which may have implications for the development of age-related diseases., (© 2012 The Authors. Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART(-)), virologically suppressed patients receiving cART (ART(+)), and HIV-uninfected controls. CD8(+) T cells were activated, as assessed by CD38(+)HLA-DR(+) expression, in ART(-) patients (p < 0.0001), which was significantly reduced in ART(+) patients (p = 0.0005). In contrast, CD38(+)HLA-DR(+) NK cells were elevated in ART(-) patients (p = 0.0001) but did not decrease in ART(+) patients (p = 0.88). NK cells from both ART(-) and ART(+) patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART(+) patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.

Objectives: To compare the impact of HIV infection and healthy ageing on monocyte phenotype and function and determine whether age-related changes induced by HIV are reversed in antiretroviral treated individuals., Design: A cross sectional study of monocyte ageing markers in viremic and virologically suppressed HIV-positive males aged 45 years or less and age-matched and elderly (≥65 years) HIV-uninfected individuals., Methods: Age-related changes to monocyte phenotype and function were measured in whole blood assays ex vivo on both CD14(++)CD16(-) (CD14(+)) and CD14(variable)CD16(+) (CD16(+)) subsets. Plasma markers relevant to innate immune activation were measured by ELISA., Results: Monocytes from young viremic HIV-positive males resemble those from elderly controls, and show increased expression of CD11b (P < 0.0001 on CD14(+) and CD16(+)subsets) and decreased expression of CD62L and CD115 (P = 0.04 and 0.001, respectively, on CD14(+) monocytes) when compared with young uninfected controls. These changes were also present in young virologically suppressed HIV-positive males. Innate immune activation markers neopterin, soluble CD163 and CXCL10 were elevated in both young viremic (P < 0.0001 for all) and virologically suppressed (P = 0.0005, 0.003 and 0.002, respectively) HIV-positive males with levels in suppressed individuals resembling those observed in elderly controls. Like the elderly, CD14(+) monocytes from young HIV-positive males exhibited impaired phagocytic function (P = 0.007) and telomere-shortening (P = 0.03) as compared with young uninfected controls., Conclusion: HIV infection induces changes to monocyte phenotype and function in young HIV-positive males that mimic those observed in elderly uninfected individuals, suggesting HIV may accelerate age-related changes to monocytes. Importantly, these defects persist in virologically suppressed HIV-positive individuals.

The therapeutic activity of arbidol was investigated against representatives of seven different virus families. Its 50% median effective concentration (EC(50) ) was 0.22-11.8 µg/ml (0.41-22 nM). Therapeutic indices of 91 were obtained for type 1 poliovirus and 1.9-8.5 for influenza A and B, human paramyxo-3, avian infectious bronchitis-, and Marek's disease viruses. Arbidol was more inhibitory for influenza A/Aichi/2/68 (H3N2) virus than rimantadine or amantadine (EC(50) 10 vs. >15 and >31.6 µg/ml); greater inhibition occurred when end-points were expressed as TCID(50) s. For respiratory syncytial virus (RSV), a reduction in plaque size but not number was observed. However, when the drug was added to infected cultures (≥5 µg/ml), a 3-log reduction in titer occurred. Arbidol did not inhibit directly influenza A/Aichi/2/68 hemagglutinin (HA) or neuraminidase (NA) activity, but inhibition of fusion between the viral envelope and chicken red blood cells occurred when added at 0.1 µg/ml prior to infection. Arbidol induced changes to viral mRNA synthesis of the PB2, PA, NP, NA, and NS genes in MDCK cultures infected with influenza A/PR/8/34. There was no indirect evidence of enhancement of interferon-α by arbidol following infection with A/Aichi/2/68. Arbidol neither reduced lung viral titers nor caused a significant reduction of lung consolidation in BALB/c mice after administration by the oral and intraperitoneal (i.p.) routes and intranasal challenge with influenza A/Aichi/2/68. A small reduction in lung consolidation, but not viral titer, occurred after i.p. administration and subsequent challenge with RSV. The results indicate the potential of arbidol as a broad-spectrum respiratory antiviral drug., (Copyright © 2011 Wiley Periodicals, Inc.)

The increased life expectancy of HIV-infected individuals due to improved treatment has revealed an unexpected increase in non-AIDS comorbidities that are typically associated with older age including cardiovascular disease, dementia and frailty. The majority of these diseases arise as the result of dysregulated systemic inflammation, and both the aged and HIV-infected individuals exhibit elevated basal levels of inflammation. In the elderly, increased inflammation and age-related diseases are associated with a state of impaired immunity called immunosenescence, which is thought to result from a lifetime of immune stimulation. It is now apparent that HIV induces premature immunosenescence within T-cells; however, the impact of HIV on aging of cells of the innate arm of the immune system is unknown. Innate immune cells play a central role in inflammation and are thus critical for the pathogenesis of inflammatory diseases. Limited evidence suggests HIV infection mimics age-related changes to innate immune cells; however, the extent of this effect and the mechanism underlying these changes remain to be defined. This review focuses on the impact of HIV infection on the function and aging of innate immune cells and discusses potential drivers of premature immunosenescence including chronic endotoxaemia, residual viraemia, telomere attrition and altered cellular signalling.

We developed a real-time PCR to quantify 16S rRNA gene levels in plasma from HIV-infected patients as a marker of microbial translocation. The assay uses shrimp nuclease (SNuc) to eliminate DNA contamination, giving high sensitivity and low variability. The 16S rRNA gene levels measured in plasma from HIV patients correlated significantly with lipopolysaccharide levels.

Background: The rise in serious complications of early syphilis, including neurosyphilis, particularly in those with HIV infection and in men who have sex with men (MSM), is of concern., Objectives: To review the manifestations and management of neurosyphilis in a population of HIV-infected MSM., Methods: Retrospective review of patients with HIV and early neurosyphilis in three centres in Melbourne, Australia, in 2000-07., Results: Eighteen male HIV patients met the criteria for diagnosis of early neurosyphilis. Thirteen patients (72.2%) had neurological symptoms: six with headache (33.3%), four with tinnitus (22.2%) and five with impaired vision (27.8%), and one patient each with ataxia, leg weakness and anal discharge with faecal incontinence. Five patients (27.8%) reported no neurological symptoms. All had serum rapid plasma reagin (RPR) titres ≥1:32 and all except one had cerebrospinal fluid positive for syphilis fluorescent treponemal antibodies-absorbed. After treatment with 14-15 days of 1.8 g intravenous benzylpenicillin 4-hourly, 12 of 17 patients (71%) demonstrated a four-fold drop in serum RPR titre over 6-12 months and were considered successfully treated. A rise in RPR was noted in three patients during the 12-month follow-up period, suggesting re-infection or recurrence., Conclusion: HIV-infected patients found to have syphilis either because of symptoms or by routine screening should be carefully assessed for neurological, ophthalmic and otological symptoms and signs. A low threshold for a diagnostic lumbar puncture to exclude the diagnosis of neurosyphilis enables appropriate administration and dose of penicillin for treatment, which appears successful in ~75% of cases.

Combination antiretroviral therapy (cART) has significantly reduced morbidity and mortality of HIV-infected patients, yet their life expectancy remains reduced compared with the general population. Most HIV-infected patients receiving cART have some persistent immune dysfunction characterized by chronic immune activation and premature aging of the immune system. Here we review biomarkers of T-cell activation (CD69, -25 and -38, HLA-DR, and soluble CD26 and -30); generalized immune activation (C-reactive protein, IL-6 and D-dimer); microbial translocation (lipopolysaccharide, 16S rDNA, lipopolysaccharide-binding protein and soluble CD14); and immune dysfunction of specific cellular subsets (T cells, natural killer cells and monocytes) in HIV-infected patients on cART and their relationship to adverse clinical outcomes including impaired CD4 T-cell recovery, as well as non-AIDS clinical events, such as cardiovascular disease.

The availability of rapid, point-of-care (POC) tests has significantly expanded the capacity of both developed and resource-constrained countries (RCCs) to diagnose HIV, with immunochromatographic tests most commonly used in these settings. This has been especially important in programs for prevention of mother-to-child transmission, in both RCCs and the developed world. However, suitable POC tests are not yet commercially available for diagnosis of neonatal HIV, where persistence of maternal antibody in the infant precludes the use of current antibody tests during the first 12 to 18 months. In addition, measurement of CD4+ T cells, CD4%, and HIV viral load still relies on sophisticated laboratory infrastructure, constraining the delivery of appropriate care to many HIV-infected patients. Continued effort is required in the development and validation of additional POC tests to support HIV patient care, and in quality assurance in manufacturing and in test performance in the field to ensure appropriate use of existing and new POC tests.

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16- monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16- monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16- subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16- monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals.

Measurement of NK cell cytolytic activity in the setting of chronic viral infection is important for determining viral pathogenicity. Mobilization of LAMP-1 (CD107a) to the NK cell surface is a surrogate marker for cytotoxic granule release and hence, NK cell cytotoxicity. We have developed a convenient, rapid, whole blood flow cytometric assay for measuring CD107a mobilization in response to CD16 cross-linking, a surrogate for NK cell ADCC activity ex vivo, which can be performed using small volumes of patient whole blood. Using this assay, we show that CD107a mobilization, in response to CD16 cross-linking, is triggered in CD56(dim) but not CD56(bright) NK cells, requiring Syk/Zap70 tyrosine kinase activity, and that there is a significant correlation between CD107a mobilization and pSyk/Zap70 in response to CD16 cross-linking. We compared whole blood from treatment-naïve, HIV-infected patients with age- and sex-matched HIV-uninfected control subjects and found a significant reduction in CD16-dependent pSyk/Zap70 (median=32.7% compared with 67.8%; P=0.0002) and CD107a mobilization (median=9.72% compared with 32.9%; P=0.046) in NK cells. Reduction of both correlated strongly with reduced CD16 surface expression on NK cells of HIV-infected individuals (P<0.01). These data suggest that ADCC is inhibited in NK cells from therapy-naïve, HIV-infected individuals at the level of early events in CD16 signal transduction, associated with low CD16R expression, and our method is a useful and reliable tool to detect pathological defects in NK cell degranulation.

Severe immunodeficiency during primary human immunodeficiency virus (HIV) infection is unusual. Here, we characterized viral and immunological parameters in a subject presenting with Pneumocystis jirovecii pneumonia in the setting of prolonged primary HIV illness and delayed seroconversion. HIV antibody was only detected by enzyme-linked immunosorbent assay 12 months after presentation, and Western blot profiles remain indeterminate. Isolated virus was of R5 phenotype, exhibited poor viral fitness, but was otherwise unremarkable. Analysis of HIV antibody isotypes showed failure to mount a detectable HIV IgG response over nearly 2 years of infection, in particular IgG(1)- and IgG(3)-specific responses, despite normal responses to common infections and vaccines. Genetic analysis demonstrated homozygosity for part of an MHC haplotype containing susceptibility genes for common variable immunodeficiency (CVID) syndrome and other antibody deficiency disorders. Thus, a primary disorder of specific antibody production may explain exceptionally slow antibody development in an otherwise severe seroconversion illness. This highlights the need for multiparameter testing, in particular use of a fourth generation HIV test, for confirming HIV infection and underscores the importance of host factors in HIV pathogenesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The quantitation of HIV viral load using an assay that measures the activity of reverse transcriptase (RT) may provide an alternative strategy for the monitoring of HIV viral load within resource-limited areas in China. Plasma viral load analyses of 215 samples from 87 patients infected with HIV were detected using the RT activity assay (ExaVir Load versions 2 and 3; Cavidi, Uppsala, Sweden) and RT polymerase chain reaction (PCR) (COBAS TaqMan 48, Amplink version 3.2; Roche Molecular Systems, Branchburg, NJ). The RT activity assay versions 3 (RT3) and 2 (RT2) could detect 95.3% and 86.9% of samples with measurable RNA by RT-PCR, respectively. A stronger correlation was observed between viral loads detected by RT3 and RT-PCR than between RT2 and RT-PCR (r = 0.95, P < 0.001, and r = 0.92, P < 0.001, respectively). The correlation between serial samples collected from 6 patients at 1, 3, 6, 12, 18, and 24 months after beginning triple combination antiretroviral therapy, using the 2 different methodologies, was also strong (r = 0.99, P < 0.001, for RT3 and RT-PCR, r = 0.98, P < 0.001, for RT2 and RT-PCR). The viral loads detected by RT activity assay were inversely correlated with CD4(+) T-cell counts. Reproducibility of the RT activity assay was assessed by testing 3 samples in triplicate by 3 different operators. Viral load testing using assays that measure HIV RT activity is an affordable, feasible, simple, and reliable alternative for HIV RNA viral load determination in laboratories in China and other developing countries., (Copyright © 2010 Elsevier Inc. All rights reserved.)

There is increasing evidence to support the inability of CD4 cell count monitoring to predict virological failure in human immunodeficiency virus-infected individuals receiving antiretroviral therapy. There is renewed interest in improving access to viral load monitoring in resource-constrained regions to monitor adherence to treatment and to switch therapy. The field is rapidly changing as new technology platforms are made available for evaluation. This article presents an up to date summary of the assays available for viral load monitoring and suggests approaches for their implementation.

HIV-infected individuals are at increased risk of coronary artery disease (CAD) with underlying mechanisms including chronic immune activation and inflammation secondary to HIV-induced microbial translocation and low-grade endotoxemia; direct effects of HIV and viral proteins on macrophage cholesterol metabolism; and dyslipidemia related to HIV infection and specific antiretroviral therapies. Monocytes are the precursors of the lipid-laden foam cells within the atherosclerotic plaque and produce high levels of proinflammatory cytokines such as IL-6. The minor CD14+/CD16+ "proinflammatory" monocyte subpopulation is preferentially susceptible to HIV infection and may play a critical role in the pathogenesis of HIV-related CAD. In this review, the central role of monocytes/macrophages in HIV-related CAD and the importance of inflammation and cholesterol metabolism are discussed.

Efficient isolation of replication-competent virus from plasma of patients infected with HIV-1 is needed to characterize important clinical parameters of virus. However, addition of plasma to in vitro cultures results in clot formation. Blood from HIV-1 infected patients was collected in the presence of three commonly used anticoagulants (ACD, heparin and EDTA) and plasma was isolated. Plasma was then used to infect HIV-1 indicator cell lines (TZM-bl and GHOST) with spinoculation in the presence or absence of additional heparin and positively charged polymers. The presence of additional heparin during inoculation significantly reduced clot formation without affecting the sensitivity of HIV-1 infection in the GHOST cell line. However, heparin reduced the frequency of HIV-1 infection of the TZM-bl cell line. Using plasma from patients with HIV RNA>1000 copies/ml (n=58), the frequency of HIV-1 isolation was 92% in GHOST (n=51) and 54% in TZM-bl (n=26) cell lines. A sensitive method was developed for rapid isolation of infectious HIV-1 from plasma of patients with HIV RNA>1000 copies/ml that includes spinoculation and the addition of heparin during infection of GHOST cells. This technique could be used for rapid evaluation of viral fitness, co-receptor usage or drug resistance without the need for viral amplification., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Background: FcRgamma is an immunoreceptor tyrosine-based activation motif (ITAM)-signalling protein essential for immunoreceptor signaling and monocyte, macrophage and NK cell function. Previous study from our laboratory showed that FcRgamma is down-regulated in HIV-infected macrophages in vitro. FcRgamma expression in immune cells present in HIV-infected individuals is unknown., Methodology/principal Findings: We compared FcRgamma expression in peripheral blood mononuclear cells isolated from HIV-1-infected individuals receiving combination antiretroviral therapy and healthy, HIV-1-uninfected individuals. FcRgamma mRNA and protein levels were measured using quantitative real-time PCR and immunoblotting, respectively. CD56(+) CD94(+) lymphocytes isolated from blood of HIV-1 infected individuals had reduced FcRgamma protein expression compared to HIV-uninfected individuals (decrease = 76.8%, n = 18 and n = 12 respectively, p = 0.0036). In a second group of patients, highly purified NK cells had reduced FcRgamma protein expression compared to uninfected controls (decrease = 50.2%, n = 9 and n = 8 respectively, p = 0.021). Decreased FcRgamma expression in CD56+CD94+ lymphocytes was associated with reduced mRNA (51.7%, p = 0.021) but this was not observed for the smaller group of patients analysed for NK cell expression (p = 0.36)., Conclusion/significance: These data suggest biochemical defects in ITAM-dependent signalling within NK cells in HIV-infected individuals which is present in the context of treatment with combination antiretroviral therapy.

The wide scale application of dried blood spots (DBS) as a collection tool for low-cost HIV drug resistance testing requires a greater understanding of the accuracy of DBS for genotype analysis and the stability of DBS under various environmental conditions. Analysis of a 50microl DBS via a single amplicon, nested PCR-based in-house assay (the Burnet genotyping assay) showed an average nucleotide concordance of 98.9% with plasma samples, although only 65% of nucleotide mixtures detected in plasma were also detected within DBS. The analysis of three DBS resulted in the detection of a greater number of nucleotide mixtures (72 and 109 mixtures detected within one and three DBS, respectively, n=10). Two DBS extraction protocols (silica particle; NucliSENS, bioMerieux and spin column extraction; High Pure, Roche) were assessed and found to be equivalent (79% and 84% recovery success respectively, n=19). FTA Elute paper (Whatman) was an inferior DBS collection medium compared to Whatman 903 paper. DBS appeared relatively tolerant to multiple freeze/thaw cycles, with 79% of DBS subjected to ten freeze/thaw cycles successfully amplified compared to 93% of DBS defrosted once (n=14). High temperature (37 degrees C) and high humidity (>90%) substantially impaired DBS recovery within two weeks of storage (38%, n=8), whilst storage at -20 degrees C or 4 degrees C adequately preserved DBS for this period (100% recovery, n=8). Therefore, whilst DBS are suitable for HIV drug resistance surveillance, the use of multiple DBS may be required to ensure accurate detection of minor HIV quasispecies and short-term storage of samples at either 4 degrees C or -20 degrees C is recommended.

Background: The integrase inhibitors (e.g. Raltegravir) are a new class of antiretroviral drugs that have recently become available for the treatment of patients with multi-drug resistant HIV-1 within Australia. The emergence of mutations that confer resistance to the integrase inhibitors has been observed in vivo; however, no commercial genotyping assay is currently available to screen for resistance to these drugs., Methods: The HIV-1 integrase gene was amplified from plasma-derived HIV-1 viral RNA via reverse transcription-polymerase chain reaction and genotype determined via population DNA sequencing. Drug resistance mutations and polymorphisms were detected using the Stanford University online HIV database. Assay sensitivity and reproducibility were determined using clinical and laboratory-derived samples., Results: Our in-house assay was capable of genotyping the integrase gene from all samples tested (n = 30) of HIV-1 subtypes B, C, D, F, CFR01&#95;AE and CRF02&#95;AG and can amplify the integrase region from plasma samples containing as few as 50 HIV RNA copies/mL. The assay is highly reproducible (average nucleotide concordance = 99.6%, n = 4) and is capable of detecting resistance-associated mutations., Conclusions: This assay is suitable for routine drug resistance screening of plasma samples from HIV-infected patients receiving integrase inhibitor antiretroviral drugs and also serves as a useful research tool.

The HIV epidemic in Fiji remains largely uncharacterized. By February 2009, there were 294 confirmed cases; the majority occurred among the 20- to 39-year old age group and resulted from heterosexual contact. There are currently no published data concerning HIV subtypes in Fiji. In this study, venous blood samples were collected as dried blood spots from 35 HIV-positive individuals in Fiji. HIV-1 subtype was determined for 27 (77%) samples and the presence of four different subtypes, with multiple introductions of two, was demonstrated. Subtype distribution was as follows: 16 (59%) were subtype C, 9 (33%) were subtype B, 1 (4%) was subtype A, and 1 (4%) was subtype G. Phylogenetic analysis showed a clear segregation of the Fijian subtype C isolates and previously published Papua New Guinea subtype C isolates as well as multiple introductions of subtype B. These findings represent the first HIV-1 subtype data from the Fiji Islands.

Background: Viral load (VL) is a critical marker for monitoring HIV disease progression and response to antiretroviral therapy. In resource-constrained settings, there is a need for a simple and inexpensive assay to monitor infected adults and children., Methods: We compared versions 2 and 3 of the ExaVir Load assay, Cavidi AB (HIV RT) with the Roche, COBAS Amplicor HIV-1 Monitor assay (HIV RNA) for quantifying HIV VL., Results: The HIV RT version 2 assay showed good sensitivity with detection in 94% of samples with HIV RNA >1000 copies per milliliter. Adult samples were tested using HIV RT version 2 (n = 35) and version 3 (n = 23) assays with plasma volumes of 1 mL (recommended), 0.5 mL and 0.25 mL in comparison with HIV RNA. The HIV RT and HIV RNA assay results were comparable when tested using different volumes. Comparison of results from pediatric samples (n = 27), tested using 1 mL and a smaller volume by HIV RT version 2 were not significantly different., Conclusions: The HIV RT assay was comparable to the HIV RNA assay with sensitivity approaching that of HIV RNA. Smaller volumes than the recommended 1 mL can be used, improving utility of this assay for pediatric monitoring.

TransFix(TM) and Cyto-Chex((R)) BCT (blood collection tube) reagents have been shown to maintain whole blood integrity for delayed immunophenotyping by flow cytometry. We evaluated the ability of these blood-stabilizing reagents to preserve HIV-seropositive blood for delayed CD4(+) T-cell quantification utilizing the Dynal((R)) Biotech T4 Quant Kit. TransFix was added to EDTA-anticoagulated whole blood and tested at a 1:10 dilution over 7 d using the Dynal (n = 21) manual method. Compared to baseline analysis, a significant decrease in mean CD4(+) counts was observed over time. Cyto-Chex BCT-preserved samples (n = 20) were tested for CD4(+) counts by Dynal over 7 d, with storage at varying temperatures: room temperature (21 degrees C), 37 degrees C, and 37 degrees C with intermittent storage at 42 degrees C. A significant decline in mean CD4(+) counts was observed in samples at all temperatures compared to baseline (p < 0.05). Increases in temperature to and above 37 degrees C resulted in a greater decline in mean CD4(+) counts over time. Our findings indicated that neither TransFix or Cyto-Chex BCT was a suitable blood stabilizer when used for delayed CD4(+) quantification with a low-cost manual CD4(+) bead-based method.

We evaluated the new low-cost ExaVir Load (version 3) reverse transcriptase viral load assay against the Roche Cobas Amplicor assay. Results for samples tested using the reverse transcriptase assay correlated well with those obtained with the Roche assay (r = 0.85; n = 202). The version 3 reverse transcriptase assay shows improved sensitivity compared to the previous version.

Human cytomegalovirus (HCMV) expresses several homologues of human interleukin 10 (hIL-10) possessing immunomodulatory properties which may promote viral infection by modulating the function of myeloid cells. We examined the phenotype and phagocytic capability of human monocytes exposed to hIL-10, an HCMV-encoded hIL-10 homologue expressed during the productive phase of infection (cmvIL-10), and a differentially spliced form of cmvIL-10 expressed during latent and productive phases of infection, (LAcmvIL-10). hIL-10 and cmvIL-10 upregulated expression of Fcgamma receptors, stimulated phagocytosis of IgG-opsonised erythrocytes and decreased MHC class II (HLA-DR) expression on purified monocytes within 24 h. In contrast, LAcmvIL-10 decreased HLA-DR expression at later times (48 h and 72 h) but did not increase Fcgamma receptor expression. We conclude that cmvIL-10 promotes differentiation of monocytes towards a pro-phagocytic phenotype and that LAcmvIL-10 does not affect monocytes by the same mechanism as cmvIL-10. The significance of these properties to cytomegalovirus pathogenesis is discussed.

Cognitive impairment and neurodegeneration still occur despite highly active antiretroviral therapy (HAART). While there are many potential reasons for this, there is increasing evidence that such impairment occurs in the absence of a clear cause. Furthermore, there are data that some neurodegenerative diseases, especially Alzheimer's or an Alzheimer-like illness, are becoming more common in the context of HAART-treated human immunodeficiency virus (HIV) disease. This review will critically examine the evidence underpinning these observations. Potential mechanisms will be discussed with particular emphasis on the effect of ageing and how it overlaps with the effects of HIV disease itself thereby leading to neurodegeneration. The nature of this overlap will then be explored for its potential role in the facilitated expression and development of neurodegenerative diseases. Lastly, there will be a brief discussion of interventions to minimize such neurodegeneration including optimization of HAART for brain entry.

Monocytes constitutively migrate from the bloodstream across the vascular endothelium for systemic immune surveillance and maintenance of macrophage populations. They also perform reverse transendothelial migration (TEM) across the endothelium, which is required for entry of tissue monocytes/macrophages into the lymphatics or back into the bloodstream. We have modeled these processes previously using HUVEC monolayers grown on three-dimensional collagen matrices. The aim of the present study was to determine whether HIV-1 infection of monocytes/macrophages in vitro affects TEM. Purified primary human monocytes and monocyte-derived macrophages (MDM) expressed important TEM proteins such as CD62L, CD18, PECAM-1, CCR2, and CCR8. Purified monocytes underwent efficient forward and reverse TEM across HUVEC, and this function was maintained by MDM after up to 15 days of culture. Monocytes exposed to HIV-1 for 2 days had unaltered forward or reverse TEM. However, HIV-1 infection of MDM for 7 days decreased reverse TEM by an average of 66.5% compared with mock-infected MDM (n=9 independent donors; P=0.004), without affecting forward TEM. Decreased reverse TEM by HIV-infected MDM required viral RT and was not a result of alterations in surface expression of CCR8 or p-glycoprotein or a general impairment in mobility, as assessed by migration toward fMLP. This study indicates that HIV-1 infection of macrophages reduces their capacity to emigrate from the subendothelial extracellular matrix in vitro, which could result in defective cell-mediated immune responses to infections and promote establishment of viral reservoirs of HIV in tissue macrophages in vivo.

The catabolic pathway of l-tryptophan (l-trp), known as the kynurenine pathway (KP), has been implicated in the pathogenesis of a wide range of brain diseases through its ability to lead to immune tolerance and neurotoxicity. As endothelial cells (ECs) and pericytes of the blood-brain-barrier (BBB) are among the first brain-associated cells that a blood-borne pathogen would encounter, we sought to determine their expression of the KP. Using RT-PCR and HPLC/GC-MS, we show that BBB ECs and pericytes constitutively express components of the KP. BBB ECs constitutively synthesized kynurenic acid, and after immune activation, kynurenine (KYN), which is secreted basolaterally. BBB pericytes produced small amounts of picolinic acid and after immune activation, KYN. These results have significant implications for the pathogenesis of inflammatory brain diseases in general, particularly human immunodeficiency virus (HIV)-related brain disease. Kynurenine pathway activation at the BBB results in local immune tolerance and neurotoxicity: the basolateral secretion of excess KYN can be further metabolized by perivascular macrophages and microglia with synthesis of quinolinic acid. The results point to a mechanism whereby a systemic inflammatory signal can be transduced across an intact BBB to cause local neurotoxicity.

An inexpensive and technically less-demanding methodology to quantify HIV-1 viral load would be of great value for resource-limited settings, where the nucleic-acid amplification technique (NAAT) is impractical and/or resource-prohibitive. In this study, an HIV-1 reverse-transcriptase enzyme-activity assay (ExaVir Load assay, version 1) was compared with the gold standard RT-PCR assay, Roche HIV-1 Amplicor Monitor, version 1.5. A total of 121 plasma specimens were used for the evaluation. ExaVir Load had a sensitivity of 97 % and a specificity of 71 % in identifying specimens with <400 copies ml(-1) in the Roche RT-PCR assay as being less than the detection limit of the assay (5500 copies ml(-1)). The mean difference (95 % limits of agreement) between Roche RT-PCR and ExaVir Load was -0.23 (-1.59 to 1.13) log(10)(copies ml(-1)) by Bland-Altman analysis. Significant negative correlations were seen between CD4(+) T-cell counts and the ExaVir Load assay (r=-0.32, P<0.05), and between CD4(+) T-cell counts and the Roche RT-PCR (r=-0.38, P<0.01). The present study with HIV-1 showed a strong correlation between the ExaVir Load assay and the RT-PCR assay. Hence, the ExaVir Load assay could be considered for use in resource-limited settings as an alternative viral-load assay to the standard NAAT-based assay after further evaluation with prospective specimens.

Background & Aims: It has been reported that GB virus C infection (GBV-C) leads to improved morbidity and mortality in patients with human immunodeficiency virus (HIV) infection. However, GBV-C has no effect on the course of liver disease in hepatitis C virus (HCV) monoinfection. The aim of the study was to determine the influence of GBV-C infection on liver disease in patients with HCV/HIV coinfection., Methods: Data on 158 HCV/HIV patients were collected from January 1996 to October 2005. Two plasma specimens, collected at least 18 months apart, were tested for GBV-C RNA by reverse transcription-polymerase chain reaction with primers to the NS5B gene and confirmed using E2 gene primers and sequencing. Antibodies to GBV-C E2 protein were also determined. Liver-related morbidity and mortality were assessed from patient records., Results: Fifty-seven of 158 (36%) patients had GBV-C RNA and 94 (59%) had evidence of exposure to GBV-C based on combined polymerase chain reaction and antibody results. Thirty-four (21%) patients had features of cirrhosis, with 20 having compensated and 14 having decompensated cirrhosis. Active GBV-C RNA was significantly associated with a reduction in cirrhosis, both compensated and decompensated in multivariate analysis (hazard ratio, 0.27; 95% confidence interval, 0.08-0.88; P = .03), as well as in analysis for cirrhosis-free survival vs duration of HCV infection (P = .006). No significant effect on liver-related or overall survival was observed., Conclusions: In these HCV/HIV-coinfected patients, GBV-C RNA was associated with a significant reduction in the severity of HCV-related liver disease.

In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and other accessory genes. Although live attenuated HIV vaccines are unlikely to be developed due to a myriad of safety concerns, opportunities exist to better understand the correlates of immune protection against HIV infection by studying rare cohorts of long-term survivors infected with attenuated, nef-deleted HIV strains such as the Sydney blood bank cohort (SBBC). Here, we review studies of viral evolution, pathogenicity, and immune responses to HIV infection in SBBC members. The studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a persistent T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Together, these results underscore the importance of the host in the eventual outcome of infection. Thus, whilst generating an effective antibody and CD8+ T-cell response are an essential component of vaccines aimed at preventing primary HIV infection, T-helper responses may be important in the generation of an effective therapeutic vaccine aimed at blunting chronic HIV infection.