Diagnostic performance of line-immunoassay based algorithms for incident HIV-1 infection.

BACKGROUND: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have previously demonstrated that a patient's antibody reaction pattern in a confirmatory line immunoassay (INNO-LIA™ HIV I/II Score) provides information on the duration of infection, which is unaffected by clinical, immunological and viral variables. In this report we have set out to determine the diagnostic performance of Inno-Lia algorithms for identifying incident infections in patients with known duration of infection and evaluated the algorithms in annual cohorts of HIV notifications. METHODS: Diagnostic sensitivity was determined in 527 treatment-naive patients infected for up to 12 months. Specificity was determined in 740 patients infected for longer than 12 months. Plasma was tested by Inno-Lia and classified as either incident (< = 12 m) or older infection by 26 different algorithms. Incident infection rates (IIR) were calculated based on diagnostic sensitivity and specificity of each algorithm and the rule that the total of incident results is the sum of true-incident and false-incident results, which can be calculated by means of the pre-determined sensitivity and specificity. RESULTS: The 10 best algorithms had a mean raw sensitivity of 59.4% and a mean specificity of 95.1%. Adjustment for overrepresentation of patients in the first quarter year of infection further reduced the sensitivity. In the preferred model, the mean adjusted sensitivity was 37.4%. Application of the 10 best algorithms to four annual cohorts of HIV-1 notifications totalling 2'595 patients yielded a mean IIR of 0.35 in 2005/6 (baseline) and of 0.45, 0.42 and 0.35 in 2008, 2009 and 2010, respectively. The increase between baseline and 2008 and the ensuing decreases were highly significant. Other adjustment models yielded different absolute IIR, although the relative changes between the cohorts were identical for all models. CONCLUSIONS: The method can be used for comparing IIR in annual cohorts of HIV notifications. The use of several different algorithms in combination, each with its own sensitivity and specificity to detect incident infection, is advisable as this reduces the impact of individual imperfections stemming primarily from relatively low sensitivities and sampling bias.

High specificity of line-immunoassay based algorithms for recent HIV-1 infection independent of viral subtype and stage of disease.

BACKGROUND: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have shown that a patient's antibody reaction in a confirmatory line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) provides information on the duration of infection. Here, we sought to further investigate the diagnostic specificity of various Inno-Lia algorithms and to identify factors affecting it. METHODS: Plasma samples of 714 selected patients of the Swiss HIV Cohort Study infected for longer than 12 months and representing all viral clades and stages of chronic HIV-1 infection were tested blindly by Inno-Lia and classified as either incident (up to 12 m) or older infection by 24 different algorithms. Of the total, 524 patients received HAART, 308 had HIV-1 RNA below 50 copies/mL, and 620 were infected by a HIV-1 non-B clade. Using logistic regression analysis we evaluated factors that might affect the specificity of these algorithms. RESULTS: HIV-1 RNA < 50 copies/mL was associated with significantly lower reactivity to all five HIV-1 antigens of the Inno-Lia and impaired specificity of most algorithms. Among 412 patients either untreated or with HIV-1 RNA ≥ 50 copies/mL despite HAART, the median specificity of the algorithms was 96.5% (range 92.0-100%). The only factor that significantly promoted false-incident results in this group was age, with false-incident results increasing by a few percent per additional year. HIV-1 clade, HIV-1 RNA, CD4 percentage, sex, disease stage, and testing modalities exhibited no significance. Results were similar among 190 untreated patients. CONCLUSIONS: The specificity of most Inno-Lia algorithms was high and not affected by HIV-1 variability, advanced disease and other factors promoting false-recent results in other STARHS. Specificity should be good in any group of untreated HIV-1 patients.

Gammaretrovirus-specific antibodies in free-ranging and captive Namibian cheetahs.

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers.

Combined effect of zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) antiretroviral therapy in suppressing in vitro FIV replication.

In view of close similarities at the molecular and clinical levels, feline immunodeficiency virus (FIV) infection of the domestic cat is subject of increasing attention as an animal model for human immunodeficiency virus (HIV) infection. A range of reverse transcriptase inhibitors effective against HIV are also active against FIV, allowing successful use of the cat model to investigate drug interactions and resistance development. Nevertheless, while combined nucleoside analog and protease inhibitor usage has proven remarkably effective in treating HIV infection, combination antiretroviral therapy of FIV infection has been hampered by lack of protease inhibitors specific for FIV. In an attempt to circumvent this problem, we have examined the feasibility of applying in the FIV system combination protocols lacking a protease inhibitor. We now report that, as observed during HIV infection, the nucleoside analog abacavir (ABC or 1592U89) is able to effectively block in vitro FIV-replication. Furthermore, we demonstrate that combined usage of ABC with the nucleoside analogs zidovudine (ZDV or AZT) and lamivudine (3TC) also blocks in vitro FIV replication in a synergistic manner. However, in contrast to its effect on HIV replication, the ribonucleotide reductase inhibitor hydroxyurea (HU) is unable to effectively control in vitro FIV replication.

Ultra-sensitive and specific detection of porcine endogenous retrovirus (PERV) using a sequence-capture real-time PCR approach.

Use of porcine xenografts presents as a possible solution to the current shortage of human allografts limiting transplantation procedures. While no definitive observation of in vivo porcine endogenous retrovirus (PERV) transmission in humans has been reported, the in vitro ability of PERV to infect human cells and the observation of PERV transmission to immunodeficient mice suggest a need for ultra-sensitive techniques to monitor porcine xenograft recipients and contacts for possible PERV transmission. In an effort to enhance current PCR-based PERV detection, the feasibility of combining nucleic acid sequence-capture with use of a quantitative real-time 5' nuclease assay was examined. Sequence-capture by means of oligonucleotide hybridization to a conserved PERV gag sequence and attachment to magnetic beads was used to extract and concentrate PERV A, B and C DNA from sample material containing high levels of background human DNA. Sequence-capture oligonucleotide design incorporated selective substitution of dUTP for dTTP in order to facilitate eventual oligonucleotide destruction. In addition, sequence-capture oligonucleotides were located outside of the amplified region in order to minimize the effects of possible PCR carry-over. Quantitative PCR was then undertaken using a real-time 5' nuclease assay incorporating primers and probe also specific for a conserved PERV gag region. Sequence-capture real-time PCR assessment of PERV levels demonstrated a dynamic range of at least five orders of magnitude, a sensitivity between 0.005 and 0.028 PERV copies per microg background human DNA and a specificity between 98.2 and 100% (95% CI). In contrast, while real-time PERV PCR in the absence of a sequence-capture step demonstrated a similar specificity between 98.4 and 100% (95% CI), the sensitivity of this conventional approach was between 0.2 and 1.0 PERV copies per microg background human DNA. In conclusion, the increased sensitivity of PERV detection obtained by the combined use of PERV-specific sequence-capture and quantitative real-time PERV PCR suggest that this approach should enhance the effectiveness and reliability of monitoring procedures currently applied to porcine xenograft recipients and contacts.

Simple estimation of incident HIV infection rates in notification cohorts based on window periods of algorithms for evaluation of line-immunoassay result patterns.

BACKGROUND: Tests for recent infections (TRIs) are important for HIV surveillance. We have shown that a patient's antibody pattern in a confirmatory line immunoassay (Inno-Lia) also yields information on time since infection. We have published algorithms which, with a certain sensitivity and specificity, distinguish between incident (< = 12 months) and older infection. In order to use these algorithms like other TRIs, i.e., based on their windows, we now determined their window periods. METHODS: We classified Inno-Lia results of 527 treatment-naïve patients with HIV-1 infection < = 12 months according to incidence by 25 algorithms. The time after which all infections were ruled older, i.e. the algorithm's window, was determined by linear regression of the proportion ruled incident in dependence of time since infection. Window-based incident infection rates (IIR) were determined utilizing the relationship 'Prevalence = Incidence x Duration' in four annual cohorts of HIV-1 notifications. Results were compared to performance-based IIR also derived from Inno-Lia results, but utilizing the relationship 'incident = true incident + false incident' and also to the IIR derived from the BED incidence assay. RESULTS: Window periods varied between 45.8 and 130.1 days and correlated well with the algorithms' diagnostic sensitivity (R(2) = 0.962; P<0.0001). Among the 25 algorithms, the mean window-based IIR among the 748 notifications of 2005/06 was 0.457 compared to 0.453 obtained for performance-based IIR with a model not correcting for selection bias. Evaluation of BED results using a window of 153 days yielded an IIR of 0.669. Window-based IIR and performance-based IIR increased by 22.4% and respectively 30.6% in 2008, while 2009 and 2010 showed a return to baseline for both methods. CONCLUSIONS: IIR estimations by window- and performance-based evaluations of Inno-Lia algorithm results were similar and can be used together to assess IIR changes between annual HIV notification cohorts.

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells.

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

Lack of evidence for PERV expression after apoptosis-mediated horizontal gene transfer between porcine and human cells.

Evidence for porcine endogenous retrovirus (PERV) infection of human cells has provoked a public health debate over the proposed use of porcine xenografts to alleviate the worldwide shortage of human allografts. Nevertheless, the potential relevance of PERV transmission by apoptosis-mediated horizontal DNA transfer, a documented means of infection-independent retrovirus delivery, appears to have been overlooked in this discussion. To examine the hypothesis that apoptotic cell death during porcine xenograft rejection is capable of fostering horizontal DNA transfer, we have now assessed in vitro cocultures, consisting of phagocytic human fibroblasts and apoptotic or necrotic porcine B-lymphoblastoid cells, for evidence of cross-species PERV exchange and eventual replication. Using real-time polymerase chain reaction (PCR) assays, designed to differentiate nuclear and cytoplasmic DNA derived from either porcine or human cells, we now report evidence for the presence of porcine DNA, including PERV, in the nucleus of human fibroblasts exposed to apoptotic porcine cells. This novel demonstration of apoptosis-mediated horizontal PERV transfer is characterized by a low efficiency of transfer and a transient nature, being present in only 0.22% of the cocultured human cells and disappearing to undetectable levels within 4 weeks of exposure to apoptotic porcine cells. In contrast, using PERV-specific real-time reverse-transcriptase PCR (RT-PCR) and ultra-sensitive product-enhanced reverse transcriptase (PERT) assays, we find no evidence for human fibroblast-derived cellular PERV RNA or coculture supernatant-based RT-activity, indicating a lack of subsequent PERV replication. Together, these results suggest that apoptosis-mediated horizontal PERV transfer does not present an overt hazard within the framework of porcine xenotransplantation. However, we also present arguments against extrapolation of these in vitro observations directly to clinical circumstances.

Chemokines and their receptors in the pathogenesis of allergic asthma: progress and perspective.

PURPOSE OF REVIEW: The importance of chemokines and their receptors to development and maintenance of allergic asthma is reflected in the burgeoning amount of literature currently devoted to this topic. Based on a series of selected references published during the last year, this review now summarizes recent advances and discusses the likely implications of these findings. RECENT FINDINGS: Of particular interest are reports describing novel interactions between chemokines and both eosinophils and mast cells, including a role for CXCL5 (epithelial cell-derived neutrophil-activating peptide-78) and intracellular CCR3. New insights into TH2-cell dominance are presented in reports dealing with a range of chemokines, including CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL5 (RANTES), CXCL9 (Mig), and CXCL10 (IP-10). The increasing importance of structural cell participation is emphasized by reports focusing on the eotaxin family (CCL11, CCL24, and CCL26), as well as CCL17 (TARC), CCL22 (MDC), CXCL9 (Mig), and CX3CL1 (Fractalkine). A developing role for nonreceptor regulatory mechanisms is also emphasized by seminal work relating to metalloproteinases, as well as reports focusing on proteoglycans and beta-Arrestin-2. Finally, significant progress in the field of asthma heritability is featured in reports relating to both known and novel genes, including those encoding CCR5 and DPP-10. SUMMARY: The critical influence of chemokine biology on the outcome of allergic asthma continues to be highlighted in recent reports describing novel mechanisms by which eosinophils are recruited into the lung and local TH2-cell dominance is maintained. Also of considerable interest is the increasing emphasis currently being realized for structural cell participation, nonreceptor regulatory mechanisms, and the influence of susceptibility genes.

Reference values for peripheral blood lymphocyte phenotypes applicable to the healthy adult population in Switzerland.

OBJECTIVES: Use of domestic reference values is known to improve the accuracy of flow cytometric analysis by integrating local variation due to race, gender, and age. In the absence of previously published estimates, we now report establishment of reference values for a wide range of peripheral blood lymphocyte phenotypes applicable to the healthy adult population in Switzerland and other regions with similar demographic characteristics. METHODS: A representative sample population was recruited from among well characterized local blood donors (n = 70) and quantitative multiparametric flow cytometry used to estimate absolute and proportional values for a range of T-, B-, and NK-cell subsets, including those associated with activation and maturity. Distribution-free methods were then applied to generate 95% reference values and to estimate the significance of gender and age-related differences. RESULTS: Reference values were obtained for the absolute and proportional levels of total CD3(+) T cells (536-1787 cells/microL, 54.90-84.03%), helper CD4(+) T cells (309-1139 cells/microL, 32.53-62.88%), cytotoxic CD8(+) T cells (137-823 cells/microL, 11.55-38.60%), activated CD3(+) T cells expressing CD25 (7-94 cells/microL, 0.50-5.95%), CD38 (102-554 cells/microL, 5.98-26.80%), HLA-DR (18-186 cells/microL, 1.25-9.68%) or CD38/HLA-DR (4-52 cells/microL, 0.30-2.30%), activated CD4(+) T cells expressing CD25 (7-52 cells/microL, 0.33-2.80%), CD38 (69-547 cells/microL, 6.13-32.20%), HLA-DR (11-55 cells/microL, 0.80-4.43%) or CD38/HLA-DR (4-22 cells/microL, 0.30-1.35%), activated CD8(+) T cells expressing CD25 (0-12 cells/microL, 0.00-0.69%), CD38 (13-124 cells/microL, 0.93-7.03%), HLA-DR (6-108 cells/microL, 0.33-6.38%) or CD38/HLA-DR (2-47 cells/microL, 0.13-2.68%), naive CD4(+) T cells expressing CD45RA(+)/CD62L(+) (84-761 cells/microL, 9.48-41.88%), naive CD8(+) T cells expressing CD45RA(+)/CD62L(+) (42-360 cells/microL, 3.68-19.23%), memory CD4(+) T cells expressing CD45RO(+) (247-807 cells/microL, 16.50-42.15%), memory CD8(+) T cells expressing CD45RO(+) (72-377 cells/microL, 3.78-22.80%), B-cells expressing CD19 (72-460 cells/microL, 4.70-19.13%) or CD20 (66-529 cells/microL, 4.63-21.00%), total CD3(-)/(CD16(+)/CD56(+)) NK-cells (77-427 cells/microL, 5.35-30.93%), and activated NK-cells expressing CD25 (0-10 cells/microL, 0-0.50%) or HLA-DR (3-99 cells/microL, 0.20-7.28%). CONCLUSION: It is anticipated that availability of localized reference values for an extended range of peripheral blood lymphocyte phenotypes should supplement previously published reference values and enhance the utility of flow cytometric analysis undertaken in Switzerland.

HIV-1 p24 antigen is a significant inverse correlate of CD4 T-cell change in patients with suppressed viremia under long-term antiretroviral therapy.

An HIV-1 p24 antigen test involving signal amplification-boosted ELISA of heat-denatured plasma was evaluated prospectively in 55 patients whose viral RNA in plasma had previously been suppressed for at least 6 months under antiretroviral combination therapy. During a median follow-up of 504 days, CD4 counts increased by a median of 62 cells per year. By univariate and multivariate linear regression analysis, the level of p24 antigen as expressed by the absorbance/cutoff ratio was a significant inverse correlate of both the CD4 count in a sample (p =.013) and its annual change in a patient (p <.0001). The p24 antigen retained significance even among 48 individuals whose HIV-1 RNA, apart from occasional blips, remained below 400 copies/mL. Batch-wise retesting of 70 samples from 5 such patients with a further improved procedure showed measurable p24 antigen in all but 1 sample and an inverse correlation with both the CD4 count (p =.0331) and percentage (p <.0001), thus confirming the prospectively generated data. Comparison of p24 antigen and HIV-1 RNA concentrations indicate that the p24 antigen detected in these samples is not associated with viral RNA-containing particles and may originate from other compartments of virus expression.

A randomized trial of simplified maintenance therapy with abacavir, lamivudine, and zidovudine in human immunodeficiency virus infection.

This randomized study evaluated the efficacy and tolerability of continued treatment with protease inhibitor plus nucleoside-analogue combination regimens (n=79) or a change to the simplified regimen of abacavir-lamivudine-zidovudine (n=84) in patients with suppressed human immunodeficiency virus type 1 (HIV-1) RNA for > or = 6 months who did not have the reverse transcriptase 215 mutation. After a median follow-up of 84 weeks, virologic failure was 6% in the continuation and 15% in the simplified group (P=.081). Previous zidovudine monotherapy or dual therapy and archived reverse transcriptase resistance mutations in HIV-1 DNA at baseline were significant predictors of failure. Study treatment was discontinued because of adverse events in 20% of the continuation and 7% of the simplified group (P=.021). Simplification to abacavir-lamivudine-zidovudine significantly decreased nonfasting cholesterol and triglyceride levels; however, this switch strategy carries a risk of virologic failure when treatment history or resistance testing suggest the presence of archived resistance mutations to the simplified regimen.

Prevalence of human T-cell leukemia virus types I and II in Switzerland.

The retroviruses human immunodeficiency virus (HIV)-1/2 and human T-cell leukemia virus (HTLV)-I/II share modes of transmission, suggesting that efforts to monitor the current HIV-1 epidemic in Switzerland should be complemented by assessment of HTLV-I/II prevalence. This study presents an updated evaluation of HTLV-I/II infection among groups within the Swiss population polarized towards either low or increased risk of infection. Archived serum and peripheral blood mononuclear cell (PBMC) samples were examined for evidence of HTLV-I/II infection by enzyme-linked immunosorbant assay (ELISA), type-specific Western blot, type-specific polymerase chain reaction (PCR), DNA sequence analysis, and virus culture. Among blood donations obtained from low-risk Swiss donors, we report a complete lack of HTLV-II infection and the occurrence of HTLV-I infection limited to a prevalence of 0.079 per 100,000 (1/1,266,466). Among high-risk HIV-positive persons and HIV-negative persons at increased risk of HIV-infection, we report a focus of HTLV-I and HTLV-II infection at prevalence rates of 62 per 100,000 (1/1,620) and 309 per 100,000 (5/1,620), respectively. The finding of low HTLV-I/II prevalence among Swiss blood donors and containment of HTLV-I/II infection within known risk-groups does not support initiation of HTLV-I/II screening for Swiss blood, tissue, and organ donations.