Viral Replication In Patients Research Articles

The proapoptotic protein Bim is not differentially regulated in CD8+ T cells from resolved or chronic HCV infection

Background: CD8 T cells play an important role in the antiviral immune response against HCV. Nevertheless, these cells fail to control the viral replication in patients with chronic infection either due to mutational escape of the virus in targeted epitopes or a dysfunctional phenotype of virus-specific CD8 T cells. Moreover, deletion of virus-specific CD8 T cells due to premature apoptosis upon their activation in the liver has been suggested. The pro-apoptotic BH3-only protein Bim has been found to be upregulated in CD8+ T cells activated by hepatocytes in mouse models as well as in patients chronically infected with HBV. Here, we addressed whether this molecule also plays a role in in HCV infection. Methods: PBMC were collected from anti-HCV positive injection drug users with chronic or spontaneously resolved HCV infection. From this cohort 13 “resolvers” and 13 “chronics” with detectable CD8 T cells by staining with HLA/peptide-multimers ex vivo were used for this study. The expression of Bim and the activation marker CD38 was analysed on HCV-, CMV- and Flu-specific CD8 T cells ex vivo and after 7 days of antigen-specific expansion via flow cytometry. Results: No significant difference in the expression level of Bim or CD38 could be observed in HCV-specific CD8+ T cells from patients with resolved infection compared to those with chronic infection ex vivo. After 7 days of antigen-specific expansion, both Bim and CD38 were upregulated on antigen-specific CD8 T cells, irrespective of the disease status. Both molecules were also upregulated in bulk CD8 T cells after unspecific activation with anti-CD3. There was also no difference between the Bim expression in HCV-specific CD8 T cells and Flu- or CMV-specific CD8 T cells. Conclusions: The pro-apoptotic molecule Bim is upregulated in CD8 T cells upon their activation, however, we find no evidence for differential regulation in HCV-specific cells from patients with chronic or spontaneously resolved HCV-infection.

Perioperative reactivation of hepatitis B virus replication in patients undergoing partial hepatectomy for hepatocellular carcinoma

Reactivation of hepatitis B virus (HBV) replication happens in patients who receive transarterial chemoembolization or systemic chemotherapy for hepatocellular carcinoma (HCC). The incidence and risk factors of HBV reactivation during the perioperative period in HCC patients receiving hepatic resection is unknown. Between May 2009 and November 2010, 164 consecutive patients with HBV-related HCC who underwent hepatic resection were prospectively enrolled in the study. Among these, 126 patients received antiviral treatment before the operation (the antiviral group) and 38 patients did not receive any antiviral treatment (the non-antiviral group). Ten patients (6.1%) developed HBV reactivation perioperatively (within 1 month after hepatectomy). The incidence of HBV reactivation in the antiviral group and non-antiviral group were 1.6% (2/126) and 21.1% (8/38), respectively (P < 0.001). On univariate analysis, preoperative HBV DNA < 1.0 × 10(3) copies/mL and non-antiviral therapy were significantly correlated with the occurrence of HBV reactivation (P = 0.044 and P < 0.001, respectively). Only non-antiviral therapy remained as a predictive factor on multivariate analysis (odds ratio, 15.46; 95% confidence interval, 2.80-85.46, P = 0.002). The recovery of liver function (defined as a decrease of alanine aminotransferase back to normal) was achieved in 86.8% (132/152) patients without HBV reactivation and in 37.5% (3/8) patients with HBV reactivation when evaluated on day 30 after hepatectomy (P < 0.001). Hepatectomy could reactivate HBV replication during the perioperative period, especially in patients who did not receive any antiviral therapy. A close monitoring of HBV DNA during the perioperative period was necessary irrespective of the preoperative HBV DNA level. Once HBV was reactivated, antiviral therapy should be given.

Mannose-binding lectin deficiency is not associated with increased risk for polyomavirus nephropathy

BackgroundPolyomavirus associated nephropathy (PVAN) affects up to 10% of kidney transplant recipients and is a major risk factor for graft loss. Mannose-binding lectin (MBL) is an important recognition molecule of the innate immune system, and its deficiency has been associated with susceptibility to various infections. In transplantation, on the other hand, high MBL levels have been associated with increased tissue damage in ischemia–reperfusion models and poorer graft and patient survival in solid organ transplant patients. To investigate the relation between MBL and BK virus infection, post-transplant (post-Tx) MBL levels were determined in a cohort of de novo kidney transplant patients with and without BK viremia. Patients and methods41 de novo kidney transplant patients with high (n=16, group 1) or low level BK viremia (n=25, group 2) and 64 patients without BK viremia (group 3) were included. In every patient, functional MBL levels were determined at 1–3 time points (days 30, 90 or 180) post-Tx using an MBL oligomer ELISA. ResultsMBL levels remained unchanged between days 30 and 180 post-Tx independent of BKV viremia. Frequencies of MBL deficiency (<500ng/mL) and MBL levels were not significantly different between the 3 groups. However, group 2 patients showed a trend towards lower MBL serum levels compared to group 1 patients, notably in patients without acute rejection (p=0.076). ConclusionMBL deficiency was not associated with higher risk for BK viremia. In contrast, we hypothesize that BK virus replication in patients with low MBL levels might imply lower risk for progression towards PVAN compared to patients with high MBL levels. This view is supported by recent data demonstrating local complement activation in BK nephropathy.

A Single-Amino-Acid Polymorphism in Reovirus Protein μ2 Determines Repression of Interferon Signaling and Modulates Myocarditis

Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/β) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/β response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-β stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/β through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/β are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded μ2 protein is a strain-specific repressor of IFN-β signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that μ2 amino acid 208 determines repression of IFN-β signaling and modulates reovirus induction of IFN-β in cardiac myocytes. Moreover, μ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-β response. Amino acid 208 of μ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, μ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-β signaling mediated by reovirus μ2 amino acid 208 is a determinant of the IFN-β response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-β response and, consequently, damage to the heart.

The clinical significance of PreS1Ag and anti-PreS1 in patients with chronic hepatitis B

To investigate the positive ratio and clinical significance of PreS1Ag and anti-PreS1 in patients with chronic hepatitis B. 428 patients with chronic HBV infection were collected, these patients were divided into e antigen-positive CHB group, e antigen-negative CHB group, inactive HBsAg carrier group and HBsAg serum conversion group. The difference of positive ratio of PreS1Ag and anti-PreS1 among all groups or between every two groups were analyzed; The relationship of PreS1Ag and anti-PreS1 with HBV M and HBV DNA were also analyzed. SPSS13.0 software was used for statistical treatment. Fourfold table chi-square test or matched-pairs chi-square test was used for enumeration data, and independent sampler t test or rank-sum test was used for measurement data. The differences of PreS1Ag among four groups were statistically significant (X2=141.7, P<0.05). The positive ratio of PreS1Ag in e antigen-positive CHB group was 95.7%, followed by 82.8% in e antigen-negative CHB group, 13.2% in inactive HBsAg carrier group and 2.2% in HBsAg serum conversion group. The difference of positive ratio of anti-PreS1 between HBsAg seroconversion group and HBsAg positive group was statistically significant (X2=6.919, P<0.05), which indicated that anti-PreS1 had good correlation with HBsAg seroconversion. The average absorbance ratio of PreS1Ag in high viral replication group (179.30) was higher than that in low viral replication group (133.87), statistical significance appeared (Z=-3.86, P<0.05). Though the difference of absorbance ratio of anti-PreS1 between two groups had no statistical significance (P>0.05), descent trend was apparent with virus replication level ascending. We analyzed the concordance of anti-HBs and anti-PreS1 by matched-pairs chi-square test, result showed no statistical significance of detection rate between them, X2=0.262, P>0.05. Serum PreS1Ag, HBeAg or HBcAg in liver tissue in reflecting hepatitis B replication had correlation with HBV DNA (X2=33.840, 24.159, 4.854 in order, P<0.05). Correlation coefficient between PreS1Ag and HBV DNA was higher (r=0.628) than that between HBeAg and HBV DNA (r=0.563). PreS1Ag was more sensitive than HBeAg in diagnosing viral replication in patients with chronic hepatitis B. Anti-PreS1 as protective antibody may be involved in clearance of hepatitis B, positive result indicated recovery of chronic hepatitis B.

Mutations in two PKR‐binding domains in chronic hepatitis C of genotype 3a and correlation with viral loads and interferon responsiveness

Interferon (IFN) induces the double-stranded RNA-dependent protein kinase (PKR) to inhibit viral replication. Two motifs of the PKR-binding domain exist in the E2 and the NS5A regions of the hepatitis C virus (HCV). These regions are called the PKR-eukaryotic transcription factor (elF2-alpha) phosphorylation homology domain (PePHD), and the IFN sensitivity-determining region (ISDR). Both regions are inhibited by PKR. Thus, several studies have reported the relationship between these regions and IFN responsiveness and the HCV viral load. However, the data obtained from these studies remain controversial. The aim of this study was to investigate the genomic heterogeneity of the PePHD and the ISDR in patients with genotype 3a and how this impacts HCV replication and the response to IFN therapy. Twenty-one male patients infected with HCV genotype 3a were studied. The PePHD was well conserved, and mutations were found in only one amino acid position in two patients. Patients with three or more mutations in the ISDR had lower viral loads than those with fewer than two mutations (192.2 ± 176.7 vs. 1279.4 ± 997.6 KIU/ml, P = 0.0277). Ten (71.4%) of 14 patients achieved a sustained virological response to IFN therapy. No specific amino acid substitutions in the PePHD and the ISDR were associated with IFN responsiveness; however, the number of mutations in the ISDR was significantly associated with the HCV viral load. The findings from this study suggest that the ISDR plays an important role in regulating viral replication in patients infected with HCV genotype 3a.

Analysis of DNA methylation in human BK virus

Human BK virus may cause nephropathy due to viral replication in patients who have undergone renal transplantation. However, the mechanism regulating replication of BKV is still not clear. Previous studies have suggested that epigenetic modifications may play a crucial role in virus replication. In this study, the DNA methylation profiles of five CpG sites located within the promoter/enhancer regions and nine CpG sites located within the early and late coding regions of the replicating BKV genome were investigated. BKV genomic DNA from mature virions and from the early and late phases of replicating BKV were examined for DNA methylation by bisulfite sequencing that covered 14 CpG sites. Our results showed that none of the examined BKV DNA from the various different stages of replication was methylated. This is the first report to analyze the methylation of BKV genomic DNA during viral replication. The results seem to indicate that methylation is not involved in regulation of BKV replication.

HIV-1 Integrase Inhibitor Resistance and Its Clinical Implications

With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development—elvitegravir and S/GSK1349572—may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification.

Polyomavirus BK Replication in Liver Transplant Candidates with Normal Renal Function

Polyomavirus-associated nephropathy (PVAN) has a predilection for kidney rather than for other solid organ transplants such as the liver. Immunosuppression is widely recognized to be a major risk factor for PVAN development. Since end-stage liver disease (ESLD) patients are immunocompromised and immunosuppression is a major cause of BK virus reactivation, we sought to evaluate BK virus replication in patients listed for liver transplantation. From April to October 2010, we enrolled 20 patients listed for liver transplantation. BK virus load was measured by quantitative real-time polymerase chain reaction on plasma and urine samples. Viremia occurred in only 1 among 20 patients. We hypothesized that in ESLD patients, the low prevalence of BK virus infection may be related to the prevalent impairment of antibacterial immunity rather than to the viral-specific one. In BK virus reactivation, not only the immunodepressive state itself, but also the specific immunologic mechanisms involved may have a role.

Finding a cure for HIV: will it ever be achievable?

Combination antiretroviral therapy (cART) has led to a major reduction in HIV-related mortality and morbidity. However, HIV still cannot be cured. With the absence of an effective prophylactic or therapeutic vaccine, increasing numbers of infected people, emerging new toxicities secondary to cART and the need for life-long treatment, there is now a real urgency to find a cure for HIV.There are currently multiple barriers to curing HIV. The most significant barrier is the establishment of a latent or "silent" infection in resting CD4+ T cells. In latent HIV infection, the virus is able to integrate into the host cell genome, but does not proceed to active replication. As a consequence, antiviral agents, as well as the immune system, are unable to eliminate these long-lived, latently infected cells. Reactivation of latently infected resting CD4+ T cells can then re-establish infection once cART is stopped. Other significant barriers to cure include residual viral replication in patients receiving cART, even when the virus is not detectable by conventional assays. In addition, HIV can be sequestered in anatomical reservoirs, such as the brain, gastrointestinal tract and genitourinary tract.Achieving either a functional cure (long-term control of HIV in the absence of cART) or a sterilizing cure (elimination of all HIV-infected cells) remains a major challenge. Several studies have now demonstrated that treatment intensification appears to have little impact on latent reservoirs. Some potential and promising approaches that may reduce the latent reservoir include very early initiation of cART and the use of agents that could potentially reverse latent infection.Agents that reverse latent infection will promote viral production; however, simultaneous administration of cART will prevent subsequent rounds of viral replication. Such drugs as histone deacetylase inhibitors, currently used and licensed for the treatment of some cancers, or activating latently infected resting cells with cytokines, such as IL-7 or prostratin, show promising results in reversing latency in vitro when used either alone or in combination. In order to move forward toward clinical trials that target eradication, there needs to be careful consideration of the risks and benefits of these approaches, agreement on the most informative endpoints for eradication studies and greater engagement of the infected community.

Short Communication: Activity of Etravirine on Different HIV Type 1 Subtypes:In VitroSusceptibility in Treatment-Naive Patients and Week 48 Pooled DUET Study Data

Etravirine (ETR) has previously shown potent in vitro activity against different primary HIV-1 isolates and demonstrated durable efficacy in treatment-experienced, HIV-1-infected patients in the Phase III DUET studies. The antiviral activity and efficacy of ETR against HIV-1 subtypes B and non-B were further investigated. The effect of HIV-1 subtype on ETR fold change in EC(50) value (FC) was analyzed in HIV-1 recombinant clinical isolates from 673 treatment-naive patients enrolled in other Tibotec studies. Subgroup analyses from the DUET studies of the effect of HIV-1 subtype on the proportion of patients with viral load (VL) <50 HIV-1 RNA copies/ml were also conducted using pooled week 48 data. Genotype/subtype and phenotype determinations were performed using the vircoTYPE HIV-1 and Antivirogram assays, respectively. In vitro results from treatment-naive patients indicated comparable median ETR FC in virus isolates from patients infected with subtype B or non-B (1.1 vs. 1.2, respectively). HIV-1 subtype data were available for 594 and 595 patients in the ETR and placebo groups of the DUET studies, respectively; 94% of patients harbored subtype B. Baseline characteristics were similar across the different subtypes, with the exception of a higher number of sensitive NRTIs used in patients with subtype non-B. At week 48, virological responses in the ETR group were higher in patients with subtype non-B versus B (73% vs. 60%, respectively). ETR was equally effective in suppressing viral replication in patients infected with HIV-1 subtype B or various HIV-1 non-B subtypes.

Study on the relations between hepatitis B virus large surface protein (LHBs) and HBV YMDD mutation

To explore the Relations between hepatitis B virus large surface protein (LHBs) and HBV YMDD mutation, by detecting the hepatitis B virus large protein (LHBs) in Serum of patients with hepatitis B virus YMDD mutation. Selected patients after lamivudine treatment, to HBV YMDD mutation detection, then detected LHBs and HBV DNA in Serum of all patients. Serum HBV-DNA was quantitively detected by using real-time polymerase chain reaction (RT-PCR), the LHBS was detected by using enzyme linked immunosorbent assay (ELISA). There was positive correlation between the A value of LHBs and HBV DNA level in 65 cases of mutation patients (r = 0.961). There was positive correlation between the A value of LHBs and HBV DNA level in 75 cases of non-mutation patients (r = 0.954). There were positive correlation between the A value of LHBs and HBV DNA level in non-mutation patients and mutation patients, showed that LHBs can partially reflect the level of viral replication in patients, but not the impact of hepatitis B virus YMDD mutation.

Long-term efficacy of interferon alpha therapy on hepatitis B viral replication in patients with chronic hepatitis B: A meta-analysis

Background/aims Interferon (IFN) alpha has been used in the treatment of chronic hepatitis B for decades. Beneficial effects including hepatitis B e antigen (HBeAg)/HBV DNA seroclearance have been documented. However, it remains unclear whether interferon has long-term efficacy on inhibiting hepatitis B viral replication. So we conducted a meta-analysis of available literature to assess the evidence obtained on the efficacy of IFN treatment in chronic HBV infection. Methods Seven clinical controlled trials, including 1550 patients and comparing IFN to no treatment, were selected. Data on the incidence of HBV DNA seroclearance, HBeAg seroclearance, and HBsAg seroclearance in IFN treated and untreated patients were extracted from each study. The evaluation of effectiveness was performed with an intention-to-treat (ITT) method. We used the relative risk (RR) and 95% confidence interval (CI) of the main outcomes as the measure of efficacy. Meta-analysis was performed using fixed-effect or random-effect methods, depending on absence or presence of significant heterogeneity. Analyses were performed with STATA version 9.0 and Review Manager Version 4.2. Results Four studies including the data of HBeAg seroclearance with significant heterogeneity were analyzed by random-effect method; six studies including the data of HBsAg seroclearance without significant heterogeneity were analyzed by fixed-effect method. A different incidence of HBeAg seroclearance and HBsAg seroclearance was observed between treated and untreated patients. The RR of HBeAg seroclearance and HBsAg seroclearance was 0.66 (95% CI: 0.44, 0.99) and 0.28 (95% CI: 0.17, 0.46), respectively. Conclusions In conclusion, the results of this meta-analysis indicate that IFN increases the incidence of HBeAg seroclearance and HBsAg seroclearance after long-term follow-up of three to seven years.

Hepatitis C virus replication in patients with occult hepatitis C virus infection†

We read with interest the review by Welker and Zeuzem1 on occult hepatitis C virus (HCV) infection. In this review, the authors claim that viral replication has not been proven in occult HCV infection. However, the presence of the antigenomic HCV RNA strand has been demonstrated in liver and in peripheral blood mononuclear cells (PBMCs) of patients with occult HCV who are negative for anti-HCV and serum HCV RNA2 (and references in Carreño et al.2). There is no doubt that detection of the antigenomic strand is an indicator of viral replication as recognized by Welker and Zeuzem in their review.1 Furthermore, ultracentrifugation of serum samples before performance of real-time reverse transcription polymerase chain reaction (RT-PCR) detects very low levels of viremia in patients with occult HCV infection, and the density of the circulating HCV particles was identical to those found in the serum of patients with chronic hepatitis C.2 In addition, family members of patients with occult HCV infection show serological markers of HCV infection even more frequently than those of patients with chronic hepatitis C.3 Other authors have reported that low-level persisting HCV remains infectious.4 Finally, HCV-specific CD4+ T cell proliferative responses to nonstructural 3 and/or 4 proteins have been detected among patients infected with occult HCV,2 which may not have occurred unless there was ongoing HCV replication, either within the liver or in immune-privileged sites. So, taking all these data into account, one may convincingly assume that HCV replication takes place in patients with occult HCV infection who no doubt are potentially infectious. However, Welker and Zeuzem1 review two studies that unconvincingly failed to detect occult HCV infection in cryptogenic hepatitis,5, 6 because of several concerns with the methodology employed. Thus, one study5 used whole blood to assess occult HCV infection, but it was demonstrated in patients with occult hepatitis C that the sensitivity of HCV RNA detection substantially decreased when using whole blood instead of PBMCs.2 Another study6 included a very small and heterogeneous population, and these authors did not perform detection of HCV RNA either in liver or in serum by PCR after ultracentrifugation. Thus, their results do not exclude the existence of occult HCV infection. On the other hand, an anti-HCV core antibody assay has been developed recently, which adds another diagnostic tool because it has been proposed as a potential new marker of the serologically silent form of occult HCV infection.7 In contrast, other studies have confirmed the existence of occult HCV infection by detection of HCV RNA in liver.8, 9 Furthermore, contrary to what is concluded by Welker and Zeuzem, HCV proteins have been detected in liver10 and in lymph nodes11 of patients who are negative for anti-HCV and serum HCV RNA. In summary, considering the data available, there is cumulative evidence strongly supporting the idea that HCV replication takes place in patients with occult HCV infection. Therefore, in order to diagnose occult HCV infection among these patients, it is mandatory to perform appropriate molecular and immunoserological tests in serum, PBMCs, and liver biopsies. Vicente Carreño*, Javier Bartolomé*, Inmaculada Castillo*, Juan Antonio Quiroga*, * Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.

Transfusion of autologous cytokine-induced killer cells inhibits viral replication in patients with chronic hepatitis B virus infection

Adoptive immune transfer plays an important role in clearance of hepatitis B virus (HBV) in chronic hepatitis B (CHB) patients. However, it is unclear whether cytokine-induced killer (CIK) cells could suppress HBV replication in CHB patients, especially if drug resistance develops. In this study, functional CIK cells were efficiently generated from 21 CHB patients and were transfused in an autologous manner. We found that CIK cells from the CHB patients displayed substantial proliferation and function. Administration of the CIK cells closely correlated with the decrease in the serum HBV load and improvement in liver function in some patients. The virological response rate in patients with baseline serum alanine aminotransferase (ALT) levels of >40 U/L was higher than that in patients with baseline serum ALT levels of < or = 40 U/L. Moreover, patients who had HBeAg loss or showed seroconversion generally had baseline serum ALT levels of >40 U/L. No serious side effects were observed. This protocol represents an alternative immune therapeutic strategy for the disease.

Intrahepatic PD-1/PD-L1 Up-regulation Closely Correlates with Inflammation and Virus Replication in Patients with Chronic HBV Infection

Chronic hepatitis B was characterized by fluctuant immune response to infected hepatocytes resulting in hepatic inflammation and virus persistence. Recently, Programmed Death-1 (PD-1) and its ligand PD-L1 have been demonstrated to play an essential role in balancing antiviral immunity and inflammation in the livers of acute hepatitis B patients, significantly influencing disease outcome. PD-1 up-regulation in peripheral T cells is associated with immune dysfunction in chronic hepatitis B patients. However, the effect of PD-1/PD-L1 on hepatic damage and chronic infective status is still unknown in patients with chronic HBV infection. Here, we report up-regulation of PD-1 and PD-L1 in liver biopsies from 32 chronic HBV patients compared to 4 healthy donors. PD-1/PD-L1 up-regulation was significantly associated with hepatic inflammation and ALT elevation. Moreover, appropriate up-regulation but not overexpression of PD-L1 in the active phase of chronic hepatitis B as well as lower expression of PD-L1 in the inactive phase in liver residential antigen presenting cells (including Kupffer cells and sinusoidal endothelial cells) may contribute to viral inhibition. Our data suggest that the intrahepatic interaction of PD-1 and PD-L1 might play an important role in balancing the immune response to HBV and immune-mediated liver damage in chronic HBV infection.

Human herpesvirus 8 DNA quantification in matched plasma and PBMCs samples of patients with HHV8-related lymphoproliferative diseases

Background The quantitative evaluation of human herpesvirus 8 (HHV8) DNA is not well described in the clinical management of HHV8-related lymphoproliferative diseases. Objectives To evaluate and to compare HHV8 viral load in different blood compartments from patients with multicentric Castleman's disease (MCD), primary effusion lymphoma (PEL) and HHV8-associated solid lymphoma (SLY) and to establish which clinical sample would be preferable for HHV8 DNA testing. Study design We assessed HHV8 DNA in plasma and peripheral blood mononuclear cells (PBMCs) paired samples from 7 PEL, 8 MCD, 2 SLY HIV+ patients at the diagnosis and during the course of the illness by using a real time PCR assay. Results HHV8 viremia was always detectable at diagnosis. HHV8 DNA levels were correlated in matched pairs of samples at diagnosis and during follow-up (Spearman correlation coefficient: r = 0.83, p < 0.001 and r = 0.73, p < 0.001, respectively). The performance characteristics of the PCR assay with both materials did not show disparity by the analysis of the receiver operating characteristic (ROC) curve ( X 1 2 = 0.50; p = 0.48). Conclusions Plasma or PBMCs are both adequate samples for HHV8 DNA quantification and Real time PCR provides a reliable method to estimate viral replication in patients with HHV8-related lymphoproliferations, where HHV8 viral load is a consistent feature.

Does isoflurane enhance rhinovirus replication and virus‐induced cytokine secretion in airway epithelial cells?

There have been no previous studies regarding the effect of volatile anesthetics on human rhinovirus (RV) infection in airway epithelial cells of patients with an upper respiratory infection (URI). We have therefore evaluated in vitro the effect of isoflurane on RV infection in airway epithelial cells. A549 cells and RV-infected A549 cells were treated with isoflurane for 2 or 4 h. Surface expression of intercellular adhesion molecule-1 (ICAM-1) was assessed by flow cytometry, and effects on the secretion of interleukin (IL)-6 and IL-8 were measured by ELISA. The effect on RV replication in the cells was determined by viral titer. Isoflurane treatment for 2 or 4 h had no significant effect on ICAM-1 expression and secretion of IL-6 and IL-8 in control cells. Isoflurane also had no significant additional effect on RV-induced ICAM-1 expression and secretion of IL-6 and IL-8. Viral titers were not significantly influenced by isoflurane. Isoflurane treatment showed no additional effects with RV on ICAM-1 expression, secretion of IL-6 and IL-8, and viral titer in A549 cells. These results suggest that isoflurane itself may not increase further RV infections, inflammations, and viral replication in patients with a viral URI.

Tipranavir: the first nonpeptidic protease inhibitor for the treatment of protease resistance

Background: Despite the availability of a growing number of potent antiretroviral agents, efforts to completely suppress viral replication in patients with HIV-1 infection are limited because of the increasing risk of resistance. Tipranavir (TPV) is the first in a class of antiretroviral agents known as nonpeptidic protease inhibitors (PIs). TPV exhibits a resistance profile distinct from that of other currently available PIs, making it a potential option for treatment experienced patients with resistance to multiple Pls. Objective: This article discusses the clinical pharmacology and efficacy of TPV in the treatment of HIV-1 infection in patients who are highly treatment experienced or harbor PI-resistant virus. Methods: A search was conducted of English language, peer-reviewed articles and abstracts indexed on the MEDLINE and Current Contents databases (1966-May 2007) using the terms tipranavir, Aptivus, nonpeptidic protease inhibitor, human immunodeficiency virus, and protease resistance. Product information and abstracts from national and international AIDS and retrovirus meetings (2005-2006) were also reviewed. Results: Use of TPV in combination with ritonavir (TPV/r) was approved by the US Food and Drug Administration based on 2 Phase III studies. In these studies, HIV-infected patients with extensive treatment experience with antiretroviral agents, including PIs, nucleoside analogues, and nonnucleoside reverse transcriptase inhibitors, were randomized to receive TPV/r or a ritonavir-boosted comparator PI. All patients had evidence of resistance to multiple PIs. The specific comparator (amprenavir, indinavir, lopinavir, or saquinavir) was selected for each patient with the aid of resistance testing. Each patient also received an optimized background regimen of antiretroviral agents, which could include enfuvirtide. At 24, 48, and 96 weeks, the TPV/r group had higher rates of virologic response (defined as ≥1 log10 decrease in HIV RNA) and viral suppression (to <400 and <50 copies/mL) than did the comparator group. Conclusions: Although TPV has a mechanism of action similar to that of earlier-generation PIs, it has activity against HIV-1 strains with resistance to multiple PIs. Currently, TPV/r is indicated for use in highly treatment experienced patients with multiple PI resistance.

Rapid Detection and Quantification of Chikungunya Virus by a One-Step Reverse Transcription–Polymerase Chain Reaction Real-Time Assay

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. A large outbreak of CHIKV disease occurred in 2005 in the Indian Ocean Islands. Many cases have been imported in European countries. Laboratory confirmation of suspected cases is mandatory for control measures during an outbreak. We report a novel, real-time, reverse transcription-polymerase chain reaction (RT-PCR) for the nonstructural protein 1 region that can quantify a wide range of viral RNA concentrations. This assay was validated by in vitro experiments in which interferon-alpha, a well-known virus inhibitor, showed a dose-dependent inhibition of virus replication on Vero cells that was assessed by viral infectivity and viral RNA production. This new real-time RT-PCR was used to measure viral load in serum samples from cases recently imported to Italy, and may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication in patients.