Serum Viral Load Research Articles

Human herpesvirus 6 reactivation and inflammatory cytokine production in patients with drug-induced hypersensitivity syndrome

Background Human herpesvirus 6 (HHV-6) reactivation has been suggested to modify the clinical features of drug induced hypersensitivity syndrome (DIHS). However, mechanisms for viral reactivation and modification of the clinical features remain unclear. Objective To determine whether cytokines play an important role in viral reactivation and modification of the clinical features. Study Design We examined the kinetics of serum cytokines and viral load in HHV-6 infections of six patients with DIHS. Results HHV-6 infection occurred three to four weeks after the onset of disease. Elevated TNF-α and IL-6 levels were observed to precede HHV-6 infection in four of the six patients. Although high levels of IL-6 were observed in samples collected prior to HHV-6 infection, the amounts of this cytokine significantly decreased to undetectable levels during viral infection in five of the six patients. Subsequently, serum IL-6 levels were increased after viral infection in five patients. IL-1β levels were also increased at the time of viral infection in three of the six patients. Neither IL-4 nor IFN-γ could be detected in any of the samples. Conclusion These results demonstrate that cytokines play an important role in HHV-6 reactivation in patients with DIHS.

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TLR3 Deletion Limits Mortality and Disease Severity due to Phlebovirus Infection

TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3(-/-) mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3(-/-) mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-gamma, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3(-/-) mice.

Viral factors correlate with hepatitis B e antigen seroconverson in patients with chronic hepatitis B

Seroconversion (SC) from hepatitis B envelope antigen (HBeAg) to anti-HBe usually indicates lower viral loads, resolved hepatitis activity and improved long-term outcomes. However, the role of viral factors in the development of SC remains largely unknown. We thus comprehensively studied these factors in 25 patients with sustained HBeAg SC and seven control patients with sustained loss of HBeAg. We determined viral factors in serum samples obtained 1 year before, 6 months before, 3 months before, at the time of, 6 months after and 1 year after HBeAg SC or HBeAg loss. Precore A1896 and basal core promoter T1762/A1764 mutants were determined by polymerase chain reaction (PCR)-based assays. Serum HBV levels were determined by a real-time PCR assay. We found that decline of serum viral load, frequently accompanied by hepatitis exacerbation, occurred within 1 year before HBeAg SC. The proportions of precore and BCP mutations also increased gradually throughout the process of HBeAg SC. The virologic features were similar between HBeAg SC group and HBeAg loss group. Before HBeAg SC or loss, genotype B patients had higher serum viral loads and lower proportions of BCP mutation compared with genotype C patients. Our findings suggested that viral factors correlate with the development of sustained HBeAg SC or loss.

Responding with Treatment to Those Acutely Infected with HIV: No Time to Lose

HIV infection when HIV RNA first appears in the blood, but before HIV-specific antibodies are detectable. Individuals with AHI have increased HIV transmissibility due to the increased viral load in both blood and genital secretions, making it critically important for prevention of secondary HIV transmission. While some patients with acute infection have a viral syndrome (known as acute retroviral syndrome or primary HIV), the mild and non-specific nature of acute retroviral syndrome complicates effective screening. A significant barrier to diagnosis of AHI is the non-specific nature of the signs and symptoms associated with the acute retroviral syndrome. Unless a clinician entertains the diagnosis in the differential and orders an appropriate diagnostic test, the diagnosis will be missed. It is vital to have clinicians consider AHI in young adults with fever and diffuse lymphadenopathy and that clinicians also know to include a viralspecific test and not just an HIV antibody as diagnostic tests. Methods to incorporate HIV RNA screening of all HIV antibody negative bloods for testing populations may reduce the number of missed diagnoses for AHI. Specimen pooling and nucleic acid amplification methodologies have proven to be a feasible and effective method of acute HIV infection screening of at-risk populations such as individuals seen in Sexually Transmitted Disease clinics, Emergency Departments, and at other locations where HIV testing is routinely provided or individuals at risk for HIV infection may seek care. Diagnosing AHI is of benefit at the level of the individual patient and at the level of the general public as part of HIV disease control efforts. Early HIV therapy presents a potential window of opportunity to improve immune function and slow the progression to AIDS, and more trials are underway now to determine the clinical benefits of early HIV therapy. Transmission of HIV is principally driven by the quantity of the HIV inoculum in either blood or genital secretions. This brief period of extremely high HIV viral load and uncontrolled viral replication in AHI last for less than eight weeks. Therapy with ARV can precipitously drop the serum and genital secretion viral load and potentially render the individual “non-infectious” should the viral load drop below detection.

Safety of anti-TNF- therapy in rheumatoid arthritis and spondylarthropathies with concurrent B or C chronic hepatitis

To assess the safety of anti-tumour necrosis factor (TNF)-alpha therapy in patients with rheumatoid arthritis (RA) or spondylarthropathies (SA) and concurrent chronic hepatitis B or C. Records concerning 480 outpatients attending the Rheumatology Department of the University Hospital of Nice (France) for RA or SA were retrospectively reviewed for the duration of disease, treatment, serological status and biological data. Six relevant cases were identified: two of RA with chronic hepatitis B; one of SA with chronic hepatitis B and three of RA with chronic hepatitis C. Five patients had received etanercept and one infliximab; two had been given adalimumab after an unsuccessful trial of etanercept. Patients with concurrent chronic hepatitis B were also given lamivudine. In none of the cases had changes in serum aminotransferases or viral load been reported. The use of anti-TNF-alpha therapy (plus lamivudine in the presence of concurrent underlying hepatitis B viral infection) appeared to be safe in that it had no effect on serum aminotransferases and/or viral load. However, repeated monitoring is necessary throughout the treatment period.

Low density lipoprotein receptor transcripts correlates with liver hepatitis C virus RNA in patients with alcohol consumption

Alcohol consumption has a major impact on the natural history of chronic hepatitis C virus (HCV) infection, although the underlying mechanisms are still debated. We designed a clinical study to evaluate the impact of alcohol abuse on both viral load and expression of low-density lipoprotein receptor (LDLR) and CD81 expression. Thirty-eight consecutive HCV-infected patients were enrolled. Group 1 (n = 18), < or =10 g alcohol/day, group 2 (n = 8), < or =30 g alcohol/day, group 3 (n = 12), >or =30 g alcohol/day. Receptors expression was measured by flow cytometry analysis in peripheral blood mononuclear cells (PBMC) and by specific real-time retrotranscription polymerase chain reaction (RT-PCR) in the liver. Serum viral load was evaluated by quantification of both HCV genomic RNA and total core antigen. The hepatic viral load was assessed by real-time RT-PCR. Serum HCV-RNA and total core antigen were significantly correlated, and were higher, albeit not significantly, in group 3 than in group 1. Alcohol consumption had no effect on expression of HCV putative receptors in PBMC, except for CD81, which was upregulated on monocytes in group 2. In the liver, viral load and levels of LDLR transcripts were significantly higher in group 3 than in group 1. Remarkably, a significant positive correlation was found between LDLR transcripts and HCV-RNA (r2 = 0.83, P < 10(-3)). Finally, in vitro experiments suggested that the effect of ethanol on LDLR expression was indirectly mediated by both tumour necrosis factor-alpha and interleukin-1beta. In conclusion, this study is the first to support a role for LDLR in the natural infection by HCV in man.

Lower serum viral loads in young patients with hepatitis‐B‐virus‐related hepatocellular carcinoma

Advanced age and high hepatitis B virus (HBV) DNA level are risk factors associated with the development of HBV-related hepatocellular carcinoma (HCC). However, little is known about the role of viral load in the carcinogenesis of HCC in young people. A total of 183 HBV-related HCC patients and 202 HBV carriers were therefore enrolled to compare serum viral loads in young (</=40 years of age) and old (>40 years of age) age groups. Other factors associated with the development of HCC were also analysed. The results showed that serum alanine aminotransferase (38.7 +/- 24.1 vs 58.4 +/- 65.4 IU/L, P = 0.006) and HBV DNA levels (log(10) titre: 4.20 +/- 1.33 vs 4.80 +/- 1.39, P = 0.053) were lower in young HCC patients than in old HCC patients. There was a positive correlation between age and serum HBV DNA level in HCC patients but a negative correlation in HBV carriers. Young HCC patients with HBV genotype B infection had higher viral loads than those with genotype C infection (log(10) titre: 4.79 +/- 1.34 vs 3.27 +/- 0.60, P = 0.001). By multivariate logistic regression analyses, high serum HBV DNA level was associated with the development of HCC in old patients [odds ratio (OR) 1.584, 95% confidence interval (CI) 1.075-2.333] rather than in young patients (OR 0.848, 95% CI 0.645-1.116). In conclusion, viral factors in association with the development of HBV-related HCC in young patients may be different from their old counterparts. The complicated interplay between host and virus could be responsible for the emergence and aggressive outcome of early-onset HCC.

Role of Hepatitis B Virus Precore/Core Promoter Mutations and Serum Viral Load on Noncirrhotic Hepatocellular Carcinoma: A Case‐Control Study

Apart from the presence of liver cirrhosis, hepatitis B virus (HBV) factors have also been shown to play a role in the development of hepatocellular carcinoma (HCC). Studying HBV-related noncirrhotic HCC may help clarify the effect of viral factors. In a hospital-based, age- and genotype-matched study, we aimed to determine the role played by basal core promoter (BCP) T1762/A1764 mutation, precore A1896 mutation, and serum viral load in noncirrhotic hepatocarcinogenesis by comparing 44 patients with HBV-related noncirrhotic HCC, 45 patients with chronic hepatitis B, and 42 patients with HBV-related cirrhotic HCC. HBV genotype, precore and BCP mutations, and viral load were determined by molecular assays. In univariate analysis, statistically significant odds ratios were obtained for male sex (P=.005) and BCP T1762/A1764 mutation (P=.0003) in patients with noncirrhotic HCC, compared with patients with chronic hepatitis B. By multiple logistic regression analysis, male sex, BCP T1762/A1764 mutation, and viral load >or=10(5) copies/mL were independently associated with the risk of noncirrhotic HCC. The virologic characteristics were similar between patients with cirrhotic HCC and those with noncirrhotic HCC. Our results suggest that BCP T1762/A1764 mutation and higher viral load may be involved in the carcinogenesis of cirrhotic and noncirrhotic HCC.

Circulating and Liver Resident CD4+CD25+ Regulatory T Cells Actively Influence the Antiviral Immune Response and Disease Progression in Patients with Hepatitis B

CD4+CD25+ regulatory T cells (Treg) have been shown to maintain immune tolerance against self and foreign Ags, but their role in persistent viral infection has not been well-defined. In this study, we investigated whether and where CD4+CD25+ Treg contribute to the development of chronic hepatitis B (CHB). One hundred twenty-one patients were enrolled, including 16 patients with acute hepatitis B, 76 with CHB, and 29 with chronic severe hepatitis B. We demonstrated that in chronic severe hepatitis B patients, the frequencies of CD4+CD25+ Treg in both PBMC and liver-infiltrating lymphocytes were significantly increased and there was a dramatic increase of FoxP3(+)-cell and inflammatory cell infiltration in the liver compared with healthy controls. In CHB patients, circulating CD4+CD25+ Treg frequency significantly correlates with serum viral load. In acute hepatitis B patients, circulating CD4+CD25+ Treg frequency was initially low and with time, the profile reversed to exhibit an increased number of circulating Treg in the convalescent phase and restored to normal levels upon resolution. In PBMC taken from infected patients, depletion of CD4+CD25+ Treg led to an increase of IFN-gamma production by HBV-Ag-stimulated PBMC. In addition, CD4+CD25+ Treg were capable of suppressing proliferation of autologous PBMC mediated by HBV Ags, which probably reflects the generation of HBV-Ag-specific Treg in circulation and in the liver of HBV-infected patients. Together, our findings suggest that CD4+CD25+ Treg play an active role not only in modulating effectors of immune response to HBV infection, but also in influencing the disease prognosis in patients with hepatitis B.

Application of real-time PCR to quantify hepatitis B virus DNA in chronic carriers in The Gambia

Background/AimThe study aimed at developing a real-time quantitative PCR assay to monitor HBV serum virus load of chronic carriers enrolled in therapeutic trials.MethodQuantitative real-time PCR assay was carried out using SYBR-Green signal detection and primers specific to the S gene. Thermal cycling was performed in an ABi 5700 sequence detection system. The assay was calibrated against an international HBV DNA standard and inter- and intra-assay reproducibility determined. Levels of viral load were monitored for 1-year in lamivudine treated carriers. Correlation between HBV DNA levels and HBeAg sero-status was determined in untreated carriers.ResultsThe qPCR assay showed good intra- and inter-assay reproducibility over a wide dynamic range (1.5 × 103 to 1.5 × 108 copies/mL) and correlated well with those from a commercial assay (r = 0.91, (p < 0.001). Viral load levels dropped dramatically but temporarily during and after a short course of lamivudine therapy. HBV DNA was a more reliable indicator of the presence of virus than HBe antigen and was detected in 77.0% (161/209) of HBeAg negative and in all HBeAg positive carriers.ConclusionThis method is reliable, accurate, and reproducible. HBV DNA Quantification by qPCR can be used to monitor the efficacy of HBV therapy and useful in understanding the natural history of HBV in an endemic area.

Overlap lamivudine treatment in patients with chronic hepatitis B receiving adefovir for lamivudine‐resistant viral mutants

Adefovir dipivoxil is effective against lamivudine-resistant hepatitis B virus (HBV) strains. Whether short-term overlap lamivudine is beneficial remains unknown, particularly in patients with decompensated chronic hepatitis B. We enrolled 30 patients who underwent 48-week adefovir treatment (10 mg daily) for exacerbation of hepatitis B, associated with lamivudine-resistant mutants. Nineteen (63.3%) patients had baseline evidence of hepatic decompensation. Lamivudine was combined for <or=1 month in eight (group I), 2-5 months in 10 (group II) and >or=6 months in 12 (group III). We analysed their serial alanine aninotransferase (ALT) levels, Child-Pugh (CP) score, serum viral load and lamivudine-resistant strains. We found that serum ALT became normalized in 20 (66.7%) and HBV-DNA decreased to <or=100 copies/mL in eight (26.7%) at the end of the 48-week treatment. The log(10) reduction of serum HBV-DNA was significantly smaller in group I patients compared with group II and III patients at week 24 and 48 of treatment [median (range): 3.0 (1.5-5.6) vs 4.5 (1.5-7.4), P = 0.032; and 3.4 (0.9-4.7) vs 5.2 (2.2-7.7), P = 0.008 respectively]. In contrast, the virologic responses at the end of the 48-week therapy were similar between group II and III patients. The improvement in serum ALT and CP score at week 48 was similar irrespective of baseline decompensation, liver cirrhosis and the duration of overlap lamivudine therapy. Our findings suggested that an overlap of lamivudine for >or=2 months might lead to better virological but not biochemical outcomes in patients receiving adefovir for lamivudine-resistance HBV. As our sample size was small and the study was not randomly controlled, further studies are needed.

Interferon gamma‐secreting HCV‐specific CD8+ T cells in the liver of patients with chronic C hepatitis: relation to liver fibrosis – ANRS HC EP07 study*

Little is known about the role of specific hepatitis C virus (HCV) CD8+ T cells in liver damage, especially for the progression of fibrosis, during the highly variable course of chronic C hepatitis. The aim of this study was to investigate the presence of HCV-specific CD8+ T cells in the liver of patients with chronic C hepatitis and to examine their clinical significance by relating the response to liver fibrosis and progression rate, serum viral load, serum aminotransferase levels, inflammatory activity and in situ characteristics of the intrahepatic infiltrate. Fifteen patients were prospectively included in the study. Intrahepatic lymphocytes were tested for interferon gamma (IFNg) production in response to HCV class I-restricted epitopic peptides using enzyme-linked immunospot analysis. Liver biopsy samples were evaluated for fibrosis, fibrosis progression rate, activity, and in situ number of CD8+ cytotoxic lymphocytes and apoptotic cells. An IFNg-specific CD8+ T-cell response was detected in the liver samples of 47% of patients which was significantly related to a lower stage of fibrosis (P = 0.02) and a lower progression rate of fibrosis (P = 0.01). It was neither related to the number of cytotoxic lymphocytes infiltrating the liver nor to hepatocyte apoptosis. In conclusion, our results indicate that the presence of HCV-specific IFNg-secreting T cells in the liver of patients with chronic C hepatitis is associated with low liver fibrosis and fibrosis progression rate, suggesting that these IFNg-secreting T cells might limit the progression of liver damage.

ＨＢｅ抗原陰性Ｂ型慢性肝炎患者におけるラミブジン投与中止時期についての検討

We studied the timing of withdrawal of lamivudine administration for HBe Ag-negative chronic patients. The amount of serum HBV DNA was measured using the Amplicor HBV monitor (AM) and the Real-time Detection direct (RTD-direct). All values became less than 2.6 log copies/ml according to the AM method after administration. Five of 16 patients were found to have no HBV according to the RTD-direct (less than 1.0 log copies/ml). Thus, withdrawal of administration was attempted for these five patients. After the withdrawal, the viral load and ALT levels had increased in all patients. These results showed the difficulty of withdrawing lamivudine administration from HBe Ag-negative chronic patients, even though they have very low viral load in serum by the treatment.

Cerebrospinal and peripheral human immunodeficiency virus type 1 load in a multisite, randomized, double-blind, placebo-controlled trial ofd-Ala1-peptide T-amide for HIV-1-associated cognitive-motor impairment

D-Ala1-peptide T-amide (DAPTA) has shown neuroprotection in vitro against gp120-induced loss of dendritic arborization and is promulgated as a CCR5 antagonist. A multisite, randomized, double-blind clinical trial of DAPTA versus placebo prior to combination antiretroviral therapy conducted with human immunodeficiency virus (HIV)-1 seropositive participants having cognitive impairment showed no overall cognitive effect, though subgroups with greater impairment and CD4 cell counts of 201 to 500 cells/mm3 at baseline showed significant improvement. The objective of this study was to examine whether intranasal administration of DAPTA at a dose of 2 mg three times per day (tid) was associated with a reduction of cerebrospinal fluid (CSF) and peripheral (plasma and serum) viral load among a subgroup of participants completing 6 months of treatment. Baseline and 6-month CSF (n = 92) and peripheral (plasma n = 33; serum n = 24) viral load were measured by the Roche Ultrasensitive assay, version 1.5, with reflexive use of the AMPLICOR assay and preservation of the blind. A DAPTA treatment indicator variable was tested using generalized linear models on change in viral load. Peripheral load (combined plasma and serum) was significantly reduced in the DAPTA-treated group. No group differences in CSF viral load were found. This retrospective study on a limited subgroup of the original trial sample indicated that DAPTA treatment may reduce peripheral viral load without concomitant CSF effects. Future studies should be undertaken to confirm the existence of this result and the CSF-periphery dissociation observed with respect to HIV-1-associated cognitive-motor impairment.

Distribution of Epstein‐Barr viral load in serum of individuals from nasopharyngeal carcinoma high‐risk families in Taiwan

The utility of EBV load as a tumor marker in nasopharyngeal carcinoma (NPC) patients suggests that it might also serve as a screening test for individuals who are at high risk for developing NPC. We previously demonstrated that unaffected individuals from high-risk families had elevated anti-EBV antibody levels compared to community controls. In this study, we measured EBV load using 2 different real-time PCR assays (targeting BamH1W and polymerase gene sequences, respectively) carried out in 2 independent research labs in serum samples from 19 untreated NPC cases, 11 healthy community controls and 100 unaffected individuals from families in which 2 or more individuals were affected with NPC. EBV genomes were detectable in 68% of NPC cases by the EBV BamH1W assay and in 74% by the EBV polymerase assay (kappa = 0.64). Patients with stage III or IV disease had significantly higher EBV load compared to those with stage I or II disease (p = 0.008). EBV DNA was detected in a single community control sample by the EBV BamH1W assay and in none of the samples by the EBV polymerase assay. Only one of 100 unaffected family members tested positive by both assays. An additional 14 were positive by only one of the 2 EBV load assays used and usually in only one of the duplicate wells tested, all with very low viral loads (3-50 copies/ml). In addition, EBV load did not correlate with EBV serology results (anti-VCA, anti-DNase, anti-EBNA-1) among these unaffected family members. In conclusion, our study suggests limited clinical utility of the EBV load test, in its current configuration, to screen individuals from high-risk families. Should a more sensitive or specific molecular assay be developed that is capable of detecting and distinguishing tumor-derived EBV genomes or gene products from true negatives, it could be evaluated as a possible screening tool for asymptomatic and early-stage NPC.

Transition of Humoral Immunity and Clinical VZV Reactivation in Hematopoietic Stem Cell Transplantation Recipients.

Reactivation of varicella-zoster virus (VZV) is common among recipients of hematopoietic stem cell trasnplantation (HSCT). In this setting, VZV infection can occur frequently as localized zoster (shingles) and sometimes as disseminated varicella (chickenpox) with or without visceral involvement, which results in a high mortality rate. Previous studies revealed that VZV-specific memory T cells play a crucial role in suppressing reactivation. Although varicella zoster immunoglobulin is used as a prophylactic and treatment modality in some contries, the role of humoral immunity against VZV infection has been poorly evaluated. The aim of the study was to identify the relationship between quantitative transition of VZV antibody titers and clinical manifestation of VZV infection in HSCT recipients. Thirteen patients with VZV infection after HSCT (7 males, 6 females; age: 21–65 years, median age: 35 years) were enrolled. Eleven patients (allo, 7; auto, 4) suffered localized zoster. Two patients (both allo) suffered systemic varicella with visceral involvement (1 with acute abdomen and 1 with interstitial pneumonia). All patients were treated with acyclovir. Seven patients who had been followed for at least 3 years after HSCT without clinical VZV infection were studied as control subjects (4 males, 3 females; age: 16–40 years, median age: 28 years; allo, 6; auto, 1). We retrospectively studied transition of anti-VZV-IgG titer by the IAHA and gpELISA method (before and at 3, 6, 12, 24, 36 months after HSCT) and VZV-DNA by realtime PCR during clinical VZV infection (onset and paired serum) using cryopreserved serum samples. All patients were seropositive for VZV before HSCT. One of the patients (the most severe case) had become seronegative before systemic varicella developed. However, VZV infections in the other 12 patients occurred despite the persistent existence of anti-VZV (IAHA:x8 to >x256; median x32). There are no statistically significant differences between anti-VZV titers in the 13 patients with VZV infection and those in the 7 controls at each time point. Acceleration of the titer (>x4) was not observed in any of the 9 recipients with VZV infection within the first 6 months after HSCT but was observed in 3 of 4 recipients with VZV infection after 6 months (Figure 1). Four available samples at onset of VZV infection (varicella, 2; zoster, 2) were all positive for VZV-DNA (94.4 to 12173.3 copies /ml serum) by realtime PCR, whereas VZV was undetectable in all paired sera (2 to 5 weeks after onset). Pre-existing anti-VZV titer (gpELISA) and serum viral load were inversely correlated (Figure 2). In HSCT recipients, pre-existing antibody does not prevent development of VZV reactivation but may contribute to decreased viral load, resulting in a mild clinical course. Despite the localized clinical feature of zoster, VZV was detected in serum samples from zoster patients. Clinicians should be aware of the risk of airborne transmission of VZV from HSCT recipients with either varicella or zoster. [Display omitted]

Human Herpesvirus 8 Infection in Patients With Cutaneous Lymphoproliferative Diseases

To investigate the prevalence of human herpesvirus 8 (HHV-8; Kaposi sarcoma-associated herpesvirus) infection in patients with lymphoproliferative skin diseases such as large-plaque parapsoriasis (LPP) and mycosis fungoides compared with inflammatory cutaneous conditions or healthy control subjects. A survey study was undertaken in 123 subjects with various clinical conditions. All patients had been seen in the Dermatology Department of the San Gallicano Dermatology Institute, Rome, Italy, in the last 2 years. Forty-five patients with inflammatory or autoimmune cutaneous diseases, 50 healthy control subjects, 10 patients with LPP, 12 patients with mycosis fungoides, and 6 patients with classic Kaposi sarcoma were included in the study. The prevalence of HHV-8 infection was investigated with serologic studies using the gold standard assay based on body cavity-based B-cell lymphoma-1 cells latently infected with HHV-8. The presence of HHV-8 conserved sequence, corresponding to open reading frame 26, was also assessed in the peripheral blood and lesion tissue samples from patients with lymphoproliferative cutaneous diseases with nested polymerase chain reaction. The presence and distribution of cell types infected with HHV-8 in the lesion tissues was determined with immunohistochemical staining with the monoclonal antibody directed against the latent nuclear antigen-1 of HHV-8 encoded by open reading frame 73. In healthy control subjects and patients with inflammatory skin diseases, 13.9% were found to have antibody against HHV-8, consistent with the seroprevalence population in Italy. A highly significant association of HHV-8 infection and LPP was found (100%) compared with mycosis fungoides (25%). The peripheral blood mononuclear cells in 8 of 10 patients with LPP were found to harbor viral sequences at nested polymerase chain reaction, whereas none of them had a detectable serum viral load. All LPP lesion tissue samples were positive for HHV-8-encoded open reading frame 26, and the presence of HHV-8-infected cells was confirmed by immunohistochemistry profiles performed on paraffin-embedded tissues from 4 of 10 patients. The positive cell types included endothelial cells and the infiltrating dermal lymphocytes, characteristic hallmarks of LPP. Analysis of T-cell receptor gamma chain rearrangements in lesion tissue from our patients confirmed the lack of a significant association between T-cell clonality and LPP. These data suggest that HHV-8 may play a role in the onset of LPP, a disease whose cause and evolution are still undefined and which has often been considered the early stage of mycosis fungoides.

One-Year Entecavir or Lamivudine Therapy Results in Reduction of Hepatitis B Virus Intrahepatic Covalently Closed Circular DNA Levels

Entecavir and lamivudine are potent nucleoside analogues that can suppress hepatitis B virus (HBV) replication. However, the effects of these two antiviral agents on intrahepatic total HBV DNA and covalently closed circular DNA (cccDNA) are not known. In this study, we aimed to assess the effect of 48 weeks of entecavir/lamivudine therapy on intrahepatic total HBV DNA and cccDNA levels. Forty chronic hepatitis B patients, participating in two Phase III entecavir trials at our centre, were randomized to receive 48 weeks of either 0.5 mg once daily of entecavir (n = 21) or 100 mg once daily of lamivudine (n = 19). Their serological, virological and biochemical responses, as well as intrahepatic HBV DNA levels were monitored. There was no significant difference between entecavir and lamivudine therapy in terms of post-treatment serological, virological and biochemical responses. Both nucleoside analogues reduced serum viral load, intrahepatic total HBV DNA, and cccDNA by about 4.8 logs, 2 logs, and 1 log respectively. An increase in the proportion of intrahepatic HBV DNA in the form of cccDNA was seen after 48 weeks of therapy. In conclusion, both entecavir and lamivudine can successfully reduce intrahepatic HBV DNA and cccDNA. CccDNA becomes the dominant form of HBV DNA during viral suppression and is possibly responsible for viral rebound after short-term antiviral therapy.

Quantitative Detection of Hepatitis C Virus (HCV) RNA in Saliva and Gingival Crevicular Fluid of HCV-Infected Patients

The search for hepatitis C virus (HCV) in body fluids other than blood is important when assessing possible nonparenteral routes of viral transmission. However, the role of oral fluids in HCV transmission remains controversial. Here we quantitatively determined HCV RNA in saliva and gingival crevicular fluid (GCF) of anti-HCV-positive patients. Most patients (14 of 18; 78%) whose saliva specimens were negative had HCV RNA in their GCF. Most patients (20 of 26; 77%) had higher HCV RNA levels in their GCF than in their saliva. Although there was not a statistically significant correlation between the serum viral load and HCV level in saliva or GCF, patients with low serum HCV loads were less likely to have detectable HCV in their saliva. These findings have important implications for medical personnel and suggest that epidemiological studies designed to understand the significance of the oral route of transmission of HCV are warranted.