Infección por virus C oculto en hepatopatías criptogenéticas Sr. Director: Desde que Brechot y colaboradores publicaron la primera serie de pacientes con Hepatitis B crónica sin hallazgo del antige-no de superficie en el suero (1) se ha venido hablando de virus B "oculto". Nosotros hemos colaborado en un trabajo de reciente publica-ción (2) en el que se ha estudiado una serie de 100 pacientes con alteración persistente de aminotransferasas y/o gamma-glutamil-transpeptidasa que no podía ser achacada a ninguna de las etiolo-gías habituales, incluyendo la negatividad de los marcadores virales (RNA-HVC, DNA-HVB) y en los que en cambio se encontró, en el 57%, por hibridación in situ, la presencia del RNA-HVC del genotipo lb en el tejido hepático y con una activi-dad necroinflamatoria y fibrótica superior a la histología de los pacientes sin RNA-HVC intrahepático. La reflexión, como clínico, que estos hallazgos implican me han hecho sumarizar las posibles consecuencias del mismo. Primero, en cuanto al diagnóstico, este hallazgo hecha por tie-rra la aseveración de que la ausencia en el plasma de marcadores del virus C (preferentemente el RNAHVC) elimina el diagnósti-co de Hepatopatía Crónica por virus C y, áun recogiendo los datos de mayor potencial necróticroinflamatorio y de fibrosis, desconocemos su evolución futura que precisará de seguimiento de estos pacientes. En cuanto a posibilidad de contagio, no tenemos aún el con-vencimiento de que el material genómico detectado sea infeccio-so, aunque con copias suficientes para identificar el genotipo 1b. Por otro lado el hecho de que, al menos en nuestra serie, los pacientes presenten elevaciones enzimáticas significativas, elimi-na el peligro de que puedan ser donantes en bancos de sangre, etc., pudiendo igualmente prevenirse posibles contagios parente-rales individuales. El hecho de no conocer aún la evolución específica de estos enfermos con virus C oculto, así como su pertenencia al genotipo Ib y que, únicamente por hibridación in situ de una nueva biopsia tras el tratamiento, pueda juzgarse de la eficacia del mismo, implican que, de momento, sólo deban emprenderse seguimien-tos y tratamientos muy controlados. A. Pérez Mota Servicio de Aparato Digestivo. Hospital Virgen de la Torre. Madrid 1. Bréchot C, Thiers V, Kremsdorf D, Nalpas B, Pol S, Paterlini-Bréchot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely "occult"?. Hepatology 2001; 34: 194-203. 2. El hepatocarcinoma es el tumor primario maligno hepático más frecuente. Una de sus manifestaciones clínicas es la produc-ción de síndromes paraneoplásicos. Entre los más frecuentes se encuentran la poliglobulia, la hipercalcemia, la hipoglucemia y la hiperlipidemia. De forma más rara, se han descrito reacciones leucemoides secundarias a hepatocarcinoma (1-3). El primer caso fue descrito por Ranke y cols. (1) en 1965. Tras realizar una bús-queda bibliográfica en Medline desde 1966-2002 existen escasos artículos publicados referentes a dicho tema y...

Chronic hepatitis C virus (HCV) infection has been associated with several extrahepatic manifestations, among these, to diseases with oral manifestations such as Sjögren's syndrome or sialadenitis. HCV-RNA has been detected in saliva and in salivary glands from patients with sialadenitis by polymerase chain reaction. However, morphological evidence of HCV replication in salivary gland cells is needed to support a role for HCV in causing sialadenitis or Sjögren's syndrome. We have used in situ hybridization and immunohistochemistry to analyze the presence of HCV-RNA of sense and antisense polarity and HCV core antigen, respectively, in salivary gland biopsies from 19 patients with chronic sialadenitis or Sjögren's syndrome (eight anti-HCV-positive; 11 anti-HCV-negative). HCV-RNA of both positive and negative polarity as well as HCV core antigen were detected in the epithelial cells of the salivary gland biopsies from all of the anti-HCV-positive patients but in none of the anti-HCV-negative cases. The percentage of HCV-infected cells ranged from 25 to 48.8% in the patients studied. In conclusion, we have shown that HCV infects and replicates in the epithelial cells from salivary glands of patients with Sjögren's syndrome or chronic sialadenitis. However, its implication in the pathogenesis of these diseases deserves future research.

TT virus (TTV) is an unenveloped, single-stranded, circular-DNA virus which resembles members of the Circoviridae, that is commonly found in humans and which lacks pathological consequences for the infected host. TTV replication has been demonstrated in bone marrow cells but not in peripheral blood mononuclear cells (PBMC), suggesting that hematopoietic cells must be activated to support TTV replication. To test this hypothesis, PBMC from two naturally TTV-infected individuals and from two healthy TTV-DNA negative donors infected in vitro with a TTV-DNA-positive serum were cultured in the presence (stimulated) or absence (unstimulated) of phytohemagglutinin, lipopolysaccharide, and interleukin-2. TTV-DNA was detected in both stimulated and unstimulated PBMC. However, TTV-DNA replicative intermediates and mRNA were detected only in stimulated PBMC. Furthermore, TTV-DNA and mRNA were detected in PBMC from two TTV negative donors reinfected with supernatants from TTV-infected stimulated cells but not when using culture supernatants from unstimulated cells. These results demonstrate that TTV replicates in PBMC only when stimulated.

In this work, we have shown that hepatitis C virus (HCV) and hepatitis B virus (HBV) can coexist in the same hepatocyte using double fluorescent in situ hybridization in liver biopsy samples from patients with chronic HCV infection with occult HBV infection. Digital image analysis of hybridization signals showed that the HBV DNA levels in coinfected hepatocytes were lower than those in cells infected only with HBV. This finding supports the hypothesis of inhibition of HBV replication by HCV. Furthermore, HCV RNA levels were lower in coinfected cells than in cells infected only with HCV, suggesting that HBV may also inhibit HCV replication.Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of animal viruses, and its genome consists of a circular partially double-stranded DNA molecule of 3.2 kb in length which contains four overlapping reading frames that code for surface proteins (HBsAg), core proteins (HBc/HBeAg), the viral polymerase, and the transcriptional transactivator X protein (HBx) (9).Hepatitis C virus (HCV) is classified in the Hepacivirus genus of the Flaviviridae family, and its genome is a positive-stranded RNA of 9.6 kb in length that encodes a large polyprotein that undergoes proteolytic processing by cellular and viral proteinases to generate the individual viral proteins (17).As HBV and HCV share similar transmission routes, coinfection with the two viruses is not a rare event (3,6,7). Clinical data obtained from chronic HBV carriers superinfected with HCV suggest that HCV may inhibit HBV replication (13,15,20). This hypothesis is supported by the fact that patients with chronic HCV infection frequently have a special form of HBV infection, termed occult HBV infection (2,8,10,12). This is characterized by the presence of low levels of HBV DNA in serum and/or in liver in the absence of detectable HBsAg in serum. This inhibition may be mediated by the host immune response (via the induction of cytokines such as interferons) or by a direct effect of HCV proteins. In this regard, it has been shown that HCV core and NS2 proteins inhibit HBV replication and gene expression in vitro (4,5,19,21,22).Direct interference mediated by HCV proteins can occur in vivo only if both HBV and HCV coexist in the same hepatocyte. However, it has not been demonstrated whether HBV and HCV infect the same cell in the liver of patients coinfected with the two viruses. To address this issue, we have used double fluorescent in situ hybridization to determine the presence of HBV DNA and HCV RNA in liver biopsy samples from six patients with chronic hepatitis C (anti-HCV positive, serum HCV RNA positive) and occult HBV infection (HBsAg negative, serum HBV DNA positive).Patients underwent a liver biopsy for diagnostic purposes. After liver samples were obtained, they were divided into two portions. One fragment was processed for histological diagnosis and for in situ hybridization, and the other was embedded in RNAlater (QIAGEN, Hilden, Germany) in less than 30 seconds after being obtained and stored at Ϫ20°C. At th...

Although occult hepatitis B virus (HBV) infection (HBV-DNA in serum in the absence of hepatitis B surface antigen [HBsAg]) is common in chronic hepatitis C, its characteristics are not well known. In this work, the presence of HBV-DNA (by polymerase chain reaction; PCR) and its distribution (by in situ hybridization) in liver biopsies and peripheral blood mononuclear cells (PBMCs) from 32 patients with chronic hepatitis C and occult HBV infection and in 20 HBsAg chronic carriers were determined. The results showed that serum HBV-DNA levels were statistically lower (P = 0.001) in patients with occult HBV infection than in HBsAg chronic carriers. The HBV infection pattern in liver cells was identical between patients with occult HBV infection and those with chronic hepatitis B. However, the mean percentage of HBV-infected hepatocytes was significantly lower (P = 0.001) in patients with occult HBV infection (5 +/- 4.44%) than in HBsAg chronic carriers (17.99 +/- 11.58%). All patients with chronic hepatitis B have HBV-DNA in their PBMCs while this occurred in 50% of the cases with occult HBV infection. In conclusion, patients with occult HBV infection have a low number of HBV-infected hepatocytes and this fact could explain the lack of HBsAg detection and low viremia levels found in these cases.

A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA-positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA-negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease.

Pegylated alpha-interferon plus ribavirin is the current therapy for chronic hepatitis C virus (HCV) infection. Serum HCV-RNA concentration before treatment has been identified as an independent predictive factor of response. We have compared the percentage of HCV-infected hepatocytes with the concentration of serum HCV-RNA in baseline samples as predictors of response. We included 97 patients with chronic HCV infection (genotype 1), treated with pegylated-interferon-alpha2b plus ribavirin. Of these 97, 38 (39%) were sustained responders and 59 (61%) were not. Statistical differences between responders and nonresponders were found regarding the percentage of infected hepatocytes (6.83+/-4.50% versus 13.44+/-10.05%; P=0.00003) but not in serum HCV-RNA concentration [1.71+/-2.70 (x10(6) IU/L) versus 1.32+/-1.86 (x10(6) IU/L); P=0.40694]. Other factors associated with response were age, gamma-glutamyl transpeptidase level, and absence of previous therapy. Logistic regression demonstrated that percentage of infected hepatocytes (odds ratio, 1.160; 95% confidence interval, 1.065-1.264) and previous therapy (odds ratio, 0.294; 95% confidence interval, 0.109-0.795) were significant predictive factors for response. Therefore, the percentage of infected hepatocytes in liver biopsy before treatment is a better predictive factor of sustained response to 48 weeks of therapy with pegylated alpha-interferon plus ribavirin than serum HCV-RNA concentration in baseline serum sample.

Several in vitro studies have shown that HIV-1 can infect CD4 negative epithelial cells of different origin including normal human oral keratinocytes, but whether this infection of mucosal epithelial cells occurs in vivo is still unclear. In this report, the presence and cell types infected by HIV-1 in paraffin embedded oral mucosa biopsies from 17 anti-HIV-1 positive patients have been examined by in situ hybridization and immunohistochemistry. As controls, oral mucosa biopsies from eight patients without HIV-1 infection markers were also analyzed. The results showed that 8 out of the 17 anti-HIV-1 positive patients had HIV-1 RNA detectable in plasma. Positive hybridization signals were observed in the mucosa biopsies from 14 of the 17 anti-HIV-1 patients (82.3%). The mean percentage of cells showing HIV-1 RNA was 2.64% +/- 1.77% (range: 1% to 5.5%). No differences in the mean percentage of HIV-1 infected cells were found between patients with and without HIV-1 RNA in plasma (3.01% +/- 1.57% vs. 3.4% +/- 1.27% respectively), or between untreated patients and patients under antiretroviral therapy (2.83% +/- 1.63% vs. 3.42% +/- 1.29% respectively). Immunohistochemical detection of S-100 antigen, cytokeratin and CD4 showed that hybridization signals appeared in cytokeratin positive cells and CD4 positive cells but not in S-100 positive cells. In conclusion, this study has demonstrated that HIV-1 infects and replicates in oral mucosa epithelial cells in vivo and that these cells could represent a reservoir of the virus that may escape to the currently used antiretroviral therapy.