*HIV and Hepatitis Coinfection Unit, National Centre of Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
†Infectious Diseases-HIV Unit, Hospital General Universitario Gregorio Marañón, Madrid, Spain
‡Departments of Pathology
§Internal Medicine, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
Correspondence to: Salvador Resino, PhD, Centro Nacional de Microbiología, Instituto de Salud Carlos III (Campus Majadahonda), Carretera Majadahonda-Pozuelo, Km 2.2, 28220 Majadahonda, Madrid, Spain (e-mail: firstname.lastname@example.org).
Supported by grants given by “Instituto de Salud Carlos III” (grant numbers PI08/0738, PI11/00245; ISCIII-RETIC RD06/006, and PI08/0928), and “Fundación para la Investigación y la Prevención del Sida en España” (grant numbers 36443/03 and 361020/10). A. Fernández-Rodríguez, M. Guzman-Fulgencio, M. García-Álvarez, and Mª A. Jimenez-sousa are supported by “Instituto de Salud Carlos III” (grant numbers UIPY-1377/08, CM09/00031, CM08/00101, and CM10/00105, respectively).
Authors’ contributions: Study concept and design: M. García-Álvarez, S Resino. Acquisition of data: J. Berenguer, T. Aldámiz-Echevarría, A. Carrero, J. Cosín, P. Miralles. Collection of samples: D. Micheloud, J. Berenguer. Assessment of liver biopsy: E. Álvarez. Administrative, technical, or material support: M. García-Álvarez, M. Guzmán-Fulgencio, M.A. Jimenez-Sousa, A. Fernández-Rodríguez. Analysis and interpretation of data: M. García-Álvarez, M. Guzmán-Fulgencio, S. Resino. Drafting of the manuscript: M. García-Álvarez, S. Resino. Critical revision of the manuscript for important intellectual content: J. Berenguer. Study supervision: S. Resino.
The authors have no conflicts of interest to disclose.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jaids.com).
Received February 15, 2012
Accepted August 10, 2012
In the era of combination antiretroviral therapy (cART), chronic hepatitis C is a leading cause of death among human immunodeficiency virus (HIV)–infected persons.1 Coinfected patients are characterized by a higher rate of fibrosis progression, cirrhosis, and end-stage liver disease than hepatitis C virus (HCV) monoinfected patients.2 However, the mechanisms through which HIV infection accelerates chronic hepatitis C progression are still unknown.
Bacterial translocation (BT) is a mechanism through which alcohol and some enteric conditions may cause liver disease.3,4 HIV infection–related depletion of mucosal CD4+ lymphocytes has been linked to disruption of gut epithelial integrity and increased mucosal translocation of bacteria and bacterial products (bacterial DNA and endotoxin) from the intestinal lumen to the systemic circulation.5,6 Lipopolysaccharide is a component of the cell wall of gram-negative bacteria, and it is only present on a proportion of enteric bacterial microbiota. Furthermore, different markers from the lipopolysaccharide and of broader spectrum have been evaluated, such as plasma levels of the DNA sequences, encoding the well-conserved 16S rRNA subunit (16S ribosomal DNA), common to most bacteria.7 Detection of bactDNA in plasma is probably a better method for BT measure,8 and it has been proposed as an independent prognostic factor of poor prognosis in noninfected patients with cirrhosis.9
The BT derived from the intestinal damage is a cause of systemic immune activation in chronic HIV infection,6,10 which can play a crucial role in the accelerated course of liver damage in HIV/HCV-coinfected patients.11,12 Furthermore, hepatic macrophages or Kupffer cells are responsible for clearing BT products; however, these cells can be infected by HIV and it might result in their impaired ability to clear these potentially fibrogenic BT products.13 However, nowadays, there is still scarce information on this topic in HIV/HCV-coinfected patients, and there are no published studies with a large number of patients.
The aim of this study was to investigate BT in HIV/HCV-coinfected patients and to explore the potential associations between the magnitude of BT and the severity of liver disease.
PATIENTS AND METHODS
A cross-sectional study was performed on 255 HIV/HCV-coinfected patients of the Hospital Gregorio Marañón, in Madrid (Spain), who underwent a liver biopsy between May 2000 and May 2007. Additionally, 100 healthy blood donors from the “Centro de Transfusión de la Comunidad de Madrid” participated as a control group, all of whom were negative for HCV, HBV, and HIV.14
Liver biopsies were performed on patients who were potential candidates for anti-HCV therapy and had not received previous interferon therapy. The inclusion criteria were no clinical evidence of hepatic decompensation, detectable HCV RNA by polymerase chain reaction (PCR), negative hepatitis B surface antigen, CD4+ lymphocyte count >200 cells per microliter, stable antiretroviral therapy, or no need for antiretroviral therapy. The exclusion criteria were active opportunistic infections or active drug or alcohol addiction.
The studies were conducted in accordance with the Declaration of Helsinki. All the patients gave their written consent for the liver biopsy and the Institutional Ethics Committee approved the study.
Clinical and Laboratory Data
The following information, on the date of liver biopsy, was obtained from medical records: age, gender, height, weight, risk category, Centers for Disease Control clinical category, nadir CD4+, CD4+ T-cells, antiretroviral therapy, complete blood counts, HIV-RNA and HCV-RNA viral load, HCV-genotype, liver biopsy scores, and liver and basic metabolic panel.
Acquired immune deficiency syndrome (AIDS) was defined according to the Centers for Disease Control classification.15 The duration of HCV infection for patients with a history of intravenous drug use was estimated starting from the first year in which needles and other injection paraphernalia were shared, which are the most important risk practices for HCV transmission.16 For non-intravenous drug use patients, we had information of the date of infection for 2 patients who were infected by blood transfusion, and for other 3 patients, who were infected by sexual contact, in which the HCV infection may be dated with certainty. Patients were questioned in relation to alcohol consumption. We considered the consumption of >50 g of alcohol per day for ≥12 months as a high intake. Liver biopsies were performed as previously described,17 and liver fibrosis and necroinflammatory activity were estimated according to Metavir score.
Quantitative Real-Time PCR for the Measurement of Bacterial DNA
Plasma samples were obtained at the same time as the liver biopsy and stored at −80°C. Next, DNA was extracted from 200 μL of plasma using the QIAamp MiniElute Virus Spin Kit (Qiagen, Hilden, Germany) in a QIAcube automated extractor (Qiagen), as recommended by the manufacturer.
The PCR was performed in a LightCycler Instrument version 1.5 (Roche Molecular Biochemicals) to amplify the DNA sequences encoding the well-conserved 16S rRNA subunit (16S rDNA) as previously described.7 The PCR protocol was as follows: 4 μL of TaqMan Master Mix (Roche Diagnostics), 0.5 μM each primer, 0.2 μM TaqMan probe, and 5 μl DNA extract (or standard) in 20-μL total reaction volume. The conditions for amplification reaction of DNA were 95°C for 10 minutes, followed by 45 cycles at 95°C for 15 seconds, 60°C for 1 minute, and at 72°C for 1 second. The primer sequence was as follows: 8F (5′-AGT TTG ATC CTG GCT CAG-3′); 515R (5′-GWA TTA CCG CGG CKG CTG-3′); and TaqMan probe, 338P (5′-FAM-GCT GCC TCC CGT AGG AGT-BHQ1-3′).7 The limit of quantification in the bacterial DNA assay was 15 copies per microliter.
Liver Disease Outcomes
Fibrosis was scored as follows: F0/F1, no fibrosis or portal fibrosis; F2, periportal fibrosis or rare portal–portal septa; F3/F4, fibrous septa with architectural distortion or cirrhosis.
Activity grade was scored as follows: A0/A1, no activity or mild activity; A2, moderate activity; A3, severe activity.
Fibrosis progression rate (FPR) was calculated dividing the fibrosis stage (0–4) by the estimated duration of HCV infection in years. Several cutoffs were selected: low FPR [<0.075 (P50th)], moderate FPR [0.075 (P50th) to 0.15 (P75th)], and high FPR [≥0.15 (P75th)].
Categorical data and proportions were analyzed by using the χ2 test. The analysis of variance test was used to compare the means between groups.
The liver disease outcomes studied are ordinal variables, and ordinal logistic regression (OLR) analysis was used to analyze the association between plasma bactDNA levels and liver disease in HIV/HCV-coinfected patients. One of the assumptions underlying OLR is that the relationship between each pair of outcome groups is the same and assumes that the coefficients that describe the relationship between, the lowest versus all higher categories of the response variable are the same as those that describe the relationship between the next lowest category and all higher categories.
For plasma bactDNA levels, a cut-off near the 75th percentile (P75th) was selected because this cutoff point separates the 25% of patients with the highest values of plasma bactDNA. Thus, we are able to study the relationship between the elevated levels of bactDNA and greater chance of having liver disease. For adjusted OLR, we included bactDNA along with epidemiological and clinical characteristics: age, gender, high alcohol intake, fasting glycemia (milligrams per deciliter), CD4+ nadir, AIDS, CD4+ cells per cubic millimeter, undetectable HIV viral load, cART, HCV-genotype 1, and HCV viral load ≥500,000 IU/mL at biopsy date.
All the tests were 2-tailed with P values <0.05 considered significant. Statistical analysis was performed by SPSS 15.0 software (SPSS Inc, Chicago, IL).
Characteristics of HIV/HCV-coinfected patients at the same time than liver biopsy are shown in Supplemental Digital Content 1 (see http://links.lww.com/QAI/A353). The median age was 39.5 years, 76% were male, and 29% showed prior AIDS-defining conditions. On the date of liver biopsy, the median CD4+ count was 483 cells per cubic millimeter, 70.6% had an HIV-RNA <50 copies per milliliter, 61.4% had HCV genotype 1, and 73.1% had an HCV-RNA > 500,000 UI/mL. Overall, 84.7% patients were on cART: 23.5% with protease inhibitor–based therapy, 47.8% with nonnucleoside analog–based therapy, and 13.3% with 3 nucleoside analog–based therapy. Furthermore, 53.3% patients showed significant fibrosis (F ≥ 2), 26.6% advanced fibrosis (F ≥ 3) and 55.1% moderate activity grade (A ≥ 2).
The prevalence of positive PCR for plasma bactDNA was significantly higher in HIV/HCV-coinfected patients than in healthy blood donors [244/255 (95.7%) versus 7/100 (7%), respectively; P < 0.001]. Furthermore, patients with CD4+ <350 cells per cubic millimeter had higher significantly plasma bactDNA levels than patients whose CD4+ ≥350 cells per cubic millimeter (157.1 ± 28.2 versus 112.8 ± 7.2, respectively; P = 0.030). Moreover, bactDNA levels were not higher in patients who had detectable HIV-RNA, and the level did not depend on the level of plasma HIV-RNA (data not shown).
Patients with markers of advanced liver disease (F3/F4, and A2/A3) and high FPR (≥0.15) had higher plasma values of bactDNA than did patients without these markers of liver disease (Fig. 1A). Note that A3 had a P value close to statistical significance (P = 0.083). In addition, it may be seen that the patients with bactDNA values >175 per microliter had a tendency to liver disease, whereas other cutoffs did not show a trend that was so clear (Fig. 1B).
Next, we evaluated the influence of plasma bactDNA on liver disease by OLR analysis. With regard to fibrosis, the chance of having an increased Metavir score was 1.20 [95% of confidence interval (95% CI) = 1.0 to 1.44, P = 0.045] times greater for every 100 copies per microliter of plasma bactDNA. For example, for every 100 copies per microliter of plasma bactDNA, the odd of F3/F4 versus F2 and F0/F1 was 1.20 and the odds of having F2 versus F0/F1 was also 1.20. Likewise, the odds of having an increased activity grade was 1.22 (95% CI = 1.1 to 1.45, P = 0.029) and of having values of FPR >0.15 or FPR 0.075 to 0.15 versus FPR <0.075 was 1.18 (95% CI = 0.98 to 1.42, P = 0.089) times greater for every 100 copies per microliter of plasma bactDNA. In addition, patients with high plasma bactDNA levels (≥175 copies per microliter) had the highest prevalence and odds of having high values of Metavir score and FPR (Table 1).
We determined the plasma levels of bactDNA in a group of 255 HIV/HCV-coinfected patients with compensated liver disease who underwent liver biopsy, to determine their suitability for undergoing interferon plus ribavirin therapy. HIV/HCV-coinfected patients had higher plasma levels of bactDNA than healthy controls, and patients with CD4+<350 cells per cubic millimeter had higher plasma bactDNA than those with CD4+ ≥350 cells per cubic millimeter. In addition, an association between higher plasma bactDNA levels with advanced liver disease and more rapid progression to fibrosis was found.
The most likely source of these bacterial products is the gastrointestinal tract, in which mucosal defenses are profoundly disrupted by HIV infection.5 Our results, and the emerging data from other investigators,7,11,15,18 indicate that the bacterial products are often circulating in the plasma of HIV-infected and HIV/HCV-coinfected patients. Our finding of an association between lower CD4+ cells and higher bactDNA plasma values support the notion that in HIV-infected patients on cART bactDNA, the plasma levels correlate with CD4+ count, the magnitude of immune CD4+ restoration, and immune activation markers.7 This is interesting because BT, CD4+ lymphocyte depletion and immune activation may contribute to liver disease progression among HIV/HCV-coinfected patients.11,19
In our study, we confirmed the association between higher plasma levels of bactDNA and both higher grades of inflammation and higher stages of fibrosis in liver biopsies from HIV/HCV-coinfected patients. Furthermore, our data show a significant association between bactDNA levels and slow FPR in these patients. Most previous studies did not analyze the role of these bactDNA levels with FPR, which is a robust measure of the risk of cirrhosis progression. Nevertheless, we consider FPR as an indirect measure that has some limitations, such as the assumption of a constant progression rate. A direct method, using serial liver biopsies and the interval between 2 adjacent biopsies, has the ability to calculate the stage-specific transition rates, but it is very difficult to compile patients with more than a single biopsy. Despite this limitation, we believe that bactDNA plasma levels might add valuable information to the factors influencing FPR and may be useful to implement targeted therapeutic interventions in HIV-infected patients with HCV infection at risk of rapid liver disease progression.
Unfortunately, our study design does not permit us to assess whether BT is either a cause or a consequence of liver disease progression. However, there is some evidence to show that BT promotes hepatic fibrogenesis, which ultimately increases portal systemic shunting (a well-known consequence of cirrhosis) and the abundance of circulating microbial products in a positive feedback loop.11 In addition, the presence of bactDNA in patients with cirrhosis is associated with hemodynamic consequences, such as aggravation of peripheral vasodilation and worsening of intrahepatic endothelial dysfunction.20
In conclusion, although the differences observed among the patients with different stages and rates of disease progression are modest; our data show that BT was associated with severe liver disease among HIV-infected patients with chronic hepatitis C. Future studies are needed to validate these results and to evaluate whether plasma levels of bactDNA are a predictive and/or surrogate marker of liver disease in HIV/HCV-coinfected patients.
The authors acknowledge the patients who participated in this study and the Centro de Transfusión of Comunidad de Madrid for the healthy donor blood samples provided.
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