A.C. Guardia

Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.

Human herpesvirus 6 in donor biopsies associated with the incidence of clinical cytomegalovirus disease and hepatitis C virus recurrence.

BACKGROUND Reactivation of cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6), as well as the recurrence of hepatitis C virus (HCV), occurs in the post liver transplantation period. However, their correlations remain questionable. The objectives of this study were to analyze the presence of CMV DNA and HHV-6 DNA in pre-transplant and post-transplant liver graft biopsies and to determine any correlations with CMV disease and HCV recurrence. METHODS Forty-one liver transplant recipients were followed up in the post-transplant period. The presence of CMV DNA and HHV-6 DNA was detected by nested PCR. RESULTS Four patients (4/41, 9.8%) were positive for CMV DNA in pre-transplant biopsies and three of them remained positive after transplantation; 11 patients became positive in the post-transplant biopsies (p=0.06). Fifteen (15/41, 36.6%) patients were positive for HHV-6 DNA in pre-transplant biopsies and 11 of these remained positive after transplantation. Another 11 patients became positive after the surgery (p=0.05). CMV disease occurred in 17 recipients; 10 of these 17 (58.8%) patients were positive for HHV-6 DNA in pre-transplant biopsies and they continued positive after transplantation (p=0.0128). Twenty-eight patients were transplanted due to hepatitis C; 12 of these patients had recurrence of the virus, and HHV-6 was positive in nine of the 12 (75%) patients (p=0.049). CONCLUSIONS Recipients with HHV-6 DNA in pre-transplant graft biopsies remained positive post transplantation, showing a possible risk for post-transplant allograft loss because there was an association between HHV-6 and recurrent HCV and CMV disease.

Cytomegalovirus, Human Herpesvirus-6, and Human Herpesvirus-7 in Adult Liver Transplant Recipients: Diagnosis Based on Antigenemia

Human herpesvirus (HHV)-6, HHV-7, and cytomegalovirus (CMV) that remain latent after primary infection can be reactivated during immunosuppression following organ transplantation in liver transplant recipients. The aim of this study was to monitor active infections for HHV-6, HHV-7, and CMV among adult liver transplantation recipients using antigenemia detected by an immunoperoxidase staining. Twenty-eight adult liver transplant patients were monitored using antigenemia in blood samples obtained at the time of transplantation, as well as weekly in the first month and once a month for 6 months. Of these patients, 28.5% showed positive CMV antigenemia; 39.2%, HHV-6 antigenemia; and 14.2%, HHV-7 antigenemia. The detection of the three viruses was considered to be independent of one another (P>.05). The results described above showed that few patients remain free of beta herpesviruses after liver transplantation. Most patients were infected sequentially and not concurrently. Antigenemia has been considered useful to detect active HHV-6 and HHV-7 infections. Antigenemia can be more efficiently interpreted when compared with polymerase chain reaction results, although other studies are necessary to establish the reference of HHV-6 and HHV-7 antigenemia.

Monitoring and Detection of Cytomegalovirus in Liver Transplant Recipients

Cytomegalovirus (CMV) is a β-herpesvirus. CMV infections are a common complication contributing to morbidity and mortality after liver transplantation. Among organ transplant recipients, CMV can reactivate from latency during the first 6 months. This prospective study performed from February 2008 to December 2009 examined liver transplant recipients during the first 6 months. Two methods were performed to detect CMV infections: antigenemia (AGM) and nested (PCR). Ninety-four patients, including 72 men (76.6%) and 22 women (23.4%) underwent liver transplantation during this period. We analyzed 575 samples including 465 for AGM and PCR. Forty-three (9.25%) showed positive AGM as detected 2 to 179 days posttransplantation with a mean of 50 days and a median of 35 days, and 93/465 (20%) showed positive PCR at 0 to 186 days posttransplantation with a mean of 31 days and a median of 38 days. Among the 43 antigenemia patients, 38 samples were positive for up to 5 cells 18 of which were PCR-positive. Five samples were positive with more than 5 cells, including 3 that were PCR-positive. Only 4.51% had AGM and were PCR-positive in the same sample. Despite only 9.25% (43/465) showing AGM, the current study suggested the utility of routine monitoring to detect early CMV infection among liver transplantation patients seeking to reduce morbidity and mortality.

Identification of Bacterial Infections and Clinical Manifestation Associated With Cytomegalovirus in Liver Transplantation Patients

INTRODUCTION Liver transplantation has become the most effective therapy for the treatment of patients with end-stage liver disease. With new immunosuppressive agents the incidence of acute rejection has been significantly reduced, but infection has become a serious problem. OBJECTIVE Our objective was to correlate cytomegalovirus (CMV) positivity of antigenemia and polymerase chain reaction (PCR) with clinical manifestations and bacterial infections among patients undergoing liver transplantation. METHODS This prospective study included patients monitored for 6 months for early detection of CMV infection. Sample collections were performed at the time of surgery and weekly until the second month followed by fortnightly in the third month, and monthly in the fourth to sixth month. CMV infection was defined by positive antigenemia (>3 cells) or 2 positive PCR tests associated or not with clinical symptoms. The methodology for the diagnosis of bacterial infection was through biochemical tests and the automated VITEK/bioMérieux (identification and antibiogram) using samples of urine and blood cultures. Chi-square test was used for dicotomic variables with significant differences when P < .05. RESULTS Sixteen patients (32%) had CMV infections, including 13 (81%) with concomitant infections. Thirty-four patients (68%) did not have CMV infections and 8 of these (24%) had bacterial infection. There was a high correlation with bacterial infections among CMV-positive patients. CONCLUSION Bacterial infections after liver transplantation were associated with CMV infection.

Co-infection and Clinical Impact of Human Herpesvirus 5 and 6 in Liver Transplantation

BACKGROUND Human herpesvirus (HHV) 5 and 6 remain latent after primary infection and can be reactivated after immunosuppression for organ transplantation. An association between HHV-5 and HHV-6 has been reported in liver transplant patients. The coinfection is associated with clinical manifestations and graft dysfunction. OBJECTIVE The aim of this study was to monitor herpesviruses in liver transplant recipients to better understand issues involving coinfection with HHV-5/6 and correlations with acute cellular rejection episodes and bacterial infections. METHODS Forty-five adult liver transplant patients of median age 47 years (range, 18-66), gave blood samples and liver biopsies in the first 6 months after their surgeries. Viremia was detected with the use of nested PCR and antigenemia; the Banff classification was used to detect allograft rejection. RESULTS IgG positive for HHV-5 was observed in 94% of subjects whose main indication (67%) for transplantation was hepatitis C. Twenty-three (51.1%) displayed cytomeg virus (CMV) infections and 12 (26.7%) HHV-6 infection. There were 6 patients (13.3%) with HHV-5/6 coinfections. Eighteen of the 23 patients had CMV disease, showing a strong correlation between a positive test and CMV disease; 6 displayed an acute cellular rejection episode in the same period (χ(2) = 6.62; P < .03). Four out of 6 patients who displayed coinfections (HHV-5/6) had concomitant bacterial infections; 3/6 experienced graft rejection episodes. During follow-up, 1 patient had HHV-6 infection diagnosed as encephalitis followed by fever on the 24th day after surgery. The median 32 days for HHV-6 detection by nested PCR positivity was shorter than 38 days for HHV-5. CONCLUSIONS HHV-5/6-infected patients displayed more allograft rejection episodes, coinfections, and concomitant bacterial infections, besides an higher risk for CMV disease.

Rhabdomyolysis as a Clinical Manifestation of Association With Ciprofibrate, Sirolimus, Cyclosporine, and Pegylated Interferon-α in Liver-Transplanted Patients: A Case Report and Literature Review

BACKGROUND Rhabdomyolysis is a syndrome characterized by impaired metabolic integrity of myocytes, causing the release of intracellular constituents into the circulation, and can be a serious side effect of drug intake. CASE REPORT This report describes a unique case of rabdomyolysis secondary in which ciprofibrate, sirolimus, cyclosporine, and pegylated interferon-α in a liver transplant patient was used. A 47-year-old male liver transplant recipient in 2009, who had hepatitis C and incidental hepatocellular carcinoma, underwent immunosuppressive therapy (cyclosporine and sirolimus). The patient is currently in treatment for viral recurrence with pegylated interferon-α and ribavirin; he had a history of hypertriglyceridemia treated with ciprofibrate. He had development of severe and generalized myalgia and fever after the eighth application of pegylated interferon-α and increasing doses of cyclosporine. Laboratorial tests showed acute renal failure and significant increase in creatine kinase. Rhabdomyolysis secondary to interaction of fibrate-cyclosporine-pegylated interferon-α was postulated. CONCLUSIONS Medical professionals should be aware of possible drug interactions and should monitor patients receiving these drugs.

Cytomegalovirus Infection in Liver Transplantation

With the global evolution of organ transplantation in humans a new class of patients with special problems related to opportunistic infections after transplantation has appeared [1, 2 ].Some of these challenges infections with members of the herpesvirus family. Among these viruses, human cytomegalovirus (HCMV) often affects immunocompromised patients, HCMV can be reactivated by immunosuppression and cause significant morbidity and mortality [3,4]. In the postoperative period, HCMV infection can result in serious complica‐ tions in patients who received grafts by modulating the immune response [5]. However in immunocompetent individuals cytomegalovirus infection can be asymptomatic or cause symptoms similar to infectious mononucleosis syndrome, such as lymphadenopathy, fever, rash, malaise, arthralgia, hepatomegaly and splenomegaly [6].

Mo1781 Molecular Detection of Helicobacter pylori in Formalin-Fixed Paraffin-Embedded (FFPE) Liver Tissue From Patients With Hepatocellular Carcinoma: Comparison of Five Methods to Extract Genomic DNA

hepatocellular carcinoma represents one of the most common human cancers in the world, the aim of this study was to investigate the presence of H. pylori DNA in the FFPE liver from Brazilian patients with HCC. For this purpose, paraffin sections of 38 liver biopsies were used and genomic DNA was extracted using phenol/chloroform method. After that, polymerase chain reaction (PCR) analysis was carried out using H. pylori specific 16S rRNA primers and PCR products of positive samples were further identified by DNA sequencing. Results showed that 14 of the 38 samples (36,8%) amplified the 16S rRNA H. pylori gene. The nucleotide sequence of the 16S rRNA amplicons demonstrated 98% similarity to H. pylori. Considering the H. pylori positive patients, in relation to viral infection, 9 of the 14 (64%) subjects were infected by hepatitis C virus (HCV), 2 (14%) were infected by hepatitis B virus (HBV); 1 (7%) presented coinfection with HCV and HBV and 2 (15%) patients had HCC without viral infection. These data confirm the identification of H. pylori in FFPE liver tissue of Brazilian patients with hepatocellular carcinoma. Additionally, patients infected with H. pylori were most frequently infected by HCV. Further studies will be performed in these samples to detect other H. pylori molecular markers such as cagA, vacA and ureA genes and its association with viral status of patients with HCC. This study was supported by grants from FAPESP ( 2009/09889-5) and FAEPEX (10111)