Nungki Anggorowati

Clinical and virological characteristics of hepatitis B or C virus co-infection with HIV in Indonesian patients†

Hepatitis virus‐related liver disease increases substantially the mortality rate of patients with HIV on highly active antiretroviral therapy (HAART). Therefore, early diagnosis of hepatitis B virus (HBV) and hepatitis C virus (HCV) is important. However, the prevalence of HBV and HCV infection in Indonesian patients infected with HIV is unknown. Therefore, this study examined the molecular and clinical characteristics of HBV and HCV in 126 patients infected with HIV, mostly on HAART, at Dr. Sardjito Hospital, Yogyakarta, Indonesia. The rates of triple infection, HIV/HCV co‐infection, HIV/HBV co‐infection, and mono‐infection were 4.8%, 34.1%, 3.2%, and 57.9%, respectively. Seven HCV genotypes were detected, with genotypes 1a, 1b, 1c, 3a, 3k, 4a, and 6n found in 23 (52%), 1 (2%), 4 (9%), 5 (11%), 7 (16%), 3 (6%), and 1 (2%) patients, respectively, indicating multiple modes of transmission. HBV‐DNA was detected in 2/10 patients with hepatitis B surface antigen; both patients were HAART naive. Univariate analysis revealed that male sex, higher education level, injection drug use, sexual contact, alanine aminotransferase ≥40 IU/L, and aspartate aminotransferase‐to‐platelet ratio index > 0.5 were associated with HCV co‐infection. In multivariate analysis, injection drug use (OR: 26.52; 95% CI: 3.52–199.54) and alanine aminotransferase ≥40 IU/L (OR: 6.36; 95% CI: 1.23–32.89) were independently associated with HCV co‐infection. HCV co‐infection was common among Indonesian patients infected with HIV, particularly among injecting drug users, and was a risk factor for disease progression of HIV. J. Med. Virol. 84:857–865, 2012.

Hepatitis B and C virus infection among hemodialysis patients in yogyakarta, Indonesia: Prevalence and molecular evidence for nosocomial transmission

Hemodialysis patients are at an increased risk of acquiring hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. However, the prevalence of hepatitis viral infection and its genotype distribution among hemodialysis patients in Indonesia are unclear. In order to investigate these issues and the possibility of nosocomial transmission, 161 hemodialysis patients and 35 staff members at one of the hemodialysis unit in Yogyakarta, Indonesia, were tested for serological and virological markers of both viruses. HBV surface antigen (HBsAg) was detected in 18 patients (11.2%) and in two staff members (5.7%). Anti‐HCV was detected in 130 patients (80.7%) but not in any staff members. Occult HBV and HCV infection were detected in 21 (14.7%) and 4 (12.9%) patients, respectively. The overall prevalence rates of HBV and HCV infection among patients were 24.2% and 83.2%, respectively. HCV infection was independently associated with hemodialysis duration and the number of blood transfusions. Phylogenetic analysis revealed that 23 of 39 tested HBV strains (59%) were genotype B, 11 (28.2%) were genotype C, and 5 (12.8%) were genotype A. HCV genotype 1a was dominant (95%) among 100 tested HCV strains. Nosocomial transmission was suspected because the genotype distribution differed from that of the general population in Indonesia, and because the viral genomes of several strains were identical. These findings suggest that HBV and HCV infection is common among hemodialysis patients in Yogyakarta, and probably occurs through nosocomial infection. Implementation of strict infection‐control programs is necessary in hemodialysis units in Indonesia. J. Med. Virol. 85:1348–1361, 2013.

Mutations within enhancer II and BCP regions of hepatitis B virus in relation to advanced liver diseases in patients infected with subgenotype B3 in Indonesia

Studies on the characteristics of mutations within the hepatitis B virus (HBV) genome, their roles in the pathogenesis of advanced liver diseases, and the involvement of host properties of HBV‐infected individuals have not been conducted in subgenotype B3‐infected populations. For addressing this issue, 40 cases with HBV surface antigen (HBsAg)—positive advanced liver diseases, including advanced liver cancer and cirrhosis (male 31, female 9, age 54.4 ± 11.6‐year‐old), were collected and compared with 109 cases with chronic hepatitis B (male 71, female 38, age 38.0 ± 13.4‐year‐old). Mutations in enhancer II (Enh II) and basal core promoter (BCP)/precore regions were analyzed by PCR‐direct sequencing method. HBV viral load was examined by real‐time PCR. For all examined regions, the prevalence of mutation was significantly higher in cases with advanced liver diseases. Multivariate analysis showed that, in patients older than 45 years, C1638T and T1753V mutations constituted independent risk factors for the advancement of liver diseases. The presence of C1638T and T1753V mutations may serve as predictive markers for the progression of liver diseases in Indonesia and other countries, where subgenotype B3 infection is prevalent. J. Med. Virol. 84:44–51, 2011.

Quantification of Pregenomic RNA and Covalently Closed Circular DNA in Hepatitis B Virus-Related Hepatocellular Carcinoma

Pregenomic RNA (pgRNA) is generated from covalently closed circular DNA (cccDNA) and plays important roles in viral genome amplification and replication. Hepatic pgRNA and cccDNA expression levels indicate viral persistence and replication activity. This study was aimed to measure hepatic pgRNA and cccDNA expression levels in various states of hepatitis B virus (HBV) infection. Thirty-eight hepatocellular carcinoma (HCC) patients, including 14 positive for hepatitis B surface antigen (HBsAg) and 24 negative for HBsAg but positive for anti-hepatitis B core (anti-HBc) antibody, were enrolled in this study. In HBsAg-negative but anti-HBc-positive group, HBV-DNA was detected in 20 of 24 (83%) noncancerous liver tissues for at least two genomic regions based on polymerase chain reaction (PCR) analysis. pgRNA and cccDNA expression levels in occult HBV-infected patients were significantly lower than those in HBsAg-positive patients (P < 0.001). pgRNA and cccDNA in cancerous tissues were also detected without significant difference from those in noncancerous tissues. In conclusion, cccDNA and pgRNA are detected and represented HBV replication not only in noncancerous but also in cancerous liver tissues. In addition, the replication is shown in not only patients with HBsAg-positive but also occult HBV-infected patients, suggesting the contribution to HCC development.

GB virus C infection in Indonesian HIV‐positive patients

GB virus C (GBV‐C), a human virus of the Flaviviridae family that is structurally and epidemiologically closest to hepatitis C virus (HCV), has been reported to confer beneficial outcomes in HIV‐positive patients. However, the prevalence of GBV‐C in HIV‐positive individuals in Indonesia is unknown. Since GBV‐C is more prevalent in anti‐HCV positive patients than in anti‐HCV negative subjects, transmission of GBV‐C and HCV could be by the same method. This study examined the prevalence and molecular characteristics of GBV‐C infection in HIV patients in Yogyakarta, Indonesia. The prevalence of GBV‐C among HIV patients (n = 125, median age 31 years) based on the 5′UTR region was 111/125 (88.8%), including 39/48 (81.3%) and 72/77 (93.5%) HIV‐infected patients with and without HCV infection, respectively. GBV‐C isolates were of genotype 2a, 3 and 6 in 58.3%, 12.6% and 28.4% of patients, respectively. Patients with genotype 3 were significantly younger than those with genotypes 2a or 6 (P = 0.001 and P = 0.012, respectively). Genotypes 3 and 6 were significantly associated with injection drug use (P = 0.004 and P = 0.002, respectively) and HCV co‐infection (P < 0.001 for both genotypes), indicating a shared transmission route with HCV. In conclusion, the prevalence of GBV‐C among HIV‐positive patients in Indonesia is high, and three genotypes were detected, namely genotype 2a, 3 and 6.

Hepatitis E virus infection in two different regions of Indonesia with identification of swine HEV genotype 3

Hepatitis E is an emerging disease with a high incidence globally. Few data are available on hepatitis E virus (HEV) infection in Indonesia. To obtain molecular information on HEV infection in two regions of Indonesia with different customs and swine breeding conditions, serum samples from 137 swine farm workers, 100 blood donors and 100 swine (27 fecal samples also obtained) in Yogyakarta (Central Java) and from 12 and 64 swine farm workers, 42 and 135 local residents and 89 and 119 swine in Tulungagung (East Java) and Mengwi (Bali), respectively, from our previous study, were compared. Serological tests for anti‐HEV antibodies by ELISA, HEV‐RNA detection by RT‐PCR and phylogenetic analysis were performed. The total prevalence of anti‐HEV antibodies in humans was higher in Bali (11.6%) than in Java (5.1%; P = 0.015). No significant differences in anti‐HEV prevalence among swine farm workers and local residents in Java were found. The finding of swine HEV genotype 3 in specimens from Yogyakarta and genotype 4 from Tulungagung and Bali is somewhat different from other reports. We suggest other factors in addition to close contact with swine might play an important role in HEV transmission of non‐endemic/related custom groups. To the best of our knowledge, this is the first report on swine HEV genotype 3 in Indonesia.

Molecular Subtypes, Apoptosis and Proliferation Status in Indonesian Diffuse Large B-Cell Lymphoma Cases

Objective: The diffuse large B-cell lymphoma (DLBCL) has two major molecular subtypes, germinal center B-cell-like (GCB) and non-GCB. These have differing behavior which affects overall patient survival. However, immunohistochemistry based molecular subtyping of Indonesian DLBCLs has been limited. This was the focus of the present study, with a focus of attention on the apoptotic index (AI) and the proliferation index (PI) of the two molecular subtypes. Materials and Methods: During the study period of 3.5 years, a total of 98 cases of DLBCL were identified. Molecular subtypes and PI were determined by immunohistochemistry and TUNEL method was used to determine the AI. Result: GCB accounted for 31 cases (31.6%) and non-GCB the remainder (68.4%). Gender showed a slight male predominance (54 cases, 55.1%), with a higher incidence in the extra-nodal region (57 cases, 58.2%). The AI and PI were significantly higher in GCB (p<0.001 in the Mann-Whitney test) and a Spearman correlation coefficient test showed that PI was positively correlated with AI (r=0.673, p<0.001). Conclusion: The findings indicate that the non-GCB subtype is more common than GCB in Indonesian DLBCL. GCB features significantly higher PI and AI, which themselves appear linked.

Histochemical and Immunohistochemical Study of α-SMA, Collagen, and PCNA in Epithelial Ovarian Neoplasm

Background: Alpha-smooth muscle actin (α-SMA) is an isoform of actin, positive in myofibroblasts and is an epithelial to mesenchymal transition (EMT) marker. EMT is a process by which tumor cells develop to be more hostile and able to metastasize. Progression of tumor cells is always followed by cell composition and extracellular matrix component alteration. Increased α-SMA expression and collagen alteration may predict the progressivity of ovarian neoplasms. Objective: The aim of this research was to analyse the characteristic of α-SMA and collagen in tumor cells and stroma of ovarian neoplasms. In this study, PCNA (proliferating cell nuclear antigen) expression was also investigated. Methods: Thirty samples were collected including serous, mucinous, endometrioid, and clear cell subtypes. The expression of α-SMA and PCNA were calculated in cells and stroma of ovarian tumors. Collagen was detected using Sirius Red staining and presented as area fraction. Results: The overexpressions of α-SMA in tumor cells were only detected in serous and clear cell ovarian carcinoma. The histoscore of α-SMA was higher in malignant than in benign or borderline ovarian epithelial neoplasms (105.3±129.9 vs. 17.3±17.1, P=0.011; mean±SD). Oppositely, stromal α-SMA and collagen area fractions were higher in benign than in malignant tumors (27.2±6.6 vs 20.5±8.4, P=0.028; 31.0±5.6 vs. 23.7±6.4, P=0.04). The percentages of epithelial and stromal PCNA expressions were not significantly different between benign and malignant tumors. Conclusion: Tumor cells of serous and clear cell ovarian carcinoma exhibit mesenchymal characteristic as shown by α-SMA positive expression. This expression might indicate that these subtypes were more aggressive. This research showed that collagen and α-SMA area fractions in stroma were higher in benign than in malignant neoplasms.