Irawan Yusuf

Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia

Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007-2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the circulation of Genotype IV in the community.

Hepatitis C virus genotype in blood donors and associated liver disease in Indonesia.

Objective: The aim of this study was to investigate the distribution of hepatitis C virus (HCV) genotype and the possible association between genotype and HCV-associated liver disease in Indonesia. Methods: 32 anti-HCV-positive asymptomatic carriers (AC), 55 chronic hepatitis (CH), 41 liver cirrhosis (LC), and 35 hepatocellular carcinoma (HCC) patients were included in this study. HCV genotyping was performed by phylogenetic analysis of the NS5B and 5′-UTR regions. Results: The HCV subtype 1b (36.5%), based on NS5B region, was the most prevalent, followed by subtypes 3k (15.4%), 2a (14.4%), 1a (12.5%) and 1c (12.5%), and 2e (4.8%). Subtypes 2f, 3a, 3b, and 4a were also found in some of the samples. HCV subtypes 3k (40.0%) and 1a (35.0%) were the two major subtypes in AC. HCV subtype 1b was not found in AC, but it was common in CH (31.3%), LC (50.0%), and HCC (57.1%). Conclusion: HCV subtype 1b was prevalent in samples of HCV-associated liver disease patients, including CH, LC and HCC. The percentage of subtype 1b was increased with the disease severity (AC < CH < LC < HCC).

Serum adiponectin and resistin in relation to insulin resistance and markers of hyperandrogenism in lean and obese women with polycystic ovary syndrome.

Objective: The role of insulin resistance in polycystic ovary syndrome (PCOS) has been established. However the role of adiponectin and resistin in the relationship between insulin resistance as markers of obesity and PCOS has not been conclusive. This study aims to determine the influence of the serum levels of adiponectin and resistin on PCOS, and assess possible correlations with the hormonal and metabolic parameters of the syndrome and obesity. Methods: This study continued a case control study that had finished recruiting 24 subjects of reproductive women with PCOS as a case group, and 24 subjects of normal ovulatory reproductive women without hyperandrogenism as a control group. Further, only 18 subjects of the control group had a body mass index (BMI) <25 kg/m2 and were included the data analysis, whereas others were excluded. Therefore, these study data were divided into three groups. Twenty-four PCOS patients from the case group were allocated to two groups, A (n = 14) patients had PCOS + BMI ≥25 kg/m2; B (n = 10) patients had PCOS + BMI <25 kg/m2. Group C was the control group of 18 reproductive women without PCOS + BMI <25 kg/m2. Blood samples were collected between day 3 and 5 of a spontaneous menstrual cycle, at 07:00 to 09:00, after overnight fasting. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone, prolactin, sex hormone-binding globulin (SHBG), glucose, insulin, adiponectin and resistin were measured. Results: Serum adiponectin levels were significantly decreased in group A compared with group B and group C. No significant difference existed in adiponectin between group B and group C. Homeostasis Model of Assessment—Insulin Resistance (HOMA-IR) value in group A was found to be significantly higher than group C, but no significant differences were found between group B and group C or between group A and group B. There was no significant difference in serum resistin between all groups, nevertheless the resistin-to-adiponectin (R:A) ratio was significantly decreased in group A compared with groups B and C. In a multiple regression model, BMI, testosterone and insulin resistance were the major determinants of hypoadiponectinemia. However, only BMI was the major determinant of the resistin represented by the R:A ratio. Conclusions: Serum adiponectin levels and the ratio of resistin to adiponectin levels are reduced in obese women with PCOS. These results suggest that, by reducing adiponectin serum level, hyperandrogenemia, together with nutritional status of obesity, might contribute to insulin resistance in the pathogenesis of PCOS.

Hepatitis B virus genotypes/subgenotypes in voluntary blood donors in Makassar, South Sulawesi, Indonesia

BackgroundHepatitis B virus (HBV) genotype appears to show varying geographic distribution. Molecular epidemiological study of HBV in particular areas in Indonesia is still limited. This study was aimed to identify the prevalence of HBV genotype/subgenotype and mutations in basal core promoter (BCP) region in voluntary blood donors in Makassar, one of the biggest cities in east part of Indonesia.A total of 214 hepatitis B surface antigen (HBsAg)-positive samples were enrolled in this study. HBV genotype/subgenotype was identified by genotype-specific PCR method or direct sequencing of pre-S region. Mutations in BCP were identified by direct sequencing of the corresponding region.ResultsHBV/B and HBV/C were detected in 61.21% and 25.23% of the samples, while mix of HBV/B and HBV/C was found in 12.62% of the samples. Based on pre-S region, among HBV/B and HBV/C, HBV/B3 (95.00%) and HBV/C1 (58.82%) were predominant. Interestingly, HBV/D was identified in two samples (22.165.07 and 22.252.07). Complete genome sequences of two HBV/D strains (22.165.07 and 22.252.07) demonstrated that both strains belong to HBV/D6, and the divergence between the two strains were 1.45%, while divergences of both 22.165.07 and 22.252.07 strains with reference strain (AM422939/France) were 2.67%. A1762T/G1764A mutation was observed in 1.96% and 5.36%, whereas T1753V mutation was found in 2.94% and 1.79% of HBV/B and HBV/C, respectively.ConclusionHBV/B and HBV/C are dominant in Makassar, similar to most areas in Indonesia. Mutations in BCP which might be associated with severity of liver disease are less common.

Drug resistance among tuberculosis patients attending diagnostic and treatment centres in Makassar, Indonesia.

SETTING Government tuberculosis (TB) diagnostic and treatment centres, Makassar, Indonesia. OBJECTIVE To determine the proportions and patterns of resistance to commonly used TB drugs (isoniazid [INH], rifampicin, ethambutol and streptomycin) among pulmonary TB patients and assess potential risk factors for drug resistance. DESIGN Cross-sectional study. RESULTS Of 657 recruited patients, 234 were culture-positive. Drug susceptibility testing (DST) results were available for 216 patients. Among these, 197 were infected with Mycobacterium tuberculosis complex (145 new and 52 previously treated). Isolates from 89 new (61.4%) and 31 previously treated (59.6%) patients were susceptible to all four drugs. Resistance to INH was high among both patient groups (28.3% of new vs. 34.6% of previously treated). Multidrug-resistant TB (MDR-TB) cases accounted for respectively 4.1% and 19.2% of these patients. Resistance to >2 drugs was high among previously treated patients (19.2%). MDR-TB cases were more likely to have a history of excess alcohol use (adjusted OR 4.01, 95%CI 1.28-12.53) and previous TB treatment (adjusted OR 6.28, 95%CI 2.01-19.64). CONCLUSIONS Regardless of previous treatment history, many culture-positive TB patients were infected with INH-resistant isolates, and a significant proportion of previously treated patients were infected with MDR-TB. Treating culture-positive TB patients, especially previously treated patients, based on DST results should therefore be considered.

Ethnic and geographical distributions of CYP2C19 alleles in the populations of Southeast Asia.

The genetic polymorphism of drug-metabolizing enzymes has a major influence on the fate of xenobiotic substances, whether as drugs or absorbed from the environment. Our understanding of this polymorphism is important in order to evaluate the genetic predisposition for exposure-related risks and, in the future, to develop individualized drug therapy. Cytochrome P450 enzymes play a central role in the metabolism of many drugs, chemicals, and carcinogens. Differences in the activity of these enzymes are responsible for the inter-individual variability in drug response and toxicity (Bertilsson, 1995). Of the cytochrome P450 enzymes, the isoform CYP2C19 is of particular interest because of its high inter-individual and inter-racial differences (Goldstein et al., 1997; Kaneko et al., 1999; Griese et al., 2001).

Low prevalence of hepatitis B virus pre-S deletion mutation in Indonesia.

The molecular epidemiological study of hepatitis B virus (HBV) in Indonesia is still limited. This study was aimed to identify the prevalence of HBV pre‐S deletion/insertion mutations, and to assess the association of pre‐S deletion mutation with liver disease progression in Indonesia. Pre‐S mutations were identified by direct sequencing. Of the 265 subjects, 32 samples (12.1%) harbored pre‐S deletion/insertion mutations. The prevalence of those pre‐S mutations was 2.7% (2/75), 12.9% (8/62), 16.7% (11/66), and 17.7% (11/62) in asymptomatic carrier, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma groups, respectively. Statistical analysis showed significant difference among them (P = 0.024). In HBV genotype B (HBV/B), pre‐S1, pre‐S1/S2, and pre‐S2 deletion mutations were detected respectively in 3 (17.6%), 4 (23.5%), and 9 (52.9%) of 17 samples. On the other hand, in HBV/C, 12 of 15 samples (80.0%) showed a pre‐S2 deletion mutation, and only 2 samples (13.3%) demonstrated a pre‐S1/S2 deletion mutation. These results suggest that in HBV/B deletion mutation tends to occur in pre‐S1 or pre‐S1/S2 region, while in HBV/C the deletion mutation usually occurs in the pre‐S2 region. Analysis of complete genome of four viruses confirmed that 3 isolates were classified into HBV/B3, and 1 isolate was HBV/C1. However, SimPlot and BootScan analyses showed that isolate 08.10.002 was an intragenotypic recombinant between HBV/B3 and HBV/B4. As conclusion, the prevalence of HBV pre‐S mutations was relatively low in Indonesian patients compared to those from Taiwan, Japan, and other Asian countries. There was a weak association between pre‐S deletion mutation and progressive liver disease. J. Med. Virol. 83:1717–1726, 2011.

Hepatitis B virus pre-S2 start codon mutations in Indonesian liver disease patients

AIM To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression. METHODS The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fishers exact test, χ² test, and t-test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced liver disease was estimated by unconditional logistic regression model adjusted with age, sex, and hepatitis B e antigen (HBeAg). P < 0.05 was considered significant. RESULTS Mutation of the hepatitis B virus (HBV) pre-S2 start codon was found in 59 samples from 268 subjects (22.0%), with higher prevalence in patients with cirrhosis 27/66 (40.9%) followed by HCC 18/63 (28.6%), chronic hepatitis 12/66 (18.2%) and asymptomatic carriers 2/73 (2.7%) (P < 0.001). Logistic regression analysis showed that pre-S2 start codon mutation was an independent factor for progressive liver disease. Other mutations, at T130, Q132, and A138, were also associated with LC and HCC, although this was not statistically significant when adjusted for age, sex, and HBeAg. The prevalence of pre-S2 start codon mutation was higher in HBV/B than in HBV/C (23.0% vs 19.1%), whilst the prevalence of T130, Q132, and A138 mutation was higher in HBV/C than in HBV/B. The prevalence of pre-S2 start codon mutation was higher in LC (38.9%) and HCC (40.0%) than CH (5.6%) in HBeAg⁺ group, but it was similar between CH, LC and HCC in HBeAg⁻ group. CONCLUSION Pre-S2 start codon mutation was higher in Indonesian patients compared to other Asian countries, and its prevalence was associated with advanced liver disease, particularly in HBeAg⁺ patients.

The Antinociceptive Effects of Pregabalin on Post-Operative Hysterectomy Patient

Postoperative pain experienced by patients who completed surgery should receive serious attention, because of effective postoperative pain management will reduce morbidity and mortality, accelerate the mobilization and reducing hospitalization time of patients. These studies aim to determine the effect of pregabalin on antinociception and substan-glutamate levels in the blood of patients with postoperative P hysterectomy. This research is an experimental study, namely the Controlled Trial Randomized clinical trials in which patients were randomly divided into two groups by the number of patients who be included 52 people (aged 20-50 years) who underwent hysterectomy surgery. They were divided into group I (n=26) orally administered pregabalin 3 mg/kg body weight and group II (n=26) placebo given orally, 1 hour before surgery. All patients are given general anesthesia that premedication with atropine sulfate and fentanyl injection, induction with propofol and atracurium injection, intubation and breath control, and maintenance of anesthesia with N2O:O2 and isoflurane. The total of postoperative use of morphine injection is assessed until 24 hours postoperative. Examination of the levels of glutamate and substan-P performed preoperative blood before administration and 1 hour postoperative pregabalin. The results showed a marked increase in the level of pain (VAS), blood pressure, heart rate and the amount of postoperative morphine consumption in patients who were given the placebo group, compared with patients given pregabalin group. It was concluded that preoperative pregabalin administration can suppress the increase in glutamate production and can reduce postoperative production of substan-P, and the subsequent decline in glutamate levels in the blood substan-P will provide antinociception effect is a decrease in the level of postoperative pain experienced by patients.

Heterogeneity of Mycobacterium tuberculosis strains in Makassar, Indonesia.

SETTING Patients with suspected pulmonary tuberculosis (TB) visiting government TB diagnostic and treatment centres in Makassar City, South Sulawesi Province, Indonesia, from February to October 2008 were included in the study. OBJECTIVE To determine the distribution of Mycobacterium tuberculosis genotypes in Makassar. DESIGN Cross-sectional study. Spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and principal genetic grouping (PGG) were used to genotype the M. tuberculosis clinical isolates. RESULTS Among 179 isolates derived from pulmonary TB patients, distribution of spoligotypes comprised the East Africa-Indian (30.2%), T (17.9%), H (12.3%) and Beijing (9.5%) lineages. Other lineages found in smaller proportions were the Latin American-Mediterranean, MANU, S and X lineages. Nineteen isolates (10.6%) could not be grouped into any of the reported lineages or shared types. Single nucleotide polymorphism analysis of katG(463) and gyrA(95) grouped these isolates primarily into PGG1 (9/19, 47%). CONCLUSION Only a few genetically identical clustered isolates were identified within the 9-month study period, and most isolates were genetically diverse. Furthermore, 15 spoligopatterns identified in our study have not been reported previously. To our knowledge, this is the first comprehensive study describing genotypes of M. tuberculosis clinical isolates in Sulawesi.