Asian Pacific Journal of Tropical Medicine

307-312

Authors: Yi-Qiang Xie, Jing-Zhe Zhang, Hua Zhang, Lei Peng, Shuang Zhou, Li-Zhuo Li, Bang-Jiang Fang, Chuan-Zhu Lyu

Objective To investigate the distribution of pathogens and drug resistance in bile and the association between the pregnane X receptor (PXR) gene polymorphisms, traditional Chinese medicine (TCM) syndromes and the risk of cholesterol gallstone disease (CGD). Methods A total of 392 samples were enrolled in this study from January 2014 to February 2015, among which 192 patients were with CGD, and 200 samples were healthy. Strains were isolated and susceptibility testing was the disk diffusion method susceptibility testing. The patients were divided into hepatochlic hygropyrexia, stagnation of liver-qi, and the accumulation of damp. The PXR gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. The association between the PXR gene polymorphisms and the risk of CGD was examined by logistic regression analysis. Results A total of 192 cases were detected in 230 of bile culture pathogens, including Gram-negative bacteria 133 (57.83%), Gram-positive bacteria 76 (33.04%), and fungi 21 (9.13%). The top five pathogens were Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Candida albicans, and Enterococcus feces, of which 110 cases was of single infection, 48 cases of mixed infection of two strains, eight cases of mixed infection of three bacteria. Among 59 Escherichia coli, the yield extended-spectrum beta-lactamases had 40 (67.80%). The hepatochlic hygropyrexia was the most TCM syndrome, followed by stagnation of liver-qi, and the accumulation of damp was least. Different pathogens and the rs6785049 genotypes distributed differently in cholelithiasis patients with different TCM syndromes (P < 0.05). In hepatochlic hygropyrexia patients the Gram-negative bacteria was most. There was significant differences between CGD group and control group in rs6785049 (P < 0.001). Comparison with wild-type portable GG, GA genotype increased the risk of the occurrence of gallstones (OR = 0.40, 95%CI: 0.16–0.79); likewise, carrying the GA+AA genotype also increased the risk (OR = 0.38, 95%CI: 0.19–0.81). There was no significant differences in rs2276707, rs3814055 site polymorphic loci alleles in CGD group and control group. Conclusions In the treatment of cholelithiasis, bile samples should be collected for bacterial culture and sensitivity test, and drugs should be strictly chosen based on the results. The rs6785049 polymorphisms in PXR gene may increase the risk of gallstones ontogeny, and gallstones can be early detected and prevented by detecting genotypes. rs6785049 polymorphisms in PXR gene may has relationship with TCM syndromes.

324-327

Authors: Valentina Virginia Ebani, Fabrizio Bertelloni, Paolo Mani

Objective To determine the presence of zoonotic tick-borne bacteria in feral pigeons (Columba livia domestica) from urban areas. Methods Spleen samples from 84 feral pigeons, found dead with traumatic injuries in urban areas, were examined by PCR to detect DNA of Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Rickettsia spp., and Chlamydophila spp. Results Twenty (23.8%) pigeons were infected by tick-borne agents, in particular 2 (2.38%) animals resulted positive for Bartonella spp., 5 (5.95%) for C. burnetii, 5 (5.95%) for Rickettsia spp., 13 (15.47%) for B. burgdorferi sensu lato. All birds scored negative for A. phagocytophilum. Moreover, 17 (20.23%) pigeons were positive for Chlamydophila spp. and among them 10 (11.9%) for Chlamydophila psittaci. Mixed infections by two or three agents were detected in 8 (9.52%) animals. Conclusions Feral pigeons living in urban and periurban areas are a hazard for the human health as source of several pathogens. The obtained results confirm pigeons as reservoirs of chlamydial agents and suggest that they may be involved in the epidemiology of zoonotic tick-borne infections too.

333-336

Authors: Loan Phuong Do, Trang Minh Bui, Nga Thi Phan

Objective To examine the multiplication efficiency Japanese encephalitis virus (JEV) genotype I (GI) and genotype III (GIII) of different cell lines which originated from human, porcine, mosquitoes in order to prove mechanism of JEV GI replacement JEV GIII since it emerging in nature recent decades. Methods The mixture of GI and GIII JEV isolates was inoculated on human rhabdomyosarcoma (RD), pig kidney epithelial (PS) and Aedes albopictus C6/36 clone (C6/36) which originated from human, porcine and mosquitoes, respectively. Plaque assays were performed to calculate virus titer and real-time RT-PCR with GI and GIII specific primer sets to quantify the number of GI and GIII RNA copies. Results The highest virus titer reached at the 3rd day of post infection when GI and GIII mixture was inoculated on RD and PS and that of C6/36 was at the 4th day. JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages, respectively. GI strain amplified and maintained more efficiently on C6/36 and PS but not RD, whereas GIII strain amplified and maintained more efficiently on RD. Conclusions There is a correlation between the multiplication efficiency of GI and GIII JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GI strains in C6/36 and PS and the limited detection of GI strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GI emerging.

344-350

Authors: Cheol Park, Ji-Suk Jeong, Jin-Woo Jeong, Sung Ok Kim, Yong-Joo Kim, Gi-Young Kim, Su-Hyun Hong, Yung Hyun Choi

Objective To investigate the effects of an ethanol extract of Kalopanax septemlobus (Thunb.) Koidz. leaf (EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action. Methods Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis. Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting, and activation of cyclin-associated kinases studied using kinase assays. Results The EEKS suppressed cell proliferation in both HepG2 and Hep3B cells, but showed a more sensitive anti-proliferative activity in HepG2 cells. Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G1 phase cell cycle arrest in HepG2 cells, along with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with EEKS also increased the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21WAF1/CIP1 and p27KIP1, without any noticeable changes in G1 cyclins and CDKs (except for a slight decrease in CDK4). Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6, which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities. Conclusions Overall, our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G1 cell cycle arrest. Further studies are required to identify the active compounds in EEKS.

357-365

Authors: Ju-Yeon Park, Jin-Young Moon, Sun-Dong Park, Won-Hwan Park, Hyuck Kim, Jai-Eun Kim

Objective To investigate the anti-inflammatory effects and the action mechanism of the fruits of Hovenia dulcis (H. dulcis) in lipopolysaccharide (LPS)-induced mouse macrophage Raw 264.7 cells. Methods The extract of H. dulcis fruits (EHDF) were extracted with 70% ethanol. Mouse macrophages were treated with different concentrations of EHDF in the presence and absence of LPS (1 μg/mL). To demonstrate the inflammatory mediators including nitric oxide, inducible nitric oxide synthase and cyclooxygenase (COX)-2 expression levels were analyzed by using in vitro assay systems. COX-derived pro-inflammatory cytokines including interleukin-1β, tumor necrosis factor-α and prostaglandin E2 were determined using ELISA kits. Cell viability, heme oxygenase-1 expression, nuclear factor-kappaB and nuclear factor E2-related factors 2 translocation were also investigated. Results EHDF potently inhibited the LPS-stimulated nitric oxide, inducible nitric oxide synthase, COX-2, interleukin-1β and tumor necrosis factor-α expression in a dose-dependent manner. EHDF suppressed the phosphorylation of inhibited kappaB-alpha and p65 nuclear translocation. Treatment of macrophage cells with EHDF alone induced the heme oxygenase-1 and nuclear translocation of nuclear factor E2-related factor 2. Conclusions These results suggest that the ethanol extract of H. dulcis fruit exerts its anti-inflammatory effects by inhibiting inhibited kappaB-alpha phorylation and nuclear translocation of nuclear factor-kappaB.

372-379

Authors: Daniel Barroso de Alencar, Fátima Cristiane Teles de Carvalho, Rosa Helena Rebouças, Daniel Rodrigues dos Santos, Kelma Maria dos Santos Pires-Cavalcante, Rebeca Larangeira de Lima, Bárbara Mendes Baracho, Rayssa Mendes Bezerra, Francisco Arnaldo Viana, Regine Helena Silva dos Fernandes Vieira, Alexandre Holanda Sampaio, Oscarina Viana de Sousa, Silvana Saker-Sampaio

Objective To evaluate the antioxidant, antibacterial and bacterial cell agglutination activities of the hexane (Hex) and 70% ethanol (70% EtOH) extracts of two species of red seaweeds Pterocladiella capillacea (P. capillacea) and Osmundaria obtusiloba. Methods In vitro antioxidant activity was determined by DPPH radical scavenging assay, ferric-reducing antioxidant power assay, ferrous ion chelating assay, β-carotene bleaching assay and total phenolic content quantification. Antimicrobial activity was tested using the method of disc diffusion on Mueller-Hinton medium. The ability of algal extracts to agglutinate bacterial cells was also tested. Results The 70% EtOH extract of the two algae showed the highest values of total phenolic content compared to the Hex extract. The results of DPPH for both extracts (Hex, 70% EtOH) of Osmundaria obtusiloba (43.46% and 99.47%) were higher than those of P. capillacea (33.04% and 40.81%) at a concentration of 1 000 μg/mL. As for the ferrous ion chelating, there was an opposite behavior, extracts of P. capillacea had a higher activity. The extracts showed a low ferric-reducing antioxidant power, with optical density ranging from 0.054 to 0.180. Antioxidant activities of all extracts evaluated for β-carotene bleaching were above 40%. There was no antibacterial activity against bacterial strains tested. However, the extracts of both species were able to agglutinate bacterial Gram positive cells of Staphylococcus aureus and Gram negative cells of Escherichia coli, multidrug-resistant Salmonella and Vibrio harveyi. Conclusions This is the first report of the interaction between these algal extracts, rich in natural compounds with antioxidant potential, and Gram positive and Gram negative bacterial cells.

385-389

Authors: Eleonora Cella, Ivan Gabrielli, Gianguglielmo Zehender, Marta Giovanetti, Alessandra Lo Presti, Alessia Lai, Giordano Dicuonzo, Silvia Angeletti, Marco Salemi, Massimo Ciccozzi

Objective To study the genetic diversity of Murray Valley encephalitis virus (MVEV) in Australia and Papua New Guinea. Methods MVEV envelope gene sequences were aligned using Clustal X and manual editing was performed with Bioedit. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Maximum likelihood analysis was performed using PhyML. The phylogenetic signal of the dataset was investigated by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST. Results The phylogenetic trees showed two main clades. The clade Ⅰ including eight strains isolated from West Australia. The clade Ⅱ was characterized by at least four epidemic entries, three of which localized in Northern West Australia and one in Papua New Guinea. The estimated mean evolutionary rate value of the MVEV envelope gene was 0.407 × 10−3 substitution/site/year (95% HPD: 0.623 × 10−4–0.780 × 10−3). Population dynamics defines a relative constant population until the year 2000, when a reduction occurred, probably due to a bottleneck. Conclusions This study has been useful in supporting the probable connection between climate changes and viral evolution also by the vector point of view; multidisciplinary monitoring studies are important to prevent new viral epidemics inside and outside new endemic areas.

395-401

Authors: Ee Leen Pang, Hwei-San Loh

Prevalence of dengue transmission has been alarmed by an estimate of 390 million infections per annum. Urban encroachment, ecological disruption and poor sanitation are all contributory factors of increased epidemiology. Complication however arises from the fact that dengue virus inherently exists as four different serotypes. Secondary infection is often manifested in the more severe form, such that antibody-dependent enhancement (ADE) could aggravate ailment by allowing pre-existing antibodies to form complexes with infecting viruses as means of intrusion. Consequently, increased viraemic titter and suppression of antiviral response are observed. Deep concerns are thus expressed in regards to escalating trend of hospitalisation and mortality rates. In Malaysia, situation is exacerbated by improper clinical management and pending vector control operations. As a preparedness strategy against the potential deadly dengue pandemic, the call for development of a durable and cost-effective dengue vaccine against all infecting serotypes is intensified. Even though several vaccine candidates are currently being evaluated in clinical trials, uncertainties in regards to serotypes interference, incomplete protection and dose adequacy have been raised. Instead of sole reliance on outsourcing, production of local vaccine should be considered in coherent to government's efforts to combat against dengue.

410-411

Authors: Beuy Joob, Viroj Wiwanitkit