Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The current standard therapy for hepatitis C patients, which is based on a combination of pegylated interferons and ribavirin, results in viral clearance in about 50% of the treated individuals. Clinical trials of a variety of specific anti-HCV drugs, including several which target virus-encoded enzymes, are on-going, and some of these studies have reported impressive reductions of HCV levels in patients. However, the development of antivirals with diverse mechanisms of action is still required to eliminate this life-threatening virus. Besides specific viral proteins, targeting host cellular factors that are key to efficient viral replication could lead to the development of novel treatment strategies. Therapies against host factors are generally considered to present a low risk of generating drug-resistant viruses. The current understanding of anti-HCV drugs in clinical development and of virus-host interactions implicated in the regulation of HCV replication is summarized.

Internal transcribed spacer regions of ribosomal DNA were sequenced, and new species-specific primers were designed to simplify the molecular identification of five morphologically related subgenus Stegomyia mosquito species--Aedes aegypti, Ae. albopictus, Ae. riversi, Ae. flavopictus, and Ae. daitensis--found in the Ryukyu Archipelago, Japan. Each newly designed primer was able to amplify a species-specific fragment with a different size. Conditions for multiplex PCR were optimized to identify all five species in a single PCR. This method is a convenient tool for entomological field surveys, particularly in arbovirus endemic/epidemic areas where some of these species coexist.

The genetic diversity and antimicrobial resistance rates of clinical Salmonella isolates (2007–2008) at the University of Malaya Medical Centre, Kuala Lumpur, were investigated and the genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic (REP)-PCR. XbaI-PFGE analysis generated 57 profiles (Dice coefficient, F=0.08–1.00), whereas REP-PCR using the REP primer generated only 35 (F=0.34–1.00). PFGE was therefore the more discriminative and reproducible method for assessing the genetic diversity of salmonellae. The antibiograms of 78 Salmonella isolates were assessed against 19 antimicrobials using the disk diffusion method. Twenty serotypes were identified, with the most common being S. Enteritidis (18%) followed by S. Typhimurium (14%), S. Paratyphi B var Java (9%), S. Weltevreden (9%), and S. Corvallis (9%). A total of 38 resistant profiles were defined, with 53.8% of the isolates being resistant to three or more antimicrobials. The highest resistance rates were observed for cephalothin (55.1%), tetracycline (47.4%), and nalidixic acid (35.9%). The presence of multidrug-resistant Salmonella strains is a cause for concern as it may limit the treatment of severe salmonellosis. One multidrug-resistant S. Enteritidis strain was a putative extended-spectrum beta-lactamase producer, based on a double disk diffusion analysis, and was resistant to ceftriaxone (MIC>32 µg/mL). The data generated by this study will contribute towards epidemiological monitoring and investigations of Salmonella infections in Malaysia.

Rabies has reemerged in China. National rabies surveillance is centralized and based mainly on six provincial stations, including one in Shandong Province, which are selected and monitored by the China CDC. Data collection includes human rabies cases (diagnosed by local hospitals on the basis of signs or symptoms), documentation of post-exposure prophylaxis, primary laboratory diagnosis of suspect animal cases, and investigation of dog or other animal bites in viral transmission. Of the 408 human rabies cases reported during the period 2003–2007, most involved middle-aged male farmers bitten by their own unvaccinated dogs, with a seasonal peak in the autumn. These data provide key pointers regarding rabies prevention and control based upon an objective evidence-based framework.

The first confirmed case of 2009 influenza A (H1N1) in Fukuoka, Japan was r eported in early-June 2009. The disease rapidly spread through this area, mainly in schools, until there were no new cases detected 3 weeks later. We describe herein the clinical characteristics of this novel infection that came to light through the investigation of this outbreak. The patient records at hospitals and local public health centers were reviewed, and we defined laboratory-confirmed cases as those of a person who had influenza-like symptoms, such as a fever of 37ºC or more, cough, sore throat, rhinorrhea, or headache. From May 19 to June 31, 2009, a total of 71 cases were identified. The median age was 11 years, and all the patient took neuraminidase inhibitors and fully recovered. The fevers lasted for 1 to 5 days (median, 2). Cough lasted for 2 to 11 days (median, 7), and in 10 cases (34.5%) cough started before the fever. The incubation period was 2 to 3 days. Infectors transmitted the disease to another person on the day of or the day before fever onset. The findings regarding the onset and duration of symptoms and the timing of disease transmission of 2009 influenza A (H1N1) may be useful for future response.

We report the appearance of Candida glabrata strains with reduced sensitivity during treatment with the echinocandin drug micafungin (MCF). Four C. glabrata strains were isolated from sputum, gastric juice, and blood taken from a patient during hospitalization. Two of these strains, one of which was obtained after treatment with MCF for suspected Candida pneumonia and the other of which was obtained during MCF treatment for candidemia, were isolated from blood and found to have a reduced susceptibility to MCF. These two clinical isolates showed a high minimum inhibitory concentration (MIC) for MCF, with this change in MIC being unique for MCF among established antifungal drugs. To further investigate the mechanism underlying this reduced sensitivity, an in vivo mouse infection model and in vitro enzymatic analysis were performed. MCF had little effect in the mouse disseminated infection model and enzymatic analysis showed the low affinity of MCF to the 1,3-β-D-glucan synthase of the clinical isolates, although the enzymes of both clinical isolates and control strain were noncompetitively inhibited by MCF. Taken together, this low affinity of MCF for the enzymes is likely to cause the reduced sensitivities. These data further indicate that MCF could induce acquired MCF-resistant strains during clinical use.

Adenovirus types 1, 2, and 3 can usually be isolated in only a short time, although occasionally it may take longer. This phenomenon has been explained empirically as being due to the viral load in the sample, although to date there has been no experimental confirmation of this. In this study we therefore tried to establish a correlation between the quantity of respiratory adenovirus genome in the clinical sample and the time required for its isolation. The correct choice of sensitive cell line is important for this purpose, thus we compared the sensitivity of three different cell lines (HeLa, A549, and RD), and found A549 to be the most sensitive to adenoviruses 1–3. Stored clinical samples (n=21) containing adenoviruses 1–3 were diluted to make solutions containing between 10 and 10⁸ copies/µL of adenovirus genome (n=242). These diluted clinical samples were then inoculated into A549 cells, which were cultivated for 21 days and the results compared to the number of viral genomes in each cultivated sample. Adenoviruses could be isolated from all samples (41/41) containing ≥10⁶ copies/µL within 6 days, whereas samples containing 10 and 10² copies/µL required cultivation for 12.6±3.8 and 11.2±3.8 days (mean±S.D.), respectively, before adenoviruses could be isolated. A cultivation time of 21 days should therefore be considered for the isolation of respiratory adenoviruses from samples containing <10³ adenovirus genome copies/µL.

This study was designed to determine the prevalence and distribution of Chlamydia trachomatis genotypes from clinical specimens in Guangzhou, China, obtained in the period 2005–2008. One hundred and ninety-four urogenital C. trachomatis samples were collected from sexually transmitted disease clinic patients, and the VS1-VS2 of OmpA gene was amplified by nested PCR and sequenced using an ABI-prism 3730 sequencer. Clinical C. trachomatis strains were genotyped and analyzed for a mutation with respect to the reference VS1-VS2 sequence. VS1-VS2 fragments with 453 bp were amplified from 194 clinical samples. Upon alignment with the sequences of the reference strains, 189 strains with discernible sequences were typed into 9 genotypes, while 5 with ambiguous sequences were considered to be mixed-serovar samples. The most prevalent genotypes were E (50, 26%), F (46, 24%), J (35, 19%), and D (24, 13%). There was no significant difference in the distribution of any of the genotypes detected during the study period, except for genotype K (P<0.01). A total of 16 (8%, 16/189) genetic variants of the OmpA VS1-VS2 of the reference strains were identified. Mutations occurred frequently for genotypes D (2/24, 8%), E (6/50, 12%), F (2/46, 4%), G (1/8, 13%), H (1/12, 8%), and K (4/11, 36%), with most of these being sense mutations that may result in amino acid substitution. Sequencing the OmpA VS1-VS2 enabled the genotype and sequence variations within each genotype to be analyzed. Genotypes E, F, J, and D continued to dominate among urogenital C. trachomatis, whereas genotype K increased significantly in Guangzhou between 2005 and 2008.

The purpose of this study was to investigate the role of matrix metalloproteinase-9 (MMP-9) in the pathogenesis of vascular leakage in patients with dengue virus infection. Serum samples from 24 children with serologically confirmed dengue virus infection (dengue fever [DF], 16; dengue hemorrhagic fever [DHF], 8; age, 9.5±2.4 years; 67% male] were analyzed for MMP-9 during the febrile and toxic stages and at follow-up. Serum samples obtained from 7 healthy children were used as controls. Serum MMP-9 levels in patients with dengue virus infection were found to be lower at the febrile (227.0±186.9 ng/ml) and toxic stages (150.9±151.7 ng/ml) than at follow-up (424.5±227.8 ng/ml) or in the control group (393.3±125.9 ng/ml, P<0.001 by one-way ANOVA). There was no significant difference between MMP-9 levels in patients with DHF and those with DF at any stage of the disease. In conclusion, MMP-9 levels are reduced during the febrile and toxic stages of dengue virus infection.

The prevalence of Ehrlichia and Anaplasma in 1,427 dogs from 32 Japanese prefectures was evaluated by PCR and DNA nucleotide sequencing. PCR screening demonstrated that 18 dogs (1.3%) were positive for Anaplasmataceae. Sequence analysis revealed that 14 of the amplicons were most closely related to Wolbachia spp., symbionts of Dirofilaria immitis, whereas three were identified as Anaplasma bovis. The remaining amplicon could not be sequenced. Almost the entire sequence of 16S rRNA (1,452 bp) from one of the positive specimens was determined, and subsequent phylogenetic analysis confirmed that the detected sequence was that of A. bovis. This is the first detection of A. bovis DNA fragments in dogs. Species-specific nested PCR showed that 15 (1.1%) of the 1,427 dogs involved in this study were positive for A. bovis. The geographical distribution of these dogs ranged from Aomori Prefecture in northern Japan to Kagoshima Prefecture in the south. The relationship between A. bovis infection and clinical disease is not yet clearly understood.

Rickettsial infection in feral raccoons (Procyon lotor) in Hokkaido, Japan was analyzed by molecular methods. Genus-specific nested polymerase chain reaction (PCR) analysis based on the Rickettsia citrate synthase (gltA) gene showed that 13 of 699 raccoons (1.9%) examined were positive for Rickettsia. Twelve of the 13 partial gltA sequence amplicons were successfully analyzed. The nucleotide sequence of one amplicon was identical to both Rickettsia heilongjiangensis and R. japonica, one was identical to R. felis, and the rest to R. helvetica. This is the first report on the detection of rickettsial agents in peripheral blood of raccoons.

We investigated whether treating Candida biofilms with micafungin, an echinocandin that inhibits the synthesis of glucan in the fungal cell wall, alters the expression of genes related to chitin synthesis and degradation in response to cell wall stress. As expected, all four genes encoding chitin synthases--CHS1, CHS2, CHS3, and CHS8--were upregulated by micafungin treatment. Interestingly, of the four genes encoding chitinases, the expression of only CHT2 and CHT3 was markedly downregulated, that of CHT1 was upregulated, and that of CHT4 remained unaltered after micafungin treatment. Thus, the suppression of only two genes associated with chitin degradation, CHT2 and CHT3, may be involved in the tolerance to the cell wall stress caused by micafungin as well as the induction of chitin synthesis. Further, micafungin markedly increased UTR2, which is calcineurin dependent, and CRZ2, which is calcineurin independent. Therefore, gene regulation possibly includes calcineurin-dependent and independent stress responses, though the regulation of genes associated with cell wall chitin has not yet been completely clarified. Our results imply that cell wall stress can be exploited to enhance the efficacy of micafungin.

The objective of this study was to determine the genetic diversity of rabies virus (RABV) in Mongolia based on the nucleotide sequences of viral N gene. A total of 24 rabies-positive samples from seven different domestic and wild animal species collected in western and central Mongolia between 2005 and 2008 were examined for their N gene sequences. The results showed that the endemic Mongolian RABVs could be divided into two different groups closely related to the Steppe-type and Arctic-like viruses isolated in Russia.

We evaluated the utility of typing the spa gene, which encodes protein A of Staphylococcus aureus, for analyzing methicillin-resistant S. aureus (MRSA) isolates from patients with health care-associated infections by comparing the results of spa typing with those of pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA). We analyzed 78 clinical MRSA isolates collected at our hospital over a period of 2 months. The clinical isolates were found to have 12 different spa types, with approximately 82% (64/78) of them being typed as t002. The same clinical MRSA isolates were classified into 15 and 19 types upon MLVA and PFGE analysis, respectively, and 19 and 28 types when spa typing was used in combination with MLVA and PFGE, respectively. The discriminatory ability of spa typing alone is low, and thus indicating that this technique is insufficient for performing the initial genotyping of MRSA in short-term epidemiological studies. Therefore, spa typing should be used in combination with MLVA or PFGE for further typing of MRSA isolates.

Tuberculosis (TB) is a disease of worldwide public-health concern. The development of resistance to an increasing number of second-line drugs and those used to treat multidrug-resistant (MDR) TB is rapidly becoming an emergency that could hinder the prevention and treatment of TB globally. This study describes the resistance profile of MDR and extensively drug-resistant (XDR) TB with a hospital-based survey in Beijing, China, conducted in the period 2007 to 2009. Drug-susceptibility tests performed on 967 Mycobacterium tuberculosis strains isolated from 967 patients showed that the rate of resistance to at least one first-line and at least one second-line drug was 70.1% and 60.7%, respectively. The overall MDR rate was 19.4%, and 14.9% of the MDR cases were XDR. In conclusion, MDR and XDR TB represent a significant number of total TB cases, therefore effective measures to manage these resistant strains are desperately needed. Development of a national TB policy in China might be a key method for solving the present problems of TB management.

Group G Streptococcus strains isolated from patients with severe invasive infections in the period 2002–2008 were surveyed and their prevalence compared with that observed in the period 1995–2001 in Japan. Strains with genotypes stg485, stg6792, stc36, stg6, and stg652 were isolated in both periods, whereas various new genotypes appeared in 2002–2008 and some genotypes found in 1995–2001 were not found subsequently, thus indicating a change in the prevalent genotyped strains causing severe invasive streptococcal infections.