Influenza C virus, an enveloped virus containing seven single-stranded RNA segments of negative polarity, belongs to the genus Influenza C Virus of the family Orthomyxoviridae. A number of questions remain to be resolved with regard to the molecular virology and epidemiology of the virus. To address them, we have established a virus-like particle (VLP) generation system and reverse genetics of the virus and succeeded in clarifying the structure-function relationship of the M1 protein of the virus. Although the approach adopted was similar to that for influenza A virus reverse genetics, the number of infectious influenza C viruses generated was much lower than that for influenza A virus. Based on a comparison of the number of influenza C VLPs with that of influenza A VLPs generated using a similar system, we proposed a virion generation mechanism unique to influenza C virus.

To investigate intranasal (i.n.) immunization efficacy of Schistosoma japonicum 97-kDa myofibrillar protein paramyosin (PM), a vaccine candidate for Asian schistosomiasis, BALB/c mice were i.n. immunized with Escherichia coli-expressed recombinant PM (rPM). I.n. immunization using rPM mixed with cholera toxin (CT) was more potent than subcutaneous (s.c.) immunization with rPM emulsified in incomplete Freund's adjuvant for induction of serum (IgG, IgE, and IgA) and mucosal (IgA in nose, lung, and intestine) antibody and delayed-type hypersensitivity (DTH) responses. The second i.n. immunization was sufficient to induce maximal serum IgG and DTH responses, which were almost completely maintained for more than 6 months. Next, to evaluate protective efficacy of the rPM against S. japonicum infection, immunized mice were infected with S. japonicum cercariae at 2 weeks after the second immunization. At 7 weeks after infection, we observed no reduction in worm burden or fecundity in both i.n. and s.c. immunized groups. Results showed that i.n. immunization with rPM/CT failed to provide protection against parasite infection, albeit the antigen was a very potent mucosal immunogen. These results may emphasize the need to innovate new mucosal adjuvants or delivery molecules to overcome such hurdles in the construction of a mucosal antiparasite vaccine platform.

Although Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. are prevalent causes of community-acquired pneumonia, rapid and sensitive diagnosis is difficult. Real-time PCR provides rapid and sensitive diagnosis, however, DNA extraction is still required, which is time-consuming, costly and includes a risk of contamination. Therefore, we aimed to develop triplex real-time PCR without DNA extraction. AmpDirect^® Plus which inhibits PCR inhibitors was used as the PCR buffer. Melting temperatures of the PCR products for the three bacteria were analyzed by SYBR green triplex real-time PCR and were found to be significantly different. Detection limits of bacteria cells diluted in nasopharyngeal aspirates (NPAs) were comparable with the detection limits of previously reported real-time PCR. Our PCR without DNA extraction and probe real-time PCR with DNA extraction showed identical results for the detection of the three bacteria from 38 respiratory specimens (sputum, endotracheal aspirates, and NPAs) collected from patients with pneumonia. No cross-reaction with other bacteria was observed. Our triplex real-time PCR successfully detected and differentiated the three bacteria. Although further field tests are required, our assay is a promising method for the rapid and cost-effective detection of the three bacteria.

A severe dengue outbreak occurred in East Timor in 2005. The dengue virus genome was detected by TaqMan RT-PCR in 40 serum samples, as follows: dengue virus type-3 (DENV-3) in 37 samples, DENV-2 in 2 samples, and DENV-1 in one sample. One DENV-1 genome, one DENV-2 genome, and 5 DENV-3 genomes were sequenced, and these specimens were aligned with the previously determined envelope (E) gene sequences. The DENV-1 strain belonged to genotype IV and was close to those previously isolated in Indonesia and Australia. The DENV-2 strain belonged to genotype I and was close to those previously isolated in Indonesia, Australia, the Far East, and India in 1993–2001. The DENV-3 strain belonged to genotype I and was close to those previously isolated in Indonesia. The results indicate that the dengue outbreak was caused mainly by DENV-3, with DENV-1 and DENV-2 as minor serotypes, and suggest that these strains of 3 serotypes of DENV entered East Timor from neighboring countries, co-circulated, and caused the dengue outbreak in 2005.

The major objective of this study was to deliver vital statistics related to cholera to health authorities so as to aid in their attempt to prioritize communicable diseases in Nepal. A laboratory-based surveillance was conducted from mid-June 2008 to mid-January 2009 at the National Public Health Laboratory, Nepal. Diarrheal samples alone were processed for Vibrio cholerae. Isolation and identification of the organisms were carried out as per standard protocol. Antimicrobial susceptibility tests were done according to the guidelines of the Clinical and Laboratory Standards Institute. The incidence of cholera was found to be 27.1%. Only V. cholerae O1 Ogawa biotype El Tor was found during the study. No variation was observed in the percentage of cases between genders (P<0.05). The 15–30 year age group was found to be more susceptible to cholera (P<0.05). The period from mid-June to mid-July had the highest incidence of cholera (P<0.05). Ampicillin, tetracycline, ciprofloxacin, and erythromycin were highly effective, while 100% resistance was observed for furazolidone, nalidixic acid, and cotrimoxazole.

Mycobacterial infection of musculoskeletal tissue is a rare disease that may cause destruction of the tissues. Both Mycobacterium tuberculosis and non-tuberculous mycobacteria affect the tissues. Although surgical debridement and antibiotic therapy are required for the treatment, it is necessary to identify the causative species before selecting the antibiotics. However, it is difficult to identify the species in clinical samples from musculoskeletal tissue. In the current study, using pathological specimens, the causative species was identified by PCR amplification and direct sequencing of mycobacterial 16S rDNA containing a hypervariable region. Twelve cases of chronic granulomatous inflammation of musculoskeletal tissues were used for the study. DNA was extracted from paraffin sections, and mycobacterial 16S rDNA was amplified by PCR. The amplicons were obtained in 5 of 12 cases (41%), even in specimens in which the microorganism was only scarcely detected by using special stains. Direct sequencing of the amplified products presented high homology with M. tuberculosis in four cases and M. avium in one. Therefore, PCR-direct sequencing of 16S rDNA containing hypervariable region using pathological specimens is useful for the diagnosis and identification of causative species in mycobacterial infection of musculoskeletal tissues.

We retrospectively analyzed serological test results for anti-hepatitis A virus immunoglobulin G (anti-HAV IgG) of sera collected from 779 military personnel during January 2001 to May 2008. The overall seroprevalence of anti-HAV IgG of the subjects was 17.5% (95% confidence interval [CI], 14.8–20.1%). When adjusted to the age-specific distribution of the army population, the age-adjusted seroprevalence was 14.6% (95% CI, 13.0–16.3%). All subjects who were 40 years and over had anti-HAV IgG. Meanwhile, the seroprevalence of anti-HAV IgG for those 24 years and younger was 4.7%. This low prevalence rate among young military personnel calls for stricter adherence to vaccination policies and stronger requirements for military HAV vaccination programs.

Kluyvera cryocrescens, formerly accepted as a benign saprophytic microorganism, is an opportunistic pathogen and its infection is very rare in humans. This report describes a preterm infant born at 30 weeks of gestational age and successfully treated for K. cryocrescens sepsis in the 3rd week of life. To our knowledge, this is the first case of K. cryocrescens sepsis in a newborn. The potential of K. cryocrescens as a serious pathogen should be recognized especially in patients such as preterm infants in whom the prognosis may be compromised without appropriate treatment.

To assess the degree to which face masks reduce the strength of cough-wind, we measured the wind pressure in front of the mouth with and without the wearing of masks. We found that any conventional masks made from paper, cotton gauze, or non-woven fabrics reduced the wind pressure to less than one-tenth that recorded when no mask was worn.

A nosocomial outbreak of Mycobacterium tuberculosis Beijing/W genotype infected 15 healthcare workers (HCWs) in a medical center in Taiwan, where there is a high prevalence of tuberculosis and a high rate of positive tuberculin skin tests. An index patient with laryngeal cancer and a lung abscess was identified by epidemiological investigation and it was found that an M. tuberculosis isolate from his lung tissue sample had an identical IS6110 restriction fragment length polymorphism pattern to the isolates from 3 HCWs. Confirmation of the identity of this strain as Beijing/W genotype was made using spoligotyping. Seven hundred and eighty-five HCWs potentially exposed to the probable index patient received contact investigation and chest X-ray screening. We used chest high-resolution computed tomography (HRCT) to clarify trivial lesions in chest X-rays. Nine HCWs with smear-negative pulmonary tuberculosis were diagnosed by HRCT. Fifteen of the 35 (42.9%) HCWs with documented exposure to the index patient developed pulmonary tuberculosis within 11 months after exposure. The outbreak was successfully controlled by active case finding and enforcement of infection control strategies. Intervention to detect the potential tuberculosis source is helpful in the prevention and control of a nosocomial tuberculosis outbreak. HRCT can be a useful tool for tuberculosis diagnosis of contacts in an outbreak situation.

Recent large-scale outbreaks in the Lao People's Democratic Republic (Lao PDR) were reported in 1993 and 1994 and from 2000 to 2002. On December 23, 2007, a drastic increase in acute watery diarrhea patients at a health center in Sekong Province was reported to the provincial health office. An outbreak investigation was initiated to understand the magnitude of the outbreak, identify new cases, identify the suspected causal agent, implement control measures, and prevent new cases. Through active village based surveillance, 370 cases and 3 deaths were reported from 31 villages between December 15, 2007 and January 29, 2008. Of these reported cases, 29% were under the age of 5. From 28 fresh stool samples taken, 17 (58.6%) were positive for Vibrio cholerae O1 Ogawa strain. Two water sources close to affected villages were found to be contaminated with the same strain of V. cholerae. Control measures implemented included health education for safe household water consumption and early identification and treatment of suspected cholera patients at village level. The cause of the outbreak was suspected to be a combination of contaminated drinking water and person-to-person transmission.

In our previous study, a novel P[8] subtype, i.e., P[8]b was identified for human rotavirus strains MMC38 and MMC71 detected in Bangladesh, of which the P types could not be determined by conventional RT-PCR genotyping methods. In the present study, a modified multiplex RT-PCR method was developed to detect P[8]b as well as common human rotavirus P types. With this method, P[8]b was detected in three strains among the 26 rotavirus specimens which had been judged as mixed P types in the previous study in Bangladesh. The VP4 nucleotide sequences of these strains showed more than 98.9% identities to those of strains MMC38 and MMC71. The newly designed RT-PCR method was considered as useful for identifying P[8]b and avoiding misclassification by the conventional RT-PCR genotyping methods.