Genetic evidence for containment of viruses in the first outbreak of influenza A pandemic (H1N1) 2009 in Kobe, Japan.

BACKGROUND: On 16 May 2009, a high school student in Kobe with no history of overseas travel was reported as the first case of influenza A pandemic (H1N1) 2009 virus infection in Japan. Subsequently, it was revealed that the infection had spread to some cities in the Kansai region and most patients were high school students. The number of patients decreased rapidly within a week; however, it began to increase in the middle of July. METHODS: We phylogenetically analyzed viral characteristics using 27 viruses isolated from patients living in Kobe. RESULTS AND CONCLUSIONS: We demonstrated that viruses isolated in the early phase of the outbreak were distinguishable from those after the reappearance of patients. These findings provide genetic evidence for the effectiveness of public health containment measures in the Kansai region in preventing the progression of the outbreak.

Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types.

We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980-2008. Most (80.2%) belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95% CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9% of all isolates). Moreover, 79.7% of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulence-associated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n=6), ST120 (n=5) and ST138 (n=5). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies.

[Psittacosis outbreak at an avian exhibition].

Psittacosis outbreak due to Chlamydophila psittaci occurred among staff members at an avian exhibition of nearly 1,000 birds in Kobe, Japan, in December 2005. Staff members not trained about zoonosis or psittacosis used little protective attire such as masks and gloves when caring for their discharges. Two of 67 staff members contracted psittacosis pneumonia. Additional two suffered from pneumonia and 19 reported symptoms such as fever and cough, although none were diagnosed with psittacosis. The roughly 970 birds were kept without quarantine and identified by leg bands. Doxycycline administrated in drinking water and food failed to eradicate chlamydia, so all birds were captured, identified by leg band, and tested for chlamydia by PCR. Six were found to carry large amounts of chlamydia. Major outer membrane protein (MOMP) DNA sequence of chlamydia in a patient's bronchoalveolar lavage fluid (BALF) was identical to that derived from a channel-billed toucan kept in a closed aviary, and staff members may have been infected by inhaling excrement while working in the aviary. The MOMP DNA sequence was useful in comparing strains. We review the difficulty of diagnosing psittacosis and the knowledge and infection control measures required against it.

Synergistic effect of HLA class II loci and cytokine gene polymorphisms on the risk of gastric cancer in Japanese patients with Helicobacter pylori infection.

It has been reported that polymorphisms of human leukocyte antigen (HLA) genes and several cytokine genes are associated with an increased risk of developing gastric cancer (GC). However, the results of studies from different geographic regions, ethnic groups and study groups are inconsistent. The aim of this study was to evaluate the influence of H. pylori infection and host genetic factors on GC susceptibility in Japanese patients with GC. We analyzed genotypes for HLA class I and II, tumor necrosis factor alpha, interleukin (IL)-1beta, IL-1 receptor, IL-4, IL-4Ralpha and IL-10 in 330 H. pylori-infected noncardia patients with GC and 190 H. pylori-infected nonulcer dyspeptic controls. Haplotype analyses indicated that the frequencies of the HLA DRB1*0405 and DQB1*0401 alleles were increased in the patients with intestinal-type GC when compared with controls (both DRB1*0405 and DQB1*0401: p = 0.015, OR = 1.57, 95% CI = 1.09-2.26), but the changes were not statistically significant after correction for multiple comparisons. None of the cytokine gene polymorphisms were associated with GC susceptibility, whether patients with GC were analyzed as a group according to the histological subtype. Of interest was the comparison of controls and patients with intestinal-type GC. The frequency of an IL-10-592AA homozygote showing concomitant carriage of the HLA DRB1*0405-DQB1*0401 haplotype was significantly higher in patients with intestinal-type GC (chi(2) = 6.369, p = 0.0116, p(c) = 0.0464, OR = 2.43, 95% CI = 1.21-4.48). Our results suggest that the HLA class II and IL-10-592A/C polymorphisms synergistically affect the susceptibility to GC development of H. pylori-infected individuals in the Japanese population.

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Campylobacter fetus.

We developed a sensitive and rapid loop-mediated isothermal amplification (LAMP) assay for detection of Campylobacter fetus. This assay provides simpler and more rapid detection of C. fetus than conventional biochemical and PCR assays. The assay correctly detected 60 C. fetus strains but not 55 non-fetusCampylobacter and 30 non-Campylobacter strains. The sensitivity of the LAMP assay in pure cultures and in a spiked bovine liver specimen was 10-fold more sensitive than that of the conventional PCR assay. The sensitivity of the LAMP assay in a spiked bovine vaginal mucus specimen was similar to that of the conventional PCR assay. The assay was markedly faster, requiring less than 40min for detection of C. fetus in a single colony on blood agar and 80min in spiked bovine specimens from the beginning of DNA extraction to final determination. Our LAMP assay is a simple and practical tool for detection of C. fetus regardless of subspecies.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli.

We developed a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Campylobacter jejuni and Campylobacter coli. The assay provides a specific LAMP product for each of these two species. The assay correctly identified 65 C. jejuni and 45 C. coli strains, but not 75 non-C. jejuni/coli strains. The sensitivity of the LAMP assay for C. jejuni and C. coli in spiked human stool specimens was 5.6 x 10(3) c.f.u. g(-1) (1.4 c.f.u. per test tube) and 4.8 x 10(3) c.f.u. g(-1) (1.2 c.f.u. per test tube), respectively. When 90 stool specimens from patients with diarrhoea were tested by LAMP and direct plating, the LAMP results showed 81.3 % sensitivity and 96.6 % specificity compared to isolation of C. jejuni and C. coli by direct plating. Further, the LAMP assay required less than 2 h for detection of C. jejuni and C. coli in stool specimens. This LAMP assay is a rapid and simple tool for the detection of C. jejuni and C. coli and will be useful in facilitating the early diagnosis of food poisoning incidents caused by these organisms.

Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis.

A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

Unusual Corynebacterium mucifaciens isolated from ear and nasal specimens.

OBJECTIVE: Corynebacterium mucifaciens was first proposed as a novel Corynebacterium species in 1997. This report describes our experience with the unexpectedly frequent isolation of C mucifaciens. STUDY DESIGN: By using agar supplemented with several antibiotics, microbiological studies were performed on specimens collected from the ear and nose. SETTING: A primary and secondary referral hospital. RESULTS: The organisms grew slowly. Five strains of C mucifaciens were found in specimens of middle ear effusion (MEE) from patients with otitis media with effusion (OME), and four strains were isolated from the nasal polyps and nasal discharge of patients with chronic sinusitis (CRS). CONCLUSIONS: C mucifaciens was isolated from nine otorhinological specimens. SIGNIFICANCE: This is the first report about the isolation of C mucifaciens from MEE, nasal polyps, and nasal discharge. Although its pathogenetic role is unknown, the characteristics of this microorganism might make it a potential cause of OME and/or CRS, so further studies are necessary. EBM RATING: C-4.

Detection of diarrheagenic viruses from diarrheal fecal samples collected from children in Kathmandu, Nepal.

Diarrhea causing viruses (Rotavirus. Adenovirus and Norovirus) were investigated in diarrheal fecal samples collected from children in Kathmandu, Nepal in Janury 2004 using both real time PCR and immuno-chromatogaphic techniques. Of the total 12 diarrheal samples investigated, 8 (66.7%) were positive for Rotavirus, 1 (8.3%) was positive for Adenovirus and none was positive for Norovirus (Norwalk like virus). The Adenovirus positive sample was also positive for Rotavirus. Similar results were obtained by immuno-chromatographic technique. All of Rotavirus detected belonged to Group-A. Results indicated that immuno-chromatographic technique was equally good in the detection of diarrhea causing viruses in fecal samples. Furthermore, it was simple, cost-effective and less time consuming (15 minutes) compared with the PCR. Immuno-chromatographic technique, therefore, appeared to be useful for rapid diagnosis of viral gastroenteritis in developing countries like Nepal.