Transmission of SARS-CoV-2 during a 2-h domestic flight to Okinawa, Japan, March 2020.

BACKGROUND: Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has rapidly spread globally. Potentially infected individuals travel on commercial aircraft. Thus, this study aimed to investigate and test the association between the use of face masks, physical distance, and COVID-19 among passengers and flight attendants exposed to a COVID-19 passenger in a domestic flight. METHODS: This observational study investigated passengers and flight attendants exposed to COVID-19 on March 23, 2020, on board a flight to Naha City, Japan. Secondary attack rates were calculated. Whole-genome sequencing of SARS-CoV-2 was used to identify the infectious linkage between confirmed cases in this clustering. The association between confirmed COVID-19 and proximity of passengers' seats to the index case and/or the use of face masks was estimated using logistic regression. RESULTS: Fourteen confirmed and six probable cases were identified among passengers and flight attendants. The secondary attack rate was 9.7%. Twelve of 14 SARS-CoV-2 genome sequences in confirmed cases were identical to that of the index case or showed only one nucleotide mutation. Risk factors for infection included not using a face mask (adjusted odds ratio [aOR]: 7.29, 95% confidence interval [95% CI]: 1.86-28.6), partial face mask use (aOR: 3.0, 95% CI: 0.83-10.8), and being seated within two rows from the index patient (aOR: 7.47, 95% CI: 2.06-27.2). CONCLUSION: SARS-CoV-2 was transmitted on the airplane. Nonuse of face masks was identified as an independent risk factor for contracting COVID-19 on the airplane.

Novel genotypes of Leptospira interrogans serogroup Sejroe isolated from human patients in Okinawa Prefecture, Japan.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of Leptospira species. It is a public health issue in the tropics, including Okinawa, the southernmost prefecture of Japan. This study reports the first isolation of L. interrogans serogroup Sejroe from two human patients in Japan, and describes its molecular characterization using multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). MLST on the two isolates, 168036 and 178129, showed that pfkB in 178129 is a novel allele, and that both isolates constitute novel sequence types (STs); ST286 for 168036 and ST287 for 178129. A minimum spanning tree based on seven alleles of L. interrogans indicates that both isolates are genetically close, but are distinct from known L. interrogans serogroup Sejroe strains. MLVA using 11 loci demonstrated that seven of the 11 loci were identical between the two isolates, whereas the identity between the isolates and the seven reference strains of L. interrogans serogroup Sejroe was zero to three loci. These results indicate that the isolates investigated in this study have novel genotypes, and are genetically closest to each other among the known L. interrogans serogroup Sejroe strains.

Emergent measles-containing vaccination recommendation for aged 6-11 months and detection of vaccine-associated measles during a large measles outbreak in Okinawa, Japan, in 2018.

Okinawa Prefecture, Japan, experienced a large measles outbreak from March to May 2018. During this outbreak, there were 99 laboratory-confirmed cases and 14 vaccine-associated measles cases. In addition to the reinforcement of routine immunization, Okinawa prefectural government introduced emergent measles-containing vaccination recommendations for infants aged 6-11 months as part of the outbreak response. Increased concern exists in Okinawa about measles in infants following a previous outbreak from 1998 to 2001, when nine children including four infants died. Of 8062 infants aged 6-11 months who received measles-containing vaccine (MCV), six developed vaccine-associated measles; incidence was 0.74 per 1000 doses (95%CI 0.27-1.62). This was similar to that of first dose routine immunization recipients at one year of age (IR 0.60, 95%CI 0.20-1.78). Among 14 vaccine-associated measles cases, throat swab samples showed the highest positive rate (92.9%) by real-time reverse transcription polymerase chain reaction (RT-qPCR), followed by urine (25.0%) and whole blood (7.7%) samples. Furthermore, one throat swab sample classified as equivocal by RT-qPCR was positive by conventional RT-PCR (RT-PCR). During an outbreak, it is critical to distinguish between cases with measles-like symptoms caused by wild circulating virus and those caused by vaccine-derived virus as accurately and urgently as possible because the public health response will be quite different. No infant deaths were observed during this outbreak, and no severe adverse events following immunization were seen among infants 6-11 months old who were given MCV as a public health response. Thus, we conclude that introduction of emergent MCV was effective and describing the characteristics of vaccine-associated measles cases during a measles outbreak will be helpful for future outbreak response efforts.

The Protobothrops flavoviridis Hemorrhagic Metalloproteinase HR2 Is Inhibited by Human Alpha 2-Macroglobulin.

Vaccinations with habu (Protobothrops flavoviridis) venom toxoid were administered to individuals living in Amami Oshima from 1965 to 2002, and its effectiveness was investigated in 1991. The results raised the possibility that normal human serum inherently contains an inhibitor of the hemorrhagic metalloproteinase HR2, considered to be one of the major components of habu venom. In this study, we investigated the interaction between the hemorrhagic metalloproteinases HR1 and HR2 from habu-venom and human alpha 2-macroglobulin (α2M). Hemorrhagic activity of HR2 was completely inhibited by human α2M. However, the hemorrhagic activity of the large molecule HR1a was not inhibited. Size exclusion chromatography revealed that human α2M captured the HR2 molecule and formed a complex with it, thus inhibiting hemorrhagic activity. These results suggest that human α2M plays an important role in the inhibition of hemorrhage induced by HR2 from habu venom.

Molecular Epidemiological Study of Mumps Epidemics of 2015 in Okinawa, Japan.

Although major mumps epidemics occurred every 4-5 years in Okinawa Prefecture in Japan, no laboratory diagnoses were conducted. A mumps epidemic started in Okinawa in October 2014, and we collected clinical samples from 31 patients in 4 areas (Hokubu, Nanbu, Miyako, and Yaeyama) from July to December 2015, for virus isolation and RT-PCR, whose positive ratios were 52% and 87%, respectively. Phylogenetic analyses showed that all isolates were classified into genotype G, and with one exception, consisted of 2 subgenotypes, Ge (55.6%) and Gw (40.7%), which have been prominent in Japan recently. One isolate was classified in another lineage, which was detected in Japan for the first time, and was similar to a Hong Kong isolate from 2014. Remarkably, the geographic distributions of the 2 major lineages were separated. The Ge viruses were isolated from the main island of Okinawa and the Yaeyama Islands, whereas the Gw isolates were mainly detected from the Miyako Islands. These results suggest that the Ge and Gw mumps viruses mainly caused the mumps epidemics of 2015 in Okinawa, and that they spread independently in separate regions. This is the first report describing the molecular epidemiology of mumps epidemics in Okinawa Prefecture.

Molecular evolution of VP3, VP1, 3C(pro) and 3D(pol) coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa, Japan.

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C(pro) and 3D(pol) coding regions performed. To assess time-scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p-distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10(-3) substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77-0.94. The p-distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. METHODS: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. RESULTS: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. CONCLUSIONS: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.

Detection and phylogenetic analysis of hepatitis E viruses from mongooses in Okinawa, Japan.

Hepatitis E virus (HEV) infection has previously been reported in wild mongooses on Okinawa Island; to date however, only one HEV RNA sequence has been identified in a mongoose. Hence, this study was performed to detect HEV RNA in 209 wild mongooses on Okinawa Island. Six (2.9%) samples tested positive for HEV RNA. Phylogenetic analysis revealed that 6 HEV RNAs belonged to genotype 3 and were classified into groups A and B. In group B, mongoose-derived HEV sequences were very similar to mongoose HEV previously detected on Okinawa Island, as well as to those of a pig. This investigation emphasized the possibility that the mongoose is a reservoir animal for HEV on Okinawa Island.

Molecular epidemiology of human metapneumovirus from 2009 to 2011 in Okinawa, Japan.

To clarify the molecular epidemiology of human metapneumovirus (HMPV) in Okinawa Prefecture, located in a subtropical region of Japan, we performed genetic analysis of the F gene in HMPV from patients with acute respiratory infection from January 2009 to December 2011. HMPV was detected in 18 of 485 throat swabs (3.7%). Phylogenetic analysis showed that 17 strains belonged to subgroup A2 and 1 strain belonged to subgroup B1. We did not observe seasonal prevalence of HMPV during the investigation period. A high level of sequence identity was observed in the strains belonging to subgroup A2 (>95%), and no amino acid substitution was found compared with other strains detected in Japan and other countries. The pairwise distance values among the present strains belonging to subgroup A2 were short. Our results suggest that the predominant HMPV strains belonging to A2 are highly homologous and seasonal epidemics were not seen in Okinawa during the investigation period.

Evaluation of the petrifilm aerobic count plate for enumeration of aerobic marine bacteria from seawater and Caulerpa lentillifera.

The enumeration and evaluation of the activity of marine bacteria are important in the food industry. However, detection of marine bacteria in seawater or seafood has not been easy. The Petrifilm aerobic count plate (ACP) is a ready-to-use alternative to the traditional enumeration media used for bacteria associated with food. The purpose of this study was to evaluate the usefulness of a simple detection and enumeration method utilizing the Petrifilm ACP for enumeration of aerobic marine bacteria from seawater and an edible seaweed, Caulerpa lentillifera. The efficiency of enumeration of total aerobic marine bacteria on Petrifilm ACP was compared with that using the spread plate method on marine agar with 80 seawater and 64 C. lentillifera samples. With sterile seawater as the diluent, a close correlation was observed between the method utilizing Petrifilm ACP and that utilizing the conventional marine agar (r=0.98 for seawater and 0.91 for C. lentillifera). The Petrifilm ACP method was simpler and less time-consuming than the conventional method. These results indicate that Petrifilm ACP is a suitable alternative to conventional marine agar for enumeration of marine microorganisms in seawater and C. lentillifera samples.

[Antimicrobial susceptibility and resistance genes among Salmonella enterica Serovar Weltevreden isolates from humans, farm animals, and the environment in Okinawa Prefecture between 1992 and 2007].

Between 1992 and 2007, a total of 86 isolates of Salmonella enterica Weltevreden were obtained from clinical human samples (n = 41), 45 farm animals and their environment on 20 farms, including poultry (n = 25), beef cattle (n = 5), swine (n = 5), dairy cattle (n = 3), mice (n = 2), pony (n = 1), fly (n = 1) and feed samples (n = 3), in Okinawa Prefecture, Japan. Only seven isolates (8.1%) of the isolates were resistance to one or more antimicrobial agents tested; six streptomycin (7.0%), six oxytetracycline (7.0%), two ampicillin (2.3%), two kanamycin, (2.3%), two chloramphenicol (2.3%), two suffamethoxazole/trimethoprim (2.3%), whereas all isolates were susceptible to gentamicin, sefuroxime, colistin, nalidixic acid, fosfomycin and ofloxacin. Drug resistance patterns showed six patterns; ABPC-SM-KM-OTC-CP-ST, ABPC-SM-ST, SM-KM-OTC, SM-OTC-CP, SM-OTC, OTC. Two ampicillin-resistant isolates harbored the blaTEM genes, streptomycin-resistant isolates (four aadA, two strA), tetracycline-resistant isolates (two tetA, three tetB), chloramphenicol-resistant isolates (two catA1), respectively.

Survey of Japanese encephalitis virus in pigs on Miyako, Ishigaki, Kume, and Yonaguni Islands in Okinawa, Japan.

Serum specimens were collected from 125 pigs on Miyako Island, 112 pigs on Ishigaki Island, and 42 pigs on Kume Island from 2005 to 2007, and 54 pigs on Yonaguni Island from 2006 to 2007. Their sera were tested for Japanese encephalitis virus (JEV) antibody by hemagglutination inhibition (HI) assay. Five serum samples (4.5%) from Ishigaki Island were positive for HI antibody, and 4 of the 5 samples were positive for 2-mercaptoethanol- sensitive antibody (IgM Ab). All samples from Miyako, Kume, and Yonaguni Islands were negative for HI antibody. Our results indicate that JEV transmission activity was extremely low on Miyako, Ishigaki, Kume, and Yonaguni Islands. The JEV genome (JEV-RNA) was detected from the sera of one pig on Ishigaki Island. The partial gene of the E region (151 nt) was analyzed phylogenetically. The analysis showed that the new JEV-RNA belonged to genotype 3 and was closely related to JEV strains isolated in Taiwan from 1985 to 1996. It was suggested that JEV previously introduced from Taiwan had been maintained on Ishigaki Island.

[Investigation and culture of microbial contaminants of Caulerpa lentillifera (Sea Grape)].

Caulerpa lentillifera is a kind of edible seaweed, known as 'sea grape' or 'green caviar'. It is used in fresh salads. However, it is sensitive to low temperature and osmotic pressure, and is easily spoilt by storage in a refrigerator or washing with tap water. That is the reason why it is difficult to prevent food poisoning, especially due to Vibrio parahaemolyticus. In this study we investigated of marine bacteria and V. parahaemolyticus in C. lentillifera and cultured them in order to develop effective control of bacteria in commercial farms. The sixteen farms in the Okinawa Islands were investigated from August to September in 2006. A total of 176 samples were collected from eleven points during the cultivation processes and from the products. About 10(3) cfu/mL of marine bacteria were detected in the seawater used in the tank culture, but after cultivation of C. lentillifera the number had increased to about 10(6) cfu/mL. The number of marine bacteria in C. lentillifera did not change significantly through the process of planting to the final product (about 10(7) cfu/g). V. parahaemolyticus was detected in seawater from all processes and C. lentillifera was isolated from 56% of seawater, 25% of seed-stocks, and 18.8% of product samples, though but thermostable direct hemolysin gene was not detected from enrichment cultures or isolated V. parahaemolyticus strains. These results indicate that for prevention of food poisoning by V. parahaemolyticus in C. lentillifera, it is important to establish a suitable sterilization procedure for each process.

Survey of the antibody against japanese encephalitis virus in Ryukyu wild boars (Sus scrofa riukiuanus) in Okinawa, Japan.

Serum specimens were collected from 99 wild boars in the Northern area of the main Okinawa Island and from 27 wild boars on Iriomote Island in Okinawa Prefecture from 1997 to 2005. Sera were tested for Japanese encephalitis virus (JEV) antibody by hemagglutination inhibition assay and IgG enzyme-linked immunosorbent assay. Sixty-four samples (64.6%) in the Northern area and 1 sample (3.7%) from Iriomote Island were positive for the JEV antibody. The difference in seroprevalence between the Northern area and Iriomote Island was statistically significant (P < 0.01, chi2 test). This difference may be due to the lack of a pig farm on Iriomote Island, whereas wild boars in the Northern area may be infected with JEV, amplified on pig farms. It is likely that there has recently been an increase in the number of wild boars living close to humans in certain areas of Japan. This in turn increases the possibility that wild boars are infected with JEV, which is amplified on pig farms, and these infected animals may play a role in carrying JEV to other regions of the country.