Seroprevalence of enterovirus D68 in Yamagata, Japan, between 1976 and 2019.

To clarify the epidemiology of enterovirus D68 (EV-D68), an enterovirus rarely identified in the 20th century, we performed seroepidemiological analysis against EV-D68 using sera collected in 1976, 1985, 1990, 1999, 2009, and 2019, as well as Yamagata isolate (EVD68/Yamagata.JPN/2023-89), in Yamagata, Japan. The neutralizing antibody (Ab)-positive rates for those under 20 years old were 61.0%, 82.5%, 84.3%, 46.7%, 50.5%, and 67.9%, in each year, whereas the rates for those above 20 years old were between 93.4% and 99.1%. Generally, geometric mean titers (GMTs）increased with age among children and the total GMT in each year was 25.4, 49.2, 37.2, 30.8, 29.5, and 33.9, from 1976 to 2019, respectively. The findings in this Yamagata-based study showed that the seroprevalence of EV-D68 over the last four decades has increased with age among children, as a susceptible group, and then reaches a plateau of over approximately 80% among adults. This study clearly revealed that EV-D68 was stably transmitted among children in the 20th century, when EV-D68 detection was quite rare.

Analytical estimation of maximum fraction of infected individuals with one-shot non-pharmaceutical intervention in a hybrid epidemic model.

BACKGROUND: Facing a global epidemic of new infectious diseases such as COVID-19, non-pharmaceutical interventions (NPIs), which reduce transmission rates without medical actions, are being implemented around the world to mitigate spreads. One of the problems in assessing the effects of NPIs is that different NPIs have been implemented at different times based on the situation of each country; therefore, few assumptions can be shared about how the introduction of policies affects the patient population. Mathematical models can contribute to further understanding these phenomena by obtaining analytical solutions as well as numerical simulations. METHODS AND RESULTS: In this study, an NPI was introduced into the SIR model for a conceptual study of infectious diseases under the condition that the transmission rate was reduced to a fixed value only once within a finite time duration, and its effect was analyzed numerically and theoretically. It was analytically shown that the maximum fraction of infected individuals and the final size could be larger if the intervention starts too early. The analytical results also suggested that more individuals may be infected at the peak of the second wave with a stronger intervention. CONCLUSIONS: This study provides quantitative relationship between the strength of a one-shot intervention and the reduction in the number of patients with no approximation. This suggests the importance of the strength and time of NPIs, although detailed studies are necessary for the implementation of NPIs in complicated real-world environments as the model used in this study is based on various simplifications.

A seroepidemiologic study of a measles outbreak, Yamagata Prefecture, Japan, 2017: The estimation of spreaders using serological assays in a measles elimination setting.

INTRODUCTION: In regions where the endemic measles virus has been eliminated, early detection of contagious patients is important for preventing the spread of measles and sustaining elimination. To investigate whether serological assays can be used for the estimation of highly infectious patients with measles, we performed a seroepidemiologic study of a measles outbreak in Yamagata Prefecture, Japan, in 2017. METHODS: We tested plaque reduction neutralization (PRN), IgG avidity, and gelatin particle agglutination (PA) assays in 31 patients with measles, subdivided into two super-spreaders, three spreaders, and 26 non-spreaders. Simultaneously, these results were compared with the cycle threshold (Ct) of a semi-quantitative real-time reverse transcription PCR for the measles virus from throat swab specimens. RESULTS: In the PRN assay, one super-spreader and two spreaders lacked protective antibodies. The IgG avidity assay showed that two super-spreaders and one spreader had low avidity. The PA assay indicated that two super-spreaders and two spreaders lacked protective antibodies. Comparison of the results of the three serological assays and Ct revealed that patients whose antibody titers were judged as low in the IgG avidity and PA assays showed low Ct (i.e., high viral load), whereas non-spreaders tended to show low viral load. CONCLUSIONS: Our preliminary seroepidemiologic analysis of a population of 31 patients with measles suggests that PA and IgG avidity assays may be used for the identification of super-spreader/spreader candidates. However, further investigations are necessary to validate the robustness of these serological assays in detecting contagious measles cases.

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli OX18 from a Fatal Hemolytic Uremic Syndrome Case.

We report a fatal case of hemolytic uremic syndrome with urinary tract infection in Japan caused by Shiga toxin-producing Escherichia coli. We genotypically identified the isolate as OX18:H2. Whole-genome sequencing revealed 3 potentially pathogenic lineages (OX18:H2, H19, and H34) that have been continuously isolated in Japan.

Longitudinal epidemiology of human coronavirus OC43 in Yamagata, Japan, 2010-2017: Two groups based on spike gene appear one after another.

Human coronavirus OC43 (HCoV-OC43) is divided into genotypes A to H based on genetic recombination including the spike (S) gene. To investigate the longitudinal transition of the phylogenetic feature of the HCoV-OC43 S gene in a community, phylogenetic analysis of the S1 region of the S gene was conducted using 208 strains detected in Yamagata during 2010 to 2017 with reference strains of the genotype. The S1 sequences were divisible into four groups: A to D. All Yamagata strains belonged to either group B or group D. In group B, 46 (90.2%) out of 51 Yamagata strains were clustered with those of genotype E reference strains (cluster E). In group D, 28 (17.8%) and 122 (77.7%) out of 157 Yamagata strains were clustered, respectively, with genotype F and genotype G reference strains. In cluster G, 28 strains formed a distinct cluster. Monthly distributions of HCoV-OC43 in Yamagata in 2010 to 2017 revealed that group B and group D appeared one after another. In group B, the cluster E strains were prevalent recurrently. In conclusion, epidemics of HCoV-OC43 in Yamagata, Japan might be attributable to two genetically different groups: group B showed a recurrent epidemic of strains belonging to a single phylogenetic cluster and group D showed epidemic strains belonging to multiple clusters.

Scrub typhus caused by Shimokoshi type Orientia tsutsugamushi showing variant 56-kDa type-specific antigen gene sequence in Tohoku region, Japan.

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56-kDa type-specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011-2012 also involved the same 56-kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.

Mycobacterium tuberculosis Transmission among Elderly Persons, Yamagata Prefecture, Japan, 2009-2015.

In many countries with low to moderate tuberculosis (TB) incidence, cases have shifted to elderly persons. It is unclear, however, whether these cases are associated with recent Mycobacterium tuberculosis transmission or represent reactivation of past disease. During 2009-2015, we performed a population-based TB investigation in Yamagata Prefecture, Japan, using in-depth contact tracing and 24-loci variable-number tandem-repeat typing optimized for Beijing family M. tuberculosis strains. We analyzed 494 strains, of which 387 (78.3%) were derived from elderly patients. Recent transmission with an epidemiologic link was confirmed in 22 clusters (70 cases). In 17 (77.3%) clusters, the source patient was elderly; 11 (64.7%) of the 17 clusters occurred in a hospital or nursing home. In this setting, the increase in TB cases was associated with M. tuberculosis transmissions from elderly persons. Prevention of transmission in places where elderly persons gather will be an effective strategy for decreasing TB incidence among predominantly elderly populations.

[COMPARISON OF QuantiFERON® TB GOLD TEST RESULTS BEFORE AND AFTER ENDOTOXIN CONTAMINATION].

PURPOSE: In response to a case of endotoxin contamination of tubes used in QuantiFERON® TB Gold (QFT-3G) testing in Japan in 2013, the effect of this contamination on QFT-3G test results was investigated. METHODS: We analyzed QFT-3G results from 4,258 participants in a tuberculosis contact investigation in Yamagata, Japan from September 2010 to April 2015. Of these, 2,488 samples were collected before the endotoxin contamination, while 1,770 samples were collected after the contamination. RESULTS: Negative control values in the group tested after the contamination were significantly lower than those in the group tested before the contamination (P < 0.0005). The proportion of positive controls that exceeded the calculated limit (10IU/ml) in the group tested after the contamination (87.8%) was lower than that in the group tested before the contamination (96.8%; P < 0.0005). The proportion of intermediate results in the group tested after the contamination (3.2%) was markedly lower than that in the group tested before the contamination (6.6%). DISCUSSION: Differences in QFT-3G test results were found to be related to a difference in blood collection before or after endotoxin contamination of blood collection tubes. Values resulting from QFT-3G testing were lower in blood samples that were collected after the contamination relative to those collected before the contamination.

Characteristics of Mycoplasma pneumoniae infection identified on culture in a pediatric clinic.

BACKGROUND: The appropriate choice of antibiotics against Mycoplasma pneumoniae infection has become difficult, as the prevalence of macrolide-resistant M. pneumoniae has increased. METHODS: Throat swab specimens were collected from children with clinically suspected M. pneumoniae infection while visiting an outpatient clinic. Cultures for M. pneumoniae were done, and all isolates were sequenced for the presence of a mutation in 23S rRNA. RESULTS: Of the 80 specimens collected between February 2012 and March 2013, 27 (34%) were positive for M. pneumoniae on culture. Macrolide-resistant mutation was detected in 24 isolates (89%): 23 isolates had an A2063G transition, and one had a C2617G mutation. Both the median age and the prevalence of pneumonia were significantly higher in M. pneumoniae-positive than in M. pneumoniae-negative children (median, 7 years vs 4 years; 88.9% vs 60.4%, respectively). The percentage of serum samples with particle agglutination titer ≥ 1:160 was 69.6% in M. pneumoniae-positive cases and 17.6% in M. pneumoniae-negative cases when the serum was collected ≥ 4 days after the onset of fever. Defervescence within 72 h after the initiation of macrolides never occurred in M. pneumoniae-positive children and also did not occur in 54% of M. pneumoniae-negative children. Switching to either minocycline or tosufloxacin resulted in fever resolution within 48 h in M. pneumoniae-positive children. CONCLUSIONS: The described clinical and laboratory characteristics of M. pneumoniae infection may be useful in guiding appropriate treatment in an outpatient clinic.

Leptospirosis in the Tohoku region: re-emerging infectious disease.

Leptospirosis is a zoonotic and disaster-related infectious disease. It is mainly endemic in subtropical or tropical countries and has not been reported since 2009 in the Tohoku region (northern Japan), including the Yamagata and Miyagi Prefectures. However, we experienced four patients with leptospirosis in the Tohoku region from 2012 to 2014; three patients (#1-3) live in the agricultural areas of the Yamagata Prefecture and one patient (#4) was a visitor to the Miyagi Prefecture. Patient 1 (81-year-old female) is a villager, with a rat bite, while Patient 2 (77-year-old male) and Patient 3 (84-year-old female) are farmers and were infected probably during agriculture work. Patient 4 (40-year-old male US citizen) was infected while traveling in Thailand. They had chief complaint of fever, headache, and myalgia and showed manifestations of hyperbilirubinemia (mean, 4.35 mg/dL), thrombocytopenia and acute kidney injury (AKI). All patients were diagnosed by polymerase chain reaction using blood and/or urine samples and a microscopic agglutination test for the anti-Leptospira antibody. All the patients were treated with infused antibiotics, including minocycline. The patients underwent hemodialysis due to severe AKI (mean serum creatinine, 4.44 mg/dL), except for Patient 2 with the normal serum creatinine level (1.12 mg/dL). All the patients recovered and were discharged. The presence of the three patients in the Yamagata Prefecture implies that leptospirosis does re-emerge in the Tohoku region. Therefore, careful survey of the pathogen is necessary for febrile patients with AKI who engage in agriculture or have a recent history of travelling in subtropical or tropical countries.

[Gene Mutations Associated with Macrolide-resistance and p1 Gene Typing of Mycoplasma pneumoniae Isolated in Yamagata, Japan, between 2004 and 2013].

To clarify the epidemiologic features of Mycoplasma pneumoniae, we examined 358 M. pneumoniae strains isolated between 2004 and 2013 in Yamagata, Japan. Analysis of macrolide-resistance-associated 23S ribosomal RNA (rRNA) domain V mutations revealed 6 kinds of mutants (81 A2063G, 43 A2063T, 1 A2063C, 1 A2064C, 4 C2617G and 1 C2617 mutation). There were only two mutants before 2009, but mutants A2063T and A2063G increased in 2009 and from 2010, respectively. The annual ratio of mutants varied from 20.4% to 76.4% between 2009 and 2013. Typing of the p1 gene revealed 4 types; 278 type 1, and 3 kinds of type 2 variant strains (10 type 2a, 5 type 2b and 65 type 2c). Type 1 strains accounted for between 85.2% and 100% of isolates from 2004 to 2011, whereas type 2 variant strains increased by 26.5% and 66.1% in 2012 and 2013, respectively. These results indicate that type 1 strains may have been replaced by type 2 variant strains in 2013. Furthermore, the ratio of type 1 strains with a 23S rRNA mutation was 65.1% in 2012 and 95.2% in 2013, but none of the type 2 variant strains had this mutation. In conclusion, type 1 strains with macrolide-resistant mutations appeared in 2006 and increased from 2009. In contrast, type 2 variant strains, which increased in 2012 and became predominant in 2013, showed no mutations.

A case of pneumonia caused by Legionella pneumophila serogroup 12 and treated successfully with imipenem.

The patient was an 83-year-old man hospitalized for Haemophilus influenzae pneumonia, who developed recurrent pneumonia after improvement of the initial episode. Legionella pneumophila serogroup 12 was isolated from the sputum, accompanied by increased serum antibody titers to L. pneumophila serogroup 12. Therefore, the patient was diagnosed as having Legionella pneumonia caused by L. pneumophila serogroup 12. Case reports of pneumonia caused by L. pneumophila serogroup 12 are rare, and the case described herein is the first report of clinical isolation of this organism in Japan. When the genotype was determined by the protocol of The European Working Group for Legionella Infections (Sequence-Based Typing [SBT] for epidemiological typing of L. pneumophila, Version 3.1), the sequence type was ST68. Imipenem/cilastatin therapy was found to be effective for the treatment of Legionella pneumonia in this patient.

[Effectiveness of interferon-gamma release assays in the tuberculosis contact investigation of elderly people].

PURPOSE: To confirm the effectiveness of interferon-gamma release assays (IGRAs) in the tuberculosis (TB) contact investigation of elderly people, we analyzed the results of the QuantiFERON TB Gold in tube (QFT-3G) test, which is a commercially available IGRA. METHODS: We analyzed the results of the QFT-3G test in 2,420 subjects who were in close contact with TB patients. We investigated subjects with latent TB infection and those showing the onset of TB among the QFT-3G-positive subjects. RESULTS: The QFT-3G-positive rate was 7.3% (95% confidence interval, 6.2%-8.3%). In addition, we demonstrated that the QFT-3G-positive rate increased with age (P < 0.001). DISCUSSION: The QFT-3G-positive rate was high, particularly in elderly people (> or = 60 years), but the rate was significantly lower than the predicted prevalence of TB infection. Therefore, it was assumed that the QFT-3G test does not always provide a positive result, even in cases of subjects with a previous TB infection. Furthermore, data from the QFT-3G-positive subjects indicated that approximately one half of subjects aged 60-69 years, approximately one-third of those aged 70-79 years, and approximately one-quarter of those aged over 80 years have had recent TB infections. In conclusion, the results of the QFT-3G test in elderly people need to be carefully evaluated according to the contact situation with TB patients; nevertheless, the QFT-3G test is useful for the screening of latent TB infection in elderly people who were in close contact with TB patients.

A Case of Legionella pneumophila Serogroup 13 Pneumonia Based on the Detection of Serogroup-Specific Genes in Culture-Negative Sputum.

Legionella pneumophila serogroup (SG) 1, the main cause of Legionnaires' disease, can be diagnosed using urinary antigen testing kits. However, lower respiratory tract specimen cultures are required to identify L. pneumophila SG 2-15. We attempted to detect L. pneumophila SG-specific genes in a culture-negative sputum specimen from a patient with pneumonia who was suspected to have Legionnaires' disease. Two multiplex PCR methods targeting L. pneumophila were modified and amplicons considered to be SG13 specific were detected. Direct sequencing revealed that the amplicons were identical to the nucleotide sequence of L. pneumophila SG13. Based on the presentation and clinical course (fever, muscle pain, disturbance of consciousness, high C-reactive protein titer, rhabdomyolysis, hypophosphatemia, and symptomatic improvement with levofloxacin treatment), in combination with the detection of L. pneumophila SG-specific genes, we suspected L. pneumophila SG13 pneumonia. L. pneumophila non-SG1 pneumonia is thought to be underestimated because of its difficult laboratory diagnosis. The modified multiplex PCR system for lower respiratory tract specimens revealed in this study is likely to improve the diagnosis of Legionnaires' disease caused by L. pneumophila SG13 and other SGs.

A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan.

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi.

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real-time PCR targeting the 16S ribosomal RNA gene, serotype-specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.

[Effectiveness of comprehensive variable number of tandem repeat (VNTR) analysis in areas with a low incidence of tuberculosis].

PURPOSE: To ascertain the effectiveness of variable number of tandem repeat (VNTR) analysis in areas with a low incidence of tuberculosis (TB), we examined the combination of comprehensive VNTR analyses and field epidemiological investigation results in Yamagata Prefecture, Japan, where estimated incidence of new TB cases per 100,000 population was 11.3 in 2011. METHODS: We collected Mycobacterium tuberculosis isolates from 184 (69.2%) of 266 pulmonary TB patients across the whole of Yamagata Prefecture between 2009 and 2011. Next, 24 loci [JATA (12), QUB-11a, ETR A, QUB-18, QUB3232, v3820, v4120, MIRU04, MIRU16, MIRU40, ETR C, Mtub30, Mtub39] in VNTR genotypes were determined. The relationships among TB patients derived from the respective clusters were surveyed using field epidemiological investigation results provided by the Public Health Center. RESULTS: Seventeen clusters were formed by 49 (26.6%) of the 184 isolates. We found 3 hospital infection cases, 3 family infection cases, and 1 nursing home infection case forming 6 clusters. Among these cases, two relationships among patients were revealed after additional epidemiological investigation at the Public Health Center. The VNTR pattern of the largest cluster, which was formed by 12 isolates, was identical with that of an incipient patient of a TB mass infection that occurred in 2007. DISCUSSION: In areas with a low incidence of TB, a combination of comprehensive VNTR analysis and field epidemiological investigation is useful to find unknown transmission routes, identify for new risk groups, and trace mass infections.

Measles Outbreak Response Activity in Japan, and a Discussion for a Possible Strategy of Outbreak Response Using Cycle Threshold Values of Real-Time Reverse Transcription PCR for Measles Virus in Measles Elimination Settings.

Measles is a highly contagious, but vaccine-preventable disease caused by the measles virus (MeV). Although the administration of two doses of measles vaccines is the most effective strategy to prevent and eliminate measles, MeV continues to spread worldwide, even in 2022. In measles-eliminated countries, preparedness and response to measles outbreaks originating from imported cases are required to maintain elimination status. Under these circumstances, real-time reverse transcription (RT) PCR for MeV could provide a diagnostic method capable of strengthening the subnational capacity for outbreak responses. Real-time RT-PCR can detect MeV RNA from patients with measles at the initial symptomatic stage, which can enable rapid public health responses aimed at detecting their contacts and common sources of infection. Furthermore, low cycle threshold (Ct) values (i.e., high viral load) of throat swabs indicate high infectiousness in patients with measles. The high basic reproduction number of measles suggests that patients with high infectiousness can easily become super-spreaders. This opinion proposes a possible strategy of rapid and intensive responses to counter measles outbreaks caused by super-spreader candidates showing low Ct values in throat swabs. Our strategy would make it possible to effectively prevent further measles transmission, thereby leading to the early termination of measles outbreaks.

Another advantage of multi-locus variable-number tandem repeat analysis that can putatively subdivide enterohemorrhagic Escherichia coli O157 strains into clades by maximum a posteriori estimation.

Enterohemorrhagic Escherichia coli O157 (O157) strains can be subdivided into clades based on their single-nucleotide polymorphisms, but such analysis using conventional methods requires intense effort by laboratories. Although multi-locus variable-number tandem repeat analysis (MLVA), which can be performed with low laboratory burden, has been used as a molecular epidemiological tool, it has not been evaluated whether MLVA can be used the clade subdivision of O157 strains like it can for that of other pathogenic bacteria. This study aimed to establish a method for subdividing O157 strains into clades using MLVA data. The standardized index of association, ISA, for O157 strains isolated in Chiba prefecture, Japan (Chiba isolates) revealed the presence of unique tandem repeat patterns in each major clade (clades 2, 3, 7, 8, and 12). A likelihood database of tandem repeats for these clades was then constructed using the Chiba isolates, and a formula for maximum a posteriori (MAP) estimation was constructed. The ratio of the number of O157 strains putatively subdivided into a clade by MAP estimation from MLVA data relative to the number of O157 strains subdivided using single-nucleotide polymorphism analysis (designated as the concordance ratio [CR]) was calculated using the Chiba isolates and O157 strains isolated in Yamagata prefecture (Yamagata isolates). The CRs for the major Chiba and Yamagata isolate clades, other than clade 2, were 89%-100%. Although the CR for clade 2 Chiba isolates was >95%, that of the Yamagata isolates was only 78.9%. However, these clade 2 CRs were not significantly different from one another, indicating that clade 2 strains can be subdivided correctly by MAP estimation. In conclusion, this study expands the utility of MLVA, previously applied predominantly for molecular epidemiological analysis, into a low-laboratory-burden tool for subdividing O157 strains into phylogenetic groups.