Longitudinal antigenic and seroepidemiological analyses of parechovirus A1 in Yamagata, Japan.

To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and PeVA1/Yamagata.JPN/2021-4785, isolated in 2021, using immune sera and 207 and 237 human serum specimens collected in 2021 and 1976, respectively. Although rabbit immune sera showed the highest neutralization antibody (NT-Ab) titers against the immunized viruses at 1:12 800-1:102 400, they were cross-reactive at 1:400-1:800. All 62 Yamagata isolates obtained between 2001 and 2021 (Yamagata strains), belonging to phylogenetic lineage 1B, reacted more strongly (mostly 4-64 times) to antiserum against PeVA1/Yamagata.JPN/2021-4785 than to antiserum against Harris, belonging to phylogenetic lineage 1 A. Human serum specimens obtained in 2021 showed higher NT-Ab titers against PeVA1/Yamagata.JPN/2021-4785, whereas those obtained in 1976 had similar NT-Ab titers against both strains. These findings suggested that Yamagata strains and Harris were antigenically cross-reactive, although there were differences. There are still high NT-Abs titers present against Harris in 2021 in particular, indicating that PeVA1 has been in circulation with high immunity in the population. In conclusion, this study suggested that PeVA1 has been endemically perpetuated with only minor antigenic changes as well as with high immunity over several decades in the community.

Seroprevalence of coxsackievirus A21 neutralizing antibodies in Yamagata, Japan, between 1976 and 2019; coxsackievirus A21 has rarely affected young children.

Although coxsackievirus A21 (CV-A21) has been associated with an acute respiratory infection (ARI) as well as poliomyelitis-like paralysis, reports of CV-A21 detection have been quite limited both globally and in Japan. CV-A21 strains were isolated from five sporadic pediatric cases with ARI in 2019 in Yamagata, Japan. Neutralizing antibodies (NT Abs) were then measured against CV-A21 using sera collected in 1976, 1985, 1999, 2009, and 2019 in Yamagata, to clarify the longitudinal epidemiology of CV-A21. The total Ab-positive rate in each year was 15.2% (35/233), 10.7% (30/281), 14.3% (28/196), 3.1% (7/236), and 1.3% (3/226), respectively. Ab-positive rates generally increased with age, especially between 1976 and 1999. Among the total Ab-positive cases, the Ab titers were relatively low; 50 cases belonged to the 1:8-1:16, 40 to 1:32-1:64, 12 to 1:128-1:256, and 1 to 1:1024< groups, respectively. No Ab-positive cases under the age of 10 were observed in any of the years analyzed. In conclusion, this study and previous works suggested that CV-A21 is a unique enterovirus, which is not transmitted readily among young children but causes sporadic ARI cases mainly among those ≥15 years of age in the community.

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.

Detection of Saffold viruses from children with acute respiratory infections in Yamagata, Japan, between 2008 and 2015.

Although Saffold virus (SAFV) was reported as a novel human cardiovirus in 2007, no causative association between SAFV and clinical disease has been proven and the longitudinal epidemiology of SAFVs is not available. To establish the relationship between SAFVs and acute respiratory infections (ARIs) and to clarify the longitudinal epidemiology of SAFVs, 7258 nasopharyngeal specimens were collected from children with ARIs in Yamagata, Japan between 2008 and 2015. The specimens were inoculated on a microplate including six cell lines as part of routine surveillance, and molecular screening was performed for SAFVs using a reverse transcription (RT)-PCR method. Throughout the study period, 95 (1.3%) SAFV genotype 2 (SAFV2), and 28 (0.4%) SAFV3 were detected, mainly between September and November. There were two outbreaks of SAFV2 in 2009 and 2013, and one outbreak of SAFV3 in 2012 and the positive rates during these outbreaks were 12.1% (53/439), 11% (35/319), and 4.4% (20/453), respectively. Sixty-three SAFV2 and 28 SAFV3 strains were detected as a single virus from children with ARIs such as pharyngitis, herpangina, and tonsillitis. These results suggested that SAFV2 and SAFV3 are possible causative agents of ARIs among children and their infections occur mainly in the autumn season in Japan.

Seroepidemiology of human parechovirus types 1, 3, and 6 in Yamagata, Japan, in 2014.

To clarify the seroepidemiology of human parechovirus type 1 (HPeV1), 3 and 6, neutralizing antibodies (NT Abs) were measured in 214 serum specimens collected in 2014 in Yamagata, Japan. The seroprevalence against HPeV1 was 100% in all age groups, while that against HPeV3 and HPeV6 was 79.4% and 66.8%, respectively, overall. The geometric mean titers of NT Abs against HPeV1, 3 and 6 were 755.2, 255.0 and 55.9, respectively, overall. Our findings indicate that HPeV1 is the most prevalent HPeV circulating in Yamagata, followed by HPeV3 and HPeV6.

[Evaluation of a Rapid Antigen Detection Kit Targeting L7/L12 Ribosomal Protein for Mycoplasma pneumoniae].

We evaluated the usefulness of a rapid antigen detection assay for L7/L12 ribosomal protein (Ribotest Mycoplasma; Asahi Kasei Pharma) for diagnosis of Mycoplasma pneumoniae (M. pneumoniae) infection. Nasopharyngeal swabs were obtained from patients with pneumonia and/or bronchitis; real-time PCR and the L 7/L12 antigen assays were performed with each sample. Serum was also taken from each patient, and the particle agglutination (PA) method was used to detect anti-M. pneumoniae antibody in these samples. Macrolide-resistance genes were detected and M. pneumoniae P1 protein subtyping was performed on PCR-positive samples. PCR assays were positive for 85 of 212 specimens (40.1%). Sensitivity and specificity of the L7/L12 antigen assays relative to the PCR standard were 74.1% (63/85) and 81.1% (103/127), respectively. For PCR-positive specimens with a large quantity of M. pneumoniae nucleic acid, sensitivity of the L7/L12 antigen assays seemed to be high. In PCR-positive specimens with fewer than 1.0 x 10(6) copies/mL of M. pneumoniae nucleic acid, sensitivity of the L7/L12 antigen assays seemed to be low. When the PA method was used as the standard, the relative sensitivity and specificity of the L7/L12 antigen assays were 41.7% (5/12) and 75.3% (58/77), respectively, for single serum and 60.9% (14/23) and 85.7% (18/21), respectively, for paired sera. The macrolide-resistance gene A2063G was detected in 20 of the 30 tested PCR-positive specimens (66.7%). Of these 20 A2063G-positive specimens, 13 (65.0%) were positive for the L7/L12 antigen assays. Tne numbers of M. pneumoniae P1 subtypes were as follows: types I (22), IIa(2), IIc(1), and untypable (5). The L7/L12 antigen assays gave positive results for 17 of 21 (81.0%) subtype I, 1 of 2 (50.0%) IIa, and 1 of 1(100%) IIc specimens.

Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71.

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.

Molecular epidemiology of Coxsackievirus A16 strains isolated from children in Yamagata, Japan between 1988 and 2011.

To clarify the longitudinal molecular epidemiology of coxsackievirus A16, phylogenetic analysis based on the VP1 region of 220 isolates in Yamagata, Japan was performed. The resultant phylogenetic tree indicates that the Yamagata isolates and reference strains can be readily genotyped into three genogroups, and 0, 12 and 208 isolates belonged to the first, second, and third genogroups, respectively. The first genogroup includes only the prototype strain, the second strains that had disappeared by the end of the 20th century and the third comprises those that have been circulating since then in local communities, such as Yamagata.

Isolation of vaccine-derived measles viruses from children with acute respiratory infection.

The measles elimination project led by the World Health Organization (WHO) has been moving toward the target of eliminating measles in the WHO Western Pacific Region. In Japan, prefectural public health institutes play a key role for the laboratory diagnosis of measles virus (MV) infection, which is based on PCR, virus isolation, and genotyping. Microscopic examination of viral-sensitive cell lines during routine virus isolation from nasopharyngeal specimens has been used to detect the morphological changes typical for the growth of respiratory viruses. Here, we describe the unexpected isolation of vaccine-derived MVs from the two unrelated 1-year-old boys with acute respiratory infection. The nasopharyngeal specimens were obtained from one patient in February 2007 and from another in December 2012. Incidentally, the two children had received measles-rubella vaccination 9 or 11 days before the sampling. The isolates from two children induced morphological changes of the viral-sensitive cell lines, such as syncythia formation (cell fusion). We finally identified the isolates as vaccine-derived MVs by sequence analysis and immunological methods with anti-measles nucleoprotein antibodies. As no typical symptoms of MV infection were observed in either patient, the vaccine-derived MVs were isolated not as causative pathogens but by chance. In fact, there was no suspected case of secondary MV infection in either patient, thereby excluding the possibility that vaccine-derived MVs spread from human to human. Our experiences suggest the possibility of vaccine-derived MV isolation by cell cultures and the difficulty in identifying MVs in specimens from patients other than clinically suspected measles cases.

Epidemiology of parainfluenza virus types 1, 2 and 3 infections based on virus isolation between 2002 and 2011 in Yamagata, Japan.

To clarify the epidemiology of viral acute respiratory infections (ARIs), 305 human parainfluenza virus types 1 (HPIV1), 154 HPIV2 and 574 HPIV3 strains were isolated from 16,962 nasopharyngeal swabs obtained between 2002 and 2011 at pediatric clinics in Yamagata, Japan. The total isolation frequency for HPIV1-3 was 6.1%. Unlike HPIV1 infections, HPIV3 showed clear seasonality with yearly outbreaks in the spring-summer season. HPIV2 tended to appear biannually in autumn-winter. Although no reliable techniques for the laboratory diagnosis of these infections have been established, the present results suggest that HPIV1-3 are an important causative agent of ARIs in children.

Acute respiratory infections due to enterovirus 68 in Yamagata, Japan between 2005 and 2010.

To clarify the epidemiology of enterovirus 68 (EV68), which is one of the most rarely identified enteroviruses, virus isolation and molecular screening using RT-PCR was performed on 6307 respiratory specimens collected at pediatric clinics in Yamagata, Japan between 2005 and 2010. In the years 2005-2009, 10, 1, 2, 0, and 2 (40) EV68-positive cases, respectively, were identified by RT-PCR. In 2010, 40 cases were identified altogether: 2 by isolation only, 26 by RT-PCR only, and 12 by both isolation and RT-PCR. Phylogenetic analysis indicated that plural genetically distinct clusters co-circulated. These results suggest that that difficulty in EV68 isolation leads to an underestimation of the prevalence of EV68 infections.

Recombinant parechovirus A3 possibly causes various clinical manifestations, including myalgia; findings in Yamagata, Japan in 2019.

BACKGROUND: Parechovirus A3 was first reported in 2004 and has been recognized as a causative agent of mild and severe infections in children. Since we first reported an outbreak of adult parechovirus A3-associated myalgia in Yamagata, Japan in 2008, this disease has since been recognized across Japan, but has not yet been reported from other countries. AIM: We analysed 19 cases of parechovirus A3 infections identified in Yamagata in 2019 to further clarify the epidemiology of this disease. METHODS: We performed phylogenetic analyses of parechovirus A3 isolates and analysed the clinical manifestations and the genomic clusters. RESULTS: There were two clusters, with cluster 2019B replacing 2019 A around October/November. Phylogenetic analysis revealed that 2019B cluster strains and Australian recombinant strains, which appeared between 2012 and 2013, were grouped in one cluster at non-structural protein regions, suggesting that the ancestor to these regions of 2019B cluster strains were Australian recombinant lineage strains. The strains from both clusters caused various infections in children including myalgia. These findings strongly support that parechovirus A3 strains cause myalgia and other paediatric infections irrespective of the virus strains involved, including recombinant strains.　　. CONCLUSIONS: We have reported repeatedly sporadic cases of myalgia and here showed that recombinant strains also cause myalgia. We hope our experiences will help better understand these infections and possibly result in detection of more cases in the world.

Detailed genetic analysis of hemagglutinin-neuraminidase glycoprotein gene in human parainfluenza virus type 1 isolates from patients with acute respiratory infection between 2002 and 2009 in Yamagata prefecture, Japan.

BACKGROUND: Human parainfluenza virus type 1 (HPIV1) causes various acute respiratory infections (ARI). Hemagglutinin-neuraminidase (HN) glycoprotein of HPIV1 is a major antigen. However, the molecular epidemiology and genetic characteristics of such ARI are not exactly known. Recent studies suggested that a phylogenetic analysis tool, namely the maximum likelihood (ML) method, may be applied to estimate the evolutionary time scale of various viruses. Thus, we conducted detailed genetic analyses including homology analysis, phylogenetic analysis (using both the neighbor joining (NJ) and ML methods), and analysis of the pairwise distances of HN gene in HPIV1 isolated from patients with ARI in Yamagata prefecture, Japan. RESULTS: A few substitutions of nucleotides in the second binding site of HN gene were observed among the present isolates. The strains were classified into two major clusters in the phylogenetic tree by the NJ method. Another phylogenetic tree constructed by the ML method showed that the strains diversified in the late 1980s. No positively selected sites were found in the present strains. Moreover, the pairwise distance among the present isolates was relatively short. CONCLUSIONS: The evolution of HN gene in the present HPIV1 isolates was relatively slow. The ML method may be a useful phylogenetic method to estimate the evolutionary time scale of HPIV and other viruses.

Isolation of Human Coronaviruses OC43, HKU1, NL63, and 229E in Yamagata, Japan, Using Primary Human Airway Epithelium Cells Cultured by Employing an Air-Liquid Interface Culture.

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.

A two-year survey of the oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and the clinical effectiveness of oseltamivir and zanamivir.

BACKGROUND: Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir. RESULTS: Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356). CONCLUSION: Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.

Comparison of virus isolation using the Vero E6 cell line with real-time RT-PCR assay for the detection of human metapneumovirus.

BACKGROUND: The use of cell culture for the diagnosis of human metapneumovirus (hMPV) infection is uncommon at present and molecular method such as reverse-transcription PCR (RT-PCR) has been widely and most commonly used as the preferred test. We aimed to compare the results of virus isolation using Vero E6 cells with real-time RT-PCR for the detection of hMPV, since such a comparison data is not available. METHODS: Between December 2007 and July 2008, we obtained 224 nasopharyngeal swab specimens from patients with acute respiratory infection and tested by the two methods. RESULTS: Forty-three (19.2%) were found positive by cell culture and 62 (27.7%) by real-time RT-PCR. Cell cultures were positive for 42 of 62 specimens found positive by real-time RT-PCR (67.7% sensitivity) and for 1 of 162 specimens found negative by real-time RT-PCR (99.4% specificity), respectively. The sensitivity of the cell culture was 76.2-87.5% (mean 81.8%) when specimens were collected within 3 days after the onset of symptoms, and the sensitivity decreased to 50% or less thereafter. Among specimens collected within 3 days after symptom onset, all of the real-time RT-PCR positive specimens having a viral load of more than 1.25x105 copies/ml were found positive by cell culture. CONCLUSIONS: Cell culture using Vero E6 cell line has 81.8% sensitivity compared with the real-time RT-PCR method, when specimens are collected within 3 days after the onset of symptoms. Thus, this method is a useful method for epidemiological and virological research even in facilities with minimal laboratory resources.

Seroprevalence of parechovirus A1, A3 and A4 antibodies in Yamagata, Japan, between 1976 and 2017.

Introduction. Although new parechovirus A (PeVA) types, including parechovirus A3 (PeVA3) and PeVA4, have been reported in this century, there have not yet been any seroepidemiological studies on PeVA over a period of several decades.Hypothesis/Gap Statement. The authors hypothesize that PeVA3 and PeVA4 emerged recently.Aims. The aim was to clarify changes in the seroprevalence of PeVA1, PeVA3 and PeVA4.Methodology. Neutralizing antibodies (NT Abs) were measured among residents in Yamagata, Japan in 1976, 1983, 1985, 1990, 1999 and 2017.Results. The total NT Ab-positive rate for PeVA1 was between 90.7 and 100 % for all years analysed, with that for PeVA3 increasing from 39.6 % in 1976 to 69.6 % in 2017, and that for PeVA4 decreasing from 93.9 % in 1976 to 49.1 % in 2017. The distribution of NT Ab titres for PeVA1, PeVA3 and PeVA4 among those aged less than 20 years old was as follows: those ≥1 : 32 for PeVA1 were between 68.0-89.2 % for all years analysed; those ≥1 : 32 for PeVA3 was 15.4 % in 1976, 44.3-54.9 % in 1983-1990 and 64.8-68.0 % in 1999-2017; and those ≥1 : 32 for PeVA4 were between 49.1-67.2 % in 1976-1990, 41.3 % in 1999 and 23.8 % in 2017.Conclusions. Our findings in this seroepidemiological study over four decades suggested that PeVA1 has been stably endemic, while PeVA3 appeared around 1970s and has spread since then as an emerging disease, and occasional PeVA4 infections were common in 1970s and 1980s but have been decreasing for several decades in our community.

Evaluation of a new rapid antigen test using immunochromatography for detection of human metapneumovirus in comparison with real-time PCR assay.

A new rapid human metapneumovirus (hMPV) detection kit using immunochromatography (SAS hMPV test) was compared to real-time PCR for 224 nasal swab specimens, 96.4% of which were obtained from children of <15 years of age. The overall sensitivity and specificity were 82.3% and 93.8%, respectively, suggesting that this test is useful for pediatricians to diagnose hMPV infection in a clinical setting.

Stability of the seven hexon hypervariable region sequences of adenovirus types 1-6 isolated in Yamagata, Japan between 1988 and 2007.

Seven hexon hypervariable regions (HVRs) of adenoviruses (Ads) were identified by comparing the regions among different serotypes; however, no one has compared HVR sequences among the identical serotypes, except for adenovirus type 3 (Ad3). To examine a variability between the HVRs for each serotype, we compared the sequences of Ad1-6 isolates, respectively, isolated between 1988 and 2007 in Yamagata, Japan. We selected 23-43 isolates randomly and sequenced 894-987 bp regions. Except for strains with insertions and deletions, the sequence identities among Ad1-6 were 99-100%, excluding that between the two Ad5 groups (approx. 94%). Even the insertions and deletions were likely to be established, as these changes were repeatedly observed. The obtained phylogenetic tree indicated that Ad isolates and reference strains branched depending on serotype. The Yamagata isolates had similar sequences or amino acid arrangements to the reference strains as well as to other strains isolated in different areas. HVRs have been stably conserved as serotype-specific regions for a long period with only minor genomic variations. Therefore, we herein recommend that these regions be hereafter referred to as "serotype-specific regions", which might be a more appropriate title with which to characterize the epidemiological nature of these sites than the current "HVRs".

Polyclonal spread of multiple genotypes of Mycoplasma pneumoniae in semi-closed settings in Yamagata, Japan.

PURPOSE: To clarify the spread of Mycoplasma pneumoniae infections in semi-closed settings such as schools and family homes using molecular typing methods. METHODOLOGY: We retrospectively searched for school- and family-based clusters of M. pneumoniae infections based on information regarding patients from whom M. pneumoniae strains had been isolated between 2011 and 2013 in Yamagata, Japan. The molecular typing profile, including the P1 type and the four-locus (Mpn13, 14, 15 and 16) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) type, was obtained from our previous study. RESULTS: We identified 11 school-based clusters involving 71 patients and 16 family-based clusters involving 38 patients, including 14 duplications between these types of clusters. A total of 95M. pneumoniae strains isolated from those patients were divided into 4 genotypes: 33 strains of type 4-5-7-2, 1; 31 of type 4-5-7-3, 1; 24 of type 3-5-6-2, 2c; and 7 of type 3-5-6-2, 2a. Of the 11 school-based clusters, 6 clusters (54.5%) consisted of multiple genotypes, and the remaining 5 clusters consisted of a single genotype. Moreover, the presence of multiple genotypes was identified in three classrooms of a school. On the other hand, in 14 (87.5%) of the 16 family-based clusters, the genotypes of the M. pneumoniae strains isolated from each family member were identical. CONCLUSION: The spread of M. pneumoniae infection in schools is likely polyclonal, since M. pneumoniae strains are brought into schools from various sites, such as family homes, which are important sites of disease transmission.