Publications by authors named "Keigo Shibayama"

183 Publications

Molecular ruler of the attachment organelle in Mycoplasma pneumoniae.

PLoS Pathog 2021 Jun 10;17(6):e1009621. Epub 2021 Jun 10.

Department of Physics, Gakushuin University, Tokyo, Japan.

Length control is a fundamental requirement for molecular architecture. Even small wall-less bacteria have specially developed macro-molecular structures to support their survival. Mycoplasma pneumoniae, a human pathogen, forms a polar extension called an attachment organelle, which mediates cell division, cytadherence, and cell movement at host cell surface. This characteristic ultrastructure has a constant size of 250-300 nm, but its design principle remains unclear. In this study, we constructed several mutants by genetic manipulation to increase or decrease coiled-coil regions of HMW2, a major component protein of 200 kDa aligned in parallel along the cell axis. HMW2-engineered mutants produced both long and short attachment organelles, which we quantified by transmission electron microscopy and fluorescent microscopy with nano-meter precision. This simple design of HMW2 acting as a molecular ruler for the attachment organelle should provide an insight into bacterial cellular organization and its function for their parasitic lifestyles.
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http://dx.doi.org/10.1371/journal.ppat.1009621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191905PMC
June 2021

On-Site Genomic Epidemiological Analysis of Antimicrobial-Resistant Bacteria in Cambodia With Portable Laboratory Equipment.

Front Microbiol 2021 13;12:675463. Epub 2021 May 13.

AMR Research Center, National Institute of Infectious Diseases, Tokyo, Japan.

The rapid emergence of carbapenemase-producing gram-negative bacteria (CPGNB) is a global threat due to the high mortality of infection and limited treatment options. Although there have been many reports of CPGNB isolated from Southeast Asian countries, to date there has been no genetic analysis of CPGNB isolated from Cambodia. Sequence-based molecular epidemiological analysis enables a better understanding of the genotypic characteristics and epidemiological significance of antimicrobial-resistant (AMR) bacteria in each country, and allows countries to enact measures related to AMR issues. In this study, we performed on-site genomic epidemiological analysis of CPGNB isolated in Cambodia using a portable laboratory equipment called Bento Lab, which combines a PCR thermal cycler, microcentrifuge, gel electrophoresis apparatus, and LED transilluminator, along with the MinION nanopore sequencer. PCR targeting of major carbapenemase genes using Bento Lab revealed that two isolates and one isolate harbored carbapenemase genes: , , and , respectively. The results of phenotypic diagnostic tests for CPGNB, such as the carbapenem inactivation method and double-disk diffusion test using a specific inhibitor of metallo-β-lactamases, were consistent with their AMR genotypes. Whole-genome sequencing analysis using MinION revealed that gene was carried on a 93.9-kb plasmid with IncFIA/IncFIB/IncFII/IncQ1 replicons, and gene was carried on a 51.5-kb plasmid with the IncX3 replicon in isolates. was encoded in two locations on the chromosome of . Plasmids carrying or in were highly structurally identical to plasmids prevalent in Enterobacterales in China and other countries, suggesting that they disseminated from a common evolutionary origin. Our findings demonstrate the potential impact of portable laboratory equipment on AMR bacteria research in hospitals and research centers with limited research facilities, and provide the first glimpse into the genomic epidemiology of CPGNB in Cambodia.
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http://dx.doi.org/10.3389/fmicb.2021.675463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158813PMC
May 2021

Disease burden of bloodstream infections caused by antimicrobial-resistant bacteria: A population-level study, Japan, 2015⿿2018.

Int J Infect Dis 2021 May 13;108:119-124. Epub 2021 May 13.

AMR Clinical Reference Centre, National Centre for Global Health and Medicine, Tokyo, Japan; Disease Control and Prevention Centre, National Centre for Global Health and Medicine, Tokyo, Japan.

Background: Antimicrobial resistance (AMR) is a global health problem. However, quantitative evaluation of its disease burden is challenging. This study aimed to estimate the disease burden of bloodstream infections (BSIs) caused by major antimicrobial-resistant bacteria in Japan between 2015 and 2018 in terms of disability-adjusted life-years (DALYs).

Methods: DALYs of BSIs caused by nine major antimicrobial-resistant bacteria in Japan were estimated using comprehensive national surveillance data of all routine bacteriological test results from more than 1400 hospitals between 2015 and 2018. The methodology of Cassini et al. was modified to enable comparison of the present results with those in other countries.

Results: It was estimated that 137.9 [95% uncertainty interval (UI) 130.7⿿145.2] DALYs per 100,000 population were attributable to BSIs caused by nine antimicrobial-resistant bacteria in 2018. Methicillin-resistant Staphylococcus aureus (MRSA), fluoroquinolone-resistant Escherichia coli (FQREC) and third-generation cephalosporin-resistant E. coli (3GREC) accounted for 87.2% overall. The burden did not decrease during the study period and was highest in people aged ⿥65 years.

Conclusion: The results revealed, for the first time, the disease burden of BSIs caused by nine major antimicrobial-resistant bacteria in Japan. The estimated disease burden associated with AMR in Japan is substantial and has not begun to decrease. Notably, the burden from FQREC and 3GREC has increased steadily, and that from MRSA is larger in Japan than in the European Union/European Economic Area, whereas the burden from other bacteria is comparatively small. These results are expected to provide useful information for healthcare policy makers for prioritizing interventions for AMR./.
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http://dx.doi.org/10.1016/j.ijid.2021.05.018DOI Listing
May 2021

Induction and Resuscitation of Viable but Nonculturable .

Microorganisms 2021 Apr 26;9(5). Epub 2021 Apr 26.

Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan.

Many pathogenic bacteria, including and , can become viable but nonculturable (VBNC) following exposure to specific stress conditions. , a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC , and its environmental survival strategy.
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http://dx.doi.org/10.3390/microorganisms9050927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145655PMC
April 2021

Association between the frequency of surgeries for video-assisted thoracic surgery and the incidence of consequent surgical site infections: a retrospective observational study based on national surveillance data.

BMC Infect Dis 2021 Apr 17;21(1):363. Epub 2021 Apr 17.

Department of Bacteriology II, National Institute of Infectious Diseases, 4-7-1 Gakuen Musashimurayama, Tokyo, 280-0011, Japan.

Background: The association between the frequency of surgeries and the incidence of surgical site infections (SSIs) has been reported for various surgeries. However, no previous study has explored this association among video-assisted thoracic surgeries (VATS). Hence, we aimed to investigate the association between the frequency of surgeries and SSI in video-assisted thoracic surgeries.

Methods: We analyzed the data of 26,878 thoracic surgeries, including 21,154 VATS, which were collected during a national surveillance in Japan between 2014 and 2018. The frequency of surgeries per hospital department was categorized into low (< 50/year), moderate (50-100/ year), and high (> 100/year). Chi-squared test or Fisher's exact test was used for discrete explanatory variables, whereas Wilcoxon's rank-sum test or Kruskal-Wallis test was used for continuous explanatory variables. Univariate analysis of the department groups was conducted to explore confounding factors associated with both SSIs and the department groups. We used a multiple logistic regression model focusing on VATS and stratified by the National Nosocomial Infections Surveillance System (NNIS) risk index.

Results: The rates of SSIs in the hospital groups with low, moderate, and high frequency of surgeries were 1.39, 1.05, and 1.28%, respectively. In the NNIS risk index 1 stratum, the incidence of SSIs was significantly lower in the moderate-frequency of surgeries group than that in the other groups (odds ratio [OR]: vs. low-frequency of surgeries: 2.48 [95% confidence interval [CI]: 1.20-5.13], P = 0.0143; vs. high-frequency of surgeries: 2.43 [95% CI: 1.44-4.11], P = 0.0009). In the stratum of NNIS risk indices 2 and 3, the incidence of SSI was significantly higher in the low-frequency of surgeries group (OR: 4.83, 95% CI: 1.47-15.93; P = 0.0095).

Conclusion: The result suggests that for departments with low-frequency of surgeries, an increase in the frequency of surgeries to > 50 per department annually potentially leads to a decrease in the incidence of SSIs. This occurs through an increase in the experience of the departmental surgeons and contributes to the improvement of VATS outcomes in thoracic surgeries.
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http://dx.doi.org/10.1186/s12879-021-06050-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052810PMC
April 2021

Subtype screening of bla genes using bipartite primers for DNA sequencing.

Jpn J Infect Dis 2021 Mar 31. Epub 2021 Mar 31.

Department of Microbiology, Ehime Prefectural Institute of Public Health and Environmental Science, Japan.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of bla genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all bla genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for bla, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of bla, which is useful for the surveillance of bacteria with bla in clinical and public health settings or environmental fields.
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http://dx.doi.org/10.7883/yoken.JJID.2020.926DOI Listing
March 2021

Isolation and characterization of from human stomach.

Proc Natl Acad Sci U S A 2021 Mar;118(13)

Department of General Medicine, Kyorin University School of Medicine, 181-8611 Tokyo, Japan

, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because has been successfully isolated from pigs, but not from humans, evidence linking human infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured directly from human stomachs. Unlike , the viability of decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, , like , is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of using complete genomes of clinical isolates revealed that the genome of each isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.
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http://dx.doi.org/10.1073/pnas.2026337118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020762PMC
March 2021

Emergence of a plasmid-borne tigecycline resistance in in Vietnam.

J Med Microbiol 2021 Mar 11;70(3). Epub 2021 Mar 11.

AMR Research Center, National Institute of Infectious Diseases, Tokyo, Japan.

Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, , encoding the resistance-nodulation-cell division (RND) efflux pump was identified on plasmids in isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified isolates harbouring the genes outside of China for the first time. Two tigecycline-resistant isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb -carrying plasmid with IncFIB and IncHI1B replicons. The gene cluster was surrounded by several mobile gene elements, including IS, and the plasmids had high sequence identity with that of isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by -carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.
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http://dx.doi.org/10.1099/jmm.0.001320DOI Listing
March 2021

Rapid and simple SNP genotyping for Bordetella pertussis epidemic strain MT27 based on a multiplexed single-base extension assay.

Sci Rep 2021 Mar 1;11(1):4823. Epub 2021 Mar 1.

Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan.

Multilocus variable-number tandem repeat analysis (MLVA) is widely used for genotyping of Bordetella pertussis, the causative bacteria for pertussis. However, MLVA genotyping is losing its discriminate power because prevalence of the epidemic MT27 strain (MLVA-27) is increasing worldwide. To address this, we developed a single nucleotide polymorphism (SNP) genotyping method for MT27 based on multiplexed single-base extension (SBE) assay. A total of 237 MT27 isolates collected in Japan during 1999-2018 were genotyped and classified into ten SNP genotypes (SG1 to SG10) with a Simpson's diversity index (DI) of 0.79 (95% CI 0.76-0.82). Temporal trends showed a marked increase in the genotypic diversity in the 2010s: Simpson's DI was zero in 1999-2004, 0.16 in 2005-2009, 0.83 in 2010-2014, and 0.76 in 2015-2018. This indicates that the SNP genotyping is applicable to the recently circulating MT27 strain. Additionally, almost all outbreak-associated MT27 isolates were classified into the same SNP genotypes for each outbreak. Multiplexed SBE assay allows for rapid and simple genotyping, indicating that the SNP genotyping can potentially be a useful tool for subtyping the B. pertussis MT27 strain in routine surveillance and outbreak investigations.
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http://dx.doi.org/10.1038/s41598-021-84409-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921669PMC
March 2021

Nationwide multicenter questionnaire surveys on countermeasures against antimicrobial resistance and infections in hospitals.

BMC Infect Dis 2021 Feb 27;21(1):234. Epub 2021 Feb 27.

Department of Healthcare Economics and Quality Management, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Background: The goals of the National Action Plan on Antimicrobial Resistance (AMR) of Japan include "implementing appropriate infection prevention and control" and "appropriate use of antimicrobials," which are relevant to healthcare facilities. Specifically, linking efforts between existing infection control teams and antimicrobial stewardship programs was suggested to be important. Previous studies reported that human resources, such as full-time equivalents of infection control practitioners, were related to improvements in antimicrobial stewardship.

Methods: We posted questionnaires to all teaching hospitals (n = 1017) regarding hospital countermeasures against AMR and infections. To evaluate changes over time, surveys were conducted twice (1st survey: Nov 2016, 2nd survey: Feb 2018). A latent transition analysis (LTA) was performed to identify latent statuses, which refer to underlying subgroups of hospitals, and effects of the number of members in infection control teams per bed on being in the better statuses.

Results: The number of valid responses was 678 (response rate, 66.7%) for the 1st survey and 559 (55.0%) for the 2nd survey. More than 99% of participating hospitals had infection control teams, with differences in activity among hospitals. Roughly 70% had their own intervention criteria for antibiotics therapies, whereas only about 60 and 50% had criteria established for the use of anti-methicillin-resistant Staphylococcus aureus antibiotics and broad-spectrum antibiotics, respectively. Only 50 and 40% of hospitals conducted surveillance of catheter-associated urinary tract infections and ventilator-associated pneumonia, respectively. Less than 50% of hospitals used maximal barrier precautions for central line catheter insertion. The LTA identified five latent statuses. The membership probability of the most favorable status in the 2nd study period was slightly increased from the 1st study period (23.6 to 25.3%). However, the increase in the least favorable status was higher (26.3 to 31.8%). Results of the LTA did not support a relationship between increasing the number of infection control practitioners per bed, which is reportedly related to improvements in antimicrobial stewardship, and being in more favorable latent statuses.

Conclusions: Our results suggest the need for more comprehensive antimicrobial stewardship programs and increased surveillance activities for healthcare-associated infections to improve antimicrobial stewardship and infection control in hospitals.
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http://dx.doi.org/10.1186/s12879-021-05921-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912490PMC
February 2021

Invasive Haemophilus influenzae type a infection and polyarthritis in a 72-year-old Japanese man: A case report.

J Infect Chemother 2021 Jul 15;27(7):1084-1088. Epub 2021 Feb 15.

Department of Internal Medicine, Hyogo Prefectural Tamba Medical Center, 2002-7 Iso, Hikami-cho, Tamba, 669-3495, Japan. Electronic address:

Haemophilus influenzae is a small, nonmotile, non-spore-forming bacterium classified into 6 serotypes (a to f) and non-typeable strains that lack a capsule. Although H. influenzae serotype a (Hia) is prevalent in Canada, the United States, Brazil, Australia, across the African continent, and several other locations, it has not been reported in Japan thus far. Our case was of a 72-year-old Japanese man who sought medical consultation after presenting with chills, fever, and polyarthritis. Cultures of blood and synovial fluid from the left knee revealed H. influenzae infection. Diagnostic imaging showed poor contrast regions in both kidneys, fluid retention around both knee joints, the left shoulder joint, and both elbow joints. Subsequently, the patient was diagnosed with invasive H. influenzae infection accompanied by polyarthritis and renal infarction. 16S ribosomal RNA gene sequencing revealed that the bacterial strain was Hia. The patient was treated with antimicrobial agents and arthroscopic curettage. We present a case of invasive Hia infection accompanied by polyarthritis and renal infarction. To the best of our knowledge, this is the first case of Hia infection in Japan. The case is very rare considering that the disease occurred in an elderly patient who developed polyarthritis.
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http://dx.doi.org/10.1016/j.jiac.2021.01.017DOI Listing
July 2021

Detection of Bartonella quintana infection among the Homeless Population in Tokyo, Japan, from 2013-2015.

Jpn J Infect Dis 2021 Jan 29. Epub 2021 Jan 29.

Department of Medical Entomology, National Institute of Infectious Diseases, Japan.

Several outbreaks of trench fever caused by Bartonella quintana, occurred in soldiers during World Wars I and II. Although the number of trench fever cases has been decreasing worldwide, the disease has been reported among the homeless population in both developing and developed countries. The current prevalence of B. quintana infection in Japan is unclear. We collected blood and body louse (Pediculus humanus humanus) samples from homeless inpatients who had body lice at the time of emergency hospitalization in Tokyo from January 2013 to March 2015. Patients were tested for B. quintana infections using culture method, polymerase chain reaction, and indirect immunofluorescence assay (IFA). Among the 29 patients tested, the presence of Bartonella spp. was confirmed by genomic sequencing of DNA extracted from the samples from 2 patients (blood culture performed for 13 out of 15 patients), and from body louse samples of 20 patients (69%). Immunoglobulin G against B. quintana was detected in 10 patients (34.5%) at a cut-off titer of 1:256 in IFA. B. quintana infection was detected in samples obtained between 2013 and 2015 in Tokyo and needs to be on the list of differential diagnoses performed for febrile homeless individuals.
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http://dx.doi.org/10.7883/yoken.JJID.2020.505DOI Listing
January 2021

Geographical distribution of Enterobacterales with a carbapenemase IMP-6 phenotype and its association with antimicrobial use: An analysis using comprehensive national surveillance data on antimicrobial resistance.

PLoS One 2020 17;15(12):e0243630. Epub 2020 Dec 17.

Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Tokyo, Japan.

Enterobacterales resistant to carbapenems, a class of last-resort antimicrobials, are ranked as an "urgent" and "critical" public health hazard by CDC and WHO. IMP-type carbapenemase-containing Enterobacterales are endemic in Japan, and blaIMP-6 is one of the notable carbapenemase genes responsible for the resistance. The gene is plasmid-encoded and confers resistance to meropenem, but not to imipenem. Therefore, IMP-6-producing Enterobacterales isolates are occasionally overlooked in clinical laboratories and are referred to as 'stealth-type'. Since previous reports in Japan were confined only to some geographical regions, their distribution across prefectures and the factors affecting the distribution remain unclear. Here, we revealed the dynamics of the geographical distribution of Enterobacterales with IMP-6 phenotype associated with antimicrobial use in Japan. We utilized comprehensive national surveillance data of all routine bacteriological test results from more than 1,400 hospitals in 2015 and 2016 to enumerate Escherichia coli and Klebsiella pneumoniae isolates with the antimicrobial susceptibility pattern (phenotype) characteristic of IMP-6 (imipenem susceptible, meropenem resistant), and to tabulate the frequency of isolates with the phenotype for each prefecture. Isolates were detected in approximately half of all prefectures, and combined analysis with the national data of antimicrobial usage revealed a statistically significant association between the frequency and usage of not carbapenems but third-generation cephalosporins (p = 0.006, logistic mixed-effect regression) and a weaker association between the frequency and usage of fluoroquinolones (p = 0.043). The usage of third-generation cephalosporins and fluoroquinolones may select the strains with the IMP-6 phenotype, and contribute to their occasional spread. We expect the findings will promote antimicrobial stewardship to reduce the spread of the notable carbapenemase gene.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243630PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745981PMC
February 2021

Complete Genome Sequence of a Japanese Clinical Isolate of Haemophilus influenzae Type a Strain TAMBA230.

Microbiol Resour Announc 2020 Nov 25;9(48). Epub 2020 Nov 25.

Department of Internal Medicine, Hyogo Prefectural Tamba Medical Center, Hyogo, Japan.

causes severe infections such as pneumonia and meningitis. Here, we report the complete genome of type a strain TAMBA230, which was isolated in 2019 from a patient exhibiting bacteremia. This represents the first case in Japan of an type a strain associated with invasive infection.
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http://dx.doi.org/10.1128/MRA.01069-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686422PMC
November 2020

Improved penicillin susceptibility of Streptococcus pneumoniae and increased penicillin consumption in Japan, 2013-18.

PLoS One 2020 22;15(10):e0240655. Epub 2020 Oct 22.

AMR Clinical Reference Center, National Center for Global Health and Medicine, Tokyo, Japan.

Objectives: To examine the association between penicillin susceptibility of Streptococcus pneumoniae and penicillin consumption in Japan.

Methods: We used Japan Nosocomial Infection Surveillance data on the susceptibility of S. pneumoniae and sales data obtained from IQVIA Services Japan K.K. for penicillin consumption. We analysed both sets of data by decomposing them into seasonality and chronological trend components. The cross-correlation function was checked using Spearman's rank correlation coefficient to examine the correlation between susceptibility and consumption.

Results: After adjusting for seasonality, the susceptibility of S. pneumoniae to penicillins gradually improved (55.7% in 2013 and 60.6% in 2018, respectively) and penicillin consumption increased during the same period (0.76 defined daily doses per 1,000 inhabitants per day [DID] in 2013, and 0.89 DID in 2018). The results showed positive cross-correlation (coefficient 0.801, p-value < 0.001). In contrast, cephalosporin consumption decreased (3.91 DID in 2013 and 3.19 DID in 2018) and showed negative cross-correlation with susceptibility of S. pneumoniae to penicillins (coefficient -0.981, p-value < 0.001).

Conclusions: The rates of penicillin-susceptible S. pneumoniae isolates did not negatively correlate with penicillin consumption at the population level. Increased penicillin consumption might not impair the penicillin susceptibility of S. pneumoniae.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240655PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580930PMC
December 2020

Corrigendum: Networking and Specificity-Changing DNA Methyltransferases in .

Front Microbiol 2020 22;11:596598. Epub 2020 Sep 22.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan.

[This corrects the article DOI: 10.3389/fmicb.2020.01628.].
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http://dx.doi.org/10.3389/fmicb.2020.596598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537328PMC
September 2020

Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae.

Nat Commun 2020 10 14;11(1):5188. Epub 2020 Oct 14.

Instituto de Biología Molecular de Barcelona (IBMB-CSIC), Parc Científic de Barcelona, Baldiri Reixac 10, 08028, Barcelona, Spain.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.
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http://dx.doi.org/10.1038/s41467-020-18777-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560827PMC
October 2020

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans.

Emerg Infect Dis 2020 10;26(10):2457-2459

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.
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http://dx.doi.org/10.3201/eid2610.200086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510715PMC
October 2020

Macrolide-Resistant Bordetella pertussis, Vietnam, 2016-2017.

Emerg Infect Dis 2020 10;26(10):2511-2513

Macrolide-resistant Bordetella pertussis emerged in Vietnam during 2016-2017. Direct analyses of swab samples from 10 patients with pertussis revealed a macrolide-resistant mutation, A2047G, in the 23S rRNA. We identified the MT104 genotype of macrolide-resistant B. pertussis (which is prevalent in mainland China) and its variants in these patients.
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http://dx.doi.org/10.3201/eid2610.201035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510698PMC
October 2020

Japan Nosocomial Infections Surveillance (JANIS): Current Status, International Collaboration, and Future Directions for a Comprehensive Antimicrobial Resistance Surveillance System.

Jpn J Infect Dis 2021 Mar 31;74(2):87-96. Epub 2020 Aug 31.

Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Japan.

Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national antimicrobial resistance (AMR) surveillance systems in the world. The JANIS Clinical Laboratory division collects comprehensive specimen-based data from diagnostic microbiology laboratories of participating hospitals to monitor the isolation rate of 11 major bacteria and specific AMR bacteria, and creates antibiograms of approximately 20 bacterial species. Data on the JANIS web database system are also annually tabulated and shared with the WHO Global Antimicrobial Resistance Surveillance System. To create a network of international AMR surveillance systems among Asian countries, Japan is developing an international web database system named ASIan Antimicrobial Resistance Surveillance Network (ASIARS-Net) based on the JANIS system; ASIARS-Net is an open-source database and confidentially available at almost no cost. JANIS continues to evolve in multiple directions; some are discussed at the end of this review.
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http://dx.doi.org/10.7883/yoken.JJID.2020.499DOI Listing
March 2021

Periodic Genotype Shifts in Clinically Prevalent Strains in Japan.

Front Cell Infect Microbiol 2020 6;10:385. Epub 2020 Aug 6.

Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan.

Nationwide increases in pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 adhesin gene; however, strains harboring type 2 or its variant gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical strains. During this analysis, we also identified strains carrying a novel variant type 1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected strains that we collected between 1976 and 2017 in Japan, and compared them with 156 genomes deposited in public databases to provide insights into the interpretation of genotyping methods, including typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of strains.
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http://dx.doi.org/10.3389/fcimb.2020.00385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424021PMC
June 2021

Networking and Specificity-Changing DNA Methyltransferases in .

Front Microbiol 2020 17;11:1628. Epub 2020 Jul 17.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan.

Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object , a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or , consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.
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http://dx.doi.org/10.3389/fmicb.2020.01628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379913PMC
July 2020

Comparison of de-duplication methods used by WHO Global Antimicrobial Resistance Surveillance System (GLASS) and Japan Nosocomial Infections Surveillance (JANIS) in the surveillance of antimicrobial resistance.

PLoS One 2020 26;15(6):e0228234. Epub 2020 Jun 26.

Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Higashimurayama, Tokyo, Japan.

A major issue in the surveillance of antimicrobial resistance (AMR) is "de-duplication" or removal of repeated isolates, for which there exist multiple methods. The World Health Organization (WHO) Global Antimicrobial Resistance Surveillance System (GLASS) requires de-duplication by selecting only the first isolate of a given bacterial species per patient per surveillance period per specimen type per age group, gender, and infection origin stratification. However, no study on the comparative application of this method has been reported. The objective of this study was to evaluate differences in data tabulation between the WHO GLASS and the Japan Nosocomial Infections Surveillance (JANIS) system, which counts both patients and isolates after removing repeated isolates of the same bacterial species isolated from a patient within 30 days, regardless of specimen type, but distinguishing isolates with change of antimicrobial resistance phenotype. All bacterial data, consisting of approximately 8 million samples from 1795 Japanese hospitals in 2017 were exported from the JANIS database, and were tabulated using either the de-duplication algorithm of GLASS, or JANIS. We compared the tabulated results of the total number of patients whose blood and urine cultures were taken and of the percentage of resistant isolates of Escherichia coli for each priority antibiotic. The number of patients per specimen type tabulated by the JANIS method was always smaller than that of GLASS. There was a small (< 3%) difference in the percentage of resistance of E. coli for any antibiotic between the two methods in both out- and inpatient settings and blood and urine isolates. The two tabulation methods did not show considerable differences in terms of the tabulated percentages of resistance for E. coli. We further discuss how the use of GLASS tabulations to create a public software and website that could help to facilitate the understanding of and treatment against AMR.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228234PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319286PMC
August 2020

A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid.

Sci Rep 2020 03 5;10(1):4139. Epub 2020 Mar 5.

Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan.

A multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales (IMP-6-CPE) occurred at an acute care hospital in Japan. This study was conducted to understand the mechanisms of IMP-6-CPE transmission by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and whole-genome sequencing (WGS), and identify risk factors for IMP-6-CPE acquisition in patients who underwent abdominal surgery. Between July 2013 and March 2014, 22 hospitalized patients infected or colonized with IMP-6-CPE (Escherichia coli [n = 8], Klebsiella oxytoca [n = 5], Enterobacter cloacae [n = 5], Klebsiella pneumoniae [n = 3] and Klebsiella aerogenes [n = 1]) were identified. There were diverse PFGE profiles and sequence types (STs) in most of the species except for K. oxytoca. All isolates of K. oxytoca belonged to ST29 with similar PFGE profiles, suggesting their clonal transmission. Plasmid analysis by WGS revealed that all 22 isolates but one shared a ca. 50-kb IncN plasmid backbone with bla suggesting interspecies gene transmission, and typing of plasmids explained epidemiological links among cases. A case-control study showed pancreatoduodenectomy, changing drains in fluoroscopy room, continuous peritoneal lavage and enteric fistula were associated with IMP-6-CPE acquisition among the patients. Plasmid analysis of isolates in an outbreak of IMP-6-CPE suggested interspecies gene transmission and helped to clarify hidden epidemiological links between cases.
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http://dx.doi.org/10.1038/s41598-020-60659-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057946PMC
March 2020

Complete Genome Sequence of Helicobacter suis Strain SNTW101c, Originally Isolated from a Patient with Nodular Gastritis.

Microbiol Resour Announc 2020 Jan 2;9(1). Epub 2020 Jan 2.

Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan.

strain SNTW101c, which was originally obtained from a patient with nodular gastritis, has been maintained in mouse stomach because of difficulty culturing it Recently, we succeeded in culturing this strain Here, we report the complete genome sequence of strain SNTW101c.
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http://dx.doi.org/10.1128/MRA.01340-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940298PMC
January 2020

National trend of blood-stream infection attributable deaths caused by Staphylococcus aureus and Escherichia coli in Japan.

J Infect Chemother 2020 Apr 1;26(4):367-371. Epub 2019 Dec 1.

AMR Clinical Reference Center, National Center for Global Health and Medicine, Tokyo, Japan; Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan.

There has been scarce evidence about deaths due to blood stream infection (BSI) in Japan so far. The main objective of this study is to understand the epidemiological trend of deaths caused by BSIs due to Staphylococcus aureus and Escherichia coli including Methicillin-resistant S. aureus (MRSA) and fluoroquinolone-resistant E. coli (FQREC) at national level. We annually estimated the number of BSI caused by S. aureus and E. coli between 2011 and 2017 across Japan using comprehensive data of bacterial culturing and drug susceptibilities collected in Japan Nosocomial Infection Surveillance (JANIS). The number of death was estimated by using BSI mortality obtained from previous studies in Japan. The number of BSI death attributable to S. aureus was estimated to 17,412 in 2011 and 17,157 in 2017, respectively, out of the whole population (126.8 million) in Japan. Among them, cases attributed to MRSA accounted for 5924 (34.0%) in 2011, and decreased to 4224 (24.6%) cases in 2017. On the other hand, the number of BSI death attributable to E. coli was estimated to 9044 in 2011 and increased to 14,016 in 2017. Among them, cases attributed to FQREC accounted for 2045 (22.6%) in 2011 and increased to 3915 (27.9%) cases in 2017. The number of BSI death attributable to MRSA has been decreasing and that attributable to FQREC has been increasing. This study provides the first annual estimate of disease burden of BSI caused by antimicrobial resistant (AMR) bacteria in Japan, and basis for formulating health policy to deal with AMR.
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http://dx.doi.org/10.1016/j.jiac.2019.10.017DOI Listing
April 2020

Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase.

Jpn J Infect Dis 2020 Mar 29;73(2):166-172. Epub 2019 Nov 29.

Department of Microbiology, Ehime Prefectural Institute of Public Health and Environmental Science.

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: bla, bla, bla, bla, bla, and bla. Of 70 bla variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.
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http://dx.doi.org/10.7883/yoken.JJID.2019.041DOI Listing
March 2020

A novel multilocus variable-number tandem repeat analysis for .

J Med Microbiol 2019 Nov;68(11):1671-1676

Present address: Division of Microbiology and Molecular Cell Biology, Nihon Pharmaceutical University, Saitama, Japan.

Human-adapted is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted . Four highly polymorphic variable number tandem repeat (VNTR) loci in the genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of . Multiplex PCR-based MLVA characterized 53 reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type. Our novel MLVA method has high discriminatory power for genotyping human . Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.
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http://dx.doi.org/10.1099/jmm.0.001095DOI Listing
November 2019

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Macromol Biosci 2019 10 20;19(10):e1900167. Epub 2019 Aug 20.

Department of Microbiology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, 216-8511, Japan.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.
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http://dx.doi.org/10.1002/mabi.201900167DOI Listing
October 2019

Molecular epidemiological analysis and risk factors for acquisition of carbapenemase-producing complex in a Japanese university hospital.

Antimicrob Resist Infect Control 2019 24;8:126. Epub 2019 Jul 24.

1Department of Infectious Diseases, Nagoya University Hospital, 65 Tsurumai, Nagoya, Aichi 466-0065 Japan.

Background: To clarify the molecular epidemiology of carbapenem-resistant complex (CREC) and the risk factors for acquisition of carbapenemase-producing complex (CPEC).

Methods: Using clinical CREC isolates detected in a Japanese university hospital over 4 years, carbapenemase production was screened with phenotypic methods. Carbapenemase genes were analysed by PCR and sequencing. Molecular epidemiological analyses were conducted with repetitive extragenic palindromic (REP)-PCR and multilocus sequence typing (MLST). CRECs were identified to the subspecies level by sequencing. Whole-genome sequencing of plasmids was conducted. A case-control study was performed to identify risk factors for acquisition of CPEC among patients with CREC.

Results: Thirty-nine CRECs including 20 CPECs carrying were identified. Patients with CPEC had longer hospital stay before detection (26.5 days vs. 12 days,  = 0.008), a urinary catheter (odds ratio [OR], 5.36; 95% confidence interval [CI], 1.14-30.9;  = 0.023), or intubation (OR, 7.53; 95% CI, 1.47-53.8;  = 0.008) compared to patients without CPEC. Four genetically closely related CPEC clusters were observed, which showed that three of four CPEC clusters corresponded to (ST 53), subsp. (ST 113 and ST 1047) and subsp. (ST 513) by MLST and sequencing. Seven representative plasmids shared structures with class I integron containing and IncHI2A replicon type.

Conclusions: A longer hospital stay, presence of a urinary catheter, and intubation are risk factors for CPEC acquisition. In addition to horizontal transmission of genetically indistinguishable CPECs, IncHI2A plasmid carrying appeared to be transferred among genetically different ECs.
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http://dx.doi.org/10.1186/s13756-019-0578-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657070PMC
June 2020
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