The recent large outbreaks of Ebola virus disease (EVD) in West Africa and the Democratic Republi... more The recent large outbreaks of Ebola virus disease (EVD) in West Africa and the Democratic Republic of the Congo (DRC) have highlighted the need for rapid diagnostic tests to control this disease. In this study, we clinically evaluated a previously developed immunochromatography-based kit, QuickNaviTM-Ebola. During the 2018 outbreaks in DRC, 928 blood samples from EVD-suspected cases were tested with QuickNaviTM-Ebola and the WHO-approved GeneXpert. The sensitivity and specificity of QuickNaviTM-Ebola, estimated by comparing it to GeneXpert-confirmed cases, were 85% (68/80) and 99.8% (846/848), respectively. These results indicate the practical reliability of QuickNaviTM-Ebola for point-of-care diagnosis of EVD.
An enzyme-linked immunosorbent assay (ELISA) that measures anti-pertussis toxin (PT) IgG antibody... more An enzyme-linked immunosorbent assay (ELISA) that measures anti-pertussis toxin (PT) IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease due to the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that diphtheria-tetanus-acellular pertussis (DTaP) vaccines did not induce anti-Vag8 IgG antibodies in mice, i.e., the antibody level could be unaffected by the vaccination. To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). Receiver operating characteristics (ROC) analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.
Legionella pneumophila serogroup 1 is the serogroup of Legionella species most frequently isolate... more Legionella pneumophila serogroup 1 is the serogroup of Legionella species most frequently isolated from infected patients in Japan and all over the world. The recent findings suggest that one of the most discriminative epidemiological methods is pulsed-field gel electrophoresis (PFGE). To develop the method of epidemiological surveillance, PFGE and monoclonal antibodies were used for the analysis of 27 clinical and 20 environmental L. pneumophila serogroup 1 isolates in Japan. Researchers reported five anti-L. pneumophila serogroup 1 monoclonal antibodies that were specific for serogroup 1 but showed different reactivities, previously. Monoclonal antibody-sensitized latex was prepared and the latex agglutination test on slide plate was performed. There was a close correlation between monoclonal antibody subtyping and PFGE analysis. The result by the two analyses suggested that the clinical isolates, the isolates from cooling towers, and the isolates from hot springs in Japan formed the distinctive genetically or antigenic clusters.
Methicillin-resistant Staphylococcus aureus (MRSA), an important cause of nosocomial infections, ... more Methicillin-resistant Staphylococcus aureus (MRSA), an important cause of nosocomial infections, produces a number of superantigenic toxins such as toxic shock syndrome toxin (TSST)-1 and staphylococcal enterotoxin (SE) C in many cases. However, toxic shock syndrome (TSS) only rarely occurs in MRSA infections. In this study, we isolated MRSA strains from the stool and sputum of a patient with TSS and characterized the molecular nature of the TSS-associated MRSA strains. The two MRSA strains were indistinguishable from each other, as demonstrated by pulsed-field gel electrophoresis. The coagulase type was type 2. The two MRSA strains manifested a multiple superantigenic toxin (MST) pattern of TSST-1, SEA, SEC, SEG, SEH, SEI, and SET in the PCR assay, unlike the previously characterized MRSA strains that lacked (e. g.) SEA. When human peripheral blood mononuclear cells were stimulated with superantigenic toxins, a combination of TSST-1, SEA, and SEC induced much higher levels of cytok...
Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of... more Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study, we investigated whether CV6209, a PAF antagonist, could modulate Shiga toxin (Stx)-induced TNF-alpha production in human monocytic cells. Cells were stimulated by Stx1 or Stx2 (5 ng/ml) with or without CV6209 addition (12-100 microg/ml) for various periods of time. CV6209 significantly suppressed Stx-induced TNF-alpha production in a dose- and time-dependent manner. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that CV6209 suppressed Stx-mediated TNF-alpha mRNA expression. Our results indicated that CV6209 had an important regulatory effect on Stx-induced TNF-alpha production and gene expression.
Although thermostable direct hemolysin–producing (tdh+) Vibrio parahaemolyticus is the leading ca... more Although thermostable direct hemolysin–producing (tdh+) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh+V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)–based procedure designated A-IS1-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate–based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh+V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively l...
SUMMARY: Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC)... more SUMMARY: Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC) in the kidney, and is involved in the reabsorption and metabolism of various proteins that have been filtered by glomeruli. Patients with diabetes, especially type 2 diabetes, or metabolic syndrome are likely to have elevated serum levels of advanced glycation end products, liver‐type fatty acid binding protein, angiotensin II, insulin and leptin, and renal metabolism of these proteins is potentially overloaded. Some of these proteins are themselves nephrotoxic, while others are carriers of nephrotoxic molecules. Megalin is involved in the proximal tubular uptake of these proteins. We hypothesize that megalin‐mediated metabolic overload in PTC leads to compensatory cellular hypertrophy and sustained Na+ reabsorption, causing systemic hypertension and glomerular hyperfiltration via tubuloglomerular feedback, and named this as ‘protein metabolic overload hypothesis’. Impaired metabolism o...
Vibrio cholerae are divided into several serogroups based on their O antigens. Epidemic cholera i... more Vibrio cholerae are divided into several serogroups based on their O antigens. Epidemic cholera is caused by V. cholerae strains belonging to serogroup 0: 1. That serogroup is further divided into three serotypes : Ogawa, Inaba, and Hikojima. Sakazaki and Tamura (6) studied these O antigen variations by cross agglutininabsorption test and found that somatic antigen fractions a and c are common to the three serotypes, and that fraction b, which is absent in type Inaba, can be used as a specific factor for recognizing type Ogawa. They also reported that type Ogawa possessed a small amount of antigen fraction c. Diagnostic antisera produced by immunization of rabbits are now available for identification of V. cholerae 0: 1 and its serotypes. Because type Ogawa possesses type-specific antigen b, anti-b serum is prepared by cross-absorption of the polyvalent Ogawa antiserum with bacteria of type Inaba. Anti-c serum is prepared by crossabsorption of polyvalent antiserum to type Inaba with Vibrio of type Ogawa. But, because type Ogawa has a little antigen c, the titer of fraction c antibody decreases by cross-absorption of polyvalent Inaba antiserum with bacteria of type Ogawa. No serum for group-specific antigen a has yet been prepared by this procedure. This paper describes experiments in which we successfully prepared monoclonal antibodies against fraction a, b, and c, and have developed a latex agglutination test for identification of V. cholerae types. V. cholerae NIH41 (type Ogawa) and NIH35A3 (type Inaba) were cultivated overnight on nutrient agar and killed by adding formalin to reach a final concentration of 1.0 v/v%. The Vibrio were washed twice in phosphate buffered saline (PBS),
In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among t... more In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture‐based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin‐detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml. In our study of previously characterized Shigatoxigenic and all non‐Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non‐Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stoo...
Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raise... more Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in in...
The recent large outbreaks of Ebola virus disease (EVD) in West Africa and the Democratic Republi... more The recent large outbreaks of Ebola virus disease (EVD) in West Africa and the Democratic Republic of the Congo (DRC) have highlighted the need for rapid diagnostic tests to control this disease. In this study, we clinically evaluated a previously developed immunochromatography-based kit, QuickNaviTM-Ebola. During the 2018 outbreaks in DRC, 928 blood samples from EVD-suspected cases were tested with QuickNaviTM-Ebola and the WHO-approved GeneXpert. The sensitivity and specificity of QuickNaviTM-Ebola, estimated by comparing it to GeneXpert-confirmed cases, were 85% (68/80) and 99.8% (846/848), respectively. These results indicate the practical reliability of QuickNaviTM-Ebola for point-of-care diagnosis of EVD.
An enzyme-linked immunosorbent assay (ELISA) that measures anti-pertussis toxin (PT) IgG antibody... more An enzyme-linked immunosorbent assay (ELISA) that measures anti-pertussis toxin (PT) IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease due to the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that diphtheria-tetanus-acellular pertussis (DTaP) vaccines did not induce anti-Vag8 IgG antibodies in mice, i.e., the antibody level could be unaffected by the vaccination. To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). Receiver operating characteristics (ROC) analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.
Legionella pneumophila serogroup 1 is the serogroup of Legionella species most frequently isolate... more Legionella pneumophila serogroup 1 is the serogroup of Legionella species most frequently isolated from infected patients in Japan and all over the world. The recent findings suggest that one of the most discriminative epidemiological methods is pulsed-field gel electrophoresis (PFGE). To develop the method of epidemiological surveillance, PFGE and monoclonal antibodies were used for the analysis of 27 clinical and 20 environmental L. pneumophila serogroup 1 isolates in Japan. Researchers reported five anti-L. pneumophila serogroup 1 monoclonal antibodies that were specific for serogroup 1 but showed different reactivities, previously. Monoclonal antibody-sensitized latex was prepared and the latex agglutination test on slide plate was performed. There was a close correlation between monoclonal antibody subtyping and PFGE analysis. The result by the two analyses suggested that the clinical isolates, the isolates from cooling towers, and the isolates from hot springs in Japan formed the distinctive genetically or antigenic clusters.
Methicillin-resistant Staphylococcus aureus (MRSA), an important cause of nosocomial infections, ... more Methicillin-resistant Staphylococcus aureus (MRSA), an important cause of nosocomial infections, produces a number of superantigenic toxins such as toxic shock syndrome toxin (TSST)-1 and staphylococcal enterotoxin (SE) C in many cases. However, toxic shock syndrome (TSS) only rarely occurs in MRSA infections. In this study, we isolated MRSA strains from the stool and sputum of a patient with TSS and characterized the molecular nature of the TSS-associated MRSA strains. The two MRSA strains were indistinguishable from each other, as demonstrated by pulsed-field gel electrophoresis. The coagulase type was type 2. The two MRSA strains manifested a multiple superantigenic toxin (MST) pattern of TSST-1, SEA, SEC, SEG, SEH, SEI, and SET in the PCR assay, unlike the previously characterized MRSA strains that lacked (e. g.) SEA. When human peripheral blood mononuclear cells were stimulated with superantigenic toxins, a combination of TSST-1, SEA, and SEC induced much higher levels of cytok...
Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of... more Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study, we investigated whether CV6209, a PAF antagonist, could modulate Shiga toxin (Stx)-induced TNF-alpha production in human monocytic cells. Cells were stimulated by Stx1 or Stx2 (5 ng/ml) with or without CV6209 addition (12-100 microg/ml) for various periods of time. CV6209 significantly suppressed Stx-induced TNF-alpha production in a dose- and time-dependent manner. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that CV6209 suppressed Stx-mediated TNF-alpha mRNA expression. Our results indicated that CV6209 had an important regulatory effect on Stx-induced TNF-alpha production and gene expression.
Although thermostable direct hemolysin–producing (tdh+) Vibrio parahaemolyticus is the leading ca... more Although thermostable direct hemolysin–producing (tdh+) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh+V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)–based procedure designated A-IS1-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate–based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh+V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively l...
SUMMARY: Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC)... more SUMMARY: Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC) in the kidney, and is involved in the reabsorption and metabolism of various proteins that have been filtered by glomeruli. Patients with diabetes, especially type 2 diabetes, or metabolic syndrome are likely to have elevated serum levels of advanced glycation end products, liver‐type fatty acid binding protein, angiotensin II, insulin and leptin, and renal metabolism of these proteins is potentially overloaded. Some of these proteins are themselves nephrotoxic, while others are carriers of nephrotoxic molecules. Megalin is involved in the proximal tubular uptake of these proteins. We hypothesize that megalin‐mediated metabolic overload in PTC leads to compensatory cellular hypertrophy and sustained Na+ reabsorption, causing systemic hypertension and glomerular hyperfiltration via tubuloglomerular feedback, and named this as ‘protein metabolic overload hypothesis’. Impaired metabolism o...
Vibrio cholerae are divided into several serogroups based on their O antigens. Epidemic cholera i... more Vibrio cholerae are divided into several serogroups based on their O antigens. Epidemic cholera is caused by V. cholerae strains belonging to serogroup 0: 1. That serogroup is further divided into three serotypes : Ogawa, Inaba, and Hikojima. Sakazaki and Tamura (6) studied these O antigen variations by cross agglutininabsorption test and found that somatic antigen fractions a and c are common to the three serotypes, and that fraction b, which is absent in type Inaba, can be used as a specific factor for recognizing type Ogawa. They also reported that type Ogawa possessed a small amount of antigen fraction c. Diagnostic antisera produced by immunization of rabbits are now available for identification of V. cholerae 0: 1 and its serotypes. Because type Ogawa possesses type-specific antigen b, anti-b serum is prepared by cross-absorption of the polyvalent Ogawa antiserum with bacteria of type Inaba. Anti-c serum is prepared by crossabsorption of polyvalent antiserum to type Inaba with Vibrio of type Ogawa. But, because type Ogawa has a little antigen c, the titer of fraction c antibody decreases by cross-absorption of polyvalent Inaba antiserum with bacteria of type Ogawa. No serum for group-specific antigen a has yet been prepared by this procedure. This paper describes experiments in which we successfully prepared monoclonal antibodies against fraction a, b, and c, and have developed a latex agglutination test for identification of V. cholerae types. V. cholerae NIH41 (type Ogawa) and NIH35A3 (type Inaba) were cultivated overnight on nutrient agar and killed by adding formalin to reach a final concentration of 1.0 v/v%. The Vibrio were washed twice in phosphate buffered saline (PBS),
In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among t... more In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture‐based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin‐detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml. In our study of previously characterized Shigatoxigenic and all non‐Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non‐Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stoo...
Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raise... more Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in in...
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Papers by Fumio Gondaira