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1.  Clinical Specimen-Direct LAMP: A Useful Tool for the Surveillance of blaOXA-23-Positive Carbapenem-Resistant Acinetobacter baumannii 
PLoS ONE  2015;10(7):e0133204.
Healthcare-associated infections are a leading cause of morbidity and mortality worldwide. Treatment is increasingly complicated by the escalating incidence of antimicrobial resistance. Among drug-resistant pathogens, carbapenem-resistant Acinetobacter baumannii (CRAb) is of increasing concern because of the limited applicable therapies and its expanding global distribution in developed countries and newly industrialized countries. Therefore, a rapid detection method that can be used even in resource-poor countries is urgently required to control this global public health threat. Conventional techniques, such as bacterial culture and polymerase chain reaction (PCR), are insufficient to combat this threat because they are time-consuming and laborious. In this study, we developed a loop-mediated isothermal amplification (LAMP) method for detecting blaOXA-23-positive CRAb, the most prevalent form of CRAb in Asia, especially in Thailand, and confirmed its efficacy as a surveillance tool in a clinical setting. Clinical samples of sputum and rectal swabs were collected from patients in a hospital in Bangkok and used for LAMP assays. After boiling and centrifugation, the supernatants were used directly in the assay. In parallel, a culture method was used for comparison purposes to evaluate the specificity and sensitivity of LAMP. As a first step, a total of 120 sputum samples were collected. The sensitivity of LAMP was 88.6% (39/44), and its specificity was 92.1% (70/76) using the culture method as the “gold standard”. When surveillance samples including sputum and rectal swabs were analyzed with the LAMP assay, its sensitivity was 100.0%. This method enables the direct analysis of clinical specimens and provides results within 40 minutes of sample collection, making it a useful tool for surveillance even in resource-poor countries.
PMCID: PMC4517775  PMID: 26218925
2.  Pneumococcal polysaccharide vaccination in rheumatoid arthritis patients receiving tacrolimus 
In rheumatoid arthritis (RA) patients receiving immunosuppressive treatments, vaccination against Streptococcus pneumoniae is recommended. The objective of the study was to evaluate the effects of tacrolimus (TAC) on immune response following administration of a 23-valent pneumococcal polysaccharide vaccine (PPSV23) in patients with established RA.
Patients with RA (n = 133) were vaccinated with PPSV23. Patients were classified into TAC (n = 29), methotrexate (MTX) (n = 55), control (n = 35), and TAC/MTX (n = 14) treatment groups. We measured the concentrations of pneumococcal serotypes 6B and 23F by using an enzyme-linked immunosorbent assay and determined antibody functionality by using a multiplexed opsonophagocytic killing assay, reported as the opsonization index (OI), before and 4 to 6 weeks after vaccination. A positive antibody response was defined as at least a twofold increase in the IgG concentration or as at least a 10-fold increase in the OI.
IgG concentrations and OIs were significantly increased in all treatment groups after PPSV23 vaccination. The TAC treatment group appears to respond in a manner similar to that of the RA control group in terms of 6B and 23F serotype concentration and function. In contrast, the MTX group had the lowest immune response. Patients who received a combination of TAC and MTX (TAC/MTX) also had a diminished immune response compared with those who received TAC alone.
TAC monotherapy does not appear to impair PPSV23 immunogenicity in patients with RA, whereas antibody production and function may be reduced when TAC is used with MTX. Thus, PPSV23 administration during ongoing TAC treatment should be encouraged for infection-prone TAC-treated patients with rheumatic diseases.
Trial registration
University Hospital Medical Information Network Clinical Trials Registry: UMIN000009566. Registered 12 December 2012.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0662-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4481124  PMID: 26036592
3.  A Bivalent Vaccine Based on a Replication-Incompetent Influenza Virus Protects against Streptococcus pneumoniae and Influenza Virus Infection 
Journal of Virology  2014;88(22):13410-13417.
Streptococcus pneumoniae is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases.
IMPORTANCE Streptococcus pneumoniae and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or Streptococcus pneumoniae. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform for the development of bivalent vaccines.
PMCID: PMC4249093  PMID: 25210171
4.  Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load 
Journal of Clinical Microbiology  2014;52(9):3325-3333.
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.
PMCID: PMC4313158  PMID: 24989600
5.  Molecular evolution of the VP1, VP2, and VP3 genes in human rhinovirus species C 
Scientific Reports  2015;5:8185.
Human rhinovirus species C (HRV-C) was recently discovered, and this virus has been associated with various acute respiratory illnesses (ARI). However, the molecular evolution of the major antigens of this virus, including VP1, VP2, and VP3, is unknown. Thus, we performed complete VP1, VP2, and VP3 gene analyses of 139 clinical HRV-C strains using RT-PCR with newly designed primer sets and next-generation sequencing. We assessed the time-scale evolution and evolutionary rate of these genes using the Bayesian Markov chain Monte Carlo method. In addition, we calculated the pairwise distance and confirmed the positive/negative selection sites in these genes. The phylogenetic trees showed that the HRV-C strains analyzed using these genes could be dated back approximately 400 to 900 years, and these strains exhibited high evolutionary rates (1.35 to 3.74 × 10−3 substitutions/site/year). Many genotypes (>40) were confirmed in the phylogenetic trees. Furthermore, no positively selected site was found in the VP1, VP2, and VP3 protein. Molecular modeling analysis combined with variation analysis suggested that the exterior surfaces of the VP1, VP2 and VP3 proteins are rich in loops and are highly variable. These results suggested that HRV-C may have an old history and unique antigenicity as an agent of various ARI.
PMCID: PMC4313092  PMID: 25640899
6.  Short-term Prediction of the Incidence of Congenital Rubella Syndrome 
PLoS Currents  2014;6:ecurrents.outbreaks.8c74272f4348781c5d01c81e6150c2f7.
Objectives In Japan, a rubella outbreak occurred from early 2012 to late 2013, primarily among adult males aged 20–49 years. We conducted this study to predict the number of congenital rubella syndrome (CRS) cases in Japan in 2014. Methods The probability of CRS when a pregnant woman is infected with rubella depends on the gestational age of the fetus. The cumulative number of CRS cases was predicted by a formula based on the parameters from two studies conducted in the U.K. and the U.S., the reported cases of rubella among women 15–49 years of age, and the reports of CRS from 2011 to week 2 of 2014. Findings While the predicted number of cases of CRS based on parameters from the U.K. study demonstrated a biphasic curve, with a low peak around week 12 and a high peak around week 50 of 2013, the predicted number of CRS cases based on the U.S. study demonstrated a single peak around week 50 of 2013. The ex post evaluation indicated that the cumulative number of CRS cases in 2014 would be 19.1–29.3. Interpretation Our prediction of the number of CRS cases may be useful for the enhanced detection of this often under-reported syndrome.
PMCID: PMC4234431  PMID: 25642379
congenital rubella syndrome; public health policy; rubella; short-term prediction; Surveillance
7.  Presence of Neutrophil Extracellular Traps and Citrullinated Histone H3 in the Bloodstream of Critically Ill Patients 
PLoS ONE  2014;9(11):e111755.
Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5–76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in “the presence of bacteria in tracheal aspirate” group (11/22, 50.0%) than in “the absence of bacteria in tracheal aspirate” group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.
PMCID: PMC4230949  PMID: 25392950
8.  Molecular Phylogenetic Analysis of Orientia tsutsugamushi Based on the groES and groEL Genes 
Vector Borne and Zoonotic Diseases  2013;13(11):825-829.
DNA sequences encoding the GroES and GroEL proteins of Orientia tsutsugamushi were amplified by the PCR and sequenced. Pairwise alignment of full-length groES and groEL gene sequences indicated high sequence similarity (90.4–100% and 90.3–100%) in O. tsutsugamushi, suggesting that these genes are good candidates for the molecular diagnosis and phylogenetic analysis of scrub typhus. Comparisons of the 56-kD type-specific antigen (TSA) protein gene and the groES and groEL genes showed that genotypes based on the 56-kD TSA gene were not related to a cluster containing the groES and groEL genes in a dendrogram, suggesting that a gene rearrangement may be associated with homologous recombination in mites.
PMCID: PMC3822374  PMID: 24107204
Orientia tsutsugamushi; groES and groEL genes; Phylogeny; Scrub typhus; Japan
10.  Global Control of Pneumococcal Infections by Pneumococcal Vaccines 
Tropical Medicine and Health  2014;42(2 Suppl):83-86.
Streptococcus pneumoniae is a major worldwide cause of morbidity and mortality. Pneumococcal carriage is considered to be an important source of horizontal spread of this pathogen within the community. Pneumococcal conjugate vaccine (PCV) is capable of inducing serotype-specific antibodies in sera of infants, and has been suggested to reduce nasopharyngeal carriage of vaccine-type pneumococci in children. PCV is generally immunogenic for pediatric patients with invasive pneumococcal disease, with an exception for the infecting serotypes. Based on evidences from the clinical trials of PCV, the health impact of childhood pneumococcal pneumonia appears to be high in developing countries where most of global childhood pneumonia deaths occur. PCV vaccination may prevent hundreds of deaths per 100,000 children vaccinated in developing countries, while PCV vaccination is expected to prevent less than 10 deaths per 100,000 children vaccinated in the developed countries. Therefore, the WHO has proposed a strategy to reduce the incidence of severe pneumonia by 75% in child less than 5 years of age compared to 2010 levels by 2025.
PMCID: PMC4204060  PMID: 25425955
Streptococcus pneumoniae; Bacterial colonization; Invasive pneumococcal disease; Pneumococcal conjugate vaccine; Serotype-specific IgG; Opsonization index; Childhood pneumonia; WHO
12.  Estimating the Risk of Parvovirus B19 Infection in Blood Donors and Pregnant Women in Japan 
PLoS ONE  2014;9(3):e92519.
Seroepidemiological study of parvovirus B19 has not taken place for some 20 years in Japan. To estimate the risk of parvovirus B19 infection in Japan among blood donors and pregnant women in this century, a seroepidemiological survey and statistical modeling of the force of infection were conducted.
Methodology/Principal Findings
The time- and age-specific seroprevalence data were suggestive of strong age-dependency in the risk of infection. Employing a piecewise constant model, the highest forces of infection of 0.05 and 0.12 per year were observed among those aged 0–4 and 5–9 years, respectively, while estimates among older individuals were less than 0.01 per year. Analyzing the antigen detection data among blood donors, the age-specific proportion positive was highest among those aged 30–39 years, agreeing with the presence of dip in seroprevalence in this age-group. Among pregnant women, up to 107 fetal deaths and 21 hydrops fetalis were estimated to have occurred annually across Japan.
Seroepidemiological profiles of PVB19 infection in Japan was characterized with particular emphasis on the risk of infection in blood donors and the burden of infection among pregnant women. When a vaccine becomes available in the future, a similar seroepidemiological study is expected to play a key role in planning the appropriate immunization policy.
PMCID: PMC3962423  PMID: 24658180
13.  Low opsonic activity to the infecting serotype in pediatric patients with invasive pneumococcal disease 
Vaccine  2012;31(5):10.1016/j.vaccine.2012.11.010.
Serotype-specific protective immunity of pediatric patients with invasive pneumococcal disease (IPD) has not been fully investigated. To determine the protective immunity to the infecting serotype, the serotype-specific immunoglobulin G (IgG) levels and opsonophagocytic assay (OPA) titers were examined in twenty-four pediatric patients whose serum was collected within one month of IPD episode in between May 2008 and June 2011 in Japan. The median age (range) of IPD patients was 17 (10–108) months and 63% were boys. The levels of serotype-specific IgG to the infecting serotype were higher than 0.2 μg/ml in all of 17 patients tested. By contrast, the OPA titers were < 8 to the infecting serotype in all of 17 patients tested. The avidities of 19F or 6B-specific IgG level higher than 5.0 μg/ml, but undetectable OPA titers, were confirmed to be lower than those with high OPA titers. Our data demonstrated that although the levels of serotype-specific IgG to the infecting serotype were higher than 0.2 μg/ml in sera of pediatric patients with IPD, the OPA titers were low during one month after the IPD episode. Impaired opsonic activities in these patients may be, in part, explained by the low avidities of serotype-specific IgG. Impaired serum opsonic activity in children immediately after the episode of IPD suggests their susceptibility to the infecting serotype of pneumococci.
PMCID: PMC3829722  PMID: 23153440
invasive pneumococcal diseases; serotype-specific IgG; opsonophagocytic activity; pneumococcal vaccine; children
14.  Dectin-2-Dependent NKT Cell Activation and Serotype-Specific Antibody Production in Mice Immunized with Pneumococcal Polysaccharide Vaccine 
PLoS ONE  2013;8(10):e78611.
Although thymus-independent type 2 antigens generally do not undergo Ig class switching from IgM to IgG, pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific IgG. How this happens remains unclear, however. In the present study, PPV immunization induced production of IgG as well as IgM specific for a serotype 3-pneumococcal polysaccharide in the sera of wild-type (WT) mice, but this phenomenon was significantly reduced in Dectin-2 knockout (KO) mice. Immunization with PPV caused IL-12p40 production in WT mice, but this response was significantly reduced in Dectin-2KO mice. Likewise, immunization with PPV activated natural killer T (NKT) cells in WT mice but not in Dectin-2KO mice. Furthermore, administration of α-galactosylceramide, recombinant (r)IL-12 or rIFN-γ improved the reduced IgG levels in Dectin-2KO mice, and treatment with neutralizing anti-IFN-γ mAb resulted in the reduction of IgG synthesis in PPV-immunized WT mice. Transfer of spleen cells from PPV-immunized WT mice conferred protection against pneumococcal infection on recipient mice, whereas this effect was cancelled when the transferred spleen cells were harvested from PPV-immunized Dectin-2KO mice. These results suggest that the detection of PPV antigens via Dectin-2 triggers IL-12 production, which induces IFN-γ synthesis by NKT cells and subsequently the production of serotype-specific IgG.
PMCID: PMC3808275  PMID: 24205278
15.  Cytokine production and signaling pathways in respiratory virus infection 
It has been confirmed that respiratory virus infections can induce abberant cytokine production in the host. These cytokines may be associated with both elimination of the virus and complications in the host, such as virus-induced asthma. Representative host defense mechanisms against pathogens, including bacteria and viruses, are mediated by the innate immune system. Cells of the innate immune system express essential molecules, namely pattern recognition receptors (PRRs), such as Toll-like receptors, nucleotide-binding oligomerization domain-like receptors, and retinoic acid-inducible gene-I-like receptors. These PRRs can recognize components of pathogens such as bacterial lipopolysaccharide, viral antigens, and their genomes (DNA and RNA). Furthermore, PRRs activate various signaling pathways resulting in cytokine production against pathogen infection. However, the exact mechanisms remain unknown. In this review, we mainly focus on the representative mechanisms of cytokine production through PRRs and signaling pathways due to virus infections, including respiratory virus infections. In addition, we describe the relationships between respiratory infections and virus-induced asthma.
PMCID: PMC3774987  PMID: 24062733
cytokine; signaling pathway; respiratory virus; innate immunity; virus-induced asthma
16.  Genetic Analysis of Non-Hydrogen Sulfide-Producing Salmonella enterica Serovar Typhimurium and S. enterica Serovar Infantis Isolates in Japan 
Journal of Clinical Microbiology  2013;51(1):328-330.
Whole-genome sequencing of non-H2S-producing Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 β-lactamase. The lack of production of H2S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistance genes.
PMCID: PMC3536232  PMID: 23135931
18.  Estimation of Influenza Incidence by Age in the 2011/12 Seasons in Japan using SASSy 
So far, it is difficult to show the incidence rate of influenza in the official sentinel surveillance in Japan. Hence we construct the system which record infectious diseases at schools, kindergartens, and nursery schools, and then can show the accurate incidence rate of influenza in children by age/grade.
So as to develop more effective countermeasures against influenza, timely and precise information about influenza activity at schools, kindergartens, and nursery schools may be helpful. At the Infectious Diseases Surveillance Center of the National Institute of Infectious Diseases, a School Absenteeism Surveillance System (SASSy) has been in operation since 2009. SASSy monitors the activity of varicella, mumps, mycoplasma pneumonia, pharyngoconjunctival fever, hand-foot-mouth disease, influenza, and many other infectious diseases in schools. In 2010, SASSy was extended to the Nursery School Absenteeism Surveillance System (NSASSy). These systems record the number of absentees due to infectious diseases in each class of all grades of schools every day. As a powerful countermeasure to the pandemic flu of 2009, SASSy was activated in 9 prefectures, in which included more than 6000 schools, and it is gradually being adopted in other prefectures. As of February 2012, 18 prefectures and 4 big cities, which together comprised 15,700 schools (about 35% of all schools in Japan), utilized SASSy. NSASSy is used in more than 4100 nursery schools, which is about 18% of all nursery schools in Japan. Some studies of similar systems were performed in the UK (1), Hong Kong (2), and the USA (3,4), examined surveillance systems for monitoring infectious disease incidence, but the systems to construct in those studies do not operate nationwide like SASSy or NSASSy, and they cannot provide influenza incidence rates in children.
All schools, kindergartens, and nursery schools in the community, enter data of the absentees due to infectious diseases into the system every day, thereby providing real-time data regarding infectious diseases prevalent in schools, to the schools around, school boards, public health centers, local governments, and medical professionals. It analyzed data for the 2011/2012 season (from September 1, 2011 to March 31, 2012) mainly, but also two seasons (2010/2011 and 2011/2012) were compared in some prefectures. In total, 12 prefectures, which comprised 2,352,839 children, were participated in 2011/2012 season. In the 2010/2011 season, 1,795,766 children of 9 prefectures were analyzed.
The incidence rate in the first grade of elementary schools is the highest both in the two seasons. The highest incidence rate in this grade distributes from 17.8% to 40.3% in 2011/2012 season, and from 11.0% to 30.7% in 2010/2011 season.
This study proved SASSy and NSASSy are quite useful for monitoring of influenza outbreak in schools and it will be gold standard of surveillance for school children in Japan. The present study also showed incidence rate of influenza in children at schools, kindergartens, and nursery schools, and proved the highest incidence was in the first grade of the elementary school. This is the first finding using such the huge number of subjects, which is more than 2 million. The intervention targeting to the weak age/grade is necessary for effective countermeasure and control of influenza and other infectious diseases.
PMCID: PMC3692790
Surveillance; Influenza; School Absenteeism
19.  Population-Based Study of Streptococcus suis Infection in Humans in Phayao Province in Northern Thailand 
PLoS ONE  2012;7(2):e31265.
Streptococcus suis infection in humans has received increasing worldwide recognition.
Methods and Findings
A prospective study of S. suis infection in humans was conducted in Phayao Province in northern Thailand to determine the incidence and the risk behaviors of the disease in this region in 2010. Thirty-one cases were confirmed. The case fatality rate was 16.1%, and the estimated incidence rate was 6.2 per 100,000 in the general population. The peak incidence occurred in May. The median age of the patients was 53 years and 64.5% were men. Consumption of raw pork products was confirmed in 22 cases and the median incubation period (range) was 2 days (0–11) after consumption of raw pork products. Isolates from 31 patients were confirmed as serotype 2 in 23 patients (74.2%) and serotype 14 in eight patients (25.8%). The major sequence types (STs) were ST1 (n = 20) for serotype 2 and ST105 (n = 8) for serotype 14. The epidemiological analysis suggested three possible clusters, which included 17 cases. In the largest possible cluster of 10 cases in Chiang Kham and its neighboring districts in May, the source of infection in four cases was identified as a raw pork dish served at the same restaurant in this district. Microbiological analysis confirmed that three of four cases associated with consumption of raw pork at this restaurant were attributable to an identical strain of serotype 2 with ST1 and pulsotype A2.
Our data suggest a high incidence rate of S. suis infection in the general population in Phayao Province in 2010 and confirm a cluster of three cases in 31 human cases. Food safety control should be strengthened especially for raw pork products in northern Thailand.
PMCID: PMC3283636  PMID: 22363601
21.  The Nasal Dendritic Cell-Targeting Flt3 Ligand as a Safe Adjuvant Elicits Effective Protection against Fatal Pneumococcal Pneumonia▿ 
Infection and Immunity  2011;79(7):2819-2828.
We have previously shown that a pneumococcal surface protein A (PspA)-based vaccine containing DNA plasmid encoding the Flt3 ligand (FL) gene (pFL) as a nasal adjuvant prevented nasal carriage of Streptococcus pneumoniae. In this study, we further investigated the safety and efficacy of this nasal vaccine for the induction of PspA-specific antibody (Ab) responses against lung infection with S. pneumoniae. C57BL/6 mice were nasally immunized with recombinant PspA/Rx1 (rPspA) plus pFL three times at weekly intervals. When dynamic translocation of pFL was initially examined, nasal pFL was taken up by nasal dendritic cells (DCs) and epithelial cells (nECs) but not in the central nervous systems, including olfactory nerve and epithelium. Of importance, nasal pFL induced FL protein synthesis with minimum levels of inflammatory cytokines in the nasal washes (NWs) and bronchoalveolar lavage fluid (BALF). NWs and BALF as well as plasma of mice given nasal rPspA plus pFL contained increased levels of rPspA-specific secretory IgA and IgG Ab responses that were correlated with elevated numbers of CD8+ and CD11b+ DCs and interleukin 2 (IL-2)- and IL-4-producing CD4+ T cells in the nasal mucosa-associated lymphoid tissues (NALT) and cervical lymph nodes (CLNs). The in vivo protection by rPspA-specific Abs was evident in markedly reduced numbers of CFU in the lungs, airway secretions, and blood when mice were nasally challenged with Streptococcus pneumoniae WU2. Our findings show that nasal pFL is a safe and effective mucosal adjuvant for the enhancement of bacterial antigen (Ag) (rPspA)-specific protective immunity through DC-induced Th2-type and IL-2 cytokine responses.
PMCID: PMC3191952  PMID: 21536790
22.  Genotypic Profile of Streptococcus suis Serotype 2 and Clinical Features of Infection in Humans, Thailand 
Emerging Infectious Diseases  2011;17(5):835-842.
To examine associations between clinical features of Streptococcus suis serotype 2 infections in humans in Thailand and genotypic profiles of isolates, we conducted a retrospective study during 2006–2008. Of 165 patients for whom bacterial cultures of blood, cerebrospinal fluid, or both were positive for S. suis serotype 2, the major multilocus sequence types (STs) found were ST1 (62.4%) and ST104 (25.5%); the latter is unique to Thailand. Clinical features were examined for 158 patients. Infections were sporadic; case-fatality rate for adults was 9.5%, primarily in northern Thailand. Disease incidence peaked during the rainy season. Disease was classified as meningitis (58.9%) or nonmeningitis (41.1%, and included sepsis [35.4%] and others [5.7%]). Although ST1 strains were significantly associated with the meningitis category (p<0.0001), ST104 strains were significantly associated with the nonmeningitis category (p<0.0001). The ST1 and ST104 strains are capable of causing sepsis, but only the ST1 strains commonly cause meningitis.
PMCID: PMC3321758  PMID: 21529392
Streptococcus suis; serotype 2; meningitis; sepsis; sequence typing; bacteria; research
23.  CD4-Independent Human Immunodeficiency Virus Infection Involves Participation of Endocytosis and Cathepsin B 
PLoS ONE  2011;6(4):e19352.
During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.
PMCID: PMC3081840  PMID: 21541353
24.  Characteristics of a Large Cohort of Patients with Autoimmune Pulmonary Alveolar Proteinosis in Japan 
Rationale: Acquired pulmonary alveolar proteinosis (PAP) is a syndrome characterized by pulmonary surfactant accumulation occurring in association with granulocyte/macrophage colony-stimulating factor autoantibodies (autoimmune PAP) or as a consequence of another disease (secondary PAP). Because PAP is rare, prior reports were based on limited patient numbers or a synthesis of historical data.
Objectives: To describe the epidemiologic, clinical, physiologic, and laboratory features of autoimmune PAP in a large, contemporaneous cohort of patients with PAP.
Methods: Over 6 years, 248 patients with PAP were enrolled in a Japanese national registry, including 223 with autoimmune PAP.
Measurements and Main Results: Autoimmune PAP represented 89.9% of cases and had a minimum incidence and prevalence of 0.49 and 6.2 per million, respectively. The male to female ratio was 2.1:1, and the median age at diagnosis was 51 years. A history of smoking occurred in 56%, and dust exposure occurred in 23%; instances of familial onset did not occur. Dyspnea was the most common presenting symptom, occurring in 54.3%. Importantly, 31.8% of patients were asymptomatic and were identified by health screening. Intercurrent illnesses, including infections, were infrequent. A disease severity score reflecting the presence of symptoms and degree of hypoxemia correlated well with carbon monoxide diffusing capacity and serum biomarkers, less well with pulmonary function, and not with granulocyte/macrophage colony-stimulating factor autoantibody levels or duration of disease.
Conclusions: Autoimmune PAP had an incidence and prevalence higher than previously reported and was not strongly linked to smoking, occupational exposure, or other illnesses. The disease severity score and biomarkers provide novel and potentially useful outcome measures in PAP.
PMCID: PMC2720118  PMID: 18202348
epidemiology; serum biomarkers; disease severity score; granulocyte/macrophage colony-stimulating factor; autoantibody
25.  Comparative Immune Responses of Patients with Chronic Pulmonary Diseases during the 2-Year Period after Pneumococcal Vaccination▿  
Clinical and Vaccine Immunology  2006;14(2):139-145.
Antibody responses to a 23-valent pneumococcal vaccine for Streptococcus pneumoniae serotypes 6B, 14, 19F, and 23F in 84 patients with chronic pulmonary diseases over a 2-year period after vaccination were examined by using a third-generation enzyme-linked immunosorbent assay. Of these patients, 28 (31%) were low responders who had developed increases of at least twofold in the levels of serotype-specific immunoglobulin G (IgG) in sera for none of the four serotypes at 1 month after vaccination. Although no specific clinical features of low responders were evident, their prevaccination levels of IgG for all serotypes were higher than those of responders. In responders, the levels of IgG specific for serotypes 14 and 23F in sera were greatly increased 1 month after vaccination and those specific for serotypes 6B and 19F were moderately increased. In contrast, no significant increases in the levels of IgG specific for serotypes 6B, 19F, and 23F in the low responders during the same period were found, but the levels of IgG specific for serotype 14 did increase. Although a rapid decline in the levels of IgG for all serotypes in responders between 1 month and 6 months after vaccination was found, the levels of IgG specific for serotypes 14 and 23F in sera remained higher than the prevaccination levels for at least 2 years after vaccination. These data suggest the need for the revaccination of responders but not low responders among patients with chronic pulmonary diseases. Revaccination as early as 3 years postvaccination is recommended for responders to increase the reduced levels of IgG in sera, especially those specific for the weak vaccine antigens.
PMCID: PMC1797796  PMID: 17167035

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