Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium that dominantly produces P44 outer membrane proteins encoded by the p44/msp2 multigene family, which are major antigens for serodiagnosis. However, A. phagocytophilum antigens from cultures with different cell lines seem to have varying reactivities with sera. In this study, we performed RNA-seq to investigate the P44 expression of A. phagocytophilum propagated in 4 cell lines. In infected HL-60 cells, the P44-2b transcript was predominant in the first RNA-seq analysis (HL-60.1). However, the P44-23 transcript was predominant in the second RNA-seq analysis at 1 month after additional passages (HL-60.2). We further analyzed the P44 expression of A. phagocytophilum cultured in THP-1, NB4, and RF/6A cells through consecutive passages in the same cell lines for 1 year after transferring A. phagocytophilum from infected HL-60 cells to the respective cell lines. In the long-term cultures, P44-18, P44-78, and P44-51 were predominantly transcribed in infected THP-1, NB4, and RF/6A cells, respectively. Therefore, the predominant shifts of different P44-expressing transcripts of A. phagocytophilum might occur during cell culture even in the same cell line at different time points of sample harvest (HL-60.1 and HL-60.2), which may be attributed to host cell adaptation/selection/interaction.

Proper surveillance of Campylobacter jejuni and Campylobacter coli, major pathogens associated with human gastroenteritis, is necessary to tackle the increasing disease burden. To detect these pathogenic species, a variety of PCR assays have been developed. This study examined the sensitivity and specificity of 12 PCR assays targeting 23S rRNA, ceuE, lpxA, hipO, mapA, ask, and cdt genes of C. jejuni and C. coli. The sensitivities of PCR assays were 85.2–100%, and 97–100%, and the specificities were 90.5–100%, and 94.3–100% for the tested C. jejuni (n = 61) and C. coli (n = 33) strains, respectively. Two PCR assays, targeting cdtC and hipO genes, were found to be 100% sensitive and/or specific for all C. jejuni strains, while 3 assays, targeting cdtB, cdtA, and ask genes, were 100% sensitive and/or specific for C. coli strains. However, PCR assays for hipO and ask genes are problematic to conduct simultaneously due to the differences in PCR conditions. Overall, multiplex PCR assays targeting cdtC and cdtB genes, encoding 2 subunits of the same toxin, were concluded to be the most reliable. The results of this study would aid in proper surveillance of C. jejuni and C. coli and adopting intervention strategies in the near future.

Acute diarrheal disease is a major health problem, and the second most common cause of death in children under 5 years of age. Conventional diagnostic methods are laborious, time consuming, and occasionally inaccurate. We used SYBR-Green real-time PCR for the detection of 10 uncommon bacterial pathogens using fecal specimens from acute diarrheal patients. In the SYBR-Green real-time PCR assay, the products formed were identified based on a melting point temperature curve analysis, and the assay was validated with the respective reference strain. In a retrospective study, we tested 1,184 stool specimens previously examined using conventional culture methods. Enterotoxigenic Bacteriodes fragilis was detected in 6.7% of the samples followed by enterotoxigenic Bacillus cereus (5.1%), Clostridium perfringens (3.9%), and Aeromonas hydrophila (3.8%). In the prospective study, A. hydrophila, Staphylococcus aureus, and C. perfringens were predominantly detected in 11 > 5 years of age, using real-time PCR. The real-time PCR assay is comprehensive, rapid, accurate, and well suited for surveillance or diagnostic purposes to detect uncommon bacterial pathogens, and should be useful in initiating appropriate care and thereby reducing patient risk.

This study aimed to identify the species and assess the antimicrobial resistance (AMR) of β-hemolytic streptococci isolated from companion animals in Japan. Strains were isolated from clinical specimens of 131 companion animals that exhibited symptoms in April–May 2017. We identified strains by 16S rRNA sequencing and assessed their antimicrobial susceptibility using the broth microdilution method. AMR genes erm(A)-erm(B)-mef(A) and tet(M)-tet(O)-tet(K)-tet(L)-tet(S) in all isolates were amplified by PCR. 16S rRNA sequencing identified β-hemolytic streptococcal species as Streptococcus canis (n = 117, 89.3%), S. agalactiae (n = 7), S. dysgalactiae subsp. equisimilis (n = 5), S. dysgalactiae subsp. dysgalactiae (n = 1), and S. equi subsp. zooepidemicus (n = 1). Overall AMR rates were 39.7% for minocycline, 19.8% for erythromycin, and 17.6% for clindamycin, with a minimum inhibitory concentration (MIC₉₀) of > 4, > 2, and > 1 μg/mL, respectively. AMR genotyping showed the presence of single or mixed types: erm(B)-mef(A) and tet(M)-tet(O)-tet(L)-tet(S). There was a significant relationship between tetracycline-resistance genotypes and open pus/skin-derived specimens. These observations identify some unique features of β-hemolytic streptococcal isolates from companion animals in Japan, such as the dominant isolation of S. canis and resistance to tetracycline, macrolide, and lincosamide antibiotics, in terms of species identification and AMR properties.

The coverage of rabies vaccinations has been reported at 70–80% of dogs in annual reports. However, there are still outbreaks of rabies among humans and dogs in Thailand, thus indicating the necessity of ensuring seroprevalence in vaccinated dogs and efficacy of human immunization. A cost effective easy competitive enzyme-linked immunosorbent assay (CEE-cELISA) was developed here for monitoring protective immunity against the rabies virus in human and dog serum samples using monoclonal antibody clone 1-46-12, which recognizes a conformational epitope of the rabies G protein. The ELISA plate is coated with the whole viral antigen from a commercial vaccine. The serotiter measured by the CEE-cELISA and by the gold standard assay (rapid fluorescent focus inhibition test), detecting the neutralizing antibody, showed a strong correlation, with an R value of 0.958 and 0.931 in humans and dogs, respectively. These correlations were detected in the serum samples from humans and dogs at antibody concentrations up to 100 and 10 IU/ml, respectively. This CEE-cELISA could be an alternative assay for evaluating mass rabies vaccination rapidly at a low cost as well as for detecting antirabies antibodies in the serum of not only humans but also other animal species.

Nagoya City initiated a public subsidy program for mumps vaccination using either the Torii or Hoshino strains in August 2010. To determine the effects of the program, we used publicly available information from Nagoya City to investigate the changes in immunization rates and numbers of patients who developed post-immunization adverse reactions, including post-vaccinal aseptic meningitis, in the 7 years since its initiation. We also investigated the numbers of mumps patients reported by sentinel sites in a national database during this period. The immunization rate in one-year-old children increased from 24.3% before the program to 91.0% after 7 years. The mean numbers of reported mumps cases per sentinel site in one-year-old to preschool children–the age groups targeted by the program– were 12.9 in the 7 years before the program and 4.93 in the 7 years after initiation of the program, showing a significant decrease of 1/2.6 (p = 0.01). The number of vaccinations during the 6.5-year period was 140,316, with only one case of aseptic meningitis reported (0.7 cases/100,000 vaccinations). No other serious adverse reactions were observed. The present findings demonstrate that the public subsidy program in Nagoya City is an effective and safe measure against mumps in children.

Loop-mediated isothermal amplification (LAMP) was assessed for rapid identification of Mycobacterium tuberculosis complex (MTC) in comparison with an immunochromatographic test (ICT) using SD Bioline Ag MPT64 Rapid^®. One hundred and fifty-one MGIT cultures positive for acid-fast bacilli were tested for MTC. DNA was extracted from a small portion of culture samples by heat lysis and subjected to LAMP analysis. Of these, 144 were positive and 5 were negative by both tests. One culture that was ICT negative but was LAMP positive was confirmed to have a mutation in the mpt64 gene. The agreement was 98.68% (95% confidence interval [CI]: 94.80–99.77), and the kappa value was 0.83% (95% CI: 0.59–1.00). Good correlation results suggested that LAMP assay is a reliable molecular test for rapid identification of MTC and is practical for use in resource-limited, high burden settings.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.

The clinical analysis of cases of bacteremia is valuable. However, limited data on bacteremia are available in Vietnam. We conducted a single-center retrospective surveillance study at the Bach Mai Hospital, Hanoi, Vietnam between 2009 and 2012. In total, 45,366 blood cultures were analyzed. The number of blood cultures per 1,000 patient-days was 9.59 sets. The percentage of solitary blood culture sets was 49.6%, and the rate of positive blood cultures was 13.9%. The major pathogens isolated in adults were coagulase-negative Staphylococcus species (16.7%), followed by Escherichia coli (6.8%), Streptococcus spp. excluding Streptococcus pneumoniae (3.8%), and Staphylococcus aureus (5.2%). Other major pathogens identified were Klebsiella spp. (4.2%) and Acinetobacter spp. (2.2%). The number of blood cultures per 1,000 patient-days was lower and the percentage of solitary blood culture sets higher than that of a Japanese study (9.6 vs. 25.2 and 49.6% vs. 32.8%, respectively). The distribution of microorganisms was unique in terms of the relative predominance of cases of Acinetobacter bacteremia. The percentage of cases of healthcare-associated bacteremia may be relatively high.

Infectious keratitis is a serious ocular infection that can lead to loss of vision. The aim of this study was to investigate the microbiological characteristics of this infection at the University Hospital of Guadalajara (Spain). We retrospectively reviewed all cases diagnosed between January 2010 and December 2016. During the 7-year study period, 297 corneal scrapes corresponding to 298 patients were performed. Antibiotic treatment prior to the culture was administered in 59 cases (19.9%). Contact lens wear was the most common risk factor (33.2%). Bacterial keratitis accounted for 64.6% of cases, viral keratitis for 3.4%, and fungal keratitis for 1%. A total of 241 bacterial strains were identified. Gram-positive isolates represented 87.1%, and gram-negative 12.7%. Coagulase-negative Staphylococcus strains were the most common microorganisms isolated (30.3%). When gram-positive microorganisms were analyzed, the sensitivity prevalence rates for vancomycin (VCM), levofloxacin, gentamicin (GM), and tobramycin (TO) were 99.4%, 84.6%, 87.9%, and 88.3%, respectively. For the gram-negative organisms, the sensitivity prevalence rates for ceftazidime, ciprofloxacin, GM, and TO were 83.3%, 93.5%, 96.3%, and 100%, respectively. Our study revealed strong predominance of gram-positive microorganisms. We suggest empirically treating bacterial keratitis originating in our area with VCM and TO, especially severe bacterial keratitis and pretreated cases in the community without a clinical response.

We hypothesized that quick Sequential Organ Failure Assessment (qSOFA) would be associated with 30-day mortality in bacteremia caused by extended-spectrum β-lactamase (ESBL)-producing bacteria and might be a selection criterion for the use of carbapenem as initial empirical therapy. A multicenter retrospective study was conducted in six hospitals. All patients who had bacteremia due to ESBL-producing bacteria were included in the study. Multivariable logistic regression analysis was performed to analyze 30-day mortality as the main outcome. A total of 203 adult patients were identified with bacteremia caused by ESBL-producing Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis. In multivariate logistic regression analysis, bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis (odds ratio [OR] 5.07, 95% confidence interval [CI] 1.64–15.56), underlying liver disease (OR 3.38, 95% CI 1.09–10.00), and underlying solid cancer (OR 3.45, 95% CI 1.27–9.69) were associated with 30-day mortality. In a subgroup analysis, empirical non-carbapenem therapy was associated with 30-day mortality in bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis. Our results suggest that the qSOFA score is not a selection criterion for the use of carbapenem in initial empirical therapy.

From 2005 to July 6, 2018, a total of 435 swine-origin influenza A H3N2 variant virus (H3N2v) infections in humans were reported in the USA. The largest H3N2v outbreak in the USA occurred in 2011–2012. This virus obtained the HA gene from the human seasonal H3N2 influenza A viruses (seasonal H3N2) via human-to-swine transmission in the mid-1990s and was classified as Cluster IV H3N2v. For early detection of public health threats associated with Cluster IV H3N2v distinct from seasonal H3N2, we developed highly specific and sensitive one-step real-time RT-PCR assays directly targeting the HA genes of Cluster IV H3N2v and seasonal H3N2. These assays are useful for the systematic surveillance and identification of Cluster IV H3N2v.

Invasive fungal infections are one of the vital complications among acute leukemia patients undergoing induction chemotherapy. Among them, Geotrichum clavatum infections present extremely rarely with atypical clinical symptoms which make them difficult to diagnose. In this paper, we report a case of infection caused by Geotrichum clavatum in a 10-year old child with acute leukemia, which is the first documented case from mainland China. With underlying childhood leukemia, the child suffered from recurrent bacterial and fungal infection and even underwent abdominal surgery during the treatment. Fortunately, the therapeutic effect was finally achieved by adjusting the treatment program to dual anti-fungal treatment with micafungin and amphotericin B. Information regarding the epidemiological, clinical, and therapeutic features, in this case, shows significant perspectives for anti-fungal treatment for immunocompromised individuals, wherefore the rate of recovery and survival can be achieved.