The aim of this study was to investigate the in vitro activities of polymyxin B (PB) and rifampin (RIF) in combination with ampicillin/sulbactam (AS) or cefoperazone/sulbactam (CS) against 20 multidrug-resistant Acinetobacter baumannii (MDR-AB) isolates by the checkerboard and E-test methods. Fractional inhibitory concentration index (FICI) values were defined as synergy, FICI ≤ 0.5; additivity, 0.5 < FICI ≤ 1.0, indifference, 1.0 < FICI < 4.0; and antagonism, FICI ≥ 4. Synergistic interaction was detected only for the RIF + AS and RIF + CS combinations. While the most frequently detected interaction type for PB + AS or PB + CS combinations was indifference, some showed antagonistic interactions. The detection rate of synergy was significantly higher by the checkerboard than by the E-test method, and the detection rate of indifference was significantly higher by the E-test than by the checkerboard method for RIF + AS combination (P ≤ 0.0001). In addition, no statistically significant difference was detected between the checkerboard and E-test methods for the detection rates of interaction types for any of the other combinations (P > 0.05), except for PB + CS combination for the detection of additivity (P = 0.018). Owing to the high percentage of synergistic interactions between RIF and AS, we considered this combination as an effective therapeutic option for MDR-AB infections.

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Immunological discordance in HIV-infected patients is associated with a higher risk of mortality and disease progression. Zinc is an essential micronutrient for immune function. A two-phase pilot study including a cross-sectional study to determine plasma zinc levels and a prospective, randomized, placebo-controlled trial to determine immunological responses after zinc supplementation was conducted in HIV-infected patients with immunological discordance in a medical school hospital. Immunological discordance was defined in patients who received antiretroviral therapy, had HIV RNA < 40 copies/mL, and a CD4⁺ cell count < 200 cells/mm³ that increased <30% from baseline after receiving ART with undetectable HIV RNA for 12 months. Of 31 patients, 12 (39%) had low plasma zinc levels (<75 μg/dL). Five of 12 patients with low plasma zinc levels and 8 of 19 patients with normal plasma zinc levels were randomized to receive zinc supplementation. The median changes in plasma zinc levels after supplementation versus placebo in patients with low plasma zinc levels were 29 versus 4.5 μg/dL, respectively. The CD4⁺ cell count significantly increased (176 versus 250 cells/mm³, P = 0.042) after zinc supplementation in patients with low plasma zinc levels. Further large-scale studies to determine long-term benefits of zinc supplementation in patients with immunological discordance are required.

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Mycobacterium simiae has been reported worldwide, particularly from the Middle East. This organism has been recognized as a causative agent of pulmonary and disseminated infections. In this study, we used molecular methods to detect this organism from patients who were suspected of having tuberculosis. A total of 117 isolates of mycobacteria were evaluated from different regions of Iran. Isolates were identified using phenotypic methods and gene sequencing of 16S rRNA, rpoB, hsp65, and ITS. Of the 117 isolates, 12 M. simiae isolates (10.2%) were identified from different clinical samples, including bronchoalveolar lavage and sputum (n = 8), blood (n = 3), and lymph node biopsy (n = 1). Three isolates (3/12, 25%) were recovered from blood samples of HIV cases when the CD4⁺ count was less than 50/μl. There was no significant relationship between infection and age or gender. Infection with nontuberculous mycobacteria (NTM), including M. simiae, is the major problem among immunocompromised patients. The results of this study illustrated the importance of molecular methods for accurate and rapid detection of NTM infections in the treatment of nonresponding patients with suspected tuberculosis.

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There is a large amount of information available regarding the chemical structure and biological activity of superoxide dismutase (SOD), which is abundantly generated by Mycobacterium tuberculosis in the early stages of growth. SOD is a strong superoxide radical scavenger, which plays a significant role in resisting oxidative stress. On the other hand, SOD mutant strains have been constructed to define the role of this molecule in the immune response to M. tuberculosis infection. These studies have suggested that the absence or attenuation of SOD can motivate innate immunity and SOD avoid destruction or growth inhibition of M. tuberculosis. For detailed investigation of how SOD proteins aid M. tuberculosis survival within macrophages, we cloned 2 SOD genes (SODA and SODC) from the M. tuberculosis H37Rv genome, overexpressed, identified, and purified the proteins, and then exposed macrophages to the proteins. Following this, we assessed NO production, the secretion of cytokine interferon-γ (IFN-γ) and intercellular adhesion molecule-1, the expression of IFN-γ receptor and Toll-like receptor 2 on the surface of macrophages, and caspase-3 enzyme activity as well as macrophage apoptosis. Our results showed that both SODA and SODC proteins considerably reduced the production of NO and oxygen radicals and impaired cell immunologic function in early infection.

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The association between Helicobacter pylori infection and tooth loss has been studied in Western countries; however, this relationship remains controversial. Although the prevalence of H. pylori infection is higher and atrophic gastritis is usually observed in patients with H. pylori infection in Japan unlike that in Western population, no study has examined the association between H. pylori infection and tooth loss. We examined 959 healthy adults who participated in a mass survey. We counted the number of residual teeth and measured both H. pylori stool antigen and serum anti-H. pylori antibodies. Serum pepsinogen levels were measured to determine the presence of atrophic gastritis. Multiple logistic regression analysis was performed after adjustments for age, body mass index (BMI), smoking and alcohol habits, and educational background. In men, H. pylori infection was a significantly reduced risk factor for loss of all the teeth even after adjustments for other factors (OR, 0.32; 95% CI, 0.12–0.81; P < 0.05). However, such an association was not found in women (0.91; 0.49–1.69). The calculated OR for the presence of atrophic gastritis among individuals with tooth loss was not significant in both men and women. H. pylori infection was associated with a decreased risk of tooth loss in healthy Japanese men.

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The aim of the present study was to evaluate renal involvement in children with Crimean-Congo hemorrhagic fever (CCHF). Forty-four children infected with CCHF virus and 30 controls were enrolled in the study. Urine neutrophil gelatinase-associated lipocalin (uNGAL) and urine protein levels were measured in the patient and control groups. Clinical and laboratory findings of the patient and control groups were compared. uNGAL levels were higher in the patient group than that in the control group (P < 0.001). Of the 44 patients, 26 (59.1%) were proteinuric. uNGAL levels in proteinuric patients were higher than those in non-proteinuric patients (P = 0.035). There was a positive correlation between uNGAL and urine protein levels in the patient group. (R = 0.614, P < 0.001). Due to renal involvement, increased proteinuria and increased uNGAL levels were observed in children with CCHF. In these children, measuring urine total protein and uNGAL levels can be useful to monitor renal involvement due to CCHF.

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To evaluate the influence of vaccination dose and clinico-demographical factors on immune status against measles, rubella, mumps, and varicella viruses among university students, we conducted a case-control study by analyzing serum antibody titers according to past immunization and infection, and perinatal histories, using a multivariate regression model. A total of 1370 medical, paramedical, and pharmaceutical students were included in the analysis. Two or more doses of measles and rubella vaccination yielded notably greater odds ratios for immuno-positivity (9.1; 95% confidence interval (CI), 2.8–28.9 and 12.2; 95% CI, 0.71–210.3, respectively), compared with 1-dose vaccination, even though the superiority did not reach statistical significance for rubella. Students having younger/older siblings were more likely to be immuno-positive for mumps (2.5; 95% CI, 1.3–4.9 and 2.7; 95% CI, 1.4–5.5, respectively). On the other hand, post-term birth or macrosomia was associated with seronegative rubella virus antibodies. We concluded that a 2-dose vaccination strategy could successfully prevent measles and rubella outbreaks by increasing immunity.

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Bacterial meningitis is a serious threat to global health, particularly in developing countries. The aim of this study was to determine the etiological agents of acute bacterial meningitis, its clinical features, and antibacterial susceptibility among Iranian children who were admitted to a referral regional children's hospital. Laboratory data as well as symptoms and signs on admission, organism identification and antibiotic susceptibility results, physical examination findings, and neurologic features of 31 patients with suspected invasive bacterial infection were evaluated, of which 20 (64.5%) had positive cerebrospinal fluid (CSF) cultures. The microorganisms most commonly associated with bacterial meningitis in children were Streptococcus pneumoniae (7 isolates, 22.5%), Haemophilus influenzae (3 isolates, 10%), and Neisseria meningitidis (3 isolates, 10%). In our study, the rate of mortality and sequelae were 10% and 35%, respectively. Ventriculomegaly hydrocephalus was found in 1 case positive for Mycobacterium tuberculosis meningitis, while other signs of meningitis were negative. According to our results, abnormalities including brain edema, subdural effusion, microabscess, and ventriculomegaly hydrocephalus were observed in 50% of the patients with confirmed bacterial meningitis; therefore, identification of predictors of early neurological complications is highly recommended. In addition, CSF culture using the BACTEC automated system is recommended for detection of the etiologic agents of bacterial meningitis, particularly in a population like ours, in which the patients receive a course of antibiotics without prescription.

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This study aimed to determine the frequency of isolation and prevalence of drug resistance in nonfermenting Gram-negative bacilli (NFGNB) other than Pseudomonas aeruginosa and predisposing factors for the acquisition of nosocomial infections caused by these emerging pathogens in a Saudi tertiary care hospital. A total of 125 nonduplicating NFGNB nosocomial strains were isolated, of these, 68 (54.4%) were Acinetobacter baumannii, 26 (20.8%) Stenotrophomonas maltophilia, 14 (11.2%) Alcaligenes faecalis, 12 (9.6%) Chryseobacterium indologenes, and 5 (4%) Ralstonia pickettii. MICs of 11 antibiotics were determined using the reference broth microdilution method. With the exception of colistin that inhibited 100% of A. baumannii isolates, trimethoprim/sulfamethoxazole that inhibited 100% of S. maltophilia isolates, and carbapenems that inhibited 100% of A. faecalis isolates, none of the tested antimicrobial agents inhibited 100% of the other NFGNB spp. Our results emphasize that clinicians and microbiologists should consider A. faecalis, C. indologenes, and R. pickettii as emerging nosocomial pathogens. In addition, local resistance data are essential for helping physicians in deciding an appropriate antibiotic for empirical therapy of infections with these emerging and unusual NFGNB.

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The recent increase in gram-negative bacteria coproducing multiple classes of β-lactamases has made it difficult to accurately identify metallo-β-lactamase (MBL) producers. In the present study, six methods for detecting MBL producers were compared using 56 gram-negative bacterial isolates that produce various β-lactamases. Sodium mercaptoacetic acid (SMA) and EDTA were used as inhibitors for a double-disk synergy test (DDST), and antimicrobial agents, including ceftazidime (CAZ), imipenem, and meropenem (MEPM), along with the distance between the disks, were compared. The Etest^®, dry-plate DPD1^®, Cica-Beta^®, and modified Hodge test were also compared. Among the six methods compared, DDST using the SMA disk showed the highest sensitivity (Se) and specificity (Sp). In DDST, the clearest appearance of growth inhibition was observed when the distance between the disks was maintained at approximately 5 mm. A combination of CAZ and SMA successfully detected only MBL-producing isolates (Se, 87.5%; Sp, 100%), and MEPM exhibited the best performance in combination with SMA in the detection of MBL producers coproducing other classes of β-lactamases such as CTX-M- and CMY-type enzymes (Se, 79.1%; Sp, 100%). DDST using SMA and CAZ and/or MEPM is a simple, specific, and cost-effective method to screen MBL producers in routine clinical laboratory testing.

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Thus far, Aeromonas aquariorum infection has been unrecorded in Korea. Herein, we report a fatal case of A. aquariorum infection in a 77-year-old male patient with liver cirrhosis. The bacterium isolated from a blood culture was initially mistaken as Aeromonas hydrophila using the Vitek2 identification system. In spite of intravenous ceftriaxone therapy, the patient was exacerbated by multiple organ dysfunction. By 4 days after admission, there was no hope for treatment or remission of symptoms and the patient was discharged. In the detailed microbiological investigations, the bacterium was identified as A. aquariorum harboring the act and alt genes, which encode cytotoxic and cytotonic enterotoxins.

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Infections due to Prototheca spp. are ubiquitous in nature, occurring in both immunocompetent and immunocompromised patients. The study cohort consisted of 14 cases of Prototheca algaemia reported over the past 5 decades and 2 recent cases from study hospitals. Prototheca wickerhamii was the most common species. The overall mortality rate was 62.5%. Prototheca algaemia, a healthcare-associated infection, was observed in immunocompromised patients and was associated with a poor prognosis.

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The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(−) virus]. The Ch(−) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(−) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.

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We describe our laboratory investigation of a massive foodborne outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 that occurred during a 2-day traditional festival held in September 2012 in Osaka Prefecture, Japan. Of 126 customers who patronized a particular Japanese restaurant during the event, 102 developed symptoms of gastrointestinal disease. We isolated strains of ETEC serotype O169:H41 from 1 food sample and from fecal samples collected from 19 of 34 patients and 2 of 4 food handlers. Pulsed-field gel electrophoresis analysis of these isolates suggested that the foodborne pathogen that caused the diarrheal outbreak was a specific clone of ETEC serotype O169:H41. Based on these findings and our interviews with the restaurant owner and employees, we concluded that a likely cause of the outbreak was an overwhelmed capacity of the restaurant kitchen in terms of preservation of sanitary procedures during the festival and the inability of the restaurant staff to handle the relatively large quantity of food to ensure a lack of contamination with ETEC. Thus, we reconfirm that ETEC strains of serotype O169:H41 remain important causes of domestic foodborne outbreaks in developed countries, including Japan.

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The antibiotic susceptibilities of 36 isolates of Japanese Francisella tularensis, an etiological agent of the zoonotic disease tularemia, were analyzed using the E test. All the isolates were susceptible to ciprofloxacin, doxycycline, erythromycin, and gentamicin but resistant to benzylpenicillin and cephalothin. The susceptibility to seven other β-lactams (aztreonam, cefotaxime, cefoxitin, ceftriaxone, cefuroxime, imipenem, and meropenem) varied among the isolates. These findings suggest that the guidelines for the antibiotic treatment of tularemia issued by the World Health Organization are appropriate for Japanese tularemia patients.

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Coxsackievirus B3 (CVB3) is an important human pathogen, which is frequently associated with aseptic meningitis and encephalitis in many parts of the world. However, the seroprevalence of this viral infection in China is not well understood; therefore, in the present study, we conducted a serological survey in which 373 serum samples obtained from healthy people belonging to 10 age groups in Yantai city, China were examined. The results revealed that CVB3 is widely distributed in the population with a seroprevalence of 52.3%. The seroprevalence of CVB3 was lowest (3.7%) in the group aged 8–12 months and >50% in all groups aged >15 years. A significant increase (P < 0.001) was observed among the successive four groups aged 1, 2–4, 5–7, and 8–10 years. Variation of geometric mean titer related to age showed similar tendencies. Our results indicated that CVB3 infections occurred mostly in preschool and early-elementary school settings, and children <5 years of age are the most susceptible populations due to their low CVB3 antibody levels.

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Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for timely treatment with effective antibiotics; however, PCR-based methods are often too expensive and technologically intensive for general use in clinical practice. In this study, the efficacy of the loop-mediated isothermal amplification (LAMP) assay for diagnosis of M. pneumoniae pneumonia in clinical practice was prospectively evaluated. From July 2011 to March 2012, 531 children hospitalized for community-acquired pneumonia were enrolled. In all patients, throat swabs were obtained on admission for the detection of M. pneumoniae DNA, and paired serum samples were obtained to assay M. pneumoniae particle agglutination (PA) antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or an increase of 4-fold or greater in the PA titer. Overall, 271 children (51.0% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia. Among these, 258 (95.2%) and 248 (91.5%) were identified by the LAMP assay and serological tests, respectively. When the results of serological tests were considered as standard, the sensitivity, specificity, and positive and negative predictive values of the LAMP assay were 94.8%, 91.9%, and 91.1% and 95.2%, respectively. The median duration of pharyngeal carriage, as measured by the LAMP assay, was 9.5 days. Thus, the LAMP assay is useful in the rapid diagnosis of M. pneumoniae pneumonia.

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Tularemia is a zoonotic disease caused by Francisella tularensis. Most patients in Japan have reportedly acquired such infections through direct contact with infected Japanese hares. We recently encountered a patient who contracted tularemia after skinning and butchering a dead hare. Because the remains of the hare were available, we attempted to determine whether the patient actually contracted infection by handling the carcass. F. tularensis-specific sequences were successfully amplified by PCR from the patient specimens as well as from the remnants of discarded hare carcass. PCR amplification of the ISFtu2 and RD1 regions indicated infection by F. tularensis subsp. holarctica, which was considered as a prevalent strain in Japan. Furthermore, high-resolution multiple-locus variable-number tandem repeat analysis (MLVA) showed that the combination of repeat numbers in sequences from the patient and hare were indistinguishable, thus indicating that the patient had been infected with F. tularensis strain that had also infected the hare. These findings demonstrated that MLVA is a useful epidemiological investigational tool to identify possible sources of certain zoonotic diseases such as tularemia.

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Streptococcus agalactiae (group B Streptococcus, GBS) is the leading cause of neonatal sepsis and meningitis and an important pathogen in elderly patients and those with underlying diseases. The diagnosis of GBS infections is primarily based on culture of GBS. Some clinical laboratories perform the Christie-Atkins-Munch-Peterson (CAMP) test for discrimination of GBS from other streptococci. Here, we developed a rapid GBS identification method, i.e., the loop-mediated isothermal amplification (LAMP) method for detecting the cfb gene encoding the CAMP factor. This method detected at least 4 copies of the cfb gene in GBS under isothermal conditions with in a short time (65℃, within 90 min). No inappropriate amplification of nucleotide by this method was observed when the chromosomal DNA of 17 streptococci and enterococci species, other than GBS, were used as templates. In this investigation, we successfully developed a LAMP method for rapid and highly sensitive detection of GBS, which provides a beneficial alternative to the conventional CAMP test.

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Antibody distribution to a novel swine-origin influenza A (H3N2) variant virus among different age groups in Japan was determined by hemagglutination-inhibition assay. Japanese children in the age group 1–14 years were at a higher risk than those in older age groups.

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