Search

CRE-JU is a novel selective agar for carbapenem-resistant Enterobacteriaceae that contains ceftazidime, cloxacillin, meropenem, and vancomycin. This study evaluated the ability of 63 carbapenem-resistant isolates and 53 non-carbapenem-resistant Enterobacteriaceae strains clinically isolated in Japan, Myanmar, Nepal, and Vietnam to grow on CRE-JU. CRE-JU showed 92.1% sensitivity and 100% specificity for detecting carbapenem-resistant Enterobacteriaceae compared with dug susceptibility profiles.

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa. We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae, Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

Resistance of Mycobacterium tuberculosis to pyrazinamide (PZA) derives mainly from mutations in the pncA gene. We developed a reverse hybridization-based line probe assay with oligonucleotide probes designed to detect mutations in pncA . The detection of PZA resistance was evaluated in 258 clinical isolates of M. tuberculosis . The sensitivity and specificity of PZA resistance obtained by this new assay were both 100%, consistent with the results of conventional PZA susceptibility testing. This assay can be used with sputa from tuberculosis patients. It appears to be reliable and widely applicable and, given its simplicity and rapid performance, will be a valuable tool for diagnostic use.

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis . Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA ( fabG1 )- inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB , eight in katG , one in the mabA-inhA regulatory region, two in embB , five in pncA , and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis . In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis .

We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6′- N -aminoglycoside acetyltransferase gene [ aac(6 ′ )-Iae ]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC ≥ 16 μg/ml), amikacin (MIC ≥ 64 μg/ml), and ciprofloxacin (MIC ≥ 4 μg/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6 ′ )-Iae gene and the AAC(6′)-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with ≥70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6 ′ )-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.

Objective To assess DNA polymorphisms in mycobacterial isolates obtained from human immunodeficiency virus (HIV)-seropositive patients with tuberculosis in Japan from 1996 to 2003. Methods Restriction fragment length polymorphisms (RFLP) from Mycobacterium tuberculosis and Mycobacterium avium isolates obtained from individual seropositive patients with tuberculosis ( n =78) were analysed with the use of IS 6110 and (CGG) 5 or IS 1245 and IS 1311 , respectively, as markers. As a control, the same procedures were applied to isolates from HIV-seronegative tuberculosis patients ( n =87). Results Of 86 mycobacterial strains, M. tuberculosis , M. avium and Mycobacterium chelonae were identified in 48 (55.8%), 36 (41.9%) and 2 (2.3%) isolates, respectively. The obtained RFLP patterns of M. tuberculosis isolates from both the HIV-seropositive and -seronegative groups were variable, suggesting no obvious clustering among the isolates. Similar results were obtained in isolates of M. avium . Conclusions This is the first report on the molecular epidemiology of Mycobacterium spp. isolated from HIV-seropositive patients in Japan. The results indicate that no particular clones of M. tuberculosis or M. avium prevail in HIV-seropositive patients in Japan. Further monitoring of mycobacterial infection associated with HIV infection in Japan should be continued.

Many viruses have been reported to be associated with rash development. Multiplex real-time PCR was used to investigate the presence of 5 viruses associated with rashes: measles virus (MV), rubella virus (RV), human parvovirus B19 (PVB19), human herpes virus 6 (HHV-6), and HHV-7. A total of 187 clinical specimens from 169 patients with erythema were collected between January 2006 and December 2011. Virus-positive specimens were as follows: MV (n = 23), PVB19 (n = 8), RV (n = 2), HHV-6 (n = 5), HHV-7 (n = 1), MV and PVB19 (n = 1), and HHV-6 and HHV-7 (n = 1). All of the MV-positive specimens were collected in 2007 and the strains whose sequence were available (21/24, 87.5%) were of genotype D5. The results indicate that multiplex real-time PCR might be a useful screening method for detecting and differentiating rash-associated viruses in clinical specimens.

Viruses are the major etiological agents of acute respiratory infections (ARIs) in young children. Although respiratory virus co-detections are common, analysis of combinations of co-detected viruses has never been conducted in Japan. Nineteen respiratory viruses or subtypes were surveyed using multiplex real-time PCR on 1,044 pediatric (patient age < 6 years) ARI specimens collected in Osaka City, Japan between January 2010 and December 2011. In total, 891 specimens (85.3%) were virus positive (1,414 viruses were detected), and 388 of the virus-positive specimens (43.5%, 388/891) were positive for multiple viruses. The ratio of multiple/total respiratory virus-positive specimens was high in children aged 0-35 months. Statistical analyses revealed that human bocavirus 1 and human adenovirus were synchronously co-detected. On the other hand, co-detections of human parainfluenza virus type 1 (HPIV-1) with HPIV-3, HPIV-3 with human metapneumovirus (hMPV), hMPV with respiratory syncytial virus A (RSV A), hMPV with influenza virus A (H1N1) 2009 (FLUA (H1N1) 2009), RSV A with RSV B, and human rhinovirus and FLUA (H1N1) 2009 were exclusive. These results suggest that young children (

MRSA-chrom, a novel chromogenic screening agar medium for methicillin-resistant Staphylococcus aureus (MRSA), was developed. There were all MRSA strains recovered in 24h as a specific blue-colored colony among 130 microbes including 42 MRSA strains. MRSA-chrom showed the highest detection ratio among 4 commercially available selective media using 50 clinical specimens.

Enterovirus 68 strains were detected in 14 specimens from children with respiratory tract infections and 1 specimen from a child with febrile convulsions during 2010 in Osaka, Japan. These strains had deletions in the 5′ untranslated region and were genetically different from reported strains. This virus is associated with respiratory tract infections in Japan.

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC≥100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.

We witnessed outbreaks of multidrug-resistant (MDR) and drug-resistant Pseudomonas aeruginosa at a hospital in Tokyo, Japan, during the period September 2004 through May 2005. The first outbreak occurred in September and October 2004. Three isolates of MDR P. aeruginosa were identified from urine samples obtained from three nonambulatory immunodeficient patients in one ward. After 3 weeks, another outbreak of P. aeruginosa occurred in the hematology ward on the same floor of the hospital. During the outbreaks, environmental surveys were conducted twice in each of the two wards, at 2-week intervals, to identify the sources of the pathogens. A total of 23 P. aeruginosa isolates, including 11 from environmental sources, were analyzed for chromosomal DNA typing by pulsed-field gel electrophoresis, for O-antigen serotyping, and for other typing. Results revealed two causative clones, as well as environmental contamination by P. aeruginosa clones on the surfaces of urine volume-measuring devices in rooms where urine is handled, which may have been sources of the pathogens during the outbreaks. To prevent further outbreaks, we performed the following: (a) environmental surface monitoring for drug-resistant P. aeruginosa, (b) active surveillance of specimens, (c) strict isolation of infected patients or carriers of MDR P. aeruginosa, (d) rigorous contact precautions, and (e) disinfection with 70% alcohol on the surfaces of apparatuses contaminated by MDR or drug-resistant P. aeruginosa and in the rooms where urine is handled. As a result, the outbreaks were contained.

To assess whether the occurrence of rifampicin (RFP) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is related to treatment of tuberculosis, we determined the RFP susceptibility of MRSA isolates obtained from tuberculosis patients and screened for mutation(s) in the rpoB gene of these isolates. The MICs of RFP for 84 MRSA isolates obtained from two hospitals in Japan were determined. DNA was sequenced in the region 1318-1602 nucleotides (nt) of the rpoB gene, which includes RFP resistance-determining clusters I (1384-1464 nt, 462-488 amino acids). The majority of MRSA isolates from tuberculosis wards, i.e., 48 of 51 (94%) [33 of 34 in a Tokyo hospital (97%) and 15 of 17 in a Chubu hospital (88%)], were resistant to RFP. Meanwhile, no isolates of 33 from the other wards were resistant to RFP. All RFP-resistant MRSA isolates had a mutation(s), including novel mutation(s) such as Val453--AEPhe, Asp471--AEAsn, and Ile527--AELeu, in rpoB. An emergence of RFP-resistant MRSAs in tuberculosis wards in Japan was strongly suggested.

To the Editor: Human enterovirus (HEV) C (family Picornaviridae, genus Enterovirus) consists of 3 types of poliovirus (1, 2, and 3), 9 types of coxsackievirus A (CV-A1, 11, 13, 17, 19, 20, 21, 22, and 24), and 9 types of enterovirus (EV) (95, 96, 99, 102, 104, 105, 109, 113, and 116) (www.picornaviridae.com/enterovirus/hev-c/hev-c.htm). EV-104 was first identified in 2009 in Switzerland in 8 children who had pneumonia or acute otitis media (1). To our knowledge, there has been only 1 other report of EV-104, detected in Italy in 3 adults and 2 children who had upper respiratory tract infection (RTI) (2). We report the detection of a novel EV-104 strain in an adult with upper RTI in Japan.