In situ hybridization (ISH) has been recognized as an important technique for identifying the causative fungi in the foci of infection observed in histopathological specimens which was processed from formalin-fixed and paraffin-embedded (FFPE) tissues. However, few basic studies have conducted an evaluation of the DNA preservation for use in ISH in comparison to polymerase chain reaction (PCR). The latter is a DNA amplification-based modality. In the present study, we analyzed 65 FFPE lung tissue specimens collected from autopsy cases for comparing the usefulness of ISH and PCR analysis. As a result, the positive identification rates for PCR were strikingly low; a majority of these results can be assumed to be false negative because the presence of fungi had been confirmed by histopathological analysis. In contrast, panfungal ISH targeting of the 28S rRNA showed a higher sensitivity than the 230-bp panfungal PCR primers did (80.0% versus 4.6%, respectively). Furthermore, over 60% of the samples we examined showed a favorable intensity of the ISH signal. Therefore, in conventional postmortem FFPE tissues, the state of DNA preservation may be more favorable for ISH than PCR analysis.

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Salmonella Typhimurium is an important nontyphoidal Salmonella serovar associated with foodborne diseases in many parts of the world. This organism is the major causative agent of nontyphoidal salmonellosis in Malaysia. We aimed to investigate the genetic profiles of the strains isolated from clinical, zoonotic, and dietary sources in Malaysia using multilocus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). By focusing on the 5 common variable number tandem repeat (VNTR) loci, we found that PFGE (D = 0.99) was more discriminative than MLVA (D = 0.76). The low MLVA score might be because of a lack of VNTR loci STTR6 (81.0%) and STTR10pl (76.2%). Both subtyping methods suggested that our S. Typhimurium strains were largely endemic with limited genetic variation. Furthermore, we observed that biphasic S. Typhimurium strains were dominant (99%) and multidrug resistance was prevalent (50%) within our sample pool. The most frequently observed phenotypes were resistance to compound sulfonamides (49%), tetracycline (51%), and streptomycin (52%). In this study, we documented the genetic relationship, antimicrobial resistance characteristics, and flagellar-phase dominance among S. Typhimurium strains found in Malaysia.

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The invasion of the yellow fever mosquito Aedes aegypti at Narita International Airport, Japan was detected for the first time. During the course of routine vector surveillance at Narita International Airport, 27 Ae. aegypti adults emerged from larvae and pupae collected from a single larvitrap placed near No. 88 spot at passenger terminal 2 on August 8, 2012. After the appearance of Ae. aegypti in the larvitrap, we defined a 400-m buffer zone and started an intensive vector survey using an additional 34 larvitraps and 15 CO₂ traps. International aircraft and passenger terminal 2 were also inspected, and one Ae. aegypti male was collected from the cargo space of an international aircraft from Darwin via Manila on August 28, 2012. Larvicide treatment with 1.5% fenitrothion was conducted in 64 catch basins and one ditch in the 400-m buffer zone. Twenty-four large water tanks were also treated at least once with 0.5% pyriproxyfen, an insect growth regulator. No Ae. aegypti eggs or adults were found during the 1-month intensive vector survey after finding larvae and pupae in the larvitrap. We concluded that Ae. aegypti had failed to establish a population at Narita International Airport.

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Epidemics of infectious diseases often occur at long-term inpatient facilities for patients with severe motor and intellectual disabilities. However, the pathogens causing these infections remain unknown in approximately half of such epidemics. Two epidemics of respiratory tract infection occurred in 2 wards in the National Hospital Organization Ehime Hospital (prevalence 1, 34 infected out of 59 inpatients in the A ward in September 2011; prevalence 2, 8 infected out of 58 inpatients in the B ward in June 2012). Human metapneumovirus (HMPV) was detected from the nasal (and some pharyngeal) swabs from 17 patients. Based on phylogenetic analysis of viral genomes, the virus was grouped in subgroup A2 (prevalence 1) and B2 (prevalence 2). We considered that the viruses had spread through the 2 wards. The average duration of high fever in the 42 patients was 6.8 days, with the majority of fevers exceeding 38℃ (79%) and being accompanied by a productive cough. Ten out of 17 patients (59%) in whom HMPV was detected had decreased lymphocyte and increased monocyte counts in the blood. Eleven cases (65%) had elevated-C reactive protein levels and fever protraction as well as images of bronchitis or pneumonia on chest radiographs approximately 1 week after onset. Anti-HMPV antibody in the blood was positive in 95% of patients (151 of 159 inpatients), indicating no relation between HMPV infection and antibody titer but revealing recurrent infections. In view of the fever protraction and frequent co-occurrence of bronchitis and pneumonia at long-term inpatient facilities for immunocompromised patients such as the ones in this study, the prevalence of HMPV must be carefully monitored, and preventive measures and early-stage treatments are required.

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A total of 12 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains were isolated during a recent outbreak in a nursery school in Ehime Prefecture, Japan. These isolates were considered to be derived from a common strain when analyzed using an IS-printing method and pulsed-field gel electrophoresis. PCR analysis revealed that the isolates harbor stx₁, stx₂, eae, and hlyA. However, assessment of the production of the Stx proteins revealed that these isolates produced Stx1 but not Stx2. We determined their stx₂ variants such as stx_2c and found that the size of the PCR product was much larger than the expected size. Sequencing of the entire stx₂ gene revealed that a 1310-bp fragment was inserted into the coding region of the Stx2A subunit and that the sequences of the insert were identical to those of IS1203v. According to the sequences around the insertion site, additional amino acid residues should be attached at the C-terminus of the A subunit, which may hamper the Stx2 complex formation. Finally, this study also suggested that such an insertion may lead to the misinterpretation of results when screening EHEC isolates for virulence genes by PCR.

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Although IFN-γ release assays (IGRAs) provide increased specificity over tuberculin skin tests, the early and sensitive detection of reactivation of latently infected Mycobacterium tuberculosis is required to control tuberculosis (TB). Recently, a multicolor flow cytometry has been developed to study CD4⁺ T cell cytokine responses (IFN-γ/IL-2/TNF-α) to purified protein derivatives (PPD) and M. tuberculosis-specific antigens (ESAT-6/CFP-10) and provided useful information regarding anti-TB immunity. However, the diagnostic relevancy remains uncertain. Here, we analyzed three additional CD4⁺ T cell cytokine responses (IL-10/IL-13/IL-17) to latent mycobacterial antigens (α-crystallin, methylated heparin-binding hemagglutinin [HBHA], and mycobacterial DNA-binding protein 1 [MDP-1]) as well as PPD and ESAT-6/CFP-10 in 12 IGRA⁺ TB cases and 8 healthy controls. No significant difference in IFN-γ response was observed between TB cases and controls, which was likely due to the high variation among the individuals. However, we found a significant increase over healthy controls in (i) the IL-2 response to HBHA in recovery stage TB cases, (ii) the number of M. tuberculosis-specific polyfunctional CD4⁺ T cells in on-treatment and recovery stage cases, and (iii) the IL-17 response to HBHA and MDP-1 in on-treatment and recovery stage cases. These results suggest that a combination of these T cell cytokine parameters could aid in accurate diagnosis of latent TB infection.

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Although Cryptococcus gattii can cause life-threatening complications, putative virulence factors of C. gattii remain controversial. Therefore, we conducted the present study to elucidate the virulence factors of the yeast and found that the mortality rate of mice infected with C. gattii R265 was significantly higher than that of those infected with C. gattii 5815; however, no difference was found in the mortality rates between mice infected with C. gattii R265 and Cryptococcus neoformans H99. In contrast, we found a significant difference in histopathological findings of the lungs between mice infected with C. gattii R265 and C. neoformans H99. The former showed alveolar expansion due to yeast proliferation with much lesser macrophage response, whereas the latter showed numerous nodules in the alveolar space consisting of macrophages and multinucleated giant cells. Furthermore, alveolar expansion was more enhanced in mice infected with C. gattii R265 than in those infected with C. gattii 5815. Our study confirmed that there is a different pathophysiology leading to death during C. gattii and C. neoformans infections. The result can provide two characteristics of C. gattii: one includes some mechanisms to escape from host recognition via macrophage and another includes a high performance of pulmonary structural alteration. These characteristics may be associated with the high virulence of C. gattii.

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Group B streptococcus (GBS; Streptococcus agalactiae) is a leading cause of neonatal invasive infections, and until recently, it was thought to be completely susceptible to penicillin. However, we recently identified several clinical GBS isolates with reduced penicillin susceptibility (PRGBS) whose minimum inhibitory concentrations of penicillin were >0.12 μg/ml, which is above the susceptibility breakpoint set by the Clinical and Laboratory Standards Institute. These PRGBS were isolated between 1995 and 2005 in Japan; whether these PRGBS existed in Japan before 1995 is unknown. In the study described here, we screened for PRGBS among 349 clinical GBS isolates obtained in Japan between 1977 and 2005 using the previously developed disk diffusion method for the detection of PRGBS. With this method, we selected 6 PRGBS candidates and confirmed that 1 isolate was PRGBS, using agar dilution method, including oxacillin, ceftizoxime, and penicillin-binding protein 2X (PBP2X) gene sequencing analysis. This isolate was obtained from sputum in 2005, and we could not detect PRGBS isolates before 1995 in this investigation.

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A survey of mumps infections from 1999 to 2010 was conducted in Yokohama City, Japan, and 17 cases—including 4 cases of aseptic meningitis—were positive for mumps virus (MuV). Based on the phylogenetic analysis of the small hydrophobic gene of the MuV genome, 3, 2, and 12 of the isolates were classified into genotypes B, L, and G, respectively. The results were supported by phylogenetic analysis of hemagglutinin-neuraminidase genes. The 3 isolates of genotype B, obtained in 2000, 2004, and 2007, were closely related to indigenous lineages and vaccine strains in Japan. Two isolates obtained from 1999 to 2000 were assigned to genotype L. Twelve isolates obtained from 2000 to 2010 were classified into genotype G, in which 8 isolates obtained from 2000 to 2006 and 4 isolates obtained in 2010 were closely related to MuVi/Gloucester.GBR/32.96 and MuVi/London.GBR/0.03, respectively. Precise analyses of nucleotide sequences suggested that the 4 viruses isolated in 2010 were not directly derived from the evolution of MuV existing before 2006 in the Yokohama area.

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The objective of this study was to determine the optimal cutoff value of serum adenosine deaminase (ADA) activity for diagnosing scrub typhus in acute febrile patients, especially in the autumn. A total of 715 febrile patients were included; 286 of them were diagnosed with acute scrub typhus. The mean serum ADA activities in patients with and without scrub typhus were 42.8 ± 13.4 IU/L and 17.2 ± 11.5 IU/L, respectively. The optimal cutoff value for serum ADA activity was determined to be 25.0 IU/L with a sensitivity of 92.3%, specificity of 86.6%, and area under the curve of 89.4% using a receiver-operating characteristic curve analysis. The possibility of scrub typhus must be considered when acute febrile patients present with high ADA activity (>25 IU/L), especially in the autumn. Determining the optimal serum ADA activity cutoff value may help clinicians diagnose acute scrub typhus prior to serological confirmation.

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Haemophilus influenzae type b (Hib) conjugate vaccine (PRP-T) and heptavalent pneumococcal conjugate vaccine (PCV7) were introduced in Japan in December 2008 and February 2010, respectively. The concurrent administration of these vaccines is routinely performed worldwide. However, the safety of the simultaneous administration of these vaccines has not been fully evaluated in Japan, because it has rarely been performed thus far. We conducted a 2-year prospective, observational, multicenter study on PRP-T and PCV7 safety from February 2009 through January 2011 in 29 facilities located in Kagoshima Prefecture, Japan. Objective severe adverse events included anaphylactoid reaction, encephalitis/encephalopathy, neurological events, severe focal reactions, systemic eruption/urticaria, fever above 39℃ within 2 days after inoculation, and other complications requiring hospitalization. The incidences of these events for PRP-T and PCV7 administration were 0.68% (76/11,197) and 0.92% (28/3,049), respectively. No deaths or subsequent complications were reported during the course of the study. There was no significant difference in the incidence of severe adverse events between the single and co-administration groups for both vaccines: PRP-T, 0.55% (31/5,662) versus 0.81% (45/5,535; P = 0.11); PCV7, 0.88% (11/1,247) versus 0.94% (17/1,802; P = 0.86). These results suggest that the simultaneous administration of vaccines including PRP-T and/or PCV7 does not increase the incidence of severe adverse events in Japanese children.

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Lymphatic filariasis is the second leading cause of long-term disability in the world. In India, filariasis is endemic in 250 districts in 20 states and union territories with about 553 million people at risk of infection. Temephos is being used as a larvicide to control the population of Culex quinquefasciatus mosquito, the vector of bancroftian filariasis. The susceptibility status of Cx. quinquefasciatus to temephos in various urban zones of Delhi and the national capital region (NCR) was evaluated using the WHO standard susceptibility test kit. Results of the test revealed that the larval mortality in urban zones ranged from 2.8 to 56.5%, while that in rural zones ranged from 45.0 to 71.0%. Among the urban zones, the lowest larval mortality was recorded from Karol Bagh zone (2.8%) and highest mortality was recorded from Shahdara South zone (56.5%). In the NCR, the larvae were collected from the areas surrounding Delhi, and the larval mortality ranged from 75.0 to 87.5%. The present study indicates the possible development of resistance against temephos in the larvae of Cx. quinquefasciatus mosquitoes in some urban areas.

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An uncomplicated cystitis caused by CO₂-dependent Proteus mirabilis was observed in a 64-year-old Japanese female patient with Sjögren's syndrome in the Aomori Kyoritsu Hospital, Aomori, Japan. The initial P. mirabilis isolate came from a midstream urine specimen containing large numbers of Gram-negative, rod-shaped organisms that failed to grow on both Drigalski agar and sheep blood agar incubated in ambient air. The organism did grow when the urine was cultured overnight on blood agar under anaerobic conditions. Hence, we believed that the organism was an anaerobe. Further investigation revealed that the isolate grew on sheep blood agar along with swarming when the atmospheric CO₂ concentrations were increased to 5%. Initially, we failed to characterize or identify the P. mirabilis isolate or determine its antimicrobial susceptibilities using the MicroScan WalkAway-40 System because the isolate did not grow in the system. However, the isolate was subsequently identified as P. mirabilis based on its morphological, cultural, and biochemical properties by using the commercially available kit systems, Quick ID-GN and ID-Test EB-20. This identification of the isolate was confirmed by sequencing the 16S rRNA gene of the organism. To our knowledge, this is the first clinical isolation of capnophilic P. mirabilis.

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Acinetobacter baumannii is an important pathogen in hospitalized patients, particularly those in the intensive care unit (ICU). A total of 21 A. baumannii (6 from 5 patients and 15 from environmental samples) were isolated in the ICU and the isolation room of a state hospital in June 2011. The possible source of the outbreak was investigated. A. baumannii isolates were identified using conventional biochemical tests, BBL Crystal Identification Systems, OXA-51 specific PCR, and 16S rDNA sequencing. All the isolates were multidrug-resistant, showing resistance to cephalosporins, carbapenems, fluoroquinolones, and the aminoglycoside group of antibiotics. Pulsed-field gel electrophoresis suggested that all A. baumannii isolates were derived from a common source.

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A simple, rapid, and low-cost identification method is required in tuberculosis high-burden countries. We report the applicability of in-house loop-mediated isothermal amplification (LAMP) targeting 16S ribosomal RNA for the rapid identification of Mycobacterium tuberculosis complex grown on Lowenstein–Jensen media. Eighty acid-fast staining-positive clinical isolates were selected and used to evaluate the LAMP assay in comparison with polymerase chain reaction and conventional culture-based tests. The LAMP assay identified 60 M. tuberculosis isolates from 80 clinical isolates using simple heat-extracted DNA directly from the colony suspension. The results were in complete agreement with those obtained using the other methods, and the utility of the direct LAMP assay from a colony was demonstrated. The LAMP assay appears to be a practical and low-cost method that can be used for the rapid identification of M. tuberculosis isolates and suitable for endemic low-resource settings.

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Highly active anti-retroviral therapy (HAART) has been freely available in Nepal since 2004. In the present longitudinal study, we followed two distinct cohorts of human immunodeficiency virus-infected participants, those receiving HAART and those under assessment of eligibility for HAART, during the period 2005–2007 in Kathmandu, Nepal. The median change in CD4+ T-cell count among participants receiving HAART after 12 months of the initiation of therapy was +118 T cells/μl (95% confidence interval [CI], +91 to +145 T cells/μl) and that among participants under assessment of eligibility for HAART was −74 T cells/μl (95% CI, −103 to −44 cells/μl). However, the median CD8+ T-cell count after 12 months remained stable in both the cohorts (P > 0.05). The CD4+/CD8+ T-cell ratio increased from 0.16 to 0.26 after 12 months of therapy (P < 0.001). The multivariate regression model revealed that participants >30 years of age, and injection drug users had significantly lower increases in the CD4+ T-cell count in response to therapy. We observed a high proportion of loss to follow-up after 12 months of therapy; however, the associated factors were unknown. In conclusion, we observed a significant improvement in the CD4+ T-cell count in participants receiving HAART; however, the CD4+/CD8+ T-cell ratio remained <0.5 after 12 months of treatment.

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To investigate the accurate prevalence of genital Chlamydia trachomatis infection in Mymensingh, a local area in central-northern Bangladesh, 40 female sex workers (FSW) and 110 sexually active women (SAW, non-FSW) of reproductive age from a local community with clinical symptoms were examined by an immunochromatography test (ICT) and plasmid-based polymerase chain reaction (PCR) during a 1-year period from July 2011 to June 2012 using the endocervical swab as a specimen. By ICT and/or PCR, the C. trachomatis detection rate was 58% and 27% in FSW and SAW, respectively, showing a significant difference (P < 0.01). Two C. trachomatis strains from FSW were determined to be serovar D by ompA-based PCR and sequencing analysis. The highest prevalence was found among women aged 15 to 35 years. A lower socioeconomic status was considered to be an important risk factor for C. trachomatis infection in FSW but not in SAW. This is the first study to determine the prevalence of C. trachomatis infections in FSW and SAW in the same local area in Bangladesh.

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Volume 66, no. 2, p. 161-164, 2013. Page 161, SUMMARY, line 3 and page 161, column 2, line 18: “September 2010 to March 2011” should read “September 2011 to March 2012.”

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