This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m₃G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m₃G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.

This study estimates the risk of vaccine-associated paralytic poliomyelitis (VAPP) in Japan between 1971 and 2000. We acquired data regarding the number of VAPP cases from the website of the Ministry of Health, Labour and Welfare, and we estimated the number of oral poliovirus vaccines (OPV) administered based on the reported immunization data. Risk was calculated as the ratio between the number of VAPP cases and the number of OPV doses administered. Both the Runs test and the Poisson model were used to analyze the occurrence of VAPP. Thirty-three cases of VAPP were recorded in Japan between 1971 and 2000; approximately one case occurred per year. There were no statistical changes in temporal trends as regards the occurrence of VAPP between 1971 and 2000. The overall risk for VAPP, including both recipient and contact VAPP, was one case per 2.0 million OPV doses administered. The risk of recipient VAPP was one per 3.7 million doses, among which the first dose posed a much higher risk of one per 2.3 million than that of the subsequent dose. These data indicated that the occurrence of VAPP is rare, but the risk has remained constant for as long as OPV has been used in Japan.

The prevalence of immunity against diphtheria among Okayama local government staff members involved in diphtheria infection control was measured. Diphtheria booster vaccination was administered to staff members with low antitoxin levels (<0.1 IU/ml) in order to reinforce of immunity. Ninety-one (36.7%) of 248 staff members, 20-69 years of age, had fully protective antitoxin levels (≧0.1 IU/ml), and the remaining 157 (63.3%) showed levels of <0.1 IU/ml. The rate of full protection was higher in females (44.9%) than in males (22.8%) and was also higher in the diphtheria-pertussis mixed vaccine (born in 1958-1967) and diphtheria-pertussis-tetanus mixed vaccine (born in 1968-) (58.3-61.0%) groups than in diphtheria vaccine (born in 1948-1957) and non-vaccinated (born until 1947) (7.4-18.9%) groups. Though antitoxin levels of 13 (68.4%) out of 19 staff members given booster vaccinations increased to 0.1 IU/ml, 50% of these individuals then showed levels of <0.1 IU/ml after 3 years. Most of the staff members with antitoxin levels of ≧0.1 IU/ml in the non-booster vaccination group maintained their immunity levels for 2-4 years, independent of their history of vaccination. To ensure that staff members of the local government have fully protective antitoxin levels against diphtheria, periodical confirmation of antitoxin levels and booster vaccination should both be systematically carried out.

The objective of the study was to investigate the trend of mupirocin resistance among methicillin-resistant Staphylococcus aureus (MRSA) in Trinidad. No premarketing susceptibility surveillance was ever done following the introduction of mupirocin in 1986. A total of 188 MRSA strains recovered over a 2-year period from various body sites were tested for mupirocin resistance via the disc diffusion method. The major sources of MRSA were surgical site infections (74.0%) and bloodstream infections (8.0%). High-level and low-level mupirocin resistance were detected in 26.1 and 44.1% of MRSA stains, respectively. Resistances to other non-β-lactam antibiotics were also high. Ninety-eight percent of all MRSA were resistant to erythromycin. This was followed by resistance rates of 96.8, 95.2, 94.1, 93.6, and 93.1%, for gentamicin, ciprofloxacin, amikacin and tobramycin, co-trimoxazole, and tetracycline, respectively. No MRSA strains were found to be resistant to vancomycin, linezolid, and quinupristin-dalfopristin. The study showed that mupirocin resistance among Trinidadian MRSA strains was relatively high compared to that seen in other countries. Because of the increasing prevalence of MRSA at the San Fernando General Hospital (SFGH) and the apparently increasing resistance to mupirocin, frequent monitoring of MRSA susceptibility patterns and infection control initiatives may be helpful in reducing the incidence of MRSA with a concomitant decrease in mupirocin resistance. This report is the first after 20 years of continuous use of the drug at the SFGH.

Cryptococcosis is a life-threatening fungal infection among human immunodeficiency virus (HIV)-positive patients and also occurs frequently in HIV-negative patients. A retrospective cohort study was conducted among patients with cryptococcosis. Clinical manifestations, laboratory findings, treatment, and outcomes for 149 HIV-positive and 29 HIV-negative patients were compared. Neurological involvement occurred more frequently in HIV-positive patients (91.9 versus 20.7%, P < 0.001), whereas pulmonary involvement was more frequently observed in HIV-negative patients (34.5 versus 2.7%, P < 0.001). Ninety percent of HIV-positive patients and 74% of HIV-negative patients had positive serum cryptococcal antigen (P = 0.119). HIV-positive patients were more likely to have a cerebrospinal fluid (CSF) preparation that is positive for India ink staining (81 versus 50%, P < 0.001) and a CSF cryptococcal antigen titer of ≥1:1,024 (61.1 versus 16.7%, P = 0.038). Most of the patients in both groups received amphotericin B as the primary therapy. Cryptococcosis-related mortality was high and did not differ between the two groups (22.2 versus 34.5%, P = 0.162). Kaplan-Meier analysis revealed that HIV-positive patients had a higher relapse rate (P = 0.011), especially among those lacking antiretroviral therapy. In conclusion, the clinical presentation of cryptococcosis among HIV-negative patients varies and differs from that of HIV-positive patients. Awareness and prompt management are crucial for establishing a diagnosis and initiating proper treatment.

Sporadic reports of Corynebacterium ulcerans infection in humans and animals have become increasingly common throughout the world. Between 2001 and 2006, five human cases, in addition to isolation of the bacterium from the carcasses of Orcinus orca and Panthera leo, were reported in Japan. While an isolate from P. leo generated only phospholipase D (PLD), the other isolates produced both PLD and diphtheria-like toxin (DLT). Pulsed-field gel electrophoresis analysis showed that isolates from P. leo and humans were genetically homologous. Southern blotting found that a human isolate was lysogenized by two corynephages coding DLT. Sequence analysis of the region of the DLT gene revealed that the integration in C. ulcerans occurred in the same manner as that in C. diphtheriae.

Cell clone #21 is a long-term producer of the infectious SARS-coronavirus, although the incorporation rate of spike (S) protein into virions is significantly lower. Sequencing analysis of the viral structural proteins revealed four and one amino acid substitutions in the S and membrane (M) proteins, respectively. We demonstrated, using a viral-like particle formation system, that the S mutations were involved in the lower incorporation of the S protein into virions, although the M mutation that disrupts the glycosylation was not present in this phenotype. Further mutational experiments identified two substitutions, Y442C and L472F, within the receptor binding domain that could be critical for the reduced S incorporation, as well as reduced binding affinity between the S protein and ACE2 receptor. Thus, these two amino acid substitutions might lead to a conformational change in the S protein, resulting in reduced incorporation into viral particles.

Out of 2,235 diarrheal stool samples collected from patients admitted to the Infectious Diseases Hospital, Kolkata, 343 cases were positive for Vibrio cholerae (341, V. cholerae O1 and 2, O139). During the year 2004, infections caused by V. cholerae serotype Ogawa and Inaba were 93 and 7%, respectively, while in 2005, the Inaba isolation rate rose to 88% as compared to 12% for Ogawa. Susceptibility to antimicrobial agents revealed that the O1 strains were resistant to multiple antibiotics (ampicillin, co-trimoxazole, furazolidone, nalidixic acid and streptomycin) with reduced susceptibility to ciprofloxacin. Increased isolation of tetracycline-resistant strains (27.3% for Ogawa and 15% for Inaba) was noted in 2005. It appears that the population might be at risk of infection by the Inaba serotype and that tetracycline may not be useful for the treatment.

Trichosporon spp. are emerging as opportunistic agents that cause systemic diseases in immunocompromised hosts. Trichosporonosis carries a poor prognosis in neutropenic patients. Trichosporon japonicum was isolated from the air and named by Sugita et al. Here we present the first case of T. japonicum isolated from a clinical specimen. Two cases of acute myeloid leukemia who had Trichosporon isolates are discussed because of their rarity and growing importance. T. asahii was isolated from the throat, feces and urine of the first patient. T. japonicum was isolated from the sputum of the second patient. Both cases produced high MICs to itraconazole, and low MICs to fluconazole and voriconazole. In virulance factor investigations there was (++) biofilm formation in T. japonicum but not in T. asahii. Conventional mycological studies were not adequate for the identification of the isolate at the species level. In our second case as in the first one, the isolate was identified as T. asahii with 99.9% accuracy by API 20C AUX. Although two T. asahii isolates from the same patient yielded identical typing profiles by arbitrary primed-PCR, the isolates of the two different patients showed different arbitrary primed-PCR typing profiles. However, the genetic identification of the other patient's strain gave the result of T. japonicum.

We herein report repeated isolation of Burkholderia cenocepacia from two cases of septicemia admitted to cardiothoracic and bone marrow transplant (BMT) units. The two blood cultures taken from each patient grew B. cenocepacia. Both patients turned afebrile after appropriate antimicrobial therapy, and the subsequent blood cultures were sterile. However, both patients had recurrence of fever after about a week, and the patient in the BMT unit died due to respiratory failure. Environmental surveillance was conducted in both units. Non-fermenting Gram-negative bacilli including Pseudomonas aeruginosa were isolated from environmental samples in the cardiothoracic ward.

In this study, 153 clinical isolates of Shigella were screened for extended-spectrum beta-lactamase (ESBL) production by double-disk synergy test. After being confirmed by the combined disk-test and inhibitor-combined E-test strips, all positive isolates were tested for the bla gene by PCR and the enzymes by isoelectric focusing. DNA sequencing of PCR products revealed that five Shigella sonnei isolates had CTX-M-3 type ESBL enzymes. All of them were resistant to ampicillin, sulfamethoxazole and cefotaxime but susceptible to ofloxacin and ceftazidime. All isolates displayed identical patterns in pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. This study reports multidrug-resistant (MDR) S. sonnei isolates producing CTX-M-3 type ESBL from successive pediatric bacillary dysentery patients, indicating widespread and rapid spread of CTX-M type ESBL in Shigella spp. To counter this emerging threat to public health, the surveillance of CTX-M type beta-lactamases should be considered, together with measures designed to prevent outbreaks of MDR Shigella in the community.

Candidiasis of the gallbladder is an uncommon cause of acute cholecystitis. Candidal cholecystitis is seen especially in patients with malignancies. In the present case, we report that acute calculous cholecystitis was caused by Candida lusitaniae in a 33-year-old patient without underlying malignancy. According to our review of the literature, this is the first report of acute cholecystitis caused by C. lusitaniae.

Human monkeypox, an infectious disease caused by monkeypox virus (MPXV), is endemic to western and central Africa. A LightCycler quantitative PCR (LC-qPCR) system was developed for the diagnosis of this disease, targeting the A-type inclusion body gene (ATI gene) of MPXV. One naïve monkey was infected with MPXV Zr-599 (Congo Basin strain) and one with MPXV Liberia (West African strain). Another three monkeys were immunized with smallpox vaccine on 0, 3, or 7 days, respectively, before infection with MPXV Zr-599. Peripheral blood cell (PBC) and throat swab (TS) specimens were serially collected. The LC-qPCR was validated for the diagnosis of monkeypox using virus isolation. Sequencing of the partial ATI gene revealed the insertion of a unique 453-nucleotide residue in the West African strains but not in the Congo Basin strains. Specific reverse primers for Congo Basin and West African strains were designed based on the unique sequence insertion. The LC-qPCR detected the MPXV genome, but not those of the other orthopoxviruses tested nor the varicella-zoster virus. Both the sensitivity and specificity of the LC-qPCR were over 90% in comparison to virus isolation when TS specimens were tested. Fourteen of the 15 virus isolation-positive PBC specimens showed positive reactions in the assay. Further, most PBC specimens collected from symptomatic monkeys in the later stage of illness showed positive reactions in the assay but negative reaction in virus isolation. It was possible to differentiate between these two groups with the LC-qPCR. Thus, the newly developed LC-qPCR is a useful and reliable diagnostic tool for MPXV infection.

The molecular epidemiology of 126 adenovirus type 3 (AdV3) isolates obtained in Hyogo Prefecture (population: 5.5 million) from 1994 to 2006 was studied. The hexon-coding region, including 7 hypervariable regions (HVRs) (1,419 bp), was sequenced. We found 5 nonsynonymous nucleotide substitutions in the HVRs. The results are strongly suggestive of positive Darwinian selection. We classified the AdV3 strains analyzed here into 3 genome types: AdV3x (n = 44), AdV3y (n = 46), and AdV3z (n = 36). AdV3x first appeared in 2001 in Hyogo Prefecture, and was detected predominantly during a large outbreak of AdV3 in 2003-2005. AdV3x was identical to a Korean strain responsible for a large outbreak of AdV3 in Korea in 1998-1999. We conclude that at least 3 genome types of AdV3 have circulated in Hyogo Prefecture, Japan, during the past 13 years (1994-2006). The findings also suggest that AdV3x was imported from Korea to Hyogo Prefecture in 2001.

Female sex workers in Korea can generally be divided into one of two categories, namely, historically rooted brothel-based prostitutes (BBPs) and a newly developed category of freelance prostitutes (FLPs). Though some social differences are known, there have been few reports of distinguishing biological characteristics between the two groups. We examined the chlamydial plasmid loads (CPLs) and the numbers of detached host cells (NDCs) from these two groups with different approaches to selling sex. Chlamydial plasmid and human genomic DNA were extracted from endocervical swabs of the subjects, and we determined the CPLs and the NDCs using real-time PCR analysis. Forty-six women in the FLP group and 21 women in the BBP group had a chlamydial infection. The CPL and NDC values were higher in the FLP than in the BBP group (P = 0.000, P = 0.0001 respectively). In the FLP group, younger-aged women had higher CPLs and more detached cells than did older women (P = 0.02, P = 0.01 respectively). However, the BBP group did not show any such age-related differences in CPL and NDC values. There was a statistically significant positive correlation between the NDC and the CPL values in the BBPs (P < 0.001) as well as in the FLPs (P < 0.001). In conclusion, different biological characteristics were observed between FLPs and BBPs, as based on different CPL and the NDC values. The high plasmid loads among the sex workers were isolated from large numbers of scraped cells within a cotton swab. Thus, minor injury may render the endocervical epithelium of FLPs more easily detachable than that of BBPs.

In order to compare the epidemiological aspects of tsutsugamushi disease (scrub typhus) between Korea and Japan, we analyzed the current state of tsutsugamushi disease outbreaks and related risk factors. The average prevalence rate of tsutsugamushi disease from 2001 to 2005 in Korea was higher than that in Japan (7.2 and 0.3 per 100,000 population, respectively) (P < 0.01). The seasonal distribution in Korea showed that the incidence of tsutsugamushi disease in autumn (94.5% of total cases) was higher than in Japan (38.2%) (P < 0.01), while the incidences of outbreaks in other seasons were much higher in Japan (P < 0.01). In Korea, more females (64.8%) were infected than males (35.2%) (P < 0.01), while there was no significant difference in Japan. The remarkable difference between the gender distribution in Korea and Japan is believed to reflect cultural differences between the two countries in terms of work, dress and ornamentation. In both countries, elderly people (over 60 years old), especially in rural areas, showed a very high prevalence rate (50%), which is possibly due to increased outdoor activities and a decreased number of young people in those areas. These differences in tsutsugamushi disease risk factors reflect the different influences of vectors/hosts, climate, and geographical and cultural characteristics between the two countries.

Melioidosis is an infection caused by Burkholderia pseudomallei that usually involves the respiratory tract. It may manifest as pneumonia, septicemia, or localized infection. We present here a case of melioidosis initially manifesting as a mass over the supraclavicular area and subsequently progressing to necrotizing fasciitis. With appropriate antimicrobial treatment and adequate surgical debridement, localized melioidosis can be treated successfully. Melioidosis should be considered in the differential diagnosis of neck masses, especially in patients who have traveled to or stayed in an endemic area.

The epidemiology of the genetic sublineages of human metapneumovirus (hMPV) and their clinical relevance are not fully understood. We compared hMPV genotypes isolated in the province of Bolzano in Northern Italy with strains from nearby Italian and Austrian regions by sequencing of NP- and L-gene fragments. Our results suggest that similar strains cycle through adjacent geographic areas, with the respective subtypes replacing each other on a seasonal basis.

Necropsied lung tissues of three fatal cases with avian influenza A virus (H5N1) infection in Vietnam were analyzed to detect H5N1 virus-infected cells. Formalin-fixed and paraffin-embedded lung tissue sections showed typical histological features of diffuse alveolar damage (DAD) in all cases. Immunohistochemistry for the influenza A virus nucleoprotein antigen revealed positive signals of bronchiolar and alveolar epithelial cells in only one patient, who exhibited DAD with an exudative phase and died on the 6th day after onset. However, no signal was detected in the other two cases of DAD with a proliferative phase. These patients died on day 16 and day 17 after onset, respectively. H5N1 virus antigens were detected predominantly in epithelial cells in terminal bronchioles and in alveoli, i.e., type I and type II alveolar pneumocytes, and in alveolar macrophages. The pathogenesis of exudative DAD caused by H5N1 infection is discussed.