Systemic candidiasis is a significant cause of morbidity and mortality in patients with hematologic disorders. The aim of this study was to determine the prevalence of systemic candidiasis and the efficiency of the polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELIZA) method for the early detection of Candida spp. in patients with hematologic malignancies. From 2004 to 2006, 194 patients with hematologic malignancies were evaluated for systemic candidiasis. Collected blood samples were assayed using the PCR-ELISA method for the presence of the bands on ethidium bromide stained gel, and for hybridization with Candida spp. as well. The female-to-male ratio was 61:133, the mean age was 33.7 years, and the mean hospitalization period was 21.2 days. Twenty-five patients (12.9%) had positive PCR-ELISA results for systemic candidiasis. The etiologic agents were Candida albicans (21 cases), C. tropicalis (3 cases), and C. krusei (1 case). The mean interval of PCR-ELISA positivity in blood samples before the manifestation of clinical signs was 12.6 days. Fungal PCR-ELISA assay became negative after 14 days when patients were treated successfully with amphotericin B, and the assay remained positive until death when the treatment failed. The PCR-ELISA method can potentially serve as a useful tool for the management of patients suffering from hematologic malignancies and at risk for systemic candidiasis.

Platelet-activating factor (PAF) is a critical mediator of severe inflammatory diseases such as pneumonia, and the PAF-receptor (PAFR) is known to be an anchor for Streptococcus pneumoniae attachment to lung epithelial cells. We conducted a DNA microarray analysis to detect critical factors that mediate fulminant pneumonia due to influenza virus and S. pneumoniae co-infection in mice. Among the factors detected, levels of PAF-acetyl hydrolase (PAF-AH), which functions as inactivated PAF, were significantly increased, and PAFR was expressed in co-infected mouse lungs, as compared to the respective levels in mice infected with either S. pneumoniae or virus alone. Significantly elevated PAF-AH enzymatic activity was observed in the co-infected mouse lung, suggesting that co-infection activated PAF-related factors. These findings suggest that PAF and related molecules play important roles in fulminant pneumonia due to influenza virus infection, especially when severe bacterial pneumonia is complicated by co-infection with influenza virus.

The mortality rate resulting from Crimean-Congo hemorrhagic fever (CCHF) has been reported in different epidemics to range from 5% to more than 70%. While ribavirin has been recommended as the drug of choice in the treatment of CCHF, no consistent study has unequivocally demonstrated its effectiveness. Using the case-control method, we attempted to evaluate the efficacy of ribavirin in reducing mortality among CCHF cases admitted to Boo-Ali Educational Hospital in Zahedan, Iran, during the years 2000 to 2006. Sixteen deaths among CCHF cases were compared with 47 cases of survival. All patients had a definitive diagnosis based on the results of IgG and IgM capture ELISA tests recorded in their files. Ribavirin therapy for patients who survived had begun on average approximately 24 h earlier than the initiation of ribavirin therapy in the cases of death (P = 0.033), and about 2 days earlier in non-bleeding survivors than in bleeding survivors (P = 0.013). Based on the results of a multivariable analysis, the most important variables found to enhance survival were the time interval between the disease onset and ribavirin prescription (P = 0.004), the time interval between bleeding onset and ribavirin prescription (P = 0.037), and the time interval between disease onset and bleeding onset (P = 0.014). Based on our findings, ribavirin appears to exert a marked effect on disease outcome, especially when it is prescribed within the first 4 days of the disease.

Shiga toxin-producing Escherichia coli (STEC) include O157:H7 and non-O157 serotypes. The public health impact of STEC infections is high because of their ability to cause severe infections. We characterized our STEC strains isolated from diarrheal and asymptomatic persons in northern and southwest Iran. The 29 STEC strains were examined for the presence of virulence genes by polymerase chain reaction (PCR) and their H type was analyzed by PCR-restriction fragment length polymorphism (RFLP) of the fliC gene. Moreover, the adherence properties of these strains were checked by HeLa cell adherence assay. The presence of non-O157 isolates under these conditions was again verified. The stx1 gene was present in 93% of the isolates, and the gene encoding intimin (eae) was not found to be present among the isolates. Almost all of the STEC isolates, with the exception of three, were non-adherent upon tissue culture assay. The serogrouping revealed the presence of seven different O types among non-O157 isolates. PCR-RFLP results for the fliC gene and classical serology examination with H antisera indicated the presence of nine different H types. In this study, three new serotypes of non-O157:H7 (i.e., O25:H3, O85:H32, and O162:H21) were found to be Shiga toxin producers. These findings reconfirm the results of our previously reported studies showing that non-O157:H7 serotypes are more prevalent under the present conditions. More detailed characterization of these isolates will require additional genetic studies.

Falciparum malaria is a fatal infection without immediate diagnosability or treatment. There are shortages of clinicians and examiners skilled in the treatment of malaria in non-endemic countries, including Japan. This study was performed to evaluate a novel rapid molecular diagnostic system consisting of loop-mediated isothermal amplification (LAMP) combined with DNA filter paper (FTA card) and melting curve analysis. Combining LAMP with melting curve analysis enabled diagnosis of Plasmodium falciparum more accurately with relative ease. FTA cards could be used to clarify problems regarding storage, infectivity, and transportation. The LAMP assay was carried out at a constant temperature of 63ºC for 90 min. The diagnostic system (malaria-LAMP) accurately diagnosed malaria (47 samples from Thailand and 50 from Zimbabwe) with 97.8% sensitivity and 85.7% specificity as compared with microscopic methods, indicating the usefulness of this combined system.

The function of protein-coding gene Rv0901 of Mycobacterium tuberculosis, which belongs to the cell wall and cell processes category, is not yet clear. To explore its features, we amplified this gene from the H37Rv genome, and His-tagged Rv0901 protein was expressed and purified. Also, a recombinant plasmid bearing Rv0901 was constructed and electroporated into a virulent Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a predicted protein of Rv0901, identified by SDS-PAGE. Rv0901 protein and recombinant M. smegmatis were used to expose mammalian cells. In addition we studied the effect of protein or recombinant M. smegmatis on cells and in animals with regard to survival ratio, apoptosis ratio, quantum of nitric oxide, and gamma interferon. Together, gene function, protein function, and animal test results suggest that Rv0901 has some relationship with the virulence and immunogenicity of M. tuberculosis.

The objectives of this study is to characterize HIV-serology-discordant couples diagnosed at a referral hospital in Thailand and to identify risk factors for HIV transmission among married couples. Firstly, cross-sectional analysis was conducted from July 2000 to October 2002. Out of 216 HIV-positive married men who knew the HIV status of their wives, the median number of sexual contacts in 63 men with HIV-negative wives was 6 times per month before the disclosure of HIV status, which did not differ from 153 men with HIV-positive wives. The majority of men with HIV-negative wives never used condoms. The median duration of marriage was 7 years for both groups. Unlike in previous reports, men with HIV-negative wives were significantly more symptomatic (P < 0.01), and their CD4+ counts and viral loads did not differ from men with HIV-positive wives. Secondarily, 71 initially discordant couples were longitudinally followed until March 2005. Four were seroconverted out of 132.24 person-years of observation. In multivariate analysis incorporating sex, age, CD4+ count and sexual contact without a condom, shorter duration of marriage (<2 years) was found to be the only risk factor significantly associated with HIV transmission (hazard ratio of 15.2, P = 0.04). Individuals substantially exposed to HIV but remaining HIV-negative are accumulated in discordant couples identified in a hospital, except in recently married couples.

To investigate the potential applicability of mucosal vaccines against mucosa-unrelated pathogens, a non-parenteral vaccination approach was taken as a prophylactic strategy against mosquito-borne Japanese encephalitis virus (JEV). Intranasal (i.n.) immunization with a mouse brain-derived formalin-inactivated JE vaccine induced a robust virus-neutralizing antibody in mice, and this induction was augmented by co-administration with cholera toxin (CT) and pertussis toxin, but not with killed Bordetella pertussis. The antibody response induced by the i.n. administration of the JE vaccine with bacterial toxins was comparable in intensity to that induced by a parenteral immunization regime, and the former was considerably more effective in terms of delayed-type hypersensitivity and local antibody response. In addition, the adjuvant effects of bacterial toxins were much more prominent for the mucosal than the parenteral route. Two other non-invasive routes, oral and transcutaneous administration, were examined, but the i.n. route was by far the most effective. Finally, the vaccine efficacy of a chimeric fusion protein between the B subunit of CT and the JEV envelope protein showed some promise for the development of non-invasive JE vaccine. Our results suggest that the mucosal vaccination approach is feasible for a non-mucosal pathogen such as JEV, but that the adjuvant, carrier molecule, and administration route must be optimized for construction of an effective vaccine platform.

The spectrum of organisms causing sepsis is different in developing countries. Data on the recent trends of organisms causing sepsis are limited. This study was conducted in a tertiary care neonatal unit in Northern India. All inborn babies with blood-culture-positive sepsis from 1995 to 2006 were divided into two epochs, viz. 1995 to 1998 (epoch I) and 2001 to 2006 (epoch II). Organisms were grouped into early (<72 h) and late onset (≥72 h) sepsis groups. The overall incidence of sepsis, the incidence of sepsis stratified by weight groups, the organism profile on different days of life, sepsis-related mortality and pathogen-specific case fatality rate were calculated and compared between the two epochs. Out of 34,362 live births during the study period, organisms were isolated in 1,491 neonates. Out of these, 89% had bacterial sepsis. The incidence of neonatal bacterial sepsis increased from epoch I to epoch II (35.8/1,000 versus 40.1/1,000 live births, P < 0.05). The incidence of early onset sepsis (EOS) did not change between the epochs, but the incidence of late onset sepsis (LOS) increased from 12 to 16.5 per 1,000 live births (P < 0.001). The incidence of bacterial sepsis decreased significantly in the 1,000- to 1,999-g birth weight groups. Klebsiella pneumoniae and Enterobacter aerogenes decreased, whereas Staphylococcus aureus increased in incidence during epoch II. Non-fermenting Gram-negative bacilli emerged as a newly identified pathogen during epoch II. Sepsis-associated mortality decreased from 42 to 20%. The incidence of bacterial sepsis has decreased significantly in 1,000- to 1,999-g infants, with a significant reduction in sepsis-related mortality. New organisms have emerged in recent years. The organism profile in recent years has changed, with a significant overlap of organisms causing EOS and LOS.

Using the adverse events monitoring system of Japan, we observed diarrhea cases in approximately 10% of patients who received oral poliovirus vaccine (OPV). This study was conducted to investigate whether diarrhea among children aged 0 to 1 is caused by OPV or by other factors such contact at the doctor's office and/or with others outside the home. We conducted a survey of the health of children after regular health check-ups and after the administration of the OPV. The data from the health check-ups were used as a control for the OPV case group. We compared the first-OPV dose vaccination group as well as the second-OPV dose vaccination group to the health check-up group. For cases of diarrhea, the odds ratio of the OPV group to the health check-up group was 1.776. Our findings strongly suggest that post-OPV cases of mild diarrhea are closely related to the administration of the OPV.

Pulsed-field gel electrophoresis (PFGE), which determines the genomic relatedness of isolates, is currently used for the epidemiological investigation of infectious agents such as bacteria. In particular, this method has been used for the epidemiological investigation of Legionella outbreaks. However, it takes 4 days to complete a Legionella-PFGE analysis. Due to partial digestion and DNA damage, the reproducibility of the obtained fragment digestion patterns is poor for this pathogen. In this study, we report an improved protocol that takes only 2 days to complete and that allows clear discrimination of the restriction profile with higher reproducibility than that previously achieved.

Cyclospora cayetanensis is a coccidian protozoa that was newly recognized in 1979 in Papua New Guinea. We report the case of a 42-year-old French man who had visited Vietnam and presented with fever and watery diarrhea that had lasted for more than 2 weeks. The patient was diagnosed with C. cayetanensis infection by examination of a stool smear using UV fluorescence microscopy. Based on this rare case, we recommend that Cyclospora infection might be considered in the differential diagnosis of traveler's diarrhea in immunocompetent patients.

Cryptococcus neoformans usually involves the central nervous system and the respiratory tract. We report a case of disseminated cryptococcosis with a liver abscess and meningoencephalitis in a patient with myelodysplastic syndrome. Computed tomography of the abdomen showed a 3-cm low-attenuated lesion in the left lobe of liver. Cultures from specimens of blood, the liver abscess, and the cerebrospinal fluid all yielded C. neoformans. The cryptococcal antigen titers for the serum and cerebral fluid were both 1:32. The patient was successfully treated with 1,335 mg of amphotericin-B followed by fluconazole. Most cryptococcal liver infections present as hepatitis, cholangitis, or microabscesses.

Herein we report our experience in containing an outbreak of nosocomial respiratory syncytial virus (RSV) infection in a psychiatric ward in central Taiwan during a non-widespread RSV seasonal occurrence. A total of 8 patients and 4 healthcare workers in the psychiatric ward developed febrile illness or upper respiratory tract infection symptoms between August 23 and 29, 2005. RSV was identified by either viral culture or reverse transcriptase-polymerase chain reaction (RT-PCR) assay. RSV was isolated from a symptomatic staff member (8.3%), and was detected in 5 (42%) by RT-PCR among 12 cases. All 5 of these RSV cases detected belonged to genotype A. In our experience, single cubicle isolation of infectious patients and a cohort of nursing care are the most important factors in the successful control of an RSV outbreak.

This report describes multiple viruses in stool specimens from oyster-associated gastroenteritis. Eleven outbreaks of oyster-associated gastroenteritis were examined for enteric viruses between January 2002 and March 2006 in Japan. Multiple norovirus genotypes were detected in all outbreaks; moreover, kobuvirus, sapovirus, and astrovirus were also detected in 6, 3, and 1 of the 11 outbreaks, respectively. Notably, multiple sapovirus genogroups were detected in the stool specimens from subjects in two oyster-associated gastroenteritis outbreaks.

Virological investigation was carried out to determine the etiology of suspected Chikungunya fever among humans reported in the Lakshadweep islands in the Indian Ocean. Three out of 23 acute sera samples showed cytopathological changes in Vero cell lines. Further, indirect immunofluorescence antibody test and reverse transcriptase-polymerase chain reaction studies demonstrated the Chikungunya virus etiology during the episode. E1 gene sequence analysis has confirmed the involvement of the Central/East African genotype of the Chikungunya virus.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis with the potential of human-to-human transmission that affects wide areas in Asia, Southeastern Europe, and Africa. Hemorrhagic manifestations constitute a prominent symptom of late stage disease with case fatality rates from 3 to 50%. We present a case of CCHF complicated by hemorrhagic pleural effusion and resulting in resolution without chest tube drainage in a 9-year-old boy. The diagnosis of CCHF was confirmed by enzyme-linked immunosorbent assay tests. Both serum and pleural fluid CCHF IgM were positive at titers of 1/1,600 and 1/6,400, respectively.

Anaplasma bovis and Anaplasma phagocytophilum infection were examined by species-specific nested polymerase chain reaction in cattle in a pastureland where sika deer appear in Hokkaido, Japan. Of the 78 cattle examined, 12 (15%) and 1 (1%) tested positive for infection by A. bovis and A. phagocytophilum, respectively. One cattle tested positive for both. A. phagocytophilum infection rates were significantly lower in cattle than in sika deer (46%), but the infection rate by A. bovis was not significantly different between cattle (15%) and sika deer (23%). The strain of A. phagocytophilum detected in this study may possess significantly lower virulence or infectivity in cattle hosts. No clinical symptoms were recorded in the positive cattle, and morulae were not detected in the blood smears.