Cyclospora cayetanensis is an intestinal coccidian parasite and known as a human pathogen causing watery diarrhea. Recently, Cyclospora organisms, morphologically indistinguishable from C. cayetanensis, were detected from the several species of primates, and three new species named, C. cercopitheci, C. colobi, and C. papionis, have been proposed to the isolates on the basis of the genetic differences. The infectivity of these species to humans is strictly unknown, and there is a possibility of infection with not only C. cayetanensis but also the species from primates among patients. Therefore, it is necessary for the accurate diagnosis of Cyclospora infection to distinguish among the Cyclospora species. In the present study, we identified species of Cyclospora isolates from patients by the PCR-based diagnostic method. Since the sequence of Cyclospora 18S ribosomal RNA gene generated with the primer pair CYC1FE and CYC4RB is found to be variable among Cyclospora species, we applied the PCR-direct sequencing using above primers to identify species of the isolates. Consequently, the diagnostic fragment was amplified by the PCR in all isolates, and the sequence of the PCR product obtained from each isolate was completely identical to that of C. cayetanensis. Therefore, we identified the isolates from patients as C. cayetanensis. On the basis of the results obtained in the present study, it is supposed that the PCR-direct sequencing using the primer pair CYC1FE and CYC4RB is a useful tool for the distinction among Cyclospora species.
In May 2000, an outbreak of acute gastroenteritis caused by human group C rotavirus (CHRV) occurred in a youth educational center located in the southern area of Okayama Prefecture. A total of 172 schoolchildren and teachers, who consisted of 51 persons belonging to F school and 121 persons belonging to K school, joined in an educational program at the center from May 24 to 26. Eighty-seven individuals (50.6%) of them showed clinical symptoms of gastroenteritis from May 24 to 30, and the outbreak peaked on May 27. The major clinical symptoms were abdominal pain (87.4%), diarrhea (50.6%), nausea or vomiting (21.8%), fever>37°C (12.6%), and headache (14.9%). The clinical symptoms of the patients in F school were more severe than those in K school. Thirtytwo fecal specimens were collected from the patients and examined for gastroenteritis viruses by electron microscopy, ELISA, reverse passive hemagglutination test, and reverse transcription (RT)-PCR. As a result, CHRVs were detected in 21 specimens (65.6%) by RT-PCR. The possible route of the CHRV infection was thought to be a person to person transmission by following reasons: (i), CHRVs were detected in stools from patients who became ill on the first day of the program; (ii), CHRVs were not detected in stools from cooks; (iii), no possible causal food was found by epidemiological analysis of the outbreak. Furthermore, phylogenetic analysis of the VP7 gene among CHRVsisolated in Okayama revealed that the virus detected in this study was more closely related to the virusisolated from a sporadic case of gastroenteritis in 1996 than that isolated from an outbreak occurred in 1999.
Ten cases of tsutsugamushi disease which were diagnosed and treated among the outpatients from 1987 through 1999 in a clinic of internal medicine locating at the northern districts of Awaji Island were investigated. The patients were consisted of 8 males and 2 females and their ages ranged from 6 to 69 years old. The infected areas were distributed in hilly districts extending over three towns. The outbreak season was from November through December. The following two facts seemed to be worthy of notice. 1) There was a time lag on the patient's onset among the different years. 2) When the incidences were more than two cases a year, the time difference of onset was within a few days each other. Then the relationship between atmospheric temperature and the date of onset was studied. Meterological observing records at Gunge Ichinomia Town near the northern districts of Awaji Island were investigated into the temperature every 10 days from 1987 to 1999. Average temperature every 10 days including the presumable day when each patient was infected with Orientia tsutsugamushi (O. T) ranged between 9.1°C and 13.3°C and in 7 of 10 cases ranged between 11.6°C and 13.3°C. The maximum temperature ranged between 11.3°C and 17.5°C, the minimum one between 6.6°Cand 9.1°C. It is likely that the seasonal occurrence of tsutsugamushi disease greatly depends upon the optimum temperature for the activity of tsutsugamushi mites in these areas. The high titer of serum IgM and IgG antibody to Karp, Gilliam Kato type O. tsutsugamushi was detected by an immunofluorescense assay (IF) in all cases. IgG antibody titers fell drastically to 20 or 10 times within a few years but 10 times titer was still detected in three cases in more than ten years. It is suggested that IgG antibody titer more than 10 times in healthy people indicates the infection in the past and titer more than 20 times implies the infection during the past few years.
Among the isolates of methicillin resistant Staphylococcus aureus (MRSA) which were isolated at Juntendo University Hospital between 1992 and 1998, there were 2 arbekacin resistant strains, 9 mupirocin resistant strain and 7 teicoplanin resistant strains. For each resistant strain, we studied pulsed-field gel electrophoresis (PFGE) pattern, coagulase type, antimicrobial susceptibility pattern and investigated a distribution of the resistant strains at the hospital wards. Two arbekacin resistant strains showed PFGE type A (A3 and A7, respectively). Nine mupirocin resistant strains were type C1; 3 strains, typeA (A1, A4, A8); 3 strains and type D, E, G for 1 one each strain. The antimicrobial susceptibility pattern was not similar to each other and the strains were isolated from different year. It is suggested that these strains were not same origin. Seven teicoplanin resistant strains had PFGE typeB (B1; 5 strains, B2/B5; 1 each strain). The antimicrobial susceptibility pattern of the strains was similar to each other and the strains were isolated from the same hospital ward between 1995 and 1997. From the above fact, the resistant strains appear to be hospital strains which have same origin.
Cryptosporidium parvum oocysts were exposed to the mixed-oxidant solution, which was electrochemically generated by Miox Water Disinfection Unit, and sodium hypochlorite in phosphate buffered saline (PBS, pH 7.2) or biologically treated wastewater at 25°C by using concentrations of residual chlorine of up to 5 mg/l and contact times of up to 8 h. The effect of two disinfectants on infectivity of the oocysts in a neonatal murine model was comparatively evaluated by determining the total number of oocysts recovered from the intestine. Exposure to the mixed-oxidant solution at 2 and 5 mg/l (residual chlorine) yielded a significant inactivation of infectivity in the dose-and exposure timedependent manner, while exposure to 5 mg/l (residual chlorine) of sodium hypochlorite for contact times of up to 4 h produced no measurable inactivation of infectivity. Morphological examination also revealed a picture of degenerating oocysts after exposure to 5 mg/l (residual chlorine) of the mixedoxidant solution, but not with sodium hypochlorite. When the oocysts were exposed to either biologically treated wastewater-or PBS-diluted the mixed-oxidant solution at 5 mg/l (residual chlorine) for4 h, the disinfectants produced a significant inactivation of infectious oocysts. The decrease number of the oocysts was 0.8 log10 in the former and 2.1 log10 in the latter. These results demonstrate that the mixed-oxidant solution may be a useful disinfectant against Cryptosporidium oocysts, but appropriate applications need to be validated.
As part of an epidemiological study of legionellosis, we investigated the growth within Acanthamoebasp. and antibiotic susceptibility of 62 strains of Legionella spp. isolated from surface soils nationwide in 2001. 1) All strains tested grew in Acanthamoeba sp., suggesting that the strains were pathogenic. The minimum bacterial number required for the growth in the amoeba was 103-108 CFU/ml and there were differences between the strains. 2) Susceptibility to 10 drugs was investigated using the Etest. The MIC90 values of imipenem, as a β-lactam, and rifampicin, as an antitubercular agent, were 0.047μg/ml and 0.064μg/ml, respectively, showing high sensitivity. In contrast, sensitivity to minocycline, as a tetracycline, and piperacillin, as a β-lactam, was low and the MIC90 values were 12μg/ml and 16μg/ml, respectively. Sensitivity to minocycline was particularly low, with a MIC value of 32μg/ml, in two strains. The above findings suggested that all soil-derived strains were pathogenic, and susceptibility of the strains tended to be slightly lower than that of clinical isolates.
A party of 57 people dined together in a restaurant in Hamamatsu City on December 11, 2001. The nextday, 22 of them developed symptoms of acute gastroenteritis, such as diarrhea, vomiting, and fever. Examination of 4 fecal specimens from these patients by ELISA for Norovirus (Norwalklike virus, NV) detected both genogroup I (GI) and genogroup II (GII) NV in all the 4 specimens. In addition, RT-PCRand real-time PCR methods for NV detected the NV gene. Approximately one month after the outbrea of the food poisoning (acute gastroenteritis) by NV, 4 individuals in the same party developed type A hepatitis. Both RT-PCR and real-time PCR methods for hepatitis A virus (HAV) detected the HAV genein their fecal specimens. The party of these patients ate purple Washington clam (saxidomus purpuratus, imported from China) steamed with red pepper. Since this food appeared to have caused the viral infections, the one with the same lot number was subjected to viral examinations, which successfully detected the NV GI, NV GII, and HAV genes. These results led to the conclusion that the clam contaminated with NV and HAV had caused the food poisoning. The DNA sequences of the NV detected in the patients and the clam had 74 to 99% homology, indicating strains of various genotypes. All the strains of HAV that were derived from the patients and the clam were genotype 1A, and these sequences had over 95% homology, but were not completely identical. This outbreak led to the demonstration of imported fishery products as a cause of type A hepatitis, and indicated the need for guiding and enlightening people on the importance of adequate cooking of bivalves.
Outbreaks of gastroenteritis caused by Norwalk-like viruses are often induced by the consumption of raw shellfish such as oysters. Incidences reach a peak during the cold season in Japan, when seawater temperatures fall below 10°C. We investigated oysters' uptake and excretion of viruses, over varying lengths of exposure, monitoring the effects of changes in temperature and flow rate of seawater, and the presence of plankton. The study was performed using a poliovirus and an experimental circulatory system, which was framed on the same principle as a model practically used for the depuration of oysters. Polioviruses present in the seawater were taken rapidly into the midgut gland of oysters. However, virus levels detected in oysters at both 10°C and 20°C were decreased to approximately 1/1, 000 to 1/10, 000 within 6hrs after the circulatory seawater was replaced by UV irradiated seawater. These results demonstrate the effectiveness of the circulatory depuration system for the elimination of poliovirus from oysters, and indicate that controlling the temperature and flow rate of the circulatory system could decrease the risk of NLV infection.
Neisseria gonorrhoeae were isolated from pharyngeal specimens of male and female patients andalso from urethral and cervical discharges of male and female patients, respectively, suspected of having gonococcal infections in a urologic clinic in Kawasaki City. Microbiological and epidemiological studies were performed in 127 male and 41 female patients. The specimens were streaked onto the modified Thayer-Martin Selective Agar and the plates were incubated at 35°C for 48h under an atmosphere of 10% CO2. In 127 male patients, N. gonorrhoeae were detected in 117 (92.1%) of the urethral specimens. In these patients, N. gonorrhoeae were detected in pharyngeal specimens from 14 (11.0%) patients, but the pathogen was also detected in urethral specimens from these patients without exception. In 41 female patients, N. gonorrhoeae were detected in 20 (48.8%) of the 41 cervical discharges. When the pharyngeal specimens were tested, N. gonorrhoeae were detected in 14 (34.1%) of the 41 specimens. N. gonorrhoeae was simultaneously detected in pharyngeal and cervical specimens from 11 of the 41 female patients and the pathogen was detected only in pharyngeal specimens from other 3 patients. There were no marked differences in antimicrobial susceptibilities between N. gonorrhoeae isolates from pharyngeal specimens and those from urethral or cervical discharges in all the patients tested. The PFGE patterns of 50 gonococcal isolates (25 pairs) from 25 patients (14 males and 11 females) in whom N. gonorrhoeae were simultaneously detected from pharyngeal and urethral or cervical specimens were analyzed. In 24 of 25 patients, N. gonorrhoeae isolated from the pharyngeal and urethral or cervical specimens in the same patients showed the same PFGE patterns. However, the 25 pairs showed the different PFGE patterns. From these results it is clarified that N. gonorrhoeae are detected in the pharyngeal specimens from considerable numbers of patients with gonorrhea, and there is a possibility that the pathogens prevailing among the patients differ in genetic sources.
Capilia TB, a lateral flow immunochromatographic slide test kit for directly identifying Mycobacterium tuberculosis complex (MTC), was evaluated by using culture-positive specimens from Mycobacteria Growth Indicator Tubes (MGIT). Sputum specimens from patients suspected of having tuberculosis were treated with NALCNaOH and cultivated in MGIT960. Liquid specimens were collected from the positive tubes and directly inoculated with Capilia TB. Liquid specimens were also directly tested with AccuProbe. Of the organisms isolated from the 100 MGIT positive tubes, M. tuberculosis complex was identified in 49 (49%) tubes with Capilia TB and not identified in 51 (51%) with CapiliaTB. Mycobacterium avium-intracellulare complex (MAC) was identified in 46 (46%) with AccuProbe MAC and other acidfast bacteria were identified in 5 (5%) by DNA-DNA hybridization method. There were one tube in which M. tuberculosis complex was detected with Capilia TB and M. tuberculosiscomplex was not detected with AccuProbe MTC, but no tubes in which M. tuberculosiscomplex was detected with AccuProbe MTC and M. tuberculosis complex was not detected with Capilia TB. Capilia TB is excellent in sensitivity and specificity and very suitable for rapid diagnosis of tuberculosis and is considered to contribute to public health intervention measures taken for the tuberculosis control in Japan.