A survey of amoebic infections and differentiation of an Entamoeba histolytica-like variant (JSK2004) in nonhuman primates by a multiplex polymerase chain reaction.

A pathogenic Entamoeba histolytica-like variant (JSK2004 strain) with genetic variations and a novel isoenzyme pattern isolated from a De Brazza's guenon in a Tokyo zoo in Japan has previously been documented. In this study, a multiplex polymerase chain reaction (PCR) assay that could distinguish the JSK2004-type E. histolytica-like variant (JSK04-Eh-V) from E. histolytica and Entamoeba dispar using three newly designed primer sets for amplifying each specific DNA fragment from their small-subunit ribosomal RNA genes was developed and established. Forty-seven primates (11 species) from the zoo were surveyed by multiplex PCR to assess the prevalence of JSK04-Eh-V infection, which was recognized in six individuals of four species, including an Abyssinian colobus monkey, a De Brazza's guenon (including the individual from whom JSK2004 was isolated), a white-faced saki, and a Geoffroy's spider monkey. In addition, the autopsied individuals of an Abyssinian colobus and Geoffroy's spider monkey that died of amoebic liver abscess were also evaluated. DNA samples were also analyzed for specific genotypes based on the nucleotide sequencing of two protein-coding (chitinase and serine-rich E. histolytica protein) genes and the protein-noncoding locus 1-2 that was used for fingerprinting of the E. histolytica strain. These studies indicated that the E. histolytica-like variant infection in this zoo was caused by the same type (i.e., JSK04-Eh-V). An axenic culture medium (yeast extract-iron-maltose-dihydroxyacetone-serum) was developed based on the yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium, which is designed for axenic culture of E. dispar. This new medium could be used for axenically culturing E. histolytica, JSK04-Eh-V, and E. dispar in a single medium.

Seroprevalence of Entamoeba histolytica infection in female outpatients at a sexually transmitted disease sentinel clinic in Tokyo, Japan.

From 2003 to 2006, we surveyed the seroprevalence of amoebic infection in female outpatients at a gynecologist's office, which was designated as a sexually transmitted disease sentinel clinic by the Tokyo Metropolitan Government, using an enzyme-linked immunosorbent assay (ELISA). The annual rate of anti-Entamoeba histolytica (HM-1:IMSScl6 strain; HM-1) antibody-positive cases as detected by ELISA increased during that period, and anti-Chlamydia trachomatis antibodies were detected in 60%, i.e., 24 of 40 anti-HM-1 antibody-positive individuals, suggesting sexual transmission of E. histolytica. We designed an ELISA with better sensitivity using the antigen extracted from the virulence-augmented E. histolytica strains (LHM-1 and LLA526 strains) by liver-passaging in hamsters. The average ratios of the S/N value (optical density [OD] of sample/OD of negative control) of ELISA with either the LHM-1 or LLA526 antigen and that of ELISA with the HM-1 antigen were significantly higher in intestinal amoebiasis cases with low S/N values than in amoebic liver abscess cases. In the present study of the seroprevalence of E. histolytica infection, the sera testing positive with low S/N values (<10) by ELISA with HM-1 antigen exhibited higher S/N values by ELISA using LHM-1 and LLA526 antigens. This modification of the antigen preparation for ELISA is expected to be effective in detecting anti-E. histolytica antibodies from such asymptomatic patients who have low antibody titers.

Profiles of a pathogenic Entamoeba histolytica-like variant with variations in the nucleotide sequence of the small subunit ribosomal RNA isolated from a primate (De Brazza's guenon).

A pathogenic Entamoeba histolytica-like variant (JSK2004) with genetic variations and a novel isoenzyme pattern was isolated from a De Brazza's guenon. A homology of 99.1% was found between the clones of E. histolytica (HM-1:IMSS) and JSK2004 in the 1,893 nucleotide bases of the small subunit rRNA (SSU-rRNA) gene. The DNA of the pathogenic amoeba species was also extracted from two sterile liver abscesses during the autopsies of an Abyssinian colobus and a Geoffroy's spider monkey occurring in the same institution in which JSK2004 was isolated, and the homology of the nucleotide sequences in the SSU-rRNA gene of the DNAs was identical to that of JSK2004.

[The risk of pertussis and diphtheria infections among pediatric healthcare workers in Japan].

For infection control in pediatric hospitals, we investigated the risk of pertussis and diphtheria infections among pediatric healthcare workers. Forty-nine Japanese pediatric healthcare workers in 12 general hospitals were screened for antibodies of pertussis toxin (PT), filamentous hemagglutinin (FHA), and diphtheria toxin (DT). The seropositive rates of anti-PT IgG (protective level, > 10 U/mL), anti-FHA IgG (> 10 U/ mL), and anti-DT (> 0.11 U/mL) were 50, 82, and 59%, respectively. During this survey period (Oct. 2003-Feb. 2004), 16 (33%) of the healthcare workers were in contact with pertussis-infant (s). However, all culture and PCR tests for Bordetella pertussis were negative. One of the 16 exposed healthcare workers, a male pediatrician, had serological evidence of a pertussis infection, but no disease symptomatic of pertussis. Our observations indicate that i) 50 and 41% of Japanese pediatric healthcare workers were seronegative for pertussis (anti-PT IgG) and diphtheria antibodies, respectively, and ii) although the healthcare workers had a high rate of contact with pertussis-infant (s), the infection rate was low. For pertussis and diphtheria infection control in pediatric hospitals, it is important for healthcare workers to be aware of their own protection levels against these diseases.

[Anti-drug pattern of drug-resistant Mycobacterium tuberculosis and analysis of mutation in drug-target genes].

The antimycobacterial susceptibility test was performed and minimal inhibitory concentration (MIC) to drugs was determined in 98 strains of Mycobacteium tuberculosis (MTB) isolated in Tokyo from 2000 to 2003, to find which were resistant to any of the four main anti-MTB drugs, isoniazid (INH), rifampicin (RFP), streptomycin (SM), and ethambutol (EMB). 27strains of them were resistant only to SM, and 16 strains were resistant only to INH. 51 strains of them were resistant to not only INH but also other drugs. 38 strains were resistant to both INH and RFP. 19 strains were resistant to all four drugs, including 7 strains resistant to new quinolon anti-biotics also. Nucleotide or amino-acid mutations in drug resistant MTB genome were determined by DNA sequencing method. Mutation of codon 516, 526, or 531 of rpoB gene was detected in 98% of MTBs resistant to RFP. Deletion or insertion of katG gene or nucleotide mutation at regulatory region of ahpC gene was detected in MTBs highly resistant to INH. Amino acid mutation of katG gene, especially at codon 315, was detected in MTBs resistant to INH intermediate. Nucleotide mutations at regulatory region of inhA gene were detected in MTBs resistant to INH at low level. Amino acid mutation at codon 43 or 88 of rpsL gene was detected in MTBs highly resistant to SM, and nucleotide mutation at 512, 513, or 516 of rrs gene was detected in MTBs resistant to SM at low level. Amino acid mutation at codon 306 of embB gene was detected in 87% of MTBs resistant to EMB.

[Drug susceptibility and analysis using pulsed-field gel electrophoresis of Streptococcus pyogenes strains isolated from the patients with streptococcal toxic shock syndrome (STSS) in Japan].

Previously, we have performed T typing of Streptococcus pyogenes strains isolated from patients with streptococcal toxic shock syndrome (STSS) in Japan, and streptococcal pyrogenic exotoxin (SPE) typing for epidemiological examination. In this study, we conducted a drug sensitivity test using these strains, and investigated the results of gene analysis by pulse-field gel electrophoresis (PFGE) of S. pyogenes strains derived from patients with STSS, the patient's family, and patients other than those with STSS. To clarify the relationship between the host and bacterial factors, we investigated the association between clinical symptoms and T typing of the isolated strains/production of streptococcal pyrogenic exotoxin. There were no strains resistant to beta-lactams, and only 1 strain was resistant to multiple agents other than beta-lactams. The PFGE pattern of T1 type strains was classified into 2 ; the pattern was consistent between the strains derived from patients with STSS and those derived from the patient's family. The PFGE pattern of T3 type strains was classified into 5 (IV) ; Pattern I, which was most frequently observed, was detected in both the strains derived from patients with STSS/non-STSS. However, Patterns II and III were detected only in the strains derived from patients with non-STSS. Patterns IV and V were detected only in the strains derived from patients with STSS. When examining the association between clinical symptoms and bacterial factors, disseminated intravascular coagulation (DIC) was associated with T1-SPE B-producing strains, and pharyngitis was associated with T3-SPE A-producing strains. In the future, the relationship between the host and bacterial factors should be further investigated.

[Epidemiological study on Streptococcus pyogenes strains isolated from the patients with streptococcal toxic shock syndrome (STSS) in Japan in 1992-2001].

To investigate clinical and microbiological features of streptococcal toxic shock syndrome (STSS), clinical, epidemiological, and bacteriological data obtained from 250 patients between 1992 and 2001 were analyzed. Among these 250 cases, 16 cases were excluded from the study because the causative microorganism were not Streptococcus pyogenes. 234 strains of S. pyogenes obtained from the aforementioned 234 cases were tested for T-type by a serological method, and for streptococcal pyrogenic exotoxin (SPE) by in vitro productivity of the toxin as well as molecular genetic methods. The number of patients was 141 (56.4%) for males, and 107 (42.8%) for females. The highest frequency of STSS was observed in those patients in their sixties in both sexes. The overall mortality rate was 43.2%. The mortality rate for male was 36.9%, and 52.3% for female. Bacteriological studies revealed that most common T types were T1 and T3. These strains consisted 54.3% of the strains collected. Among strains of T1 type, 98.8% possessed genes of spe A, and 46.1% were shown to produce SPE A in vitro. Among strains of T3 type, 82.9% possessed spe A gene, and all of these strains were shown to produce the toxin in vitro. It is concluded that certain strains of S. pyogenes, such as those with T1, or T3 type, and those with spe A gene or in vitro production of SPE A, are the most frequent cause of STSS. Although infections caused by such bacteria are quite common, STSS rarely occurs in most such patients. Additional factors, such as host factors, may play a crucial role in the pathogenesis of STSS.

[A case of invasive Clostridium perfringens infection complicated intravascular hemolysis].

We experience a case of a 83-year-old male who was admitted complaining of chills, cramp, high fever and respiratory distress. His blood revealed marked hemolysis. Gram positive Rods was observed in the hemoliesed blood taken on admission. About 2 hours after admission, he suddenly fell into a critical condition. He died about 6 hours after admission in spite of resuscitation. Clostridium perfringens was detected from the blood and liver obtained by autopsy. We suspected that he died of acute intravascular hemolysis caused by alpha-toxin produced by C. perfringens. In conclusion, for a patient who has a high fever with strong hemolysis such as our case, C. perfringens infection should be considered.