Development of a sensitive immunochromatographic kit using fluorescent silica nanoparticles for rapid serodiagnosis of amebiasis.

We have previously shown that the C-terminal region of the intermediate subunit of Entamoeba histolytica galactose- and N-acetyl-D-galactosamine-inhibitable lectin (C-Igl) is a useful antigen for serodiagnosis of amebiasis. An immunochromatographic kit was developed using fluorescent silica nanoparticles coated with C-Igl prepared in Escherichia coli. Samples for examination were added to the freeze-dried particles and then applied to the immunochromatographic device, in which a test line on the membrane was also coated with C-Igl. Fluorescent intensity was measured using a hand-held reader. In an evaluation of the kit using a human monoclonal antibody, the minimum amount of C-Igl specific antibody showing positive results was 100 pg. In the evaluation of serum samples with different antibody titers in indirect immunofluorescent antibody tests in the kit, 20 µL of serum was sufficient to obtain positive results at 30 min. Serum samples from symptomatic patients with amebic colitis and amebic liver abscess and those from asymptomatic E. histolytica-cyst carriers showed positive results in the kit. Based on evaluation using sera from healthy controls and patients with other infectious diseases, the sensitivity and specificity of the kit were 100 and 97.6%, respectively. Therefore, we conclude that the newly developed kit is useful for rapid serodiagnosis of amebiasis.

Clinical features of 62 imported cases of dengue fever in Japan.

To describe the clinical features of dengue cases in Japan, a retrospective study was conducted on 62 laboratory-confirmed Japanese dengue cases presented to Tokyo Metropolitan Komagome Hospital between 1985 and 2000. Age distribution was from 18 to 62 years old (mean, 31.5 years). All cases were imported from abroad and diagnosed as dengue fever. Clinical manifestations included fever (100%), headache (90%), and skin rash (82%). Laboratory examinations revealed leukocytopenia (71%), thrombocytopenia (57%), elevated levels of serum aspartate aminotransferase (78%), and lactate dehydrogenase (71%). Antibody responses were consistent with that of secondary flavivirus infection in 60% of cases. Severity of symptoms in patients with primary dengue antibody response and those with secondary flavivirus antibody responses didn't show statistical significance. Dengue virus infection should be taken into consideration in the differential diagnosis of febrile patients who recently entered Japan from tropical or subtropical countries.

Characterization of seven genotypes (A to E, G and H) of hepatitis B virus recovered from Japanese patients infected with human immunodeficiency virus type 1.

To investigate the prevalence of hepatitis B virus (HBV) genotypes and characteristics of HBV isolates among Japanese patients infected with human immunodeficiency virus type 1 (HIV), serum samples collected between September 1990 and March 2002 from 471 HIV-infected patients (age, 38.8 +/- 11.4 [mean +/- standard deviation] years; male, 90%) were tested for hepatitis B surface antigen (HBsAg) and HBV DNA. Positivity for HBsAg and HBV DNA was seen in 42 patients (8.9%), 41 of whom had contracted HIV infection through sexual activity and 1 had hemophilia. Genotypes of HBV were determined by comparative and phylogenetic analyses of the S gene sequence (396 nucleotides [nt]). The distribution of HBV genotypes among the 42 HBV-viremic patients was: A (50%), B (5%), C (24%), D (5%), E (2%), H (10%), A plus D (2%), A plus G (2%). The hemophilia patient had HBV genotype D. Genotypes E, G, and H which had not been reported in Japan, were found in one patient each who had traveled to Zambia, the US, and South America, respectively. Genotypes A and D, which are rare in Japan, were found in patients who had no history of traveling abroad. The entire genome of the HB-JI411 (genotype E [3,212 nt]), HB-JI444G (genotype G [3,248 nt]), and HB-JI260 (genotype H [3,218 nt]) isolates had the highest identity of 98.3%, 99.9%, and 98.5%, respectively, with reported HBV isolates of the same genotype. Most Japanese patients coinfected with HIV and HBV had HBV genotypes that are found rarely or had not been reported in Japan.

Evaluation of recombinant fragments of Entamoeba histolytica Gal/GalNAc lectin intermediate subunit for serodiagnosis of amebiasis.

We have recently identified a 150-kDa surface antigen of Entamoeba histolytica as an intermediate subunit (Igl) of galactose- and N-acetyl-D-galactosamine-inhibitable lectin, which is a cysteine-rich protein consisting of 1,101 amino acids (aa) and containing multiple CXXC motifs in amino acid sequences. In the present study, full-length Igl except for the signal sequences (aa 14 to 1088) and three fragments of Igl-the N-terminal part (aa 14 to 382), the middle part (aa 294 to 753), and the C-terminal part (aa 603 to 1088)-were prepared in Escherichia coli, and the reactivity of these recombinant proteins with sera from patients with amebiasis was examined by means of enzyme-linked immunosorbent assay (ELISA). Sera from 57 symptomatic patients with amebic liver abscess or amebic colitis, sera from 15 asymptomatic cyst passers, sera from 40 individuals with other protozoan infections, and sera from 50 healthy controls were used. The sensitivity and specificity of the recombinant full-length Igl in the ELISA were 90 and 94%, respectively. When three fragments were used as antigens in the ELISA, the sensitivities were 56% in the N terminus, 92% in the middle part, and 97% in the C terminus. The specificities of the three antigens were 96% in the N terminus and 99% in both the middle and C-terminal fragments. These results demonstrate that Igl is well recognized in not only symptomatic but also asymptomatic patients with E. histolytica infection and that the carboxyl terminus of Igl is an especially useful antigen for the serodiagnosis of amebiasis.

Remarkable genetic polymorphism among Entamoeba histolytica isolates from a limited geographic area.

In order to understand genetic polymorphisms among Entamoeba histolytica strains in a limited geographic area and among restricted social populations, we studied nucleotide polymorphism in DNA regions that do not encode proteins (locus 1-2 and locus 5-6) and in genes coding for chitinase and for serine-rich E. histolytica protein. Thirty E. histolytica isolates from domestically infected Japanese amebiasis patients (male homosexuals and residents in institutions for the mentally handicapped) and four reference strains were examined. PCR revealed remarkable polymorphisms in both the number and size of the PCR fragments containing these loci. Polymorphisms in lengths, types, and numbers of internal repeat units were observed in locus 1-2 and the repeat-containing region of serine-rich E. histolytica protein among the Japanese isolates. In contrast, polymorphism at locus 5-6 was observed almost exclusively in the number of repeats of a 16-nucleotide unit. The repeat-containing region of chitinase appeared to be the least polymorphic among the four loci with a single dominant genotype representing 66% (20 out of 30) of all of the isolates. Isolates obtained from male homosexuals showed a more complex genetic polymorphism than those from residents in institutions. Considering all four polymorphic loci together, all 19 Japanese isolates from male homosexuals were distinct. In contrast, all isolates obtained from mass-infection cases at a single institution had an identical genotype, suggesting that these cases were caused by a single E. histolytica strain. No significant correlation was found between genotypes and zymodemes or between genotypes and clinical presentations, e.g., colitis or liver abscess. Certain genotypes were observed with higher frequencies in male homosexuals or residents of institutions. These data indicate that genotyping of the E. histolytica isolates by using these four polymorphic loci could serve as a tool to fingerprint individual isolates. We propose that genotyping of ameba isolates should help to determine geographic origins of isolates and routes of transmission.

[Cyclosporiasis: four case reports with a review of the literature].

Four patients infected with Cyclospora cayetanensis who sought medical care at the Tokyo Metropolitan Komagome Hospital are herein presented. All were Japanese males, and their ages ranged from 22 to 50 years of age. One patient, who was HIV-positive with a CD4+ lymphocyte count of 141/microliter, demonstrated no AIDS-defining illness. This patient acquired HIV in some Southeast Asian country/countries through heterosexual contact. This patient presented with watery diarrhea with a frequency of up to 18 times a day for more than two months. The other three cases were not considered to be debilitated hosts. Diarrhea occurring from one to ten times a day continued for 6 to 26 days in all of these three patients. The presumed origin of the infection was considered to be Southeast Asian countries and the season of onset of diarrhea was March to July in all four cases. Treatment with a sulfamethoxazole/trimethoprim compound was performed for both the HIV-infected patient and the other non-debilitated patient. Both symptomatic and parasitologic improvements were quickly observed in these patients. A prospective study was performed using fecal specimens from the diarrheal patients to identify the presence of C. cayetanensis during the period from 1996 to 2001. Protozoa-positive specimens were found in 3 of 410 (0.7%) specimens from patients who had traveled overseas, is one of 148 (0.7%) for HIV-infected (the C. cayetanensis-positive patient also acquired the protozoa in Southeast Asia), and in none of 513 (0%) patients who developed diarrhea in Japan. In summary, C. cayetanensis infection is rare in Japan and most patients infected with this pathogen tend to be overseas travelers and HIV-infected individuals at present.

[A fatal case of shigellosis in an elderly patient].

A 71 year-old male, with no recent history of travelling abroad and a past history of lung cancer two years prior to presentation, which had been successfully treated, developed a sudden onset of watery diarrhea more than ten times a day on February 26, 2001, which gradually became bloody. The next day he visited the Department of Integrated Medicine of the Tokyo Metropolitan Komagome Hospital by ambulance because his consciousness was deteriorating and he was hospitalized. He was hypotensive on admission, and a dopamine preparation was used throughout. The peripheral WBC was 3,800/microliter and the lymphocyte count was 76/microliter which thus suggested the presence of cellular immune suppression. HIV was not tested. He died seven hours after admission. His stool culture yielded a growth of Shigella flexneri 2a, and a blood culture on admission was sterile. No verocyte toxin-producing Esherichia coli was not detected. The causes of death in cases with shigellosis have been reported in the literature to be an electrolyte imbalance, septicemia and disseminated intravasucular coagulation (DIC) in developed countries. Our present case was considered to be a debilitated patient complicated with hemolytic uremic syndrome due to an infection with Shigella bacteria which resulted in death despite performing intensive treatments.