Atsuro Hashimoto

Comparison of PCR, (1→3)‐β‐D‐glucan and galactomannan assays in sera of rats with experimental invasive aspergillosis

We compared PCR, galactomannan detection assay using a latex agglutination test and (1→3)‐β‐D‐glucan detection assay in detecting infection in rats experimentally infected with Aspergillus fumigatus. On day 2 after inoculation, (1→3)‐β‐D‐glucan and nested PCR were positive for 80%, while galactomannan detection assay was positive for 60%. In addition, the positive result of nested PCR (87.5%) was higher than those of galactomannan detection assay (75%) and (1→3)‐β‐D‐glucan (71.4%) on day 3 after inoculation. The sensitivity of nested PCR was superior to those of galactomannan detection assay and (1→3)‐β‐D‐glucan detection assay. The three diagnostic tests were compared with histopathological findings, and the sensitivity of three diagnostic tests was correlated with histopathological changes. In addition, the elevated levels of (1→3)‐β‐D‐glucan paralleled the development and progression of pulmonary aspergillosis. Our results indicate that a combination of two or three of these tests seems to provide a rapid diagnosis of invasive aspergillosis and assist in the evaluation of the development and severity of invasive aspergillosis. J. Clin. Lab. Anal. 12:257–262, 1998.

Rapid detection and identification of mycobacteria by combined method of polymerase chain reaction and hybridization protection assay

A new method for the rapid detection and identification of mycobacteria, combining polymerase chain reaction (PCR) with DNA probe, was developed. The mycobacterial 16S rRNA encoding gene was amplified by nested PCR, and the PCR product was identified by hybridization protection assay using acridinium ester labelled DNA probe. The optimum temperature for hybridization of PCR product and DNA probe was estimated as 55 degrees C. The specificity of the combined method was excellent in the detection and identification of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex (MAC). The detection limit was 10 fg DNA for M. tuberculosis, and 100 fg DNA for MAC. Results of preliminary clinical investigation of this method using 207 clinical specimens demonstrated an efficient detection and identification of M. tuberculosis and MAC. These results indicate that the new combined method may be a useful test for the rapid detection and identification of M. tuberculosis and MAC in clinical specimens.

Efficacy of Recombinant Human Granulocyte-Colony Stimulating Factor Alone and in Combination with Antifungal Agents against Disseminated Trichosporonosis in Neutropenic Mice

The ability of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to protect and potentiate the activity of antifungal agents against a lethalTrichosporon beigelii infection in myelosuppressed mice was evaluated in this study. Mice were rendered neutropenic by 2 consecutive-day intraperitoneal injections of cyclophosphamide (200 mg/kg). Recombinant hG-CSF, given subcutaneously at daily doses of 15, 60, and 120 μg/kg for 6 days, shortened the period of neutropenia and increased the number of circulating neutrophils in a dose-dependent manner. When rhG-CSF was administered to neutropenic mice before challenge withT. beigelii (5×106 CFU), it protected against the lethal infection, resulting in improved survival and decreased numbers of fungal cells in the lung, liver, spleen and kidney. However, when the inoculum size increased to 7×106 CFU, a poorer result was obtained using a dose of 60 μg/kg of rhG-CSF, suggesting that the activity of rhG-CSF is dependent on the severity of theT. beigelii infection. Combined with fluconazole (10 mg/kg) or amphotericin B (1 mg/kg), rhG-CSF improved the median survival time from 16 days with fluconazole (59%) and 12 days with amphotericin B (41%) alone to 20 days (93%) and 16 days (55%) in neutropenic mice treated with rhG-CSF plus fluconazole or amphotericin B, respectively. However, the combination of rhG-CSF and itraconazole did not produce significant improvement in survival or clearance of fungal cells from the organs of neutropenic mice. These findings show that rhG-CSF may be a useful immunomodulator againstTrichosporon infections in neutropenic mice and the therapeutic outcome improves when used in combination with fluconazole or amphotericin B.

Clinical and Immunological Evaluation of Infection in Patients on Hemodialysis

Infection is a major complication associated with increased morbidity and mortality in patients on hemodialysis. We analyzed the incidence and type of infection occurring in 4841 patients on hemodialysis between 1986 and 1993 in our hospital and 11 other hemodialysis centers. Infection was noted in 193 patients (4.98 infections/1000 patients/year). Pneumonia (n=71) and bacteremia (n=24) were the 2 most common infections, followed by tuberculosis (n=14), herpes zoster infections (n=12) and infections at the vascular access site (n=12). The most commonly isolated organism in pneumonia, bacteremia and vascular access site infections wasStaphylococcus aureus. Analysis of the prognosis of patients with pneumonia showed a mortality rate of 50% in patients greater than 60 years old, which was significantly higher than that of younger patients (6.7%,P<0.01), whereas the mortality rate in patients with bacteremia was not different between the 2 age groups (60.0% vs. 57.9%, respectively). We also analyzed changes in immunological function and nutritional status in 16 patients on hemodialysis and 21 healthy control subjects. Although the phagocytic and bactericidal activities of neutrophils and monocytes were not different between the groups, superoxide production, the percentage of natural killer cells and the degree of blastoid transformation with phytohemagglutinin stimulation were significantly lower in hemodialysis patients. Low levels of Nidermans index and serum albumin and transferrin indicated poor nutritional status in these patients. Furthermore, the degree of Nidermans index and serum albumin significantly correlated with impairment of immunological function, such as reduced blastoid transformation and the number of lymphocytes. Our results suggest that analysis of the patterns of infection in patients on hemodialysis should provide better management and that improvement of malnutrition may ameliorate impaired immunity in hemodialysis patients.