Takashige Miyazaki

An evaluation of serodiagnostic tests in patients with candidemia : Beta-glucan, mannan, candida antigen by Cand-Tec and D-arabinitol

The serodiagnostic tests, beta‐glucan, mannan, candida antigen by Cand‐Tec, and D‐arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta‐glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test‐D® and Endospecy®), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand‐Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D‐Arabinitol was the least sensitive, because a D‐arabinitol/creatinine ratio greater than 2.0 μmol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti‐fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.

Comparison between Wako‐WB003 and Fungitec G tests for detection of (1→3)‐β‐D‐glucan in systemic mycosis

The limulus factor G reacts with (1→3)‐β‐D‐glucan, a major structural component of fungal cell walls. The Fungitec G test is a colorimetric assay that measures the concentration of (1→3)‐β‐D‐glucan and is used as a serodiagnostic test for deep mycosis. Wako‐WB003 is another assay for (1→3)‐β‐D‐glucan that determines the change in turbidity of the gelatin reaction of limulus factor G with (1→3)‐β‐D‐glucan. In five rabbits inoculated intravenously with 1 × 107 CFU of Candida albicans, the concentration of (1→3)‐β‐D‐glucan measured by the fungitec G test increased gradually reaching a peak of 660.9 ± 427.9 pg/ml (mean ± SD) 4 days after inoculation, but to 42.225 ± 41.275 ng/ml on day 6 in the Wako‐WB003 test. In one rabbit challenged intravenously with 5 × 106 CFU of C. albicans, (1→3)‐β‐D‐glucan increased to 101.5 pg/ml on day 4 on the fungitec G test, whereas the level remained below the detection limit of the Wako‐WB003 test throughout the course of the disease. We also detected high concentrations of (1→3)‐β‐D‐glucan in 11 patients with candidemia, 4 with suspected candidemia, 1 with invasive pulmonary aspergillosis, and 12 patients with aspergilloma. The concentration of (1→3)‐β‐D‐glucan measured by the Fungitec G test was > 150, > 1006.8, 312.1, and 55.6 ± 37.4 pg/ml (range, 20.1–138.0 pg/ml), and by the Wako‐WB003 test > 153.000, > 17.70, 153.000 and 2.645 ± 7.248 ng/ml (range, < 25.20 ng/ml) in these patients, respectively. In contrast, the concentration of (1→3)‐β‐D‐glucan in 9 patients with pulmonary cryptococcosis and 6 with superficial candida colonization ranged from < 13.2 and < 15.3 pg/ml in the Fungitec G test and < 0.53 and < 0.12 ng/ml in Wako‐WB003 test. There was a weak relationship between the concentration of (1→3)‐β‐D‐glucan measured by the Fungitec G test and Wako‐WB003 test (r = 0.521). Our results indicate that the sensitivity of the Wako‐WB003 test is lower than that of the Fungitec G test. J. Clin. Lab. Anal. 11:73–77.

In vitro and in vivo antifungal activities of liposomal amphotericin B, and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.

The Eiken Latex test for detection of a cryptococcal antigen in cryptococcosis

A latex agglutination test for cryptococcal antigen, the Eiken Latex test (Eiken, Tokyo, Japan), was compared with a monoclonal antibody-based agglutination assay, Pastorex® Cryptococcus (Diagnostics Pasteur, Marneur-la-Coquette, France). In a murine model of disseminated cryptococcosis, the kinetics of the antigen titers by the Eiken Latex were similar to those by the Pastorex® Cryptococcus, but sensitivity was much higher. In HIV-negative patients with pulmonary cryptococcosis, a cryptococcal antigen was detected in 6 of 10 patients by the Eiken Latex test and in only 3 of those patients by the Pastorex® Cryptococcus. The results indicate that the Eiken Latex is more sensitive for the detection of the cryptococcal antigen, even in non-disseminated cryptococcosis. The sensitivity and specificity of the Eiken Latex were examined using 195 sera from 25 patients with pulmonary cryptococcosis and 170 patients with non-cryptococcosis. The cutoff value of ≥ 1:8 showed a sensitivity of 76% (19/25) and a specificity of 98.9% (168/170).

Micronutrient Status and Glutathione Peroxidase in Bedridden Patients on Tube Feeding

Deficiency of micronutrients, especially selenium, is common in critically ill patients. We investigated the micronutrient status (selenium, zinc, copper and manganese) and glutathione peroxidase (GSH-Px) activity in 30 tube-fed patients and 21 hospitalized non-tube-fed control patients. Serum levels of selenium, copper and manganese in tube-fed patients were significantly lower than in control patients (selenium: 4.85 ± 1.38 μg/dl versus 8.67 ± 1.45 μg/dl; copper: 44.7 ± 36.9 μg/dl versus 92.1 ± 21.2 μg/dl; manganese 0.59 ± 0.41 μg/dl versus 1.52 ± 0.59 μg/dl). However, zinc and log GSH-Px in the serum were similar in the two groups. Serum selenium concentration correlated with the daily intake of selenium in tube-fed patients, but zinc, copper and manganese concentrations did not correlate with the daily intake of the respective trace elements in tube-fed patients. Blood GSH-Px activity correlated positively with serum selenium concentrations in the control patients, but not in tube-fed patients. These results demonstrate that selenium content of enteral feed appears to be insufficient to maintain normal serum levels in elderly bedridden patients. Our findings emphasize the importance of monitoring micronutrient status in patients on enteral feeding to avoid trace element deficiencies

Production of monoclonal antibodies against Legionella pneumophila serogroup 1

Four monoclonal antibodies to Legionella pneumophila Philadelphia 1 were produced by the fusion of immunized BALB/c lymphocytes to a murine myeloma cell line. Two (Lpl‐1 and Lpl‐3) of the four monoclonal antibodies reacted with 14 L. pneumophila serogroup 1 strains, and the other (Lpl‐2 and Lpl‐4) reacted with only three out of 20 strains tested. These four monoclonal antibodies did not bind to any strains of L. pneumophila serogroup 2–7 and other Legionella species. In addition, it has been shown that these monoclonal antibodies may be useful not only for subserotyping of L. pneumophila but also for retrospective diagnosis using immunopathological methods.

Combination therapy with miconazole and flucytosine for deep-seated mycoses

This study evaluated combination therapy with miconazole and flucytosine for treating deep seated mycosis. Both mycological and clinical efficacy against candidemia and pulmonary cryptococcosis were satisfactory: All four isolatedCandida spp. were eradicated and all four patients with candidemia were cured, while six of nine patients with pulmonary cryptococcosis exhibited clinical improvement. The efficacy of combination therapy appeared to be low against aspergillosis, since only one of two patients with invasive pulmonary aspergillosis and two of five patients with pulmonary aspergilloma exhibited clinical improvement. Nevertheless, the clinical efficacy against aspergillosis was higher than that obtained with monotherapy in our previous study. Adverse reactions were observed in 36% (13 of 36) and abnormal laboratory findings in 22% (8 of 36) of patients treated with combination therapy; these rates were higher than those reported for monotherapy. These increases were not due to changes in serum concentrations of the two drugs due to simultaneous administration. The findings of this study suggest that combination therapy with miconazole and flucytosine may be an alternative therapy for refractory mycoses for carefully chosen patients.

Rapid Diagnostic Methods for Fungal Infections-Using Chromogenic Limulus Tests and Enzymatic Measurement of D-Arabinitol

Conventional chromogenic limulus test (toxicolor test) reacts with glucan of bakers yeast as well as endotoxin, because toxicolor test contains factor G which is sensitive to glucan and polysaccharide of fungus. A new endotoxin specific limulus test (endospecy test) was developed to improve reaction which responded only to endotoxin. The toxicolor test showed the positive reaction to the culture media of candida albicans 7N strain in RPMI 1640 medium, but the endospecy test did not. D-arabinitol, measured by fluometric enzymatic method, also was positive to culture media. These results suggested that a combination of the toxicolor and the endospecy test and enzymatic measurement of D-arabinitol could be applied to rapid diagnostic methods of fungal infection.