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Invasive fungal infections have emerged as important causes of morbidity and mortality in neutropenicand some other immunocompromised hosts; Candida and Aspergillus are among the major pathogens in this patient population. The clinical diagnosis of these infections is not specific and the traditional mycological methods for them not sensitive, with limits in the early detection of the pathogen. The potential additives or complements to the laboratory diagnosis of invasive candidiasis and aspergillosis are two non-culture-based methods, serodiagnostic methods and molecular ones. The former methods include the detection of pathogen-specific antigens, antibodies, metabolites and cell wall components. Several have already become standard laboratory tools and some others are under active investigation for developing new, more accurate detection systems. In this review, I will discuss the current status and future potential of serodiagnostic methods, highlighting both their technical and clinical implications.

We examined a disparity in D-arabinitol values between two commercial assay kits, LABOFIT and ARABINITEC-AUTO. The determined values by the former were increased by 26%(y = 0.2643x) because of concomitant D-mannitol, whereas those by our newly developed ARABINITEC-AUTO was increased only by 2%(y = 0.0242x). Of 109 samples, 5 samples were found to contain more than 100 mumol/l of D-mannitol. A clear relation(r = 0.89) was noted between the degree of disparity between measurements by the two methods and D-mannitol concentrations in samples. Thus, we have proved that the disparity is mainly caused by D-mannitol.

Purposes: Serum D-arabinitol were measured 1) differentiate between invasive candidasis and colonization in the urinary tract 2) evaluate its efficacy for early diagnosis and treatment monitor of invasive candidasis., Materials and Methods: 1. Laboratory study: Serum D-arabinitol levels were measured in both candida infected rats and control rats. The cut-off levels were determined by the comparison of the two groups. Serum levels of D-arabinitol and CAND-TEC were measured every day per after candida was inoculated. Furthermore, the candida infected rats were treated with fluconazole (0.5 mg/body), and the changes of serum levels of D-arabinitol and CAND-TEC were observed. 2. Clinical study: Serum D-arabinitol, CAND-TEC and beta-D-glucan were measured to evaluate its efficacy for early diagnosis in the group with candida positive urine cultures., Results: The cut-off levels of D-arabinitol were set 2.0 micrograms/ml, at which sensitivity was high, and specificity acceptable. The serum level of D-arabinitol raised as candida infection was established and lowered after treatment was started. CAND-TEC was negative in all materials. High levels of D-arabinitol and beta-D-glucan was measured in invasive candidasis, and lowered++ by fluconazole treatment. CAND-TEC had a low positive rate., Conclusion: Serum D-arabinitol level was sensitive for the early diagnosis of invasive candidasis and its efficacy as a monitor for treatment effects was implied.

Visceral candidiasis is nonspecific in the clinical presentation and a microbiological diagnosis is often difficult to make. Currently, several techniques are available for detection of the antigen and antibody and/or fungal product. We report the results of an investigation in immunodiagnosis and blood biochemical diagnosis of visceral candidiasis and suggest the cutoff limits of positive, from these findings and those reported by others. The efficiency and specificity were 36.4% approximately 60% and 94% approximately 100% for the mannan detection kit from Kyokuto Co., Ltd. and 57.1% approximately 100% and 25% approximately 69.6% for the CAND-TEC, and 28.6% and 91.7% for PASTOREX CANDIDA. For kits using a blood biochemical assay; the efficiency and specificity were 10% approximately 56.7% and 91.4% approximately 100% with detection of D-arabinitol (cutoff limits; 20 mumol/ml < or =), and 70% approximately 75% and 91.7% approximately 100% for Fungal Index (cutoff limits; 60pg/ml < or =). We investigated the evaluation of LA test to detect candidal mannan antigen in sera obtained from experimental gastric candidiasis of mice with or without treatment. There was a good correlation between the change of the titer of mannan antigen and the severity of infections, and gastric lesions healed histopathologically 3 weeks after disappearance of mannan antigen in sera obtained from mice treated with an antifungal agent. These findings indicate that the antifungal therapy is necessary for more than 3 weeks after the candidal mannan antigen disappeared from sera.

Two different flucytosine (5-FC) treatment regimens, one by itself and the other in combination with fluconazole (FLCZ) were compared in chemotherapy against mycotic infections in 60 patients with hematological diseases. The patients in a randomized fashion were assigned to the two treatments. In the combination regimen, the two drugs were used in half doses. beta-D-Glucan and D-arabinitol in the sera of patients were measured to document mycotic infections, and bacterial examinations were also performed. The efficacy of the combination therapy was 60.0% (18/30) and that of 5-FC alone was 65.5% (19/29). The stratified evaluation indicated that no factor was found to contribute to the efficacy in the two treatments with statistical significance. The side effects occurred in few cases and none of those was serious; including, one case of subjective symptom in each groups and two episodes of liver dysfunction in combination treatment. Changes in beta-D-glucan concentrations in the sera reflected well the pathophysiology of mycotic infections and clinical improvement. These results suggested that a combination of 5-FC and FLCZ at half doses provided a clinical benefit comparable to 5-FC alone at the ordinary dose, and the safety was considered satisfactory.

The measurement of D-arabinitol in serum has been reported to be useful for the diagnosis of invasive candidiasis. However, excessive proliferation of Candida species in intestinal tract often leads false positive result of serum D-arabinitol. Based on the evidence that amphotericin B (AMPH) is scarcely absorbed from intestinal tract and inhibits the proliferation of Candida species only in intestinal tract, we have developed a simple differentiation method of intestinal candida colonization from invasive candidiasis by measuring serum level of D-arabinitol in combination with oral administration of low dose AMPH. AMPH, 600 mg/day for 2 days was orally administered to five patients with hematological malignancies who showed more than 1.7 mumol/mg of D-arabinitol/creatinine ratio (D/C ratio) in serum without any evidence of invasive candidiasis. D/C ratios were markedly decreased and normalized after the oral administration of low dose AMPH. While, in a patient with invasive candidiasis in whom Candida species was detected by blood cultures, D/C ratio remained unchanged in spite of oral administration of AMPH. These observations suggest that this method is a simple and reliable diagnostic method to distinguish intestinal candida colonization from true invasive candidiasis.

We encountered a case of an 83-year-old patient with candida endocarditis. He had been diagnosed as having aortic regurgitation with moderately calcified aortic valve 5 years previously, and had received medication. He was admitted to our hospital because of pancreatic cancer. He had high fever from the time of his admission and antibiotics produced no effect. Candida albicans was detected in arterial blood culture. We also detected antibody against Candida albicans, and investigated serum mannan and the D-arabinitol creatinine ratio several times. We performed echo-cardiographic examination and recorded a vegetation at the aortic valve. Rising antibody titers against Candida albicans, mannan antigenemia and an elevated creatinine ratio were also observed. So we concluded that these examinations were also effective in the diagnosis of candida infection. Finally, this patient died of cerebral hemorrhage and was autopsied. Macroscopic findings showed mass-like vegetation involving the aortic valve, and microscopic findings showed candida organisms scattered within the vegetation.

In this study, we measured D-arabinitol, candida antigen and beta-glucan in the rabbit model of systemic candidiasis. It was recently reported that the factor G, one of the coagulation enzymes from the horseshoe crad amebocyte lysate, reacts with beta-glucan or polysaccharide from fungus, and not with endotoxin. The newly developed endotoxin specific chromogenic Limulus assay (endospecy test), which is devoid of factor G, did not react with any rabbit serum. But the beta-glucan measured by the conventional Limulus assay (toxicolor test), which contains the factor G, was elevated (from 10 pg/ml to 100 pg/ml) in all cases of rabbit model of system candidiasis. Serum D-arabinitol, measured by Sone's enzymatic fluorometric method, increased in all cases, but Cand-Tec (candida detection system by latex agglutination) showed only one positive case in five innoculated rabbits.

A clinical study was carried out on 11 patients with Candida urinary tract infection; 4 with chronic cystitis and 7 with acute pyelonephritis including 2 patients associated with candidemia. The patients received 1.5 to 6.0 g of 5-fluorocytosine (5-FC) orally daily for 6 to 22 days. The response was excellent in 6, moderate in 4 and poor in 1 case, overall efficacy rate being 90.9%. No patients showed any adverse reaction. The antibody against the Candida species was measured by an indirect hemagglutination test and the serum level of D-arabinitol, a major metabolite of Candida species, was determined by gas-liquid chromatography. A 4-fold or greater rise in anti-Candida antibody was detected in 4 of the 6 patients with pyelonephritis, including one patient associated with candidemia, 14 to 31 days following the treatment. Serum D-arabinitol levels in all of the 4 patients with pyelonephritis exceeded the detection limit of 0.2 microgram/ml before treatment. The levels decreased promptly during treatment and normalized 10 to 20 days after the start of treatment. These results suggest that 5-FC is an effective drug for Candida urinary tract infections. The determinations of anti-Candida antibody titer and serum D-arabinitol level may be clinically useful for the diagnosis of Candida pyelonephritis. In particular, the serum levels of D-arabinitol appear to be a useful parameter to follow-up the effect of anti-fungal treatment on Candida pyelonephritis.