D-arabinitol — a marker for invasive candidiasis

Medical Mycology 1999, 37, 391–396 Accepted 5 October 1998 Review article D-arabinitol – a marker for invasive candidiasis B. CHRISTENSSON, G. SIGMUNDSDOTTIR & L. LARSSON Department of Infectious Diseases and Medical Microbiology, Lund University Hospital, Lund, Sweden Keywords arabinitol method, candidiasis, D-arabinitol Introduction Invasive candidiasis is a serious condition that affects mainly immunocompromised individuals. At present, there is no ideal diagnostic method in terms of sensitivity and specificity for the diagnosis of invasive candidiasis in humans. Results from blood and tissue cultures are still considered the ‘gold standard’, although a number of non-culture methods have been developed, including antibody and antigen tests, DNA amplification tests, and detection of Candida metabolites. A method that has gained considerable attention in recent years concerns the measuring of D-arabinitol (DA) in body fluids. This five-carbon sugar alcohol (pentitol) is produced in 6itro by most pathogenic and medically important Candida spp. [1 – 3]. Various methods for measuring DA in blood and urine have been developed and applied since 1979, but there is still controversy regarding the analytical procedures and their diagnostic value. Correspondence: Bertil Christensson, M.D., Ph.D., Department of Infectious Diseases, University Hospital, SE-221 85 Lund, Sweden. Tel.: + 46 46 171000; fax: +46 46 137414; e-mail: bertil. © 1999 ISHAM The aim of this review is to describe the different methods that have been applied to measure DA in body fluids and discuss their usefulness for diagnosing invasive Candida infections in humans. Methods Gas chromatography Chemical derivatization renders DA volatile and thus detectable by gas chromatography (GC). In earlier GC studies, packed columns were used for measuring DA in serum, but the peaks assigned as DA actually represented the sum of the pentitols and therefore included xylitol, adonitol, DA and L-arabinitol (LA) [4]. Employing a capillary GC column allowed separation of these pentitols, although the two arabinitol enantiomers still appeared as a single chromatographic peak. In the late 1980s, chiral stationary phase columns were developed that are useful for the separation of the D- and L-enantiomers of arabinitol. A column containing (S)-N-1(naphthyl)ethyl-4-allyloxybenzamide was found to separate various halogenated derivatives of DA from LA [5], and another column coated with a-perpentylated The five-carbon sugar alcohol D-arabinitol (DA) is a metabolite of most pathogenic Candida species, in 6itro as well as in 6i6o, and can be determined by gas chromatography or enzymatic analysis. Endogenous DA and L-arabinitol (LA) are present in human body fluids, and serum DA and LA increase in renal dysfunction. In prospective clinical studies, elevated DA/LA or DA/creatine ratios in serum or urine have been found in immunocompromised, usually neutropenic, patients with invasive candidiasis. In addition, positive DA results have been obtained several days to weeks before positive blood cultures, and the normalization of DA levels has been correlated with therapeutic response in both humans and animals. However, to date, only a few prospective studies have been conducted in which adequate analytical methods were used. Thus, further investigation of various patient groups is needed to establish the applicability of the ‘arabinitol method’ in the diagnostic battery for invasive Candida infections. 392 Christensson et al. Enzymatic methods Bernhard et al. [11] were the first to show that the excess arabinitol found in serum from patients with invasive candidiasis was in fact DA. These investigators incubated serum with a strain of C. tropicalis that stereospecifically consumed DA, and they used GC to determine the difference in arabinitol concentration between the untreated and treated serum samples; the difference was then used to calculate the DA concentration. However, this method requires a 24-h incubation step, and it was found that the presence of antifungal agents in the clinical sample interfered with the analysis, hence methods using purified enzymes were later adopted instead. Soyama & Ono [12] used a spectrophotofluorimetric method that entailed adding D-arabinitol dehydrogenase (D-ADH) from Enterobacter aerogenes and nicotinamide adenine dinucleotide (NAD) to the sample containing DA and sub- sequently measuring the rate of NAD reduction. Later, the same authors [13] added resazurin to react with NADH (reduced NAD) and found that the initial increase in the concentration of one of the products, resorfin, correlated with the concentration of DA. However, D-ADH also oxidizes D-mannitol, which is present in normal serum, thus falsely high DA concentrations were obtained. Still, using this method, Tokunaga et al. [14] showed a satisfactory correlation (r=0·94) with arabinitol concentrations, as determined by GC using a packed column. Wong & Brauer [7] used a more specific D-ADH substrate, purified from Klebsiella pneumoniae, for GC determination of arabinitol both before and after treating samples with the enzyme. Switchenko et al. [15] measured NADH dependent end products by applying a semi-automated spectrophotometric assay in which a highly specific C. tropicalis DADH was used to avoid cross-reactions with other polyols. Serum samples from patients suffering from fatal illnesses (none with Candida infections), with or without renal dysfunction, were subjected to this enzymatic assay, and the results correlated well with DA concentrations determined by GC (r= 0·94) [15]. To our knowledge, the only commercially available enzymatic assay is the fluorimetric test developed by Soyama & Ono [14,16]. Sources of D-arabinitol Endogenous D -arabinitol As mentioned above, both DA and LA are present in the body fluids of healthy individuals. However, it has been assumed that only LA arises from mammalian metabolism, since the polyol dehydrogenase enzymes detected in mammalian tissues catalyse oxidation of LA but not DA [17]; hence the origin of endogenous DA is still unclear. It has been proposed that DA present in normal human serum could originate from either dietary DA or from Candida organisms present in the gastrointestinal tract. DA is readily absorbed in the gut, which is why Wong et al. [18] were able to detect 85% of a 1-g oral dose of DA in urine within 24 h in a healthy human volunteer. However, it has also been proposed that endogenous DA might be derived from the central nervous system [19]. This suggestion was based on the findings that the absolute amounts of DA were about 10 times higher in normal cerebrospinal fluid (CSF) than in normal serum [19], whereas the concentrations of LA in CSF were about the same as in serum. Thus, it is likely that DA is also included in mammalian metabolism, and that at least part of the endogenous DA found in normal serum and urine may originate from the brain or spin (...truncated)