Prospective evaluation of a combination of fungal biomarkers for the diagnosis of invasive fungal disease in high-risk haematology patients.

We prospectively evaluated a combination of fungal biomarkers in adult haematology patients with focus on their clinical utility at different time points during the course of infection. In total, 135 patients were monitored once to twice weekly for serum (1-3)-ß-d-glucan (BG), galactomannan (GM), bis-methyl-gliotoxin and urinary d-arabinitol/l-arabinitol ratio. In all, 13 cases with proven or probable invasive fungal disease (IFD) were identified. The sensitivity of BG and GM at the time of diagnosis (TOD) was low, but within 2 weeks from the TOD the sensitivity of BG was 92%. BG >800 pg/mL was highly specific for IFD. At a pre-test probability of 12%, both BG and GM had negative predictive values (NPV) >0.9 but low positive predictive values (PPV). In a subgroup analysis of patients with clinically suspected IFD (pre-test probability of 35%), the NPV was lower, but the PPV for BG was 0.86 at cut-off 160 pg/mL. Among IFD patients, 91% had patterns of consecutively positive and increasing BG levels. Bis-methyl-gliotoxin was undetectable in 15 patients with proven, probable and possible IA. To conclude, BG was the superior fungal marker for IFD diagnosis. Quantification above the limit of detection and graphical evaluation of the pattern of dynamics are warranted in the interpretation of BG results.

Identification of organic acids as potential biomarkers in the urine of autistic children using gas chromatography/mass spectrometry.

There is a need to identify metabolic phenotypes in autism as they might each require unique approaches to prevention. Biological markers can help define autism subtypes and reveal potential therapeutic targets. The aim of the study was to identify alterations of small molecular weight compounds and to find potential biomarkers. Gas chromatography/mass spectrometry was employed to evaluate major metabolic changes in low molecular weight urine metabolites of 14 children with autism spectrum disorders vs. 10 non-autistic subjects. The results prove the usefulness of an identified set of 21 endogenous compounds (including 14 organic acids), whose levels are changed in diseased children. Gas chromatography/mass spectrometry method combined with multivariate statistical analysis techniques provide an efficient way of depicting metabolic perturbations of diseases, and may potentially be applicable as a novel strategy for the noninvasive diagnosis and treatment of autism.

Clinical experience of urine D-arabinitol/L-arabinitol ratio in the early diagnosis of invasive candidiasis in paediatric high risk populations.

In 2 prospective studies, we previously reported on the early and accurate diagnosis of invasive candidiasis by determining the D-arabinitol/L-arabinitol (DA/LA) ratio in urine in neutropenic children with cancer at the paediatric oncology unit (POU) and in premature infants at the neonatal intensive care unit (NICU) at our hospital. In this retrospective study at the same units, we report how the DA/LA assay was implemented in clinical practice immediately after the prospective study periods. We found that, in the POU, the recommendation of regularly monitoring urine DA/LA ratios in patients at risk and considering antifungal therapy in the case of elevated ratios had been followed. A significant decrease in the incidence of culture positive invasive candidiasis may have been attributed to the introduction of the DA/LA assay. At the NICU, where the DA/LA assay was recommended only as an adjunct to other diagnostic tools, morbidity in invasive candidiasis remained unchanged. While regular monitoring of the urine DA/LA ratio probably facilitates the early detection of invasive candidiasis in paediatric oncology, it remains to be determined if the test can be used in a similar way in neonatal intensive care.

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry.

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.

Development and application of a rapid diagnostic method for invasive Candidiasis by the detection of D-/L-arabinitol using gas chromatography/mass spectrometry.

A rapid non-culture-based diagnostic method utilizing d-/l-arabinitol (DA/LA) ratios as a chemical marker of invasive candidiasis was developed and explored. The enantiomers-ratios detection was made possible by the use of gas chromatography coupled with mass spectrometry (GC/MS). The mean DA/LA ratios +/- standard deviation (range) in urine (n = 40) and serum (n = 20) were 2.08 +/- 0.78 (0.57 to 3.55) and 1.79 +/- 0.75 (0.74 to 3.54), respectively, from patients without evidence of fungal infection or colonization; in patients (n = 7) with culture-proven invasive candida infections, the figures were 9.91 +/- 3.04 (7.24 to 16.27) and 13.58 +/- 7.31 (5.57 to 25.88) in urine and serum, respectively. The differences in DA/LA ratios between the candidemic patients and the non-candidemic patients were statistically significant (p < 0.01) in both serum and urine samples. The DA/LA ratios were not significantly affected in patients with oral or vaginal candidiasis and candiduria.

Gas chromatographic determination of D-/L-arabinitol ratio in healthy Polish children.

The D-/L-arabinitol enantiomers ratio (a marker of disseminated candidiasis of Candida species) in urine was determined by gas chromatography (GC) in 198 healthy Polish children ranging in age from 0 to 18 years. The urine samples were dry and trifluoroacetic anhydride (TFAA)-treated. Enantiomers derivatives were separated on a chiral column (beta-Dex 120, 60 m x 0.25 mm I.D.). A glass "solid-phase" injector and electron capture detector (ECD) were used. The ECD response was linear with correlation coefficients 0.999. The limit of detection was 0.02 micromol/l. Good results in terms of reproducibility, accuracy (RSD<10%, bias<6%), and linearity were obtained from real urine samples containing up to 400 micromol/l D-arabinitol. TFA-arabinitol derivatives in biological samples were stable from 1 to 5 days (depending on the arabinitol contents), while TFA-arabinitol standard derivatives were stable for 2 weeks. The identity of D- and L-arabinitol were confirmed by GC-MS analysis. The mean D-/L-arabinitol ratios ranged from 2.48 to 1.65 in the examined groups. The D-/L-arabinitol ratio was found to be exponentially regressive with age. A few cases of diagnosis of disseminated candidiasis by the GC method and confirmed by blood culture are described. The described GC method was also used for monitoring antifungal treatment of patients with disseminated candidiasis.

Urine D-arabinitol/L-arabinitol ratio in diagnosing Candida infection in patients with haematological malignancy and HIV infection.

Adult patients with hematologic malignancies along with HIV infected patients were prospectively studied to determine the performance of urine D-arabinitol/L-arabinitol (DA/LA) ratio in diagnosing invasive candidiasis. Ten evaluable febrile neutropenic patients had proven invasive candidiasis and elevated DA/LA ratios were found in 5. Invasive candidiasis with normal DA/LA ratios was most frequently due to Candida krusei infection. This Candida species is a non-producer of arabinitol. Only 4 of 81 febrile neutropenic patients given either antifungal prophylaxis or empiric antifungal treatment had elevated DA/LA ratios. Only 1 of 15 HIV positive patients with either oropharyngeal or esophageal candidiasis had elevated DA/LA ratios. Widespread use of fluconazole prophylaxis in bone marrow transplantation patients at the study hospital has led to an increased prevalence of C. krusei infection. This is the likely reason for the low sensitivity of the test in proven and suspected invasive Candida infections reported here.

Measurement of the D-arabinitol/L-arabinitol ratio in urine of neutropenic patients treated empirically with amphotericin B.

The aim of the present study was to evaluate the diagnostic significance of the D-arabinitol/L-arabinitol ratio in urine of neutropenic patients with suspected fungal infection. D-arabinitol/L-arabinitol ratios were determined in 373 serial urine samples of 104 patients with haematological malignancies receiving empirical amphotericin B treatment for suspected invasive fungal infection. Twenty-eight (8%) urine samples obtained from 17 (16%) patients were positive (ratio > or =4). Eight (47%) patients had positive urine samples at the initiation of empirical amphotericin B treatment and the rest from 7 to 30 days after empirical therapy was started. Several urine samples were positive in six patients. Only one of the five patients with candidemia had elevated D-arabinitol/L-arabinitol ratios (persistent Candida krusei fungaemia). Four patients with transient candidemia and seven patients with invasive mould infections were negative. Patients who died during the study period had significantly higher D-arabinitol/L-arabinitol ratios than patients who survived (P=0.0002). Pneumonia was the most common manifestation of infection (53% of patients with elevated D-arabinitol/L-arabinitol ratios) and was associated with an especially high mortality (67%). The present study shows that elevated urine D-arabinitol/L-arabinitol ratios are common in febrile, neutropenic patients. However, the urine arabinitol test did not detect transient candidemia at elevated levels during the course of infection. Furthermore, D-arabinitol/L-arabinitol ratios were often elevated in the late phase of infection only. This contests the use of this test in guiding the initiation of antifungal therapy. The detection of elevated arabinitol levels in neutropenic patients during empirical amphotericin B treatment is associated with poor prognosis.

Urine D-arabinitol/L-arabinitol ratio in diagnosis of invasive candidiasis in newborn infants.

Infants treated in neonatal intensive care units suffer an increased risk for invasive candidiasis, but the diagnosis is sometimes difficult. D-arabinitol is a metabolite of most pathogenic Candida species. An elevated urine D-arabinitol/L-arabinitol (DA/LA) ratio is a sensitive sign of invasive candidiasis in children with cancer, but the method has not been previously evaluated for newborn infants. We therefore enrolled 117 infants in a neonatal intensive care unit, and 411 urine samples were obtained on filter paper. The DA/LA ratio was measured by gas chromatography-mass spectrometry. For 81 infants with no suspicion of superficial or invasive candidiasis, the urine DA/LA ratio was 2.7 +/- 0.7 (mean +/- standard deviation [SD]). The upper cutoff level was set at 4.8 (mean plus 3 SD). Of 22 infants with mucocutaneous candidiasis and not given systemic antifungal treatment, two had elevated DA/LA ratios, which normalized after removal of intravascular catheters. Eight other infants were given empiric antifungal treatment but had negative cultures; five of these had repeatedly elevated DA/LA ratios. Six infants with culture-positive invasive candidiasis all had one or more samples with elevated ratios. For seven infants, three with suspected and four with confirmed invasive candidiasis (for which follow-up samples were available), ratios normalized during antifungal treatment. In conclusion, urine DA/LA ratio determination is a rapid test and can be used for newborns. It is possibly more sensitive than fungal blood cultures in the diagnosis of invasive candidiasis and can also be used for monitoring the effect of antifungal treatment.

Gas chromatographic-mass spectrometric analysis of urinary sugar and sugar alcohols during pregnancy.

A refined and simplified method has been developed for the simultaneous analysis of urinary sugar and sugar alcohols after urease treatment by using capillary gas chromatography-mass spectrometry (GC-MS). Since carbohydrate metabolism during pregnancy is considered to be diabetogenic, our interest has been concentrated on understanding the mechanism of the metabolic deviation by assessing the glucose excursion and glucose fluxes. The present study suggests that changes of the levels of glucose, sorbitol, fructose, myo-inositol, and 1,5-anhydro-D-glucitol (1,5-AG) may reflect a mild alteration in carbohydrate metabolism that goes undetected by conventional diabetic indicators.

PCR and other non-culture methods for diagnosis of invasive Candida infections in allogeneic bone marrow and solid organ transplant recipients.

In this prospective study 197 serum and 152 urine samples were collected from 40 bone marrow and solid organ transplant recipients with clinically suspected invasive fungal infection before, during and after empirical treatment with lipid formulation of amphotericin B or fluconazole. Serum was analysed by Candida polymerase chain reaction (PCR) and urine by measurement of D/L-arabinitol ratio. One serum from each patient was also tested for concentration of (1-->3)-beta-glucan and two commercial Candida antigens. Invasive fungal infection was diagnosed in four candidosis and one aspergillosis patients (13%). Positive PCR, elevated D/L-arabinitol ratio, (1-->3)-beta-glucan concentration and antigens were detected in nine, 15, 17, and seven patients, respectively. The agreement between PCR and D/L-arabinitol assays was poor. However, 56% agreement was observed between positive PCR and beta-glucan and/or antigen assays, and 60% agreement between positive D/L-arabinitol and beta-glucan and/or antigen assays. Combination of several non-culture assays is needed to diagnose invasive fungal infection in high-risk transplant recipients. No single test was sufficient for diagnosis.

Diagnosis of invasive candidiasis in neutropenic children with cancer by determination of D-arabinitol/L-arabinitol ratios in urine.

Determination of D-arabinitol/L-arabinitol ratios (referred to as D/L-arabinitol ratios) in urine as a tool for the diagnosis of invasive candidiasis was investigated in a prospective study comprising 100 children with cancer. The analyses were made by gas chromatography. Positive D/L-arabinitol ratios were found for 10 of 10 children with confirmed invasive candidiasis, 12 of 23 patients undergoing empiric antifungal chemotherapy, and 4 of 67 children not receiving antifungal treatment. D/L-Arabinitol ratios were positive 3 to 31 days (median, 12 days) before the first culture-positive blood sample was drawn or empiric therapy was initiated. The regular monitoring of D/L-arabinitol ratios in urine holds great promise as a sensitive method for diagnosing invasive candidiasis in immunocompromised children with cancer. Moreover, it may be possible to use an early rise in D/L-arabinitol ratios as a basis for the institution of antifungal chemotherapy and as a means of avoiding unnecessary treatment with potentially toxic antifungal agents.

Diagnosis of disseminated candidiasis by measurement of urine D-arabinitol/L-arabinitol ratio.

Relative amounts of D-arabinitol (fungal origin) and L-arabinitol (part of normal human metabolism) in urine were determined by gas chromatography and mass spectrometry from 61 hospitalized patients with hematological malignancies. Seventeen neutropenic patients with acute leukemia (with 53 samples) had disseminated yeast infections and received empiric antifungal therapy before confirmation of the diagnosis. Control groups consisted of 22 hematologic patients (76 samples) with either mucosal (n = 10) or urinary (n = 12) Candida colonization and 22 neutropenic patients (34 samples) with no clinical or laboratory signs of invasive yeast infection. Reference values were also obtained from 50 healthy adults (50 samples). The mean urine D-arabinitol/L-arabinitol ratio +/- standard deviation (range) was 16.91 +/- 41.79 (1.41 to 254.75) in patients with disseminated infection, 2.73 +/- 2.48 (1.11 to 19.00) in colonized hematologic patients, 2.12 +/- 0.84 (1.16 to 5.84) in neutropenic controls, and 1.95 +/- 0.34 (0.97 to 3.44) in healthy adults (P < 0.001 between patients with disseminated infection and all control groups). The sensitivity and specificity of the assay for detecting disseminated yeast infection were, respectively, 88 and 91% per patient (upper limit of normal, 4.00). Seventy-one percent of patients already expressed elevated values at the onset of empiric antifungal therapy. The diagnosis of disseminated infection was confirmed on average 21.7 days after the first elevation of the D-arabinitol/L-arabinitol ratio. The method contributes to diagnosis of disseminated yeast infection and helps in monitoring patients at risk, to support the initiation of antifungal therapy at an early stage of the disease.

Gas chromatographic determination of D-arabinitol/L-arabinitol ratios in urine: a potential method for diagnosis of disseminated candidiasis.

A gas chromatographic procedure was developed to determine the relative amounts of D- and L-arabinitol in urine. Samples were filtered, diluted, purified through extractions, evaporated, and treated with trifluoroacetic anhydride; the arabinitol derivatives thus obtained were separated on a chiral stationary phase and registered by using an electron-capture detector. Urine samples from a patient with disseminated candidiasis had higher D-arabinitol/L-arabinitol ratios (referred to as D/L-arabinitol ratios)--up to 19.0--than samples from 96 study individuals with no signs of deep Candida infections (range, 1.1 to 4.5). D/L-Arabinitol ratios in urine samples from hospitalized patients without Candida infections were slightly higher than those in samples from healthy individuals; ratios in urine from children were slightly higher than those in adult urine samples. The D/L-arabinitol ratios in several urine samples culture positive for Candida albicans, but from patients without symptoms of disseminated candidiasis, did not differ from those in the urine of healthy individuals. The described gas chromatographic method is straightforward and can be implemented clinically to determine urine D/L-arabinitol ratios as a means of diagnosing disseminated candidiasis.

Diagnosis and monitoring of disseminated candidiasis based on serum/urine D/L-arabinitol ratios.

Disseminated candidiasis, a devastating disease with high morbidity and mortality in immunosuppressed patients, is difficult to diagnose because of the protean nature of symptoms and the lack of rapid and reliable laboratory diagnostic procedures. The subject of this review is the status of gas chromatographic-mass spectrometric techniques for the determination of D-arabinitol, a unique metabolite of pathogenic Candida species, in serum and urine. The enantiomers are separated by chiral chromatography followed by specific and sensitive detection using chemical ionization and selected ion monitoring. Using D/L-arabinitol ratios, instead of individual concentrations, eliminates the need for knowing the volume of samples and for calibration curves. A new filter paper technique requires only an unmeasured drop of whole blood (venous or finger/heel puncture) or urine; paper spots are mailable. Parallel determinations of D/L-arabinitol ratios in serum and urine in normal subjects and cancer patients with both normal and increased D/L-arabinitol ratios revealed constant (1.2-1.3 range) ratios of serum D/L-arabinitol/urine D/L-arabinitol for all populations studied. Analyzing two body fluids taken at the same time increases reliability by reducing false positives.

Separation and quantification by gas chromatography-mass spectrometry of arabinitol enantiomers to aid the differential diagnosis of disseminated candidiasis.

To differentiate increased arabinitol due to fungal (only D-arabinitol) and non-fungal origin, O-trifluoroacetyl derivatives of the enantiomers were separated using alpha-perpentylated cyclodextrin columns and measured by selected ion monitoring. Mean +/- S.D. D/L in normal serum: 1.40 +/- 0.42. D/L ratios greater than 2.24, defined as normal mean + 2S.D., were considered outside normal range. D/L was greater than 2.2 in 10 of 12 confirmed candidiasis cases with one false negative and one borderline. Renal dysfunction without candidiasis yielded normal D/L despite high arabinitol concentrations. D/L in normal urine was nearly identical to that in serum despite 60 times larger concentration. D/L ratios, determined by peak heights or areas, could be used without the need to determine concentrations.

Determination of polyol profiles in human urine by capillary gas chromatography.

A new rapid capillary gas chromatographic method is described for the profile analysis of urinary polyols as their trifluoroacetyl derivatives. Ten urinary polyols: erythritol, threitol, fucitol, ribitol, arabinitol, xylitol, mannitol, glucitol, galactitol and myo-inositol were completely determined for the first time. Iditol was newly found in normal urine. Urinary polyols were verified by gas chromatography/mass spectrometry.

Stereoisomeric configuration of arabinitol in serum, urine, and tissues in invasive candidiasis.

Because routine analytical methods cannot differentiate D- from L-arabinitol, a combined microbiological and gas chromatographic method was developed to study the stereoisomeric configuration of the arabinitol in humans and rats with invasive candidiasis. D-Arabinitol was defined as the difference between arabinitol concentrations measured with and without incubation with 5.0 X 10(5) blastospores of Candida tropicalis strain CT 12 at 37 C for 24 hr. The yeast consumed at least 95% of the D-arabinitol and none of the L-arabinitol added to normal serum and urine. D-Arabinitol as a fraction of D,L-arabinitol was 0.43 +/- 0.15 (mean +/- SD) in the urine of 10 normal humans, 0.82 +/- 0.12 in the serum or urine of five patients with cancer and invasive candidiasis (P less than .001), and 1.0 in the kidneys of rats with candidiasis. Because most or all of the excess arabinitol in body fluids or tissues in candidiasis was the D isomer, which is produced by fungal metabolism, stereospecific quantitation of arabinitol should improve the sensitivity of this approach to diagnosis of candidiasis.

Estimation of neutral sugars and sugar alcohols in biological fluids by gas-liquid chromatography.

Gas-liquid chromatographic procedures for measuring sugar and polyol concentrations in biological fluids are reviewed. Such methods require the preparations of derivatives such as methyl ethers, trimethylsilyl ethers or acetyl esters. Prior to derivatisation samples must be deproteinised and dried. Complex mixtures of sugars and sugar alcohols may be resolved. Quantitative analyses are precise, sensitive and linear. If internal standardisation is used recoveries approaching 100% are obtained.