Corine Heymans

Rapid Detection of Candida albicans in Clinical Blood Samples by Using a TaqMan-Based PCR Assay

ABSTRACT We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.

Tissue concentrations and bioactivity of amphotericin B in cancer patients treated with amphotericin B-deoxycholate.

We have studied amphotericin B concentrations in tissues of 13 cancer patients who died after having received 75 to 1,110 mg (total dose) of amphotericin B-deoxycholate for suspected or proven disseminated fungal infection. Amphotericin B concentrations were measured by high-pressure liquid chromatography (HPLC) and by bioassay, the latter being done on tissue homogenates as well as on tissue methanolic extracts. The fungistatic and fungicidal titers of the tissue homogenates were also tested against three strains of Candida albicans and one strain of Aspergillus fumigatus. Tissue concentrations of amphotericin B measured by HPLC varied with the tested tissues as well as with the total dose of amphotericin B-deoxycholate administered and ranged from 0.4 to 147.1 micrograms/g. A mean of 38.3% (range, 23.0 to 51.3%) of the total dose was recovered by HPLC from all of the tested organs. Bioassay of tissue methanolic extracts reached 58 to 81% of the concentration measured by HPLC, whereas only 15 to 41% was recovered from the homogenates. Overall, 27.5% of the total dose was recovered from the liver, 5.2% was recovered from the spleen, 3.2% was recovered from the lungs, and 1.5% was recovered from the kidneys. The median concentration in bile was 7.3 micrograms/ml, suggesting that biliary excretion could contribute to amphotericin B elimination to an estimated range of 0.8 to 14.6% of the daily dose. Fungicidal titers were seldom measured in tissues, but fungistatic titers were observed and were linearly correlated with amphotericin B concentration measured by HPLC. In conclusion, only a small proportion of the amphotericin B administered as amphotericin B-deoxycholate to patients seems diffusible and bioactive.

Low Mannose-Binding Lectin Concentration Is Associated with Severe Infection in Patients with Hematological Cancer Who Are Undergoing Chemotherapy

BACKGROUND Mannose-binding lectin (MBL) is a serum lectin involved in innate immune response. Low serum MBL concentration may constitute a risk factor for infection in patients receiving myelosuppressive chemotherapy. METHODS We conducted a prospective, observational study that assessed MBL concentration as a risk factor for infection in patients with hematological malignancy who were hospitalized to undergo at least 1 chemotherapy cycle. MBL deficiency was defined using an algorithm that considered the serum MBL concentration and the MBL genotype. The primary end point was the ratio of duration of febrile neutropenia to the duration of neutropenia. Secondary end points included the incidence of severe infection (e.g., sepsis, pneumonia, bacteremia, and invasive fungal infection). Logistic regression analysis was conducted, and Fishers exact test was used to analyze binary outcomes, and Kaplan-Meier estimates and log rank tests were used for time-to-event variables. RESULTS We analyzed 255 patients who received 569 cycles of chemotherapy. The median duration of neutropenia per cycle was 7 days (interquartile range, 0-13 days). Sixty-two patients (24%) were found to have MBL deficiency. Febrile neutropenia occurred at least once in 200 patients. No difference in the primary outcome was seen. The incidence of severe infection was higher among MBL-deficient patients than among non-MBL-deficient patients (1.96 vs. 1.34 cases per 100 days for analysis of all patients [P=.008] and 1.85 vs. 0.94 cases per 100 days excluding patients with acute leukemia [P<.001]). CONCLUSIONS MBL deficiency does not predispose adults with hematological cancer to more-frequent or more-prolonged febrile episodes during myelosuppressive chemotherapy, but MBL-deficient patients have a greater number of severe infections and experience their first severe infection earlier, compared with nondeficient patients.

Real-Time PCR for Determining Capsular Serotypes of Haemophilus influenzae

ABSTRACT A two-step real-time PCR assay targeting all six capsulation loci of Haemophilus influenzae (i.e., serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131 H. influenzae clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of H. influenzae a to c (101 CFU/PCR) was higher than that for type e (103 CFU/PCR) and types d and f (104 CFU/PCR), and a broader dynamic range was obtained (5 to 8 log10 units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of H. influenzae invasive infections.

In vitro evaluation of various antifungal agents alone and in combination by using an automatic turbidimetric system combined with viable count determinations.

A new method combining automatic turbidimetry and sequential viable count determinations was developed to evaluate the in vitro activity of various antifungal agents alone and in combination against three clinical isolates of Candida spp. (two Candida albicans and one C. tropicalis) at two inocula (10(-5) and 10(-6) CFU/ml). Specific parameters were derived from the time-kill curves: the maximal rate of killing, the lowest biomass, and the overnight biomass. Their intra-assay and between-assay reproducibilities were high, with respective standard deviations of 0.4 and 0.25 to 1.4 log CFU/ml. Amphotericin B alone showed a linear relationship between rate of killing or lowest biomass and the log of concentration from 0.03 to 4 mg/liter that was similar for the three strains tested. 5-Fluorocytosine (flucytosine) alone showed a dose-related reduction of overnight biomass for concentrations up to 8 mg/liter with no further increase at higher concentrations for one strain of C. albicans and a paradoxical decrease for one strain of C. tropicalis. Ketoconazole alone was found to be only fungistatic with no increased activity at concentrations up to 16 mg/liter. Amphotericin B plus flucytosine interacted synergistically in 46 to 60% of the combinations tested against C. tropicalis depending on the initial inoculum. Indifference was observed for the two strains of C. albicans. Amphotericin B or flucytosine plus ketoconazole was usually indifferent against the three tested strains.

Circulating concentrations of interleukin-6 in cancer patients and their pathogenic role in tumor-induced hypercalcemia

Circulating interleukin-6 (IL-6) concentrations correlate with disease activity in severe inflammatory conditions, in sepsis and in some hematological malignancies. On the other hand, IL-6 is a potent stimulator of osteoclastogenesis and has been implicated as a contributory factor in the genesis of osteopenic conditions. We measured circulating IL-6 levels by a sensitive (detection limit of 10 U/ml) and specific bioassay in 103 patients with advanced cancer, including 41 with tumor-induced hypercalcemia before any specific hypocalcemic therapy. We related IL-6 concentrations to clinical features and to biochemical parameters of bone metabolism, including blood Ca, Ca2+, Pi, intact parathyroid hormone, parathyroid hormone-related protein, osteocalcin, 1,25-(OH)2-vitamin D and, as markers of bone resorption, the fasting urinary excretion of calcium (Ca/creatinine) and hydroxyproline. IL-6 levels were increased, i.e. detectable, in 23% of the patients, 8/41 (20%) hypercalcemic and 16/62 (26%) normocalcemic patients (NS); the distribution of the values was similar in the two groups. The presence of increased IL-6 concentrations was not related to any clinical characteristic, notably not to the survival nor to the existence of bone metastases, whether in hypercalcemic or normocalcemic patients; e.g., only 3/12 (25%) hypercalcemic subjects without bone metastases had elevated IL-6 levels. We found no significant correlations between IL-6 concentrations and any of the biochemical parameters studied. Hypercalcemic subjects with increased IL-6 had higher urinary Ca/creatinine levels than patients with normal IL-6 levels (P<0.005) but this was not the case in normocalcemic subjects. Mean concentrations of inflammatory or other bone metabolism markers were not significantly different between patients with normal or with elevated IL-6 levels. In summary, circulating IL-6 levels were increased in 23% of 103 patients with advanced cancer, but the frequency of increased IL-6 concentrations was not related to the presence of hypercalcemia or to any marker of calcium metabolism or bone turnover. The pathogenic importance of circulating IL-6 in patients with solid tumors remains to be demonstrated and our data indicate that increased circulating levels of IL-6, possibly reflecting the activation of the immune system, only contribute in a minor way to the osteolytic process in patients with tumor-induced hypercalcemia.

Inaugural bilateral aspergillus endophthalmitis in a seriously immunocompromised patient

A. Georgala, B. Layeux, J. Kwan, I. Ahmad, J. Libert, F. Willermain, P. Koch, C. Heymans, M. Husson and M. Aoun Department of Internal Medicine – CHU Brugmann, Bruxelles, Belgium, Department of Clinical Oncology, Institut Jules Bordet, Université Libre de Bruxelles, Belgium, Department of Haematology, Institut Jules Bordet, Université Libre de Bruxelles, Belgium, Microbiology Laboratory ⁄ Section of Mycology, Institut Jules Bordet, Université Libre de Bruxelles, Belgium, Department of Ophthalmology, CHU-St Pierre, Bruxelles, Belgium and Department of Infectious Diseases, Institut Jules Bordet, Université Libre de Bruxelles, Belgium