Margit Hummel

DNA Microarray-Based Detection and Identification of Fungal Pathogens in Clinical Samples from Neutropenic Patients

ABSTRACT The increasing incidence of invasive fungal infections (IFI) in immunocompromised patients emphasizes the need to improve diagnostic tools. We established a DNA microarray to detect and identify DNA from 14 fungal pathogens (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, Candida dubliniensis, Candida glabrata, Candida lusitaniae, Candida tropicalis, Fusarium oxysporum, Fusarium solani, Mucor racemosus, Rhizopus microsporus, Scedosporium prolificans, and Trichosporon asahii) in blood, bronchoalveolar lavage, and tissue samples from high-risk patients. The assay combines multiplex PCR and consecutive DNA microarray hybridization. PCR primers and capture probes were derived from unique sequences of the 18S, 5.8S, and internal transcribed spacer 1 regions of the fungal rRNA genes. Hybridization with genomic DNA of fungal species resulted in species-specific hybridization patterns. By testing clinical samples from 46 neutropenic patients with proven, probable, or possible IFI or without IFI, we detected A. flavus, A. fumigatus, C. albicans, C. dubliniensis, C. glabrata, F. oxysporum, F. solani, R. microsporus, S. prolificans, and T. asahii. For 22 of 22 patients (5 without IFI and 17 with possible IFI), negative diagnostic results corresponded with negative microarray data. For 11 patients with proven (n = 4), probable (n = 2), and possible IFI (n = 5), data for results positive by microarray were validated by other diagnostic findings. For 11 of 11 patients with possible IFI, the microarray results provided additional information. For two patients with proven and probable invasive aspergillosis, respectively, microarray results were negative. The assay detected genomic DNA from 14 fungal pathogens from the clinical samples, pointing to a high significance for improving the diagnosis of IFI.

Prospective clinical evaluation of a LightCyclerTM‐mediated polymerase chain reaction assay, a nested‐PCR assay and a galactomannan enzyme‐linked immunosorbent assay for detection of invasive aspergillosis in neutropenic cancer patients and haematological stem cell transplant recipients

Invasive aspergillosis (IA) is a considerable clinical problem in neutropenic patients with haematological malignancies but its diagnosis remains difficult. We prospectively evaluated a LightCyclerTM polymerase chain reaction (PCR) assay, a nested‐PCR assay and a galactomannan (GM) enzyme‐linked immunosorbent assay (ELISA) to validate their significance in diagnosing IA. During 205 treatment episodes in 165 patients from six centres, a nested‐PCR assay and GM testing was performed at regular intervals. Positive nested‐PCR results were quantified by a LightCyclerTM PCR assay. Patient episodes were stratified according to the 2002 European Organization for Research and Treatment of Cancer/Mycosis Study Group consensus criteria and the PCR and serology results were correlated with the clinical diagnostic classification. Sensitivity and specificity rates for the nested‐PCR assay were up to 63·6% [95% confidence interval (CI): 30·8–89%) and 63·5% (95% CI: 53·4–72·7%) respectively, and 33·3% and 98·9% (95% CI: 7·5–70·1% and 94·2–99·9%) for GM respectively. The LightCyclerTM PCR assay yielded positive results in 21·4%, lacking discrimination by quantification across the different clinical categories. In this prospective comparison, PCR was superior to GM with respect to sensitivity rates. In patients at high risk for IA, positive results for Aspergillus by PCR of blood samples are highly suggestive for IA and contribute to the diagnosis.

Therapy with antifungals decreases the diagnostic performance of PCR for diagnosing invasive aspergillosis in bronchoalveolar lavage samples of patients with haematological malignancies

OBJECTIVES Invasive aspergillosis (IA) is a life-threatening infection in severely immunocompromised patients, especially those receiving intensive chemotherapy or undergoing haematopoietic stem cell transplantation. As the clinical diagnosis of IA is mostly based on biomarkers (galactomannan, β-d-glucan, PCR assays) indicating Aspergillus as the underlying pathogen, the effect of antifungal treatment on the performance of these parameters is still controversial. We evaluated the effect of antifungal treatment on the performance of an Aspergillus-specific PCR assay in bronchoalveolar lavage (BAL) samples. PATIENTS AND METHODS Two-hundred-and-twenty-six BAL samples from 226 patients with haematological malignancies at high risk for IA classified according to the 2008 European Organization for the Research and Treatment of Cancer criteria were analysed retrospectively for the diagnostic performance of a nested Aspergillus PCR assay in relation to the number and type of mould-active antifungals received prior to BAL sampling. RESULTS Sensitivity of BAL PCR for patients without antifungal treatment prior to BAL sampling was 0.69, whereas specificity was 0.87. While no significant change in diagnostic performance by the addition of one antifungal was observed, receiving two or more antifungals prior to BAL sampling led to a significant decrease in the diagnostic performance of BAL PCR testing (P < 0.009). CONCLUSIONS Treatment with mould-active antifungals prior to BAL sampling significantly decreases the performance of the Aspergillus PCR assay in haematological patients if BAL was performed after administration of more than one antifungal agent. Performing BAL sampling for Aspergillus PCR diagnostic despite pre-treatment with one antifungal or while on prophylaxis is feasible.

Detection of Aspergillus DNA in Cerebrospinal Fluid from Patients with Cerebral Aspergillosis by a Nested PCR Assay

ABSTRACT Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples.

Diagnostic yield of bronchoscopy with bronchoalveolar lavage in febrile patients with hematologic malignancies and pulmonary infiltrates.

Infectious complications are a major cause of morbidity and mortality in immunosuppressed patients. Febrile patients with hematologic malignancies and pulmonary infiltrates have high mortality rates, especially if mechanical ventilation is required. The diagnostic value of fiberoptic bronchoscopy (FOB) with bronchoalveolar lavage (BAL) in these patients is controversial. We retrospectively analyzed the microbiological results of BAL samples obtained during 249 FOB examinations from 199 febrile patients with hematologic malignancies and pulmonary infiltrates (underlying diseases: acute leukemia 103 patients, lymphoma 84 patients, other malignancies 12 patients). Two hundred forty-six examinations could be evaluated. Seventy-three out of 246 BAL samples were sterile; 55 samples showed microbiological findings classified as contamination or colonization. One hundred eighteen samples showed positive microbiological results of bacteria and/or fungi classified as causative pathogens. Thereof, in 70 samples, only bacterial pathogens were detectable (Gram-positive, 35; Gram-negative, 30; mixed Gram-positive and Gram-negative, 5). Thirteen samples showed both fungi and bacterial pathogens. In 33 samples, only fungi were detectable, thereof, in 15 samples Aspergillus species, in 16 samples Candida species, and in 2 both. In two samples, a viral pathogen could be detected. Three nonlethal complications (bleeding, arrhythmia) occurred that required early termination of FOB. In 94 (38.2%) patient episodes, antibiotic treatment was modified as a result of microbiological findings in BAL samples. Our results show that FOB with BAL is a valuable diagnostic tool with low complication rates in high-risk febrile patients with hematologic malignancies and pulmonary infiltrates, contributing crucial results for the individual case, and also improving epidemiologic knowledge.

Effective treatment and prophylaxis of hyperuricemia and impaired renal function in tumor lysis syndrome with low doses of rasburicase

Background:  Tumor lysis syndrome (TLS) is a complication that can cause renal failure by precipitation of uric acid (UA) and phosphate crystals in renal tubules. Rasburicase proved to be effective in rapidly reducing UA levels. Costs of rasburicase average up to 4500€. To assess if lower doses of rasburicase are effective, we treated patients with lower doses than recommended.

Detection of Aspergillus DNA by a nested PCR assay is able to improve the diagnosis of invasive aspergillosis in paediatric patients

Fungal infections are a leading cause of morbidity and mortality in severely immunocompromised patients and have been increasing in incidence in recent years. Invasive aspergillosis (IA) is the most common filamentous fungal infection and is, in adults as well as in children, difficult to diagnose. Several PCR assays to detect Aspergillus DNA have been established, but so far, studies on molecular tools for the diagnosis of IA in children are few. We evaluated the results of a nested PCR assay to detect Aspergillus DNA in clinical samples from paediatric and adolescent patients with suspected IA. Blood and non-blood samples from immunocompromised paediatric and adolescent patients with suspected invasive fungal infection were sent for processing Aspergillus PCR to our laboratory. PCR results from consecutive patients from three university childrens hospitals investigated between November 2000 and January 2007 were evaluated. Fungal infections were classified according to the EORTC classification on the grounds of clinical findings, microbiology and radio-imaging results. Two hundred and ninety-one samples from 71 patients were investigated for the presence of Aspergillus DNA by our previously described nested PCR assay. Two, 3 and 34 patients had proven, probable and possible IA, respectively. Sensitivity (calculated from proven and probable patients, n=5) and specificity (calculated from patients without IA, n=32) rates of the PCR assay were 80 and 81 %, respectively. Our nested PCR assay was able to detect Aspergillus DNA in blood, cerebrospinal fluid and bronchoalveolar lavage samples from paediatric and adolescent patients with IA with high sensitivity and specificity rates.

Diagnostic Performance of an Aspergillus-Specific Nested PCR Assay in Cerebrospinal Fluid Samples of Immunocompromised Patients for Detection of Central Nervous System Aspergillosis

Central nervous system (CNS) invasive aspergillosis (IA) is a fatal complication in immunocompromised patients. Confirming the diagnosis is rarely accomplished as invasive procedures are impaired by neutropenia and low platelet count. Cerebrospinal fluid (CSF) cultures or galactomannan (GM) regularly yield negative results thus suggesting the need for improving diagnostic procedures. Therefore the performance of an established Aspergillus-specific nested polymerase chain reaction assay (PCR) in CSF samples of immunocompromised patients with suspicion of CNS IA was evaluated. We identified 113 CSF samples from 55 immunocompromised patients for whom CNS aspergillosis was suspected. Of these patients 8/55 were identified as having proven/probable CNS IA while the remaining 47 patients were classified as having either possible (n = 22) or no CNS IA (n = 25). PCR positivity in CSF was observed for 8/8 proven/probable, in 4/22 possible CNS IA patients and in 2/25 NoIA patients yielding sensitivity and specificity values of 1.0 (95% CI 0.68–1) and 0.93 (95% CI 0.77–0.98) and a positive likelihood ratio of 14 and negative likelihood ratio of 0.0, respectively, thus resulting in a diagnostic odds ratio of ∞. The retrospective analysis of CSF samples from patients with suspected CNS IA yielded a high sensitivity of the nested PCR assay. PCR testing of CSF samples is recommended for patients for whom CNS IA is suspected, especially for those whose clinical condition does not allow invasive procedures as a positive PCR result makes the presence of CNS IA in that patient population highly likely.

Aspergillus polymerase chain reaction (PCR) diagnosis.

In recent years novel molecular methods, notably polymerase chain reaction (PCR) assays, have been developed to improve the diagnosis of life-threatening invasive aspergillosis in patients at high risk, especially patients with malignant hematological disease. Early diagnosis and treatment are essential for adequate therapeutic management. Management, however, often remains difficult since most of the diagnostic tools used clinically at present either lack specificity or acceptable sensitivity. The clinical value, advantages and remaining problems of PCR approaches to detect the emerging fungal pathogen are reviewed.

Recurrent chemotherapy‐induced tumor lysis syndrome (TLS) with renal failure in a patient with chronic lymphocytic leukemia – successful treatment and prevention of TLS with low‐dose rasburicase

Abstract:  Introduction: Rasburicase is a recombinant urate oxidase that is produced by a genetically modified Saccharomyces cerevisiae and has been approved for prophylaxis and treatment of tumor lysis syndrome in 2001. In several studies, rasburicase, given at a dose of 0.15–0.2 mg/kg for up to 7 d, proved to be highly effective in lowering urate levels. Case report: We report the case of a patient with chronic lymphatic leukemia (CLL) who experienced tumor lysis syndrome (TLS) with acute renal failure after fludarabine/cyclophosphamide chemotherapy and after bendamustine treatment. During the first episode of TLS, after fludarabine/cyclophosphamide (creatinine 3.3 mg/dL, urate 24.6 mg/dL), the patient received rasburicase 0.2 mg/kg for 3 d. Urate levels decreased below the lower limit of normal and renal function recovered. After bendamustine therapy, given for disease progression 8 months later, TLS with acute oliguric renal failure re‐occurred (creatinine 3.1 mg/dL, urate 20.8 mg/dL). The patient was treated with hyperhydration and two doses of rasburicase (0.056 mg/kg), resulting in a prompt decrease of the urate level and recovery of renal function. Both episodes of TLS were successfully treated with rasburicase in a lower dose than recommended by the manufacturer. During a second bendamustine course, TLS was successfully treated by low doses of rasburicase (0.056 mg/kg for 2 d). Conclusion: This is the first report of TLS in CLL after bendamustine chemotherapy reported in the literature. Treatment and prevention of TLS by low doses of rasburicase is possible and cost‐effective.