Heyko Skladny

Detection of Aspergillus Species in Blood and Bronchoalveolar Lavage Samples from Immunocompromised Patients by Means of 2-Step Polymerase Chain Reaction: Clinical Results

Bronchoalveolar lavage (BAL) samples from 67 patients who were at high risk for invasive aspergillosis were examined using a recently developed 2-step polymerase chain reaction (PCR) that detects </=10 fg of Aspergillus DNA in blood and BAL samples in vitro. Thirteen of these patients had PCR and diagnostic results positive for Aspergillus infection. Four patients with possible invasive aspergillosis also had positive PCR results, and the remaining 50 had negative PCR results. In addition, 907 blood samples from 218 high-risk patients were screened. Thirty-three patients with positive PCR results had invasive aspergillosis; 148 patients had PCR and diagnostic results that were negative, and 34 patients with positive PCR results had nonconclusive clinical data. Both blood and BAL testing were performed for 45 patients. All 8 patients with proven invasive aspergillosis showed concordance of positive PCR results. Our data suggest that this PCR method has possible clinical value for confirming and improving the diagnosis of invasive aspergillosis in high-risk patients.

Development of a LightCycler PCR Assay for Detection and Quantification of Aspergillus fumigatus DNA in Clinical Samples from Neutropenic Patients

ABSTRACT The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitively Aspergillus fumigatus DNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from an A. fumigatus-specific sequence of the mitochondrial cytochrome b gene. The assay is linear in the range between 13.2 fg and 1.3 ng of A. fumigatus DNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.

Clinical evaluation of a polymerase chain reaction assay to detect Aspergillus species in bronchoalveolar lavage samples of neutropenic patients.

Summary. The increasing incidence of invasive aspergillosis, a life‐threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. Using a recently developed two‐step polymerase chain reaction (PCR) assay to detect 10 fg of Aspergillus DNA, corresponding to 1–5 colony‐forming units (CFU)/ml of spiked samples in vitro, we prospectively examined 197 bronchoalveolar lavage (BAL) samples from 176 subjects, including 141 neutropenic, febrile patients with lung infiltrates, at risk for invasive fungal disease. Underlying diseases of these patients were haematological malignancies; 93 patients suffered from acute leukaemias. Thirty‐one of these immunocompromised patients (17·6%) were PCR positive, correlating with positive BAL culture, positive histology from lung surgery or from autopsy, positive computerized tomography scans or positive galactomannan enzyme‐linked immunosorbent assay. Six patients (4·3%) of this group had positive PCR results without any correlation to clinical or other diagnostic data, probably owing to contamination of the samples by ubiquitous Aspergillus spores. The samples of two patients (1·4%) with a subsequent histologically proven mould infection were PCR negative. All 102 immunocompromised patients (72·3%) with a negative PCR showed no evidence of invasive fungal disease. From 35 patients without immunodeficiency, four (11·4%) showed positive results, without evidence of invasive or non‐invasive pulmonary aspergillosis. In this haematological population, the sensitivity and specificity values of the test reached 93·9% and 94·4%, the positive predictive value 83·8%, the negative predictive value 98·1%. Our data support the considerable clinical value of this PCR assay for confirming and improving diagnosis of pulmonary aspergillosis in high‐risk patients.

Variable numbers of BCR-ABL transcripts persist in CML patients who achieve complete cytogenetic remission with interferon-α

Summary .A substantial minority of patients with chronic myeloid leukaemia (CML) achieve a complete response to treatment with interferon‐α (IFN‐α), defined as the disappearance of Philadelphia chromosome positive metaphases or, for patients who are Philadelphia chromosome negative but BCR‐ABL positive, the disappearance of the leukaemic clone as assayed by Southern blot. We have measured the levels of BCR‐ABL transcripts in 20 such patients by quantitative PCR. Results were standardized for both quality and quantity of cDNA by quantification of ABL as an internal control. All 20 patients had evidence of residual disease; the median number of transcripts was 750/μg RNA (range 10–22 000) and the median BCR‐ABL/ABL ratio was 0–17% (range 0–0008–3–6%). Our findings show that CML has not been eradicated in any patient and that the quantity of residual disease in complete responders may vary by as much as four orders of magnitude.

Molecular response of CML patients treated with interferon‐α monitored by quantitative Southern blot analysis

We analysed 459 samples from 206 chronic myeloid leukaemia (CML) patients at diagnosis and during or after treatment with interferon‐α (IFN‐α) by quantitative Southern blot analysis for BCR rearrangement. In a minority (2%) of Ph‐positive patients, no BCR rearrangement was detectable due to breakpoints outside the major breakpoint cluster region (M‐bcr) or possibly due to M‐bcr deletions. Results from 235 samples were compared with the proportion of Ph‐positive metaphases found in contemporaneous bone marrow specimens analysed by conventional cytogenetics. The rank correlation between both methods was 0.82 (P<0.001). The proportion of CML cells in samples determined by Southern blot analysis (BCR ratio) was significantly different between cytogenetically‐defined minor, partial, and complete response groups (P<0.001). Empirically‐derived cut‐off points in the BCR ratio were introduced in order to define molecular response groups for comparison to standard cytogenetic response groups: a BCR ratio of 0% was defined as complete molecular response and ratios of 1–24%, 25–50%, and >50% were defined as partial, minor, and no molecular response, respectively. Using these cut‐off points the concordance between both methods was 67% (P<0.0001), a major cytogenetic response could be predicted or excluded in more than 90% of cases (P<0.0001). Our findings demonstrated that quantitative Southern blot was as sensitive as cytogenetics and as peripheral blood samples are suitable for this technique it should be considered as the method of choice for routine monitoring IFN‐α therapy in CML patients.

Systemic Infections with Candida sp. and Aspergillus sp. in Immunocompromised Patients with Hematological Malignancies: Current Serological and Molecular Diagnostic Methods

Within recent years, novel serological and molecular methods have been developed to improve the early diagnosis of invasive fungal infections especially in patients with malignant hematological diseases being at highest risk of developing these life-threatening infections. Early diagnosis is essential for adequate therapeutic management, which, however, often remains difficult since the diagnostic tools clinically used either lack specificity or sensitivity, or both at worst. The clinical value, the advantages and remaining problems of recently developed, more sensitive and specific serological assays and molecular methods are reviewed.

Impact of human endogenous retroviral elements on cellular genes: strategy for isolation of LTR-driven chimeric transcripts.

Impact of human endogenous retroviral elements on cellular genes: strategy for isolation of LTR-driven chimeric transcripts

Cloning and characterization of the gene encoding the major sigma factor of Stigmatella aurantiaca

The gene (sigA) encoding the major sigma factor of the myxobacterium, Stigmatella aurantiaca, was cloned and sequenced. The deduced polypeptide contains 706 amino acids (aa) and has a deduced M(r) of 79,910. It exhibits four different aa sequence motifs which correlate with the conserved domains of the major sigma factors of Myxococcus xanthus (sigma 80), Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The sigma factor (sigma A) was detected in crude lysates of vegetative cells and in cells of different developmental stages from S. aurantiaca with an antiserum to M. xanthus sigma 80 by Western blot analysis. The SigA polypeptide copurified with RNA polymerase from vegetative S. aurantiaca cells. The aa sequence of its N terminus matches a sequence located 25 codons downstream from the proposed start codon. The sigA gene was expressed in E. coli and the corresponding gene product cross-reacted with the SigA antiserum as a polypeptide of 100 kDa, which is identical in size to the sigma A detected in vegetative cells of S. aurantiaca.

PCR-Mediated Detection of Aspergillus Species in Bronchoalveolar Lavage Samples of Febrile Neutropenic Patients with Acute Leukemias: Clinical Results

The incidence of systemic fungal infections in neutropenic patients with malignant hematological diseases is increasing (Beck-Sague and Jarvis 1993; Bodey, Bueltmann, et al. 1992). Patients at highest risk for these life-threatening infections are those with prolonged periods of neutropenia after intensified chemotherapy, usually patients with acute leuke-mia or after bone marrow transplantation (Denning 1998). Particularly increasing are invasive infections with Aspergillusspecies, showing high mortality rates and, if the patient survives, also considerable complications often limiting further antileukemic therapies (Denning 1998). The paragon of antifungal therapy, Amphotericin B, is toxic; chemoprophylaxis against Aspergillus species is therefore controversially discussed and not generally recommended or practiced (Schwartz, Behre et al. 1999).

SigB, an alternative sigma factor of the myxobacterium Stigmatella aurantiaca, is synthesized during development and heat shock

Alternative sigma factors have been detected in the myxobacterium Stigmatella aurantiaca during indole-induced sporulation, fruiting body formation and heat shock using an antiserum raised against sigma factor SigB. The time course of sigB gene expression was analysed by RT-PCR and by determining beta-galactosidase activity during development in a merodiploid strain that harboured a sigB-lacZ fusion gene. Inactivation of the sigB gene by insertion of the neo gene resulted in the loss of one sigma factor as shown by Western analysis. Neither fruiting body formation nor sporulation, nor the production of possible SigB targets, such as DnaK, GroEL or HspA, were affected.