NUP98 is rearranged in 3.8% of pediatric AML forming a clinical and molecular homogenous group with a poor prognosis.

Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.

[Caspofungin: mode of action and therapeutic applications]. / La caspofungine: du mécanisme d'action aux applications thérapeutiques.

SUBJECT: Since the last two decades, the incidence of invasive fungal infections has drastically increased. It becomes urgent to enlarge the panel of antifungal drugs with more potent activity and less toxicity. Since the target of all previously available antifungal agents is the synthesis of ergosterol located in the fungal membrane, the fungicidal activity of echinocandins is based on the inhibition of the glucan synthesis. Caspofungin (CAS) (Cancidas MSD France), a cyclic hexapeptide semisynthetic derivative of pneumocandin B, is the first available drug belonging to this new class. MAIN ISSUES: CAS has a fungicidal activity covering a wide range of pathogens, including Candida spp. Data from animal and human studies demonstrate that the drug is 96% plasmatic protein bound and the proposed route of elimination is hepatic. For the treatment of systemic, oesophageal and oropharyngeal candidiasis, CAS has the same efficacy as amphotericin B or as triazoles. Among 50% of patients suffering of invasive aspergillosis with intolerance or resistance to classical treatments, CAS induces a successful response and even more in combination with these drugs. For patients with fever and neutropenia, the efficacy of CAS is non inferior than Ambisome. CAS is generally well tolerated. The most common adverse effects are fever, nausea, vomiting and complication at the infusion point of the vein. CAS has a better tolerability than amphotericin B and a similar one compared to fluconazole (FCZ) but with less drug interactions. PERSPECTIVES: For rare but severe localisations (i.e.: endocarditis, cerebral, arthritis, etc.), CAS combinations with classical antifungal drugs could be tested in order to improve the life time in patients suffering from systemic fungal infections.

Usefulness of genotyping with microsatellite markers to investigate hospital-acquired invasive aspergillosis.

To assess whether invasive aspergillosis (IA) was hospital acquired, Aspergillus fumigatus isolates, obtained during a one-year study from inpatients with haematological diseases and IA and from their environment, were genotyped using microsatellite markers. The analysis of 62 environmental isolates showed an extremely diverse A. fumigatus population with 43 genotypes represented only once. Eight genotypes were found more than twice at different times and/or at different locations showing that a given isolate can persist over time and is not dependent on a specific location. Twenty-seven isolates were obtained from 12 patients with IA. Of eight patients with multiple isolates, four were infected with isolates of different genotypes. Five patients (42%) had hospital-acquired IA according to the following definitions: patients infected with an isolate found in the environment, or patients infected with the same genotype. Although genotyping results are highly suggestive of hospital-acquired IA, this cannot be proved definitively because of the high diversity of the A. fumigatus population and the limited environmental sampling. A better knowledge of the A. fumigatus population outside hospitals is needed. For this purpose, genotyping using microsatellite markers seems appropriate.

Fatal primary cutaneous aspergillosis in a bone marrow transplant recipient: nosocomial acquisition in a laminar-air flow room.

A case of primary cutaneous aspergillosis occurring in an allogeneic bone marrow transplant recipient in a laminar airflow room is reported. The patient developed grade III graft-versus-host-disease and epidermolysis. Although the patient had remained in his laminar airflow room from the graft onward, he subsequently developed primary cutaneous aspergillosis. The aspergillosis became invasive and the patient died. The patient was probably contaminated by air containing conidia when he left the sterile room for endoscopy, and the fluidized bed used may have contributed to the local development of the disease. This nosocomial aspergillosis stresses the necessity of performing invasive procedures under laminar airflow protection to prevent Aspergillus contamination in immunocompromised hosts at risk for aspergillosis.

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum.

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.

Serum Aspergillus galactomannan antigen testing by sandwich ELISA: practical use in neutropenic patients.

The double sandwich ELISA detecting Aspergillus galactomannan (GM) was prospectively evaluated for the diagnosis of invasive aspergillosis (IA) in 50 haematological patients at risk for IA. Serum samples were collected once weekly as long as the risk factors persisted. Six patients had proven or probable IA (3 A. fumigatus, 1 A. flavus, 1 A. niger, 1 A. ustus) and the GM titres were parallel to the clinical evolution of IA. Six of nine patients with suspected IA had at least two consecutive serum GM titres above 1 ng/ml and died with increasing titres, whereas the GM-negative patients survived. Positive GM titres did not anticipate the isolation of fungi. Unfortunately, positive GM titres did not anticipate the initiation of antifungal therapy, based on clinical suspicion. Moreover, if a true-positive result was defined as two consecutive positive serum samples, four patients out of 35 without proven, probable or suspected IA were positive. Then, the rate of false-positive results was high (12%) in the range previously reported. Nevertheless, the GM ELISA appears useful to assess IA and to follow the efficacy of antifungal treatment.

Candida albicans genotyping in studies with patients with AIDS developing resistance to fluconazole.

We characterized Candida albicans strains responsible for recurrent oropharyngeal candidosis (OPC) in four patients with AIDS who developed clinical and mycological resistance to fluconazole (FCZ). Karyotype and restriction fragment length polymorphism analyses were performed on the clonal populations to differentiate relapse from reinfection, and the results were assessed with those of serotype and FCZ MICs. Despite the polymorphism in chromosomal bands larger than 2.2 Mbp related to an intraclonal variation, karyotype analysis showed a single strain type attributable to each patient. On the other hand, EcoRI and HinfI restriction fragments revealed a polymorphism for one patient between the first sample and the subsequent ones, relevant to the acquisition of a new strain causing the following episodes of OPC. This result coincided with switching of the serotype and with the acquisition of a resistance to FCZ. For the other three patients, the similarity of the DNA electrophoretic patterns and the serotype of the samples suggested that recurrence can be due to the initial strain that generates FCZ resistance. Although useful for epidemiological studies, molecular typing methods seem to be inadequate to detect the acquisition of FCZ resistance.

Microsatellite markers for typing Aspergillus fumigatus isolates.

The use of microsatellites as highly polymorphic DNA markers for the typing of isolates of Aspergillus fumigatus was investigated. Four CA repeats were selected by screening an A. fumigatus DNA library with a (CA)10 oligonucleotide. Primers flanking these CA repeats were designed to amplify each locus. One primer of each pair was labeled with a fluorophore, and the PCR products were analyzed with an automatic sequencer and the GeneScan software. For each primer set and for a given isolate, one band was detected and was assigned to an allele because A. fumigatus is haploid. With 50 clinical isolates, 50 environmental isolates, and 2 reference strains we obtained 12, 11, 10, and 23 different alleles for the four CA microsatellites, respectively (discriminatory power, 0.994). The results were identical by whatever DNA extraction technique was used. Interestingly, no clustering between environmental and clinical isolates was observed, suggesting that every isolate is potentially pathogenic. Microsatellite markers appear suitable for use in large epidemiological studies of invasive aspergillosis.

Comparison of serum galactomannan antigen detection and competitive polymerase chain reaction for diagnosing invasive aspergillosis.

To improve the diagnosis of invasive aspergillosis (IA), we retrospectively compared competitive polymerase chain reaction (PCR) and sandwich ELISA for detection of serum galactomannan (GM) antigen. We studied 281 serum samples collected weekly during the period at risk for IA from 41 selected hematology patients. Twenty-two patients had confirmed, probable, or suspected IA, according to clinical and mycologic data. Fifteen of them had positive GM titers (87 samples) and 12 had positive PCRs (20 samples). Nineteen of the 20 PCR-positive samples were also GM-positive. Of the 19 patients without IA (83 samples), one had 3 GM-false-positive samples. Neither test anticipated the initiation of antifungal therapy on the basis of clinical suspicion. Both tests were more likely to be positive before death. This study suggests that PCR on serum samples is not more sensitive than GM detection. However, PCR can improve the specificity of the GM test. Together, these noninvasive tests should improve the diagnosis of IA.

Contribution of molecular typing methods and antifungal susceptibility testing to the study of a candidemia cluster in a burn care unit.

We investigated a cluster of cases of Candida septicemia diagnosed in four burn patients. Twenty clinical isolates of Candida albicans and two of Candida parapsilosis, plus eight isolates of C. albicans recovered from nurses' clothes, were analyzed by antifungal susceptibility testing and three genotyping methods (restriction fragment length polymorphism analysis with EcoRI and HinfI, arbitrarily primed PCR, and karyotyping). The high MICs of the azoles for all of the C. albicans isolates tested suggest either a natural resistance of the endogenous flora or the transmission of isolates with acquired resistance. The genotyping methods demonstrated the involvement of four different strains, cross-infections with one C. albicans strain and one C. parapsilosis strain, and identity between some of the strains from the patients and nurses. The origins of the strains remain unclear. Our results show that the use of a combination of at least two different methods such as those used in the present study is recommended for C. albicans typing.

Comparison of restriction fragment length polymorphism, microsatellite length polymorphism, and random amplification of polymorphic DNA analyses for fingerprinting Aspergillus fumigatus isolates.

Aspergillus fumigatus fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.