Detection of Aspergillus DNA by a nested PCR assay is superior to blood culture in an experimental murine model of invasive aspergillosis.

For diagnosing invasive aspergillosis (IA), an increasing clinical problem in immunocompromised patients, molecular tools are gaining in importance. Detection of Aspergillus DNA in blood samples was investigated by a nested PCR assay in a murine model of experimentally induced IA. Ex vivo, the detection threshold of the PCR assay was determined in blood and organ homogenates of mice. After intravenous injection of Aspergillus fumigatus conidia on different days, growth of colonies was determined in cultures of blood and organs from immunocompetent and immunosuppressed mice and Aspergillus DNA was detected from blood samples by a nested PCR assay. The detection threshold of the PCR assay was as low as 1 c.f.u. ml(-1). The assay proved to be more sensitive than cultures of blood, with sensitivity rates between 17.6 and 87.5 % depending on the fungal burden. In conclusion, the nested PCR assay is superior to cultural methods in detecting Aspergillus spp. in murine blood samples.

Multiple groups of endogenous betaretroviruses in mice, rats, and other mammals.

Betaretroviruses exist in endogenous and exogenous forms in hosts that are widely distributed and evolutionarily distantly related. Here we report the discovery and characterization of several previously unknown betaretrovirus groups in the genomes of Mus musculus and Rattus norvegicus. Each group contains both mouse and rat elements, and several of the groups are more closely related to previously known betaretroviruses from nonmurine hosts. Some of the groups also include members from hosts which were not previously known to harbor betaretroviruses, such as the gray mouse lemur (Microcebus murinus) and Seba's short-tailed bat (Carollia perspicillata). Some of the mouse and rat elements possess intact open reading frames for gag, pro, pol, and/or env genes and display characteristics of having retrotransposed recently. We propose a model whereby betaretroviruses have been evolving within the genomes of murid rodents for at least the last 20 million years and, subsequent to (or concomitant with) the global spread of their murid hosts, have occasionally been transmitted to other species.

Structure and expression of mobile ETnII retroelements and their coding-competent MusD relatives in the mouse.

ETnII elements are mobile members of the repetitive early transposon family of mouse long terminal repeat (LTR) retroelements and have caused a number of mutations by inserting into genes. ETnII sequences lack retroviral genes, but the recent discovery of related MusD retroviral elements with regions similar to gag, pro, and pol suggests that MusD provides the proteins necessary for ETnII transposition in trans. For this study, we analyzed all ETnII elements in the draft sequence of the C57BL/6J genome and classified them into three subtypes (alpha, beta, and gamma) based on structural differences. We then used database searches and quantitative real-time PCR to determine the copy number and expression of ETnII and MusD elements in various mouse strains. In 7.5-day-old embryos of a mouse strain in which two mutations due to ETnII-beta insertions have been identified (SELH/Bc), we detected a three- to sixfold higher level of ETnII-beta and MusD transcripts than in control strains (C57BL/6J and LM/Bc). The increased ETnII transcription level can in part be attributed to a higher number of ETnII-beta elements, but 70% of the MusD transcripts appear to have been derived from one or a few MusD elements that are not detectable in C57BL/6J mice. This element belongs to a young MusD subgroup with intact open reading frames and identical LTRs, suggesting that the overexpressed element(s) in SELH/Bc mice might provide the proteins for the retrotransposition of ETnII and MusD elements. We also show that ETnII is expressed up to 30-fold more than MusD, which could explain why only ETnII, but not MusD, elements have been positively identified as new insertions.

Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients.

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.

Insertional polymorphisms of ETn retrotransposons include a disruption of the wiz gene in C57BL/6 mice.

ETn (early transposon) elements are moderate repetitive sequences present in hundreds of copies in the mouse genome. Their length ranges from 4.4 to 7.1 kb, and, like transposons, they contain long terminal repeats (LTRs) on both sides and are flanked by target site duplications (Kaghad et al. 1985). ETn-related elements can be grouped into three distinct families. Members of the ETn I and ETn II families mainly contain sequences of unknown origin in their core region. Only very short stretches of retrovirus-like sequences are present, and there are no ORFs. ETn I and ETn II elements differ primarily in the 3- half of both the 5- and 3- LTR, and in the 5- end of the core region (see Fig. 1). As a consequence, only ETn II elements contain a primer binding site for tRNALys. In contrast to ETn I and ETn II, members of the recently described MusD family (Mager and Freeman 2000) contain ORFs for (at least parts of) D-type virus Gag, Pro, and Pol proteins. However, in other regions they are structurally similar to ETn II elements and contain an intact primer binding site. It has been shown that MusD sequences are evolutionarily older than ETn II elements, suggesting that the latter might have arisen by recombinatory replacement of the MusD gene-coding sequences with sequences of unknown origin (Mager and Freeman 2000). ETn elements are still active as retrotransposons. In the past years, several germ line and somatic mutations caused by fresh ETn integrations have been found (Table 1). From 19 mutations, sufficient sequence is available in seven cases to show that the insertion was an ETn II element. In eight cases, the sequence data available indicate either an ETn II or a MusD element. ETn I has not been found to be the cause of any mutations, prompting the suggestion that ETn II is the "mobile" family, whereas ETn I elements have lost the capacity to retrotranspose.

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

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.