Multiplex real-time PCR for the diagnosis of malaria: correlation with microscopy.

Malaria is generally diagnosed by microscopy and rapid antigen testing. Molecular methods become more widely used. In the present study, the contribution of a quantitative multiplex malaria PCR was investigated. We assessed: (i) the agreement between PCR-based identification and microscopy and (ii) the correlation between the parasite load as determined by quantitative PCR and by microscopy. For 83 patients positive by microscopy for Plasmodium spp., the first EDTA-blood sample was tested by multiplex PCR to confirm smear-based species identification. Parasite load was assessed daily using both microscopy and PCR. Among the 83 patients tested, one was positive by microscopy only and 82 were positive by microscopy and PCR. Agreement between microscopy and PCR for the identification at the species level was 89% (73/82). Six of the nine discordant results corresponded to co-infections by two or three species and were attributed to inaccurate morphological identification of mixed cases. The parasite load generally decreased rapidly after treatment had been started, with similar decay curves being obtained using both microscopy and PCR. Our PCR proved especially useful for identifying mixed infections. The quantification obtained by PCR closely correlated with microscopy-based quantification and could be useful for monitoring treatment efficacy, at least in clinical trials.

Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus.

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.

Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus.

A metalloprotease (MEP) secreted by Aspergillus fumigatus was isolated from an alkaline protease-deficient mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxy-methyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fumigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.

Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus.

The gene encoding the secreted alkaline protease, a suspected virulence factor of Aspergillus fumigatus, was inactivated by gene disruption. The disruption was performed by transformation of a pathogenic strain of the fungus with a linear DNA fragment carrying the gene from which the central part was replaced by the selectable Escherichia coli hygromycin B dominant resistance marker. Two transformants were shown to produce no alkaline protease. Restriction fragment analysis of the DNA of these two transformants was consistent for chromosomal integration of the disrupted gene by homologous recombination. Both isogenic alkaline protease-producing and non-producing A. fumigatus strains invaded lung tissues, causing comparable mortality in immunosuppressed mice. A significant residual proteolytic activity observed in alkaline protease non-producing strain cultures could play a role in the invasion of the tissues by the fungus.

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus.

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.

Characterization by DNA restriction endonuclease analysis of Listeria monocytogenes strains related to the Swiss epidemic of listeriosis.

Listeria monocytogenes strains responsible for outbreaks of listeriosis were studied by using serotyping and phage typing. An additional approach based on restriction endonuclease analysis (REA) of the chromosomal DNA was used to characterize L. monocytogenes strains collected from various sources during and after a Swiss outbreak of listeriosis (1983 to 1987). Among the 169 wild-type strains of Listeria spp. that were examined, 161 (95%) belonged to the species L. monocytogenes, of which 109 were of human origin. Ten different REA profiles were obtained from the 120 L. monocytogenes serotype 4b strains tested. All 57 serotype 4b strains that were identified as Swiss epidemic strains by phage typing clustered in two closely related REA profiles. In particular, 10 L. monocytogenes 4b strains isolated from the brand of soft cheese responsible for the outbreak and from its direct environment were indistinguishable from isolates from 40 patients by both phage typing and REA analysis. However, 5 of the 17 non-phage-typeable L. monocytogenes strains and 18 L. monocytogenes strains with a phage type different from those of the Swiss epidemic types showed the same profile. REA enabled the characterization of non-phage-typeable strains and, thus, seems a promising tool for L. monocytogenes typing, especially during epidemiological investigations.