Invasive pulmonary aspergillosis in an ICU patient with Legionnaires' disease: A diagnostic challenge.

Aspergillus fumigatus can cause a wide range of diseases, from hypersensitivity to invasive infection. Invasive disease usually occurs in severely immunocompromised patients with deep and prolonged neutropenia. It is a less well-recognized complication in critically ill patients without traditional risk factors. We describe a case of early invasive pulmonary aspergillosis (IPA) secondary to Legionella pneumophila serogroup 1 pneumonia in a patient on an intensive care unit (ICU). In addition to commonly accepted risk factors for IPA in ICU patients, we hypothesis that L. pneumophilia pneumonia could enhance this type of infection. We also reviewed all published cases of coinfection with L. pneumophila and A. fumigatus to assess whether Legionnaires' disease could be a risk factor for IPA.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

[Evaluation of the performances of the iQ(®)200 ELITE automated urine microscopy analyser and comparison with manual microscopy method]. / Évaluation des performances de l'automate de cytologie urinaire Iris iQ(®)200 ELITE et comparaison avec la méthode manuelle microscopique.

Microscopy urinalysis is one of the last manual analyses in laboratories and it is difficult to control. The automation of this analysis permits our laboratory to standardise this test. We performed an evaluation of the iQ(®)200 ELITE's performances and compare the data to the manual microscopy method which is considered as "the gold standard". The repeatability achieved at different levels of leukocytes and erythrocytes showed CVs below 10% for pathological values. Inter-run repeatability is 5,57% and carry-over is negligible if we take into consideration the linearity of the formed particles. Compared to the manual method, sensitivity (Se) of the analyser for leukocytes and erythrocytes are respectively 94.85 and 100%, the negative predictive value (NPV) are 92.91 and 100%, respectively. The weaknesses of the specificity (Sp) and positive predictive value (PPV) of erythrocytes (24.22 and 41.92%) are due to the lack of sensibility of the manual method. Also, the weak PPV of yeasts, casts and crystals are due to the better detection of those particles with the iQ(®)200's image recognition system than with manual method. NPV for all the urine formed particles are excellent, they are between 98 and 100%. The iQ(®)200 ELITE permits us to standardise and control this test for a future accreditation of the laboratory. We also significantly increase the turn around time.

High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology.

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts.

Pneumocystis carinii organisms constitute a large group of heterogeneous atypical microscopic fungi that are able to infect immunocompromised mammals by an airborne route and to proliferate in their lungs, inducing Pneumocystis carinii pneumonia. This pneumonia remains a crucial epidemiological challenge, since neither the source of Pneumocystis carinii infection in humans nor the process by which humans become infected has been clearly established. Polymerase chain reaction (PCR) assays have shown that profoundly immunosuppressed patients without pneumocystosis can be subclinically infected with Pneumocystis. Other PCR-based studies have suggested that healthy immunocompetent hosts are not latent carriers of the parasite. However, recent reports have indicated that Pneumocystis carinii can persist for limited periods in the lungs of convalescent rats after recovery from corticosteroid-induced pneumocystosis, and also that immunocompetent mammals can be transiently parasitized by Pneumocystis carinii after close contact with hosts with Pneumocystis carinii pneumonia. Can transiently parasitized hosts be a source of infection for immunosuppressed hosts? In order to investigate this important clinical question, the ability of immunocompetent BALB/c mice, which were carrying subclinical levels of Pneumocystis carinii, to transmit the infection by the airborne route to highly susceptible, uninfected mice with severe combined immunodeficiency was studied. The results indicated that the immunocompetent mice, transiently parasitized by Pneumocystis carinii organisms after close contact with Pneumocystis carinii-infected mice, were able to transmit the infection to Pneumocystis carinii-free mice with severe combined immunodeficiency.

Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii.

Genetic divergence at the SODA (manganese-dependent superoxide dismutase, MnSOD) locus were compared in six Pneumocystis carinii formae speciales isolated from mouse, rabbit, human, macaque and pig. A degenerate oligonucleotide primer strategy was designed to amplify 85-90% of the full-length SODA gene from P. carinii genomic DNA isolates. DNA sequence analysis revealed an A/T bias in the nucleotide composition (71-77.2%) and the presence of seven small introns (41-142 bp), interrupting each P. carinii open reading frame (ORF) at the same position. The MnSOD deduced amino acid sequences from all P. carinii isolates shared residues which were conserved within the MnSOD family and which are required for enzymatic activity and binding of the cofactor metal. Phylogenetic analysis including MnSOD sequences from representatives of the fungal phyla Basidiomycota and Ascomycota indicated that the P. carinii formae speciales form a monophyletic group that is related to the budding yeasts (subphylum Saccharomycotina, previously called class Hemiascomycetes) in the Ascomycota. In the whole Pneumocystis group, P. carinii f. sp. hominis, P. carinii f. sp. macacae and P. carinii f. sp. oryctolagi MnSOD sequences clustered together, as did the rat-derived P. carinii and P. carinii f. sp. muris sequences.

Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome.

Mycobacterial interspersed repetitive units (MIRUs) are 40-100 bp DNA elements often found as tandem repeats and dispersed in intergenic regions of the Mycobacterium tuberculosis complex genomes. The M. tuberculosis H37Rv chromosome contains 41 MIRU loci. After polymerase chain reaction (PCR) and sequence analyses of these loci in 31 M. tuberculosis complex strains, 12 of them were found to display variations in tandem repeat copy numbers and, in most cases, sequence variations between repeat units as well. These features are reminiscent of those of certain human variable minisatellites. Of the 12 variable loci, only one was found to vary among genealogically distant BCG substrains, suggesting that these interspersed bacterial minisatellite-like structures evolve slowly in mycobacterial populations.

Epidemiological and taxonomic impact of Pneumocystis biodiversity.

A cluster of antigenic, genomic, karyotypic, isoenzymatic and morphological differences have been reported among Pneumocystis populations. Multilocus enzyme electrophoresis revealed strong linkage disequilibrium suggesting that Pneumocystis genotypes from different hosts have been genetically isolated from each other for a very long time. At least in some cases, genetic diversity is associated with phenotypic differences as revealed by in vitro, ultrastructural and cross infection studies. Thus, biodiversity in Pneumocystis has obvious epidemiological implications. Cross infection experiments revealed that Pneumocystis host species-related genetic differences are associated with close host species specificity, which suggests that transmission cannot take place between hosts of different species and that immunocompromised patients contract the infection primarily from infected humans. But these affirmations do not preclude other reservoirs for human pneumocystosis and research has to be extended to natural populations of synanthropic or wild mammals. Transmission of human pneumocystosis was also approached by typing human Pneumocystis isolates from patients or carriers, which should allow the follow up of parasite strains in human populations. As the strains of Pneumocystis found in different host species were considered for a long time to be morphologically indistinguishable, only one species of Pneumocystis was accepted for almost one century. At present, the scientific community is progressively accepting that the terminology 'P. carinii' is hiding a heterogeneous group of microorganisms. As available data made it impossible to establish if genetic divergence derives from clonal reproduction or speciation, no new species names have been attributed to Pneumocystis populations, but a trinomial nomenclature, including the Latin name of the host, was adopted in 1994. It has to be outlined finally that works on biodiversity of Pneumocystis populations are basically important as they have revealed a new group of eukaryotic, pathogenic, heterogeneous microorganisms with fungal affinities, difficult to cultivate until now and widely spread in ecosystems. These researches are opening a virgin field for microbiology research.

[Evaluation of a dot-blot technique for the detection of Epstein-Barr virus antibodies (IgG anti-viral capsid antigen)]. / Evaluation d'une technique de dot-blot pour la détection d'anticorps dirigés contre le virus d'Epstein-Barr (IgG anti-antigène de capside virale).

Infectious mononucleosis syndrome is caused by several infectious agents, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Toxoplasma gondii. The ImmunoDot EBV VCA-IgG test (Biomedical Diagnostics) is a dot-blot enzyme-immunoassay, which determines the presence of heterophile antibodies and IgG antibodies directed against Epstein-Barr viral capsid antigen (anti-VCA), CMV and T. gondii. This test is simple, unitary, ready-to-use, and rapid (40 minutes), requiring approximately 10 microliters of serum or plasma or 20 microliters of whole blood. The aim of this study was to compare the results of this technique for the determination of anti-VCA IgG antibodies with those obtained by the reference technique using indirect immunofluorescence on 185 serum specimens. A sensitivity of 99.2%, a specificity of 92.4%, a correlation with indirect immunofluorescence of 97.3%, a the absence of cross-reactions with CMV, T. gondii, and herpes simplex viruses and the absence of interference with antinuclear antibodies were the main results of this comparative study. In association with another test (Monolert 2TM) detecting IgM and IgG antibodies against Epstein-Barr nuclear antigen, the dot-blot method represents a useful screening strategy for EBV response.

[Study of in vitro activity of 3 cephalosporins and their combination with 2 beta-lactamase inhibitors against 4 enterobacteria strains producing extended spectrum beta-lactamase]. / Etude de l'activité in vitro de trois céphalosporines et de leurs associations avec deux inhibiteurs de bêta-lactamase vis-à-vis de quatre espèces d'entérobactéries productrices de bêta-lactamase à spectre étendu.

The in vitro antibacterial activity of cefotaxime, cefepime and cefpirome was evaluated alone and in association with two beta-lactamase inhibitors: clavulanic acid and sulbactam against 65 extended broad spectrum beta-lactamase producing enterobacteriaceae strains [Klebsiella pneumoniae (35), Proteus mirabilis (9), Escherichia coli (11), Enterobacter aerogenes (10)]. The lowest MICs were observed for cefepime and cefpirome used alone. In association with inhibitors cefotaxime was less effective against E. aerogenes. Most isolates of K. pneumoniae, P. mirabilis, E. coli were susceptible to all the different tested associations.

Isoenzyme diversity in Pneumocystis carinii from rats, mice, and rabbits.

Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised patients. To investigate the genetic diversity of P. carinii populations, multilocus enzyme electrophoresis was used to analyze five enzyme systems (malate dehydrogenase, glucose phosphate isomerase, leucine aminopeptidase, malic enzyme, and 6-phosphogluconate dehydrogenase). Only five different multilocus associations (zymodemes) were recorded for the 70 isolates studied. While only one multilocus combination was found in mice and rabbits, three different multilocus associations were recorded in rats. Population genetic tests and phylogenetic analysis strongly suggest that P. carinii genotypes are host-specific, in agreement with molecular study results, and that no genetic exchange occurs between genotypes from different host species. This hypothesis could be verified only by the evolutionary genetic approach, which relies here on multilocus analysis.

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions.

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.

Relapsing Plasmodium vivax malaria with atypical parasite forms and phagocytosis by peripheral neutrophils.

A case of atypical Plasmodium vivax malaria is presented. The clinical follow-up has allowed to characterize three consecutive malaria clinical episodes within one year. At the first attack, 39% of the infected red blood cells were parasitized by gametocytes. Furthermore, rare crisis forms, exceptional "pseudoparthenogenesis" forms, a few equatorial trophozoites, malaria pigment-containing leucocytes and phagocytized parasites were also found in the thin blood smears. At the second malaria episode, morphological aspects were quite similar, but the gametocyte percentage decreased and that of the equatorial trophozoite forms increased. Only at the third attack, was the morphology typical of P. vivax. The Plasmodium species and the absence of mixed infection were unequivocally confirmed using polymerase chain reaction. Atypical strains of P. vivax are relatively frequent. Nevertheless, to our knowledge, neither so high a gametocyte percentage, nor extensive P. vivax peripheral phagocytosis were previously reported.