Laurence Millon

Quantitative Polymerase Chain Reaction Detection of Circulating DNA in Serum for Early Diagnosis of Mucormycosis in Immunocompromised Patients

BACKGROUND The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. METHODS We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. RESULTS No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. CONCLUSION Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.

Practical Approach for Typing Strains of Leishmania infantum by Microsatellite Analysis

ABSTRACT Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies.

Use of Real-Time PCR To Process the First Galactomannan-Positive Serum Sample in Diagnosing Invasive Aspergillosis

ABSTRACT Positive galactomannan (GM) antigenemias are included as a microbiological item in the diagnosis of probable or possible invasive aspergillosis (IA). Because false-positive GM results frequently occur, at least two positive results on two different samples are required. Waiting for clinical specimens can delay the initiation of treatment. As an alternative, we wondered whether detection of circulating Aspergillus DNA on the first positive GM serum sample could aid in diagnosing IA. Therefore, we retrospectively screened the first GM-positive serum samples from 29 patients from our hematology unit for Aspergillus DNA using real-time PCR. We compared the real-time PCR results with the final classification of proven, probable, and possible IA according to consensual criteria. No clear correlation between PCR results and the classification with the medical files could be shown. However, a positive PCR result was associated with a poor prognosis (Fishers test; P = 0.01). Our preliminary data suggest that a positive PCR result could indicate a more advanced stage of the disease. Therefore, concomitant positive PCR and GM results may justify the initiation of antifungal therapy in neutropenic patients. In contrast, a negative PCR on the first positive GM sample may argue for postponing costly antifungal administration until additional arguments for the diagnosis of IA are presented.

Cutaneous manifestations of human toxocariasis.

Human toxocariasis is a parasitic disease characterized by the presence of larvae of the genus Toxocara in human tissues. T canis and T cati, the adult roundworms of which are found in dog and cat intestines, respectively, are the most common causative agents of the disease. Toxocaral larvae usually cause two severe syndromes: visceral larva migrans and ocular larva migrans, depending on the location of the larvae. Two other syndromes, covert toxocariasis and common toxocariasis, which are less typical and not as severe, have also been described. During the last two decades, cutaneous manifestations such as chronic urticaria, chronic pruritus, and miscellaneous eczema, in patients with Toxocara antibodies, have been studied by different authors. In some cases, these cutaneous manifestations are the only signs indicating the presence of the disease, and they are cured after antihelmintic treatment when there is good patient compliance. In this review, we focus on these particular skin manifestations regarding their clinical description, diagnosis, and treatment.

Hypersensitivity pneumonitis and metalworking fluids contaminated by mycobacteria

Metalworking fluids (MWF) are responsible for hypersensitivity pneumonitis (HP). The aim of the present study was to identify the antigen (Ag) responsible for MWF-associated HP, and to optimise serological diagnosis by definition of a threshold allowing discrimination between HP patients and asymptomatic exposed workers. 13 patients, who were workers at a car engine manufacturing plant, were suspected of MWF-associated HP. Microbial analysis of 83 used MWFs was carried out. Sera from 13 MWF-associated HP patients, 12 asymptomatic exposed workers and 18 healthy unexposed controls were tested to determine their immunological responses to three Ags, including Mycobacterium immunogenum. M. immunogenum was identified in 40% of used fluids by culture and confirmed by DNA sequencing. The threshold for differentiating MWF-associated HP patients from asymptomatic exposed workers was five arcs of precipitation (sensitivity 77% and specificity 92%), as determined by electrosyneresis (ES). Using ELISA methods with protein extract from M. immunogenum, a threshold leading to 92% sensitivity and 100% specificity was established. The detection of specific antibodies against M. immunogenum Ag at high levels in case sera suggests that M. immunogenum-contaminated MWF is responsible for MWF-associated HP. To discriminate MWF-associated HP patients from asymptomatic exposed workers, we suggest a five-arc threshold for ES and a 1.6-AU threshold for ELISA methods.

Indoor mold concentration in Eastern France

UNLABELLED Our prospective case-control study of 118 dwellings in Eastern France examined fungal contamination in unhealthy dwellings (n = 32) (homes with visible mold contamination and adverse health outcomes reported by the occupants), dwellings occupied by allergic patients (with medical diagnostic and positive prick-tests for molds) (n = 27) and matched control dwellings (n = 59). Unhealthy dwellings present higher airborne concentrations of Aspergillus, Penicillium, and Cladosporium than control dwellings, irrespective of the room sampled. Bedroom walls were more highly contaminated by molds than others. Dwellings occupied by allergic patients differed significantly for airborne concentrations of Penicillium only, but not for wall surface contamination, whereas bathroom walls were more highly contaminated than other rooms. Molecular identification of 12 Penicillium species showed Penicillium chrysogenum and Penicillium olsonii to be the two main species. From the total average of molds, by impaction method, useful thresholds can be given: below 170 CFU/m(3), between 170 and 560 CFU/m(3), 560 and 1000 CFU/m(3) and above 1000 CFU/m(3), respectively for dwellings with low, moderate, high, and very high concentrations. The latter would be considered a potential health hazard. PRACTICAL IMPLICATIONS A single measure of airborne concentrations of molds by impaction allows to establish useful thresholds by social services to estimate in a objective way the housing moldiness. Excluding the summer period, reproducibility of this kind of measure on 3 months, in the fixed limits, is 94.3%. The differences in terms of biodiversity of the unhealthy housing and those accommodating allergic patients imply a specific approach to decrease fungi airborne concentrations. The biodiversity of Penicillium raises the problem of the use of the single extract of Penicillium chrysogenum for skin-tests. The extent of the contaminated surfaces must be measured to assess the potential risk linked to spore contamination. Indeed, surface sampling mostly allows qualitative assessment of the environment.

Characteristics of dwellings contaminated by moulds

Dwellings showing a presence of moulds are considered to be unhealthy both by the inhabitants and by sanitary authorities. Although the thresholds of pathogenicity have not yet been established, the toxic, allergic and infectious risk of indoor moulds is better understood today. A study on indoor fungi contamination for 128 dwellings was done between October and May in France. It concerned 69 dwellings, the occupants of which either complained to the sanitary authorities about problems of moulds and humidity or consulted a doctor who related their symptoms to housing conditions. Fifty-nine other dwellings, the occupants of which were healthy, constituted the control group. We present the statistical analysis of questionnaires, which aimed to clarify characteristics of dwellings associated with high concentrations of airborne moulds. Air samples were taken with an impactor in 500 rooms. On visiting dwellings, investigators obtained answers to 25 questions concerning characteristics of inhabitants and living space, as well as the presence of mould indicators. Indoor and outdoor temperature and indoor relative humidity of air measurements were taken. The total concentration of fungi in the air was significantly higher in ground floor apartments versus those on other floors (p = 0.047), in small and highly occupied dwellings (p = 0.03 and 0.003), in dwellings with electric heating (p = 0.04), without a ventilation system (p = 0.003), with water damage (p = 0.003), and finally, in those where the investigator noted an odour of moisture or visible moulds (p < 0.001). The efficacy of the latter criteria in the evaluation of insalubrity is discussed.

Indoor fungal contamination of moisture-damaged and allergic patient housing analysed using real-time PCR.

Aims:  The aim of our study was to compare, using real‐time (Rt) PCR, quantitative levels of five fungal species in three kinds of dwellings.

Indoor fungal contamination: Health risks and measurement methods in hospitals, homes and workplaces

Abstract Indoor fungal contamination has been associated with a wide range of adverse health effects, including infectious diseases, toxic effects and allergies. The diversity of fungi contributes to the complex role that they play in indoor environments and human diseases. Molds have a major impact on public health, and can cause different consequences in hospitals, homes and workplaces. This review presents the methods used to assess fungal contamination in these various environments, and discusses advantages and disadvantages for each method in consideration with different health risks. Air, dust and surface sampling strategies are compared, as well as the limits of various methods are used to detect and quantify fungal particles and fungal compounds. In addition to conventional microscopic and culture approaches, more recent chemical, immunoassay and polymerase chain reaction (PCR)-based methods are described. This article also identifies common needs for future multidisciplinary research and development projects in this field, with specific interests on viable fungi and fungal fragment detections. The determination of fungal load and the detection of species in environmental samples greatly depend on the strategy of sampling and analysis. Quantitative PCR was found useful to identify associations between specific fungi and common diseases. The next-generation sequencing methods may afford new perspectives in this area.

Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549.

Inhalation of conidia is the main cause of invasive pulmonary aspergillosis (IPA) and the respiratory epithelium is the first line of defence. To explore the triggering factor for the inflammatory response to Aspergillus fumigatus, the species mainly responsible for IPA, this study analysed the differential expression of three inflammatory genes in A549 cells after challenge with live and killed conidia. The influence of steroids, one of the main risk factors for developing IPA, was also investigated. Quantification of mRNAs of the inflammatory mediator genes encoding interleukin (IL)-8, tumour necrosis factor (TNF)-alpha and granulocyte-monocyte colony-stimulating factor (GM-CSF) was carried out using real-time PCR. Ingestion rates were studied for the conidia of A. fumigatus and Penicillium chrysogenum using a fluorescence brightener. Similar results were obtained for both species, with ingestion rates ranging from 35 to 40 %. Exposure of A549 cells to live A. fumigatus conidia only induced a four- to fivefold increase in the mRNA levels of the three genes, starting 8 h after the initial contact. Both inactivation of live A. fumigatus conidia and treatment by dexamethasone (10(-7) M) prevented the overexpression of TNF-alpha, IL-8 and GM-CSF. Fungal growth, rather than conidia ingestion, appears to be the main stimulus for the production of inflammatory mediators by epithelial cells, and this production is inhibited by steroid therapy. These results underline the role that the epithelium plays in the innate response against IPA.