Emeline Scherer

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.

Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients: a 3-Year Multicenter Retrospective Study

ABSTRACT Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV+; the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.

Ribosomal and mitochondrial DNA target for real-time PCR diagnosis of invasive aspergillosis

ABSTRACT The aim of the present study was to assess the diagnostic efficacy of a combination of two quantitative Aspergillus PCR assays, targeting a mitochondrial and a ribosomal target, in patients with risk factors for invasive aspergillosis (IA) and positive galactomannan (GM) antigen. Forty-four patients with hematological malignancies and risk factors for IA according to revised European Organization for Research on Treatment of Cancer and the Mycoses Study Group criteria (EORTC/MSG) criteria and presenting at least two sequential GM-positive sera were included in the study. Mitochondrial PCR was carried out prospectively on all GM-positive serum samples. Ribosomal PCR was carried out retrospectively on frozen stored sera. The sensitivities of mitochondrial and ribosomal PCRs were 58% and 50%, respectively. The diagnostic test performance was improved by using a combination of both PCR assays and by considering a patient PCR positive when at least two positive results were obtained. The sensitivity, specificity, and positive and negative likelihood ratios were 65%, 94%, and 11.8 and 0.37, respectively. A significant association between fatal outcome at 90 days and positive results of ribosomal PCR assays was observed (adjusted hazard ratio = 8.2; 95% confidence interval [CI] = 1.0 to 65.8; P = 0.048). Our results showed that the combination of two PCR assays targeting mitochondrial and ribosomal Aspergillus DNA improves the sensitivity of PCR in the diagnosis of IA in hematological patients with risk factors and positive GM results. This study also confirms that a positive PCR result is associated with a poor prognosis in these patients and should lead to specific antifungal therapy being introduced immediately.

Contribution of a Cyclonic-Based Liquid Air Collector for Detecting Aspergillus Fumigatus by QPCR in Air Samples

Fungal infections represent a constant threat in hospitals, especially for high-risk patients hospitalized in hematology and bone marrow transplant units. While routine air sampling to predict the fungal contamination risk is discussed by some authors,(1,2) French hospital guidelines specify that each hospital is responsible for the air it provides to patients and that air control efficiency must be monitored by specific methodologies.(3) Traditional monitoring involves impacted culture media that are incubated for several days so that the fungal species may grow and be identified on the basis of macroscopic and microscopic criteria.(4) A variety of portable air impactors are commercially available and validated,(5,6) but these systems are limited by their link with culture techniques, involving a time-lapse to results of around 7 days and by a maximal sampling rate of 100 L/min. As optimal environmental monitoring requires large sample sizes and fast, accurate detection of microorganisms contained within the samples,(7,8) new techniques concurring with these characteristics are regularly evaluated. Quantitative polymerase chain reaction (QPCR) represents a very attractive alternative to culture techniques as it allows Aspergillus fumigatus, which is the preponderant etiologic agent of invasive aspergillosis, to be specifically detected in under 2 days. Reducing the time to results is key to permit intervention to prevent Aspergillus exposure in immunocompromised patients. In this way, QPCR was previously used to amplify Aspergillus fumigatus DNA on various substrates, such as tap water,(9) carpets,(7) air filters,(10) and impacted lowmelt agar plates.(11) A new device, the Coriolis μ air sampler (Bertin Technologies, Montigny, France), based on a cyclonic system, was recently proposed for collecting large volumes of air on liquid medium quickly. This device was previously assessed for different areas other than fungal aero-contamination monitoring: analysis of pollen grain distribution(12,13) and detection of Pneumocystis jiroveci DNA by QPCR.(14) A preliminary study was carried out to test this cyclonic-based liquid device, combined with QPCR, and to assess its performance for detecting A. fumigatus DNA.

Fungal food choices of Dermatophagoides farinae affect indoor fungi selection and dispersal

House dust mite (HDM) feces and molds are the main allergens involved in allergic asthma. Differences exist between the housing fungal biome of allergic patients and standard or unhealthy housing. House dust mite (HDM) feed off spores and transport them on their bodies, but do they have fungal food preferences? We observed Dermatophagoïdes farinae in vitro with 16 mold menus and repeated the experiment 10 times. This observation led us to define Alternaria alternata, Cladosporium sphaerospermum, and Wallemia sebi as “tasty” molds and Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum as “repulsive” molds. The food preferences of D. farinae may play a role in the following two phenomena: a decrease in spore numbers due to HDM consumption and a scattering of spores that stick to the bodies of HDMs. The extent of these two phenomena should be estimated in future studies for other common domestic HDM species.

Wheezing phenotypes and risk factors in early life: The ELFE cohort

Objective Different phenotypes of wheezing have been described to date but not in early life. We aim to describe wheezing phenotypes between the ages of two months and one year, and assess risk factors associated with these wheezing phenotypes in a large birth cohort. Methods We studied 18,041 infants from the ELFE (French Longitudinal Study of Children) birth cohort. Parents reported wheezing and respiratory symptoms at two and 12 months, and answered a complete questionnaire (exposure during pregnancy, parental allergy). Results Children with no symptoms (controls) accounted for 77.2%, 2.1% had had wheezing at two months but no wheezing at one year (intermittent), 2.4% had persistent wheezing, while 18.3% had incident wheezing at one year. Comparing persistent wheezing to controls showed that having one sibling (ORa = 2.19) or 2 siblings (ORa = 2.23) compared to none, nocturnal cough (OR = 5.2), respiratory distress (OR = 4.1) and excess bronchial secretions (OR = 3.47) at two months, reflux in the child at 2 months (OR = 1.55), maternal history of asthma (OR = 1.46) and maternal smoking during pregnancy (OR = 1.57) were significantly associated with persistent wheezing. These same factors, along with cutaneous rash in the child at 2 months (OR = 1.13) and paternal history of asthma (OR = 1.32) were significantly associated with increased odds of incident wheezing. Having one sibling (ORa = 1.9) compared to none, nocturnal cough at 2 months (OR = 1.76) and excess bronchial secretions at 2 months (OR = 1.65) were significantly associated with persistent compared to intermittent wheezing. Conclusion Respiratory symptoms (cough, respiratory distress, and excessive bronchial secretion) were significantly associated with a high risk of persistent wheezing at one year. Smoking exposure during pregnancy was also a risk factor for persistent and incident wheezing.

Assessment of pets (cats and dogs) in homes using electrostatic dust collectors and QPCR: new tools to evaluate exposure and risk of allergies

Abstract Contradictory results are found in the literature concerning fungi, bacteria, and pet exposure and the risk of developing asthma. All these allergens have been thoroughly studied separately in cohort studies, and a variety of sampling and analytical methods are used. It is already possible to characterize fungi, mites, and bacteria by QPCR. The aim of our study is to evaluate QPCR systems to quantify the presence of cats and dogs in homes. Twenty-four houses were sampled with an Electrostatic Dust Collector which was analyzed by QPCR. Questionnaires on the presence of pets in homes were completed. The results from QPCR were correlated for real presence of cats and dogs, and highlighted indirect exposure. This study provides a useful screening tool that will be used in future large cohort studies, such as the ELFE cohort study.

Correction: Wheezing phenotypes and risk factors in early life: The ELFE cohort

[This corrects the article DOI: 10.1371/journal.pone.0196711.].

QPCR detection of Mucorales DNA in bronchoalveolar lavage fluid to diagnose pulmonary mucormycosis

Early diagnosis and treatment are essential to improving the outcome of mucormycosis. The aim of this retrospective study was to assess the contribution of quantitative PCR detection of Mucorales DNA in bronchoalveolar lavage fluids for early diagnosis of pulmonary mucormycosis. ABSTRACT Early diagnosis and treatment are essential to improving the outcome of mucormycosis. The aim of this retrospective study was to assess the contribution of quantitative PCR detection of Mucorales DNA in bronchoalveolar lavage fluids for early diagnosis of pulmonary mucormycosis. Bronchoalveolar lavage fluid samples (n = 450) from 374 patients with pneumonia and immunosuppressive conditions were analyzed using a combination of 3 quantitative PCR assays targeting the main genera involved in mucormycosis in France (Rhizomucor, Mucor/Rhizopus, and Lichtheimia). Among these 374 patients, 24 patients had at least one bronchoalveolar lavage fluid sample with a positive PCR; 23/24 patients had radiological criteria for invasive fungal infections according to consensual criteria; 10 patients had probable or proven mucormycosis, and 13 additional patients had other invasive fungal infections (4 probable aspergillosis, 1 proven fusariosis, and 8 possible invasive fungal infections). Only 2/24 patients with a positive PCR result on a bronchoalveolar lavage fluid sample had a positive Mucorales culture. PCR was also positive on serum in 17/24 patients. In most cases, a positive PCR result was first detected using sera (15/17). However, a positive PCR on bronchoalveolar lavage fluid was the earliest and/or the only biological test revealing mucormycosis in 4 patients with a final diagnosis of probable or proven mucormycosis, 3 patients with probable aspergillosis, and one patient with a possible invasive fungal infection. Mucorales PCR performed on bronchoalveolar lavage fluid could provide additional support for earlier administration of Mucorales-directed antifungal therapy, thus improving the outcome of lung mucormycosis cases.

Development of a quantitative PCR detecting Cunninghamella bertholletiae to help in diagnosing this rare and aggressive mucormycosis

Mucormycosis is an invasive mold infection, frequently fatal in immunocompromised patients. We report the case of a patient with chronic lymphocytic leukemia admitted to the hematology unit for febrile aplasia. Pulmonary lesions suggesting a fungal infection expanded/increased despite a combination of posaconazole and liposomal amphotericin B. The fungal biomarkers performed repeatedly were negative. At D65 after chemotherapy a bronchial biopsy was positive for Cunninghamella bertholletiae. The patient died despite appropriate antifungal management. A qPCR targeting Cunninghamella was developed a posteriori, and a retrospective analysis showed that a sample was positive more than 30 days before culture-based identification could be made.