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.

Assessment of the role of Trichomonas tenax in the etiopathogenesis of human periodontitis: A systematic review.

OBJECTIVE: This systematic review was to assess the presence of Trichomonas tenax in patients with periodontitis and to elucidate its potential role in the onset and development of this disease. METHOD: Systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and by consulting the five databases: Medline, Science Direct, Web of Science, Dentistry and Oral Science Sources and Cochrane Central Register of Controlled Trials. Following Koch's postulates revisited by Socransky as PICO framework, this collection data was only including full text of clinical trials concerning patients with periodontitis, case-reports and in vitro research published between 1960 and March 2019. RESULTS: On the 376 studies identified, only 25 fulfilled our eligible criteria. Most of these studies were in vitro research articles designed to evaluate potential virulence factors, and others were clinical trials (case-control studies, randomized controlled trial) and case-reports. The analysis of these papers has shown that i) Trichomonas tenax is more frequently detected in dental biofilm from sites with periodontitis than in healthy sites; ii) this live flagellate seems capable of producing diverse enzymes that could participate in periodontal breakdown and has the capacity to adhere to epithelial cells, its lysed form could induce the synthesis of IL-8 from macrophage cell lines; iii) the impact of non-surgical treatment of periodontitis have not been thoroughly evaluated on the presence of T. tenax. CONCLUSIONS: This systematic review has reported the presence of T. tenax more frequently in diseased than healthy sites and the capacity of this flagellate to synthesis enzymes which could participate to the degradation of periodontal tissues. Nevertheless, these data do not meet all the postulates and are not enough to provide firm conclusions about the role of T. tenax in the etiopathogenesis of periodontitis.

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification.

OBJECTIVES: Besides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis. METHODS: This evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37). RESULTS: This new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica. CONCLUSION: Given the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.

Update on Actinomucor elegans, a mucormycete infrequently detected in human specimens: how combined microbiological tools contribute efficiently to a more accurate medical care.

Actinomucor elegans is a fungus belonging to mucormycetes and is still probably underdiagnosed due to misidentification. Based on a recent first case of Actinomucor elegans sinusitis in Europe, in an immunocompromised patient under voriconazole treatment, this paper aims to summarize knowledge about A. elegans mucormycoses. Even if the diagnosis of mucormycosis was made using traditional mycology techniques, precise identification of the fungus could only be achieved using molecular tools. In this observation, the galactomannan dosage was positive until the introduction of treatment and surgical debridement. The patient experienced no relapse after one year. By reviewing the four previous A. elegans reported cases and describing the mycological characteristics of this species, we highlight the need to use a combination of tools to improve the diagnostic strategy in such rare and life-threatening clinical situations.

[From guinea pig to man: Tinea outbreak due to Trichophyton mentagrophytes var. porcellae in pet shops in Nancy (France)]. / Du cochon d'Inde à l'Homme : épidémie de teigne à Trichophyton mentagrophytes var. porcellae dans les animaleries nancéiennes.

Dermatophytes are responsible for widespread superficial fungal infections, currently representing a real public health problem. Some of the fungi involved in these mycoses are transmitted by pets, illustrating great host specificity within this fungal group. Thus, a new variety of zoophilic dermatophyte has been described in recent years by the Mycology Laboratory of the University Hospital of Nancy, within the complex T. mentagrophytes. This variant was named T. mentagrophytes var. porcellae, following the observation of a significant number of patients with dermatomycoses of exposed parts of the body and having had contact with a guinea pig. The current work follows this first description and aims to assess the frequency of T. mentagrophytes var. porcellae in guinea pigs within three pet shops in the region of Nancy (France). In total, almost two thirds of collected guinea pigs were carriers of this new dermatophyte. This study highlights the risks associated with the adaptation of dermatophytes to potential new hosts that may spread to new species. Thus, in this context, sanitary measures could be proposed to the pet shops, usually not informed of the risks facing the growing enthusiasm of the population for new pets, in order to limit contamination.

Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

Molecular diagnosis of onychomycosis.

Onychomycosis is a frequent cause of nail infections due to dermatophytes. Molds and yeast may also be responsible of these pathologies. Antifungal treatments are frequently given without a mycological diagnosis, partly because of the requisite time for obtaining the biological results. The mycological diagnosis requires a direct microscopic examination and a culture in order to accurately identify the fungal genus and species. Nevertheless, this conventional diagnosis is often time consuming due to the delay of fungal cultures and presents disadvantages that make it not sufficient enough to give a precise and confident response to the clinicians. Therefore additional tests have been developed to help distinguish onychomycosis from other nail disorders. Among them, molecular biology techniques offer modern and rapid tools to improve traditional microbiological diagnosis. In this review, we first present the conventional diagnosis methods for onychomycosis and then we describe the main molecular biology tools and the currently available commercial kits that allow a rapid detection of the pathology.

Characteristics of Pneumocystis pneumonia in Nancy from January 2007 to April 2011 and focus on an outbreak in nephrology.

BACKGROUND: Pneumocystis jirovecii is responsible for pneumonia in immunocompromised populations. Pneumocystis pneumonia has first been discovered as a common and life-threatening opportunistic infection in HIV-infected patients. OBJECTIVES: The aim of this study is to characterize the epidemiological aspects of Pneumocystis pneumonia and then to highlight an outbreak of this infection in a nephrology unit with molecular tools. PATIENTS/METHODS: A multilocus sequence typing method has been used to study the epidemiology of strains isolated during this episode. RESULTS: From January 2007 to April 2011, 39 cases of P. jirovecii pneumonia have been observed. In two thirds of cases, underlying diseases as transplantations, hematologic or solid malignancies, or immunodepressed treatment were the main risk factors and in one third of cases, there were HIV positive patients. This distribution is due to an outbreak of 13 cases in a nephrology unit, where the MLST resulted in two strains profiles regrouping each one 6 and 4 cases among the 10 available isolates. CONCLUSIONS: New categories of risk patients of Pneumocystis infection have emerged with severe clinical manifestations and mostly with a fatal outcome. The origin of the transmission is still unknown but a local transmission has been showed in our nephrology unit.

Scytalidium and scytalidiosis: what's new in 2012?

Fungi belonging to the genus Scytalidium are widespread around the world. Among them, two species are responsible for human superficial infections mimicking dermatophytosis: Neoscytalidium dimidiatum and Scytalidium hyalinum. Whereas these ascomycetous fungi are endemic in tropical or subtropical countries, both species have a different geographical distribution. Scytalidiosis represents approximately 40% of dermatomycoses in these areas. A few cases of invasive infections due to Scytalidium sp. have also been reported, assessing the ability of these fungi to behave as opportunists. Here we have reviewed the data on N. dimidiatum and S. hyalinum concerning their classification, clinical features, diagnosis and treatment. We also have presented the example of a specific consultation dedicated to nails in Martinique, in order to optimize the diagnosis and treatment of onychomycosis, many of which being due to Scytalidium sp. Even if Scytalidium cases are still rare in temperate countries, imported cases may increase in the future due to immigration and travel.

Identification of the five human Plasmodium species including P. knowlesi by real-time polymerase chain reaction.

Recently, Plasmodium knowlesi has been recognised as the fifth Plasmodium species causing malaria in humans. Hundreds of human cases infected with this originally simian Plasmodium species have been described in Asian countries and increasing numbers are reported in Europe from travellers. The growing impact of tourism and economic development in South and Southeast Asia are expected to subsequently lead to a further increase in cases both among locals and among travellers. P. knowlesi is easily misidentified in microscopy as P. malariae or P. falciparum. We developed new primers for the rapid and specific detection of this species by low-cost real-time polymerase chain reaction (PCR) and added this method to an already existing panel of primers used for the molecular identification of the other four species in one reaction. Reference laboratories should now be able to identify undisputably and rapidly P. knowlesi, as it is a potentially fatal pathogen.

Disseminated Scopulariopsis brevicaulis infection in an allogeneic stem cell recipient: case report and review of the literature.

A fatal case of disseminated Scopulariopsis brevicaulis infection in an allogeneic stem cell transplant recipient is described. The patient was initially thought to have pulmonary aspergillosis, on the basis of clinical signs and antigenaemia, but Aspergillus was not isolated by culture. Scopulariopsis brevicaulis was subsequently isolated from skin and then from sputum and stool. Further investigation revealed that the infection had spread from a primary pulmonary site to the skin. A review of the literature underscores the difficulty of diagnosing infections caused by such emerging fungal pathogens and the poor outcome of immunocompromised patients with non-Aspergillus mould infections.

Molecular diagnosis of disseminated adiaspiromycosis due to Emmonsia crescens.

Emmonsia crescens is a saprophytic fungus that is distributed worldwide, causing diseases mostly in rodents. It has also been described, though rarely, as an etiologic agent of pulmonary pathology in humans, potentially leading to death. A case of pulmonary adiaspiromycosis is reported in a 30-year-old immunocompetent man. The patient presented with a history of several weeks of weakness, cough, fever, and weight loss of 10 kg. Clinical and radiographic findings showed pulmonary lesions consistent with tuberculosis or histoplasmosis, but no pathogen was found with classical microbiological procedures. The diagnosis of adiaspiromycosis due to Emmonsia crescens was initially made using molecular biology techniques. Histological observations subsequently confirmed the presence of adiaspores in granulomas. To our knowledge, this is the first case of adiaspiromycosis diagnosed by PCR and sequencing. The patient was treated with itraconazole and was seen at 1 month with symptomatic improvement. Here we will discuss this rare fungal infection and its difficult treatment and diagnosis. As represented in this case, molecular biology is a powerful method to optimize diagnostic tests and therefore improve the care of the infected patient.

Development of a PCR assay followed by nonradioactive hybridization using oligonucleotides covalently bound to CovaLink NH microwells for detection of four Plasmodium species in blood samples from humans.

We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.

Genetic identification of the main opportunistic Mucorales by PCR-restriction fragment length polymorphism.

Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis.

Polymorphisms and intronic structures in the 18S subunit ribosomal RNA gene of the fungi Scytalidium dimidiatum and Scytalidium hyalinum. Evidence of an IC1 intron with an His-Cys endonuclease gene.

The fungi Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are mainly encountered in (sub)tropical areas as plant pathogens and agents of human dermatomycosis. Because the classification and differentiation of these two species is unclear, we studied 22 S. dimidiatum and 15 S. hyalinum isolates in order to identify potential species-specific insertions and polymorphisms in the 18S subunit ribosomal gene. The presence of an IE intron in S. dimidiatum, together with a single polymorphism (A in S. dimidiatum, G in S. hyalinum) in the coding region, allowed us to differentiate these two species in most cases. Moreover, in one S. dimidiatum isolate we found a group IC1 intron containing a putative truncated His-Cys endonuclease gene. This enzyme shows strong similarity to the intronic homing endonuclease of Physarum polycephalum. Based on these results and our previous findings, we propose an evolutionary pathway for 18S rDNA S. dimidiatum insertions, implying independent events.