Clinical evaluation of a dermatophyte RT-PCR assay and its impact on the turn-around-time: A prospective study.

Onychomycosis is an important public health problem whose prevalence continues to grow and impact public health at several levels. Nevertheless, today the main diagnostic methods used in routine practice have many drawbacks. The aim of this study was to evaluate, for the first time, the clinical performance of a new multiplex polymerase chain reaction (PCR) (Novaplex®) in the identification of the causative agent on nail samples, and its impact on the turnaround time, compared to our traditional laboratory methods. From June 2022 to December 2022, all nail samples sent to our laboratory for suspected onychomycosis were included in this prospective study. We collected for each sample the results obtained with the Novaplex® PCR method and with the traditional direct microscopy examination and culture. Each discordant result was checked using a third method, which is another PCR method (DermaGenius® kit) as a resolver. For culture-positive samples, a turnaround time was calculated and compared to the one obtained with the Novaplex® method. A total of 131 samples were included. Among them, 5 were positive (3.8%) on direct microscopy, 33 were positive (25.2%) after culture, and 98 were negative (74.8%). All positive (n = 33) and negative (n = 69) cultures were also positive/negative with the Novaplex® PCR. Twenty-nine samples were positive with the Novaplex® method but negative with culture (discordant results). The percentage agreement between the culture and the Novaplex® methods was 77.9% (102 out of 131). While tested with the resolver (DermaGenius® PCR), 28 out of 29 discordant results were similarly found positive. The percentage agreement between the two PCR methods (Novaplex® and DermaGenius®) was 96.6%. The Novaplex® PCR method evaluated proved to be very reliable and allowed the direct identification of 62 out of 131 positive samples (47.3%) with the following distribution: 79.0% of Trichophyton rubrum complex, 11.3% of Trichophyton mentagrophytes complex, 6.5% of both Trichophyton rubrum complex and Trichophyton mentagrophytes complex, and 3.2% of Candida albicans. The median time [± 95% CI] for positive culture (between incubation and validation of the final identification) was 15 [12-23] days, while the turnaround time for the Novaplex® method adapted to our clinical laboratory routine is ≤7 days. Laboratory confirmation of onychomycosis is crucial and should always be obtained before starting treatment. The evaluated PCR method offered a rapid, reliable, robust, and inexpensive method of identification of the causative agent compared to traditional methods.

The aim of this study was to evaluate the clinical performance of a multiplex PCR in the identification of the causative agent of onychomycosis on nail samples, and its impact on the turnaround time, compared to our traditional laboratory methods. This new method is rapid, reliable, robust, and inexpensive.

Superficial fungal infections in the south of France-is fusariosis the next emergent onychopathy?

In France, onychomycoses represent about 30% of superficial mycoses seen by dermatologists. In recent years, an increased number of mycoses have been observed due to non-dermatophytic moulds. The purpose of this study was to evaluate the epidemiological profile of identified superficial fungal infections in the Laboratory of Parasitology-Mycology of the University Hospital of Nice over a 2-year period. A retrospective study was performed from the nail, skin, and scalp samples of patients analyzed from January 2018 to December 2019. In this study, 3074 samples (54.2% nails, 39.7% skin, and 6.1% scalp) were analyzed representing 1922 patients. Among them, 809 (42.1%) patients were sampled by dermatologists and 1113 (57.9%) were sampled by our experts in the clinical unit of the University Hospital of Nice. In total, 1159 (37.7%) samples had a positive culture (1195 strains identified) including 712 (59.6%) dermatophytes, 345 (28.9%) yeasts, and 138 (11.5%) other filamentous moulds. Trichophyton rubrum was the main dermatophyte (563; 47.1%) followed by T. interdigitale (84; 7.0%), and T. soudanense (25; 2.1%). Yeasts were mostly represented by Candida albicans (155; 13.0%). Among the other moulds, Fusarium sp. was the most isolated (61; 5.1%). Dermatophytes stay predominant in superficial fungal infections where the anthropophilic species T. rubrum was found in almost half of the positive cultures. Interestingly, moulds represented an important part of infections in our population. This study highlights the increasing share of Fusarium sp. superficial fungal infection in our patients' population, perhaps requiring a major therapeutic adaptation in the years to come.

We assessed the epidemiological profile of superficial fungal infections in the Laboratory of Parasitology­Mycology of the Hospital of Nice, over a 2-year period. Among our samples, dermatophytes remain predominant, mainly the species Trichophyton rubrum and we had a large proportion of Fusarium.

Invasive Fatal Scopulariopsis brevicaulis Infection in Canaries.

Scopulariopsis brevicaulis, a soil saprophyte, is the most common dermatomycotic mould and causes deep fungal infection. Ten canaries died in a flock of 200 and, at necropsy, S. brevicaulis was isolated from lung and beak samples. Macroscopically, the colonies were flat, velvety or powdery, white, tan, dark brown, grey or black. Microscopically, the isolated fungus had hyaline and septate hyphae, finger-like conidiophores on which annelids produced chains of conidia. On histopathological examination, multiple irregular thin red hyphae were seen in lung tissue of the canaries. Although S. brevicaulis may be involved in onychomycosis, pulmonary mycosis or invasive infection in humans, this infection has not been reported in canaries. This study shows that S. brevicaulis can cause invasive and fatal infection in canaries.

Reactive oxygen and nitrogen species are crucial for the antifungal activity of amorolfine and ciclopirox olamine against the dermatophyte Trichophyton interdigitale.

Onychomycosis is a nail infection caused by Trichophyton interdigitale and other fungi, which can be treated with topical amorolfine (AMR) and ciclopirox olamine (CPX). Although these drugs are widely used, little is known about the role of reactive oxygen (ROS) and nitrogen (RNS) in their mechanism of action. To better understand the effects of AMR and CPX in dermatophytes, we evaluated whether they act through the production of ROS and peroxynitrite (PRN). We tested a set of strains, all susceptible to AMR and CPX, and these antifungals significantly reduced T. interdigitale viability within 24 h. This effect occurred concomitantly with reduced ergosterol, increased production of ROS and PRN, and consequently increased lipid peroxidation. Together, these mechanisms lead to cell damage and fungal death. These fungicidal effects were abolished when PRN and superoxide scavengers were used in the assays, demonstrating the role of these species in the mechanism of action. We also studied the antioxidant system when T. interdigitale was exposed to AMR and CPX. Interestingly, superoxide dismutase and catalase inhibition lead to altered ROS and PRN production, lipid peroxidation, and ergosterol levels. In fact, the combination of AMR or CPX with a superoxide dismutase inhibitor was antagonistic. Together, these data demonstrate the importance of ROS and PRN in the antifungal action of AMR and CPX against the evaluated T. interdigitale strains. LAY SUMMARY: Onychomycosis is a nail infection, which can be treated with amorolfine and ciclopirox olamine. Here we demonstrate that these drugs exhibit antifungal activity also through the production of oxidative and nitrosative radicals.

Ability of hydroxypropyl chitosan nail lacquer to protect against dermatophyte nail infection.

The development of a topical agent that would strengthen the nail, improve the natural barrier, and provide better drug penetration to the nail bed is needed. In this study, we examined the effects of a hydroxypropyl chitosan (HPCH)-based nail solution using a bovine hoof model. Following application of the nail solution, changes in the hardness of the hoof samples were measured using the Vickers method. Tensile and flexural strengths were tested by stretching or punching the samples, respectively. The ultrastructure was examined using scanning electron microscopy (SEM), and samples stained with periodic acid-Schiff (PAS) stain were used to determine the fungal penetration depth. The comparators included 40% urea and 70% isopropyl alcohol solutions. The HPCH nail solution increased hoof sample hardness in comparison to the untreated control sample (mean, 22.3 versus 19.4 Vickers pyramid number [HV]). Similarly, the HPCH solution increased the tensile strength (mean, 33.07 versus 28.42 MPa) and flexural strength (mean, 183.79 versus 181.20 MPa) compared to the untreated control. In contrast, the comparators had adverse effects on hardness and strength. SEM showed that the HPCH solution reduced the area of sample crumbling following abrasion compared to the untreated control (7,418 versus 17,843 pixels), and the PAS-stained images showed that the HPCH solution reduced penetration of the dermatophyte hyphae (e.g., penetration by Trichophyton mentagrophytes was <25 µm at day 9 versus 275 µm in the untreated control). Unlike chemicals normally used in cosmetic treatments, repeated application of the HPCH nail solution may help prevent the establishment of new or recurring fungal nail infection.

Comparison of the antifungal efficacy of terbinafine hydrochloride and ciclopirox olamine containing formulations against the dermatophyte Trichophyton rubrum in an infected nail plate model.

Onychomycosis is a fungal infection mostly induced by dermatophytes such as Trichophyton rubrum. Due to slow nail growth, the treatment takes 3-9 months depending on the nail size and infected area. Hence, high efficacy of the active ingredient without systemic side effects is of major interest. To test the efficacy of an antifungal formulation, an appropriate in vitro model reflecting the in vivo situation as close as possible is required. In this study, a variety of antifungal formulations, i.e., commercial ones (Ciclopoli and Lamisil cream), those used in compounding pharmacies (Pentravan) as well as poloxamer 407-based systems, have been evaluated in an infected nail plate model. The active pharmaceutical ingredients (APIs) were ciclopirox olamine and terbinafine hydrochloride. The poloxamer 407-based formulations consisted of poloxamer 407, double distilled water, propylene glycol, isopropyl alcohol, medium chain triglycerides and either 1% ciclopirox olamine or 1% terbinafine hydrochloride as API, respectively. Former studies have shown high permeation rates of terbinafine hydrochloride from similar poloxamer 407-based formulations with dimethyl isosorbide instead of propylene glycol. The present contribution shows superior inhibition of T. rubrum growth from poloxamer 407-based formulations in comparison to the commercial Lamisil cream. Moreover, poloxamer 407-based formulations were equally effective as the nail lacquer Ciclopoli even though the poloxamer formulations contained only 1% of the drug instead of 8% in the marketed lacquer. Poloxamer 407-based systems containing ciclopirox olamine proved to be about as effective as similar terbinafine hydrochloride systems.

Isolation of Fusarium sp. from a claw of a dog with onychomycosis.

An 8-year-old male Golden Retriever had lameness and claw abnormality in the second digit of the left forelimb. Radiography revealed osteomyelitis in the distal phalanx bone of the affected limb. Microscopic examination of the claw revealed numerous hyphae in the claw matrix. Fungal DNA fragments coding the ribosomal internal transcribed spacer region (ITS) were detected from the claw matrix as well as fungal colonies of the clinical isolates by PCR. Nucleotide sequencing revealed that the amplicons shared > 99% homology with Fusarium sp. Therapy including oral itraconazole resulted in regrowth of a new claw, in which no hyphae were detected. To the authors' knowledge, this is the first case report of canine onychomycosis in which Fusarium sp. was isolated from the affected claw.

First description of onychomycosis caused by Chrysosporium keratinophilum in captive Bennett's wallabies (Macropus rufogriseus rufogriseus).

Seven Bennett's wallabies (Macropus rufogriseus rufogriseus) presented within a period of several months with onychodystrophy, onychomadesis, and severe digital tumefaction. Histopathologic findings included a pseudocarcinomatous hyperplasia of the claw matrix surrounding a cavity filled with keratin and septate hyphae stained with periodic acid Schiff reagent. The fungal species Chrysosporium keratinophilum was identified on cultures. The wallabies were orally treated with ketoconazole (15 mg/kg s.i.d.) for 20 wk. Material and enclosures were cleaned and sprayed with 0.2% enilconazole solution once a month over a period of 4 mo. No improvement of advanced cases was observed, but no new case appeared for the next 6 mo. The positive mycological culture and the invasion of tissues on histopathologic examination suggested that the fungal species C. keratinophilum was implicated in this claw disease. This is the first report of onychomycosis caused by C. keratinophilum in animals.

The lamellar wedge.

In horses with chronic laminitis, an abnormal horn structure called the lamellar wedge develops within the lamellar region of the foot. This pathologic structure adversely affects normal foot function, and influences return to previous performance levels. Understanding the pathologic process that leads to the development of this structure is essential for correct supportive foot management of the horse with chronic laminitis. The ability to prevent or reduce the formation of the lamellar wedge may eventually lead to better outcomes in cases of laminitis.

Scanning electron microscopy and fungal culture of hoof horn from horses suffering from onychomycosis.

Horn samples were taken from the hooves of eight horses with clinical signs of equine onychomycosis in at least one hoof capsule. None of the horses had a documented mycological history. The predominant alterations of the horn capsules were sand cracks, white line disease, brittleness (especially around the nail holes), parakeratosis and bruising. The horn samples were stored in sterile tubes for transportation and transferred onto Sabouraud Dextrose Agar and dermatophyte test agar for mycological examination within 6 h. Fungal cultures were incubated for 30 days at room temperature. Fungal identification was based on colonial morphology and microscopic examination of conidia. Horn samples were also stored at -80°C until used for scanning electron microscopy (SEM). The fungal culture revealed that the hoof horn from all eight horses was infected with keratinophilic fungi. The keratinopathogenic fungi Trichophyton spp and Scopulariopsis brevicaulis were also detected in six horses. SEM revealed severe alterations of the horn structure in horn samples infected with keratinopathogenic fungi compared to horn samples from a sound hoof. The most evident changes were deterioration of the tubular structure of the horn wall, disruption of the horny layers, superficial lysis of cornified cells and the presence of fungal elements. Samples without dermatophyte or Scopulariopsis infection, in contrast, were similar to healthy hoof horn.

A suggested pathogenic role for Trichosporon montevideense in a case of Onychomycosis in a Japanese monkey.

Onychomycosis is a fungal infection of fingernails or toenails caused by several species of fungi and yeasts. A Japanese monkey, Macaca fuscata, displayed severe onychomycosis in his 4 limbs. Diagnosis and etiological agent identification were performed by conventional and DNA-mediated methods. The accumulated findings of this case revealed Trichosporon montevideense, which has long been considered to be a nonpathogenic yeast. Here, we present the first report of an involvement of T. montevideense in an onychomycosis case.

[Canine onychomycosis produced by Microsporum gypseum. A case report]. / Onicomicosis canina producida por Microsporum gypseum. A propósito de un caso.

One case of severe canine onychomycosis is described. The aetiological agent was identified as Microsporum gypseum. The incidence of this fungus in this kind of pathology is discussed, with special attention to the successful treatment with topic enilconazole and systemic griseofulvin.

Case report. Onychomycosis due to Microsporum canis.

A case of distal subungual onychomycosis of the big toe due to Microsporum canis is reported in a 69-year-old male asthma patient who had been treated with systemic corticosteroids for the last 3 years. The nail infection was contracted from a cat who was a healthy carrier. The patient was treated successfully with intermittent itraconazole therapy.

Human onychomycosis caused by Trichophyton equinum transmitted from a racehorse.

We report fingernail onychomycosis caused by Trichophyton equinum in a farmer who breeds racehorses. In addition to the thumbnail, T. equinum had infected one of the racehorses. Oral terbinafine cured the infection in the farmer.

Onychomycosis in white line disease in horses: pathology, mycology and clinical features.

This paper describes onychomycosis in horses and reports the pathological findings, associated fungi and incidence of concurrent white line disease. In addition to these observations, relevance between post mortem and clinical findings of onychomycosis are discussed in 3 necropsied horses. Samples were collected from 100 hooves from a total of 51 Thoroughbreds suffering from white line disease. Of these, 15 hooves from 13 horses were also complicated with severe hoof wall fissure formation. Preparations from the same samples were used both for histopathology and for culture to identify the associated fungi. Onychomycosis was diagnosed when it could be confirmed histologically. Fungal identification was based on morphological characteristics in culture and the associated fungi were determined by comparison with culture morphology. As a result, 10 samples collected from horses were diagnosed as suffering from onychomycosis. Seven of these showed complicating severe inner hoof wall fissures and the soil fungus Scedosporium apiospermum or the teleomorph of this fungus, i.e., Pseudallescheria boydii, was isolated. The fungus was found to be ubiquitous in the fissure cavities, the terminal horn of the white line and the terminal horn-like laminae of the metaplastic white line-like tissue. It can be concluded that onychomycosis frequently causes white line disease and/or makes it worse. Associated with deterioration of the submural condition, the main associated fungus for onychomycosis in this series, was Genus Scedosporium and the most susceptible region was the terminal horn of the hypertrophied white line and/or the terminal horn-like laminae of the metaplastic white line-like tissue.

Pathomorphological findings in a case of onychomycosis of a racehorse.

The hooves of a racehorse which were affected with white line disease and hoof wall disorders on both forelimbs were histopathologically investigated using thin ground section and standard paraffin section techniques. On both hooves, large quantities of fungus were found to have invaded the white line tissues, especially in the terminal horn which were markedly damaged. The fungus was also present among the cellular debris in the fissures of horny tissues. The morphological characteristics of the fungus were brown (its natural color), PAS-positive, mold-like shape with septa inside the tissues, and unicellular spores outside the tissues. These findings suggest that onychomycosis was a primary and/or secondary cause of white line disease in this subject.

[The abnormal white line and hollow wall in horses, a comparative mycological study]. / De afwijkende 'witte lijn' en holle wand bij het paard, een vergelijkend mycologisch onderzoek.

There are few descriptions of an abnormal white line in the horse, and it is not known what causes this disorder. Thirty horses and ponies with this disorder were examined mycologically, as were 30 horses and ponies with normal hooves. Scopulariopsis brevicaulis was detected in 60% of the animals with an abnormal white line with or without hollow wall or brittle toe.