Antifungal Activity and Type of Interaction of Melissa officinalis Essential Oil with Antimycotics against Biofilms of Multidrug-Resistant Candida Isolates from Vulvovaginal Mucosa.

(1) Background: Vulvovaginal candidosis (VVC) is a major therapy issue due to its high resistance rate and virulence factors such as the ability to form biofilms. The possibility of combining commonly used antifungals with natural products might greatly improve therapeutic success. (2) Methods: A total of 49 vulvovaginal isolates, causative agents of recurrent VVC, were tested for their susceptibility to fluconazole, nystatin, and Melissa officinalis essential oil (MOEO). This examination included testing the antibiofilm potential of antifungals and MOEO and the determination of their types of interaction with mature biofilms. (3) Results: Antimicrobial testing showed that 94.4% of the Candida albicans isolates and all the Candida krusei isolates were resistant to fluconazole, while all strains showed resistance to nystatin. The same strains were susceptible to MOEO in 0.156-2.5 mg/mL concentrations. Additionally, the results revealed very limited action of fluconazole, while nystatin and MOEO reduced the amount of biofilm formed by as much as 17.7% and 4.6%, respectively. Testing of the combined effect showed strain-specific synergistic action. Furthermore, the lower concentrations exhibited antagonistic effects even in cases where synergism was detected. (4) Conclusions: This study showed that MOEO had a very good antibiofilm effect. However, combining MOEO with antimycotics demonstrated that the type of action depended on the choice of antifungal drugs as well as the applied concentration.

Etiology, Predisposing Factors, Clinical Features and Diagnostic Procedure of Otomycosis: A Literature Review.

Otomycosis (OM) is a superficial fungal infection of the external auditory canal (EAC) with a worldwide prevalence ranging from 9% to 30%. Commonly, otomycoses are caused by Aspergillus (A.) niger complex and Candida spp. Other causative agents are yeasts of the genera Cryptococcus spp., Rhodotorula spp., Geotrichum candidum, dermatophytes (Trichophyton mentagrophytes), and non-dermatophytes molds (Fusarium spp., Penicillium spp., Mucorales fungi). The widest range of different species causing OM are found in the territories of Iran, India, China, Egypt, Mexico, and Brazil. Fungal infection of the EAC varies from mild to severe forms. It can be acute, subacute, or chronic, and is often unilateral, while the bilateral form is more common in immunocompromised patients. From an epidemiological point of view, tropical and subtropical climates are the most significant risk factor for the development of otomycosis. Other predisposing conditions include clothing habits, EAC hygiene practices, long-term antibiotic therapy, diabetes, and immunodeficiency. Since it is often difficult to distinguish otomycosis from an infection of a different origin, laboratory-based evidence, including standard procedures (microscopy and cultivation), is essential for diagnosis. For the treatment of this superficial fungal infection, there are no official therapeutic guidelines and protocols. However, many antifungals for local application, such as polyene, imidazoles, and allylamines, can be applied, as well as systemic antimycotics (triazoles) in severe forms of infection.

A case of mycotic keratitis due to Fusarium sp. with an undesirable outcome.

Fungal keratitis, an infective disease of the cornea, represents a serious diagnostic and therapeutic problem that, if not recognized on time, could lead to irreversible eye damage. Herein we report a case of fungal keratitis due to Fusarium spp. infection. The 60-year-old man was admitted to our clinic due to an atraumatic acute onset of the disease, with a decrease in the visual acuity, photophobia, redness, and severe pain in the right eye. Clinical observation revealed an ulcer that affected 1/3 of the cornea and a hypopion in the anterior chamber. After the first results of microbiological analyzes, local and systemic antifungal therapy was applied. Due to the fact that the patient voluntarily left the treatment, there was a drastic worsening of the local findings as a full thickness total corneal infiltrate with more intense anterior chamber reaction. Finally, evisceration was performed. Given the fact that fungal keratitis is more prevalent in developing countries, official protocols and available effective antifungals are crucial for adequate treatment and a favorable outcome of this infection.

Molecular Survey of Dirofilaria and Leishmania Species in Dogs from Central Balkan.

Dirofilariosis and leishmaniosis are severe parasitic diseases in dogs, and their causative agents can also be pathogenic to humans. In this study, we conducted a multicentric survey in the regions of Serbia and North Macedonia with the goal to establish an epidemiological scenario of dirofilariosis and leishmaniosis in the territory of Central Balkan. Using molecular analyses, a total of 535 dogs from Northern Serbia (NS), Southern Serbia (SS) and North Macedonia (NM) were screened for the presence of Dirofilaria spp. and Leishmania spp. We confirmed that Central Balkan is an endemic region for Dirofilaria (D.) immitis, as it was found to be the dominant species in this area, with the highest prevalence of 8.75% in NM, followed by NS (6.68%) and a significantly lower prevalence in SS (1.51%). Two dogs (2.5%) from NM were positive for Leishmania (L.) infantum infection. None of the dogs from Serbia tested positive for Leishmania spp. High prevalence and dominance of D. immitis species, and the rising threat of L. infantum spread to the territory of Serbia, suggest that preventive measures are of a great necessity to combat the spread of these vector-borne zoonoses.

Clinical Presentations, Cluster Analysis and Laboratory-Based Investigation of Aspergillus Otomycosis-A Single Center Experience.

Species of Aspergillus (A.) niger complex and A. flavus complex are predominant molds that are causative agents of otomycoses. The goal of this study was to investigate the clinical presentation, diagnostic procedure, and appearance of relapse in patients with Aspergillus-otomycosis, as well as to determine the biofilm production ability of species isolated in relapse. Thirty patients with laboratory evidenced Aspergillus-otomycosis followed by two check-ups (30 and 60 days after initiation of treatment with antimycotics for local application) were included in the study. For isolation and identification of Aspergillus spp. the standard mycological procedure was applied. Results showed very high sensitivity of microscopy, but 16.7% Aspergillus species required the optimal temperature of 27-28 °C for cultivation. Applied statistical cluster analysis showed a defined specific cluster/group of patients with A. niger complex-otomycosis. Sixty days after diagnosis and treatment initiation, six patients had a relapse, with the same species of Aspergillus genus being the cause. To establish the ability of biofilm production, the modified method described by Pierce and Kvasnicková was performed, and all six species isolated in the relapse episode had the ability to produce biofilm. Official criteria and recommendations are needed due to the possibility of misdiagnosis, which leads to the prolongation and complication of the disease.

New cases of human dirofilariosis on the Balkan Peninsula - "Masked intruders" uncovered by a surgeon.

Dirofilaria spp. nematodes are accidental parasites of humans causing mild to serious, superficial or visceral infections. Superficial dirofilariosis is rather common in Europe and is typically manifested as subcutaneous form. Herein we report 46 new cases of human dirofilariosis (19 patients with subcutaneous, 18 patients with ocular, 4 patients wih genital, 2 patients with submucosal, 2 patients with pulmonary and 1 patient with intramuscular form of infection) that were recorded from the beginning of 2015 to May 2021 on the Balkan Peninsula with a goal to update the prevalence of this parasitosis and point out potential problems in diagnosis and treatment. Besides, given the high possibility of misinterpretation as tumor, our second aim was to encourage the inclusion of this pathogen in the differential diagnosis of subcutaneous nodules. Although quite common forms, subcutaneous and ocular dirofilariosis can be very often misdiagnosed in clinical practice due to the absence of specific clinical manifestations. Therefore, raising awareness of clinicians about this zoonosis is needed as well as closer collaboration between physicians and veterinarians.

Recurrent Vulvovaginal Candidosis and Cluster Analysis of Clinical Signs and Symptoms: A Laboratory-Based Investigation.

Recurrent vulvovaginal candidosis (RVVC) represents a major health problem that significantly affects a patient's quality of life (QoL). This infection presents with a plethora of clinical manifestation, and this is the first study that carries out a cluster analysis of these signs and symptoms (SS). The goals are to evaluate the distribution of species causing RVVC, their in-vitro susceptibility to antifungals, and the patient's QoL. Additionally, the clinical characteristics are analyzed using cluster analysis. Prospective analysis of data was performed for women diagnosed with RVVC in the period from January 2016 to December 2019 based on the analysis of data from a single-center's records. The standard mycological methods and antifungal susceptibility testing were done. Clinical characteristics and QoL were examined by appropriate questions. The cluster analysis was used to identify clusters of SS. A total of 320 women were diagnosed. The dominant species was Candida (C.) albicans. Non-albicans Candida (NAC) yeast was found in 24.4%, and the most common was C. glabrata. Interestingly, Saccharomyces (S.) cerevisiae was detected in 2%. All of the isolated species, except C. parapsilosis and C. kefyr, demonstrated reduced susceptibility to antifungals. We confirmed the emergence of the NAC species and S. cerevisiae with reduced susceptibility to antifungals. Cluster analysis represented by a dendrogram revealed three SS clusters: irritation, uncommon, and discharge, but further studies are needed to examine the relationship between clusters, Candida strains, and outcomes.

Human dirofilariosis of buccal mucosa - First molecularly confirmed case and literature review.

When presented in unusual manner, human dirofilariosis is hard to recognize and often leads to misdiagnosis and unjustified use of various medications. Herein, we report a rare case of Dirofilaria infection localized in buccal mucosa. A 45-year-old male, residing in the Central Serbia, developed striking edema of buccal mucosa followed by leucocytosis, with both neutrophylia and eosinophylia. Thirty days after first symptoms occurred, nodule was formed along the lateral edge of right maxilla. Therefore, patient underwent surgical extirpation and subsequent histopathological analysis of the specimen revealed the presence of a nematode. Based on morphological characteristics of detected parasite, the diagnosis of submucosal Dirofilaria repens-like infection was made. The diagnosis was confirmed, being the first case in the world analyzed by molecular methods. So far, only 13 such cases have been reported worldwide and our paper brings attention to rare form of this zoonosis and updates the data about human dirofilariosis in buccal mucosa.

Species distribution and epidemiological characteristics of superficial fungal infections in Southeastern Serbia.

BACKGROUND: Superficial fungal infections (SFI), one of the most prevalent diseases in the world, are infections of keratin-rich structures of human body mostly caused by dermatophytes and yeasts. OBJECTIVES: The goal of this study was to determine the possible changes in the epidemiology of SFI on the territory of Southeastern Serbia and to investigate epidemiological characteristics and the influence of SFI on the patient's quality of life. METHODS: From 2012 to the end of 2017, samples of 1643 patients (568 males and 1075 females, mean age 40.32 ± 22.44 years) with suspected SFI from Southeastern Serbia were examined using the standard mycological methods. The questionnaires were used to investigate epidemiological characteristics. RESULTS: Superficial fungal infections were diagnosed in 20.5% (n = 336) of patients. In the group of dermatophytes, the most prevalent was Microsporum canis (63.9%, n = 76) followed by Trichophyton mentagrophytes (21.8%, n = 26). Non-albicans Candida species were dominant aetiological agents of superficial candidosis (62.3%). BMI ≥25 kg/m2 (P = 0.019) was determined as an independent risk factor for SFI. There was a statistically significant difference in the EQVAS score between the groups of patients and the control group (P < 0.001). CONCLUSIONS: Results of conducted study indicate that SFI prevalence has not changed in the previous period. However, increase of Candida-SFI prevalence, especially Candida onychomycosis, was established.