Lactophenol Cotton Blue Staining Research Articles

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Occurrence and antimicrobial resistance patterns of bacterial and fungal pathogens from patients with urinary tract infections in Ojo, Lagos Nigeria

Urinary tract infections (UTIs) is a common bacterial and fungal infection affecting millions of people globally. The aim of this study was to investigate the distribution and antimicrobial susceptibility patterns of bacteria and fungi from patients with UTI. Urine samples were collected aseptically from patients with symptoms of UTI in sterile universal containers from five hospitals in a period of three months. A total of 200 urine samples from 75 males and 125 females were analyzed and subjected to culture, biochemical identification and antimicrobial susceptibility testing using the Kirby-Bauer technique. Fungal isolates were identified based on cultural characteristics, lactophenol cotton blue stain, chlamydospore formation, and colony colour on CHROM agar Candida medium. Most samples were positive for one microorganism each and a few had two isolates, thus giving a frequency of 210 microbial isolates. The most commonly isolated bacteria were Escherichia coli, 71(34%), followed by Staphylococcus aureus, 63(30%), Enterobacter spp., 32(15.2%), Klebsiella spp., 17(8.1%), Proteus mirabillis, 15(7.1%) and Candida albicans 12(5.7%), being the only fungus isolated. Antimicrobial susceptibility testing showed that Escherichia coli showed 57.7% susceptibility to streptomycin. Klebsiella spp. were 70.5% susceptible to septrin, but with susceptibility as low as 47.1% to chloramphenicol. Enterobacter spp. showed 71.9% susceptibility to septrin, 68.8% to gentamycin, 59.4% to streptomycin and chloramphenicol, 53.1%. Proteus spp showed 53.5%, and 66.7% to pefloxacin, septrin and streptomycin respectively. Staphylococcus aureus showed susceptibility of 56(88.9%), 55(87.3%), and 47 (74.6%) to gentamicin, ciprofloxacin. and pefloxacin respectively. Candida albicans was highly susceptible to nystatin, fluconazole and miconazole by 7(58.3%) but was highly resistant to ketoconazole, 41.7%. The findings of this study provide valuable information on the distribution of bacterial and fungal agents that are common etiology of UTIs, and likely antimicrobial drugs for the treatment of UTIs.

Characterization and Isolation of Fungi from Domestic Pigeon Droppings in the Governorate of Erbil and its Suburban Area

A total of 200 samples of domestic bird droppings were collected from 54 regions in Erbil Governorate and its suburbs, and 23 of the isolates were fungi obtained by using the Niger bird seed medium and Sabouraud dextrose agar medium. The fungal genera were identified through macroscopic, microscopic, and phenotypic properties on agar plates and slides by staining with Lactophenol cotton blue stain. Findings belong to nine genera (Aspergillus, Alternaria, Chrysosporium, Cunninghamella, Helminthosporium, Mucor, Penicillium, Rhizomucor, and Rhizopus). Among the isolates, six species belonging to Aspergillus were identified which also had the highest frequency (44.4 %) in total genera, while the lowest frequency was (0.1 %) for Alternaria sp., Chrysosporium sp., Cunninghamella sp. Results reveal that 17 sites' samples of bird droppings produced negative results when tested on Niger bird seed agar, while only two locations produced no results when tested on Sabouraud dextrose agar. Thus, Sabouraud dextrose agar (SDA) is thought to be better than Niger bird seed agar (NBSA) for isolating fungi since it is considered a generally rich media for them.

Microbial Analysis and Antibiogram of Bacteria and Fungi Isolated from Meat Sellers Tables in Dutsin-Ma Metropolis, Katsina State

The goal of the research project was to carry out microbiological analysis and antibiogram of bacteria and fungi associated with meat vendor tables in the city of Dutsin-Ma. This study made use of twenty samples in total. Five distinct locations at Wednesday Market and Darawa were chosen, and two samples from each table were taken using swab sticks. For analysis, the samples were then brought to the Federal University of Dutsin-ma microbiology laboratory department. The agars that were initially employed to isolate the bacteria and fungus were MacConkey, nutrient, mannitol salt, bile esculin, salmonella-shigella, eosin methylene blue, and potato dextrose agars. Bacterial species were identified using gram staining and biochemical tests while lactophenol cotton blue staining was used for the identification of fungal species. Bacterial isolates identified include Escherichia coli, Salmonella species, Klebsiella pneumonia, Bacillus subtilis, Streptococcus species and Staphylococcus aureus while the fungal isolates are Aspergillus flavus, A. niger, A. fumigatus, and Oidiodendron griseum. The antimicrobial susceptibility test findings showed that while some of the bacterial isolates were responsive to certain drugs, others were resistant. The results of this study's investigations therefore demonstrated how heavily polluted meat vendors' tables are with a wide variety of bacteria. This can be explained by the unsanitary procedures used in the meat sale industry as well as other environmental issues. Therefore, it is imperative that the government authorities tasked with these duties make sure that the vendors of these meat products adhere to standard and hygienic procedures. Raising public knowledge of the risks of foodborne illnesses associated with eating raw meats is another important goal of public health awareness campaigns.

Cutaneous Mucormycosis Due to Rhizopus oryzae in Immunocompromised Pediatric Patient

Abstract Introduction Mucormycosis is an infection predominantly in immunocompromised patients by fungi of the Mucorales order, the most frequent being Rhizopus spp., Mucor spp. and Lichteimia spp. Characterized by vascular invasion, necrosis in the lung, CNS, paranasal sinuses, gastrointestinal system, and skin. Case Presentation 2-year-old female patient, native and resident of Valle de Chalco, State of Mexico. Recent diagnosis of B lineage acute lymphoblastic leukemia, in remission induction phase. Medical/surgical history: December 10 2021: Bone marrow aspirate -January 10 2022: Remission induction chemotherapy with vincristine and daunorubicin -January 17 2022: Central venous catheter placement -Family history: Parents are reported healthy. Positive drug addiction for smoking. They have two doses of vaccine for SARS COV2 -Epidemiologic history: Protective isolation was indicated in this patient -Allergies, rash, transfusion and traumatic: Denied -Adequate nutrition, adequate hygiene, complete vaccination schedule. Neurodevelopment according to age -Hospitalized on January 17 for fever and neutropenia, treatment with fourth-generation cephalosporin as initial management. -On January 20, 2022, she presented an erythematousviolaceous papular lesion, raised in the center, without central necrosis, without elevation, and painful on palpation on the right forearm -Treatment with fourth generation cephalosporin and glycopeptide was started, a biopsy culture of the lesion was taken and Central and peripheral blood cultures -On physical examination, an erythematous-violaceous maculopapular lesion was observed on the right forearm, without necrosis, without exudate and painful on palpation. Vital Signs in normal ranges. Relevant labs Leukopenia was found with profound neutropenia and thrombocytopenia. Positive acute phase reactants, with positive procalcitonin and C-reactive protein. Final Diagnosis For the study of the tissue, debridement of the wound, samples of the spindle center and external edges for biopsy and Lactophenol cotton blue staining were performed. Final diagnosis is Cutaneous Mucormycosis. The culture reports Rhizopus oryzae. For treatment, start Liposomal Amphotericin B 5 milligrams per kilogram per dose every 24 hours. It was increased to 10 milligrams per kilogram per dose for progression of the lesion. Teaching Points Mucormycosis is produced by opportunistic fungi of the order Mucorales. It presents an acute and fatal course that requires early diagnosis and treatment to avoid complications and death (1) A timely diagnosis is very important, since with surgical and antifungal treatment the survival rate increases to 70% (2) Isavuconazole showed activity against mucormycosis with efficacy similar to Amphotericin B. Isavuconazole can be used for the treatment of mucormycosis and is well tolerated (3,4).

Aspergillus fumigatus identification and antibiotic sensitivity in Iraqi patients

Pulmonary aspergillosis is a severe infection in immunocompromised caused by Aspergillus fungi, with Aspergillus fumigatus being the most frequently cultured of this genus, although A. flavus, A. niger, A. terrus also include If left untreated, invasive aspergillosis can have mortality approaching 100%. A total of 105 samples were collected from TB patients with symptom of lung infection . These sputum samples were obtained from patients treated in Public health department Chest and Respiratory diseases clinic, from February 2023 to April 2023. All samples were transferred to the laboratory for culturing to isolation and identification fungi after labeling each sample with questionnaire form. Lacto phenol cotton blue staining method was used in identification of fungal isolates, fungi isolated by inoculating sample on culture media such as sabouraud dextrose agar (SDA), and disc diffusion. The sample mean ± standard deviasion (SD) of age was 48.5±14.2 years (ranged 9-75 years). Male to female ratio was 2:1.5, age groups number and percentage were 32(30.5%), 13(12.4%), 14(13.3%), 14(13.3%), 16(15.2%) and 16(15.3%) in the age groups <=29, 30-39, 40-49, 50-59, 60-69 and >=70. Laboratory investigations indicated that 40 (38.1%) of the total samples diagnosed with a fungus species, 16 (15.3%) of them present with Candida species followed by 14 (13.3%) with A.fumigatus, 5 (4.8%) with A. niger, 3 (2.8%) with A.terreus and 2 (1.9%) A.flavus, while negative results were indicated in 65 (61.9%) of collected samples. A.fumigatus. isolates were subjected to susceptibility testing using different antifungals. The antifungal drugs NYS, KEA, CMZ, ICZ and PCZ were the most effect. Resistant outcomes were reported in FCZ, while intermediate results were showed in MIC and AMB. The results showed an increase in the prevalence of Aspergillus fumigatus in Iraqi patients. This prevalence may be alarming on public health.

Isolation and Characterization of Proteolytic Enzyme Produced from Fungi

Protease is an enzyme that conducts proteolysis and receives high attention as the most important enzyme in many industries. The enzymes find applications in waste treatment, bioremediation processes, detergents, and the leather industry. However, the search for microbial sources of novel proteases in nature is a promising area of interest for researchers. In the present research work, protease-producing microorganisms were isolated from soil samples collected from two different locations in UNIZIK, from the surface and at depths of 15 cm. The isolated organisms were screened for their protease-generating abilities by using the skim milk agar experiment. The isolates that gave high positive results in screening were identified as Aspergillus sp. and Penicillium sp. by analysing the colonies morphology and using the lacto-phenol cotton blue (LPCB) staining technique. Of these isolates, Aspergillus sp. that was isolated from the soil surface demonstrated the highest protease activity, as indicated by the clear zone around the colony that was incubated at room temperature for 96 hours. The results showed that among the isolates, two fungi had great potential to be used for the production of protease enzymes and upscaled for industrial production.

Sources and Prevalence of Aflatoxin B1 in Different Rice Paddies of Punjab and Sindh, Pakistan

Rice is a major cash crop used all over world. The hygroscopic nature of rice kernel with warm and fumic conditions is favorable to enhance the growth and development of toxigenic fungi which produce mycotoxins. This study estimates the prevalence of aflatoxin B1 in different rice paddies collected from twelve regions of the Punjab and Sindh provinces of Pakistan. All the samples were analyzed for their phenotypic characteristics (appearance, odor, and grain length). Competitive Enzyme Linked Immuno Sorbent Assay (ELISA) was used to evaluate AFB1, both before and after applying the steam decontamination procedure to detoxify aflatoxin contamination. Cultural assay was used to find the source of aflatoxin. PDA and SDA agar were used to detect aflatoxin producing fungal species. Lactophenol Cotton Blue (LCB) staining was used for microscopic identification of fungal contaminants. Paddies from most regions appeared yellowish in color and odorless with the exception of Hafizabad, Jhang, Sheikhupura, and Gujarat (in Punjab), where they appeared blackish yellow in color and with a pungent smell. According to the results, 43 (61.42%) samples were detected with aflatoxin B1 out of 70 samples, with an average of 15.86±1.7 ug/kg, before treating with seam. After treatment with steam, 7 (10%) samples were detected with aflatoxin B1, with an average of 2.55±1.51 ug/kg. Most regions showed the presence of P. chrysogenum with the exception of R.Y. Khan, which showed the presence of A. niger. Steam, on average, reduced aflatoxin to 51.42%. The current study indicates that steam is an effective treatment to eradicate aflatoxin at industrial level. New approaches may be explored to target the contaminants in order to ensure food safety.

Attention-Guided Transfer Learning for Identification of Filamentous Fungi Encountered in the Clinical Laboratory.

This study addresses the challenge of accurately identifying filamentous fungi in medical laboratories using transfer learning with convolutional neural networks (CNNs). The study uses microscopic images from touch-tape slides with lactophenol cotton blue staining, the most common method in clinical settings, to classify fungal genera and identify Aspergillus species. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently. IMPORTANCE This study utilizes transfer learning with CNNs to classify fungal genera and identify Aspergillus species using microscopic images from touch-tape preparation and lactophenol cotton blue staining. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently.

Isolation, Identification, Chemical and Biological Synthesis of silver nanoparticles andits Antimicrobial Activity

Abstract: Extracellular biosynthesis of silver nanoparticles (AgNPs) by Aspergillus Fumigatus isolated from Soil was reported in the present study. The biosynthesis of AgNPs was monitored by ultraviolet-visible spectroscopy and its antimicrobial activity, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC). The isolated fungus was identified by observing morphological characteristics and Lactophenol cotton blue staining and confirmed as Aspergillus fumigatus from the report given by National Fungal Culture Collection of India (NFCCI). Based on the study, it is concluded that the AgNP’s which is produced by biological methods is showing the antimicrobial activity

Microbiota from Intestinal Gut of Dawkinsia filamentosa (Valenciennes,1844) as Indicator of Environmental Condition

Aims: To isolate and identify the gut associated micro-organisms present in the gastrointestinal tract of freshwater fish Dawkinsia filamentosa. 
 Place and Duration of Study: Sampling site: The fish samples were collected from the river Gadana, located at the foot hills of Western Ghats, Alwarkurichi, Tenkasi district, Tamil Nadu, India. Place: Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Azhwarkurichi, Tenkasi, Tamilnadu, India (Latitude 8o20’N; Longitutde 77o10’E). Duration: The research was done during December 2021 and April 2022.
 Methodology: The fish samples were anaesthetized and surface sterilized followed by aseptic dissectionby using sterile scalpel and needle. The gut contents were removed and homogenized well and isolation of Gut micro biome was done by means of serial dilution. The identification of gastrointestinal bacteria was done by performing various biochemical tests such as Gram staining, Motility, Indole test, Methyl red test, Voges Proskauer test, Citrate utilization test, Triple sugar Iron test, Catalase test, Oxidase test, Urease test, Nitrate test, Starch hydrolysis and Carbohydrate Fermentation Test. The fungi present in gastrointestinal tract is identified with lacto phenol cotton blue staining followed by microscopic observations.
 Results: The overall bacterial load of gastrointestinal material ranged from 47 x10-7 to 219 x10-5 cfu/g. A total of six bacterial strains and four fungi strains were isolated. Several biochemical tests were used to identify the bacterial isolates. The six bacterial isolates were tentatively identified as Pseudomonas sp., Aeromonas sp., Staphylococcus sp., Bacillus sp., Enterobacter sp., and Vibrio sp. and the four fungi species isolated from the gutwere Aspergillus flavus, Aspergillus niger, Beauveria basiana, and Penicillium sp.
 Conclusion: The current research revealed that, Dawkinsia filamentosa has a variety of microbiota in its gut. Microbial species from freshwater environments may be able to augment fish feed with these bacteria in commercial aquaculture operations. Additionally, these findings have aided in the development of possible remedies, enhanced knowledge of host-microbe interactions in other vertebrates, and enhanced aquaculture practices. The benefits of identifying gut microbiota in fishes is, it helps to study about the host nutrient catabolism and immune defence mechanism against the disease-causing pathogens.

Antibacterial activity of Rhizopus specie isolated from rhizosphere of Mentha Piperita

Soil is loaded by microbes which are capable of producing antibiotics. These compounds were used therapeutically and sometimes prophylactically in the control of infectious disease. Soil microbes can be grouped into Bacteria, Actinomycetes, Fungi, Algae, Protozoa and Nematodes. Fungi are unique eukaryotes which ingest food from outside and take nutrients through its cell walls and consists of tiny tubular mycelia known as hyphae. The fungi are well known in medical field due to the production of a wide variety of secondary metabolites. The current research study was focused on isolation, identification and antibacterial activity of fungi isolated from rhizosphere of Mentha Piperita (Mint), Abasyn University, Peshawar. The research study was conducted in the Microbiology Research Laboratory (MRL) of Abasyn University Peshawar. The soil sample was collected from rhizosphere by using sterile techniques. Serial dilution method was used to dilute the concentration of microbes present in soil sample. The Potato Dextrose Agar media was prepared and by pour plate method the sample was streaked on the plate. The plates were incubated at 280C for 72hours. The fungus was isolated by sub culturing technique and then identified by morphological and microscopic examination using Lactophenol cotton blue stain (LPCB).

MYCOLOGICAL PROFILE OF DERMATOPHYTES

Aims: Dermatophytoses are commonest type of fungal infections seen in human beings, invading keratinised tissue skin, hair and nails by keratinophilic fungi. Dermatophytoses can be treated effectively by antifungals. Settings and Design: Retrospective study Methods and Material: A total of 200 patients which were referred from the department of dermatology to the department of microbiology were collected from July 2020 to June 2021. After cleaning with povidone iodine and 70% alcohol, the skin was scrapped, hair was plucked and nails were clipped. Skin, Hair and Nail were treated with 10%, 20% and 40% KOH respectively to identify septate hyphae and arthrospores. The specimen was cultured on Sabouraud's dextrose agar (SDA) with and without cycloheximide. Microscopic description of the growth was done by Lactophenol cotton blue (LPCB) staining. Antifungal susceptibility testing was done by E strip method using Rosewell Park memorial institute (RPMI) media for Ketoconazole, Itraconazole, Fluconazole, Voriconazole, Posaconazole, Griseofulvin and Terbinane. Results: Out of 200 samples, the commonest age group affected was between (21-30 yrs) 39.5%. Female to male ratio was 2.5:1.6. Tinea corporis was 63% and tinea cruris 27%. The commonest species isolated was Trichophyton rubrum 47.5%. Antifungal susceptibility test – Most susceptible to Itraconazole 81.4% and resistant to Griseofulvin 67.9%. Conclusions: Dermatophytosis is prevalent in high temperature zones. Most common isolates were T. rubrum and Trichophyton mentagrophytes in tinea cruris and tinea corporis respectively, they were most susceptible to Itraconazole and resistant to Griseofulvin. Resistance pattern of antifungal drugs helps in deciding empirical therapy for patient's better outcome.

Screening for antibacterial producing fungi species isolated from plant rhizosphere in Keffi, Nasarawa, State, Nigeria

Antibiotics were considered to be organic compounds produced by microorganisms which were toxic to other microorganism. This study is aimed at isolation and screening for antimicrobial producing fungi isolated from soil in Keffi. Standard microbiological method was used in isolation and screening for antimicrobial of activity of the crude extracts. Fungi were identified using lactophenol cotton blue stain, and view under x10 and x100 objective microscope lens. highest fungi were isolated from groundnut rhizosphere (58.3 %) and the least was from beans rhizosphere (25.0 %). The screening for antimicrobial producing fungi, isolate fgm8 shows activity against the test organism with 11.02+0.12mm inhibited zone against E. coli (5.33+10mm) against S. aureus and (9.03+0.03 mm) against C. albica. The antimicrobial activity of the fungi grow in complex medium with nitrogen sources showed different ranges of activity against the test organism were Rhizopus sp. only showed inhibition zone of 12.11+1.34 mm against C. albicans. These fungi species should be used to develop starter culture that will be used in local food production, as they show high antimicrobial activity against some pathogenic bacteria that cause infection and food spoilage.

Identification of Fungus Causing Onychomycosis in Chicken Meat Traders in Traditional Markets

Onychomycosis is an infection of the nails caused by dermatophyte, non-dermatophyte, and yeast. Infection is characterized by discoloration of the nails and thickening of the nail plate. The proliferation of fungi can occur in jobs that are often in direct contact with dirty, damp and wet work sites such as chicken meat traders. Because chicken traders make direct contact with chicken meat which causes the hands and nails of traders to become wet and moist. The purpose of this study was to identify the pathogenic fungi that cause onychomycosis on the fingernails of chicken meat traders at traditional market in Batubulan Village. Identification was carried out by the culture on Saboraoud Dextrose Agar (SDA) media and followed by microscopic examination of the growing fungal colonies with Lactophenol Cotton Blue staining. The results of this examination were that all samples were grown on Saboraud Dextrose Agar (SDA) media. The dermatophyte group was identified as many as 17 and the Yeast group was identified as many as 14. Most of the fungi identified on the fingernails of chicken butchers were non-dermatophytes, 22 of which were Aspergillus sp.

Hyalohyphomycete or zygomycete? Challenges with microscopic diagnosis

Background: Invasive mould infections are associated with profound morbidity. Direct microscopic examination of specimens is a useful tool for laboratories to advise clinicians on the presumptive mould identity. Nonetheless, pitfalls abound. Here, we highlight how microscopy may not always be straightforward. Case description: A 69-year-old male with T cell lymphoma was admitted for chemotherapy. Shortly after conditioning, he developed hand cellulitis which progressed to a 10 cm necrotic ulcer despite vancomycin and piperacillin-tazobactam. Skin biopsy was performed. Direct Gram stain and calcofluor white stain revealed narrow hyphae with suggestion of septa and 45 degree branching. Given the clinical syndrome (eschar in an immunocompromised host), we communicated our suspicion of Fusarium to the clinician, who started voriconazole. Overnight, a cottony mould grew. Lactophenol cotton blue stain revealed irregular, broad, ribbon-like hyphae with sporangia. The clinician was informed of the possibility of Mucorales. Antifungals were switched to liposomal amphotericin B. Internal transcribed spacer sequencing subsequently confirmed the mould as Rhizopus arrhizus. Antifungal susceptibility testing revealed MICs of 1 μg/ml and >8 μg/ml to amphotericin B and voriconazole respectively. Discussion: Direct microscopy findings should always be correlated with colonial morphology. Clinicians should be promptly informed of critical findings so appropriate treatment can be instituted.

Cutaneous Sporotrichosis of Face with Verrucous Lesions over Nose, Successfully Treated with a Combination of Itraconazole and Terbinafine: A Case Report.

Dear Editor, A 13-year-old boy presented with nasal erythematous, crusted, confluent papules, and nodules persisting for approximately 35 days [Figure 1]. The patient denied contact with sick cats but mentioned playing in the bushes. The clinical diagnosis was cutaneous sporotrichosis, and he started treatment with itraconazole 100 mg per day, which was administered orally for 10 days and then increased to 200 mg per day. The exudate from the skin lesions was directly examined by a potassium hydroxide test, which was negative for fungal presence. At 25°C, a mycosal media culture of the exudate revealed colonies resembling Sporothrix sp, which appeared membranous, beige, wrinkled, and with a blackened halo [Figure 2]. At 37°C, phenotypic reversal for creamy colonies with beige and colorless verse was observed, thus demonstrating dimorphism. The lactophenol cotton blue stain revealed hyaline septate hyphae [Figure 3]. On day 44 of itraconazole treatment alone, the lesions became severely crusted, demonstrating no improvements. Thus, 250 mg of oral terbinafine was added to the treatment regimen. On day 60 of itraconazole use, a skin biopsy was performed. Grocott silver stain revealed rare samples of fungal structures [Figure 4]. Histopathology showed acanthosis of the epidermis, areas with perivascular mononuclear inflammatory infiltrate and fibrosis [Figure 5]. On day 226 of itraconazole and day 182 of terbinafine use, clinical improvement was noted, indicating the end of oral antifungal drugs [Figure 6]. The patient underwent Laser Erbium YAG, an ablative laser, to achieve favorable aesthetic results for the scar and uniformization of the skin of the nasal region.Figure 1: Infiltrated erythematous nodules of sporotrichosis on the nose with verrucous appearanceFigure 2: Culture at 25°C, showing colonies of membranous, beige, wrinkled with a blackened halo, resembling Sporothrix speciesFigure 3: The hyaline septate hyphae of Sporothrix spp. with one pyriform microconidia in daisy arrangement (lactophenol cotton blue stain, ×40)Figure 4: Grocott silver stain presenting a fungal structures (40x)Figure 5: Acanthosis of the epidermis, areas with perivascular mononuclear inflammatory infiltrate, and intense remodeling of dermal collagen with fibrosis. (H and E, 10x)Figure 6: Clinical improvement after terbinafine and itraconazole use. Only residual erythema, papules and nodulesSporotrichosis is a subcutaneous mycosis caused by a thermodimorphic fungus, Sporothrix spp., most commonly S. brasiliensis, S. schenckii, and S. globosa.[1] The lesions manifest in different clinical forms: mainly lymphocutaneous, fixed, cutaneous, disseminated, and extracutaneous. Recently, the incidence of various diseases caused by Sporothrix has been increasing in Brazil, particularly a high disease rate in urban areas. Felines have been reported as a new source of infection that spreads diseases.[2] Itraconazole is a first-line treatment for Sporothrix, but some strains, mainly S. brasiliensis, are resistant.[3–5] Moreover, itraconazole content sometimes does not match the concentrations indicated by manufacturers.[3] Itraconazole has certain pharmacokinetic concerns because of several drug interactions. For example, a high gastric pH reduces its bioavailability. Terbinafine is an alternative drug that can effectively treat cutaneous sporotrichosis, and it is affordable and has fewer interactions with other drugs.[1,4,5] To improve therapeutic outcome, itraconazole has been combined with a second drug and demonstrated good results, for example, with supersaturated potassium iodide (SSKI) solution.[6] SSKI is an alternative for cutaneous forms, which is primarily used for infants. However, compared with terbinafine, SSKI has side effects and issues, such as a lack of standardized commercial formulation and unknown mechanisms of action.[1,4] Studies describing the use of itraconazole with terbinafine are limited. In vitro, terbinafine and itraconazole have synergistic effects because both disrupt ergosterol synthesis in the cell membrane of fungi at different stages. This mechanism is beneficial due to accelerated response against chromoblastomycosis, onychomycosis, and sporotrichosis, where facial lesions can cause aesthetic stigmas and affect the quality of life of patients. In conclusion, the combination of terbinafine and itraconazole can effectively inhibit disease progression faster and thus prevent excessive fibrosis and scars.[5,7–10] Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Phylogenetic analysis of pathogenic Candida spp. in domestic pigeons.

The current study was conducted to survey the prevalence of pigeon candidiasis in diseased pigeons suspected to candidiasis by isolation, microscopic examination, and polymerase chain reaction (PCR) method and to characterize Candida spp. phylogenetically. For this purpose, samples were obtained from 100 suspected pigeons from September 2018 to February 2019 in Ahvaz, Iran. Cloacal and oropharyngeal swab samples were collected from each diseased pigeon with diarrhea resistant to the antibiotics, crop stasis, white diphtheritic membrane in the mouth, regurgitation, and vomiting. Sabouraud dextrose agar was used as a culture medium. Selected colonies were stained with lactophenol cotton blue stain. In the culture and direct microscopic observation, 19.00% of birds were suspected to candidiasis. Twenty-two isolates were identified. All 22 isolates were confirmed as Candida spp. By PCR method. The PCR test confirmed the presence of Candida spp. in 19.00% of pigeons. Based on the sequencing results of some PCR products, the isolates belonged to Candida albicans and Candida glabrata. The results revealed a 99.78% accordance when compared with other sequences of C. albicans which were formerly deposited in GenBank® from Colombia, Indonesia, China, and Sudan. The results revealed a 99.54% accordance when compared with other sequences of C. glabrata which were formerly deposited in GenBank® from the Netherlands and Spain. The symptoms such as diarrhea resistant to antibiotics, crop stasis, white diphtheritic membrane in the mouth, regurgitation, and vomiting were the most prevalent clinical symptoms in positive pigeons.

Isolation and Screening of Aspergillus niger and Bacillus coagulans as Potential Candidates for Amylase and Glucose Isomerase Production

There is an increasing demand for high fructose corn syrup as an alternative to glucose especially for use by people with diabetes due to its low glycemic index. The high cost of this product coupled with its high demand has attracted the attention of many researchers to search for an alternative and sustainable production route. Therefore, the focus of this work was isolating and screening Aspergillus niger and Bacillus coagulans as possible producers of amylase and glucose isomerase respectively, which are needed to produce high fructose corn syrup. Nine (9) samples of loamy soil were taken from three (3) separate locations (BG = Botanical Garden, RD = Refuse Dumpsite, FB = Flower Bed) to isolate the bacterium and fungus. Based on the lacto-phenol cotton blue stain preparation under a microscope, colonies that were thought to be Aspergillus niger colonies were observed and further identified. Cultural, microscopic, and biochemical characteristics were used to confirm the identification of colonies that were thought to be Bacillus coagulans. The probable isolates of Aspergillus niger and Bacillus coagulans were then screened for amylase and glucose isomerase production respectively. Out of the total of nine (9) soil samples analysed, 5 (55.6 %) were positive for Aspergillus niger. A higher isolation rate of Aspergillus niger, 2 (66.7 %) was recorded in soil samples from botanical garden (BG) and flower bed (FB). The lowest isolation rate, 1 (33.3 %) was observed in soil obtained from the refuse dump. Out of all the isolates screened for their potential to produce amylase, the isolate from the botanical garden showed the highest zone of starch hydrolysis (28 mm), and the isolate from the flower bed showed the least zone of hydrolysis of starch (13 mm). On the other hand, out of the nine (9) soil samples analysed, 6 (66.7 %) were positive for Bacillus coagulans and the highest occurrence of Bacillus coagulans, 3 (100 %) was recorded with soil from botanical garden (BG1), while the least occurrence, 1 (33.3 %) was observed in soil from flower bed (FB2). The probable isolates of Bacillus coagulans screened for glucose isomerase production revealed that all the six (6) isolates produced glucose isomerase, with isolate from refuse dump (RD2) producing the highest concentration of glucose isomerase (4.7014 g/L).

Cutaneous Infection by Fusarium solani in an Immunocompetent Patient

Fusarium species are widely distributed throughout the environment, and rarely cause infection in healthy individuals. Herein, we report a rare case of cutaneous infection by Fusarium solani in a healthy patient. A 64-year-old patient presented with a tender, erythematous, deep-seated nodule above the left ankle after an intralesional triamcinolone injection for lipoma was administered at a private clinic 1 month ago. Fungal hyphae were identified in subcutaneous tissue by skin biopsy using Gomori methenamine silver stain. Lactophenol cotton blue staining, fungal culturing, and 28S rRNA sequencing confirmed the presence of F. solani. After diagnosis, the patient was successfully treated with oral itraconazole.