Lactophenol Cotton Blue Mount Research Articles

Onychomycosis: Prevalence, Fungal Pathogens, Risk Factors, and Antifungal Susceptibility Profile among People Living with Diabetes Mellitus in Kano, Northwestern Nigeria

Abstract Context: Onychomycosis is a fungal infection of the nail unit that over time results in dystrophy and disfigurement. It affects the quality of life and increases the prevalence and severity of foot ulcers in patients with diabetes. Aim: The study investigated the prevalence of onychomycosis, the associated fungal pathogens, and their antifungal susceptibility profile in a cohort of people living with diabetes (PLWDM) in Kano, Nigeria. Settings and Designs: This cross-sectional hospital-based study recruited and enrolled 300 PLWDM from two major diabetes treatment centers in Kano. Materials and Methods: Participants were screened for thickened, brittle, crumbly, or ragged nails, and 30 met the inclusion criteria. Sociodemographic data, clinical history, and other risk factors for onychomycosis were also evaluated. Toenail and fingernail, nail clippings, and scrapings were collected and cultured on Sabouraud dextrose agar (SDA). The identification of yeasts and mold isolates was performed using gram stain, germ tube test, CHROMagar, lactophenol cotton blue (LPCB) mount, and slide cultures. Antifungal susceptibility testing was achieved by the agar well-diffusion method. Statistical Analysis Used: The data generated were analyzed using descriptive statistics and associations between variables checked by the chi-square test or Fisher’s exact test. Results: The prevalence of onychomycosis was 63.3% (19/30), and fingernails were mostly affected. Fungal pathogens recovered include dermatophytes, yeasts, and non-dermatophyte molds, with a predominance of Aspergillus spps. (7/19 (36.8%)), Trichophytonmentagrophytes (4/19 (21.1%)), Trichophytonrubrum (4/19 (21.1%)), and Candidaalbicans (2/19 (10.5%)). A preponderance of the isolates shows a good susceptibility profile to itraconazole, ketoconazole, and clotrimazole, but demonstrated high resistance to fluconazole. Age and use of oral hypoglycemic agents were some of the risk factors identified. Conclusion: The study shows a high prevalence of onychomycosis among PLWDM in Kano and calls for routine screening to minimize or eliminate all possible complications.

Mucormycosis Associated with Global Coronavirus Disease-19 Pandemic: A Study of Fungal Profile from a Tertiary Health-care Center

Background and Aim: In this era of the coronavirus disease (COVID) pandemic, new health issues appear quickly and cause an outbreak in India. Here, we focus on invasive fungal infections (IFA), namely mucormycosis, aspergillosis, and candidiasis. Hence, in this article, we focus on conventional techniques for early diagnosis of fungus to help clinicians start empirical treatment for the patient and take necessary steps thereby. Materials and Methods: This observational study was conducted for 1 month, from May 2021 to June 2021, on 200 patients admitted to our mucormycosis ward. Samples were first examined microscopically using potassium hydroxide (KOH) wet mounts followed by culture on sabouraud dextrose agar and the gross appearance of the growth was observed. Further identification of species was achieved through the use of lactophenol cotton blue mounts and slide culture technique. Results: Out of 200 patients, 65.5% were male and 34.5% female. We divided these patients into three age groups, i.e., 20–30 years (15%), 40–60 years (54%), and 60–80 years (31%). Seventy-four percent of patients presented with a history of diabetes mellitus, 50% had a history of steroids taken in their past, and 48% had a history of oxygen therapy taken at the time of COVID-19 positivity (52%) samples positive for mucormycosis. Forty-one percent were KOH-positive for Mucorales and 35.5% were culture positive. The most commonly isolated fungus was Rhizopus, followed by Mucor, Lichthemia, Aspergillus spp., and Candida spp. Conclusion: To improve test sensitivity and enable early diagnosis, other serum biomarkers such as galactomannan, beta-D-glucan, cryptococcal antigenemia, and blood quantitative polymerase chain reaction for Aspergillus or Mucorales should be implemented.

Prevalence of Dermatophytic Infections and Their Antifungal Susceptibility Pattern in the Rajshahi Region

Background: Dermatophytes are the most significant fungi because of their widespread involvement in the population and their prevalence worldwide. Bangladesh's hot and humid climate, overcrowded population, poverty, malnutrition, and ignorance make dermatophytosis a common cutaneous infection. These infections occur in both healthy and immunocompromised patients. Dermatophytes are responsible for most cutaneous fungal infections, and the estimated lifetime risk of acquiring a dermatophyte infection is 10-20%.
 Objective: To isolate and identify different dermatophytes and their antifungal susceptibility pattern in the Rajshahi region. Materials and Methods: Different clinical samples (e.g., skin scrapings, nail clipping, and hair plucking) were collected under aseptic precautions. The isolation and identification of dermatophytes were performed through a microscopic examination using 10% KOH mount, mycological culture, and species identification by lactophenol cotton blue mount from positive culture. In addition, all dermatophytes isolates were subjected to antifungal susceptibility testing using the agar-based disk diffusion method in Mueller Hinton agar media.
 Results: Out of 171 samples, Trichophyton rubrum was the predominant dermatophyte species with 76(71.7%), followed by T.mentagrophyte were 15(14.2%), E. floccosum were 12(11.3%), and M. canis were 03(2.8%). voriconazole, clotrimazole, and itraconazole were more effective drugs. Griseofulvin was the least effective drug, followed by fluconazole.
 Conclusion: This study indicates dermatophytosis is a common skin disease in northern Bangladesh. Due to the increasing trend of antifungal drug resistance among dermatophytes, treatment should be based on antifungal sensitivity testing.
 TAJ 2022; 36: No-1: 119-125

Isolation, identification and antifungal susceptibility of dermatophytes from clinical specimens in a tertiary care hospital in South Kerala

Dermatophtyosis is common, more prevalent in tropical and subtropical countries including India. Though not life threatening it can cause great discomfort, especially in immunocompromised conditions. In the last few years a number of new less toxic antifungal drugs have become available for clinical use. Lack of clinical response may occur in 20%.125 clinically suspected cases of dermatophytosis were studied.The samples were subjected to direct microscopy and culture. Identification of the causative pathogen was done by performing slide culture, lacto phenol cotton blue mount, hair perforation tests and urease tests. In this study we evaluated the efficacy of seven antifungal medications using CLSI broth microdilution method (M38-A). The antifungals used were sertaconazole, terbinafine, griseofulvin, fluconazole, itraconazole, voriconazole and amphotericin B.Out of 125 samples, dermatophytosis manifested clinically more in the age group of 21-30 yrs. In our study, KOH positivity rate was 60.8% and culture positivity rate was 32%. Dermatophytosis was more common in females. Tinea corporis was the most common lesion (54.4%) followed by Tinea cruris (25.6%), Tinea pedis (8%), Tinea unguium(7.2%) and Tinea faciei (0.8%). was the commonest aetiological agent (50%) followed by (35%), (7.5%), (5%) and (2.5%). The range of their minimum inhibitory concentration endpoint by broth microdilution technique for the seven antifungal drugs were as follows, sertoconazole <0.06-4 μg/ml , griseofulvin 0.25-1 μg/ml, terbinafine <0.06-2 μg/ml, voriconazole 0.06-0.5 μg/ml, itraconazole <0.06-1 μg/ml, fluconazole 2-8 μg/ml and amphotericin B 2-8 μg/ml. This study gives an insight about the aetiological agents of dermatophytosis in this part of South Kerala, India. Both direct microscopy and culture are important tools for diagnosis of the superficial fungal infections. Majority of strains in the study were inhibited by relatively low concentration of the antifungals tested.

Clinicomycological Profile of Cutaneous Dermatophytosis: A Cross-sectional Study from Western India

Introduction: The prevalence of cutaneous mycoses has been on the rise worldwide with an increased incidence of recurrent and recalcitrant infections. This study was conducted to assess the clinical patterns and etiological agents of dermatophytosis, at a tertiary care hospital in western India. Material and Method: One hundred clinically diagnosed cases of dermatophytosis attending the OPD of a tertiary care hospital were included in this cross-sectional study. Thorough history taking and detailed clinical examination were done for each patient. For studying the mycological profile, direct microscopy for fungal elements in KOH and fungal culture on Sabouraud dextrose agar (SDA) was done for each patient. The growth on SDA plates was further mounted on lactophenol cotton blue mounts for species identification. Results: Our study found a male preponderance, and the majority of infections (36%) were in the 21–30 years age group. The most common clinical pattern was the mixed type of infections followed by T. corporis. Direct microscopy for fungal elements was positive in 71% but the culture positivity was only 35%. The most common species isolated in our study was T. mentagrophytes (62.8%). Conclusion: The present study highlights the bearing of factors like socioeconomic status and occupation on the clinical profile of dermatophytosis and reveals a changing trend in the etiological agent of dermatophyte species in this part of western India.

Microbiological Genus Characterisation, Clinical Features, and Outcome in Patients of COVID-19 Associated Mucormycosis: A Single-centre Cross-sectional Study

Introduction: Mucormycosis is a known invasive fungal infection, its prevalence increased during the Coronavirus Disease-2019 (COVID-19) pandemic, particularly during the second wave. The most common clinical form is rhino-orbitalcerebral, but other forms such as pulmonary, gastrointestinal, cutaneous, and disseminated forms also exist. There have been very few Indian studies exploring the various aspects of COVID19 Associated Mucormycosis (CAM). Aim: To describe and characterise the genus of the causative agents of mucormycosis, an invasive fungal infection, and to analyse the clinical features and outcomes in the study participants. Materials and Methods: This was a cross-sectional study conducted in a dedicated COVID-19 Care Hospital at Dr. Vasantrao Pawar Medical College in Nashik City, Maharashtra, India. The study duration was one year, from February 2021 to January 2022. The universal sampling strategy included all clinically diagnosed cases of mucormycosis (n=104). The study variables included demographic factors, presence of risk factors, organ involvement, and in-hospital mortality. Diagnosis was based on fungal Potassium Hydroxide (KOH) mount and fungal culture. Genus characterisation was performed using Lactophenol Cotton Blue (LPCB) mount. Data were analysed using Statistical Package for the Social Sciences (SPSS) version 16.0, and the Chi-square test was applied to study the association between qualitative variables. Results: The majority of patients with the disease were male (77, 74%). The most commonly involved site was isolated paranasal sinuses (48, 46.1%), followed by paranasal sinus with maxilla involvement (24, 23.1%). The KOH report was positive in 100 (96.2%) of patients. Fungal growth in culture was reported in 69 (66.3%) patients, among them Rhizopus spp. (66, 95.6%) being the most common fungal genus isolated. Among 25 patients in the age category >60 years, 21 (84%) had a positive culture (p-value=0.041, significant). Diabetic patients had a significantly higher incidence of mucormycosis compared to non diabetics based on culture results (p-value=0.004). The mortality rate in this study was 26 (25%). Conclusion: Mucormycosis was more prevalent in males and predominantly affected the paranasal sinuses. Risk factors such as age category, sex, and a history of diabetes were significantly associated with the presence of mucormycosis. KOH can be used as a quick diagnostic test to initiate prompt treatment.

P417 Evaluation of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)-Bruker Biotyper Sirius for identification of invasive molds

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesSusceptibility to various antifungal drugs varies between different species and subspecies within the same genus. Phenotypic identification of fungi has limitations for species-level identification. Correct identification of species and subspecies in invasive mold infections is important to initiate the appropriate antifungal therapy. Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) with its proteomic analysis overcomes this limitation and helps in administering the correct anti-fungal therapy. A total of seven mold isolates from invasive fungal infections were evaluated for identification by MALDI-TOF MS and conventional morphological methods.MethodsTotal of seven isolates from invasive mold infections were identified by the conventional method of culturing specimens on Sabouraud's dextrose agar and Potato Dextrose agar with incubation at room temperature and 37°C in Biological oxygen demand (BOD) incubator. Micro-morphological identification of the fungus was done by Lacto Phenol Cotton Blue (LPCB) mount. Same isolates were processed on MALDI-TOF MS Bruker Biotyper Sirius (Bruker Daltonics, Bremen Germany) following recommended extraction protocol using ethanol absolute, acetonitrile, and 70% formic acid.ResultsAs per the below Figure.ConclusionIn four out of seven isolates phenotypic identification upto species level based on LPCB micromorphology was confirmed on MALDI-TOF MS. In the remaining three isolates we could only give a genus level identification based on LPCB mount. These three isolates were further identified upto the level of species after processing on MALDI as Aspergillus tamarii, Phaeoacremonium cinerum, and Fusarium equiseti. All mold isolates were identified with good quality mass spectra. In our experience, mold identification by MALDI-TOF MS using the Bruker Biotyper Sirius platform definitely has an edge over conventional phenotypic methods in species-level differentiation of various molds, impacting targeted antifungal management.

P206 Phenotypic and molecular characterization of emerging Basidiomycete Schizophyllum commune isolated from clinical samples from India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjective Schizophyllum commune is an environmental basidiomycete capable of causing human infections. Its identification is difficult as often it produces sterile, cottony white colonies without any conidia formation. S. commune is characterized by clamp connections and hyphal spicules. Though it produces basidiocarps with basidiospores it is difficult to induce in vitro. Molecular techniques are essential to confirm its identification. Here, we present the description of Schizophyllum commune isolates collected at our center over the last 5 years.MethodsAll the isolates used in this study was received from various part of India and accessioned at the National culture collection of pathogenic fungi (NCCPF), Chandigarh India. Isolates were grown on Sabouraud dextrose agar and malt-extract agar (MEA) at 25°C for 5-7 days. Lacto phenol cotton blue mounts were prepared and a microscopic examination was done. For observing basidiocarps, MEA plates were incubated for 6-8 weeks. DNA extraction was done using the phenol-chloroform method and ITS region was amplified using pan-fungal primers followed by Sanger sequencing.ResultsAmong total of 22 Schizophyllum commune cultures, 19 were isolated from lower respiratory samples, two from corneal scrapings, and one from the brain. Upon microscopic examination, the major identification feature of this fungi, i.e., clamp connection and spicules were observed only in 8 (36%) isolates. The remaining 14 (64%) were identified up to species level using ITS sequencing. Basidiocarps could be induced only in two isolates.ConclusionAlthough rarely involved in human disease, Schizophyllum commune is being isolated from the clinical specimens. As microscopic identification is difficult and needs expertise, molecular identification is required for early diagnosis and treatment.

P022 Luliconazole—a novel potent imidazole activity against Aspergillus niger and Aspergillus flavus causing otomycosis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Background and ObjectivesSuppurative otitis media (SOM) is characterized by the inflammation of the middle ear and mastoid, tympanic membrane perforation as well as discharge. The tympanic membrane perforation may result in increased exposure of the middle ear to pathogens. Aspergillus niger and Aspergillus flavus, are the most common causative agents of otomycosis with worldwide distribution, when it spreads from the external auditory canal to adjacent anatomical structures, it is classified as Aspergillus invasive otitis externa. Aspergillus otomycosis treatment is initiated by thorough cleaning of the ear canal, accomplished with suction, and drying with cotton swabs. In developing countries, SOM is a major cause of preventable hearing loss, its incidence ranges from 7% to 46% and is common amongst children of lower socioeconomic status. Treatment of SOM is directed at debridement and drying the ear with topical antifungal agents. Extensive surgical debridement and systemic antifungal therapy are needed in cases of refractory otomycosis or Aspergillus invasive otitis externa. Despite this management, treatment failure may result from suboptimal therapeutic management caused by antifungal agent toxicity. Luliconazole is currently confirmed for the topical therapy of dermatophytosis. Moreover, it is found that luliconazole has in vitro activity against some molds and yeast species. The aim of the present study was to evaluate the efficacy of luliconazole in comparison to routinely used antifungals on clinical isolates of A. niger and A. flavus.MethodsThe study was carried out in the Department of Microbiology, SRIHER, Chennai. A total of 55 (29 A. niger and 26 A. flavus) strains of Aspergillus isolates obtained from clinical otomycosis cases were confirmed based on macroscopic and microscopic identification by Lacto Phenol Cotton Blue mount and slide culture technique. Antifungal susceptibility patterns of all the Aspergillus isolates to itraconazole, voriconazole, posaconazole, and luliconazole were determined by broth microdilution method as per Clinical Laboratory Standards Institute (CLSI) M38-A2 guidelines.ResultsThe lowest minimum inhibitory concentration (MIC) geometric mean (GM) (0.00309 μg/ml) was attributed to luliconazole followed by posaconazole (0.18409 μg/ml), voriconazole (1.02727 μg/ml) and itraconazole (11.0091 μg/ml). Also, among the azoles tested, luliconazole had the lowest MIC50 and MIC90 values of 0.00098 μg/ml and 0.00781 μg/ml respectively. Among the triazoles tested posaconazole had a lower MIC50 and MIC90 values of 0.125 μg/ml and 0.25 μg/ml. Being the drug of choice for invasive aspergillosis voriconazole had a slightly higher MIC50 and MIC90 value of 1 μg/ml and 2 μg/ml. Luliconazole was found to be more effective even for pan azole-resistant isolates (n = 3) with lower MIC values.ConclusionThe results of this study showed that luliconazole had an excellent in vitro activity against all Aspergillus isolates with a lower MIC GM, MIC50, and MIC90 values than the triazoles tested. Hence, this novel imidazole antifungal agent can be regarded as appropriate Candidate for the treatment of otomycosis caused by A. niger and A. flavus strains. Also, luliconazole showed better efficacy with lower MIC values for pan azole resistant isolates, suggesting that it could be a potential antifungal for treating aspergillosis caused by pan azole-resistant isolates.

P275 Dematiaceous fungi as a rare cause of fungal sinusitis in a tertiary care center

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectivesTo discuss the occurrence and diagnosis of dematiaceous fungi as a causative organisms of fungal sinusitis in patients at a tertiary care center in North India. Since there is limited data on its prevalence, this study was aimed to know the non-Aspergillus causes of fungal sinusitis focusing on the dematiaceous fungi.MethodsA total of 451 nasal biopsy samples, from the department of pulmonary medicine ward and ICU were received over a period of 3 years, from January 2019 to December 2021. The samples were subjected to conventional mycological diagnostic techniques including direct epifluorescence and light microscopy, culture on solid media and visual identification of growth in culture using lactophenol cotton blue mounts.ResultsOut of 451 samples, no fungi were isolated from 299 samples (66.29%), Aspergillus spp from 63 samples (13.96%), dematiaceous fungi from 10 samples (2.21%), and other fungi from 79 samples (17.5%). Among the dematiaceous fungi isolated, 7 isolates were identified as Alternaria spp. (70%) and 3 isolates were identified as Curvularia species (30%) and described.ConclusionMost reported cases of allergic and invasive sinusitis were attributed to Aspergillus spp. However, in the current study, dematiaceous ‘black’ fungi like Alternaria and Curvularia, were also identified as causes of fungal sinusitis in both immunocompromised and immunocompetent individuals, showing an increasing pathogenic spectrum. Hence a high index of clinical suspicion and appropriate laboratory diagnosis assists in initiating appropriate treatment such as surgical debridement, reducing immunosuppression, and antifungal treatment with newer azoles.

P053 Invasive Mucormycosis (Zygomycosis) -Pre-Covid Era and Covid Era: a retrospective study at a tertiary care centre, chennai

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo compare the prevalence and clinical presentation of Mucormycosis in pre-COVID era (January 2017-December 2019) and COVID era (January 2020-till date).To compare the AFST pattern of the zygomycetes causing Mucormycosis in pre-COVID era and COVID era.MethodsThis is a retrospective, hospital-based descriptive study. This study included patients admitted during the pre-COVID and COVID era at a tertiary care center, Chennai. The cases were categorized into two: (1) possible mucormycosis cases which included direct microscopy [Potassium hydroxide (KOH) and histopathological examination] positives and (2) confirmed cases which included direct microscopy and culture positives. Direct microscopic examinations like KOH wet mount and histopathological examination (H and E stain and special stains) were performed. Samples were cultured on Sabourauds dextrose agar and identification was done by analyzing the microscopic morphology using lactophenol cotton blue mount. AFST was performed for culture positive isolates with amphotericin B, itraconazole, posaconazole, voriconazole and isavuconazole by microbroth dilution method according to CLSI M38-A2.ResultsDuring the Pre-COVID era, out of the 365 samples received in the laboratory, 35 were possible mucormycosis cases. Only 17 were confirmed cases, out of which 16 grew Rhizopus oryzae and 1 grew Apophysomyces elegans. During the COVID era, among 886 samples received in the laboratory, 143 were possible mucormycosis cases, and 31 were confirmed cases that grew Rhizopus oryzae (26), Rhizomucor pusillus (2), Mucor sp (2), and Basidiobolus ranarum (1). Though the risk factors were common during the pre-COVID and COVID era, additional risk factors like steroid therapy (19.2%), and COVID infection (28.7%) were seen during the COVID era. Though clinical presentations were common during both pre-COVID and COVID era, additional complications like epistaxis (0.57%), orbital cellulitis (32.7%), and loss of smell (8.04%) were seen during COVID era. The prevalence of complications was more during COVID era compared to pre-COVID era. Treatment received during the pre-COVID era was only amphotericin B, whereas during the COVID era majority of the patients received posaconazole (74.5%) followed by liposomal amphotericin B (25.5%). The antifungal susceptibility test showed the following mean minimum inhibitory concentration (MIC) values: amphotericin B (1.8 μg/ml), itraconazole (3.6 μg/ml), posaconazole (0.31 μg/ml), and voriconazole (1.61 μg/ml) during the pre-COVID era while the mean MIC values during the COVID era had the following variations: amphotericin B (0.97 μg/mL), itraconazole (13.6 μg/ml), Posaconazole (13.4 μg/ml), voriconazole (14.5 μg/ml), and isavuconazole (1.10 μg/ml).ConclusionHigh incidence of Mucormycosis during the COVID-19 era may be related to common risk factors of COVID and mucormycosis. Though most of the risk factors and clinical presentations were similar during the pre-COVID and COVID era, serious complications like loss of vision and the percentage of complications were more during COVID era which may be attributed to the increased invasiveness of Zygomycetes during COVID infection. The high mean MIC value of amphotericin B during pre-COVID era and higher mean MIC value of posaconazole during COVID era may be contributed to the higher usage of these antifungals. Usage of the antifungal agents is the main contributor toward the resistance. Newer azole like isavuconazole which had a low mean MIC in our study, can be considered as a good therapeutic option for the future management of resistant infections. Hence timely management of the patients with an appropriate antifungal agent by performing AFST will help in the reduction of resistance in the future.

P213 Lasiodiplodia theobromae: an emerging human pathogen

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Lasiodiplodia theobromae is a dematiaceous fungus, rarely reported to cause infections in humans. This case-series was conducted to identify the potential risk-factors and spectrum of diseases caused by L. theobromae.MethodsWe performed a retrospective analysis of all cases from which the isolates of the L. theobromae were accessioned at the National culture collection of pathogenic fungi (NCCPF), over the last 10 years (January 2012- March 2022). The isolates were retrieved and identified by conventional (colony morphology, color, and microscopic appearances) and molecular (sequencing of ITS1 region of ribosomal DNA) methods. In vitro antifungal susceptibility testing (AFST) was performed by microbroth dilution recommended by the Clinical and Laboratory Standards Institute (CLSI). (M38-A2). The clinical features, demographic details and outcome were recorded.ResultsIn 10 years, a total of 20 patients were identified. The median age of all cases was 39.5 (range: 13-71 years) and 75% were males. The most common clinical presentation was keratomycosis (12,68.6%), followed by soft tissue/sub-cutaneous infection (3,15%), rhinosinusitis (3,15%), onychomycosis (1, 5%), and pneumonia (1, 5%). Most patients were immunocompetent (85%). Among immunocompromised, two patients had acute myeloid leukemia and developed fungal rhinosinusitis while one patient of post-renal transplant on immunosuppressants developed subcutaneous tissue infection in the interscapular region. The fourth patient had decompensated alcoholic hepatitis, encephalopathy, sepsis, and developed pneumonia while he was on mechanical ventilation. Among the various potential risk factors associated with keratomycosis, the most common were, accidental trauma wounds (9, 45%), and mosquito bite in the eye in one patient. The culture on Sabouraud's dextrose agar (SDA) revealed the growth of black mycelial fungus (Fig. 1a). The lactophenol cotton blue (LCB) mount revealed the presence of only dematiaceous septate hyphae which failed to sporulate (Fig. 1b). All the isolates were confirmed by sequencing of ITS (internal transcribed spacer) region of the rDNA using universal primers ITS1 and ITS2. In vitro antifungal susceptibility testing (AFST) was performed using a broth microdilution (BMD) method which revealed variable MIC (μg/ml), i.e., amphotericin (1-2), voriconazole (0.25-2), itraconazole (8-16), posaconazole (1-2). All patients improved on therapy except one patient who succumbed to death due to pneumonia.Conclusion Lasiodiplodia theobromae is an emerging cause of human infections in both immunocompetent and immunosuppressed individuals. It is often difficult to identify due to lack of sporulation making morphological identification challenging. Hence, prompt suspicion and rapid diagnosis with guided therapy are necessary for a better outcome.

P265 Phaeohyphomycosis caused by Rhinocladiella similis mimicking Sporotrichosis

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjective: A case of phaeophyphomycosis caused by Rhinocladiella similis with a clinical picture simulating sporotrichosis is described.MethodsA 34-year-old male patient, employed in a textile factory presented with multiple subcutaneous nodules on the lateral aspect of the dorsum of the left hand and left forearm. There was a history of mechanical trauma 1 year back. On examination, three to four verrucous ulcerative nodules clustered on the dorsum of the left hand were observed. A preliminary diagnosis of sporotrichosis was made and a punch biopsy was performed from the lesion and subjected to KOH examination, fungal culture, and histopathological examination. ITS sequencing was done to confirm the identity of the isolate.ResultsThe skin punch biopsy from the lesion on the dorsum of the hand showed marked hyperkeratosis, papillomatosis, parakeratosis, acanthosis, and irregular elongation of rete ridges. No fungal structures and neutrophils were seen. On examination with 40% Potassium hydroxide round yeast-like cells were seen. An initial diagnosis of sporotrichosis was established. After 5 days, white to grayish colonies with aerial hyphae were observed on Sabouraud's dextrose agar which eventually turned greenish black on further incubation. Lactophenol cotton blue (LPCB) mount of the slide culture showed thin, hyaline, septate hyphae with oval conidia arranged in a bottle brush pattern around erect conidiophores and also directly arising from the hyphae. The pathogen was confirmed by ITS sequencing as R. similis. Patient was started on itraconazole 200 mg twice daily for 12 weeks. The patient is on regular follow-up and has shown gradual regression of the lesions indicating response to therapy.Conclusion Rhinocladiella similis infections have been reported chiefly from Brazil causing chromoblastomycosis. There have been no reports of infections due to this pathogen from the Indian subcontinent. This report presents the first case of R. similis as an agent of phaeohyphomycosis and calls for the need of a thorough evaluation of these cases so as to manage cases appropriately. It also underlines the need to study and redefine the ecological niche of this pathogen as well as its clinical spectrum.

P137 Remote inoculation mycosis: Rip Van Winkle wakes up

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectiveAfter immunosuppression, a remotely inoculated organism may be activated to produce clinical disease. We describe such a case with infection due to Medicopsis romeroi, a rare mold.Patient and MethodsA 54-year-old male, diabetic, hypertensive, status post-live related donor renal transplantation, done in February 2021. The patient was on standard triple immunosuppressant regimen. He developed a painless nodule on his thumb over 5 months which did not respond to multiple courses of antibiotics. The swelling was excised and sent for various tests. Review of USG after infectious disease referral, showed a small foreign body, like a wooden splinter in the wall of the lesion (Fig. 1). On inquiry, a 3 mm wooden splinter in the lesion was noted during surgery and there was an injury at the same site, 20 years ago when the patient used to work on a farm. Organisms derived from soil or thorn injury including bacterial and fungal organisms were considered in the differential diagnosis. Bacterial organisms were considered less likely as there was no response to antibiotics.ResultsHistopathology showed brownish septate hyphae with constrictions at the areas of septations (Fig. 2). The excised tissue grew a dematiaceous mold. In Lactophenol Cotton Blue (LPCB) mount branched, septate hyphae with sparse conidia were seen. MALDI- TOF MS was unable to identify the mold. Sequencing identified it as M. romeroi. There are no ECOFFs or break points (BP) available for M. romeroi. Minimum inhibitory concentration (MIC) of Voriconazole (VCZ) is reportedly low and hence was chosen for treatment with an appropriate dose adjustment of Tacrolimus.ConclusionThis case underscores that remote inoculation, when the patient was immunocompetent, could have introduced a mold, which remained latent and reactivated after immunosuppression. Sending excised tissue for appropriate tests is rewarding. Medicopsis romeroi is a rare mold with only 12 cases reported so far. It is difficult to identify except with sequencing. There is no standard guidance on treatment. Surgical excision along with prolonged treatment with one of the new azoles is beneficial.

P225 Subcutaneous phaeohyphomycosis by Phaeoacremonium species in a renal transplant recipient

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivePhaeohyphomycosis refers to infections due to a large group of heterogenous organisms called ‘dematiaceous’ or ‘melanized’ fungi. The infection exhibits a wide spectrum of presentations such as deep local infections, pulmonary infection, cerebral infection, and disseminated disease, which are associated with high mortality. The incidence of phaeohyphomycosis among solid organ transplant recipients is 0.7%. Hereby, we report a case of phaeohyphomycosis in a post renal transplant patient who presented with a subcutaneous swelling over the right elbow region.MethodA detailed history was obtained from the patient after informed consent. The cystic fluid was subjected to microbiological including bacterial (Gram stain, culture on blood agar incubated at 37°C) and fungal investigations [Calcofluor potassium hydroxide (KOH) mount, culture on Sabouraud dextrose agar (SDA) incubated at 37°C and 25°C and brain heart infusion agar (BHI) incubated at 25°C]. The identification of isolate was done phenotypically by preparing lactophenol cotton blue (LCB) mount from slide culture and molecular technique using Sanger's sequencing.ResultA 61-year-old diabetic and post renal transplant (7 years) male presented with complaints of swelling over the right elbow region for 5 months duration. Swelling was insidious in onset, gradually progressive, painless, cystic, non-pulsatile, non-reducible, ∼9 × 7 cm in size, lobular in shape with irregular surface having normal overlying skin which minimally restricting the movement of the elbow. There was no history of trauma over the elbow region. Reddish purulent pus was received for mycological investigation. Calcofluor KOH mount demonstrated septate hyphae. Culture on SDA incubated at 37°C and 25°C grew cream-colored colonies, turning grayish beige to olive-brown and developed clusters of aerial hyphae with reverse tan to brown in color after 2 weeks of incubation. LCB mount showed hyaline to brownish hyphae, cylindrical phialides growing along the hyphae with slightly tapering towards the apex, hyaline, oblong, conidia, gathering in clusters at end of phialide. Phenotypically, the fungus was identified as Phaeoacremonium species and molecular identification and antifungal susceptibility are awaited. Patient was advised for surgical excision but he left against medical advice. Currently, he is not receiving any antifungals.ConclusionPhaeohyphomycosis is a rare and unique entity among fungal infections. Cutaneous phaeohyphomycosis predominantly occurs on the extremities with a localized solitary nodule or abscess. A high index of suspicion and the surgical approach including excision or debridement are mainly chosen.

S1.5d Spectrum of etiological agents of mycotic keratitis: A n 11- year review

S1.5 Mycotic Keratitis, September 21, 2022, 11:00 AM - 12:30 PMBackgroundTo assess the spectrum of etiological agents of mycotic keratitis over an 11-year-period at a tertiary eye care center in southern India.MethodsA retrospective review was made of microbiological data relating to corneal scrapings performed over a period of 11 years (January 2011-December 2021) on 1200 individuals who presented with suspected microbial keratitis. Each individual underwent corneal scraping and the scraped materials were subjected to meticulous microbiological analysis that included direct microscopy (Gram-stain and lactophenol cotton blue wet mount) and culture on multiple solid and liquid culture media.ResultsA total of 404 fungal isolates were recovered from the corneal scrapings of 1200 patients with suspected microbial keratitis. Of the 404 fungal isolates, Fusarium spp (133) were the predominant isolates, followed by Aspergillus flavus (104), Curvularia spp (24), Aspergillus fumigatus (17), Bipolaris sp (7), Alternaria sp (2), Colletrotrichum sp (2) Cylindrocarpon lichenicola (2) Exserohilum sp (1) and Drechslera sp (1); there were also 111 filamentous fungus isolates that defied identification in spite of various efforts made to induce sporulation. Of the 404 culture-proven cases of mycotic keratitis, 381 patients confirmed ocular trauma while engaged in agricultural activity.Conclusion Fusarium spp., followed by Aspergillus spp., were the most common organisms found in mycotic keratitis patients in this specific geographical area. Additional efforts are required to spread awareness among villagers about the dangers of not promptly treating mycotic and other forms of microbial keratitis so that blindness and visual disability caused by corneal scarring in rural areas can be reduced.

P205 Uncommon Aspergillus species encountered in human infections

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objective Aspergillus species are ubiquitously found in environment and have propensity to infect both immunocompromised as well as immunocompetent individuals. Though the most common species implicated in human illness include Aspergillus fumigatus and A. flavus, many uncommon Aspergillus species are increasingly being reported. Moreover, these Aspergillus species have the potential to afflict varied organ systems and can thereby, be implicated in a wide array of infections. Here, we describe three patients infected by rare Aspergillus species viz. A. sclerotium, A. cristatus, and A. japonicum causing brain abscess, corneal ulcer and chronic dacrocystitis.MethodsUncommon Aspergillus species were isolated from clinical samples (nasal scraping, corneal scraping, and brain biopsy). Samples were inoculated on Sabouraud dextrose agar (incubated at 37°C and 25°C) and brain heart infusion agar (incubated at 25°C), followed by slide culture and lactophenol cotton blue (LCB) mount. The isolates were further subjected to molecular identification using polymerase chain reaction (PCR) targeting ITS region, followed by sequencing. Demographic details, clinical characteristics, risk factor, and management profile associated with these Aspergillus species were evaluated. We conducted systematic review using the search terms ‘A. sclerotium’ AND ‘humans’, ‘A. cristatus’ AND ‘humans’ and ‘A. japonicum’ AND ‘humans’.ResultsWe isolated three newer Aspergillus species from nasal scraping, corneal ulcer and brain biopsy. Direct microscopy using potassium hydroxide-calcofluor mount from all these clinical samples showed hyaline septate hyphae in background of pus cells. After 3-5 days of incubation, yellow-green mycelia were observed on the obverse in all the isolates. However, their microscopic features did not relate with the typical lactophenol cotton blue (LCB) picture of A. flavus, owing to which these were sub-cultured on Czapek Dox agar. After 4-5 days of incubation, LCB of these isolated again failed to divulge any specific picture and these isolates were subjected to molecular identification.Nasal scraping from 31-year-old immunocompetent male who presented with post-traumatic chronic dacrocystitis and recurrent orbital cellulitis yielded A. japonicum. He responded well with empirical antibiotics and oral voriconazole.Corneal scrapings from 56-year-old immunocompetent male who presented with post-traumatic blurring of vision yielded A. cristatus. He was treated with nystatin and voriconazole with good response.Brain biopsy from 45-year-old male with hypertension and subarachnoid hemorrhage, who presented with fever, headache, vomiting and periorbital swelling yielded A. sclerotium. He was treated with amphotericin B and had favorable outcome.The systematic literature review didn't yield any results of infection caused by these species in humans.ConclusionThis is the first report of isolation of A. sclerotium, A. japonicum, and A. cristatus from human tissue, though these fungi have been isolated from ambient air and environment. The present report highlights the emergence of uncommon Aspergillus species causing invasive infections even in immunocompetent patients and the requisite of molecular modalities to aid in identification of these rare, emerging species.

P071 Isolate profiling and antifungal susceptibility determination for terbinafine and itraconazole among dermatophytes in a tertiary care hospital of western rajasthan

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectiveTo determine the species distribution of causative agents causing dermatophytosis, and their antifungal susceptibility pattern for terbinafine and itraconazole in trichophyton spp among samples collected in patients with dermatophytosis clinical suspicion during the period of December 1, 2020 to January 31, 2022.Materials and MethodsThis is a prospective study conducted in the Department of Microbiology of a tertiary care super specialty and referral Centre of western Rajasthan from December 1, 2020 to January 31, 2022.Skin scraping, nail clipping, and hair pluckings were collected in mycology lab from clinically suspected cases of dermatophytosis presenting to the department of dermatology for conventional identification, and antifungal susceptibility testing.The specimens were subjected to direct KOH and calcofluor white microscopy and conventional fungal culture on SDA at 25 ͦ C and 37 ͦ C.The cultures positive for dermatophytes were speciated by microslide culture lactophenol cotton blue mount, hair perforation test, and urease test.The isolates identified as Trichophyton spp were taken up for antifungal susceptibility testing against terbinafine and itraconazole by microbroth dilution according to CLSI-M38 A2 guidelines. Further terbinafine resistance gene evaluation for detection of C1191A and T1189C single nucleotide polymorphism in Squalene epoxide by Amplified Refractory Mutation System-Polymerase chain Reaction (ARMS-PCR) is undergoing for trichophyton spp.ResultsOver the 14-month study period, the laboratory processed total of 174 specimens: 134 skin scraping, 36 nail clipping, and 4 hair pluckings. Of them, 106 (61.62%) specimens were microscopy positive and 111 (63.79%) were culture positive. Out of the 111 culture-positive agents isolated, 94 (84.68%) were found to be dermatophytes. On isolate profiling of 94 dermatophytes T. mentagrophyte was found to be most common 45 (48%) followed by T. rubrum 27 (29%), T. tonsurans 20 (21%), T. verrucosum 1 (1.1%), and Microsporum spp 1(1.1%). Antifungal susceptibility of 93 Trichophyton spp against terbinafine showed resistance among 58.06% isolates with 83.33% isolates among terbinafine resistant cases showing ≥4 μg/ml minimum inhibitory concentration. There was no resistance detected for itraconazole with microbroth dilution.ConclusionA total of (54.02%) skin, hair, nail infections were found to be caused by dermatophytes.On isolate profiling, T. mentagrophyte, T. rubrum, and T. tonsurans were found to be predominant species among our isolates showing altered trend of local isolates from T. tonsurans being second most common spp isolated in past.On antifungal susceptibility >55% isolates showed resistance for Terbinafine with >80% having higher MIC of ≥4 μg/ml on the contrary there was no observed resistance for itraconazole.There is a need for encouraging dermatologists for prescribing routine fungal microscopy, culture, and AFST for dermatophytes in Western Rajasthan, to reduce the indiscriminate use of antifungals.

P393 Clinical spectrum and molecular characterization of Rhytidhysteron spp. (now Gloniopsis) from India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjective Rhytidhysteron spp. are emerging dematiaceous fungi implicated in subcutaneous infections in immunocompromised as well as immunocompetent individuals. Diagnosis of Rhytidhysteron spp is delayed owing to non-sporulation and misidentification. The present study was conducted to characterize Rhytidhysteron spp. isolated from India by molecular techniques.MethodWe retrieved a total of eight isolates of Rhytidhysteron spp from our National Culture Collection for Pathogenic Fungi (NCCPF) that were received from various centers across India. Colony morphology on Sabouraud's dextrose agar (SDA) and potato dextrose agar (PDA) was recorded. Molecular identification was performed by sequencing internal transcribed spacer (ITS) and 28 s region of rDNA. Phylogenetic tree was constructed using the Neighbor-Joining method along with sequences of standard strains retrieved from the NCBI database.Results Rhytidhysteron spp was confirmed by sequencing from cutaneous specimens of eight cases. Six patients were immunocompetent while two were post-renal transplant recipients. Phenotypic features on SDA and PDA revealed growth of greyish-black mycelia on the obverse with black pigmentation on reverse after 7 days of incubation. On microscopic examination of lactophenol cotton blue mount, only dematiaceous septate hyphae without any spores were noted. On phylogenetic analysis of 28 s region, four of our isolates were closely clustered with Gloniopsis calami (Fig.1b), whereas two isolates with G. pneumonia and one each with G. percutanea, and Gloniopsis spp. (Fig.1b). On ITS phylogeny, the same four isolates closely clustered with G. calami (Fig.1a) as in 28 s region, one each isolate clustered with G. pneumonia, and G. percutanea while two isolates formed a separate clade with Rhytidhysteron rufulum (Fig.1a).ConclusionThis fungus is often difficult to identify due to lack of sporulation making morphological identification challenging. Therefore, molecular sequencing is a must for its identification. However, identification using the sequence of ITS and 28 s rDNA does not clearly correlate. Hence to confirm the identity additional genes needs to be sequenced and analyzed.

P246 Neglected keratitis caused by Exserohilum rostratum from the arid region of north-west India leading to vision loss—a case report

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesTo report a case of complete loss of vision due to delay in diagnosis of fungal keratitis caused by Exserohilum rostratum in an immunocompetent patient from the arid area of north-west India.MethodA 65-year-old female farmer was admitted to ophthalmology with a history of pain, redness, watering, and foreign body sensation in the left eye for 2 months. She had a history of trauma by splinters 2 months back. On ocular examination, a large corneal ulcer of about 7 × 8 mm size at 2-8‘o’ clock position in the left eye was present with diffuse corneal edema. She had no history of diabetes mellitus, hypertension, tuberculosis, COVID-19, and steroid eye drops instillation. There was no relevant previous history of any ocular surgery also. She was negative for hepatitis-B and human immune deficiency virus on serology. All her hematological parameters were within normal limits.Patient was treated with moxifloxacin, carboxy methyl cellulose eye drops, and Neosporin eye ointment for around 2 months at primary health care facilities and later referred to our hospital for further management.Corneal scraping of the patient was sent to our laboratory for potassium hydroxide mount and culture identification.ResultsFungus was identified as E. rostratum on the basis of gross, macroscopic, and microscopic morphology. Gram's staining was bacteriologically negative while true fungal hyphae were seen. In KOH mount pigmented, septate, and branched true hyphae were seen. Bacterial culture was reported sterile.Lactophenol cotton blue mount of culture revealed dematiaceous hyphae along with 4-9 septate elongated, ellipsoid macroconidia of 14-90 μm with prominent dark conspicuous hilum and geniculate conidiophore arranged sympodially. On the basis of these characteristics, it was diagnosed as E. rostratum.After the diagnosis patient was switched over to topical natamycin 5% two hourly and oral itraconazole 200 mg BD from moxifloxacin and neosporin. To which the patient responded symptomatically. Ulcer healed in a month leaving behind a lateral scar. However, vision is permanently compromised and the patient is advised for therapeutic penetrating keratoplasty (TPK).Conclusion Exserohilum rostratum is generally regarded as a pathogen in hot and humid climates. However, the isolation of this organism in our area highlights the pathogenic potential of this emerging fungus in arid climates also. Ophthalmologists need to be made aware of the significance of prompt mycological identification to prevent vision loss.