Diagnosis Of Fungal Infections Research Articles

S1.2c Diagnosis of fungal infections in animals: Combining the old and the new to maximize results

S1.2c Diagnosis of fungal infections in animals: Combining the old and the new to maximize results

P420 Fungal detection by means of HCR using 2D-Covalent Organic Framework Nanosheet

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMThe timely diagnosis of fungal infections is of prime importance for prescribing appropriate anti-fungal drugs. Current methods for fungal diagnosis involve culture-based methods, antibody-based detection using lateral flow assays and RT-PCR. In the present work, we devised a non-enzymatic amplification using 2D-Covalent Organic Framework (COF) nanosheet for the detection of fungal DNA.Objectives(1) Validation of exfoliated 2D COF Nanosheet as an efficient DNA detection tool via Hybridization chain reaction (HCR) triggered fluorescent assay. (2) Sequence retrieval and probe generation of fungal sample and detection of extracted target DNA via fluorescent assay.MethodA novel COF was synthesized and characterization was done using FTIR, BET, TGA, XRD, and SEM. Probes for the detection of fungi (Candida, Aspergillus, and Mucor) were designed using NUPACK software. HCR was monitored for different time and probe concentrations and standardized reaction was used for the detection of fungal RNA.ResultsFTIR, BET, TGA, XRD, and SEM confirmed the structure and formation of COF-nanosheet. H1, H2 probes at a concentration of 1μm and in presence of Target DNA (0.001μm-1μm) showed HCR reaction at 1.5 h. Fluorescence quenching was observed when probes were mixed with both, bulk COF and COF-nanosheets but increased quenchingConclusionFungal detection can be done by means of HCR using the Covalent nanosheets.

Mixed Fungal Biofilms: From Mycobiota to Devices, a New Challenge on Clinical Practice.

Most current protocols for the diagnosis of fungal infections are based on culture-dependent methods that allow the evaluation of fungal morphology and the identification of the etiologic agent of mycosis. Most current protocols for the diagnosis of fungal infections are based on culture-dependent methods that enable the examination of the fungi for further identification of the etiological agent of the mycosis. The isolation of fungi from pure cultures is typically recommended, as when more than one species is identified, the second agent is considered a contaminant. Fungi mostly survive in highly organized communities that provoke changes in phenotypic profile, increase resistance to antifungals and environmental stresses, and facilitate evasion from the immune system. Mixed fungal biofilms (MFB) harbor more than one fungal species, wherein exchange can occur that potentialize the effects of these virulence factors. However, little is known about MFB and their role in infectious processes, particularly in terms of how each species may synergistically contribute to the pathogenesis. Here, we review fungi present in MFB that are commensals of the human body, forming the mycobiota, and how their participation in MFB affects the maintenance of homeostasis. In addition, we discuss how MFB are formed on both biotic and abiotic surfaces, thus being a significant reservoir of microorganisms that have already been associated in infectious processes of high morbidity and mortality.

DNA chip-based diagnosis of onychomycosis and tinea pedis.

Onychomycosis (OM) and tinea pedis (TP) are common fungal infections. Currently, diagnosis is based on direct microscopy and culture that have a low to moderate sensitivity and/or require up to 3-4 weeks until results are obtained. PCR techniques have emerged for the diagnosis of fungal infections, but little is known about their sensitivity and specificity in diagnosing. Here, we compared the diagnostic value of a DNA-chip technology, that detects 56 fungal pathogens, in a single-center prospective diagnostic study with microscopy and culture in suspected OM/TP. Microscopy, culture and DNA microarray assays were performed on scraping material from patients with suspected OM (n = 67) or TP (n = 73). To test whether swabs can be used as an alternative for scraping, PCR yields were compared in a further 13 patients with OM and 11 patients with TP. DNA microarrays had the highest sensitivity. Combination of DNA-chip technology with microscopy further increased the sensitivity, and results from this combined laboratory diagnosis can be obtained within 24 hours. Comparison of sampling techniques (scraping, dry or wet swab) for DNA-chip assays showed similar results in suspected OM or TP. DNA-chip technologyshows high sensitivity for OM and TP diagnosis, especially when combined with microscopy.

Amphotericin B in Pediatrics: Analysis by Age Stratification Suggests a Greater Chance of Adverse Events from 13 Months of Age Onwards.

Amphotericin B deoxycholate (AMB-D) remains an antifungal agent with great therapeutic value in pediatric patients. The currrent consensus is that its use in neonates is safer than in older children. However, childhood presents different periods of development that deserve to be evaluated more precisely. Our goal was to assess the usage profile of AMB-D in stratified pediatric age groups, adapted according to the National Institute of Child Health and Human Development classification. This retrospective cross-sectional observational study was conducted at a Brazilian tertiary children's hospitalbetween January 2014 and December 2019. Data of patients who received at least two doses of intravenous AMB-D while hospitalized were extracted from electronic health files. Information on patient demographics, underlying diseases and comorbidities, laboratory examinations, fungal infection diagnosis, and AMB-D use were gathered following specific criteria.Nonparametric tests were applied, such as the chi-square test to compare proportions and Fisher's exact test to assess the association between categorical variables or contingency tables. One hundred and twenty-seven (127) medical records were stratified as preterm neonatal (birth <37 weeks postmenstrual age), term neonatal (birth-27 days), infants (28 days-12 months), toddlers (13 months-2 years), early childhood (3-5 years), middle childhood (6-11 years), and early adolescence (12-18 years). The criteria for the indication of AMB-D followed empirical use as the main indication (n = 74; 58.26%), proven and probable fungal infection (n = 39; 30.71%), and medical suspicion (n = 14; 11.02%). Candida spp. was the main etiologic agent isolated in cultures, with the highest frequency of C. albicans (n = 18; 40%), followed by Candida parapsilosis (n = 14; 31.11%), and Candida tropicalis (n = 6; 13.33%). Very few acute infusion-related adverse effects were observed during the administration of AMB-D in pediatric patients. We found an unfavorable impact of AMB-D use in patients from 13 months of age onwards suggesting this group as a turning point for a greater chance of adverse events, and not soon after the neonatal period. Clinical or observational studies based on age stratification are essential to accurately elucidate whether potentially toxic drugs can be used safely in the pediatric population. Our search for a turning point was shown to contribute to the accuracy of the study, as it provided data on the impact of D-AMB in specific pediatric age groups.

Significance of biomarkers in stewardship program in pediatric patients infected with Aspergillus species

BackgroundThe potential use of biomarkers in the diagnosis of fungal infections is a challenge. The aim of this study was to evaluate the role of a biomarker-guided antifungal stewardship program for hospitalized pediatrics suffering from invasive aspergillosis (IA).MethodsPediatric patients with suspected probable or proven IA were enrolled in this study. Demographic data were collected from their records. Clinical samples were examined by wet mount KOH smear, culture, galactomannan Ag test, and real-time PCR. Patients’ sera were evaluated for procalcitonin (PCT) and soluble-triggering receptor expressed on myeloid cells -1 (sTREM-1) levels by ELISA Kits.ResultsA total of 73 children were entered in this study with a mean age of 5 years and the male to female ratio 39/34. The most predisposing factors were hematologic disorders (71.2%). The area under the curves (95% confidence interval) for each biomarker were 0.9 (0.85% to 97%) for lactate de hydrogenase (LDH), 0.9 (0.85% to 0.94%) for C-reactive protein, 0.8 (0.75% to 0.84%) for PCT, 0.8 (0.73% to 0.85%) for erythrocyte sedimentation rate, 0.7 (0.6% to 0.8%) for sTREM-1, and 0.5 (0.45% to 0.58%) for white blood cell count. During the study period, 27.4% patients died. The LDH and sTREM-1 levels were significant increase in died patient (p < 0.05).ConclusionsAccording to our data, evaluation of biomarkers along with radiologic and clinical signs and symptoms of pediatric patients can lead to proper antifungal therapy and decreased side effects, antifungal resistance, and cost. The combined measurements could be better than a single marker in the prediction of IA.

Alternate Special Stains for the Detection of Mycotic Organisms in Oral Cyto-Smears-A Histomorphometric Study.

In the wake of the COVID-19 pandemic, fungal infections of the maxillofacial region have become prevalent, making their accurate diagnosis vital. Histopathological staining remains a simple, cost-effective technique for differentiation and diagnosis of the causative fungal organisms. The present study aims to evaluate the staining efficacy of Periodic Acid-Schiff (PAS), Alcian Blue, Safranin-O and Gomori’s Methenamine Silver (GMS) on fungal smears. This research work also attempts to study the morphometric characteristics of Candida albicans, Aspergillus flavus, Rhizopus oryzae. Candida albicans, Aspergillus flavus and Rhizopus oryzae, 10 smears each, were stained using PAS, Alcian Blue, Safranin-O and GMS. The morphological characteristics and staining efficacy were examined, and semi-quantitative scoring was performed. Candida albicans, Aspergillus flavus and Rhizopus oryzae were stained for the first time with Safranin-O. The morphometric traits were then analyzed using an image analysis software. Safranin-O provided the most reliable staining efficacy amongst the stains and optimum morphological definition for all three organisms. Safranin-O was found to be superior to PAS and GMS, ensuring detection of even the most minute mycotic colonies. The hyphae of Aspergillus flavus to be the largest, and the spores and fruiting body of Rhizopus oryzae were found to be the largest amongst the three organisms compared. Early and accurate diagnosis of fungal infections can significantly reduce morbidity in orofacial fungal infections.

Malignant course of invasive pulmonary aspergillosis in the new coronavirus infection COVID-19

Respiratory infections caused by fungal pathogens are the main cause of death in immunocompromised patients. During the COVID-19 pandemic, the registration of respiratory fungal pathology, especially pulmonary aspergillosis, has increased significantly.&#x0D; We present a histopathologically confirmed case of fatal invasive pulmonary aspergillosis complicating COVID-19. A 65-year-old patient with long-term diabetes backgrounded with a severe course of COVID-19 (the virus was identified ICD-10 code U07.1) described. During the illness bilateral polysegmental pneumonia of fungal-bacterial (Klebsiella pneumoniae) etiology with necrosis and sequestration of the affected tissue the lower lobe of the right lung with the formation of a fungal ball and the development of a right-sided pneumothorax was developed. In other parts of both lungs, focal destruction of the interalveolar septa was determined with the formation of small cavities filled with detritus and accumulations of segmented neutrophils, with the proliferation of fungal mycelium, positively stained in the PAS reaction. Fungal hyphae and conidial heads were also found in the lumens of individual bronchi and vessels with invasion of their walls. Long-term (more than one month) course of the disease, clinical and radiological dynamics, and detection of a forming connective tissue capsule along the periphery of the necrosis zone during microscopy testifies in favor of the subacute nature of the infection.&#x0D; The presented clinical case, as well as a review of current publications and meta-analyses on invasive pulmonary aspergillosis, point to diagnostic problems and poor outcomes of invasive pulmonary aspergillosis in patients with COVID-19.&#x0D; Pulmonary aspergillosis associated with COVID-19 is a serious and potentially life ― threatening complication in patients with severe COVID-19 receiving immunosuppressive treatment. Early diagnosis of fungal infections is crucial to ensure the survival of such patients. Targeted biopsy examination with microscopy and/or seeding of a lung biopsy allows not only to establish the diagnosis of aspergillosis, but also to determine the presence of tissue invasion.&#x0D; Further research should include approaches aimed at developing an effective diagnosis of fungal tissue invasion and respiratory tract damage, determining the patients immune status in order to conduct personalized immunotherapy.

Challenges in Serologic Diagnostics of Neglected Human Systemic Mycoses: An Overview on Characterization of New Targets.

Systemic mycoses have been viewed as neglected diseases and they are responsible for deaths and disabilities around the world. Rapid, low-cost, simple, highly-specific and sensitive diagnostic tests are critical components of patient care, disease control and active surveillance. However, the diagnosis of fungal infections represents a great challenge because of the decline in the expertise needed for identifying fungi, and a reduced number of instruments and assays specific to fungal identification. Unfortunately, time of diagnosis is one of the most important risk factors for mortality rates from many of the systemic mycoses. In addition, phenotypic and biochemical identification methods are often time-consuming, which has created an increasing demand for new methods of fungal identification. In this review, we discuss the current context of the diagnosis of the main systemic mycoses and propose alternative approaches for the identification of new targets for fungal pathogens, which can help in the development of new diagnostic tests.

"Inverted Snowing-Cloud" Sign in Endogenous Candida Endophthalmitis.

Candida spp. is the most common cause of endogenous fungal endophthalmitis. The diagnosis of this rare disease is based on clinical findings supported by positive blood culture. Recently, it has been shown that optical coherence tomography (OCT) characteristic findings are beneficial in making a correct diagnosis of fungal infection in cases with endogenous endophthalmitis. The current photo-essay aims to highlight the role of OCT in diagnosis of Candida endogenous endophthalmitis where OCT imaging of one of the retinal lesions disclosed a pre-retinal hyper reflective lesion with overlying punctate vitreous opacities. We propose “inverted snowing-cloud” sign for this OCT pattern considering the resemblance of the vitreous opacities to snowflakes.

Pulmonary and Extrapulmonary Manifestations of Fungal Infections Misdiagnosed as Tuberculosis: The Need for Prompt Diagnosis and Management.

Fungal infections commonly present with myriad symptoms that mimic other clinical entities, notable amongst which is tuberculosis. Besides histoplasmosis and chronic pulmonary aspergillosis, which can mimic TB, this review has identified several other fungal infections which also do. A total of 80 individual cases misdiagnosed as TB are highlighted: aspergillosis (n = 18, 22.5%), histoplasmosis (n = 16, 20%), blastomycosis (n = 14, 17.5%), cryptococcosis (n = 11, 13.8%), talaromycosis (n = 7, 8.8%), coccidioidomycosis (n = 5, 6.3%), mucormycosis (n = 4, 5%), sporotrichosis (n = 3, 3.8%), phaeohyphomycosis (n = 1, 1.3%) and chromoblastomycosis (n = 1, 1.3%). Case series from India and Pakistan reported over 100 cases of chronic and allergic bronchopulmonary aspergillosis had received anti-TB therapy before the correct diagnosis was made. Forty-five cases (56.3%) had favorable outcomes, and 25 (33.8%) died, outcome was unclear in the remainder. Seventeen (21.3%) cases were infected with human immunodeficiency virus (HIV). Diagnostic modalities were histopathology (n = 46, 57.5%), culture (n = 42, 52.5%), serology (n = 18, 22.5%), cytology (n = 2, 2.5%), gene sequencing (n = 5, 6.3%) and microscopy (n = 10, 12.5%) including Gram stain, India ink preparation, bone marrow smear and KOH mount. We conclude that the above fungal infections should always be considered or ruled out whenever a patient presents with symptoms suggestive of tuberculosis which is unconfirmed thereby reducing prolonged hospital stay and mortalities associated with a delayed or incorrect diagnosis of fungal infections.

Differences in Levels of Human 1,3β-D-Glucan from Bronchoalveolar Lavage (BAL) Fluid in The Immunocompromised and Immunocompetent Groups Patients with Suspected Lung Cancer

Background: Invasive candidiasis occurs in immunocompromised individuals as an opportunistic infection in patients with lung cancer. Although culture and histopathology remain the standard diagnosis of fungal infections, other tests are still needed to provide faster results. Human 1,3-β-D-Glucan (BDG) uses ELISA to detect candidiasis. β D-Glucan level from BAL fluid is positive when the cut-off is p 130 pg / mL. This study aims to determine differences in the levels of Human 1,3β-D-Glucan from Bronchoalveolar Lavage (BAL) fluid between the immunocompromised and immunocompetent groups in patients with suspected lung cancer at RSU Dr. Saiful Anwar Malang.Method: A cross-sectional study was conducted on 33 lung cancer patients who had risk factors for invasive candidiasis in Dr. Saiful Anwar Hospital Malang.Result: 33 study patients had significant differences in BAL levels of Human 1,3β-D-Glucan in the immunocompromised and immunocompetent groups (p = 0.009). In the different tests, there was a slight difference in the levels of BAL Human 1,3β-D-Glucan but it was not statistically significant based on age and sex in the immunocompromised and immunocompetent groups (p = 0.632, p = 0.338, p = 0.472, p = 0.667).Conclusion: Patients suspected of lung cancer have risk factors for invasive candidiasis with higher BDG levels due to immunoparalysis. There were significant differences in the BAL Human levels of 1,3β-D-Glucan in the immunocompromised and immunocompetent groups.

Utility of Turbidimetric Beta-D-Glucan in Lung Transplant Recipients

<h3>Purpose</h3> Beta-D-Glucan (BDG) is a cell wall polysaccharide and its detection in serum can be indicative of fungal infection. Evidence for BDG testing in solid organ transplantation has been derived from data using a colorimetric assay. We introduced a turbidimetric assay for BDG (Wako) into our center and assessed its utility in the diagnosis of fungal infections in lung transplant recipients. Furthermore, BDG can be used as an antimicrobial stewardship tool if results are available promptly, as a high negative predictive value can give clinicians confidence in stopping or avoiding empiric antifungal therapy. <h3>Methods</h3> Data were collected on 115 BDG tests undertaken from February 2020 through to December 2020 on 66 post lung transplant recipients with suspected or confirmed fungal infection. Tests were ordered following multidisciplinary team discussions with transplant physicians and clinical microbiologists. <h3>Results</h3> Of 115 tests, 50 were positive of which 12 contributed to the early diagnosis of a fungal infection, 36 were used to follow response to treatment in a confirmed fungal infection and 2 were falsely positive. Infections diagnosed were invasive aspergillosis (8), invasive candidiasis (1), Pneumocystis jirovecii pneumonia (1), cryptococcal meningitis (1) and invasive Scedosporium apiospermun infection (1). Of the 65 negative tests, 43 contributed to an invasive fungal infection being ruled out and empiric antifungal therapy being discontinued or avoided without any adverse consequences. 5 negative tests did not result in de-escalation from antifungal therapy. 17 tests were repeat tests undertaken as follow-up. <h3>Conclusion</h3> In our cohort, the turbidimetric BDG had a sensitivity and specificity of 100% and 97% respectively, with a positive predictive value of 96% and negative predictive value of 100%. The high efficiency of this test is likely influenced by the diagnostic stewardship applied in ordering the test, raising the pre-test probability. Turn-around times were 1 day average with a range of 1-7 days. The high negative predictive value allowed negative BDG results to be used as an antimicrobial stewardship tool. A direct saving of €9,053 was made as a result of empiric antifungal therapy being stopped following a negative BDG result. In conclusion, the turbidimetric assay for BDG is efficacious for confirming or ruling out fungal infection in post-lung transplant patients.

DIAGNOSIS OF CANDIDA FROM PERIPHERAL BLOOD FILM: A CASE REPORT

Candidemia is the most frequent invasive fungal infection in hospitalized patients. This report describes the case of a nine month old baby on whom three colostomy were performed due to intestinal obstruction. After third colostomy, patient presented with fecal peritonitis. Peripheral blood lm examination revealed yeast form of candida. Subsequently candida species was isolated on blood culture and subculture on cornmeal agar and biochemical tests conrmed the presence of Candida albicans. A diagnosis of fungal infection based on the peripheral blood lm has a potential to lead to early diagnosis and prompt treatment of a life threatening infection.

Comparative evaluation of methods for detecting fungal bodies of the paranasal sinuses

The aim of the work was to study the diagnostic value of clinical, microbiological and histopathological methods for detecting the fungal balls of the paranasal sinuses. Material and methods. An analysis of investigation and treatment of 17 patients was carried out, in whom structureless caseous masses were found during endoscopic sinus surgery, which were possible to suspect the presence of a fungal ball. The examination of the material removed during the operation included microscopy, culture and histopathological examination. Results. Histopathological examination confirmed the clinical diagnosis of fungal infection in all 17 patients with isolated lesions of the sphenoid sinus. Primary microscopy revealed fungal mycelium in 70.5 % of cases). Mycological study found the growth of filamentous fungi in 29.4 % of histopathologically confirmed cases.

T2 Candida Panel: A Game Changer in Diagnosis of Fungal Infections

T2 Candida Panel: A Game Changer in Diagnosis of Fungal Infections

Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis.

Molecular diagnostic assays can expedite the diagnosis of fungal infections, and subsequently help in early interventions and appropriate management of patients. The aim of this study was to develop a single set of primers for a real-time quantitative polymerase chain reaction (qPCR) assay to detect and identify commonly reported, clinically relevant molds i.e., Aspergillus spp, Mucorales and Fusarium spp., up to genus level by melting curve analysis. This assay was evaluated in whole blood from patients with suspected invasive aspergillosis (IA), and in tissue biopsy, bronchoalveolar lavage (BAL) fluid and other site-specific samples from patients with suspected invasive mucormycosis (IM). The limit of detection (LoD) was determined as 10 copies/μl for all three molds. The mean coefficient of variation (CV) across all sets of intra- and inter-assay data was 0.63% (ranging from 0.42 to 1.56%), showing high reproducibility of the assay. Sensitivity and specificity of the assay were 93.3 and 97.1% respectively for diagnosis of IA, and 99.29 and 83.84% respectively for diagnosis of IM. Fusarium was not detected in any of the clinical samples included and the few laboratory confirmed cases of fusariosis did not meet the inclusion criteria of the study. Hence no ROC curve or cutoff value could be generated for the same. This newly developed qPCR assay therefore appears to be a promising tool in detection of IA and IM.

Long-Reads-Based Metagenomics in Clinical Diagnosis With a Special Focus on Fungal Infections.

Identification of the causative infectious agent is essential in the management of infectious diseases, with the ideal diagnostic method being rapid, accurate, and informative, while remaining cost-effective. Traditional diagnostic techniques rely on culturing and cell propagation to isolate and identify the causative pathogen. These techniques are limited by the ability and the time required to grow or propagate an agent in vitro and the facts that identification based on morphological traits are non-specific, insensitive, and reliant on technical expertise. The evolution of next-generation sequencing has revolutionized genomic studies to generate more data at a cheaper cost. These are divided into short- and long-read sequencing technologies, depending on the length of reads generated during sequencing runs. Long-read sequencing also called third-generation sequencing emerged commercially through the instruments released by Pacific Biosciences and Oxford Nanopore Technologies, although relying on different sequencing chemistries, with the first one being more accurate both platforms can generate ultra-long sequence reads. Long-read sequencing is capable of entirely spanning previously established genomic identification regions or potentially small whole genomes, drastically improving the accuracy of the identification of pathogens directly from clinical samples. Long-read sequencing may also provide additional important clinical information, such as antimicrobial resistance profiles and epidemiological data from a single sequencing run. While initial applications of long-read sequencing in clinical diagnosis showed that it could be a promising diagnostic technique, it also has highlighted the need for further optimization. In this review, we show the potential long-read sequencing has in clinical diagnosis of fungal infections and discuss the pros and cons of its implementation.

Isolation method of Saccharomyces cerevisiae from red blood cells based on the optically induced dielectrophoresis technique for the rapid detection of fungal infections.

Saccharomyces cerevisiae (S. cerevisiae) has been classically used to treat diarrhea and diarrhea-related diseases. However, in the past two decades, fungal infections caused by S. cerevisiae have been increasing among immunocompromised patients, and it takes too long to isolate S. cerevisiae from blood to diagnose it in time. In this paper, a new method for the isolation and selection of S. cerevisiae from red blood cells (RBC) is proposed by designing a microfluidic chip with an optically-induced dielectrophoresis (ODEP) system. It was verified by theory and experiments that the magnitude and direction of the dielectrophoresis force applied on RBCs and S. cerevisiae are different, which determine that the S. cerevisiae can be isolated from RBCs by the ODEP system. By designing the specific light images and the dynamic separation mode, the optimal operating conditions were experimentally achieved for acquiring higher purity of S. cerevisiae. The purity ranges were up to 95.9%-97.3%. This work demonstrates a promising tool for efficient and effective purification of S. cerevisiae from RBCs and provides a novel method of S. cerevisiae isolation for the timely diagnosis of fungal infections.

Evaluation of Sensitivity and Specificity of Direct Microscopical Examination of Suspected Mucor Mycosis Samples by Potassium Hydroxide (KOH) during Covid-19 Pandemic Era

Introduction: Mucormycosis is a common fungal infection in India even before the COVID-19 pandemic. The incidence of Mucor mycosis has entered public consciousness in response to an outbreak of cases in India during COVID-19 pandemic. Mortality is unacceptably high, reaching 80% in some case series. Prompt diagnosis is crucial because treatment initiation is time-critical due to the rapid progression of the infection. Direct KOH mount is a simple, rapid, inexpensive point-of-care test, which requires minimum technical aids. Interpretation of KOH smears requires some amount of experience. The present study was to evaluate the sensitivity and specificity of Direct Microscopical examination by KOH preparation of suspected Mucor mycosis samples taking the fungal culture as gold standard. Material and Methods: 230 samples were received in Microbiology Department from May 2021 to August 2021 from the patients suspected of Mucor mycosis. Samples were processed for KOH preparation using different concentrations of KOH like 20% and 40% till all tissues get digested. Samples were cultured also in Sabourauds dextrose agar (SDA) medium. Results of microscopy and culture were statistically analyzed using SPSS software version 23. Result: Sensitivity of KOH preparation was 67%, specificity was 87%. Positive predictive value was 76% and negative predictive value was 81% and accuracy was 80% when compared with the fungal culture. Discussion: Direct microscopic examinations using KOH preparation are the simplest, cheapest method used for the diagnosis of mycotic infections. This study is valuable in establishing a reliable method for early information on diagnosis of Mucor mycosis that may be crucial for determining appropriate therapy for the successful treatment of Mucor mycosis patients. Conclusion: KOH examination plays an important role in the diagnosis of Mucor mycosis suspected patients during COVID-19 pandemic era when compared with fungal culture which is a gold standard test in diagnosis of Fungal infection. It is the simplest and cheapest method that can be developed easily with minimal infrastructure or human resources in resource-limited countries during the COVID-19 era.