Fungal Culture Results Research Articles

Clinico-Mycological Study of Dermatophytosis among Diabetic and Non-Diabetic Patients in a Tertiary Level Hospital: A Comparative Study

Abstract Background: Dermatophytosis is a major public health concern in India, especially in recent years, with an alarmingly rising trend, particularly in relation to recurrent and chronic infection. The number of studies examining the relationship between an individual’s glycemic status and the development of dermatophytosis, and a comparison of the evolving species trend between diabetic and non-diabetic patients infected with dermatophytes, is limited. Aims and Objectives: To study and compare the clinical and mycological aspects of dermatophytosis among diabetic and non-diabetic patients and to compare the culture isolates in both groups. Materials and Methods: The study included 378 patients of clinically suspected dermatophytosis, divided into two groups of 189 known diabetics and non-diabetics each. We subsequently analysed and compared the demographics, clinical data, potassium hydroxide (KOH) mount, calcofluor white (CW) staining and fungal culture results of all patients in both groups. Results: Among the 378 patients, diabetic patients had a significantly greater extent of involvement and higher rates of recurrence when compared to non-diabetics. The overall positivity rate was higher in CW staining (77.8%) as compared to KOH (57.7%) in both non-diabetics and diabetics. Trichophyton mentagrophytes was the most common isolate (47.6%) in both diabetic (55.0%) and non-diabetic (61.4%) patients, followed by Trichophyton rubrum (31.8% and 29.6%, respectively). The positivity percentages of fungal culture, KOH and CW staining increased to 100%, 77.9% and 95.7%, respectively, when scrapings were taken from ≥2 sites. Conclusion: The pattern of dermatophytosis is comparatively more chronic and severe in diabetics compared to non-diabetics. T. mentagrophytes was the most common culture isolate in both groups. CW staining can potentially be used as the initial method of choice for the diagnosis of dermatophytosis as it has significantly outperformed the conventional KOH mount. Scrapings can regularly be taken from ≥2 sites to avoid false negative results.

Prolonged treatment of dermatophytosis caused by Trichophyton indotinea with terbinafine or itraconazole impacts better outcomes irrespective of mutation in the squalene epoxidase gene.

Over the past decades, the increasing incidence of recurrent dermatophytosis associated with terbinafine-resistant Trichophyton has posed a serious challenge in management of dermatophytosis. Independent reports of failure of treatment and high minimum inhibitory concentrations (MIC) of antifungals are available, but data correlating MIC and clinical outcomes is still sparse. Therefore, the present study was conducted to evaluate the outcomes of systemic treatment of dermatophytosis and its correlation with MIC of the etiological agents isolated from such patients. Retrospective analysis of 587 consecutive patients with dermatophytosis was done from March 2017 to March 2019. Demographic and clinical details of the patients were noted, along with the results of direct microscopy and fungal culture. The isolates were identified by sequencing the internal transcribed spacer region of rDNA. Antifungal susceptibility testing was performed following the CLSI M38 protocol. Mutation in the squalene epoxidase (SE) gene was detected by DNA sequencing and ARMS-PCR. Based on the culture-positivity and prescribed systemic antifungal, patients were categorised into Group I culture-positive cases treated with systemic terbinafine and Group II culture-positive cases treated with systemic itraconazole, each for a total period of 12 weeks. In the present study, 477 (81.39%) were culture-positive; however, 12 weeks follow-up was available for 294 patients (Group I-157 and Group II-137) who were included for statistical analysis. In both groups [Group I-37/63 (51.4%) and Group II-14/54 (58.3%)], a better cure rate was observed if the initiation of therapy was performed within <6 months of illness. Treatment outcome revealed that if therapy was extended for 8-12 weeks, the odds of cure rate are significantly better (p < .001) with either itraconazole (Odd Ratio-15.5) or terbinafine (Odd Ratio-4.34). Higher MICs for terbinafine were noted in 41 cases (cured-18 and uncured-23) in Group I and 39 cases (cured-16 and uncured-23) in Group II. From cured (Group I-17/18; 94.4% and Group II-14/16; 87.5%) and uncured (Group I-20/23; 86.9% and Group II-21/23; 91.3%) cases had F397L mutation in the SE gene. No significant difference in cure rate was observed in patients with Trichophyton spp. having terbinafine MIC ≥ 1or <1 μg/mL (Group I-p = .712 and Group II-p = .69). This study revealed that prolonging terbinafine or itraconazole therapy for beyond 8 weeks rather than the standard 4 weeks significantly increases the cure rate. Moreover, no correlation has been observed between antifungal susceptibility and clinical outcomes. The MIC remains the primary parameter for defining antifungal activity and predicting the potency of antifungal agents against specific fungi. However, predicting therapeutic success based solely on the MIC of a fungal strain is not always reliable, as studies have shown a poor correlation between invitro data and invivo outcomes. To address this issue, further correlation of antifungal susceptibility testing (AFST) data with clinical outcomes and therapeutic drug monitoring is needed. It also highlights that initiation of the treatment within <6 months of illness increases cure rates and reduces recurrence. Extensive research is warranted to establish a better treatment regime for dermatophytosis.

IL-6 and TIMP-1 Correlated to Airway Pathogen Colonization and Predict Disease Severity in Patients with Non-Cystic Fibrosis Bronchiectasis.

Non-cystic fibrosis bronchiectasis is associated with airway pathogen colonization. We planned to investigate the inflammatory markers in patients with different airway pathogens and their correlation with disease severity. We enrolled patients aged between 20 and 75 from October 2021 to August 2022. All patients had sputum evaluation for bacterial and fungal cultures before enrollment, and were classified into four groups according to the culture results. Forty-four patients with non-CF bronchiectasis and six controls were enrolled and categorized as follows: Group 1, no pathogens identified in sputum cultures (n = 14); Group 2, positive fungal culture results (n = 18); Group 3, positive P. aeruginosa culture results (n = 7); and Group 4, positive culture results for both fungi and P. aeruginosa (n = 5). Group 4 had significantly higher serum defensin α1, IL-6 and tissue inhibitors of MMP (TIMP)-1 levels than group 1 patients. The serum levels of IL-6 and TIMP-1 were positively correlated with the FACED score and negatively correlated with distance-saturation product. Significantly higher levels of serum IL-6 and TIMP-1 were found in the patients who had concomitant fungal and P. aeruginosa colonization, and were closely related to clinical severity and may have important roles in disease monitoring.

Clinico-mycological study of onychomycosis and in vitro antifungal susceptibility of Trichophyton rubrum.

Onychomycosis, a fungal nail infection, is primarily caused by dermatophytes, yeasts, and non-dermatophyte moulds (NDMs). The incidence of this disease and the predominance of specific pathogens vary across different regions and evolve. This study aimed to elucidate the epidemiology of onychomycosis and the pattern of causative pathogens in Beijing, and to ascertain the in vitro antifungal susceptibility profiles of Trichophyton rubrum against itraconazole (ITR), terbinafine (TER), and fluconazole (FLU). Involving 245 patients of onychomycosis with positive fungal culture results, the study implemented internal transcribed spacer (ITS) sequencing of ribosomal DNA (rDNA) on all collected samples. The mean age of the participants was 37.93 ± 13.73 years, with a male-to-female ratio of 1.53:1. The prevalence of toenail infections was significantly higher than that of fingernails. Distal and lateral subungual onychomycosis (DLSO) were the most frequent clinical classifications. PCR results indicated that dermatophytes were the most prevalent pathogens, followed by yeasts and NDMs, among which T. rubrum was the most dominant dermatophyte. TER demonstrated high sensitivity to T. rubrum. However, in clinical settings, some patients with onychomycosis exhibit a poor response to TER treatment. The relationship between in vitro antifungal sensitivity and clinical effectiveness is complex, and understanding the link between in vitro MIC values and clinical efficacy requires further investigation.

Misdiagnosis of cutaneous facial sporotrichosis: An analysis of five cases.

Facial cutaneous sporotrichosis presents with diverse clinical manifestations, often leading to misdiagnosis. This study aims to present the clinical characteristics of five misdiagnosed cases of facial cutaneous sporotrichosis, aiming to enhance understanding of this disease and prevent misdiagnosis and mistreatment. Clinical data, histopathology, and fungal culture results of these five cases were comprehensively analyzed. Among these five patients, three presented with lymphocutaneous sporotrichosis, while two had the fixed cutaneous type. Due to misdiagnosis, initial treatments were ineffective for all patients. Upon histopathological examination and fungal culture confirming sporotrichosis, treatment with itraconazole for 3 months led to complete resolution of lesions. While one patient experienced a relapse due to noncompliance with the prescribed medication. Facial sporotrichosis, with its diverse clinical manifestations and obscure trauma history, is prone to misdiagnosis. Timely and thorough examinations are crucial for precise diagnosis and management. Itraconazole treatment demonstrated notable efficacy, and patient compliance is also essential for favorable outcomes.

Clinicopathological study of otomycosis in a tertiary hospital in South-west Nigeria.

Otomycosis is common in environments with hot, humid weather, and it may be challenging to manage. To profile common clinical presentations, the pathogenic fungi, the treatment modalities with responses, and explore clinical factors associated with having positive fungal culture in Otomycosis. Retrospective review of patients with Otomycosis. Demographic and clinical parameters, otoscopic findings and mycological study results were recorded. The treatment modalities used and treatment response were summarized. Comparative statistical analyses of associated factors to positive fungal culture were performed with Chi square test, and Student's t-test, using SPSS version 22.0. Total of 71 patients with M: F=1:1.8, mean age 38.5±19.8 years. Average duration of symptoms was 5.4 ±4.6 weeks; common presenting complaint was itchy ear (33.8%). Majority of patients (85.9%) had unilateral ear involvement, 50.0% applied ototopic medications before presentation, 8.5% had multiple co-morbidities. 20 patients had positive fungal culture results; common fungal isolate was Aspergillus niger 9 (45.0%).Clinical factors associated with positive culture of fungus were age, non-previous use of ototopic drugs, and presence of co-morbidity. The most common treatment was local ear debridement and use of topical antifungal creams. Majority (91.5%) of the patients responded with resolution of fungal infection. Complications rate was 8.4%. Otomycosis commonly present with itchy ears, the pathogenic fungi commonly being Aspergillus species. The factors associated with positive fungal culture were age, non-usage of ototopic agents and presence of co-morbidity. Treatment modality used was local debridement and topical antifungal agents, which produced favourable response in most patients.

Value of Imprint Cytology for the Rapid Diagnosis of Mucormycosis in the COVID-19 Pandemic Setting - A Pilot Study.

The second wave of the coronavirus disease 2019 (COVID-19) pandemic recorded a surge in rhino-orbital-cerebral mucormycosis (ROCM) infection in COVID-19-positive patients with diabetes and on concomitant steroid therapy. The rapidly progressive and devastating nature of the disease necessitated prompt diagnosis and early intervention to improve patient outcomes. Histopathology and fungal culture remain essential tools; however, these investigations have long and variable turn-around times (TATs) and may delay the initiation of treatment. Frozen section is not widely available and should be avoided in COVID-19-positive cases due to the risk of aerosol production and droplet exposure. In cases with high clinicoradiologic suspicion for mucormycosis, imprint cytologic evaluation provides a rapid diagnosis. Familiarity with fungal cytomorphology, awareness of morphologic pitfalls, and implementation of a standardized reporting format aid in diagnostic accuracy. Eighteen COVID-19-positive patients, who were admitted to our hospital with clinical suspicion of mucormycosis during June and July 2021, were included in the study. We used nasal or oral imprint cytology for the initial, rapid detection of Mucor. Cytology findings were correlated with histopathology and fungal culture results. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The sensitivity, specificity, PPV, and NPV were 100%, 100%, 100% and 100%, respectively. This study showed that imprint cytology can be a rapid, cost-effective, first-line diagnostic modality in Mucor diagnosis.

Loop-Mediated Isothermal Amplification (LAMP): Potential Point-of-Care Testing for Vulvovaginal Candidiasis.

The aim of this study is to establish a loop-mediated isothermal amplification (LAMP) method for the rapid detection of vulvovaginal candidiasis (VVC). We developed and validated a loop-mediated isothermal amplification (LAMP) method for detecting the most common Candida species associated with VVC, including C. albicans, N. glabratus, C. tropicalis, and C. parapsilosis. We evaluated the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and Kappa value of the LAMP method to detect different Candida species, using the conventional culture method and internal transcribed spacer (ITS) sequencing as gold standards and smear Gram staining and real-time Rolymerase Chain Reaction (PCR) as controls. A total of 202 cases were enrolled, of which 88 were VVC-positive and 114 were negative. Among the 88 positive patients, the fungal culture and ITS sequencing results showed that 67 cases (76.14%) were associated with C. albicans, 13 (14.77%) with N. glabratus, 5 (5.68%) with C. tropicalis, and 3 (3.41%) with other species. Regarding the overall detection rate, the LAMP method presented sensitivity, specificity, PPV, NPV, and Kappa values of 90.91%, 100%, 100%, 93.4%, and 0.919, respectively. Moreover, the LAMP had a specificity of 100% for C. albicans, N. glabratus, and C. tropicalis, with a sensitivity of 94.03%, 100%, and 80%, respectively. Moreover, the microscopy evaluation had the highest sensitivity, while the real-time PCR was less specific for C. albicans than LAMP. In addition, CHROMagar Candida was inferior to LAMP in detecting non-albicans Candida (NAC) species. Based on the cost-effective, rapid, and inexpensive characteristics of LAMP, coupled with the high sensitivity and specificity of our VVC-associated Candida detection method, we provided a possibility for the point-of-care testing (POCT) of VVC, especially in developing countries and some laboratories with limited resources.

621. Evaluation of a Multiplex Endpoint PCR for the Detection of 21 Fungi Commonly Causing Fungal Pneumonia Using Barcoded Magnetic Bead Technology

Abstract Background Fungal pneumonia caused by Aspergillus, Mucorales, and other fungi are potentially life-threatening and often are difficult to diagnose due to similar clinical presentations and poor sensitivity by culture. The BioCode MDx-3000 from Applied BioCode uses Barcoded Magnetic Bead (BMB) technology to digitally detect 21 fungi in a multiplex PCR. We evaluated the BioCode assay using clinically simulated samples and retrospective frozen bronchoalveolar lavage samples. Methods For limit of detection (LOD), 10-20 replicates of each organism were run at 1E4-10 spores/mL. Reproducibility was performed by running three replicates of each target (1E3 spores/mL) within one run, three separate times. Specificity was determined by testing for 37 fungal analytes not targeted by the panel. Matrix specific inhibition was also tested by spiking each organism in the following matrices: whole blood, plasma, serum and BAL. 90 retrospective BAL samples from patients with fungal culture results were run to assess clinical performance. Results LOD of 21 fungal organisms ranging from 19.83 – 271.76 spores/ml. Detection rate was 100% (36/36) in all samples mixed with two fungi; 95% (20/21) in all samples mixed with three fungi. No detection or cross-reactivity was observed in samples containing any of the 37 non-targeted fungi. All replicates were detected in Inter- and intra- reproducibility experiments. No difference or inhibition was observed in detection of targeted fungi spiked in different matrices. Testing retrospective BAL samples showed an overall positive concordance of 61.4% and negative concordance of 98.1%. In 23 BALs positive by both calcofluor white stain (hypha elements) and culture, the BioCode assay correctly detected fungal targets in 78% (18/23) of the samples. Additionally, the assay produced positive detection in 25% of BALs that were culture negative. Limit of Detection and Concordance Limit of detection for the 21 fungal targets. Concordance for each target in retrospective BALs compared to culture. N/A indicates not tested. Conclusion The BioCode assay showed an overall good analytical sensitivity and specificity. Analysis of retrospective BAL samples demonstrated the assay showing good concordance with the culture results. Further studies are warranted to determine the assay’s clinical sensitivity and specificity in aid in diagnosis of fungal pneumonia. Disclosures Kristen Haag, BS, Applied BioCode, Inc.: I am an employee of Applied BioCode|Applied BioCode, Inc.: Stocks/Bonds Michael Aye, PhD, Applied BioCode: Stocks/Bonds Martín Cardona, BS, Applied BioCode, Inc.: I am an employee of Applied BioCode. Sean Zhang, PhD, Applied BioCode: Grant/Research Support|IMMY Diagnostics: Grant/Research Support|KARIUS: Advisor/Consultant|Pearl Diagnostics: Grant/Research Support|Scanogen: Grant/Research Support|T2 Biosystems: Advisor/Consultant|Vela Diagnostics: Grant/Research Support

Antifungal susceptibility profiles for fungal isolates from corneas and contact lenses in the United Kingdom

ObjectiveTo report the identification and results of susceptibility testing for fungal isolates from the cornea or contact lens care systems.Materials and methodsIn this retrospective epidemiological study, we searched the results of fungal cultures from cornea or contact lens systems referred for identification and susceptibility testing to the United Kingdom National Mycology Reference Laboratory between October 2016 and March 2022. For each fungal isolate, we recorded the genus and species of the fungus and the minimum inhibitory concentration (MIC) to six antifungal agents available to treat corneal infection (amphotericin, econazole, itraconazole, natamycin, posaconazole, and voriconazole).ResultsThere were 600 isolates from 585 patients, comprising 374 (62%) from corneal samples and 226 from contact lenses and care systems, of which 414 (69%) isolates were moulds (filamentous fungi) and 186 (31%) were yeasts. The most frequent moulds isolated were Fusarium spp (234 isolates, 39%) and Aspergillus spp (62, 10%). The most frequent yeasts isolated were Candida spp (112, 19%), predominantly Candida parapsilosis (65, 11%) and Candida albicans (33, 6%), with 35 isolates (6%) of Meyerozyma guilliermondii. In vitro susceptibility was greatest for natamycin (347 moulds tested, mode 4 mg/L, range 0.25–64 mg/L; 98 yeasts tested, mode 4 mg/L, range 0.5–32 mg/L), with susceptibility for 94% for moulds and 99% yeasts. Of the 16 isolates interpreted as highly resistant to natamycin (MIC ≥16 mg/L), 13 were Aspergillus flavus complex.ConclusionsIn vitro susceptibility supports the use of natamycin for the empiric treatment of fungal keratitis in the UK.

Citrate phosphate dextrose adenine (CPDA-1) can be sampled from blood collection bags in a multi-dose manner.

To evaluate the sterility of citrate phosphate dextrose adenine (CPDA-1) anticoagulant when sampled from blood collection bags in a multi-dose manner. 10 pre-filled CPDA-1 blood collection bags; 46 bacterial and 28 fungal culture result reports. 10 CPDA-1 blood collection bags were split into 2 equal groups and stored at either room temperature (24 °C) or refrigerator temperature (5 °C) for 30 days. Two bags in each group were designated as controls. Beginning on day 0 a 1.0 mL aliquot was withdrawn from each experimental bag and submitted for bacterial culture (aerobic and anaerobic) every 5 days, and fungal culture every 10 days. All 10 bags were sampled on day 30. Bacterial and fungal culture results were compiled and interpreted. 46 CPDA-1 aliquots were cultured, resulting in 2 positive microbial isolates: Bacillus was cultured from a previously unopened experimental bag on day 0, and Candida was cultured from a refrigerated experimental bag on day 30. Both positives are thought to represent post-sampling contamination, though these suspicions cannot be confirmed in the bag yielding Candida due to a lack of subsequent data. All other samples were negative for microbial growth. CPDA-1 blood collection bags stored at either 24 °C or 5 °C can be used in a multi-dose manner for up to 20 days when each sample is obtained aseptically. These results support the clinician's ability to utilize the contents of 1 bag multiple times rather than discarding the bag after a single use.

Single-Point Nail Sampling to Diagnose Onychomycosis Caused by Non-Dermatophyte Molds: Utility of Polymerase Chain Reaction (PCR) and Histopathology

The three most commonly used methods for diagnosing non-dermatophyte mold (NDM) onychomycosis are culture, polymerase chain reaction (PCR), and histopathology. Toenail samples from 512 patients (1 sample/patient) with suspected onychomycosis were examined using all three diagnostic tests. A statistically significant association was found between PCR and histopathology results, as well as between fungal culture and histopathology results. All PCR-positive and culture-positive dermatophyte samples were confirmed by histopathology. However, 15/116 (12.9%) of culture-positive NDM samples had negative histopathology results, while all PCR-positive NDM samples were confirmed by histopathology. The overall rate of dermatophyte detection was higher using PCR compared to culture (38.9% vs. 11.7%); the lower rate of NDM detection by PCR (11.7% vs. 38.9%) could be attributed to the restriction of the assay design to seven pre-selected targets. When repeat sampling in the clinic is not possible, a combination of NDM detection by PCR and positive histopathology of hyphae may be a proxy for NDM infection, particularly where the NDM occurs without a concomitant dermatophyte. There was a high degree of correlation between negative PCR and negative histopathology. A negative PCR result with negative histopathology findings may be a reliable proxy for the diagnosis of non-fungal dystrophy.

Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii.

Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples.

Low positivity rate and high percentage of nondermatophyte molds in an analysis of 35,257 fungal nail culture results from a United States national commercial laboratory, 2019-2022

Low positivity rate and high percentage of nondermatophyte molds in an analysis of 35,257 fungal nail culture results from a United States national commercial laboratory, 2019-2022

A Retrospective Study of Clinical and Immunological Features of a Pediatric Population with Talaromyces marneffei Infection.

Talaromyces marneffei (T. Marneffei) infection is considered as an indicator of immunosuppression in immunocompromised individuals, leading to multiple organ damage. Our study aimed to evaluate both the clinical characteristics and immunological features of pediatric patients infected with T. marneffei from our institute, providing novel insights into diagnosis and treatment for this life-threatening disease. Thirteen pediatric patients with T. marneffei infection were enrolled in Guangzhou Women and Children's Medical Center during 2012 to 2020. Clinical data and laboratory findings were collected and further analyzed. Pearson correlation coefficient was calculated to determine the relationship between serum immunoglobulins (Igs) levels and white blood cell count, or the absolute lymphocyte count. Patients were diagnosed as having T. Marneffei infection mainly based on the results of fungal culture and Gram stain of specimens. The most common presentations were fever (69%), pneumonia (38%) and immunodeficiency (38%). The total levels of Igs (IgE, IgA, and IgM) were positively correlated with both white blood cell count and absolute lymphocyte count. Serum Ig expression Pattern in patients diagnosed with T. marneffei infection might serve as an effective prognostic marker which would help with the development of early interventions for children with this fatal disease.

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n=11 and n=4, respectively) and IUK patients (n=22 and n=8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

The Prevalence of Positive Donor Corneoscleral Rim Culture and its Association with Ocular Infection After Transplantation.

The aim of the study was to determine the prevalence of positive corneoscleral donor rim cultures and to report keratitis and endophthalmitis after keratoplasty. Eye bank records and medical records of patients who underwent keratoplasty between September 1, 2015, and December 31, 2019, were retrospectively reviewed. Patients who had routine donor-rim culture taken during surgery and followed up for at least 1 year in the post-operative period were included in the study. A total of 826 keratoplasty procedures were performed. A total of 120 (14.5%) cases had a positive donor corneoscleral rim culture. Positive bacterial cultures were obtained from 108 (13.7%) of the donors. Bacterial keratitis was observed in one patient (0.83% of recipients) who had a positive bacterial culture. Positive fungal cultures were obtained from 12 (1.45%) donors, of whom one (8.33% of recipients) developed fungal keratitis. Endophthalmitis was observed in one patient whose culture result was negative. Both bacterial and fungal culture results were similar in penetrating and lamellar surgical procedures. Although the donor corneoscleral rims have a high positive culture result, the rate of bacterial keratitis and endophthalmitis is low, the risk of infection is high in patients with a fungal positive donor rim. Closer follow-up of patients with fungal positive donor corneo-scleral rim result and initiation of aggressive antifungal treatment when infection occurs will be beneficial.

Invasive fungal disease in critically ill and Immunocompromised patients.

To study the frequency and type of invasive fungal disease in critically ill and immunocompromised patients. The prospective, cross-sectional, descriptive study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from January 2017 to December 2020, and comprised pathological samples from immunocompromised and critically ill patients for fungal culture. Data regarding demographics, comorbidities, results of direct microscopy and fungal culture was recorded. Data was analysed using SPSS 22. Of the 8285 patients' specimens, 4722(57%) belonged to males and 3563(43%) to females. The mean age of the patients was 48.32±5.42 years (range: 14-98 years). Out of total 8285, 3465(41.82%) were related to blood, 2640(32%) endobronchial washing, 837(10%) sputum, 623(7.5%) tissue, 332(4%) body fluids, 288(3.5%) bronchoalveoar lavage and 100(1.2%) cerebrospinal fluid. Aspergillus flavus (20.7%) and candida albicans (14.5%) were the two most commonly isolated fungal species. A high index of suspicion for invasive fungal disease should be maintained in immunocompromised and critically ill patients.

The impact of amphotericin B-fortified preservation media on donor rim cultures and posttransplant infection.

To determine the impact of amphotericin B supplementation to donor cornea preservation solutions on the rates of positive donor rim fungal cultures and postkeratoplasty fungal infections. This was a retrospective analysis of cases undergoing corneal transplantations at a single tertiary referral center from 2016 to 2021. Patients undergoing corneal transplantations with and without amphotericin B supplementation to the storage media were reviewed for donor rim culture results and postoperative infection. The primary outcome measures were positive donor rim fungal culture results and postkeratoplasty fungal infection. A total of 1238 corneal transplants were analyzed. Of these, 849 were stored in preservation solution without amphotericin B, while 389 had amphotericin B included. There was a lower incidence of positive donor rim fungal cultures in cases with amphotericin B supplementation (1.8%) compared to the cases without amphotericin B (2.9%), although this difference was not statistically significant (P = 0.24). Of the 389 cases with amphotericin B supplementation, one (0.25%) went on to develop clinically significant infection, while three of 849 (0.35%) cases without amphotericin B developed infection. The sample size was too small to determine the effect of amphotericin B on the incidence of postkeratoplasty fungal infection. The addition of amphotericin B to donor cornea preservation solution resulted in a downward trend of positive donor rim fungal cultures and postkeratoplasty fungal infections, although these differences did not reach statistical significance. Further studies with larger sample sizes are necessary to appropriately determine the impact of amphotericin B supplementation in the storage solution on positive donor rims and postkeratoplasty fungal infections.

Rapid and Visual RPA-Cas12a Fluorescence Assay for Accurate Detection of Dermatophytes in Cats and Dogs.

Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes. Wood’s lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.