bluuebunny/crossref_metadata_2025_split huggingface dataset:
P379 Antifungal activity of α defensins 5 like peptide against Cryptococcus neoformans var. grubii and in silico designing of its peptidomimetics
Authors: Singh Karuna , Neelabh  , Uttam Gunjan 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
Combinatorial and retroviral therapies have been found effective in the treatment of cryptococcosis, but safer and more potent antifungal therapeutics are the need of the hour. In this regard, the natural antifungal peptides (AFPs), the essential components of the innate immune system have drawn the attention of researchers and drug developers. AFPs are 12-50 amino-acids-long host defense peptides of cationic nature. Although AFPs have certain limitations like low stability, susceptibility to proteolysis, loss of activity in the presence of metal ions and salts, short half-lives, and peptide aggregation in oral formulation, but these can be addressed by designing and synthesizing the peptidomimetics.

Methodology
The whole blood of the rat was collected by cardiac puncture. The blood was mixed with 0.83% aqueous ammonium chloride. The neutrophils were separated from lysed WBCs by centrifugation. The purification of the peptide was carried out by SDS-PAGE, size exclusion, cation exchange chromatography, and HRLCMS. The MSA of the characterized peptide was performed using a T-coffee program. The in vitro antifungal activity of crude was tested against C. n. grubii (ATCC 6352) using flow cytometry. The structure of peptides was altered through ChemDraw by replacing the elements with their bioisosteres to form mimetics. The peptide and its mimetics were docked upon the target of C. n. grubii, i.e., O-acetyl transferase (modeled through Phyre2 web server) using PatchDock, and FireDock web servers.

Results
The peptide isolated from rat PMNs had a molecular weight of 3 kDa. It has lysine and arginine which are the characteristic residues of an antifungal peptide. The eluted peptide has the highest (83%) similarity with α defensins 5 (accession no NP_001013071 XP_214 386). The mild antifungal activity of peptide was evident against C. n. grubii. The structure of O-acetyl transferase was modeled with 100% confidence and 92% query coverage (Table 1). The mimetic 1 (M1) showed the best interaction with O-acetyl transferase with −54.47 Kcal/mol binding energy and 4 hydrogen bonds (Table 2).

Conclusion
The designed M1 of α defensins 5 like peptide could be a promising lead in antifungal drug development. As mimetic 1 was designed using an in-silico approach, its synthesis and wet lab validation are desirable.
P393 Clinical spectrum and molecular characterization of Rhytidhysteron spp. (now Gloniopsis) from India
Authors: Ahmad Haseen , Kaur Harsimran , Gupta Parakriti , Shankaranarayana Shamanth , Agnihotri Sourav , Mehta Preeti , Salve Suneeta , Yoganand Raksha , Tarai Bansidhar , Tejashree A , Hemashettar Basavaraj , Balajee G , Gopal Lokeshwari , Gupta Sunita , Ghosh Anup , Rudramurthy Shivaprakash 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objective
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.

Method
We 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).

Conclusion
This 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.
P401 Identification and characterization of cryptic species of Aspergillus isolated from clinical samples
Authors: Janani R. Sruti , Singh Gagandeep , Xess Immaculata , Das Bimal Kumar 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Background and Objective
Molds are emerging as a major cause of life-threatening infections in immunocompromised patients. There is an increasing recognition of the cryptic Aspergillus species, which are organisms that are morphologically indistinguishable yet can be differentiated by molecular methods. These organisms have been known to show a higher minimal inhibitory concentration for the majority of the antifungal agents in vitro. Therefore, correct identification of these cryptic species is very important to administer a proper antifungal agent.
In this study, we wish to identify and characterize the cryptic species of Aspergillus from all clinical samples.

Methods
Patient routine samples like broncho alveolar lavage, endo-tracheal aspirate, sputum, pus, tissue, and CSF from various wards, OPDs, and ICUs of All India Institute of Medical Sciences, New Delhi which were processed in the Mycology laboratory, department of Microbiology showing growth of Aspergillus species were included in this study. Identification of the isolates was done using phenotypic methods and by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). For the analysis of cryptic species, the isolates underwent PCR and then sequencing of the ß-tubulin gene was done. Antifungal susceptibility testing was done using micro broth dilution as per the CLSI method.

Results
Of the 92 isolates, using morphological methods, 53 were identified as A. fumigatus, 33 were identified as A. flavus, 3 as A. nidulans, 2 as A. terreus, and 1 as A. niger MALDI-TOF (Vitek MS database) misidentified 2 isolates of A. nidulans and 1 isolate of A, stellatus as A. fumigatus and 1 isolate of A. tubingensis as A. niger. The ß-tubulin sequence analysis for the identification of cryptic species revealed that 2 isolates (2.08%) were cryptic, one was A. stellatus morphologically identified as A. nidulans and another one was A. tubingensis morphologically identified as A. niger.

Conclusion
Currently the available data on cryptic Aspergillus species is very limited. Because of its varied susceptibility pattern, it is important to identify the Aspergillus isolates to its species level. There is also a need for expansion of the number of strains for each species in MALDI-TOF MS databases for convenient, faster, and correct identification.
P409 Study of epidemiology, risk factors and antifungal sensitivity pattern of fungal pneumonia in critically ill cirrhotics
Authors: Singh Paras , Kale Pratibha , Khillan Vikas , Sarin Shiv Kumar 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
Liver cirrhosis causes immune dysregulation and increased susceptibility to fungal infections. We studied the epidemiology and risk factors, and compared the rapid diagnostic methods and biomarkers for fungal pneumonia in critically ill cirrhotics.

Methods
Single-center, prospective cohort study of 100 critically ill cirrhotics with fungal pneumonia between January to September 2021. Comparative analysis was done for culture, realtime PCR and biomarkers, bronchoalveolar lavage and serum Galactomannan, and serum procalcitonin measured on days 1, 3, and 7. The final outcome considered was mortality within 1 month after diagnosis or discharge.

Results
Aspergillus flavus was the most common species (70/100,70%). Risk factors were, neutropenia (P .03), steroids prior to ICU admission (P .02), prolonged hospitalizations ˃21 days (P .05), and culture positivity was 80%. The culture was not inferior to realtime PCR for the diagnosis of fungal pneumonia. BAL Galactomannan was an early prognostic marker with a median rise above ˃ 3.5 index value. The Median PCT level was higher from day 1in the fungal pneumonia non-survivor group (3.29 vs. 0.8 ng/ml) with higher 30-day mortality (72%). Higher PCT was associated with bacterial co-infection (48%), antibiotic (74%), antifungal therapy, and renal failure and mortality.

Conclusion
Fungal pneumonia complicates cirrhotics with neutropenia, prolonged hospitalization, and steroids as risk factors. Aspergillus flavus predominate in consensus with Asian epidemiology. Culture methods are reliable and a combination of molecular tests with BAL Galactomannan is useful for rapid diagnosis. Serum PCT is raised in patients with fungal pneumonia and is associated with higher mortality. In our study the baseline PCT at admission to ICU was higher in the non-survivor group, and levels on D3 and D7 were persistently higher. High serum procalcitonin level is an independent prognostic biomarker of mortality risk in fungal pneumonia.
P412 QuicGM Aspergillus Galactomannan Ag lateral flow assay as a practical aid to diagnose invasive Aspergillosis in high-risk hematology patients
Authors: Aerts Robina , Mercier Toine , Maertens Johan , Lagrou Katrien 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Background
Early diagnosis of invasive aspergillosis (IA) is an important factor to improve survival, but it remains challenging. QuicGM Aspergillus Galactomannan Ag Lateral Flow Assay (LFA) is based on a fluorescence immunochromatography test using a monoclonal antibody to identify Aspergillus galactomannan antigen in serum and bronchoalveolar lavage fluid.

Methods
In this study, we analyzed the diagnostic performance of QuicGM in 120 serum samples of high-risk hematology patients [41 cases of proven (n = 4) and probable (n = 37) IA and 79 equally high-risk controls] and compared it to performance of the galactomannan (GM) [Platelia Aspergillus enzyme immunoassay (ELISA) (Bio-Rad, Marnes-la-Coquette, France)]. Probable cases defined by a positive Biorad GM in serum were also included in the cohort.

Results
Using the same cut-off of 0.5, QuicGM was positive in 22 cases versus GM in 21 cases. However, QuicGM was false positive in 11 controls with GM only in 1 control. These false positive samples remained positive upon repeated testing with the QuicGM. The test results of QuicGM and GM correlated well with a Spearman's correlation coefficient of 0.80 (P &lt;.001). McNemar test showed no significant difference between both tests (P = 1) and Cohen's kappa coefficient was 0.65 (CI 0.49-0.81), suggesting a substantial strength of agreement.
QuicGM sensitivity (using 0.5 cutoff) was 54% versus 51% for GM. Specificity was 86% for QuicGM versus 99% for GM. Positive predictive value (PPV) was only 67% for QuicGM versus 95% for GM. Combining GM and QuicGM in serum increased sensitivity to 59%. Youden index of QuicGM in the cohort was 0.39. Using this cutoff, sensitivity was 66% and specificity was 80%. When using a higher cutoff of 0.6 for the QuicGM, specificity increased to 94% and PPV to 78%, at the cost of a decrease in sensitivity to 44%.

Discussion
Including probable cases defined by a positive Biorad GM in serum could have led to a bias in favor of the GM.
With the cutoff suggested by the company, there was a notable decrease in specificity and PPV for the QuicGM. The need to adjust this cut-off must be questioned. With the implementation of LFAs in the EORTC-MSGERG criteria, one usually strives for the same cutoff of 0.5, to assure a practical approach. However, we must remain thoughtful about slight differences in these tests. They all have their own IgG and IgM built. Caution is needed more in the setting of diagnosis than in screening.
More knowledge must be obtained regarding how different hospital laboratories have implemented the LFAs.

Conclusion
Diagnostic performances of QuicGM and Biorad GM correlate well.
Further research is necessary regarding the optimal cutoff index.
P435 Evaluation of remote triazole capillary blood testing to facilitate remote therapeutic drug monitoring (TDM): A validation study
Authors: Nwankwo Lisa , Donovan Jackie , Ni Melody , Loebinger Michael , Armstrong-James Darius , Shah Anand 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Background
The advent of COVID-19 has meant that patients with chronic diseases needed to shield, however, investigations were needed to guide continual management of their disease. Remote monitoring options were evaluated to ensure the standard of care is not compromised.

Purpose and Hypothesis
The aim was to validate remote (return-posted) capillary triazole blood testing and evaluate the potential role of remote TDM in chronic antifungal therapy.

Materials and Methods
A single-center prospective cross-sectional study of remote finger prick capillary blood testing compared with gold standard venesection was performed. Remote finger prick capillary blood testing was validated compared to local standard venesection using comparative statistical analysis Comparative statistical analysis: Paired t-test, correlation and Bland-Altman were used to determine if there was agreement or association between the sampling methods.

Results
A total of 66 patients receiving triazole therapy were recruited and 57 pooled pairs of remote capillary and venous triazole concentrations and metabolites were prospectively analyzed, with the rest of the blood samples not being analyzed due to insufficient sample, hemolysis, or undetectable triazole level of &lt;0.2 mg/l. There was a significant difference in the comparison of the two methods of sampling with paired t-test at P &lt;.0001. Bland-Altman analysis yielded wide bias (−49.07%) and wide limits of agreement (−85.5% to −12.64%). On average capillary triazole, concentrations were 37% lower than venous concentrations (Fig. 1). There was however a very strong correlation between capillary and venous tests (Pearson's correlation coefficient r = 0.9219, P &lt;.0001, Fig. 2).

Conclusions
Remote capillary triazole sampling does not appear interchangeable with venous sampling, but being strongly correlated and on average 2/3rd of the venous value, could be a predictor of venous triazole level, or be useful for intra-patient longitudinal monitoring. When incorporated into an outpatient clinical pathway it can improve shared decision-making and patient experience. Further research is required to determine appropriate target reference ranges if the new lower capillary levels can be used routinely, especially in the climate of COVID-19 where social distancing measures limit patient access to hospitals and clinics for routine investigations.
P441 Molecular identification, genotyping, and antifungal susceptibility of Trichosporon species isolated from clinical samples of patients at various parts of the Indian subcontinent
Authors: Parashar Abhila , Rastogi Vijaylatha , Prakash Hariprashad , Rudramurthy Shivaprakash M. 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
(1) To study mycological characteristics of strains belonging to Trichosporon and its related genera obtained from clinical samples of patients from India. (2) Molecular identification by intergenic spacer (IGS) region 1 sequencing of the rDNA locus. (3) Genotyping of the major causative agent, T. asahii, and its in vitro drug susceptibility testing.

Materials and Methods
A total of 55 clinical isolates of Trichosporon species were collected from NCCPF (National culture collection of pathogenic fungi) PGIMER, Chandigarh along with different health institutions of India. These isolates were recovered from urine, blood, sputum, nail, tissue biopsy, pleural fluid, hair, BAL, and wound discharge over a period of 12 years (2006–2018). The isolates were molecularly characterized and genotyped using IGS-1 region sequencing. In vitro drug susceptibility testing of the isolates was performed against amphotericin-B, fluconazole, itraconazole, voriconazole, and posaconazole according to the CLSI M27-A3 guidelines (CLSI 2008).

Results
Predominant underlying risk factors identified were presence of an indwelling catheter, use of broad-spectrum antibiotics, and presence of comorbid conditions such as diabetes, hypertension, and anemia. A total of 47 (85%) of the 55 isolates were identified as T. asahii, 6 were T. inkin (11%), and 2 were Cutaneo Trichosporon dermatis (3.6%). Trichosporon asahii genotype III (22; 41%) was the most common type, followed by genotype IV (12; 22%), I (8; 15%), and VII (2; 4%). In addition to the 15 known T. asahii genotypes, one novel genotype was identified in this study. Indian T. asahii isolates showed high MIC ranges to amphotericin B (0.06-4 μg/l) and fluconazole (0.25-64 μg/l). Relatively low MIC ranges were found in the case of voriconazole (0.03-1 μg/l), Posaconazole (0.06-1 μg/l), and itraconazole (0.06-1 μg/l). Voriconazole appeared to be the most active drug in maximum T. asahii isolates. The MICs for all the drugs were comparatively lower in the case of non-T. asahii strains.

Conclusion
Trichosporon asahii remains the most common etiology of Trichosporonosis in India and presents a challenge for both diagnosis and treatment. With increasing drug resistance, therapeutic options are limited, and antifungal regimens with triazoles especially voriconazole appear to be the best. Accurate timely identification, removal of indwelling catheters/central venous lines, and voriconazole-based treatment along with control of underlying conditions were associated with favorable outcomes. Identification of the novel genotype has epidemiological implications and requires further work up.
P455 Identification of Cryptic species of Aspergillus using Beta-tubulin gene in a tertiary care center in South India
Authors: Karunai Almas Fathima Upaisal , Jyoti Kindo Anupma , Kumar S. Prasanna , Thirunarayan M. , Sree V. Lakshmi 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
To identify cryptic species of the genus Aspergillus using Beta-tubulin gene by sequence typing.

Methods
Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud's Dextrose Agar/Oatmeal Agar.
Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape, and arrangement of the conidia.
DNA Extraction using phenol-chloroform method: DNA was extracted and purified directly from 4-day-old culture and used as a template for polymerase chain reaction (PCR) amplification.
DNA Amplification: A fragment of the target gene were amplified using PCR.
The primer pairs used for Beta-tubulin gene was Bt2a (Forward primer) and Bt2b (Reverse primer).
Beta-tubulin.
DNA Purification: The PCR product was purified with multi-screen filter plates.
Gel electrophoresis: The amplified DNA product was subjected to agarose gel electrophoresis and specific band formation was to be observed for the species of Aspergillus.
DNA Sequencing: The purified product will be used as a template for sequencing. An applied biosystem 3730 sequencer will be used to obtain DNA sequences.
Phylogenetic Analysis: Parsimony analysis of individual and combined matrices will be conducted using PAUP version 4.0b10 software/free software at phylogeny.fr by the maximum parsimony (MP) and Neighbor-joining methods.
The results will be represented using a dendrogram.

Results
A total of 30 Aspergillus isolates were collected and identified using Phenotypic methods.
The DNA extraction and PCR amplification were done and sent for Sequencing. The results of Sequence typing are awaited.

Conclusion
Cryptic species are morphologically indistinguishable forms of Aspergillus and their identifications can be confirmed exclusively by using molecular techniques which have led to the description of previously unknown or rare species among different Aspergillus species complex. According to studies, the frequency of such species was found to be 10% in TRANSNET study U.S and 12% in FILPOP study Spain. Most of these species are less susceptible and certain species are multidrug-resistant (azoles and amphotericin B) and the emergence of antifungal-resistance among the Aspergillus species forms the importance in identifying Cryptic species.
P460 The Aspergillus Lateral Flow Assay for the diagnosis of invasive Aspergillosis
Authors: Liu Chonglong , Peng Jie , Zhang Yuan , Wang Ying , Wang He , Su Yan , Zhou Zeqi 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
Invasive Aspergillosis (IA) is a life-threatening opportunistic mycosis that occurs in some people with a compromised immune system. The detection of Aspergillus galactomannan (GM) rapidly gained widespread acceptance as part of the diagnostic workup of a patient suspected of IA. Recently, a Novel QuicGM™ Aspergillus Galactomannan Ag Lateral Flow Assay (LFA) from Dynamiker Biotechnology (Tianjin) Co., Ltd. was released as a screening test of IA. It is a fluorescent immunochromatographic cassette test which using a monoclonal antibody against GM and Europium nanoparticles (Eu NP) (Fig. 1). It is a semi-quantitative test which was easy to operate, rapid with portable detection devices, and can be widely accepted by clinical and primary medical (Fig. 2). We conducted a comparative study between the Dynamiker QuicGMTM Aspergillus Galactomannan Ag (LFA) and the Bio-Rad Platelia™.

Methods
A total of 66 serum samples and 54 BALF samples were collected, 15 serum samples from the College of American Pathologists (CAP) certificated lab were also included in this study. The samples were tested in parallel using the Bio-Rad Platelia™ Aspergillus Ag (ELISA) and Dynamiker QuicGM™ Aspergillus Galactomannan Ag (LFA).

Results
The total coincidence rate of the two assays was 98.48% (Kappa value was 0.97 &gt;0.75) and 90.74% (Kappa value was 0.81 &gt;0.75) in 66 serum samples and 54 BALF samples respectively.

Conclusions
The performance of the Dynamiker QuicGM™Aspergillus Galactomannan Ag (LFA) and Bio-Rad Platelia™ Aspergillus Ag (ELISA) were highly consistent, and the performance of Dynamiker Aspergillus Galactomannan kit were all in line with the statement of the manufacturer. The results are accurate and reliable, and it could be used as an aid for the early rapid screening test of IA.