Mold Infections Research Articles

In situ polymerization of N-methylol acrylamide (NMA) for bamboo anti-mold modification

Bamboo is an eco-friendly and sustainable material but with limited application due to its susceptibility to mold infection. N-methylol acrylamide (NMA) is a cross-linking monomer with bifunctional groups, here NMA was used to modified bamboo to enhance its anti-mold property through in situ polymerization and cross-linking reaction. When the monomer concentration was 6% and above, no mold germinated at the NMA treated bamboo surface. SEM/EDX, FTIR was used to explore the anti-mold mechanism of NMA modified bamboo. The primary reason for the improvement in the anti-mildew property was the coating effect of PNMA on starch as well as the antimicrobial properties of PNMA. Thermal stability of bamboo before and after modification was almost unchanged. Results also indicated that the mechanical properties of modified bamboo are improved by around 50%, which was mainly attributed to more compact structure of cell wall obtained in the neutral reaction condition. The NMA in situ polymerization was proved to be a potential modification for bamboo to broaden its application.

Uçucu Yağ Uygulamalarının Depolama Devresinde Şeker Pancarı (Beta vulgaris L.) Kalitesine Etkileri

This study was carried out in order to determine the effects of dill, clove and hyssop essential oils and fungicides applied on sugar beet roots on beet storage quality during the storage period. The roots of Esperanza (KWS) variety, which was produced in the experimental areas in 2019, were used in the study. Roots were treated with 100, 500 and 1000 ppm doses of dill (Anethum graveolens L.), clove (Szygium aromaticum) and hyssop (Echinophora tenuifolia) essential oils, synthetic fungicide (80% Thiram) and Tween-80 right after harvest and the roots were placed in the storage. Weight loss of roots determined at 15-day intervals from the beginning of the storage period, dry matter ratio, brix, polar sugar, reducing sugar and alpha amino nitrogen contents, phytotoxicity and fungal infection development at the end of the 3-month storage period. The applications made in the research significantly affected the post-harvest weight and quality losses in beet. Depending on the applications, the weight losses at the end of the 3-month storage period varied between 9.43-19.90%, and the weight losses in essential oil applications were lower than the control. The highest dry matter content and brix values were obtained from clove essential oil and fungicide and Tween-80 applications. While Tween-80 and fungicide applications increased the polar sugar content compared to the control, 1000 ppm clove essential oil caused a significant decrease in the polar sugar content. In the study, reducing sugar and alpha amino nitrogen contents of roots applied 1000 ppm essential oil E. tenuifolia essential oil were lower. E. tenuifolia essential oil showed the highest antifungal activity against white mold and green mold infections. It was concluded that with the application of essential oil to the roots after the harvest, both weight and quality losses and the development of fungal diseases can be reduced, however, some active substances may cause phytotoxicity in the roots.

Liposomal amphotericin B-the present.

Most invasive fungal infections are opportunistic in nature but the epidemiology is constantly changing, with new risk groups being identified. Neutropenia is a classical risk factor for fungal infections, while critically ill patients in the ICU are now increasingly at risk of yeast and mould infections. Factors to be considered when choosing antifungal treatment include the emergence of rarer fungal pathogens, the risk of resistance to azoles and echinocandins and the possibility of drug-drug interactions. Liposomal amphotericin B has retained its place in the therapeutic armamentarium based on its clinical profile: a broad spectrum of antifungal activity with a low risk of resistance, predictable pharmacokinetics with a rapid accumulation at the infection site (including biofilms), a low potential for drug-drug interactions and a low risk of acute and chronic treatment-limiting toxicities versus other formulations of amphotericin B. It is a suitable choice for the first-line empirical or pre-emptive treatment of suspected fungal infections in neutropenic haematology patients and is an excellent alternative for patients with documented fungal disease who can no longer tolerate or continue their first-line azole or echinocandin therapy, both in the haematology setting and in the ICU. Moreover, it is the first-line drug of choice for the treatment of invasive mucormycosis. Finally, liposomal amphotericin B is one of the few antifungal agents approved for use in children of all ages over 1 month and is included in paediatric-specific guidelines for the management of fungal disease.

Survey on the rate of microbial infection, contaminants of heavy metals and nitrite in bird’s nest form some southerwest provinces

This survey aimed at assessing the level of contamination with some microorganisms, some heavy metals and nitrite in semi-processed and raw bird's nest samples in a number of provinces and cities including Binh Phuoc, Binh Duong, Đong Nai, Binh Thuan, Tay Ninh, Ho Chi Minh, Kien Giang, An Giang, Bac Lieu, Can Tho, Gia Lai, Quang Nam, Quang Ngai, Phu Yen. Test results showed that E. coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, arsenic, and cadmium were not detected in the collected samples; Salmonella, were detected in 03 semi-processed of total 84 samples, accounting for 3.57%. The mold infection rate accounted for 22.92% (11/48 samples); there were 14 raw bird's nest samples exceeding the permited limit of nitrite content, in 80 tested samples, accounting for 17.50% (14/80 samples); 02 semi-processed samples collected at the processing facility detected lead and mercury content exceeded the permitted limit, accounting for 2.50% (2/80 samples).  

Dosage, Safety and Efficacy of Posaconazole As Primary Antifungal Prophylaxis in High-Risk Recipients of Allogeneic Stem Cell Transplantation

Introduction: The use of anti-mold prophylaxis with anti-aspergillus activity is recommended for patients (pts) undergoing allogeneic hematopoietic stem cell transplantation (Allo-HSCT) with high risk of fungal infection (Maertens, J Antimicrob Chemother 2018). Yet, few data are available regarding the use of posaconazole. Methods: This retrospective study included all Allo-HSCT adult recipients having benefited from posaconazole therapy in our center as a primary anti-fungal prophylaxis for high-risk of fungal infection. The latter was defined as use of i)an alternative donor, ii)sequential conditioning regimen, iii)a regimen associated with severe mucositis, iv) post-transplant cyclophosphamide (PT-Cy) or v) previous allo-HSCT. Posaconazole was programmed to be initiated at day (D) 0 or D5/6 (in case of PT-Cy) and administered up to day 100. We report here on the residual concentration of posaconazole during the neutropenic phase then on the tolerance and efficacy of such prophylaxis in this cohort. Results: Seventy high-risk pts (males 63.3%, median age 54 years old, range 20-73) received Allo-HSCT between April 2020 and December 2021 for a myeloid (n=51), lymphoid (n=17) or non-malignant hematological (n=2) disease and received posaconazole as a primary prophylaxis. The Sorror score at transplant time was ≥ 3 for 24 pts (33.8%). Twenty-two patients had already received posaconazole as a primary prophylaxis before Allo-SCT including 26 during the neutropenic phase of their induction for AML or MDS, 3 for prolonged neutropenia, 2 during treatment with alemtuzumab for T-cell leukemia/lymphoma, and 1 during a previous AlloHCST. Four patients had also received other antifungal agentsbefore Allo-SCT (caspofongine (n=1), voriconazole (n=1) or liposomal amphotericin B (n=2)) because of a suspected mold infection which has been finally excluded. All pts had at least one high-risk criterion of fungal infection. These included the use of an alternative donor in 42 pts (haplo-identical donor n= 41, umbilical cord blood n=1), sequential conditioning regimen in 9, with PT-Cy in 57, second or third allo-HSCT in 8 and conditioning regimen associated with severe mucositis in 11 (TBI 8-12gy n=3, thiotepa-based n=8). All but one pts received posaconazole using the gastroresistant tablet form at a dose of 300mg/day. One patient received the drinkable suspension form at a dose of 200mg 3 times a day. Following prophylaxis initiation, serum posaconazole concentrations were available in 51 and 16 patients at medians of 9 and 39 days, respectively. A protective residual level of posaconazole (> 0.5mg/L) was achieved in 57% of the patients (n=29) at first assessment and 87.5% (n=12) at second evaluation. Posaconazole was stopped early due to related-toxicity in 9 cases (12.6%), including liver toxicity in 7 pts, QT prolongation in 1 and skin hyperesthesia in 1. Treatment was restarted without recurrence of toxicity in 2 cases. Posaconazole was stopped early in 2 patients due to non-related toxicity, including one veno-occlusive disease and one hepatic acute GVHD. Posaconazole was temporary discontinued in 4 patients due to a suspected fungal infection before being restarted. Most patients (n=59/70, 84.2%) did not present fungal infections while being under posaconazole therapy. Only 11 cases were documented with this complication (Table 1), including 8 invasive pulmonary aspergillosis (IPA; possible n=3; probable n=5), one possible invasive fungal infection and 2 mucormycoses (1 with possible IPA). Fungal infections occurred despite documentation of a protective residual level of posaconazole (> 0.5mg/L) in 5 of 6 evaluable patients. Of note, 9/11 patients had received PTCY as GVHD prophylaxis. Posaconazole was stopped (before D7 in 3) and other antifungal drugs started (voriconazole or liposomal amphotericin B) allowing to fungal infection cure in 5 pts. However, 7 pts died including 2 of an uncontrolled fungal infection. Conclusion: Posaconazole is a well-tolerated and efficient primary prophylaxis in recipients of Allo-HSCT at high-risk of fungal infection. As some fungal infections occured very early after D0 while prophylaxis started only a D5/6 after PTCY, our results suggests to propose posaconazole as early as the beginning of the conditionning together with dose monitoring. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

60-Year-Old Patient with Invasive Mold Infection Consequent to an Encounter with a Barnyard Animal

Abstract Introduction/Objective Lichtheimia and Scedosporium species are ubiquitous fungi associated with a range of clinical presentations. We report a complex soft tissue coinfection caused by Lichtheimia spp. and Scedosporium spp. following a puncture wound by a rooster spur. Methods/Case Report A 60-year-old immunosuppressed man with a history of heart transplant presented with a non-healing wound 2-months after sustaining a lower leg puncture secondary to a rooster spur. The patient was previously treated with Augmentin for presumed cellulitis without improvement. To characterize the ongoing disease process, the wound was sampled with tissue portions routed for both histopathologic review and culture. In short order, the biopsy demonstrated invasive hyaline pauciseptate fungal hyphae consistent with organisms of the Mucorales order. In congruence, culture rapidly revealed a mucoraceous mold, subsequently identified as a Lichtheimia spp. Thus, the patient was treated with liposomal amphotericin B. However, days later, a second fungus - a Scedosporium spp. - was isolated from the original specimen. Consequently, isavuconazonium sulfate therapy was added. Re-review of the initial biopsy enabled distinction of two populations of fungal hyphae plausibly representing both molds. Despite numerous debridements and optimal medical management, the patient was ultimately referred for amputation. Results (if a Case Study enter NA) NA. Conclusion When encountered in an immunocompromised host, providers understandably respond to an invasive mucoraceous mold with alarm followed by quick, decisive action. However, septate molds, such as the Scedosporium spp., can also be aggressive. Further, the presence of more than one invasive mold presents a treatment challenge as the mucormycetes may require surgery coupled with amphotericin B therapy; whereas, the a Scedosporium spp. may be wholly resistant to amphotericin B. This case illustrates the critical role that an alert pathologist plays in guiding management through integration of data from different diagnostic modalities. It also constitutes an emphatic reminder that an illness may be caused by more than one actionable, invasive fungus.

Artificial Intelligence in the Diagnosis of Invasive Mold Infection: Development of an Automated Histologic Identification System to Distinguish Between Aspergillus and Mucorales.

Histopathological identification is usually required since the sensitivity of fungal culture is not sufficient for accurate diagnosis. On the other hand, pathological diagnosis, especially of molds, often is not accurate, even when performed by an experienced pathologist. This is particularly true in the differentiation between mucormycosis and aspergillosis, which have different drugs of choice and medical management. The diseases can easily become severe in a short period of time in accordance with the severity of the underlying disease or predisposing factors. Therefore, correct diagnosis is extremely important and should be entrusted to the pathologist. To develop an artificial intelligence (AI)-based automated histological diagnostic system for mold infection to support the diagnosis by general pathologists, especially for distinguishing between Aspergillus and Mucorales. We used two indicators for the diagnostic system; namely, the angle of independent hyphae and tortuosity of each hypha. We collected 147 and 67 image samples respectively from standard cases of aspergillosis and mucormycosis. All the images were successfully analyzed by automatic recognition of the two indicators. The independent areas divided by the threshold curve generated by two-dimensional plots of the data clearly include the test data obtained from the cases of Aspergillus and Mucorales. The present study demonstrates the usefulness of our newly developed AI-based diagnostic system. Further investigation is required for its practical use.

Acute Lymphoblastic Leukemia and Invasive Mold Infections: A Challenging Field.

Acute lymphoblastic leukemia (ALL) patients comprise a highly immunocompromised group due to factors associated either with the treatment or the disease itself. Invasive mold infections (IMIs) are considered to be responsible for higher morbidity and mortality rates in patients with hematologic malignancies, including ALL. Defining the exact incidence of IMIs in ALL patients has been rather complicated. The available literature data report a highly variable incidence of IMIs, ranging from 2.2% to 15.4%. Although predisposing factors for IMIs in the setting of ALL are ill-defined, retrospective studies have indicated that a longer duration of neutropenia, treatment with high-dose corticosteroids, and a lack of antimold prophylaxis are associated with an increased risk of IMIs. Additionally, the influence of novel ALL treatments on the susceptibility to fungal infections remains obscure; however, initial data suggest that these treatments may induce prolonged neutropenia and thus an increased risk of IMIs. Administering primary antimold prophylaxis in these patients has been challenging since incorporating azole antifungal agents is troublesome, considering the drug-to-drug interactions (DDIs) and increased toxicity that may occur when these agents are coadministered with vincristine, a fundamental component of ALL chemotherapy regimens. Isavuconazole, along with several novel antifungal agents such as rezafungin, olorofim, and manogepix, may be appealing as primary antimold prophylaxis, given their broad-spectrum activity and less severe DDI potential. However, their use in ALL patients needs to be investigated through more clinical trials. In summary, this review outlines the epidemiology of IMI and the use of antifungal prophylaxis in ALL patients.

Ibrexafungerp, a Novel Triterpenoid Antifungal in Development for the Treatment of Mold Infections.

Molds are ubiquitous in the environment, and immunocompromised patients are at substantial risk of morbidity and mortality due to their underlying disease and the resistance of pathogenic molds to currently recommended antifungal therapies. This combination of weakened-host defense, with limited antifungal treatment options, and the opportunism of environmental molds renders patients at risk and especially vulnerable to invasive mold infections such as Aspergillus and members of the Order Mucorales. Currently, available antifungal drugs such as azoles and echinocandins, as well as combinations of the same, offer some degree of efficacy in the prevention and treatment of invasive mold infections, but their use is often limited by drug resistance mechanisms, toxicity, drug-drug interactions, and the relative paucity of oral treatment options. Clearly, there is a need for agents that are of a new class that provides adequate tissue penetration, can be administered orally, and have broad-spectrum efficacy against fungal infections, including those caused by invasive mold organisms. Ibrexafungerp, an orally bioavailable glucan synthase inhibitor, is the first in a new class of triterpenoid antifungals and shares a similar target to the well-established echinocandins. Ibrexafungerp has a very favorable pharmacokinetic profile for the treatment of fungal infections with excellent tissue penetration in organs targeted by molds, such as the lungs, liver, and skin. Ibrexafungerp has demonstrated in vitro activity against Aspergillus spp. as well as efficacy in animal models of invasive aspergillosis and mucormycosis. Furthermore, ibrexafungerp is approved for use in the USA for the treatment of women with vulvovaginal candidiasis. Ibrexafungerp is currently being evaluated in clinical trials as monotherapy or in combination with other antifungals for treating invasive fungal infections caused by yeasts and molds. Thus, ibrexafungerp offers promise as a new addition to the clinician's armamentarium against these difficult-to-treat infections.

Checkpoint inhibitors as immunotherapy for fungal infections: Promises, challenges, and unanswered questions.

Opportunistic fungal infections have high mortality in patients with severe immune dysfunction. Growing evidence suggests that the immune environment of invasive fungal infections and cancers share common features of immune cell exhaustion through activation of immune checkpoint pathways. This observation gave rise to several preclinical studies and clinical case reports describing blockade of the Programmed Cell Death Protein 1 and Cytotoxic T-Lymphocyte Antigen 4 immune checkpoint pathways as an adjunct immune enhancement strategy to treat opportunistic fungal infections. The first part of this review summarizes the emerging evidence for contributions of checkpoint pathways to the immunopathology of fungal sepsis, opportunistic mold infections, and dimorphic fungal infections. We then review the potential merits of immune checkpoint inhibitors (ICIs) as an antifungal immunotherapy, including the incomplete knowledge of the mechanisms involved in both immuno-protective effects and toxicities. In the second part of this review, we discuss the limitations of the current evidence and the many unknowns about ICIs as an antifungal immune enhancement strategy. Based on these gaps of knowledge and lessons learned from cancer immunology studies, we outline a research agenda to determine a "sweet spot" for ICIs in medical mycology. We specifically discuss the importance of more nuanced animal models, the need to study ICI-based combination therapy, potential ICI resistance, the role of the immune microenvironment, and the impact of ICIs given as part of oncological therapies on the natural immunity to various pathogenic fungi.

Non-Aspergillus mould lung infections.

Non-Aspergillus filamentous fungi causing invasive mould infections have increased over the last years due to the widespread use of anti-Aspergillus prophylaxis and increased complexity and survival of immunosuppressed patients. In the few studies that have reported on invasive mould infection epidemiology, Mucorales are the most frequently isolated group, followed by either Fusarium spp. or Scedosporium spp. The overall incidence is low, but related mortality is exceedingly high. Patients with haematological malignancies and haematopoietic stem cell transplant recipients comprise the classical groups at risk of infection for non-Aspergillus moulds due to profound immunosuppression and the vast use of anti-Aspergillus prophylaxis. Solid organ transplant recipients also face a high risk, especially those receiving lung transplants, due to direct exposure of the graft to mould spores with altered mechanical and immunological elimination, and intense, associated immunosuppression. Diagnosing non-Aspergillus moulds is challenging due to unspecific symptoms and radiological findings, lack of specific biomarkers, and low sensitivity of cultures. However, the advent of molecular techniques may prove helpful. Mucormycosis, fusariosis and scedosporiosis hold some differences regarding clinical paradigmatic presentations and preferred antifungal therapy. Surgery might be an option, especially in mucormycosis. Finally, various promising strategies to restore or enhance the host immune response are under current evaluation.

Morphologic identification of clinically encountered moulds using a residual neural network.

The use of morphology to diagnose invasive mould infections in China still faces substantial challenges, which often leads to delayed diagnosis or misdiagnosis. We developed a model called XMVision Fungus AI to identify mould infections by training, testing, and evaluating a ResNet-50 model. Our research achieved the rapid identification of nine common clinical moulds: Aspergillus fumigatus complex, Aspergillus flavus complex, Aspergillus niger complex, Aspergillus terreus complex, Aspergillus nidulans, Aspergillus sydowii/Aspergillus versicolor, Syncephalastrum racemosum, Fusarium spp., and Penicillium spp. In our study, the adaptive image contrast enhancement enabling XMVision Fungus AI as a promising module by effectively improve the identification performance. The overall identification accuracy of XMVision Fungus AI was up to 93.00% (279/300), which was higher than that of human readers. XMVision Fungus AI shows intrinsic advantages in the identification of clinical moulds and can be applied to improve human identification efficiency through training. Moreover, it has great potential for clinical application because of its convenient operation and lower cost. This system will be suitable for primary hospitals in China and developing countries.

S3505 A Case of Invasive Gastrointestinal Mold Infection

IntroductionL: Invasive mold infections can be devastating and are mostly seen in patients with hematologic malignancies, profound neutropenia, solid organ or hematopoietic cell transplantation, and poorly controlled diabetes mellitus. Mucormycosis is caused by a group of ubiquitous molds called Mucormycetes and characterized by tissue infarction and necrosis that results from invasion of the vasculature by hyphae. We present a rare case of a patient with invasive gastrointestinal mold infection. Case Description/Methods: A 40-year-old male with chronic inflammatory demyelinating polyneuropathy (CIDP) was admitted for autologous stem cell transplantation (ASCT). Initial hospitalization was complicated by neutropenic fever and C. difficile enteritis treated with Fidaxomicin. He was admitted one month later for nausea, vomiting and push enteroscopy revealed two circumferential deep small bowel ulcerations and a jejunal stricture too distal to be reached. Ulcer biopsies showed granulation tissue and was positive for Cytomegalovirus. He was treated with Valganciclovir and initiated on TPN. One week later, he had a third admission for intractable nausea and vomiting due to a small bowel obstruction. He failed conservative management and underwent diagnostic laparoscopy that revealed five strictures in the distal small bowel which were all resected with two small bowel resections and re-anastomosed. Histopathology of resected jejunum showed necrotizing granulomatous inflammation with aseptated fungal hyphae concerning for Mucormycosis, which was not identified on fungal PCR. He was treated with liposomal Amphotericin B then transitioned to oral Isavuconazole before making a full recovery. (Figure) Discussion: The most common presentation of Mucormycosis is a rhino-orbital-cerebral infection. Gastrointestinal infection is rare in living patients and has been estimated to occur in around 7% of all Mucormycosis cases and carries mortality as high as 85%. Our patient had a history of refractory CIDP and underwent chemotherapy for ASCT that was complicated by development of C. difficile and CMV gastrointestinal infections and small bowel obstruction necessitating endoscopic intervention which led to exploratory laparoscopy for surgical resection and the diagnosis of invasive Mucormycosis. Although invasive GI Mucormycosis is rare, in severely immunocompromised patients with unexplained gastrointestinal symptoms and multiple risk factors as presented in our patient, this infectious organism can be considered in the differential.Figure 1.: (A, B) Circumferential ulcerations seen in the small bowel on push enteroscopy. (C) Mucosal ulceration with underlying necrotizing granulomatous inflammation in the deep muscularis propria/pericolonic adipose tissue with non-septated fungal hyphae present.

S1.4b Challenges in diagnosing and management of invasive fungal infections during the pandemic

S1.4 Fungal infections in Asia, bringing it out of the dark, September 21, 2022, 11:00 AM - 12:30 PMInvasive fungal diseases have been increasing in Asian countries. Recent advances in novel medical care such as solid organ and stem cell transplantations, chemotherapy for cancer treatment, and corticosteroid therapy, resulted in the increased prevalence of invasive mycoses. Invasive aspergillosis, mucormycosis, and endemic mycoses are among the most common mold infections in Asia. Non-classical and novel risk factors of invasive fungal diseases have been increasingly recognized in Asia. In contrast to the classical neutropenic patients, most of the patients with invasive mycoses who had non-classical risk factors are mostly non-neutropenic and may present with an atypical clinical manifestation. These novel risk factors include biologic agents or small molecule kinase inhibitors used for cancer treatment, and severe viral pneumonia such as influenza pneumonia or coronavirus disease 2019 (COVID-19) pneumonia. Recently, COVID-19-associated aspergillosis (CAPA) and COVID-19-associated mucormycosis (CAM) have been described. These particular mold infections had high mortality. Treatment of CAPA and CAM are similar to those who had mold infections without COVID-19. However, the interaction between triazole and drugs used for the treatment of COVID-19 must be taken into consideration. Timely and accurate diagnoses are crucial for the management of invasive fungal infections. Conventional fungal cultures from sterile clinical samples or blood are useful but they are time-consuming. Nevertheless, the diagnosis of invasive mold infections is challenging as the imaging is non-specific and the serological tests are not widely available in Asian countries. In some circumstances such as those with non-classical risk factors, serology revealed relatively low sensitivities. Molecular diagnostic tests are also the unmet needs among Asian countries for timely and accurate diagnosis of invasive fungal diseases. Several factors should be considered for the appropriate choice of antifungal agents, including antifungal coverage, adverse effects, underlying conditions, drug-drug interactions, and cost. Recently, novel antifungal agents such as novel triazoles or new classes of antifungal agents have been studied and may be a promising choice for the treatment of invasive fungal diseases.

P168 Metastatic mo ld infections after COVID-19: the mo ld time -bomb

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectiveCOVID-19-positive patients are at risk of invasive mold infection. The portal of entry is presumably the lungs, but infection may disseminate to involve other organs as described in this case series.Patients and MethodsPatients who presented with de novo mold infection (6 proven and 1 probable) involving kidney, spine, eye, knee joint, subcutaneous tissue within 10-180 days of COVID-19 infection were included in this series (Table 1).ConclusionThese cases suggest that metastatic infection by molds involving various organs could result from a presumed primary source in the lungs. However, the precise connection between pulmonary and metastatic infection is difficult to establish.COVID-19 patients should be carefully followed for such metastatic mold infections which need proper diagnosis and management.

S2.3c High-resolution digital DNA melting: a breakthrough in diagnosing IMI?

S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PMInvasive mold infections (IMIs) such as Aspergillosis, Mucormycosis, Fusariosis, and Lomentosporiosis have emerged as important pathogens in immunocompromised patients, with mortality rates as high as 50% to nearly 80% for these infections. Outcomes can be substantially improved with early initiation of appropriate antifungal therapy, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive microbiologic findings. Conventional fungal culture and PCR techniques are limited by low sensitivity, long turn-around time, and are insufficient for differentiating between infection and colonization. Other non-culture-based tests, such as the Galactomannan test, are available only for invasive aspergillosis and are limited by imperfect test performance. Better and more rapid diagnostics are needed to enable early diagnosis and targeted treatment of IMIs and improve survival. We have developed a new technology called digital high resolution melting analysis (dHRM) to enable a rapid and robust diagnosis of IMI. This technology accomplishes fast genotyping of fungal genomic sequences in clinical samples by: (1) conducting broad-based amplification of fungal genes in a digital PCR format, and (2) conducting high-resolution melting of the DNA amplicons in each digital reaction. High resolution melting measures the fluorescence of a saturating intercalating dye as dPCR-amplified fungal DNA fragments are rapidly heated and disassociated, producing sequence-defined melt curves in a closed reaction format. These melt curves serve as unique fungal ‘fingerprints’, which allows us to correctly identify disease-causing pathogen(s) with an accuracy of 99%-100% through the use of machine learning methods trained to tolerate variations in reaction conditions. This approach provides a simple, low-cost, fast, and robust method for pathogen identification. Here, we will present the performance of this new technology on clinical bronchoalveolar lavage (BAL) samples from patients with and without IMI. A total of 75 patient BAL samples were tested, coming from 30 patients with proven (n = 1), probable (n = 25), or putative (n = 4) invasive pulmonary aspergillosis (IPA) infections, and from 45 patients classified as not having IPA (n = 4 possible IPA and n = 41 no IPA). Our results show that dHRM was able to reliably differentiate between different Aspergillus spp. and was also able to identify Aspergillus spp. in BAL samples from patients with probable IPA where qPCR had resulted in negative. In a few instances, it also detected rare molds in culture-negative samples and multiple Aspergillus spp. within one BAL sample, a phenomenon that has been previously described in cultures. Interestingly, there was no clear correlation between dHRM results and BAL galactomannan levels, suggesting that dHRM may serve as an independent method allowing for a combined diagnostic approach. While further validation and optimization are needed, these results suggest that dHRM is a promising new diagnostic technology capable of providing clinically relevant information within 2-3 h.

P300 Importance of direct microscopy in respiratory mold infection: a diagnostic dilemma

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM IntroductionOpportunistic fungal infections are on the rise due to a changing healthcare paradigm worldwide. India is the second most populated country in the world and is experiencing an upward trend in fungal lung diseases.ObjectiveRapid diagnosis of respiratory mold infections by direct microscopy.Material and MethodsWe retrospectively analyzed the mycology of the received respiratory samples; sputum, BAL, or ETT secretion between January 1, 2021 to March 31, 2022. All the samples were processed as per standard mycological procedures. For each sample, two KOH wet mounts were prepared and examined. Samples were inoculated over SDA and blood agar and incubated at 28°0C and 37°C in BOD. Once there was growth, it was processed as per standard protocol.ResultsWe received a total of 109 respiratory samples from suspected respiratory fungal infections of which early morning sputum samples 84 (77%) were the predominant followed by BAL samples 25 (23%). Most of the enrolled patients were male 73(67%) with a mean age of 48 years. Among 109 samples, 83 (76%) were shown to be fungal elements by KOH wet mount while 26 (24%) were KOH negative. A total of 53 (48.6%) samples were culture-positive Aspergillus flavus30(27%), was found to be the most common species isolated followed by A. fumigatus 16 (14.6%), miscellaneous11(10%), Aspergillus. species 5 (4.5%), and A. terreus 2 (1.8%).ConclusionConsidering the findings of KOH mount in our study, we would like to emphasize that this is a very simple, rapid, and cost-effective laboratory method with high diagnostic sensitivity when correlated with clinical findings, radiological findings, and host factors.Further, it has been found to be very dependable for making decisions to start empirical antifungal therapy early by clinicians.

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.

S2.3d Mixed biofilm of Aspergillus fumigatus and Stenotrophomonas maltophilia: Microscopic visualization of galactosaminogalactan and galactomannan polysaccharides in the extracellular matrix

S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PM Introduction Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are commonly co-isolated from the airways of cystic fibrosis patients, especially in mixed biofilms. A mixed Af-Sm biofilm model, developed by our lab, demonstrated that Sm exhibits an antibiosis effect on Af by (1) inhibiting fungal growth, (2) rendering the fungal mycelia highly branched, and (3) increasing the fungal cell wall thickness.1 The presence of three Af cell wall polysaccharides, galactomannan (GM), galactosaminogalactan (GAG), and α-1,3-glucan, have been described in the extracellular matrix (ECM) of Af biofilms.2 GM and GAG are known to be released out from the cell wall of Af.3 GAG contains N-acetylgalactosamines (GalNAc) which undergo partial deacetylation extracellularly, to be converted to GalN, and thus transform this polysaccharide into a polycation with strong surface binding properties.4Aim of the studyAnalyze the structure of fungal cell wall polysaccharides in the formation of Af biofilm alone or mixed with Sm.MethodsThe Af-Sm co-culture was prepared on an 8-chambers Lab-Tek slide as described previously1. Anti-GAG and anti-GM monoclonal antibodies produced at the Aspergillus Unit at Pasteur Institute of Paris, were used to focus on the 3D structure of GAG and GM in the ECM of the biofilm. These polysaccharides were analyzed by fluorescence confocal microscopy at 24 h.Results and ConclusionGM was found, as expected, in the hyphal Af cell wall but was very little in the ECM. GAG was also found in the cell wall but mainly formed a beautiful fibrillary network between the hyphae, showing the importance of this polysaccharide in cell-cell interaction and in the structuration of Af biofilm. GAG could be the surface receptor for Sm, which would promote strong adhesion between Sm and Af in the biofilm.Sources1. Melloul E et al. Front Microbiol. 2018;2. Beauvais A et al. Microbiol Spectr. 2015;3. Fontaine T et al. J Fungi. 2020;4. Briard B et al. Mycologia 2016.

S2.1d Incidence of mixed fungal infections in post-COVID-19 outbreak of Mucormycosis

S2.1 Update on Mucormycosis, September 21, 2022, 3:00 PM - 4:30 PM IntroductionPost-COVID-19 rhino orbital mucormycosis has emerged as an important life-threatening complication adding to mortality. Fungal infections are a major health challenge, especially in the immunocompromised.Mucormycosis is a severe, frequently fatal fungal infection that has a unique predisposition to infect patients with diabetes. The most probable reasons for the emergence of these cases could be the extensive use of steroids in the management of coronavirus disease 2019 (COVID-19) patients and extensive dysregulated immune response due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A prompt diagnosis is vital for the effective management of invasive rhino-orbital fungal infections due to their propensity for angioinvasion and destructive spread with brain involvement.Materials and MethodsA total of 150 surgical pathology specimens received with a clinical suspicion of invasive fungal infection during the post-COVID-19 outbreak of mucormycosis were retrieved from the archives of the Department of histopathology. The cases were reviewed for the presence of Aspergillus fruiting bodies by senior pathologists and microbiologists on a multi-headed microscope. The morphological features of the fruiting bodies were noted and correlated with the fungal KOH and culture. The tissue reaction pattern, presence of oxalate crystals, and morphology of the fungal hyphae were also noted in each case showing Aspergillus fruiting bodies.ResultsA total of 8 out of 150 cases (5.3%) showed the presence of Aspergillus fruiting bodies. The histopathological diagnosis given in these 8 cases were—Aspergillus (1), combined Aspergillus and Mucorales (7). Two types of fungal hyphae were noted in all seven cases of combined infection. Granulomatous tissue reaction was noted in two out of seven cases of combined infection. Calcium oxalate crystals were noted in the single case of Aspergillosis and were absent in all cases of mixed infection.ConclusionTo conclude Aspergillus fruiting bodies are found in a small but significant number of cases of post-COVID-19 Rhino-Orbital invasive mold infections so while reporting the surgical specimens with clinical suspicion of post-COVID-19 mucormycosis one should be aware of the possibility of mixed fungal infections and look for Aspergillus fruiting bodies as a tell-tale sign of mixed Mucorales and Aspergillus infection. However, their presence does not estimate the true incidence of mixed fungal infections for which immunohistochemistry and polymerase chain reaction are needed.