Recent advances towards point-of-care devices for fungal detection: Emphasizing the role of plasmonic nanomaterials in current and future technologies.

Fungal infections are a significant global health problem, particularly affecting individuals with weakened immune systems. Moreover, as uncontrolled antibiotic and immunosuppressant use increases continuously, fungal infections have seen a dramatic increase, with some strains developing antibiotic resistance. Traditional approaches to identifying fungal strains often rely on morphological characteristics, thus owning limitations, such as struggles in identifying several strains or distinguishing between fungal strains with similar morphologies. This review explores the multifaceted impact of fungi infections on individuals, healthcare providers, and society, highlighting the often-underestimated economic burden and healthcare implications of these infections. In light of the serious constraints of traditional fungal identification methods, this review discusses the potential of plasmonic nanoparticle-based biosensors for fungal infection identification. These biosensors can enable rapid and precise fungal pathogen detection by exploiting several readout approaches, including various spectroscopic techniques, colorimetric and electrochemical assays, as well as lateral-flow immunoassay methods. Moreover, we report the remarkable impact of plasmonic Lab on a Chip technology and microfluidic devices, as they recently emerged as a class of advanced biosensors. Finally, we provide an overview of smartphone-based Point-of-Care devices and the associated technologies developed for detecting and identifying fungal pathogens.

Rapid detection of pathogenic fungi from coastal population with respiratory infections using microfluidic chip technology.

BACKGROUND: Currently, culture methods are commonly used in clinical tests to detect pathogenic fungi including Candida spp. Nonetheless, these methods are cumbersome and time-consuming, thereby leading to considerable difficulties in diagnosis of pathogenic fungal infections, especially in situations that respiratory samples such as alveolar lavage fluid and pleural fluid contain extremely small amounts of microorganisms. The aim of this study was to elucidate the utility and practicality of microfluidic chip technology in quick detection of respiratory pathogenic fungi. METHODS: DNAs of clinical samples (mainly derived from sputa, alveolar lavage fluid, and pleural fluid) from 64 coastal patients were quickly detected using microfluidic chip technology with 20 species of fungal spectrum and then validated by Real-time qPCR, and their clinical baseline data were analyzed. RESULTS: Microfluidic chip results showed that 36 cases infected with Candida spp. and 27 cases tested negative for fungi, which was consistent with Real-time qPCR validation. In contrast, only 16 cases of fungal infections were detected by the culture method; however, one of the culture-positive samples tested negative by microfluidic chip and qPCR validation. Moreover, we found that the patients with Candida infections had significantly higher rates of platelet count reduction than fungi-negative controls. When compared with the patients infected with C. albicans alone, the proportion of males in the patients co-infected with multiple Candidas significantly increased, while their platelet counts significantly decreased. CONCLUSIONS: These findings suggest that constant temperature amplification-based microfluidic chip technology combined with routine blood tests can increase the detection speed and accuracy (including sensitivity and specificity) of identifying respiratory pathogenic fungi.

Talaromycosis from Wuhan: two-case report and literature review.

Background: Talaromycosis is a serious opportunistic infectious disease caused by Talaromyces marneffei, which mostly occurs in immunocompromised patients. The disease is mainly prevalent in tropical countries and regions of Southeast Asia and South Asia, but non-endemic areas also have patients with Talaromycosis. The disease has no characteristic clinical manifestations and is difficult to diagnose. Delayed diagnosis often leads to death. Case presentation: Both patients had cellular immunodeficiency. Case 1 had a history of acquired immune deficiency syndrome, and case 2 had a history of renal transplantation and glucose-6-phosphate dehydrogenase deficiency. They all had fever, anemia, fatigue, and skin lesions. Case 1 had gastrointestinal bleeding, enlarged lymph nodes, and hepatosplenomegaly. Case 2 had cough and dyspnea. Both patients had thrombocytopenia and hypoalbuminemia; an increased neutrophil ratio, procalcitonin, and C-reactive protein; and abnormal liver function and coagulation dysfunction. Case 1 sputum culture, blood culture, and bronchoalveolar lavage fluid were positive for T. marneffei. T. marneffei was detected in the blood culture of case 2, with infection of Candida parapsilosis and Pneumocystis jirovecii. Chest computed tomography scan mainly showed pulmonary exudative lesions. Although these two patients were actively treated, they died of poor efficacy. Conclusion: Talaromycosis has an insidious onset, long course, atypical clinical symptoms, imaging performance and laboratory results, difficult diagnosis, and high mortality. Therefore, it is important to promptly consider and treat Talaromycosis in immunocompromised patients upon infection in order to reduce mortality.

Lung infections due to emerging fungal pathogens.

PURPOSE OF REVIEW: This review highlights the epidemiology, pathogenesis and clinical management of pulmonary infections caused by emerging fungal organisms. RECENT FINDINGS: Emerging fungal infections have arisen as a result of population and environmental changes. An enlarging pool of immunocompromised hosts on triazole antifungal prophylaxis has led to an increased incidence of non- Aspergillus molds, such as Fusarium , Scedosporium and Lomentospora spp. Advances in diagnostic capabilities led to the identification of the Emergomyces genus and non- dermatitidis Blastomyces species, which have a significant disease burden in Africa and the Middle East. Climate change has contributed to changing the distribution of previously confined endemic mycoses, like coccidioidomycosis and talaromycosis. These emerging organisms pose important diagnostic and therapeutic challenges. SUMMARY: Newly recognized pathogenic fungi and established endemic mycoses with expanding geographic boundaries have become important agents of pulmonary disease. There is a dearth of clinical evidence on the appropriate management of these infections.

Talaromyces marneffei endocarditis initially detected by Next Generation Sequencing: A case report.

BACKGROUND: Talaromyces marneffei (T. marneffei) is a thermal dimorphic fungus, which can cause lung or blood stream infection in patients, often life-threatening. However, endocarditis caused by T. marneffei has not been reported. For elderly patients with implanted cardiac devices or artificial valves, the prevention and treatment of infective endocarditis should not be ignored. METHODS: This is a descriptive study of a T. marneffei endocarditis by joint detection of cardiac ultrasound examination, peripheral blood DNA metagenomics Next Generation Sequencing (mNGS), and in vitro culture. RESULTS: We describe an 80-year-old female patient with an unusual infection of T. marneffei endocarditis. After intravenous drip of 0.2 g voriconazole twice a day for antifungal treatment, the patient showed no signs of improvement and their family refused further treatment. CONCLUSION: Infective endocarditis is becoming more and more common in the elderly due to the widely use of invasive surgical procedures and implantation of intracardiac devices. The diagnosis and treatment of T. marneffei endocarditis is challenging because of its rarity. Here, we discussed a case of T. marneffei endocarditis, and emphasized the role of mNGS in early diagnosis, which is of great significance for treatment and survival rate of patients.

Fungal infection mimicking soft tissue lesion: Role of on-site evaluation, special stain, and cellblock.

BACKGROUND: Fine-needle aspiration (FNA) cytology is a basic diagnostic method used for the investigation of superficial and deep lesions. The implementation of rapid on-site evaluation (ROSE) in cytological analysis can help in reducing the inadequacy rate and obtaining proper samples for further tests/analysis. CASE PRESENTATION: We report a case of 44-year-old male, who presented to our outpatient department with complaints of swelling in his right arm for the last 34 years. FNA with ROSE using 1% aq. toluidine blue helped identify the pathology (fungal lesion) in the patient with further confirmation by cellblock, periodic acid Schiff & Gomori methenamine silver stains. CONCLUSION: The role of FNA was significant in the above case, special stains prove their efficacy when sufficient sample is available. The differential diagnosis of fungal etiology should be considered in subcutaneous soft tissue lesions. There has been a major leap in diagnostic cytopathology with the advent of molecular testing. However, FNA still holds its charm.

Invasive fungal pathogens from the tropical and temperate areas - a challenge in pathology and diagnosis.

Fungi play a vital role in ensuring a physiological balance in the surrounding environments, interacting closely with humans, plants, and animals. While most of the time their contribution is beneficial, occasionally, they can become harmful, especially in patients with weakened immune systems. The work at hand aims to present the most common fungal pathogens involved in invasive infections, focusing on fungi that are present in the tropical and temperate areas of the world. While in the former, due to the humid climate, most fungal infections are caused by dimorphic fungi such as Coccidioides spp., Blastomyces spp., Histoplasma spp., Emergomyces spp. and Paracoccidioides spp., in the latter, after Candida spp., the most frequent fungi that are involved in disseminated mycosis are Aspergillus spp., Fusarium spp. and species from the order Mucorales. Nowadays, the etiology, severity, and number of cases of fungal diseases are starting to rise significantly. There are no exact reasons reported for this increase, but several factors are thought to be incriminated: the expansion of the range of medical conditions that constitute risk factors for developing the disease, an improvement in the available diagnostic methods, the commodity offered by modern traveling services associated with the lack of an available vaccine against fungal infections, as well as climatic influences. All the above-mentioned aspects consequently caused infections that used to be endemic to be spread worldwide. Therefore, it is of critical importance to understand the epidemiology, clinical manifestations of fungi induced diseases, virulence factors, and diagnosis for each of those pathogens.

Designing a Digital Health Solution: A Platform for Automated Surveillance of Fungal Infection.

Surveillance of invasive fungal infection (IFI) requires laborious review of multiple sources of clinical information, while applying complex criteria to effectively identify relevant infections. These processes can be automated using artificial intelligence (AI) methodologies, including applying natural language processing (NLP) to clinical reports. However, developing a practically useful automated IFI surveillance tool requires consideration of the implementation context. We employed the Design Thinking Framework (DTF) to focus on the needs of end users of the tool to ensure sustained user engagement and enable its prospective validation. DTF allowed iterative generation of ideas and refinement of the final digital health solution. We believe this approach is key to increasing the likelihood that the solution will be implemented in clinical practice.

The value of calcofluor white in the diagnosis of invasive fungal diseases.

PURPOSE: To evaluate the value of calcofluor white in the diagnosis of invasive fungal disease (IFD). METHODS: A total of 84 patients with possible pulmonary fungal infection who underwent bronchoscopy with bronchoalveolar lavage fluid (BALF) were included. All BALF specimens were subjected to Calcofluor white (CFW), potassium hydroxide (KOH) and Gram stains. RESULTS: CFW has the most sensitivity than KOH and Gram staining. The specificity of CFW was 92.00 %, which was lower than that of Gram staining. The PPVs for CFW, KOH and Gram staining were 94.44 %, 84.62 % and 80.00 % respectively. The NPVs for CFW, KOH and Gram staining was 47.92 %, 32.39 % and 30.38 % respectively. The AUCs of these three methods were 0.748, 0.550 and 0.510 respectively. CONCLUSION: CFW is superior to KOH and Gram staining in the diagnosis of invasive fungal diseases.

Dematiaceous fungal infections: clinical and pathologic conundrums.

Dematiaceous fungi are defined by pigment within their cell walls. They are increasingly recognised human pathogens, causing a wide range of clinical presentations, from localised subcutaneous infections to disseminated disease in rare cases. We report our institutional experience with diagnosis of dematiaceous fungal infections from 2005 to 2022 and highlight four instructive cases that clinically and pathologically mimicked other diseases for which the diagnosis was confirmed by fungal culture (one case) or supported by PCR with 28S rRNA and internal transcribed spacer primers (three cases). Two patients were immunocompromised and two had presumed exposure to the organism. In each highlighted case, fungal infection was not clinically suspected, and the pathologist was critical in making the diagnosis and ensuring appropriate clinical management, which was supplemented by fungal stains and novel molecular methods.

eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account.

BACKGROUND: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management. RESULTS: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA. CONCLUSIONS: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg.

Old and new strategies in therapy and diagnosis against fungal infections.

Fungal infections represent a serious global health threat. The new emerging pathogens and the spread of different forms of resistance are now hardly challenging the tools available in therapy and diagnostics. With the commonly used diagnoses, fungal identification is often slow and inaccurate, and, on the other hand, some drugs currently used as treatments are significantly affected by the decrease in susceptibility. Herein, the antifungal arsenal is critically summarized. Besides describing the old approaches and their mechanisms, advantages, and limitations, the focus is dedicated to innovative strategies which are designed, identified, and developed to take advantage of the discrepancies between fungal and host cells. Relevant pathways and their role in survival and virulence are discussed as their suitability as sources of antifungal targets. In a similar way, molecules with antifungal activity are reported as potential agents/precursors of the next generation of antimycotics. Particular attention was devoted to biotechnological entities, to their novelty and reliability, to drug repurposing and restoration, and to combinatorial applications yielding significant improvements in efficacy. KEY POINTS: • New antifungal agents and targets are needed to limit fungal morbidity and mortality. • Therapeutics and diagnostics suffer of delays in innovation and lack of targets. • Biologics, drug repurposing and combinations are the future of antifungal treatments.

Embolic stroke and misidentification candida species endocarditis: Case presentation and literature review.

Fungal endocarditis is a rare but serious form of infective endocarditis associated with high morbidity and mortality. Among fungal pathogens, Candida species are the most frequently isolated and commonly found in individuals with predisposing factors, such as prosthetic heart valves. The clinical presentation of endocarditis is highly variable and nonspecific, often including symptoms and signs of embolization. In this paper, we present a case of fungal prosthetic valve endocarditis in which the initial presentation was an acute ischemic stroke. The initial misidentification of Candida famata was attributed to limitations in the presumptive methodology used through selective chromogenic culture identification. However, the surgical specimen underwent mass spectrometry, leading to the correct identification of Candida guilliermondii instead of Candida famata. Furthermore, we conducted a non-systematic narrative review of the literature on Candida endocarditis. Our findings underscore the importance of considering fungal endocarditis in the differential diagnosis of patients with possible extracardiac complications, particularly those with a history of heart valve replacement. Early diagnosis and a comprehensive treatment strategy tailored by species identification and antifungal susceptibility testing are crucial in improving patient outcomes.

Aspergillus infections of lateral skull base: a case series.

PURPOSE: While extensive research with accurate classification has been done in mycoses of the paranasal sinuses and anterior skull base, a similar understanding of lateral skull base fungal pathologies is lacking due to relative rarity and diagnostic difficulties. We introduce a series of eleven cases and two different invasive entities of Aspergillus temporal bone diseases-fungal skull base osteomyelitis (SBO)/malignant otitis externa (MOE) and chronic invasive granulomatous fungal disease (CIGFD). METHODOLOGY: A retrospective observational study was conducted at the neuro-otology unit of a tertiary care referral center between July 2017 and November 2022. Diagnosed cases of lateral skull base osteomyelitis with atypical symptoms and lack of response to culture-directed antibiotics were evaluated for fungal origin. Patient data, including history, laboratory findings, serum galactomannan assay, CT and MRI imaging findings, clinical examination findings, and co-morbidities, were analyzed. The treatment course and response were assessed. RESULTS: A total of 11 cases were included in the study. Of these, 9 were cases of Aspergillus-induced skull base osteomyelitis (SBO) and 2 of Aspergillus-induced chronic invasive granulomatous fungal disease (CIGFD). CIGFD presented with persistent ear discharge and slowly progressive post-aural swelling, while all patients of fungal SBO had lower cranial nerve palsies. CIGFD responded to excision and antifungals, while SBO responded well to conservative anti-fungal treatment. CONCLUSION: In cases of lateral SBO not responding to antibiotic therapy, the possibility of fungal etiology should be considered. Aspergillus spp. seems to be the major fungal pathogen.

Nanotechnology-based fungal detection and treatment: current status and future perspective.

Fungal infections impose a significant impact on global health and encompass major expenditures in medical treatments. Human mycoses, a fungal co-infection associated with SARS-CoV-2, is caused by opportunistic fungal pathogens and is often overlooked or misdiagnosed. Recently, there is increasing threat about spread of antimicrobial resistance in fungus, mostly in hospitals and other healthcare facilities. The diagnosis and treatment of fungal infections are associated with several issues, including tedious and non-selective detection methods, the growth of drug-resistant bacteria, severe side effects, and ineffective drug delivery. Thus, a rapid and sensitive diagnostic method and a high-efficacy and low-toxicity therapeutic approach are needed. Nanomedicine has emerged as a viable option for overcoming these limitations. Due to the unique physicochemical and optical properties of nanomaterials and newer biosensing techniques, nanodiagnostics play an important role in the accurate and prompt differentiation and detection of fungal diseases. Additionally, nano-based drug delivery techniques can increase drug permeability, reduce adverse effects, and extend systemic circulation time and drug half-life. This review paper is aimed at highlighting recent, promising, and unique trends in nanotechnology to design and develop diagnostics and treatment methods for fungal diseases.

Invasive fungal infection in ANCA-associated vasculitis: Between the Devil and Deep blue sea. Case series and review of the literature.

INTRODUCTION: Patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) are susceptible to opportunistic infections, including invasive fungal infections (IFI). This is due to many factors, including prolonged immunosuppressive therapy. The treatment of AAV with such IFIs is challenging. METHODS: A descriptive analysis of 5 patients with AAV complicated by concomitant invasive fungal infections was performed. We also have done a comprehensive literature review of IFIs in AAV using PubMed and Google Scholar databases. RESULTS: All 5 patients initially received immunosuppressive medication but subsequently acquired IFI. One patient had sphenoid sinus involvement, and four had lung parenchymal involvement. Aspergillus infection was diagnosed in three patients, Cryptococcus infection in one patient and mixed infection with Aspergillus and Mucor infection in one patient. All our patients were on low doses of corticosteroids for several months to years or had received high-dose pulse steroids with cyclophosphamide in the last few weeks before being diagnosed with IFI. It was difficult to distinguish disease activity from IFI in all the cases. Two of the five patients died despite antifungal therapy. The literature review revealed a prevalence of IFIs ranging from 1 to 9.6% (excluding pneumocystis pneumonia). Aspergillosis was the predominant type of IFI, affecting 46 of 86 patients. Most of these patients (40/46) had pulmonary involvement. The prognosis for patients with IFI was consistently poor, as evidenced by 19 deaths out of 29 reported outcomes. CONCLUSION: Overall, IFIs have a poor prognosis in patients with AAV. Differentiating disease activity from IFI is difficult because of similar organ distribution, imaging lesions, and histopathological characteristics. A high suspicion index and good-quality microbiology are needed for early treatment and prevention of mortality.

Invasive Eumycotic mycetoma of the nasal septum in a post-dialysis patient: A case report.

BACKGROUND: Mycetoma is a slowly progressive chronic granulomatous disease of the skin, subcutaneous tissue, and underlying or adjacent cartilage or bone. Most commonly involves the foot region. Other parts such as the knee, arm, leg, head, neck, thigh, perineum, chest, abdominal walls, facial bones, mandible, paranasal sinuses, eyelid, vulva, orbit, and scrotum are seldom affected. METHODS: This is a rare presentation of Eumycotic mycetoma involving the nasal septum. Surgical debridement is done under local anesthesia. Histopathological examination of debrided specimen guided in the diagnosis and treatment. RESULTS: Histopathological examination is the one that confirms the diagnosis and rules out the other granulomatous conditions and fungal rhinitis causing septal perforation. CONCLUSIONS: In an immunocompromised state, we know that mucormycosis and zygomycosis are known to cause aggressive complications like orbital invasion and palatal perforation by vascular route. However, other fungal infections also can lead to septal perforations whenever there is lessened resistance by the mucosal barrier due to trauma (nasal intubations).