Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Background: Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. Methods: This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. Results: mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. Conclusions: mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.

Manifestation and associated factors of systemic and local allergy among patients with allergic fungal rhinosinusitis: An observational study.

Allergic fungal rhinosinusitis (AFRS) is a subtype of chronic rhinosinusitis, characterized by excessive immune responses to environmental molds or fungi. The diagnosis and classification of AFRS into systemic and local types remain clinically challenging due to overlapping characteristics. This study investigated the prevalence of AFRS, its manifestation and associated factors in systemic and local AFRS. A total of 200 patients diagnosed with fungal rhinosinusitis underwent both skin provocation tests (SPT) and nasal provocation tests (NPT) to confirm AFRS and classify systemic and local types. Patients were considered to have AFRS if either the SPT or NPT was positive. Among these, patients with systemic AFRS were those who had a SPT positive. Local AFRS was when patients had a negative SPT and a positive NPT. Medical history, serum total IgE level, nasal endoscopy examinations, and CT scans were also recorded. Most patients were female (65.8%), with a mean age of 55.6 years (SD = 14.4). Based on the SPT and NPT results, 31% of patients (n = 62) were diagnosed with AFRS. Among these, 54.8% (n = 34) had systemic AFRS, while 45.2% (n = 28) had local AFRS. Patients with AFRS exhibited significantly higher levels of total IgE, eosinophils, and more pronounced signs and symptoms compared to those without AFRS. However, no statistically significant differences were observed between patients with systemic AFRS and those with local AFRS. AFRS was prevalent in our study. Among patients with AFRS, both systemic AFRS and local AFRS were also prevalent. While allergic indicators and clinical presentations can aid in AFRS diagnosis, minimal distinctions were observed between systemic and local AFRS. A comprehensive assessment incorporating both local and systemic allergic responses through provocation tests, such as a combination of skin and nasal tests, is imperative for optimizing AFRS diagnosis and management.

A case study: Lecanicillium infection detected in the human body.

Lecanicillium dimorphum and Lecanicillium psalliotae are fungi that exist naturally in plants or insects, and are generally considered non-pathogenic to humans. However, in this case, we cultured Lecanicillium from the synovial fluid of a patient, and identified it through genome sequencing and sequence alignment as Lecanicillium dimorphum or Lecanicillium psalliotae. Due to the conservation of sequences, we can only identify the genus and not the species. There are very few reports on the human infection and pathogenicity of these two fungi, and this case also cannot completely prove that the pathogenic agent is this fungus. But this case also holds clinical significance, as the discovery of Lecanicillium in a human sample can alert the clinician to the presence of an uncommon mold with unclear clinical significance.

Evaluation of pretreatment methods for filamentous fungal detection.

In order to obtain the best mass spectrometry identification results for using the most appropriate methods in clinical practice, we explore the optimal pretreatment methods for different species and morphologies of filamentous fungi. 98 fungal strains were treated with formic acid sandwich method, dispersion method, extraction method, and other methods using a medium element mass spectrometer (EXS3000) as a platform. Each strain had three targets, and the identification rates and confidence differences under different pre-treatment methods were compared to evaluate the identification effects of these methods. The mass spectrometry identification rates of 98 filamentous fungi obtained after pre-treatment with formic acid sandwich method, dispersion method, and extraction method were 85.71%, 82.65%, and 75.51%, respectively. The identification rate of the formic acid sandwich method was significantly higher than the other two methods (P < 0 005) has the best identification ability and the obtained confidence is also higher than the other two methods. The use of formic acid sandwich method for mass spectrometry identification of filamentous fungi can achieve ideal identification results, which is suitable for mass spectrometry identification of filamentous fungi in conventional laboratories.

Disseminated Talaromyces marneffei infection initially presenting as cutaneous and subcutaneous lesion in an HIV-Negative renal transplant recipient: a case report and literature review.

BACKGROUND: The incidence of Talaromyces marneffei (T. marneffei) infection has increased in recent years with the development of organ transplantation and the widespread use of immunosuppressive agents. However, the lack of clinical suspicion leading to delay or misdiagnosis is an important reason for the high mortality rate in non-human immunodeficiency virus (HIV) and non-endemic population. Herein, we report a case of disseminated T. marneffei infection in a non-HIV and non-endemic recipient after renal transplant, who initially presented with skin rashes and subcutaneous nodules and developed gastrointestinal bleeding. CASE PRESENTATION: We describe a 54-year-old renal transplantation recipient presented with scattered rashes, subcutaneous nodules and ulcerations on the head, face, abdomen, and right upper limb. The HIV antibody test was negative. The patient had no obvious symptoms such as fever, cough, etc. Histopathological result of the skin lesion sites showed chronic suppurative inflammation with a large number of fungal spores. Subsequent fungal culture suggested T. marneffei infection. Amphotericin B deoxycholate was given for antifungal treatment, and there was no deterioration in the parameters of liver and kidney function. Unfortunately, the patient was soon diagnosed with gastrointestinal bleeding, gastrointestinal perforation and acute peritonitis. Then he rapidly developed multiple organ dysfunction syndrome and abandoned treatment. CONCLUSIONS: The risk of fatal gastrointestinal bleeding can be significantly increased in kidney transplant patients with T. marneffei infection because of the long-term side effects of post-transplant medications. Strengthening clinical awareness and using mNGS or mass spectrometry technologies to improve the detection rate and early diagnosis of T. marneffei are crucial for clinical treatment in non-HIV and non-endemic population.

Deciphering Unexpected Vascular Locations of Scedosporium spp. and Lomentospora prolificans Fungal Infections, France.

Scedosporium spp. and Lomentospora prolificans are emerging non-Aspergillus filamentous fungi. The Scedosporiosis/lomentosporiosis Observational Study we previously conducted reported frequent fungal vascular involvement, including aortitis and peripheral arteritis. For this article, we reviewed 7 cases of Scedosporium spp. and L. prolificans arteritis from the Scedosporiosis/lomentosporiosis Observational Study and 13 cases from published literature. Underlying immunosuppression was reported in 70% (14/20) of case-patients, mainly those who had solid organ transplants (10/14). Osteoarticular localization of infection was observed in 50% (10/20) of cases; infections were frequently (7/10) contiguous with vascular infection sites. Scedosporium spp./Lomentospora prolificans infections were diagnosed in 9 of 20 patients ≈3 months after completing treatment for nonvascular scedosporiosis/lomentosporiosis. Aneurysms were found in 8/11 aortitis and 6/10 peripheral arteritis cases. Invasive fungal disease--related deaths were high (12/18 [67%]). The vascular tropism of Scedosporium spp. and L. prolificans indicates vascular imaging, such as computed tomography angiography, is needed to manage infections, especially for osteoarticular locations.

Molecular Identification of Fonsecaea monophora, Novel Agent of Fungal Brain Abscess.

A 3-year-old patient in India experiencing headaches and seizures was diagnosed with a fungal infection, initially misidentified as Cladophialophora bantiana. Follow-up sequencing identified the isolate to be Fonsecaea monophora fungus. This case demonstrates the use of molecular methods for the correct identification of F. monophora, an agent of fungal brain abscess.

[The preliminary application of mNGS in the diagnosis of invasive fungal sinusitis].

Objective: By conducting a retrospective analysis of the clinical data of 14 patients diagnosed with invasive fungal rhinosinusitis (IFRS) confirmed by metagenomics next generation sequencing (mNGS) technology, we aim to explore the rapid diagnosis value of mNGS in IFRS. Methods: The clinical data of 14 IFRS patients admitted to TianJin First Central Hospital were retrospectively analyzed from February 2021 to October 2023. The study cohort comprised 8 males and 6 females, with ages ranging from 14 to 77 years. All patients were diagnosed as IFRS by performing mNGS sequencing technology of nasal sinus lesion biopsy specimens. Clinical data such as laboratory examination, imaging examination, histopathological examination results, treatment plan and prognosis were summarized and analyzed. Results: All 14 patients were diagnosed as IFRS, with mNGS detecting pathogens such as Rhizopus (7 cases), Aspergillus (5 cases), Trichoderma (1 case), and Scedosporium apiospermum (1 case). Follow-up evaluations were conducted for a period ranging from 2 months to 2 years post-treatment. At the end of follow-up, 11 out of 14 IFRS patients achieved a complete cure with no signs of recurrence, while the symptoms of the remaining 3 patients significantly improved with comprehensive treatment. Conclusion: mNGS emerges as a highly effective diagnostic tool for IFRS, providing valuable microbiological evidence for clinical diagnosis and demonstrating promising clinical utility.

A rare cause of malignant otitis externa and skull base osteomyelitis.

An elderly male with type 2 diabetes presented with a 2-month history of otalgia and severe headaches. He was diagnosed with malignant otitis externa (MOE) and was commenced on empirical treatment with oral ciprofloxacin. Pseudomonas is the most common cause of MOE. A baseline CT scan was undertaken that demonstrated skull base osteomyelitis (SBO) due to findings of bone erosion at the mastoid tip and an infiltrating soft tissue mass eroding the clivus. Eight weeks later, he returned with worsening and bilateral symptoms of otitis externa, hearing loss, temporomandibular pain and dysfunction. Worsening and now bilateral malignant otitis externa were confirmed with an MRI scan that also demonstrated a small fluid collection in his left temporal region. The collection was aspirated and grew scedosporium apiospermum. He was diagnosed with fungal SBO and was commenced on treatment with the antifungal voriconazole, with significant improvement in symptoms and radiological findings. Fungal osteomyelitis is more likely in immunosuppressed patients, particularly those with type 2 diabetes. Fungal aetiology should be suspected in patients with progressive symptoms, despite treatment. A microbiology diagnosis of fungal SBO or MOE can be challenging to obtain and can lead to diagnostic delay. A sampling of the external auditory canal can aid in diagnosing MOE; however, scedosporium may also be isolated as a commensal organism. Aspirations from accessible fluid collections, infratemporal fossa needle sample and bone biopsy can provide material for diagnosis. Scedosporium is a rare cause of disease in humans, however, fungal infections are increasing in humans, due to an increase in susceptible populations. Scedosporium apiospermum is a rare cause of SBO and should be considered in patients not responding to standard treatment.

Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.

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.

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.

Expert consensus on the diagnosis and treatment of end-stage liver disease complicated by infections.

End-stage liver disease (ESLD) is a life-threatening clinical syndrome and when complicated with infection the mortality is markedly increased. In patients with ESLD, bacterial or fungal infection can induce or aggravate the occurrence or progression of liver decompensation. Consequently, infections are among the most common complications of disease deterioration. There is an overwhelming need for standardized protocols for early diagnosis and appropriate management for patients with ESLD complicated by infections. Asia Pacific region has the largest number of ESLD patients, due to hepatitis B and the growing population of alcohol and NAFLD. Concomitant infections not only add to organ failure and high mortality but also to financial and healthcare burdens. This consensus document assembled up-to-date knowledge and experience from colleagues across the Asia-Pacific region, providing data on the principles as well as evidence-based current working protocols and practices for the diagnosis and treatment of patients with ESLD complicated by 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.

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.

Quantitative Assessment of Fungal Biomarkers in Clinical Samples via an Interface-Modulated Optical Fiber Biosensor.

Invasive fungal infections pose a significant public health threat. The lack of precise and timely diagnosis is a primary factor contributing to the significant increase in patient mortality rates. Here, an interface-modulated biosensor utilizing an optical fiber for quantitative analysis of fungal biomarkers at the early stage of point-of-care testing (POCT), is reported. By integrating surface refractive index (RI) modulation and plasmon enhancement, the sensor to achieve high sensitivity in a directional response to the target analytes, is successfully optimized. As a result, a compact fiber-optic sensor with rapid response time, cost-effectiveness, exceptional sensitivity, stability, and specificity, is developed. This sensor can successfully identify the biomarkers of specific pathogens from blood or other tissue specimens in animal models. It quantifies clinical blood samples with precision and effectively discriminates between negative and positive cases, thereby providing timely alerts to potential patients. It significantly reduces the detection time of fungal infection to only 30 min. Additionally, this approach exhibits remarkable stability and achieves a limit of detection (LOD) three orders of magnitude lower than existing methods. It overcomes the limitations of existing detection methods, including a high rate of misdiagnosis, prolonged detection time, elevated costs, and the requirement for stringent laboratory conditions.

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.