Diagnostic Mycology Research Articles

LABORATORY INVESTIGATIONS FOR FUNGAL INFECTION OF ORAL CAVITY : AN EAGLE EYE VEIW

The incidence of invasive infections caused by fungi has been increasing for the last few decades. This is primarily due to the significant increases in the populations of at-risk patients; this includes those receiving immunosuppressive chemotherapy for various malignancies, solid organ transplant recipients and those receiving prolonged steroid treatment. New diagnostic approaches have been developed based on non-culture-based methods, which may allow early diagnosis and treatment of fungal infections. Laboratory procedures in diagnostic mycology are directed mainly toward the direct demonstration of the pathogenic fungi in clinical specimens by microscopy along with successful isolation of pathogenic fungi by using various culture techniques. They also help in the prediction of possible therapeutic outcome by determining antifungal susceptibility and can also be used in epidemiological studies by tracing the source of infection. The purpose of our review is to provide the reader with comprehensive and up-to-date information on diagnostic methods.

Diagnostic Mycology Laboratories Should Have a Central Role for the Management of Fungal Disease.

The absence of awareness of fungal diseases as part of the differential diagnosis in at-risk populations has severe consequences. Here, we show how the active role of laboratories can improve patients' survival. Recently, major advances have been made in non-culture-based assays for fungal diseases, improving accuracy and turnaround time. Furthermore, with the introduction of proficiency control systems, laboratories are an easily monitored environment with good analytical accuracy. Diagnostic packages for opportunistic infections can overcome many deficiencies caused by the absence of awareness. In Guatemala, to make diagnosis accessible, we set up a diagnostic laboratory hub (DLH) providing screening for cryptococcosis, histoplasmosis and tuberculosis to a network of 13 healthcare facilities attending people living with HIV (PLWHIV). In two years, we screened 2127 newly HIV-diagnosed patients. The frequency of opportunistic infections was 21%, rising to 30.3% in patients with advanced HIV disease (<200 CD4); 8.1% of these patients had more than one infection. With the implementation of this diagnostic package, mortality decreased by 7%, a key goal of many public health interventions. Screening for serious infection in high-risk populations can partially overcome training or experiential deficiencies among clinicians for life-threatening fungal diseases.

Evaluation of a custom Sensititre YeastOne plate for susceptibility testing of isavuconazole and other antifungals against clinically relevant yeast and mould species in three Australian diagnostic mycology laboratories

This study aimed to validate the performance of the custom formulated Sensititre YeastOne One (SYO) microdilution plate which includes isavuconazole (AUSNMRC1) to perform susceptibility testing on clinically relevant yeast and mould species across three Australian reference laboratories. The minimum inhibitory concentration (MIC) results were compared with the IVD approved SYO YO10 microdilution plate and isavuconazole gradient strips. A total of 127 isolates were tested on both the YO10 and AUSNMRC1 plates. The overall essential agreement (EA) and categorical agreement (CA) for the eight common drugs was 99.9% and 98.8%, respectively. The EA was 96.9% for the isavuconazole MICs obtained using the AUSNMRC1 plate and gradient strip. The MIC results for all nine antifungals on the AUSNMRC1 panel were highly reproducible for all quality control and reference strains and the overall EA and CA for 45 clinical strains tested across all three participating laboratories were >93% and 94.1%, respectively. These findings demonstrate the SYO AUSNMRC1 plate provides a commercial means to determine isavuconazole MICs by broth microdilution testing.

Expert recommendations for prevention and management of Candida auris transmission.

Candida auris was first described as a yeast pathogen in 2009. Since then, the species has emerged worldwide. In contrast to most other Candida spp., C.auris frequently exhibits multi-drug resistance and is readily transmitted in hospital settings. While most detections so far are from colonised patients, C.auris does cause superficial and life-threatening invasive infections. During management of the first documented C.auris transmission in a German hospital, experts from the National Reference Centers for Invasive Fungal Infections (NRZMyk) and the National Reference Center for Surveillance of Nosocomial Infections screened available literature and integrated available knowledge on infection prevention and C.auris epidemiology and biology to enable optimal containment. Relevant recommendations developed during this process are summarised in this guidance document, intended to assist in management of C.auris transmission and potential outbreak situations. Rapid and effective measures to contain C.auris spread require a multi-disciplinary approach that includes clinical specialists of the affected unit, nursing staff, hospital hygiene, diagnostic microbiology, cleaning staff, hospital management and experts in diagnostic mycology / fungal infections. Action should be initiated in a step-wise process and relevant interventions differ between management of singular C.auris colonised / infected patients and detection of potential C.auris transmission or nosocomial outbreaks.

Epidemiology of fungal diseases in Africa: A review of diagnostic drivers.

Background and Purpose: There has been a significant increase in the burden of fungal diseases in the last few decades which has imposed a global threat to the health of humans, animals, and plants. Epidemiology of fungal diseases is not completely understood in Africa. Most of these diseases are under-reported or not reported at all mainly due to the challenges related to the availability of and access to fungal diagnostics and the lack of human resources in clinical and diagnostic mycology across the continent. Therefore, it is imperative to highlight the epidemiology of the endemic and epidemic of emerging and re-emerging fungal diseases as well as their diagnostic challenges in Africa based on the available data. Moreover, it is important to underline the existing gaps in this regard as well.Materials and Methods: For the purposes of the study, Medline and Google Scholar were searched to retrieve articles on these prominent fungal diseases, as well as their etiologies and available diagnostics.Results: It was found that histoplasmosis and other AIDS-associated mycoses have been reported in Africa, including blastomycosis, coccidioidomycosis, and paracocci-dioidomycosis. Other reported infections were fungal neglected tropical diseases, especially sporotrichosis, dermatophytosis, mycetoma, and chromoblastomycosis as well as emerging fungal diseases, such as Emergomyces africanus, Candida auris, and Blastomyces emzantsi. In Africa, the major drivers of fungal diseases include human immunodeficiency infection, tuberculosis, and poverty.Conclusion: Serious fungal diseases are common in Africa; however, the true burden remains unknown.

Recent Advances and Novel Approaches in Laboratory-Based Diagnostic Mycology.

What was once just culture and microscopy the field of diagnostic mycology has significantly advanced in recent years and continues to incorporate novel assays and strategies to meet the changes in clinical demand. The emergence of widespread resistance to antifungal therapy has led to the development of a range of molecular tests that target mutations associated with phenotypic resistance, to complement classical susceptibility testing and initial applications of next-generation sequencing are being described. Lateral flow assays provide rapid results, with simplicity allowing the test to be performed outside specialist centres, potentially as point-of-care tests. Mycology has responded positively to an ever-diversifying patient population by rapidly identifying risk and developing diagnostic strategies to improve patient management. Nowadays, the diagnostic repertoire of the mycology laboratory employs classical, molecular and serological tests and should be keen to embrace diagnostic advancements that can improve diagnosis in this notoriously difficult field.

Point of care tests for invasive fungal infections: a blueprint for increasing availability in Africa.

Invasive fungal infections (IFIs) such as cryptococcosis, disseminated histoplasmosis, and chronic pulmonary aspergillosis are significant causes of morbidity and mortality in Africa. Lack of laboratory infrastructure and laboratory personnel trained in diagnostic mycology hamper prompt detection and management of IFIs on the continent. Point-of-care tests (POCT) obviate the need for complex infrastructure, skilled technicians, and stable electricity and have had major impacts on the diagnosis of bacterial, viral, and parasitic infections in low- and middle-income countries. Over the last 10 years, POCTs for IFIs have become increasingly available and they have the potential to revolutionize the management of these infections if scaled up in Africa. At the beginning of 2021, the World Health Organization (WHO) Essential Diagnostic List (EDL) included a cryptococcal antigen test for the diagnosis of cryptococcosis, Histoplasma antigen test for the diagnosis of disseminated histoplasmosis, and Aspergillus-specific test for the diagnosis of chronic pulmonary aspergillosis. All of these are available in formats that may be used as POCTs and it is hoped that this will improve the diagnosis of these life-threatening IFIs, especially in low- and middle-income countries. This perspective review discusses commercially available POCTs and outlines strategies of a blueprint to achieve their roll-out in Africa. The strategies include raising awareness, conducting research that uncovers the exact burden of IFIs, increasing advocacy, integrating diagnosis of IFIs into existing public health programs, adoption of the WHO EDL at country levels, and improving logistics and supply chains.

Utility of non-serum liquid media against conventional human serum in germ tube production test

: , a predominant yeast pathogen can be easily distinguished from other non-albicans species in a diagnostic laboratory based on their property to produce germ tubes using human serum as a routine substrate. As usage of human serum is accompanied with risk of transmission of blood-borne viral infections, several other media were employed by many clinical laboratories. The aim of this study was to evaluate the efficacy of four commonly used non-serum liquid media relative to fresh human serum in germ tube (GT) productivity among various Candida isolates. A total of 146 Candida isolates (96 ; 47 ; 3 ) identified by conventional methods were assessed for germ tube production in five types of media namely peptone water, Mueller Hinton broth, Trypticase soy broth, Brain Heart infusion broth and fresh human serum . Results obtained in different media for germ tube production in all Candida isolates were analysed for sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.All the Candida isolates showed 100% specificity and PPV in all the five-liquid media. Highest performance in germ tube production (96%) in isolates was found in Trypticase soy broth followed by fresh human serum (95%). Trypticase soy broth was shown to have 96% sensitivity and 97% accuracy for germ tube formation comparable to human serum with 95% sensitivity and 96.5% accuracy. Trypticase soy broth can be suggested as a choice medium for GT production in diagnostic mycology.

Onygenalean Fungi as Major Human and Animal Pathogens

During the last two decades, many onygenalean pathogens were discovered, redefined, or re-classified from existing taxa into clusters of micro-species, among which the original genotypes often appeared to be uncommon and exceptional. The impact of these developments on the diagnosis and treatment of fungal diseases remains to be determined in most instances. This exciting collection of invited articles provides a full flavor of ongoing changes in the knowledge about taxonomy, genetics, ecology, epidemiology, and clinical spectra of human and animal pathogens classified in the order Onygenales. Recent developments have set the stage for an ambitious translational research agenda. Diagnostic mycology laboratories now need MALDI-TOF-MS spectra, PCR probes, and other specific tools to assist them in the rapid diagnosis of new species. Similarly, an educational set of type materials of new species needs to be readily available for enhanced expertise among the wider medical mycology community. As several new species were discovered retrospectively, it is crucial to expand the re-sampling to other fungal culture collections and archived paraffin tissues. Finally, clinical and laboratory investigations are needed to get an accurate assessment of the prevalence and impact of new pathogens as the cause of major fungal diseases.

DIGGING DEEPER: WHEN AN HRCT IS NOT ENOUGH FOR AN ILD DIAGNOSIS

SESSION TITLE: Monday Fellow Case Report Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: 69-year old Native American male, never-smoker, presents with 3-month history of shortness of breath. His symptoms persisted despite courses of steroids and antibiotics. He underwent a bronchoscopy at the referring hospital with inconclusive results and was subsequently transferred to our center on steroids for a working diagnosis of ILD. He has no smoking history, no pets, no recent travel, and no occupational exposures. He pursued a hobby mowing his farm in Oklahoma. CASE PRESENTATION: His admission computed tomography (CT) scan of the chest showed a mosaic pattern of hyperlucent lung, normal appearing parenchyma, and ground glass ("headcheese sign") on a background of traction bronchiectasis and reticulations. The findings were concerning for an acute on chronic process, and specifically subacute on chronic hypersensitivity pneumonitis given the constellation of findings. Based on the CT results, empiric prednisone 60mg/day was started. Lab workup for connective tissue disease and hypersensitivity pneumonitis was unremarkable. Given cardiac comorbidities, he was deemed not a surgical lung biopsy candidate, so a repeat bronchoscopy was pursued. TBBX specimens demonstrated macrophages with intracytoplasmic yeast forms with some budding and a hint of capsule formation. Mucicarmine stain showed mucicarmine positive forms consistent with infection by cryptococcus. Confirmatory cryptococcal serum antigen was positive, 1:160. The patient had no signs of being immunocompromised, and HIV status was negative. He was started on fluconazole and a lumbar puncture was performed looking for CNS involvement, which was negative. DISCUSSION: Cryptococcus is a fungal infection that usually affects immunocompromised hosts. It can be readily detected with high sensitivity and specificity in both serum and CSF studies. In immunocompetent patients, treatment consists of a prolonged course of fluconazole (6 to 12 months). CONCLUSIONS: Both infectious and noninfectious diseases of the lung can present radiographically and clinically similar. A comprehensive workup is important to achieve the correct diagnosis in a timely fashion. Reference #1: McMullan BJ, Halliday C, Sorrell TC, et al: Clinical utility of the cryptococcal antigen lateral flow assay in a diagnostic mycology laboray. PLoS One 7(11):e49541, 2012. Reference #2: Tanner DC, Weinstein MP, Fedorciw B, et al: Comparison of commercial kits for detection of cryptococcal antigen. J Clin Microbiol 32(7):1680–1684, 1994. DISCLOSURES: No relevant relationships by Alastair Moore, source=Web Response No relevant relationships by Haala Rokadia, source=Web Response No relevant relationships by Christopher Wood, source=Web Response

National mycology laboratory diagnostic capacity for invasive fungal diseases in 2017: Evidence of sub-optimal practice

A survey of laboratory testing capabilities for systemic fungal pathogens was undertaken in the UK, to identify where improved compliance with published standards and guidelines is required and to inform antifungal stewardship (AFS). The survey captured information from laboratories in the UK on diagnostic capacity for invasive fungal diseases (IFD), including identification, serology, molecular diagnostics and susceptibility testing. The survey was circulated in March 2017 through key networks. Of 154 laboratories providing diagnostic mycology services in the UK, 80 (52%) responded to the survey. Results indicated that 85% of respondents identified fungal isolates from high risk patients to species level, and that many laboratories (78%) could access local susceptibility testing for yeasts, whereas 17% could for Aspergillus species. However, direct microscopy was only used in 49% as a first line investigation on samples where it would be appropriate. A low number of respondents identified yeasts cultured from intravascular line tips to species level (63%) and even fewer fully identified urine isolates from critically ill patients (42%) or the immunocompromised (39%). Less than half of respondents advised therapeutic drug monitoring (TDM) for flucytosine. Few laboratories had access to local β-glucan (4%) or galactomannan (20%) testing. The survey highlights that the current level of fungal diagnostics in the UK is below accepted best practice with an urgent need to improve across many diagnostic areas including the timely accessibility of fungal biomarkers, susceptibility testing and provision of TDM testing. Improvements are important to facilitate the delivery of diagnostic driven AFS strategies as well as appropriate management of IFD.

Recent advances and novel approaches in laboratory-based diagnostic mycology.

The field of diagnostic mycology represents much more than culture and microscopy and is rapidly embracing novel techniques and strategies to help overcome the limitations of conventional approaches. Commercial molecular assays increase the applicability of PCR testing and may identify markers of antifungal resistance, which are of great clinical concern. Lateral flow assays simplify testing and turn-around time, with potential for point of care testing, while proximity ligation assays embrace the sensitivity of molecular testing with the specificity of antibody detection. The first evidence of patient risk stratification is being described and together with the era of next generation sequencing represents an exciting time in mycology.

Microbial investigations of fungal infections: An overview

The incidence of fungal infections has increased considerably in recent years. Fungal infections remain an important cause of morbidity and mortality. New diagnostic approaches have been developed based on non-culture-based methods, which may allow early diagnosis and treatment of fungal infections. Laboratory procedures in diagnostic mycology are directed mainly toward the direct demonstration of the pathogenic fungi in clinical specimens by microscopy along with successful isolation of pathogenic fungi by using various culture techniques. They also help in the prediction of possible therapeutic outcome by determining antifungal susceptibility and can also be used in epidemiological studies by tracing the source of infection. An active interaction between the clinician, microbiologist, and pathologist facilitates the exact diagnosis and accurate interpretation of culture and histopathological results. This article focuses on specific conventional and rapid advanced techniques concerning a practical approach to the lab investigations in fungal infections.

The Development of Diagnostics Tools and Techniques in the Isolation and Detection of Fungal Pathogens

Until of the recent, the use of molecular tools in diagnostic mycology laboratories supersedes the old conventional methods. The diagnostics laboratories consider two important parameters; sensitivity and specificity, and the use of PCR and it is related have it all. This review went through the timeline of how fungal pathogen detection techniques have developed and still in the progress. The most recent other review articles and research papers were selected at random and critical analyses are made on the previous studies and papers. It is ascertain that molecular tools are more sensitive and specific and hence accurate results are achieved. It is clear that even though molecular techniques are reliable enough, the methods and the purity of DNA extraction and the amount respectively determines the fingerprint that gives identity to the respective pathogens.

Identification of Molds by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Although to a lesser extent than diagnostic bacteriology, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently revolutionized the diagnostic mycology workflow. With regard to filamentous fungi (or molds), the precise recognition of pathogenic species is important for rapid diagnosis and appropriate treatment, especially for invasive diseases. This review summarizes the current experience with MALDI-TOF MS-based identification of common and uncommon mold species of Aspergillus, Fusarium, Mucorales, dimorphic fungi, and dermatophytes. This experience clearly shows that MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly with common species or typical strains of filamentous fungi.

Diagnosis of dermatophytosis: an evaluation of direct examination using MycetColor® and MycetFluo®

Dermatophytes are an important cause of superficial fungal infection. Direct examination of skin, nail, or hair samples remains essential in diagnosis, as it provides a quick response to the clinician. However, mycological analysis, including direct examination and culture, often lacks sensitivity. The use of stains or fluorochromes may enhance the performance of direct examination. We analyzed 102 samples from patients with suspected dermatophytosis in 4 different diagnostic mycology laboratories. Two reagents, MycetColor® and MycetFluo®, which use Congo red and calcofluor dye, respectively, were evaluated for the direct microscopic examination of skin, hair, and nail specimens. The results were compared to those of culture and conventional direct examination. Both reagents were able to clarify the specimens and also to specifically stain fungal elements. Microscopic examination of the specimens was greatly facilitated with MycetFluo®, which allowed a higher number of positive cases to be detected compared to the other methods.

Laboratory diagnosis of fungal infection

There are numerous species of fungi of which only a few are pathogenic to humans. Laboratory procedures in diagnostic mycology are directed mainly towards the direct demonstration of the pathogenic fungi in clinical specimens by microscopy along with successful isolation of pathogenic fungi by using various culture techniques. They also help in the prediction of possible therapeutic outcome by determining antifungal susceptibility and can also be used in epidemiological studies by tracing the source of infection. No single procedure may help in definitive diagnosis, since each has its own limitations. Accurate diagnosis relies heavily on a combination of microbiological, histopathological and serological evidence. There is also a need to characterize fungi quickly and accurately which can be done by rapid diagnostic techniques like antigen detection and molecular techniques. This article contains specific conventional and advanced techniques concerning a practical approach to the laboratory investigations of fungal infections.

Enhancing Molecular Approaches for Diagnosis of Fungal Infections

Molecular tests can improve the diagnosis of fungal infections. Despite the increasing application for fungal detection, molecular tests are still not accepted as a diagnostic criterion to define invasive fungal diseases. This limitation is largely due to a lack of a standardized method. Method standardization can be achieved by following a consensus protocol developed by a working group, by performing a molecular test in a centralized laboratory or by using a commercial assay that provides a standardized method and quality-controlled reagents. Forming a consortium or a working group consisting of large-scale diagnostic mycology laboratories can accelerate the process of validating and implementing a commercial molecular assay for clinical use through a joint effort between industry partners and clinicians. Development of molecular tests not only for the detection of fungi but also for the identification of antifungal drug resistance directly in blood, bronchoalveolar lavage fluid, cerebrospinal fluid, and formalin-fixed and paraffin-embedded tissues greatly enhances fungal diagnostic capacities. Advances of developing quantitative assays and RNA detection platforms may provide another avenue to further improve fungal diagnostics.

State-of-the-Art Procedures and Quality Management in Diagnostic Medical Mycology

Detection and correct identification of fungi causing invasive disease is a major challenge for diagnostic laboratories. However, early and reliable diagnosis is essential to keep mortality to a minimum, whereas delayed or missed diagnosis is typically associated with fatal outcome in the case of invasive fungal infections. Therefore, rapid and reliable results are of importance for successful management. In order to guarantee correct reports, accurate processing of all samples and appropriate methods for detecting the fungal agent as well as quality assurance is mandatory. This article gives an overview of current methods and elucidates quality management measures in medical mycology laboratory.

Poct in clinical mycology

Point-of-care testing (POCT) in diagnostic mycology is less well-defined than for pathogens of wide public health impact. Fungal infections are relatively uncommon and largely affect specific host groups: the immunocompromised and critically ill. Early diagnosis and therapy has been shown to improve patient outcomes in invasive candidiasis including candidaemia. Commercial PCR-based platforms are available to directly detect <i>Candida</i> in blood cultures but also in whole blood up to 4 days prior to the ‘flagging' of positive cultures. However systematic evaluation of impact of this technology on outcomes has not been undertaken. Detection of <i>Aspergillus</i> DNA and galactomannan in blood and other body fluids has good potential for early diagnosis of invasive aspergillosis. In resource-limited countries and settings where cryptococcosis is prevalent, detection of serum cryptococcal antigen (CRAG) is sensitive for early diagnosis, including in asymptomatic patients; early treatment reduces mortality. The CRAG lateral flow assay is one such tool to detect CRAG in serum, urine and saliva with high sensitivity and specificity. Therapeutic drug monitoring of drug levels is increasingly important. Not only do voriconazole and posaconazole blood levels have relevance, but blood fluconazole levels should also be measured, especially in severely ill patients to ensure meaningful dosing.