Lateral-Flow Device for Diagnosis of Fungal Infection

Invasive aspergillosis (IA) is a life-threatening complication of haematological malignancy and haematopoietic stem cell transplantation caused by the ubiquitous fungus Aspergillus. Current diagnosis of IA is multifaceted relying on data from clinical, radiological, and microbiological sources. The detection of Aspergillus biomarkers provides strategies both to pre-empt and to exclude disease, but the choice of biomarker assays is limited, requiring specialist equipment and training. This review examines recent advances in the accurate diagnosis of IA through the development of an Aspergillus lateral-flow device (LFD) incorporating a monoclonal antibody, JF5, which detects an antigenic marker of active infection. Recent trials using bronchoalveolar lavage fluids and serum samples from humans and from animal models of disease, have demonstrated the utility of the LFD as a rapid and user-friendly adjunct test for the quick and accurate diagnosis of pulmonary infections.     

Galactomannan-Based and PCR-Based Assays in Bronchoalveolar Lavage to Diagnose Invasive Aspergillosis: Current Status and Future Prospects

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in patients receiving chemotherapy or undergoing allogeneic haematopoietic stem cell transplantation for acute leukemia. The existing tools to diagnose IPA lack specificity or sensitivity, or both; the search for improved diagnostic tools for IPA has focused on novel serologic and molecular methods. Aspergillus Galactomannan enzyme-linked immunosorbent assay (GM) analyses showed sensitivity rates in serum samples ranging in a wide span; testing GM in bronchoalveolar lavage (BAL) originated from the primary site of the infection seems to be more sensitive in patients with IPA. Other novel diagnostic markers to detect fungal DNA directly in clinical samples, rapidly, early, sensitively and specifically, are provided by polymerase chain reaction (PCR) based assays; higher sensitivity and specificity rates have been observed for BAL samples in IPA, even under antifungal treatment. The clinical place value of a diagnostic approach combining PCR and GM in BAL is unclear.     

Detection of (1, 3)-β-d-glucan in bronchoalveolar lavage and serum samples collected from immunocompromised hosts

The incidence of invasive fungal infections (IFI) has increased in recent years, especially among immunocompromised hosts (ICH). In 2003, the Fungitell^® assay received FDA clearance for the presumptive diagnosis of IFI using serum and detects (1-3)-β-d-glucan, which is a major cell wall component of certain fungi (e.g., Candida, Aspergillus, and Pneumocystis). The goal of the current study was to assess the performance of the assay on bronchoalveolar lavage (BAL) fluid and serum to identify IFI in ICH. Patients were classified as having proven, probable, possible, or no IFI according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) guidelines. Among 109 patients for whom the results of Fungitell were compared to the EORTC/MSG criteria, Fungitell showed a low positive predictive value for the identification of IFI from both BAL (20.0 %) and serum (26.7 %). However, the negative predictive value of Fungitell was significantly higher for both sample types (BAL, 83.0 %; serum, 84.8 %). Interestingly, the results of Fungitell were positive in BAL and serum in 7/8 (87.5 %) patients diagnosed with Pneumocystis pneumonia (PcP) by real-time, non-nested PCR. These data indicate that the Fungitell assay has a low positive predictive value for the diagnosis of IFI in ICH, regardless of the specimen type that is tested. However, testing of serum samples by Fungitell may permit a rapid and noninvasive initial screening approach in patients with presumed PcP.     

Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis: Current Status

Purpose of Review

Diagnosis during early stages of invasive aspergillosis (IA) and targeted antifungal treatment has the potential to improve survival significantly. Despite advances in the diagnostic arsenal, invasive mold infections remain difficult to diagnose—especially at early stages before typical radiological signs develop. Varying availability and time-to-results are important limitations of current approved biomarkers and molecular assays for diagnosis of IA. Here, we will give an update on the Aspergillus-specific lateral-flow device (LFD) test. We further review promising findings on feasibility of point-of-care (POC) detection of urinary excreted fungal galactomannan-like antigens.

Recent Findings

POC LFD assays for detection of Aspergillus antigens are currently in development. The Aspergillus-specific LFD test, which is based on the JF5 antibody (Ab), detects an extracellular glycoprotein antigen secreted during active growth of Aspergillus spp. The test has shown promising results in various studies. In addition, a monoclonal Ab476-based LFD for POC detection of urinary excreted fungal galactomannan-like antigens has been developed but needs further validation.

Summary

Important advances have been made in the development of LFD assays for IA. Most promising is the Aspergillus-specific LFD test; commercial availability is still pending, however. The search for reliable POC tests for other molds, including mucorales, continues.

     

Use of Mycological, nested PCR, and Real-time PCR Methods on BAL Fluids for Detection of Aspergillus fumigatus and A. flavus in Solid Organ Transplant Recipients

Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.     

Case Series Study of Invasive Pulmonary Aspergillosis

Diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. The objective of the study was to assess the value of microbiological tests to the diagnosis of IPA in the absence of non-specific radiological data. A retrospective study of 23 patients with suspicion of IPA and positivity of some microbiological diagnostic tests was performed. These tests included conventional microbiological culture, detection of Aspergillus galactomannan (GM) antigen and in some patients (1 → 3)-β-d-glucan (BDG) and Aspergillus fumigatus DNA using the LightCycler^® SeptiFast test. In 10 patients with hematological malignancy, 6 cases were considered ‘probable’ and 4 ‘non-classifiable.’ In 8 patients with chronic lung disease, 7 cases were classified as ‘probable’ and 1 as ‘proven,’ and in 5 patients with prolonged ICU stay (>7 days), there were 2 ‘proven’ cases, 2 ‘non-classifiable’ and 1 putative case. Microbiological culture was positive in 17 cases and 18 Aspergillus spp. were isolated (one mixed culture). A. fumigatus was the most frequent (44.4%) followed by A. tubingensis. The Aspergillus galactomannan (GM) antigen assay was positive in 21 cases (91.3%). The GM antigen and the (1 → 3)-β-d-glucan (BDG) assays were both performed in 12 cases (52.2%), being positive in 9. The SeptiFast test was performed in 7 patients, being positive in 4. In patients with non-classifiable pulmonary aspergillosis and one or more positive microbiological tests, radiological criteria may not be considered a limiting factor for the diagnosis of IPA.     

Reproducibility of Positive Results for the Detection of Serum Galactomannan by Platelia™ Aspergillus EIA

Galactomannan (GM) was recently included in consensus guidelines as an indirect mycological criterion for the diagnosis of invasive aspergillosis. Currently, there is an enzyme immunoassay available to detect GM in biological samples, the Platelia? Aspergillus EIA. In this study, the reproducibility of positive results obtained using this assay was evaluated using serum samples from neutropenic patients. A trend toward lower values was observed, and 55 %(27/49) of positive results were negative after retesting. A low reproducibility of positive results for the detection of GM in serum was observed.     

Cloning,expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody

Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni⁺-NTA affinity chromatography (95–98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.     

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

Aims: The current study was aimed to develop a loop‐mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Methods and Results: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC‐labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP–LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non‐parahaemolyticus Vibrio isolates and 35 non‐Vibrio bacterial isolates. The sensitivity of LAMP–LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml^?1. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 × 10³ CFU g^?1 or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. Conclusions: The established LAMP–LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. Significance and Impact of the Study: The developed LAMP–LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.     

Plant pathogens but not antagonists change in soil fungal communities across a land abandonment gradient in a Mediterranean landscape

We assessed whether the presence and abundance of plant pathogens and antagonists change in soil fungal communities along a land abandonment gradient. The study was carried out in the Cilento area (Southern Italy) at a site with three different habitats found along a land abandonment gradient: agricultural land, Mediterranean shrubland and woodland. For all microbiological substrates the colony forming units were about 3.1 × 10⁶ g⁻¹ soil for agricultural land and about 1.1 × 10⁶ g⁻¹ soil for Mediterranean shrubland and woodland. We found the following genera in all habitats: Cladosporium, Mortierella, Penicillium and Trichoderma. In agricultural land, the significantly most abundant fungus genera were Aspergillus, Fusarium, Cylindrocarpon and Nectria; in Mediterranean shrubland, Rhizopus and Trichoderma; and in woodland, Bionectria, Mortierella, Cladosporium, Diplodia, Paecilomyces, Penicillium and Trichoderma. We found a total of 8, 8 and 9 species of fungal antagonist, and 16, 6 and 6 species of fungal plant pathogens in agricultural land, Mediterranean shrubland and woodland respectively. Fungal plant pathogens decreased significantly over a land abandonment gradient, while we no found significant differences among fungal antagonists in the three habitats. We conclude that a decrease in the number of fungal pathogen species occurs when formerly cultivated areas are abandoned. On the other hand, fungal antagonists seem not to be affected by this process.     

Sorption of lead and copper from an aqueous phase system by marine-derived Aspergillus species

A total of 47 cultures of Aspergillus representing 13 species were screened for their ability to tolerate 7.5 mM Pb²⁺ and 2 mM Cu²⁺, all of which were positive, with growth of 31 of the cultures being enhanced by low concentrations of lead. The isolates of Aspergillus versicolor, A. niger and A. flavus were tolerant to concentrations as high as 10 - 12.5 mM Pb²⁺ and 3 – 4 mM Cu²⁺. Selected cultures displayed a good sorption capacity of 32 - 41 mg Pb²⁺ and 3.5 - 6.5 mg Cu²⁺ g^-1 dry weight of mycelia, which was improved by alkali pretreatment of the biomass and negatively affected by mild dry heat treatment. The sequestration of the metal occurred mainly by sorption to the cell-surface with very little intracellular uptake. FTIR analysis indicated the involvement of hydroxyl, amino, and carbonyl groups in Pb²⁺ and Cu²⁺ biosorption by fungal biomass of the different species of Aspergillus.     

Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi

The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 10 min; amplification temperature, 61 °C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL⁻¹ when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1–1000 cells mL⁻¹. All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.     

Isolation and molecular characterization of mycotoxigenic fungi in agarwood

Agarwood (Oudh), is often used by people in the Kingdom of Saudi Arabia. The Oudh has been mentioned in the Hadith and is traditionally used for its aroma (perfuming smell) and potential medicinal applications. The aim of the study was to isolate mycotoxigenic fungi that grow on agarwood and the factors and storage conditions that enhance their growth potential. In addition to the detection of associated mycotoxins like: Aflatoxin B1 (AFB₁) and ochratoxin A (OTA) from agarwood. Agarwood samples were collected from local markets of Jeddah governorate, Kingdom of Saudi Arabia. Standard dilution plate method was used for the isolation of fungi. Isolated fungi were identified based on morphological characteristics and confirmed using molecular biology techniques. AFB₁ and OTA were detected by High Performance Liquid Chromatography (HLPC). The results indicated that the most commonly isolated fungal genera were in the following descending order: Aspergillus, Penicillium, Fusarium and Rhizopus. Among Aspergillus genera, A. flavus and A. ochraceus were detected based on their morphology and confirmed by PCR using specific primers. It was also noted that AFB₁ was released by 15.3 and 55.0% of A. flavus and A. parasiticus isolates respectively with levels reaching up to 14.60 µg/L. The moisture content in the samples ranged from 3% to 10% affected fungal growth. AFB₁ was detected in 22 out of 50 of the samples. The maximum level of AFB₁ (50.7 µg/kg) was detected in samples with higher moisture content (12%) stored at a temperature of 32 °C. Aspergillus fungi were found to be the most predominant fungal genera found on agarwood. Moisture content (9–10%) and storage temperature (32 °C) stimulated fungal growth and their ability to produce mycotoxins. For this reason, storage conditions at the marketing place should be adequate in order not to provide a conducive environment for fungal growth which is associated with the mycotoxin production. In order to prevent fungal growth and mycotoxin production, it would be recommended to store agarwood at temperatures not exceeding 25 °C and moisture content of up to a maximum of 5–6%.     

Aspergillus-specific antibodies – Targets and applications

Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.     

Marine-Derived Aspergillus Species as a Source of Bioactive Secondary Metabolites

This report reviews biologically active secondary metabolites from marine-derived members of the fungal genus Aspergillus. Pharmacological activities and biological roles of the secondary metabolites from marine-derived Aspergillus spp. were addressed in respect of pharmaceutical potential.     

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense^1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores⁴. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.     

Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick

The dinoflagellate Karlodinium veneficum that is usually present at relatively low cell abundances is a globally-distributed harmful algal bloom-forming species, which negatively affects marine ecosystems, fisheries, and human health. Hence, an efficient detection platform for the rapid and sensitive identification of K. veneficum is highly demanded. In this study, a method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for the rapid detection of K. veneficum. The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K. veneficum by molecular cloning and multiple alignments of the related sequences. The developed RPA can gain an approximately 300 bp specific band from K. veneficum. Successful amplification for RPA could be achieved at a temperature range of 35 °C–45 °C. RPA for 30 min could produce enough products that could generate clearly visible electrophoresis bands and were adequate for subsequent LFD analysis. The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature. The developed RPA-LFD was exclusively specific for K. veneficum and displayed no cross-reactivity with other algal species that are commonly distributed along the Chinese coast. In addition, the lowest detection limit of RPA-LFD was 10 ng μL⁻¹ of genomic DNA and 0.1 cell mL⁻¹, which was 100-fold sensitive than conventional PCR. In conclusion, the developed RPA-LFD assay in this study can be used as a rapid and sensitive method to monitor K. veneficum in the future.     

Effects of Qufeng Xuanfei Decoction in Animal Model of Post-Infectious Cough

This study evaluated the effects and potential mechanisms of Qufeng Xuanfei decoction in animal model of post-infectious cough. Sixty SD rats were randomly divided into six groups (10 animals per group): control, disease model, low- (4.62 g kg^?1), medium- (9.24 g kg^?1), and high-dose (13.86 g kg^?1) decoction, and positive treatment groups (dextromethorphan hydrobromide, 8 mL kg^?1). To model post-infectious cough, all but control group animals were challenged with exposure to 50 g sawdust and 10 cigarette smokes for 30 min day^?1 for a total of 10 days, followed by subsequent exposures to lipopolysaccharide (20 µg) and capsaicin (10^?4 M) aerosols. The drugs were given by oral gavage for 15 days after which lung pathology, cell counts and cell differentials in bronchoalveolar lavage (BAL), and concentrations of neuropeptides [substance P (SP), neurokinins A (NKA) and B (NKB), and calcitonin gene-related peptide (CGRP)] in BAL (ELISA) were assessed. Compared with control group animals, significant inflammation and damage to bronchial epithelium were observed in the disease model group. A marked decrease in BAL percentages of all types of inflammatory cells was observed in the decoction-treated groups, with most changes in the medium-dose decoction group (p < 0.001 vs. disease model group). Further, airway inflammation and damage, as well as the levels of SP, NKA, NKB, and CGRP in BAL decreased the most in the medium-dose group (p < 0.001 vs. disease model group). In conclusion, medium-dose Qufeng Xuanfei decoction efficiently decreases the levels of neuropeptides, attenuates airway inflammation, and promotes recovery from disease.