Adaptation to an amoeba host drives selection of virulence-associated traits and genetic variation in saprotrophic Candida albicans.

Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic predators suggests that the pressure from predatory selection can significantly influence the development of fungal virulence and evolutionary processes. Thus, the purpose of this study was to investigate the adaptation of saprotrophic Candida albicans strains during their interactions with Acanthamoeba castellanii. We conducted a comprehensive analysis of survival after co-culture by colony counting of the yeast cells and examining yeast cell phenotypic and genetic characteristics. Our results indicated that exposure to amoebae enhanced the survival capacity of environmental C. albicans and induced visible morphological alterations in C. albicans, particularly by an increase in filamentation. These observed phenotypic changes were closely related to concurrent genetic variations. Notably, mutations in genes encoding transcriptional repressors (TUP1 and SSN6), recognized for their negative regulation of filamentous growth, were exclusively identified in amoeba-passaged isolates, and absent in unexposed isolates. Furthermore, these adaptations increased the exposed isolates' fitness against various stressors, simultaneously enhancing virulence factors and demonstrating an increased ability to invade A549 lung human epithelial cells. These observations indicate that the sustained survival of C. albicans under ongoing amoebic predation involved a key role of mutation events in microevolution to modulate the ability of these isolates to change phenotype and increase their virulence factors, demonstrating an enhanced potential to survive in diverse environmental niches.

Antibacterial and antivirulence factor activities of protein hydrolysates from Phatthalung Sangyod rice (Oryza sativa L.) seeds against zoonotic and foodborne pathogens.

Background and Aim: Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals. Materials and Methods: After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 µg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli, P. aeruginosa, B. cereus, and S. aureus, using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum. Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Results: Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 µg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa, B. cereus, and E. coli, at 256 and >256 µg/mL, respectively. We observed that at least 256 µg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16-128 µg/mL, it exhibited antibiofilm activity against S. aureus. Furthermore, at 256 µg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 µg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 µg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 µg/mL. Conclusion: Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals.

Evaluation of the yeast phase-specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin sandwich ELISA to detect Talaromyces marneffei antigen in human urine.

Talaromyces (Penicillium) marneffei (TM) is an important, but neglected, thermally dimorphic fungus. It is the pathogenic cause of talaromycosis, which is strongly associated with the immunodeficiency state present in individuals with advanced HIV disease. The purpose of this study was to develop a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of T. marneffei cytoplasmic yeast antigen (TM CYA) in human urine. Monoclonal antibody (MAb) 4D1 specifically binds to TM CYA. Galanthus nivalis agglutinin (GNA), a mannose -binding lectin, recognizes and binds to mannose residues of TM CYA. For the sandwich ELISA, the microplate was coated with GNA as the capturing molecule for absorbing immune complexes of MAb 4D1-TM CYA. The MAb 4D1-GNA sandwich ELISA did not detect a cross-reaction with other antigens from other fungi or bacteria. Seventy-four urine samples from patients with blood culture -confirmed talaromycosis and 229 urine samples from people without talaromycosis residing in the endemic area were subjected to the MAb 4D1-GNA sandwich ELISA. At an optical density (OD) cutoff value of 0.356, the sensitivity was 89.19% [95% confidence interval (CI): 79.80% -95.22%]; the specificity was 98.69% (95% CI: 96.22% -99.73%). The diagnostic performance of the MAb 4D1-GNA sandwich ELISA was highly consistent with those of blood culture and the Platelia Aspergillus galactomannan (GM) ELISA kit. Collectively, the MAb 4D1-GNA sandwich ELISA is a promising technique for the rapid diagnosis of T. marneffei infection, which would facilitate the early treatment of patients with talaromycosis and it may be used to monitor treatment responses.

Antifungal activity of protein hydrolysates from Thai Phatthalung Sangyod rice (Oryza sativa L.) seeds.

Background and Aim: Fungal zoonoses are an economic and public health concern because they can cause various degrees of morbidity and mortality in animals and humans. To combat this issue, alternative natural antifungals, such as products derived from rice protein hydrolysates or rice antifungal protein/peptide are being considered because they are highly bioactive and exhibit various functional properties. Thailand is a leading rice producer and exporter. Among the various cultivated rice varieties, Sangyod rice (Oryza sativa L.) is exclusively indigenous to Thailand's Phatthalung province; it has a Thai geographical indication tag. Here, we investigated whether the Phatthalung Sangyod rice seeds have bioactive antifungal peptides. Materials and Methods: Antifungal activity in four Sangyod rice seed extracts (SYPs) - namely, (1) the crude lysate, SYP1; (2) the heat-treated lysate, SYP2; (3) the heat- and pepsin digested lysate, SYP3; and (4) the heat- and proteinase K-digested lysate, SYP4 - was analyzed. Protein concentrations in these SYPs were determined using the Bradford assay. The total phenolic compound content was determined using the modified Folin-Ciocalteu method in a 96-well microplate. Then, the SYP protein pattern was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, using the agar well diffusion method, the antifungal properties of these SYPs were tested against ten medically important pathogenic fungi. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration values were determined for the active SYPs - SYP2-4. Finally, the clinical safety of SYP4 was determined using a hemolytic assay (using canine red blood cells [RBCs]). Results: The crude lysate SYP1 did not show antifungal activity against any of the ten tested pathogenic fungi. Surprisingly, hydrolysates SYP2, SYP3, and SYP4 displayed antifungal properties against the ten tested pathogenic fungi. Thus, heat and enzymatic hydrolysis seem to transform the bioactivity of the crude protein extract - SYP1. Further, SYP4 shows the most effective antifungal activity. It completely inhibited Cryptococcus neoformans, Talaromyces marneffei yeast phase, Trichophyton mentagrophytes, and Trichophyton rubrum. A partial inhibitory action on Candida albicans and Microsporum gypseum was possessed while showing the least activity to C. neoformans. SYP4 was nontoxic to canine RBCs. Hemolysis of canine RBCs was undetectable at 1 × MIC and 2 × MIC concentrations; therefore, it can be safely used in further applications. Conclusion: These results indicate that heat and proteinase K hydrolyzed SYP is a very potent antifungal preparation against animal and human fungal pathogens and it can be used in future pharmaceuticals and functional foods.

Extracellular vesicles derived from Talaromyces marneffei contain immunogenic compounds and modulate THP-1 macrophage responses.

Pathogenic eukaryotes including fungi release extracellular vesicles (EVs) which are composed of a variety of bioactive components, including peptides, nucleic acids, polysaccharides, and membrane lipids. EVs contain virulence-associated molecules suggesting a crucial role of these structures in disease pathogenesis. EVs derived from the pathogenic yeast phase of Talaromyces (Penicillium) marneffei, a causative agent of systemic opportunistic mycoses "talaromycosis," were studied for their immunogenic components and immunomodulatory properties. Some important virulence factors in EVs including fungal melanin and yeast phase specific mannoprotein were determined by immunoblotting. Furthermore, fluorescence microscopy revealed that T. marneffei EVs were internalized by THP-1 human macrophages. Co-incubation of T. marneffei EVs with THP-1 human macrophages resulted in increased levels of supernatant interleukin (IL)-1ß, IL-6 and IL-10. The expression of THP-1 macrophage surface CD86 was significantly increased after exposed to T. marneffei EVs. These findings support the hypothesis that fungal EVs play an important role in macrophage "classical" M1 polarization. T. marneffei EVs preparations also increased phagocytosis, suggesting that EV components stimulate THP-1 macrophages to produce effective antimicrobial compounds. In addition, T. marneffei EVs stimulated THP-1 macrophages were more effective at killing T. marneffei conidia. These results indicate that T. marneffei EVs can potently modulate macrophage functions, resulting in the activation of these innate immune cells to enhance their antimicrobial activity.

Differential lipase virulence in Malassezia furfur dimorphism isolated from pityriasis versicolor patients and healthy individuals.

BACKGROUND: Malassezia furfur is a member of the human skin microbiomes that can cause various skin diseases. Dimorphism plays a role as the yeast phase predominates during skin colonisation whereas mycelial forms are observed in the scales of patients with pityriasis versicolor (PV). However, due to their condition-dependence for growth, it is difficult to culture M. furfur and this is an additional challenge for studying the pathogenicity of this fungus. OBJECTIVE: To describe different media suitable for culturing Malassezia from the yeast phase into mycelial forms, with a particular focus on nutritional supplements and pH conditions. METHODS: Clinical M. furfur isolates from patients with PV and healthy individuals were used to investigate Malassezia dimorphism as well as the activity and expression of lipase enzymes. RESULTS: Our experimental media were significantly more likely to promote mycelial growth in strains from healthy individuals compared to those from patients with PV. Lipase activity was increased in the mycelial phase cells compared to yeast forms for all strains tested. Assessment of the relative transcriptional expression of lipase within M. furfur revealed that LIP-coding genes were upregulated in mycelium relative to yeast forms for the strains tested. However, the increases in LIP3, LIP5 and LIP6 gene expressions were significantly greater in strains from healthy individuals compared to those from patients with PV. CONCLUSION: Overall, this study validated effective growth conditions to study M. furfur virulence factors and demonstrated that lipase is associated with M. furfur dimorphism.

Expression of Cytokine Profiles in Human THP-1 Cells during Phase Transition of Talaromyces marneffei.

Talaromyces marneffei, a dimorphic fungus, exhibits temperature-dependent growth, existing in a filamentous form at 25 °C and as a yeast at 37 °C. Several studies have highlighted the important roles of macrophages in defense against T. marneffei infection. However, the immune responses to the interaction of macrophages with T. marneffei cells during phase transition require further investigation. This study reports the expression of cytokine profiles in human THP-1 cells during infection by T. marneffei. THP-1 cells were infected with T. marneffei conidia at different multiplicity of infections (MOIs). Surviving conidia transformed into yeasts after phagocytosis by macrophages, and the number of yeasts gradually increased over 36 h. The transcription and secretion levels of pro- and anti-inflammatory cytokines were examined at different times by qRT-PCR and ELISA. Transcription levels of IL-8, IL-12, IL-1ß, and TNF-α increased significantly at 12 or 24 h and then slightly decreased at 36 h. In contrast, the transcription levels of IL-6, IL-10, and TGF-ß gradually increased at all MOIs. The levels of IL-6 and IL-10 secretion corresponded to their levels of transcription. These results indicated that as the number of intracellular yeasts increased, the infected macrophages first underwent slight M1 polarization before shifting to M2 polarization. This polarization transition was confirmed by the fungicidal ability and the expression of macrophage surface markers. By inducing the M2-type polarization of macrophages, the intracellular T. marneffei cells can successfully evade the immune response. Our study provides a novel insight into the immune characterization during the transition of T. marneffei infection and could further contribute to possible diagnostic and therapeutic interventions for this infection.

Interaction of Talaromyces marneffei with free living soil amoeba as a model of fungal pathogenesis.

Talaromyces (Penicillium) marneffei is an important dimorphic mycosis endemic in Southeast Asia and Southern China, but the origin and maintenance of virulence traits in this organism remains obscure. Several pathogenic fungi, including Cryptococcus neoformans, Aspergillus fumigatus, Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum and Paracoccidioides spp. interact with free living soil amoebae and data suggests that fungal pathogenic strategies may emerge from environmental interactions of these fungi with ubiquitous phagocytic microorganisms. In this study, we examined the interactions of T. marneffei with the soil amoeba Acanthamoeba castellanii. T. marneffei was rapidly ingested by A. castellanii and phagocytosis of fungal cells resulted in amoeba death after 24 h of contact. Co-culture also resulted in a rapid transition for conidia to the fission-yeast form. In addition, well-established virulence factors such as melanin and a yeast specific mannoprotein of T. marneffei were expressed during interaction with A. castellanii at 37°C. Our findings support the assumption that soil amoebae environmental predators play a role in the selection and maintenance of particular features in T. marneffei that impart virulence to this clinically important dimorphic fungus in mammalian hosts.

Immunogenicity of snake α-neurotoxins and the CD4 T cell epitopes.

Snakebite envenomation is an important medical problem in numerous parts of the world causing about 2.7 million envenomations and between 81,000 and 138,000 deaths ayear. Antivenoms (AVs) are time proven effective therapeutics for snakebite envenomation. However, AVs, especially those against elapid neurotoxic venoms (cobras, kraits and mambas), are difficult to produce and are generally of low neutralizing potency. The most lethal component of most elapid venoms is the postsynaptic neurotoxins or the α-neurotoxins, which are responsible for death in most victims. It is generally believed that the low neutralizing potency of the AVs is due to the small molecular sizes, and thus the low immunogenicity, of the α-neurotoxins. Therefore, modifications of the toxins have been made to increase their size, and/or to detoxify them, hoping to improve the toxin's immunogenicity and AV potency. However, these maneuvers have not been applied to commercial AV production. The α-neurotoxins belong to a group of small proteins called three-finger toxins (3FTxs). The 3FTxs contain about 60-77 amino acid residues with four to five disulfide linkages and three anti-parallel ß-sheets, which extend from a globular hydrophobic core resembling three fingers. The members of the 3FTxs exhibit a number of important pharmacological activities, e.g., inhibition of neuromuscular transmission and acetyl cholinesterase activities. Recent immunization experiments with a 26 amino acid peptide containing the consensus sequence of the α-neurotoxins, and a mixture of elapid α-neurotoxins using highly effective adjuvants and immunization protocols have resulted in neutralizing antibodies in rabbit and horse, respectively. In the present report using bioinformatics, we show that 23 3FTxs which include α-neurotoxins, cardiotoxins and non-conventional toxins, and the 26 amino acid peptide, were all predicted to contain high to medium score CD4 T-cell epitopes for human and mouse MHC IIs. This information corroborates the results obtained from animal experiments that the α-neurotoxins, in spite of their small sizes and toxicity, are in fact immunogenic. Thus, the uses of effective adjuvants and immunization procedures, rather than chemical/physical modifications of the toxin structures, are crucial to the production of potent AVs against elapid neurotoxic venoms.

Talaromyces marneffei Infection: Virulence, Intracellular Lifestyle and Host Defense Mechanisms.

Talaromycosis (Penicilliosis) is an opportunistic mycosis caused by the thermally dimorphic fungus Talaromyces (Penicillium) marneffei. Similar to other major causes of systemic mycoses, the extent of disease and outcomes are the results of complex interactions between this opportunistic human pathogen and a host's immune response. This review will highlight the current knowledge regarding the dynamic interaction between T. marneffei and mammalian hosts, particularly highlighting important aspects of virulence factors, intracellular lifestyle and the mechanisms of immune defense as well as the strategies of the pathogen for manipulating and evading host immune cells.

Fungal Keratitis in Northern Thailand: Spectrum of Agents, Risk Factors and Putative Virulence Factors.

Fungal keratitis (FK) is a serious ocular infection that can result in various degrees of vision loss, including blindness. The aim of the study was to identify and retrospectively review all FK cases diagnosed between August 2012 and December 2020 at a tertiary care hospital in northern Thailand with a specific focus on epidemiologic features, including season, patient sex and age, the spectrum of pathogens, and presence of certain putative virulence factors. Of 1237 patients with corneal ulcers, 294 (23.8%) were confirmed by direct microscopic examination and/or fungal culture. For the positive cases, direct examinations of Calcofluor white (CW) stains and KOH mounts were found in 97.3% (286/294) and 76.5% (225/294), respectively (p < 0.05). Of the cases diagnosed by microscopy and culture, fungi were isolated in 152 (51.7%), with Fusarium spp. being the most frequently identified (n = 69, 45.5%) followed by dematiaceous fungi (n = 45, 29.6%) and Aspergillus spp. (n = 18, 11.8%). The incidence of FK was higher in the rainy season of July to October. The mean age was 54.4 ± 14.4 (SD) years, with a range of 9-88 years. Males (75.8%) were affected significantly more than females (24.2%) (p < 0.05). Of 294 patients, 132 (44.9%) were middle-aged adults (41-60 years) and 107 (36.4%) were older than 60 years. Trauma to the eye by soil or vegetative matter were the most common preceding factors (188/294; 64.0%). We assessed two virulence factors. First, 142 of the 152 culture-positive FK cases were due to molds, indicating that hyphal morphogenesis is extremely important in disease. We also demonstrated that fungal melanization occurs in the molds during the course of FK by applying a melanin-specific monoclonal antibody (MAb) that labeled fungal elements in corneal samples of patients, and melanin particles derived from the hyphae were also recovered after treatment of the samples with proteolytic enzymes, denaturant and hot concentrated acid. In summary, we demonstrate that northern Thailand has a high rate of FK that is influenced by season and males engaged in outside activities are at highest risk for disease. Moulds are significantly more commonly responsible for FK, in part due to their capacity to form hyphae and melanins. Future studies will examine models of fungal corneal interactions and assess additional factors of virulence, such as secreted enzymes, to more deeply decipher the pathogenesis of FK.

An inexpensive point-of-care immunochromatographic test for Talaromyces marneffei infection based on the yeast phase specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin.

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1-GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 µg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA-MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use.

A Novel, Inexpensive In-House Immunochromatographic Strip Test for Cryptococcosis Based on the Cryptococcal Glucuronoxylomannan Specific Monoclonal Antibody 18B7.

The aim of this study was to develop a novel lateral flow immunochromatoghaphic strip test (ICT) for detecting cryptococcal polysaccharide capsular antigens using only a single specific monoclonal antibody, mAb 18B7. The mAb 18B7 is a well characterized antibody that specifically binds repeating epitopes displayed on the cryptococcal polysaccharide glucuronoxylomannan (GXM). We validated the immunoreactivities of mAb 18B7 against capsular antigens of different cryptococcal serotypes. The mAb 18B7 ICT was constructed as a sandwich ICT strip and the antibody serving in the mobile phase (colloidal gold conjugated mAb 18B7) to bind one of the GXM epitopes while the stationary phase antibody (immobilized mAb18B7 on test line) binding to other remaining unoccupied epitopes to generate a positive visual readout. The lower limit of detection of capsular antigens for each of the Cryptococcus serotypes tested was 0.63 ng/mL. No cross-reaction was found against a panel of antigens isolated from cultures of other pathogenic fungal, except the crude antigen of Trichosporon sp. with the lower limit of detection of 500 ng/mL (~800 times higher than that for cryptococcal GXM). The performance of the mAb 18B7 ICT strip was studied using cerebrospinal fluid (CSF) and serum and compared to commercial diagnostic kits (latex agglutination CALAS and CrAg IMMY). The sensitivity, specificity and accuracy of the mAb18B7 ICT with CSF from patients with confirmed cryptococcal meningitis were 92.86%, 100% and 96.23%, respectively. No false positives were observed with samples from non-cryptococcosis patients. With serum samples, the mAb 18B7 ICT gave a sensitivity, specificity and accuracy of 96.15%, 97.78% and 96.91%, respectively. Our results show that the mAb 18B7 based ICT was reliable, reproducible, and cost-effective as a point-of-care immunodiagnostic test for cryptococcosis. The mAb 18B7 ICT may be particularly useful in countries where commercial kits are not available or affordable.

Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei.

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T. marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells' transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T. marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

An in vitro α-neurotoxin-nAChR binding assay correlates with lethality and in vivo neutralization of a large number of elapid neurotoxic snake venoms from four continents.

The aim of this study was to develop an in vitro assay for use in place of in vivo assays of snake venom lethality and antivenom neutralizing potency. A novel in vitro assay has been developed based on the binding of post-synaptically acting α-neurotoxins to nicotinic acetylcholine receptor (nAChR), and the ability of antivenoms to prevent this binding. The assay gave high correlation in previous studies with the in vivo murine lethality tests (Median Lethal Dose, LD50), and the neutralization of lethality assays (Median Effective Dose, ED50) by antisera against Naja kaouthia, Naja naja and Bungarus candidus venoms. Here we show that, for the neurotoxic venoms of 20 elapid snake species from eight genera and four continents, the in vitro median inhibitory concentrations (IC50s) for α-neurotoxin binding to purified nAChR correlated well with the in vivo LD50s of the venoms (R2 = 0.8526, p < 0.001). Furthermore, using this assay, the in vitro ED50s of a horse pan-specific antiserum against these venoms correlated significantly with the corresponding in vivo murine ED50s, with R2 = 0.6896 (p < 0.01). In the case of four elapid venoms devoid or having a very low concentration of α-neurotoxins, no inhibition of nAChR binding was observed. Within the philosophy of 3Rs (Replacement, Reduction and Refinement) in animal testing, the in vitro α-neurotoxin-nAChR binding assay can effectively substitute the mouse lethality test for toxicity and antivenom potency evaluation for neurotoxic venoms in which α-neurotoxins predominate. This will greatly reduce the number of mice used in toxicological research and antivenom production laboratories. The simpler, faster, cheaper and less variable in vitro assay should also expedite the development of pan-specific antivenoms against various medically important snakes in many parts of the world.

A pan-specific antiserum produced by a novel immunization strategy shows a high spectrum of neutralization against neurotoxic snake venoms.

Snakebite envenomation is a neglected tropical disease of high mortality and morbidity largely due to insufficient supply of effective and affordable antivenoms. Snake antivenoms are mostly effective against the venoms used in their production. It is thus crucial that effective and affordable antivenom(s) with wide para-specificity, capable of neutralizing the venoms of a large number of snakes, be produced. Here we studied the pan-specific antiserum prepared previously by a novel immunization strategy involving the exposure of horses to a 'diverse toxin repertoire' consisting of 12 neurotoxic Asian snake toxin fractions/ venoms from six species. This antiserum was previously shown to exhibit wide para-specificity by neutralizing 11 homologous and 16 heterologous venoms from Asia and Africa. We now show that the antiserum can neutralize 9 out of 10 additional neurotoxic venoms. Altogether, 36 snake venoms belonging to 10 genera from 4 continents were neutralized by the antiserum. Toxin profiles previously generated using proteomic techniques of these 36 venoms identified α-neurotoxins, ß-neurotoxins, and cytotoxins as predominant toxins presumably neutralized by the antiserum. The bases for the wide para-specificity of the antiserum are discussed. These findings indicate that it is feasible to generate antivenoms of wide para-specificity against elapid neurotoxic venoms from different regions in the world and raises the possibility of a universal neurotoxic antivenom. This should reduce the mortality resulting from neurotoxic snakebite envenomation.

Diagnostic laboratory immunology for talaromycosis (penicilliosis): review from the bench-top techniques to the point-of-care testing.

The pathogenic fungus Talaromyces (formerly Penicillium) marneffei is a thermally dimorphic fungus that can cause disseminated infection in patients with secondary immunodeficiency syndrome, in particular in the setting of advanced HIV infection. The areas of highest incidence are in Southeast Asia, Southern China, and Indian subcontinents. Talaromycosis (formerly penicilliosis) is identified as an AIDS-defining illness, and it has recently been recognized in non-HIV-associated patients with impaired cellular-mediated immunity. Microbiological culture is the gold standard method for the diagnosis of T. marneffei infection and usually requires up to 2-4 weeks for detectable growth to occur, which may result in a delay of appropriate treatment. Immunodiagnosis has become an alternative method for confirming talaromycosis. This article reviews various immunological tests for the diagnosis of talaromycosis, including a proposed novel rapid point-of-care assay using a new T. marneffei yeast phase-specific monoclonal antibody.

Production of melanin pigments in saprophytic fungi in vitro and during infection.

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin-specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin-specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.

An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja.

In order to facilitate/expedite the production of effective and affordable snake antivenoms, a novel in vitro potency assay was previously developed. The assay is based on an antiserum's ability to bind to postsynaptic neurotoxin (PSNT) and thereby inhibit the PSNT binding to the nicotinic acetylcholine receptor (nAChR). The assay was shown to work well with antiserum against Thai Naja kaouthia which produces predominantly the lethal PSNTs. In this work, the assay is demonstrated to work well with antiserum/antivenom against Bungarus candidus (BC), which also produces lethal presynaptic neurotoxins, as well as antivenom against Sri Lankan Naja naja (NN), which produces an abundance of cytotoxins. The in vitro and in vivo median effective ratios (ER50s) for various batches of antisera against BC showed a correlation (R2) of 0.8922 (p < 0.001) while the corresponding value for the anti-NN antivenom was R2 = 0.7898 (p < 0.01). These results, together with the known toxin profiles of various genera of elapids, suggest that this in vitro assay could be used with antisera against other species of Bungarus and Naja and possibly other neurotoxic snake venoms worldwide. The assay should significantly save numerous lives of mice and accelerate production of life-saving antivenoms.

Development and characterization of an immunochromatographic test for the rapid diagnosis of Talaromyces (Penicillium) marneffei.

Talaromyces (Penicillium) marneffei is a thermally dimorphic fungus that can cause opportunistic systemic mycoses in patients infected with the human immunodeficiency virus (HIV). It has also been reported among patients with other causes of immunodeficiency, such as systemic lupus erythematosus, cancer, organ transplanted patients receiving immunosuppressive drug and adult onset immunodeficiency syndromes. Recent studies indicate that the clinical manifestations, laboratory findings and treatment strategies of talaromycosis (penicilliosis) marneffei are different between patients with and without HIV infection. Therefore early and accurate diagnosis of talaromycosis marneffei is crucial to the proper management and treatment. Since current diagnostic methods are currently inadequate, the aim of this study was to develop an immunochromatographic test (ICT) for the detection of T. marneffei yeast antigens in urine samples. The highly T. marneffei-specific monoclonal antibody 4D1 (MAb 4D1) conjugated with gold colloid at pH 6.5 was used as signal generator. The nitrocellulose membrane was lined with T. marneffei cytoplasmic yeast antigen (TM CYA) to serve as the test line, and rabbit anti-mouse IgG was the control line. Subjecting the assembled test strip to urine samples containing T. marneffei antigen produced a visible result within 20 minutes. The sensitivity limit of the assay was 3.125µg/ml of TM CYA. The ICT was used to test urine samples from 66 patients with blood culture confirmed talaromycosis marneffei, 42 patients with other fungal or bacterial infections, and 70 normal healthy individuals from endemic area of T. marneffei. The test exhibited sensitivity, specificity and accuracy of 87.87%, 100% and 95.5%, respectively. This rapid, user-friendly test holds great promise for the serodiagnosis of T. marneffei infection.