Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis

Pythiosis is an emerging and life-threatening infectious disease of humans and animals living in tropical and subtropical countries and is caused by the fungus-like organismPythium insidiosum. Antifungals are ineffective against this pathogen. Most patients undergo surgical removal of the infected organ, and many die from advanced infections. Early and accurate diagnosis leads to prompt management and promotes better prognosis for affected patients. Immunohistochemical assays (IHCs) have been developed using rabbit antibodies raised againstP. insidiosumcrude extract, i.e., culture filtrate antigen (CFA), for the histodiagnosis of pythiosis, but cross-reactivity with pathogenic fungi compromises the diagnostic performance of the IHC. Therefore, there is a need to improve detection specificity. Recently, the elicitin protein, ELI025, was identified inP. insidiosum, but it was not identified in other human pathogens, including true fungi. The ELI025-encoding gene was successfully cloned and expressed as a recombinant protein inEscherichia coli. This study aims to develop a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) and to compare its performance with the previously reported anti-CFA-based IHC. Thirty-eightP. insidiosumhistological sections stained positive by anti-ELI-based and anti-CFA-based IHCs indicating 100% detection sensitivity for the two assays. The anti-ELI antibody stained negative for all 49 negative-control sections indicating 100% detection specificity. In contrast, the anti-CFA antibody stained positive for one of the 49 negative controls (a slide prepared fromFusarium-infected tissue) indicating 98% detection specificity. In conclusion, the anti-ELI based IHC is sensitive and specific for the histodiagnosis of pythiosis and is an improvement over the anti-CFA-based assay.

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Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii

Infection and Immunity ◽

10.1128/iai.00850-16 ◽

2016 ◽

Vol 85 (4) ◽

Cited By ~ 5

Author(s):

Brenda L. Tesini ◽

Terry W. Wright ◽

Jane E. Malone ◽

Constantine G. Haidaris ◽

Martha Harber ◽

...

Keyword(s):

Pneumocystis Carinii ◽

Surface Protein ◽

Pneumocystis Pneumonia ◽

Negative Control ◽

Cross Reactivity ◽

Pneumocystis Jirovecii ◽

Content Type ◽

Life Threatening ◽

Potential Vaccine ◽

Preventive Methods

ABSTRACT Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina. Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii. Potential orthologs of Pca1 have been identified in P. jirovecii. Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.

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Immunological Cross-Reactivity of Proteins Extracted from the Oomycete Pythium insidiosum and the Fungus Basidiobolus ranarum Compromises the Detection Specificity of Immunodiagnostic Assays for Pythiosis

Journal of Fungi ◽

10.3390/jof7060474 ◽

2021 ◽

Vol 7 (6) ◽

pp. 474

Author(s):

Tiwa Rotchanapreeda ◽

Pattarana Sae-Chew ◽

Tassanee Lohnoo ◽

Wanta Yingyong ◽

Thidarat Rujirawat ◽

...

Keyword(s):

False Positive ◽

Specific Protein ◽

Cross Reactivity ◽

Clinical Implementation ◽

Pythium Insidiosum ◽

Microscopic Features ◽

Life Threatening ◽

And Control ◽

Positive Results ◽

Detection Specificity

Pythiosis, a life-threatening disease caused by Pythium insidiosum, has been increasingly diagnosed worldwide. A recently developed immunochromatographic test (ICT) enables the rapid diagnosis of pythiosis. During the 3-year clinical implementation of ICT in Thailand, we collected the laboratory reports of 38 animals with suspected pythiosis and detected ICT false-positive results in three horses and a dog with basidiobolomycosis. P. insidiosum and Basidiobolus ranarum cause infections with indistinguishable clinical and microscopic features. This study investigated cross-reactive antibodies by probing P. insidiosum and B. ranarum crude extracts and cell-free synthesized I06 protein (encoded in P. insidiosum genome, not other fungi) against a panel of pythiosis, basidiobolomycosis, rabbit anti-I06 peptide, and control sera by Western blot analyses. ICT false-positive results occurred from the cross-reactivity of anti-B. ranarum antibodies to the 15, 50, 60, and 120 kDa proteins of P. insidiosum, not double infections caused by both pathogens. Notably, ICT could help to screen pythiosis, and the positive test requires confirmation by culture or molecular method. The detection specificity of ICT requires improvement. The crude extract containing multispecies antigens needs replacement with a refined P. insidiosum-specific protein. We proposed that the 55 kDa I06 protein is an excellent candidate for developing a more specific serodiagnostic test for pythiosis.

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In Vitro Susceptibility of Thai Pythium insidiosum Isolates to Antibacterial Agents

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02099-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 2

Author(s):

Navaporn Worasilchai ◽

Ariya Chindamporn ◽

Rongpong Plongla ◽

Pattama Torvorapanit ◽

Kasama Manothummetha ◽

...

Keyword(s):

Antibacterial Agents ◽

Antifungal Agents ◽

Radical Surgery ◽

Synergistic Effects ◽

In Vitro Susceptibility ◽

Pythium Insidiosum ◽

Content Type ◽

Life Threatening ◽

Report Data

ABSTRACT Human pythiosis is a life-threatening human disease caused by Pythium insidiosum. In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 μg/ml), minocycline MICs (1 to 4 μg/ml), tigecycline MICs (1 to 4 μg/ml), azithromycin MICs (1 to 16 μg/ml), and clarithromycin MICs (0.125 to 8 μg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.

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Human-Associated Lachnospiraceae Genetic Markers Improve Detection of Fecal Pollution Sources in Urban Waters

Applied and Environmental Microbiology ◽

10.1128/aem.00309-18 ◽

2018 ◽

Vol 84 (14) ◽

Cited By ~ 23

Author(s):

Shuchen Feng ◽

Melinda Bootsma ◽

Sandra L. McLellan

Keyword(s):

Urban Areas ◽

Human Microbiome ◽

Cross Reactivity ◽

Fecal Pollution ◽

Pollution Sources ◽

Rrna Gene ◽

Human Pathogens ◽

Fecal Indicator ◽

Content Type ◽

Nonspecific Amplification

ABSTRACT The human microbiome contains many organisms that could potentially be used as indicators of human fecal pollution. Here we report the development of two novel human-associated genetic marker assays that target organisms within the family Lachnospiraceae . Next-generation sequencing of the V6 region of the 16S rRNA gene from sewage and animal stool samples identified 40 human-associated marker candidates with a robust signal in sewage and low or no occurrence in samples from nonhuman hosts. Two were chosen for quantitative PCR (qPCR) assay development using longer sequences (the V2 to V9 regions) generated from clone libraries. Validation of these assays with these markers, designated Lachno3 and Lachno12, was performed using fecal samples ( n = 55) from cat, dog, pig, cow, deer, and gull sources, and the results were compared with those of established host-associated assays (the Lachno2 marker and two human Bacteroides markers, the HB and HF183/BacR287). Each of the established assays cross-reacted with samples from at least one other animal species, including animals common in urban areas. The Lachno3 and Lachno12 markers were primarily human associated; however, the Lachno12 marker demonstrated low levels of cross-reactivity with samples from select cows and nonspecific amplification with samples from pigs. This limitation may not be problematic when testing urban waters. These novel markers resolved ambiguous results from previous investigations of stormwater-impacted waters, demonstrating their utility. The complexity of the microbiome in humans and animals suggests that no single organism is strictly specific to humans, and the use of multiple complementary markers in combination will provide the highest resolution and specificity for assessing fecal pollution sources. IMPORTANCE Traditional fecal indicator bacteria do not distinguish animal from human fecal pollution, which is necessary to evaluate health risks and mitigate pollution sources. Assessing water in urban areas is challenging, since the water can be impacted by sewage, which has a high likelihood of carrying human pathogens, as well as pet and urban wildlife waste. We demonstrate that the Lachno3 and Lachno12 markers are human associated and highly specific for the detection of human fecal pollution from urban sources, offering reliable identification of fecal pollution sources in urban waters.

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Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum

mSystems ◽

10.1128/msystems.00196-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Pattarana Sae-Chew ◽

Thidarat Rujirawat ◽

Yothin Kumsang ◽

Penpan Payattikul ◽

Tassanee Lohnoo ◽

...

Keyword(s):

Protein Synthesis ◽

Genetic Engineering ◽

Pythium Insidiosum ◽

Content Type ◽

Free Protein ◽

Functional Studies ◽

Life Threatening ◽

Cell Free Protein Synthesis

ABSTRACT Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica. The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen. IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.

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Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

Journal of Virology ◽

10.1128/jvi.01079-15 ◽

2015 ◽

Vol 89 (15) ◽

pp. 7457-7464 ◽

Cited By ~ 19

Author(s):

B. M. Westerhuis ◽

K. S. M. Benschop ◽

G. Koen ◽

Y. B. Claassen ◽

K. Wagner ◽

...

Keyword(s):

Risk Factor ◽

Neutralizing Antibodies ◽

Therapeutic Option ◽

Antiviral Treatment ◽

Severe Disease ◽

Human Memory ◽

Cross Reactivity ◽

Human Mabs ◽

Content Type ◽

Life Threatening

ABSTRACTThe familyPicornaviridaeis a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies.IMPORTANCEHPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPeV1-specific neutralization and cross-neutralized HPeV2. One MAb also cross-neutralized other HPeVs. Surprisingly, this MAb also neutralized CV-A9. These MAbs provide a unique tool for further research and for the diagnosis (antigen detection) and possible treatment of HPeV infections.

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Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates

Applied and Environmental Microbiology ◽

10.1128/aem.01454-17 ◽

2017 ◽

Vol 83 (23) ◽

Cited By ~ 1

Author(s):

Andreas Bauwens ◽

Monika Marejková ◽

Barbara Middendorf-Bauchart ◽

Rita Prager ◽

Annelene Kossow ◽

...

Keyword(s):

Escherichia Coli ◽

Czech Republic ◽

Sequence Similarity ◽

Enterohemorrhagic Escherichia Coli ◽

Type Ii Secretion ◽

Human Pathogens ◽

The Czech Republic ◽

Content Type ◽

E Coli ◽

Life Threatening

ABSTRACT Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H− strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx 2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfA O157OI-141, and lpfA O157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H− strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H− strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H− (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H− patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

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Gamma knife radiosurgery in the management of cavernous sinus meningiomas

Journal of Neurosurgery ◽

10.3171/jns.2000.93.supplement_3.0068 ◽

2000 ◽

Vol 93 (supplement_3) ◽

pp. 68-73 ◽

Cited By ~ 112

Author(s):

Pierre-Hugues Roche ◽

Jean Régis ◽

Henry Dufour ◽

Henri-Dominique Fournier ◽

Christine Delsanti ◽

...

Keyword(s):

Mr Imaging ◽

Tumor Growth ◽

Gamma Knife ◽

Cavernous Sinus ◽

Gamma Knife Radiosurgery ◽

Surgical Removal ◽

Content Type ◽

Cavernous Sinus Meningioma ◽

The Mean ◽

Fine Print

Object. The authors sought to assess the functional tolerance and tumor control rate of cavernous sinus meningiomas treated by gamma knife radiosurgery (GKS). Methods. Between July 1992 and October 1998, 92 patients harboring benign cavernous sinus meningiomas underwent GKS. The present study is concerned with the first 80 consecutive patients (63 women and 17 men). Gamma knife radiosurgery was performed as an alternative to surgical removal in 50 cases and as an adjuvant to microsurgery in 30 cases. The mean patient age was 49 years (range 6–71 years). The mean tumor volume was 5.8 cm3 (range 0.9–18.6 cm3). On magnetic resonance (MR) imaging the tumor was confined in 66 cases and extensive in 14 cases. The mean prescription dose was 28 Gy (range 12–50 Gy), delivered with an average of eight isocenters (range two–18). The median peripheral isodose was 50% (range 30–70%). Patients were evaluated at 6 months, and at 1, 2, 3, 5, and 7 years after GKS. The median follow-up period was 30.5 months (range 12–79 months). Tumor stabilization after GKS was noted in 51 patients, tumor shrinkage in 25 patients, and enlargement in four patients requiring surgical removal in two cases. The 5-year actuarial progression-free survival was 92.8%. No new oculomotor deficit was observed. Among the 54 patients with oculomotor nerve deficits, 15 improved, eight recovered, and one worsened. Among the 13 patients with trigeminal neuralgia, one worsened (contemporary of tumor growing), five remained unchanged, four improved, and three recovered. In a patient with a remnant surrounding the optic nerve and preoperative low vision (3/10) the decision was to treat the lesion and deliberately sacrifice the residual visual acuity. Only one transient unexpected optic neuropathy has been observed. One case of delayed intracavernous carotid artery occlusion occurred 3 months after GKS, without permanent deficit. Another patient presented with partial complex seizures 18 months after GKS. All cases of tumor growth and neurological deficits observed after GKS occurred before the use of GammaPlan. Since the initiation of systematic use of stereotactic MR imaging and computer-assisted modern dose planning, no more side effects or cases of tumor growth have occurred. Conclusions. Gamma knife radiosurgery was found to be an effective low morbidity—related tool for the treatment of cavernous sinus meningioma. In a significant number of patients, oculomotor functional restoration was observed. The treatment appears to be an alternative to surgical removal of confined enclosed cavernous sinus meningioma and should be proposed as an adjuvant to surgery in case of extensive meningiomas.

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Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽

10.3390/pathogens10030365 ◽

2021 ◽

Vol 10 (3) ◽

pp. 365

Author(s):

Fengli Liu ◽

Yanxin Cao ◽

Maokai Yan ◽

Mengxu Sun ◽

Qingshui Zhang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Colloidal Gold ◽

Preventive Intervention ◽

Virus Detection ◽

Cross Reactivity ◽

Duck Enteritis Virus ◽

Detection Sensitivity ◽

Immunochromatographic Assay ◽

Efficient Detection ◽

Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

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Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽

10.1128/msphere.00715-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Suresh Ambati ◽

Emma C. Ellis ◽

Jianfeng Lin ◽

Xiaorong Lin ◽

Zachary A. Lewis ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Effective Dose ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Antifungal Drugs ◽

Extracellular Matrices ◽

Content Type ◽

Order Of Magnitude ◽

Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

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