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.

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.

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.

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.

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.

Protein A/G-based immunochromatographic test for serodiagnosis of pythiosis in human and animal subjects from Asia and Americas.

Pythiosis is a life-threatening infectious disease of both humans and animals living in Asia, Americas, Africa, and parts of Australia and New Zealand. The etiologic pathogen is the fungus-like organism Pythium insidiosum The disease has high mortality and morbidity rates. Use of antifungal drugs are ineffective against P. insidiosum, leaving radical surgery the main treatment option. Prompt treatment leads to better prognosis of affected individuals, and could be achieved by early and accurate diagnosis. Since pythiosis has been increasingly reported worldwide, there is a need for a rapid, user-friendly, and efficient test that facilitates the diagnosis of the disease. This study aims to develop an immunochromatographic test (ICT), using the bacterial protein A/G, to detect anti-P. insidiosum IgGs in humans and animals, and compare its diagnostic performance with the established ELISA. Eighty-five serum samples from 28 patients, 24 dogs, 12 horses, 12 rabbits, and 9 cattle with pythiosis, and 143 serum samples from 80 human and 63 animal subjects in a healthy condition, with thalassemia, or with other fungal infections, were recruited for assay evaluation. Detection specificities of ELISA and ICT were 100.0%. While the detection sensitivity of ELISA was 98.8%, that of ICT was 90.6%. Most pythiosis sera, that were falsely read negative by ICT, were weakly positive by ELISA. In conclusion, a protein A/G-based ICT is a rapid, user-friendly, and efficient assay for serodiagnosis of pythiosis in humans and animals. Compared to ELISA, ICT has an equivalent detection specificity and a slightly lower detection sensitivity.

Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.

Pythiosis is a life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. Morbidity and mortality rates of pythiosis are high. The treatment of choice for pythiosis is surgical debridement of infected tissue. Early and accurate diagnosis is critical for effective treatment. In-house serodiagnostic tests, including immunodiffusion (ID), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICT) and hemagglutination (HA) have been developed to detect antibodies against P. insidiosum in sera. This study compares the diagnostic performance of ID, ELISA, ICT, and HA, using sera from 37 pythiosis patients and 248 control subjects. ICT and ELISA showed optimal diagnostic performance (100% sensitivity, specificity, positive predictive value and negative predictive value). ICT was both rapid and user-friendly. ELISA results were readily quantitated. ID is relatively insensitive. HA was rapid, but diagnostic performance was poor. Understanding the advantages offered by each assay facilitates selection of an assay that is circumstance-appropriate. This will promote earlier diagnoses and improved outcomes for patients with pythiosis.

Luminol encapsulated liposome as a signal generator for the detection of specific antigen-antibody reactions and nucleotide hybridization.

Liposomes prepared with biotinylated phospholipids and luminol entrapped were shown to be of 187 nm in size, 59% of which were unilamellar and with 43% luminol trapping efficiency. Liposome prepared from biotinylated phospholipids with a longer hydrophilic PEG2000 spacer, but not with the shorter hydrophobic caproyl one, bound efficiently and specifically with immobilized streptavidin in a microplate assay. The interactions of dinitrophenol and tobramycin with their respective antibodies, and the hybridization of 20-mers oligonucleotides were studied using the liposome as a signal generator. These reactions were shown to be specific with limits of detection of 0.58 microM, 0.96 microM and 18 nM, respectively.

Development of an immunochromatographic test for rapid serodiagnosis of human pythiosis.

Human pythiosis is an emerging and life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. High rates of morbidity and mortality for patients with pythiosis are exacerbated by the lack of early diagnosis and an effective treatment. Here, we developed and evaluated an immunochromatographic test (ICT) for the diagnosis of human pythiosis, in comparison to a standard serological test of immunodiffusion (ID). Culture filtrate antigen of P. insidiosum was used to detect human anti-P. insidiosum antibody. Sheep anti-human immunoglobulin G-colloidal gold conjugate was used to generate an ICT signal. Thirty-three sera from patients with vascular (n = 27), ocular (n = 4), and cutaneous (n = 2) pythiosis and 181 control sera from healthy blood donors (n = 100), as well as patients with a variety of infectious (n = 56) and noninfectious (n = 25) diseases, were included in the test evaluation. The turnaround time for generating a result by the ICT was less than 30 min, while that for ID was approximately 24 h. Based on the results for all sera of pythiosis patients and the control groups, the ICT showed 88% sensitivity and 100% specificity and ID showed 61% sensitivity and 100% specificity. By both tests, false-negative results for sera from all ocular pythiosis patients were obtained. In addition, the ID test yielded false-negative results for sera from eight patients with vascular pythiosis and one patient with cutaneous pythiosis. It was concluded that the ICT is a rapid, user-friendly, and reliable serological test for the early diagnosis of vascular and cutaneous pythiosis.