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 ∼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

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

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

The ICT strip system of <i>T</i>. <i>marneffei</i>.

<p>(a) Cartoon of the components of the <i>T</i>. <i>marneffei</i> ICT strip. (b) A schematic diagram of the ICT strips for the detection of TM CYA antigen in urine. (A: Pre-run strip, B: Positive result, C: Negative result) WP, wicking pad; AP, analytical pad; SP, sample pad; CRP, conjugate releasing pad; C, control line; T, test line.</p

Urine samples from patients infected with bacterial and fungal infections used in the study.

<p>Urine samples from patients infected with bacterial and fungal infections used in the study.</p

The ICT system does not detect antigens from other common fungal pathogens.

<p>Urine samples from healthy volunteers were spiked with TM CYA (PC), TM CMA, <i>C</i>. <i>albicans</i> (Ca), <i>C</i>. <i>neoformans</i> (Cn), <i>P</i>. <i>insidiosum</i> (Pi), <i>H</i>. <i>capsulatum</i> (Hc), <i>Penicillium</i> sp. (Ps), <i>A</i>. <i>fumigatus</i> (Af) at 50 μg/ml each. NC, normal urine negative control; C, control line; T: test line.</p

Development and characterization of an immunochromatographic test for the rapid diagnosis of <i>Talaromyces (Penicillium) marneffei</i>

<div><p><i>Talaromyces</i> (<i>Penicillium</i>) <i>marneffei</i> 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 <i>T</i>. <i>marneffei</i> yeast antigens in urine samples. The highly <i>T</i>. <i>marneffei-</i>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 <i>T</i>. <i>marneffei</i> 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 <i>T</i>. <i>marneffei</i> 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 <i>T</i>. <i>marneffei</i>. 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 <i>T</i>. <i>marneffei</i> infection.</p></div

MAb 4D1 similarly labels prepared <i>T</i>. <i>marneffei</i> cytoplasmic yeast antigens and proteins released into <i>T</i>. <i>marneffei</i> culture supernatants.

<p>SDS-PAGE (A) and Western immunoblots (B) prepared with <i>T</i>. <i>marneffei</i> cytoplasmic mycelial antigen (TmM), yeast antigen (TmY) and the concentrated supernatants from day 7 (TmS1) and day 14 (TmS2) yeast cell cultures. MAb 4D1 was used in (B). The numbers on the left indicate relative molecular weights of markers (M).</p

Determination of the limit of detection (LOD) of the ICT strip.

<p>(A) By visual inspection and (B) by UVP visionWorks LS scanner. Urine samples from healthy individuals were spiked with the indicated concentrations in μg/ml of TM CYA. NC, urine from healthy individuals as negative control; C: control line, T: test line.</p