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

Pakornswit Sathongdejwisit,Kritsada Pruksaphon,Sirida Youngchim,Kavi Ratanabanangkoon,Nitat Sookrung,Joshua D Nosanchuk,Akarin Intaramat,Pisinee Aiumurai

doi:10.3390/diagnostics11050758

Abstract

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.

Highlights

Cryptococcus neoformans is an opportunistic pathogenic fungus that causes cryptococcosis in immunocompromised patients, especially in advanced HIV/AIDS [1]
The purified monoclonal antibodies (mAbs) 18B7 was investigated for immunoreactivity with GXM cryptococcal capsular antigen from strain H99
The optimized working concentration of mAb 18B7 was at 20 ng/mL

Summary

Introduction

Cryptococcus neoformans is an opportunistic pathogenic fungus that causes cryptococcosis in immunocompromised patients, especially in advanced HIV/AIDS [1]. The lung is the initial site of infection, C. neoformans frequently disseminates and is notorious for causing cryptococcal meningitis [3]. Recommendations for antiretroviral therapy (ART) in HIV infected patients, have improved the health of afflicted patients and reduced the incidence of cryptococcosis, but cryptococcosis continues to kill ~180,000 people every year [2]. The definitive (confirmatory) methods are direct examination of the fungus in body fluids with India ink preparation, histopathological staining of infected tissues and microbiological cultures, with culture being the “gold standard” For the conventional definitive method, India ink preparation and fungal culture demonstrated a high degree of diagnostic specificity, but the diagnostic sensitivity is insufficient.

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