Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test

Abstract

Sporotrichosis is an infection of the skin caused by traumatic inoculation of the fungus Sporothrix schenckii. Definitive diagnosis relies on direct visualization of the fungus or its isolation on culture medium, although both have low sensitivity. Alternatively, the detection of the antibody response offers a more rapid alternative for diagnosis. Although the available immunoassays possess good sensitivity and specificity, cross-reactivity is still a problem. This study aimed to evaluate the effect of sodium metaperiodate and 6 M urea solutions on the serological diagnosis of sporotrichosis using an enzyme-linked immunosorbent assay (ELISA) test. Ninety-six-well plates were sensitized with exoantigens from the yeast phase of S. schenckii. Sera of patients with confirmed sporotrichosis, sera of patients with paracoccidioidomycosis, and sera of individuals with a sporotrichin-negative skin test were tested. Two strategies were used; the first consisted of treating the antigen with sodium metaperiodate solution for different incubation times, and the second consisted of treating the serum with 6 M urea solution for different incubation times. ROC curve analysis revealed that the best discrimination parameters were obtained using 6 M urea solution incubated for 5 min and serum dilution at 1/600. The use of 6 M urea solution improves the performance of the ELISA test in the diagnosis of sporotrichosis.

Sporotrichosis: A Review of a Neglected Disease in the Last 50 Years in Brazil

“…Among the classic laboratory procedures (Table 2) for the sporotrichosis diagnosis, the isolation in culture medium is the gold standard, which must be followed by macro and micromorphological identification, in addition to the in vitro thermoconversion test [45,46,[58][59][60][61][62][63]. The ELISA (Enzyme Linked ImmunonoSorbent Assay) method, used for several years for the diagnosis of sporotrichosis, has been a useful tool for the sporotrichosis serological diagnosis [64][65][66][67], although some of these tests may cross-react with other fungal diseases [66]. Molecular diagnostic methods including conventional Polymerase Chain Reaction (PCR), Random Amplified Polymorphic DNA (RAPD), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), and gene sequencing (calmodulin, beta-tubulin, translation elongation factor-1-alpha (TEF1) and translation elongation factor-3 (TEF3) EF-1α) [9][10][11][13][14][15]20,21,32,52,53,56,68], or more recently, methods that employ the Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technique [69,70], have also been used for the diagnosis of sporotrichosis or for isolates genotyping (Table 3) more efficiently than other phenotypic diagnostic methods.…”

Seroepidemiological survey on sporotrichosis-infection in rural areas of the south of Minas Gerais State, Brazil

“…The treatment with 6M urea was able to eliminate the low-affinity Ag-Ac bonds, aiming the detection of antibodies with high affinity and decreasing false-positive results [27,28]. The study conducted by Coelho et al [20] showed efficient discrimination among sera from patients with sporotrichosis, patients with paracoccidioidomycosis, and from sporotrichin-negative individuals using the same ELISA technique [27]. In the present work, the seroprevalence of sporotrichosis-infection was done using the same technique described by Coelho et al Namujju et al [28] were able to associate high-and low-affinity antibodies in African women at risk of infection by certain human papillomavirus (HPV) strains using the ELISA test similar to that employed by Coelho et al [20].…”

“…The ELISA technique was performed according to the protocol proposed by Coelho et al [20]. Briefly, 96-well polystyrene plates were sensitized with crude extract of S. schenckii fungus at a concentration of 5 μg/mL in 0.1 M carbonatebicarbonate buffer, pH 9.6 overnight at 4°C.…”