Evaluation of Five Non-Culture-Based Methods for the Diagnosis of Meningeal Sporotrichosis

Abstract

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture's limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

Keywords: ELISA; Sporothrix; cerebrospinal fluid; immunological diagnosis; molecular diagnosis; qPCR.

Figure 1

Representative amplification profile of CSF samples in two DNA-based methods for Sporothrix spp. detection. (a) Nested PCR-Agarose gel with positive CSF samples, demonstrating 152 bp fragments compatible with Sporothrix spp. Values 1 and 10 = Molecular weight (100 pb Plus–Invitrogen), 2 to 6 = positive CSF samples, 7 = positive control of DNA extracted from Sporothrix brasiliensis culture (CBS 120339), 8 = PCR mix negative control, and 9 = PCR mix negative control with water addition. (b) Amplification curves of the qPCR observed in positive CSF samples using the CY3 fluorescence channel. Each colored curve represents a different sample. The yellow horizontal line represents the threshold of the reaction. +: positive control. −: negative control.

Figure 2

Detection by enzyme-linked immunosorbent assay (ELISA) of (a) IgG and (b) IgM responses against the mycelial phase S. brasiliensis exoantigens in cerebrospinal fluid samples from patients with disseminated sporotrichosis and meningitis (cases), patients without sporotrichosis (controls), and patients with sporotrichosis but without criteria for meningitis (tests). The doted horizontal lines indicate the cutoff values for each single ELISA.