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Open Access Research article

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

Katya C Rocha15*, Cinthia Pinhal1, Sônia Cavalcanti1, Monica SM Vidal1, Matheus Toscano1, Dewton Moraes-Vasconcelos1, Alberto JS Duarte1, Fernando LA Fonseca235, Luiz Carlos de Abreu2, Vitor E Valenti4 and Anete SG Grumach12

Author affiliations

1 Laboratório de Dermatologia and Immunodeficiências, Departmento de Dermatologia, Faculdade de Medicina, Universidade de São Paulo. Av. Dr. Arnaldo, 455. 01246903, São Paulo, SP, Brazil

2 Departamento de Morfologia e Fisiologia, Faculdade de Medicina do ABC. Av. Príncipe de Gales, 821. 09060-650, Santo André, SP, Brazil

3 Departamento de Ciências Biológicas, Universidade Federal de São Paulo, Rua Prof. Artur Riedel, s/n, Diadema, SP, Brazil

4 Departamento de Fonoaudiologia, Faculdade de Filosofia e Ciências, Universidade Estadual Paulista, UNESP. Av. Higyno Muzzi Filho, 737. 17.525-900, Marília, SP, Brazil

5 Departamento de Patologia, Faculdade de Medicina do ABC. Av. Príncipe de Gales, 821. 09060-650 Santo André, SP, Brazil Av. Príncipe de Gales, 821. 09060-650, Santo André, SP, Brazil

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Citation and License

BMC Infectious Diseases 2012, 12:278  doi:10.1186/1471-2334-12-278

Published: 30 October 2012

Abstract

Background

Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis.

Methods

Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of 3H-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05.

Results

The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis.

Conclusion

Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Keywords:
Lymphocytes; Antigens; Biases, Statistical; Cryptococcus; Methods; Cryptococcus neoformans