94 Am. J. Trop. Med. Hyg., 60(1), 1999, pp. 94–98 Copyright  1999 by The American Society of Tropical Medicine and Hygiene DETECTION OF LIGHT SUBUNIT NEUROFILAMENT AND GLIAL FIBRILLARY ACIDIC PROTEIN IN CEREBROSPINAL FLUID OF TRYPANOSOMA BRUCEI GAMBIENSE–INFECTED PATIENTS V. LEJON, L. E. ROSENGREN, P. BUSCHER, J.-E. KARLSSON, AND H. N. SEMA* Department of Parasitology, Institute of Tropical Medicine, Antwerp, Belgium; Department of Neurology, Institute of Clinical Neuroscience, Sahlgren’s University Hospital, University of Goteborg, Goteborg, Sweden; Bureau Central de la Trypanosomiase, Centre de Developpement Integre Bwamanda, Bwamanda, Democratic Republic of the Congo; Department of Biochemistry, University of Antwerp, Wilrijk, Belgium Abstract. Light subunit neurofilament (NFL) and glial fibrillary acidic protein (GFAP) concentrations were de- termined in cerebrospinal fluid (CSF) of 34 patients with human African trypanosomiasis (HAT), five serologically positive but parasitologically unconfirmed individuals, and four healthy controls without evidence of HAT. In patients with second stage HAT (n = 30), NFL levels were abnormally elevated in 10 cases and GFAP levels in five. The astrogliosis observed in HAT and experimental models of HAT is confirmed in our study by the presence of increased GFAP levels in the CSF. The abnormal NFL CSF levels reflect structural damage of nerve cells in 33 % of the second-stage patients studied. To our knowledge, this is the first time neuronal damage in HAT patients is demon- strated by using biochemical markers of brain damage in the CSF. Human African trypanosomiasis (HAT) or sleeping sick- ness is a tropical disease caused by Trypanosoma brucei gambiense. Two consecutive disease stages can be dis- cerned: 1 the first, a hemolymphatic phase, is followed by a second, meningoencephalitic phase, which is associated with the spread of trypanosomes into the central nervous system (CNS). The meningoencephalitis observed in African try- panosomiasis is characterized by parasite invasion of the me- ninges and choroid plexus, infiltration of the leptomeninges and perivascular areas by IgM producing plasma cells, Mott cells, and T helper/inducer cells, diffuse microglial hyper- plasia, astrogliosis in the white matter, and demyelination of neurons. 2–6 A correct stage determination is indispensable since it is directly related to the choice of an optimal treat- ment with minimal risk for the patient. This is currently car- ried out by examining the cerebrospinal fluid (CSF) for cell number, protein concentration, and presence of trypano- somes. Other CSF parameters associated with second-stage HAT, such as the occurrence of IgM, 7,8 trypanosome-specific an- tibodies, 8–10 and autoantibodies against CNS components such as myelin, 11 galactocerebroside, 12,13 and the 200-kD and 160-kD neurofilament proteins, 14 have been described. These autoantibodies may be induced by cerebrospecific antigens leaking from injured nervous tissue, or otherwise may be actively involved in the pathogenesis of the nervous system damage in HAT by initiating or maintaining demyelination and nerve cell injury. 2,15,16 The glial fibrillary acidic protein (GFAP) is a major struc- tural protein of astrocytes. It composes the glial intermediate filament, 17,18 which forms the morphologic basis of astro- gliosis. 19 Immunohistochemical studies of GFAP in experi- mental and in vitro models of HAT show extensive astro- gliosis accompanied by production of prostaglandin, inter- leukin-1 (IL-1), IL-6, macrophage inflammatory protein-1, tumor necrosis factor-, and interferon-, which contribute to CNS pathology. 6,20–25 The observed astrocyte activation and associated cytokine production may be a key element in CNS inflammatory processes. * Deceased. The neurofilament is a major structural element of neu- rons. Its principal role is to maintain axon caliber and neu- ronal size and shape. 26 It is composed of a triplet protein of which the light subunit (NFL) is the essential component of the neurofilament core. Experimental research has shown that NFL can be used as a marker for neuronal degeneration in animal brain. 27 Both of these intermediate filament proteins can be as- sayed in the CSF. 28,29 Increased levels of GFAP in the CSF have been observed in patients with disease associated with astrogliosis such as dementia, 28,30 progressive encephalopa- thy in children, 31 multiple sclerosis, 32 and Lyme neurobor- reliosis. 33 Pathologically high CSF levels of NFL have been observed in inflammatory diseases and neurodegenerative disorders such as multiple sclerosis, amyotrophic lateral sclerosis, and dementia 29,34 reflecting the neuronal, mainly axonal damage in these disorders. In the present study, GFAP and NFL concentrations were determined in the CSF of second-stage HAT patients to in- vestigate 1) astrocyte activation and neuronal damage in sec- ond-stage HAT and 2) the usefulness of these proteins as second-stage disease markers. MATERIALS AND METHODS Glial fibrillary acidic protein assay. The GFAP concen- trations were measured with a slightly modified sandwich ELISA. 28 Briefly, microtiter plates (96-well, flat-bottom) were coated with chicken anti-GFAP IgG (isolated from yolk) and postcoated with bovine serum albumin. The mi- crotest plates were kept frozen at -20°C until use. Duplicate samples of CSF, trypanosome extracts, or reference GFAP were added and incubated for 2 hr at room temperature. Fol- lowing washing with 0.9% NaCl, 0.05% Tween 20, the wells were incubated with rabbit anti-GFAP IgG for 1 hr at room temperature. The plates were then washed with 0.9% NaCl, 0.05% Tween 20, peroxidase-conjugated donkey anti-rabbit IgG (Amersham, Buckinghamshire, United Kingdom) was added (diluted 1:2,000 in phosphate-buffered saline, 0.1% bovine serum albumin), and incubated for 1 hr at room tem- perature. Following washing with 0.9% NaCl, 0.05% Tween