Academic journal article Bulletin of the World Health Organization

Human African Trypanosomiasis: A Latex Agglutination Field Test for Quantifying IgM in Cerebrospinal Fluid

Academic journal article Bulletin of the World Health Organization

Human African Trypanosomiasis: A Latex Agglutination Field Test for Quantifying IgM in Cerebrospinal Fluid

Article excerpt

Introduction

In 1968, Mattern (1) reported that IgM concentrations exceeding 10% of the total cerebrospinal fluid (CSF) protein concentration are a common feature of human African trypanosomiasis (HAT) in the meningoencephalitic, second stage. The phenomenon was observed in all 230 second-stage HAT patients examined, while was absent in 940 neuropsychiatric patients and in 74 of 75 neurosyphilis cases. Whittle et al. (2) also found high IgM levels in the CSF in 87% of second-stage patients, but not in first-stage cases. Concentrations up to 250 mg/l and 500 mg/l have been reported in Trypanosoma brucei gambiense-infected patients (3, 4). Compared to a normal upper limit of 0.36 mg/l (5), CSF IgM levels in African trypanosomiasis patients are extremely high, which could be the consequence of local production of IgM in the central nervous system (6).

Despite its relevance to stage determination in such patients, IgM detection in CSF is not currently carried out in the field, owing to the lack of simple and robust tests. Two practical assays for IgM quantification, i.e. radial immunodiffusion (RID) and latex agglutination, are commercially available. However, RID is laborious, whereas Rapi Tex IgM (Behring, Frankfurt, Germany), originally developed for detection of IgM in the serum of neonates, has a detection limit of 33 mg/l, far above normal CSF IgM values, and the stability of the reagent is limited.

Reported here is a rapid and sensitive latex agglutination assay (LATEX/IgM), which uses a stable lyophilized reagent. Its performance was assessed on CSF samples from HAT and non-HAT patients and compared with results obtained using nephelometry, RID and Rapi Tex IgM.

Materials and methods

CSF samples from 34 untreated, parasitologically confirmed T.b. gambiense patients ([T.sup.+]), 5 seropositive but parasitologically unconfirmed persons ([T.sup.-] [S.sup.+]), and 4 controls without evidence of HAT ([T.sup.-] [S.sup.-]) were included in the study. All CSF samples originated from Equateur Province, Democratic Republic of Congo, and were obtained from the WHO Central Serum Bank for Sleeping Sickness.

Cell counts were performed within 15 min of CSF collection using Fuchs-Rosenthal counting chambers; any trypanosomes that were found during this procedure were also recorded. CSF samples contaminated with blood were rejected. In order to remove cells, CSF was centrifuged at ambient temperature and the supernatant subsequently stored between -l [degrees] C and -20 [degrees] C for a maximum of 14 days and then at -70 [degrees] C. Total protein concentration was determined using the Bio-Rad modified Coomassie brilliant blue total protein test kit (7). Trypanosomiasis patients with more than 5 cells per [micro]l or protein concentrations above 300 mg/l or with trypanosomes in their CSF were considered to be in second stage.

The IgM concentration was measured by nephelometry (Behring, Frankfurt, Germany) and RID (Human IgM UL and LL Nanorid kits, the Binding Site; Birmingham, England) following the protocols prescribed by the manufacturers. The incubation time of RID was 72 h, following procedure 3 (measurement of ring diameters before completion). Quantification of IgM by means of latex agglutination was carried out with Rapi Tex IgM (Behring, Frankfurt, Germany) and LATEX/IgM following the protocols described hereafter.

Preparation of LA TEX/IgM reagent. A suspension of 7.5 ml carboxyl-modified polystyrene latex (Estapor K1.08, 10% w/v suspension, particle diameter 0.9 [micro]m) is mixed with 6 ml of water; 1.5 ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Pierce, Rockford, IL, USA) freshly dissolved in water to a concentration of 10 mg/ml is added and the suspension is mixed and left for 15 min at room temperature. Subsequently, 3 ml of anti-human IgM (Pasteur Diagnostics, Marnesla-Coquette, France: antisera for immunoelectrophoresis, anti-human IgM ([Mu]) in 0. …

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