E. Magnus

Intrathecal immune response pattern for improved diagnosis of central nervous system involvement in trypanosomiasis

Diagnosis of central nervous system (CNS) involvement in human African trypanosomiasis is crucial in determination of therapy. Cerebrospinal fluid (CSF) and serum immunoglobulin concentrations, blood-CSF barrier dysfunction, pattern of intrathecal immunoglobulin synthesis, trypanosome-specific antibody synthesis, and CSF lactate concentrations were analyzed in 272 patients with Trypanosoma brucei gambiense infection. As part of the 2- or 3-class immune response, the predominant intrathecal IgM synthesis was the most sensitive (95%) marker for inflammation of the brain. We propose to replace the World Health Organization (WHO) criteria (white blood cell count >5 cells/microL and presence of trypanosomes in CSF) with a new approach for stage determination in trypanosomiasis: CNS involvement is diagnosed only in patients with >20 cells/microL or with intrathecal IgM synthesis, independent of the presence of trypanosomes in CSF. Compared with the use of these new criteria, the WHO criteria incorrectly classified 49 of 234 patients in the meningoencephalitic stage and 7 of 38 patients in the hemolymphatic disease stage. We also show that trypanosomiasis-related immunoglobulin patterns are of value in differential diagnosis.

Evaluation of variant specific trypanolysis tests for serodiagnosis of human infections with Trypanosoma brucei gambiense

Twelve T.b. gambiense clone populations of distinct Variable Antigen Type (VAT) were combined in immune lysis tests with 340 sera of trypanosome infected patients from 8 different African countries and 267 non trypanosomiasis control sera. The diagnostic specificity of the test was 100%. At a serum dilution of 1:4 the overall test sensitivity with single VATs varied from 39.1 to 98.2% and from 12.1 to 86.8% at 1:32. At a serum dilution of 1:32 some combination tests with 2 VATs still scored above 96%. The VAT recognition patterns were clearly correlated with the geographical origin of the sera, reflecting a diversity in variable antigen repertoires.

Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon.

Antibodies to the variable antigen type (VAT) designated LiTat 1.3 are common in sera from parasitologically confirmed patients with gambian sleeping sickness. For this reason, LiTat 1.3 has been considered a suitable antigen for detecting Trypanosoma brucei gambiense in the Card Agglutination Test for Trypanosomiasis (CATT; Testryp-CATT, Smith Kline-RIT). However, surveys in the T.b. gambiense endemic focus of Fontem in Cameroon have suggested that expression of LiTat 1.3 might be rare or absent. We show here that the gene for LiTat 1.3 was indeed absent from some T.b. gambiense stocks isolated from this focus, and a LiTat 1.3-like gene was present in others. The divergent gene differed from the cloned version of LiTat 1.3. In addition, antibodies to LiTat 1.3 could not be detected in rabbits infected with either of the two kinds of T.b. gambiense from the Fontem area. We suggest that the absence of LiTat 1.3 expression in this focus may have important implications for the epidemiology and control of sleeping sickness, especially if heavy reliance is placed on the CATT.

Follow‐up of Card Agglutination Trypanosomiasis Test (CATT) positive but apparently aparasitaemic individuals in Côte d'Ivoire: evidence for a complex and heterogeneous population

Summary The aetiological diagnosis of human African trypanosomiasis (HAT) is based on the detection of the parasite, but currently available parasitological tests have low sensitivity and are hampered by fluctuating parasitaemia. The identification of seropositive individuals on whom to focus parasitological examination is based on antibody detection by means of the Card Agglutination Trypanosomiasis Test (CATT/T.b.gambiense). A complicating phenomenon is the occurrence of serologically positive but parasitologically unconfirmed results (isolated CATT positivity). This work presents a two‐year longitudinal serological, parasitological and molecular follow‐up of CATT‐positive individuals including repeated examinations of each individual, to study the evolution over time of seropositivity at both the population and the individual levels. At the population level, the rate of seropositivity decreased during the first months of the survey, and afterwards showed remarkable stability. At the individual level, the results reveal the extreme heterogeneity of this population, with subjects showing fluctuating results, others with a short transient CATT positivity, and subjects that maintain their seropositivity over time. The stability of seropositivity and the pattern of results obtained with both immunological and parasitological examinations support the view that individual factors, such as immune response to infection, might be involved in the isolated CATT positivity phenomenon.

IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test

An increased IgM concentration in cerebrospinal fluid (CSF), occurring as a consequence of massive intrathecal IgM synthesis, is a marker of interest for diagnosis of the meningo‐encephalitic stage in human African trypanosomiasis. However, in current practice, IgM in CSF is not determined because of the lack of a simple and robust test that is applicable in African rural regions where the disease prevails. We describe the development of a sensitive semiquantitative card agglutination test, LATEX/IgM, for IgM quantification in CSF. The test is simple and fast and the lyophilized reagent remains stable even at 45 °C. CSF end‐titres obtained with LATEX/IgM parallel the IgM concentrations determined by nephelometry and enzyme‐linked immunosorbent assay. Detection of intrathecal IgM synthesis is the most sensitive marker for CNS involvement in sleeping sickness. At a cut‐off value of ≥ 8, the sensitivity and specificity of LATEX/IgM for intrathecal IgM synthesis are 89.4 and 92.7%. As a consequence, patients with LATEX/IgM end‐titres ≥ 8 are likely to have intrathecal IgM synthesis, thus central nervous system involvement and therefore should be treated accordingly. Further studies should concentrate on the relationship between the LATEX/IgM end‐titres, presence of intrathecal IgM synthesis and occurrence of treatment failures in patients treated with pentamidine.

A semi-quantitative ELISA for detection of Trypanosoma brucei gambiense specific antibodies in serum and cerebrospinal fluid of sleeping sickness patients.

A semi-quantitative ELISA, using variable surface glycoprotein of T.b. gambiense as antigen, was developed for the detection of antibodies of different immunoglobulin isotypes in serum and cerebrospinal fluid of sleeping sickness patients. Using the assay, the antibody profiles of paired serum and cerebrospinal fluid samples of 28 patients have been studied. Total concentrations of various Ig isotypes were determined as well. In serum and cerebrospinal fluid a drastic increase in IgG, basically IgG1, as well as in IgM levels was observed. The concentration of IgA remained relatively normal. The antitrypanosomal antibodies detected in serum and cerebrospinal fluid were mainly of the IgG (IgG1 and IgG3) and IgM isotypes. Measurement of immunoglobulin and trypanosome specific antibody concentrations in serum and CSF allows calculation of intrathecal antibody synthesis and is a possible tool for determining the clinical stage of sleeping sickness.

Comparative evaluation of freeze-dried and liquid antigens in the direct agglutination test for serodiagnosis of visceral leishmaniasis (ITMA-DAT/VL).

Objective  The direct agglutination test (DAT) for visceral leishmaniasis (VL) with liquid (LQ) antigen is known to be only moderately reproducible because of inter‐observer and batch‐to‐batch variability as well as its sensitivity to temperature and shaking during transport. We evaluated a DAT with freeze‐dried (FD) antigen and compared it with the LQ antigen version.

Susceptibility of Grammomys surdaster thicket rats to Trypanosoma brucei gambiense infection.

Human African Trypanosomiasis is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. Historically, a treatment relapse rate of about 5% is observed in patients treated with melarsoprol, an arsenical derivative used for treatment of both gambiense and rhodesiense second stage sleeping sickness. More recently, relapse rates up to 30% are noted in gambiense sleeping sickness foci in Angola, Sudan and Uganda. Therefore, WHO established a Network on Treatment Failure and Drug Resistance in Sleeping Sickness. One of its objectives is to improve isolation of T. b. gambiense from relapsing cases for research on drug resistance mechanisms. Trypanosoma b. gambiense isolation techniques suffer from low success rates and long periods needed to adapt the parasite to its new host. Usually, rodents are inoculated with patients blood or cerebrospinal fluid and sub‐passaged until the strain becomes sufficiently adapted to yield high parasitaemia within few days after inoculation. Until now, the best recipient for T. b. gambiense is Mastomys natalensis, with a success rate of about 50%. In this study, Grammomys surdaster (former Thamnomys surdaster) was investigated as a potential recipient for isolation of T. b. gambiense. Comparative experimental infections of Swiss mice, Wistar rats and G. surdaster thicket rats with T. b. gambiense clearly show that this trypanosome grows faster in G. surdaster. Inoculation of the same rodent species with patients blood and cerebrospinal fluid in Kinshasa (R.D. Congo) confirms the observation that the thicket rats are more susceptible to T. b. gambiense infection than typical laboratory rodents.

Evaluation of an EDTA version of CATT/Trypanosoma bruceigambiense for serological screening of human blood samples

CATT/Trypanosoma brucei gambiense, a direct card agglutination test designed for field surveys on human African trypanosomosis, is currently used with freshly collected heparinized blood samples. When testing serum samples, it has been observed earlier that, at lower sample dilutions, a complement-mediated inhibition phenomenon may cause false negative test results. This can be avoided by adding an anticomplementary agent such as di-sodium ethylenediaminetetraacetate dihydrate (EDTA) to the reaction. As the sensitivity of the blood assay might be improved in the same way, this possibility has been examined under both laboratory and field conditions, by adding EDTA to the test buffer or, as an anticoagulant, to the blood samples. The CATT-EDTA versions proved up to 7% more sensitive but also 1-2% less specific than the current test. CATT buffer supplemented with EDTA remained stable for at least 2 years at +45 degrees C.

Increased sensitivity of the card agglutination test CATT/Trypanosoma brucei gambiense by inhibition of complement

CATT/Trypanosoma brucei (T.b.) gambiense is an antibody detection test currently used in field surveys on Gambian sleeping sickness. The screening test is usually performed on a drop of freshly collected heparinized blood, followed by a more specific confirmation test on diluted blood, plasma or serum. This approach may be biased by the occurrence of a complement-mediated prozone phenomenon causing lower test sensitivity at lower sample dilutions. A simple remedy is by addition of a Ca2+ chelating agent such as EDTA.