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. METHODS: Blood samples of clinical VL suspects and healthy endemic controls were collected in Sudan, Nepal and India. Both test versions were performed in duplicate in the respective countries and in the reference laboratory. Interbatch variability and stability tests were conducted and agreement was examined within and between centres on a dichotomic scale by Cohen's kappa as well as on a continuous scale through Bland-Altman plots. RESULTS: The FD antigen remains fully active even after storage at 45 degrees C for 24 months. Using a cut-off titre of 1:6400, the agreement between the FD and the LQ formats was excellent. CONCLUSION: The major advantages of FD antigen are its better stability at higher temperatures and its longer shelf life, which make it much more suitable than the LQ version for use in the field.

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 patient's 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 patient's 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.

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 the micro-CATT, CATT/Trypanosoma brucei gambiense, and LATEX/T b gambiense methods for serodiagnosis and surveillance of human African trypanosomiasis in West and Central Africa.

OBJECTIVE: To evaluate the performance of serological tests using dried blood on filter-papers (micro-card agglutination test for trypanosomiasis (micro-CATT)) performed under field and laboratory conditions and using whole blood ((CATT/T.b. gambiense) (wb-CATT) and latex agglutination (LATEX/T.b. gambiense) (wb-LATEX)) for the serodiagnosis and surveillance of human African trypanosomiasis in West and Central Africa. METHODS: We evaluated the micro-CATT, wb-CATT and wb-LATEX methods in Côte d'Ivoire and the Central African Republic by screening 940 people. Sensitivity and specificity were calculated for each serological test; only patients with the confirmed presence of trypanosomes in the blood or lymph aspirate were considered true positives. Positive and negative predictive values were also calculated. FINDINGS: Each of the tests showed a lower sensitivity in the Central African Republic than in Côte d'Ivoire. CONCLUSION: The results confirmed the efficiency of the classic wb-CATT to detect sleeping sickness patients. The micro-CATT method can be used for human African trypanosomiasis surveillance if the test is performed on the same day as the blood collection, or if samples are stored at 4 degrees C. Otherwise, micro-CATT can be used when absolute sensitivity is not required. wb-LATEX should only be used for high-specificity screening.

Novel primer sequences for polymerase chain reaction-based detection of Trypanosoma brucei gambiense.

Progress in diagnosis, treatment, and epidemiology of human African trypanosomiasis (sleeping sickness) depends on the existence of specific and sensitive diagnostic tools. Inherent shortcomings of serologic and parasitologic diagnostic methods can be overcome by molecular techniques. Therefore, we have developed a new polymerase chain reaction (PCR) test using primers derived from the recently identified sequence of the Trypanosoma brucei gambiense-specific glycoprotein (TgsGP). The specificity of the TgsGP-PCR was evaluated on DNA extracted from 73 different trypanosome populations belonging to diverse taxonomic groups that were isolated from various host species, and from different geographic origins. The TgsG-PCR was shown to be specific for T. b. gambiense and was suitable for detection of trypanosome DNA in blood samples of patients with confirmed sleeping sickness.

Evaluation of an EDTA version of CATT/Trypanosoma brucei gambiense 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.

The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense.

In the search for new diagnostic methods that would distinguish Trypanosoma brucei rhodesiense from T. b. brucei and T. b. gambiense, we have developed two polymerase chain reaction (PCR) primer sets. The first primer set was derived from the serum resistance-associated (SRA) gene of T. b. rhodesiense that confers resistance to lysis by normal human serum (NHS). The specificity of the SRA-based PCR was tested on 97 different trypanosome populations originating from various taxonomic groups, host species, and geographic regions. Only one of 25 T. b. rhodesiense samples was negative in this PCR, and none of 72 other samples were positive in this assay. Interestingly, a reference T. brucei strain (TREU927/4) currently used for genome sequencing was negative for the SRA gene; however, this strain was resistant to lysis by NHS. The second primer set was derived from a specific variant surface glycoprotein (VSG) expression site where the SRA gene is expressed (R-ES). This primer set identified the strain as T. b. rhodesiense in 17 of 17 SRA gene-positive strains in which it was tested. These data strongly suggest that expression of the SRA gene is generally involved in resistance to lysis by NHS in T. b. rhodesiense strains.