Biomedical subjects

P Büscher

Publications and source records attributed to P Büscher.

At least 19 recordsLinked to original sources

PubMed2006

Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes.

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.

Animals↗

PubMed2006

Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis: a potential human reservoir of parasites?

The serological and parasitological tests used for Trypanosoma brucei gambiense human African trypanosomiasis (HAT) diagnosis have low specificity and sensitivity, respectively, and in the field, control program teams are faced with subjects with positive serology but negative parasitology who remain untreated. The aim of this work was to explore, using PCR tool, the significance of these aparasitemic serological suspects. Since discordant PCR results have been observed earlier with different extraction methods, two DNA extraction methods were compared (the Chelex 100 resin and the DNeasy Tissue kit). The study was conducted on 604 blood samples: 574 from parasitologically confirmed patients, aparasitemic serological suspects and endemic controls collected in Côte d'Ivoire and 30 from healthy volunteers collected in France. No significant differences were observed between the PCR results obtained with the two extraction methods. Concerning PCR, problems of reproducibility and discordances with both serological and parasitological test results were observed, mainly for the aparasitemic serological suspects. In addition to previous results that pointed to the existence of non-virulent or non-pathogenic trypanosome strains and of individual susceptibility leading to long term seropositivity without detectable parasitaemia but positive PCR, the results of this study support the notion of a long lasting human reservoir that may contribute to the maintenance or periodic resurgences of HAT in endemic foci.

Agglutination Tests↗

PubMed2006

Mineralization and mechanical properties of the canine mandible distraction wound following acute molding.

To investigate the influence of an acute single step callus manipulation immediately after distraction on mechanical properties and mineralization of the regenerate, custom made distraction devices were bilaterally placed in the mandibular angle of 15 beagle dogs, allowing to simultaneously compress and stretch the regenerate after completed linear distraction. The animals were divided in three groups (n=5): Group 1 and 2 underwent manipulation of the regenerate, group 3 remained in a linear position. After 42 (group1) and 90 (group 2 and 3) days of consolidation the animals were sacrificed. The mechanical properties were assessed in an Instron testframe and bone density quantified by quantitative computed tomography and three- dimensionally assessed (Scion Image processing and analysis software). After 6 weeks of consolidation 25% of the specimens reached a stiffness which was >/=90% of the mean values of the unoperated reference hemi-mandibles. After a 13 week consolidation period, 62.5% were as stiff as the referenced specimens. Manipulated regenerates, allowed to heal under stable conditions for 13 weeks, had the same mechanical properties as specimens that underwent pure linear distraction. A temporary but not significant delay of osseous healing had to be postulated for the stretched zone after 6 weeks, indicating this area to be more critical than the compressed area.

Animals↗

PubMed2006

[Validity, cost and feasibility of the mAECT and CTC confirmation tests after diagnosis of African of sleeping sickness].

OBJECTIVES: To evaluate the validity, cost and feasibility of two parasitological tests for the confirmation of Human African Trypanosomiasis (HAT): the mini Anion-exchange Centrifugation Technique (mAECT) and Capillary Tube Centrifugation (CTC). METHODS: During a sleeping sickness screening campaign in 2004 we screened 6502 people in Kwamouth, DRC. Those with a positive result in the Card Agglutination Test for Trypanosomiasis (CATT) had a gland puncture, fresh blood examination, stained thick blood film, mAECT, CTC and CATT titration. Sensitivity and specificity of the confirmation tests were calculated using the combination of all parasitological tests as a reference standard. Each method was costed and its feasibility was assessed with structured interviews of the technicians. RESULTS: Sensitivity of classical parasitological methods was 44.8% (36.8-53.0), of CTC 56.5% (48.3-64.5) and of mAECT 75.3% (95% CI: 67.7-81.9). Cost per test was 2.82 Euro for mAECT and 0.76 Euro for CTC. Time per test was 29.78 min for mAECT and 18.25 min for CTC. These two tests were judged feasible in field conditions. CONCLUSION: CTC and mAECT used alone or in combination would bring a considerable improvement to HAT active case finding when used as confirmation tests in CATT-whole blood-positive persons. They proved feasible in operational conditions if a 220 V power supply can be guaranteed. As mAECT is more sensitive but also considerably more expensive, efficiency as well as feasibility considerations will have to guide the choice of the best algorithm.

Adolescent↗

PubMed2006

Detection of trypanosome-specific antibodies in saliva, towards non-invasive serological diagnosis of sleeping sickness.

OBJECTIVE: The detection of trypanosome-specific antibodies in saliva is technically feasible, and, if clinically validated, could become an attractive option for non-invasive diagnosis of sleeping sickness. We wanted to optimize the test format of an enzyme-linked immunosorbent assay (ELISA)-based antibody detection system. METHODS: Different ELISA formats for antibody detection in serum and saliva were developed and standardized. Saliva and serum samples were collected from 78 patient and 128 endemic control samples, and sensitivity and specificity of saliva ELISAs, serum ELISAs and the card agglutination test for trypanosomiasis (CATT), were evaluated. RESULTS: All ELISA formats showed sensitivity and specificity above 90%. Saliva ELISAs showed a similar test performance as serum ELISAs and the CATT on whole blood or serum. CONCLUSIONS: This study confirms the potential of trypanosome-specific antibody detection in saliva.

Agglutination Tests↗

PubMed2006

Molecular dipstick test for diagnosis of sleeping sickness.

Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 microl of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.

Animals↗

PubMed2005

Recombinant RoTat 1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary camels.

The transcript encoding a predominant Trypanosoma evansi variable surface glycoprotein RoTat 1.2 was cloned and expressed as a recombinant protein in Spodoptera frugiperda and Trichoplusia ni (insect) cells. Its potential as an antigen for specific detection of antibody in serum of dromedary camels affected by surra, was evaluated. In ELISA, the reactivity of the recombinant RoTat 1.2 VSG was similar to that of native RoTat 1.2 VSG. An indirect agglutination reagent was therefore prepared by coupling the recombinant RoTat 1.2 VSG onto latex particles. The performance of the latex agglutination test was evaluated on camel sera, and compared with the performance of CATT/T. evansi and LATEX/T. evansi tests, using the immune trypanolysis assay with T. evansi RoTat 1.2 as a reference test. The relative sensitivity and specificity of the latex coated with recombinant RoTat 1.2 VSG, using a 1:4 serum dilution, were respectively, 89.3 and 99.1%. No differences were observed between the performance of latex coated with recombinant RoTat 1.2 VSG and LATEX/T. evansi or CATT/T. evansi. Here, we describe the successful use of the recombinant RoTat 1.2 VSG for detection of specific antibodies induced by T. evansi infections.

Animals↗

PubMed2005

Review Article: cerebrospinal fluid in human African trypanosomiasis: a key to diagnosis, therapeutic decision and post-treatment follow-up.

Human African trypanosomiasis is a lethal parasitic infection with neurological involvement. Examination of the cerebrospinal fluid (CSF) plays an essential role in diagnosis, selection of treatment and post-treatment follow-up. This paper reviews clinical presentation, diagnosis and treatment of the disease, with emphasis on CSF characteristics and interpretation of the CSF results for therapeutic decision and post-treatment follow-up.

Animals↗

PubMed2004

Evaluation of the card agglutination test (CATT/T. evansi) for detection of Trypanosoma evansi infection in water buffaloes (Bubalus bubalis) in Egypt.

A card agglutination test (CATT/T. evansi) was evaluated for detection of antibodies against Trypanosoma evansi (T. evansi) in experimentally and naturally infected buffaloes. Four calves were inoculated with a strain of T. evansi isolated from a dromedary camel. Parasitological examination of the calves revealed trypanosomes in the blood from days 4 to 9 post-inoculation (PI). General emaciation appeared from day 26 PI and aggravated until the end of the experiment (day 88 PI). Antibodies against T. evansi were detectable from day 8 PI till the end of the experiment. Parasitological examination of 200 water buffalo blood samples obtained from slaughterhouses revealed negative results. Serological examination of these animals showed that 48 (24%) water buffaloes had anti-T. evansi antibodies.

Agglutination Tests↗

PubMed2004

Trypanosoma vivax: a simplified protocol for in vivo growth, isolation and cryopreservation.

A rodent adapted clone of Trypanosoma vivax was used to infect cyclophosphamide treated mice and rats. Fresh blood containing trypanosomes, was centrifuged in a density gradient of three Percoll solutions, 1.07, 1.06, 1.05 g/ml, respectively, carefully layered on top of each other. The yields of this simple procedure for trypanosome purification were about six times higher than those obtained with the conventional anion-exchange columns. Cryopreservation of trypanosomes using glycerol yielded 90% viable parasites, whereas using dimethylsulfoxide, a more commonly used cryoprotectant, the viability was only 35%.

Animals↗

PubMed2003

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum.

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.

Agglutination Tests↗

PubMed2003

Evaluation of LATEX/T.b.gambiense for mass screening of Trypanosoma brucei gambiense sleeping sickness in Central Africa.

We compared the Card Agglutination Test for Trypanosomiasis (CATT), which consists of lyophilized bloodstream form trypomastigotes of Trypanosoma brucei gambiense (T.b.g.) variable antigen type LiTat 1.3, with LATEX/T.b.g., which consists of a lyophilized suspension of latex particles coated with variable surface glycoproteins of T.b.g. variable antigen types LiTat 1.3, 1.5 and 1.6. This study was carried out during two mass screening surveys in 1998 in Campo, a sleeping sickness focus in Cameroon, with a low prevalence (0.3%) and in 1999 in Batangafo which belongs to the Central African focus of Ouham which has a higher prevalence (3%). In Campo, we compared the CATT performed on whole blood with the LATEX/T.b.g. on diluted blood. In Batangafo, both tests were performed on diluted blood. In all circumstances, the specificity of the LATEX/T.b.g. was higher than of CATT. The use of LATEX/T.b.g. on diluted blood instead of CATT results in an important decrease of workload and as a consequence, of costs related to parasitological examinations. In the case of Campo the workload was up to 12 times less than when using CATT 1.3 on whole blood and the cost divided by 3. In Batangafo the workload was decreased by nearly 20% with the LATEX/T.b.g. Finally, it should be noted that in Batangafo, one of the parasitologically confirmed sleeping sickness patients was negative in CATT and positive in LATEX/T.b.g. and that the reading of the test result in LATEX/T.b.g. is easier than in CATT.

Africa, Central↗

PubMed2003

The challenge of Trypanosoma brucei gambiense sleeping sickness diagnosis outside Africa.

Sleeping sickness is a lethal African disease caused by parasites of the Trypanosoma brucei subspecies, which is transmitted by tsetse flies. Occasionally, patients are reported outside Africa. Diagnosis of such imported cases can be problematic when the infection is due to Trypanosoma brucei gambiense, the chronic form of sleeping sickness found in west and central Africa. The low number of trypanosomes in the blood and the non-specific, variable symptoms make the diagnosis difficult, particularly when the index of suspicion is low. When the trypanosomes have penetrated into the central nervous system, neuropathological signs become apparent but even at this stage, misdiagnosis is frequent. Rapid and correct diagnosis of sleeping sickness can avoid inappropriate or delayed treatment and even death of the patient. In this article, an overview on diagnosis of imported cases of T b gambiense sleeping sickness is given, and possible pitfalls in the diagnostic process are highlighted. Bioclinical parameters that should raise the suspicion of sleeping sickness in a patient who has been in west or central Africa are discussed. Techniques for diagnosis are reviewed. A clinician suspecting sleeping sickness should contact a national reference centre for tropical medicine in his or her country, or the WHO, Geneva, Switzerland, or the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, for clinical consultation and provision of specific diagnostic tests. Appropriate drugs for sleeping sickness treatment are also provided by WHO and the CDC.

Animals↗

PubMed2003

How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and multiplex-endonuclease genotyping approach.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

Animals↗

PubMed2003

Towards saliva-based screening for sleeping sickness?

A pilot study was carried out on the detection of trypanosome-specific antibodies in saliva for diagnosis of sleeping sickness. All twenty-three saliva samples of parasitologically confirmed Trypanosoma brucei gambiense patients tested positive in an indirect enzyme-linked immunosorbent assay, whereas all 14 saliva samples of a negative control group remained negative. Trypanosome-specific antibody levels in patient saliva correlated with antibody levels in serum, but were about 250-fold lower. Eight of 23 undiluted saliva samples of sleeping sickness patients tested positive in CATT/T. b. gambiense and two of 23 in LATEX/T. b. gambiense. All fourteen saliva samples of the negative control group were also positive in CATT/T. b. gambiense, as were four of 14 in LATEX/T. b. gambiense. CATT and LATEX were thus inappropriate for antibody detection in saliva. These results indicate that trypanosome-specific antibody detection in saliva is possible. This could lead to the development of a simple, non-invasive, reliable saliva field test for diagnosis of sleeping sickness.

Animals↗

PubMed2003

Stage determination and therapeutic decision in human African trypanosomiasis: value of polymerase chain reaction and immunoglobulin M quantification on the cerebrospinal fluid of sleeping sickness patients in Côte d'Ivoire.

In human African trypanosomiasis (HAT), two disease stages are defined: the first, or haemo-lymphatic stage, and the second, or meningo-encephalitic stage. Stage determination forms the basis of therapeutic decision and is of prime importance, as the drug used to cure second-stage patients has considerable side-effects. However, the tests currently used for stage determination have low sensitivity or specificity. Two new tests for stage determination in the cerebrospinal fluid (CSF) were evaluated on 73 patients diagnosed with HAT in Côte d'Ivoire. The polymerase chain reaction (PCR) detecting trypanosome DNA (PCR/CSF) is an indirect test for trypanosome detection whereas the latex agglutination test detecting immunoglobulin M (LATEX/IgM) is an indicator for neuro-inflammation. Both tests were compared with classically used tests, double centrifugation and white blood cell count of the CSF. PCR/CSF appeared to be the most sensitive test (96%), and may be of use to improve stage determination. However, its value for therapeutic decision appears limited, as patients whose CSF was positive with PCR were successfully treated with pentamidine. This result confirms those of previous works that showed that some patients with trypanosomes in the CSF could be treated successfully with pentamidine. LATEX/IgM, which depending on the cut-off, showed lower sensitivity of 76% and 88%, but higher specificity of 83% and 71% for LATEX/IgM 16 and LATEX/IgM 8 respectively, appears more appropriate for therapeutic decision making.

Animals↗

PubMed2003

Human African trypanosomiasis: quantitative and qualitative assessment of intrathecal immune response.

Quantitative and qualitative techniques for assessment of the intrathecal humoral immune response in human African trypanosomiasis were compared, and their diagnostic potential for detection of the meningo-encephalitic stage of the disease was evaluated. Total and trypanosome specific immunoglobulin G (IgG) and IgM intrathecal synthesis were studied in paired cerebrospinal fluid (CSF) and blood samples of 38 trypanosomiasis patients and in three controls using Reiber's formulae. The presence of CSF-specific oligoclonal IgG and of trypanosome-specific antibodies was determined using iso-electric focusing followed by immunoblotting and antigen-driven immunoblots. The intrathecal IgG fraction (16% positive) and oligoclonal IgG detection (24% positive) were insensitive for detection of an intrathecal humoral immune response. Trypanosome-specific IgG synthesis, reflected by the IgG antibody index (AI) (26% positive), was confirmed by the presence of oligoclonal specific IgG (47% positive), but the latter was more sensitive. Although the detection technique failed for oligoclonal IgM, the intrathecal IgM fraction (42% positive) and the IgM AI (32% positive) indicated that the meningo-encephalitic stage of the disease is characterized by a dominant intrathecal IgM response, which was higher than the IgG response. The highest combination of diagnostic sensitivity and specificity for the meningo-encephalitic stage of trypanosomiasis was observed for quantitative IgM determinations.

Albumins↗

PubMed2002

Blood-cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness.

Diagnosis of central nervous system (CNS) involvement in sleeping sickness is crucial in order to give an appropriate treatment regimen. Neurological symptoms occur late, therefore field diagnosis is based on white blood cell count, total protein concentration and presence of trypanosomes in cerebrospinal fluid (CSF). More sensitive and specific parameters are now available. Blood-CSF barrier (B-CSFB) dysfunction, intrathecal total and specific immunoglobulin synthesis were evaluated in 95 patients with and without obvious meningoencephalitis, and compared to field criteria.B-CSFB dysfunction is a rather late event in the course of CNS involvement and correlates with the presence of trypanosomes, neurological signs and intrathecal polyspecific and specific immune response. IgM intrathecal response and particularly IgM antibody index are early markers of CNS invasion. We showed that 29% of patients with CSF abnormalities but without trypanosome detection in the field had no neuro-immunological response. In contrast, patients with normal CSF according to field diagnosis showed an intrathecal immune response in 31% of the cases.Field diagnosis can therefore fail to determine neurological involvement but can also provide false positive results. Improved criteria including B-CSFB dysfunction and IgM detection are needed in order to provide an adapted treatment regimen.

Albumins↗