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Objectives Diagnosis of the neurological stage of human African trypanosomiasis is performed by examination of cerebrospinal fluid ( CSF) for the presence of trypanosomes and numbers of white blood cells ( WBC). Both CSF parameters are also used to assess treatment outcome during follow-up. In view of the importance of CSF examination, and the practical problems encountered with it, we compared the sensitivity of two trypanosome concentration techniques and the repeatability of two cell counting methods, as well as occurrence of systematic differences between them. Methods Patients were recruited at Dipumba hospital, in Mbuji-Mayi in the Democratic Republic of the Congo. In 94 CSF samples, trypanosome detection was performed with modified single centrifugation ( MSC) and double centrifugation ( DC). On 189 CSF samples with ≤30 cells/μl, cell counting was performed in duplicate in a Fuchs-Rosenthal counting chamber and in a disposable Uriglass counting chamber. Results Modified single centrifugation detected trypanosomes in significantly ( P < 0.0001) more patients (85) than DC (46). Cell counts did not differ systematically in the two methods. Variability in the differences between duplicate cell counts was significantly higher ( P = 0.002) in Uriglass ( SD of differences 2.03) than in Fuchs-Rosenthal ( SD of differences 1.62). Conclusions For analysis of CSF in the context of sleeping sickness stage determination and follow-up after treatment, we strongly recommend the MSC for parasite detection and the application of disposable counting chambers. When the first cell count is ≤20 cells/μl, we recommend repeating the counting procedure on the same CSF specimen and taking the average of both countings. [ABSTRACT FROM AUTHOR]

Objective To evaluate the accuracy of a peptide, corresponding to the variant surface glycoprotein ( VSG) LiTat 1.5 amino acid ( AA) sequence 268-281 and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. Methods A synthetic biotinylated peptide (peptide 1.5/268-281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from patients with HAT and 102 endemic HAT-negative controls. Results The area under the curve ( AUC) of peptide 1.5/268-281 was 0.954 (95% confidence interval 0.918-0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982-1.000) and 0.997 (0.973-1.000), respectively, and significantly higher than the AUC of peptide 1.5/268-281. On a model of VSG LiTat 1.5, peptide 1.5/268-281 was mapped near the top of the VSG. Conclusions A biotinylated peptide corresponding to AA 268-281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full-length native VSG LiTat 1.3 and VSG LiTat 1.5. [ABSTRACT FROM AUTHOR]

Objectives To provide a better understanding of sleeping sickness transmission and spread in mangrove areas to optimize its control. Methods In the Forecariah mangrove area, Guinea, 19 sleeping sickness cases and 19 matched controls were followed up in their living areas (at home, in fields and at water points). All occupational sites and pathways were mapped and then placed in their environmental context. Results The sleeping sickness cases displayed a significantly broader and more diverse spatial occupation than the controls. They covered double the daily walking distances of controls and had on average two more occupational sites, most of which were located in mangrove forests. Activities with a higher transmission risk (rice culture, attendance of pirogue jetties) were identified as well as high-risk areas and pathways. Conclusions An entomological control strategy targeting transmission risk areas is proposed. Its implementation in a control programme would reduce by 86% the efforts needed for a classical vector control programme throughout the area. Medical surveys set up at specific locations, such as pirogue jetties and high-risk paths, should also enable better targeting of the population at highest risk. [ABSTRACT FROM AUTHOR]

Objectives In human African trypanosomiasis (HAT, sleeping sickness), staging of disease and treatment follow-up relies on white cell count in the cerebrospinal fluid (CSF). As B lymphocytes (CD19 positive cells) are not found in the CSF of healthy individuals but occur in neurological disorders such as multiple sclerosis, B lymphocyte count may be useful for field diagnosis/staging and therapeutic follow-up in HAT. Methods Seventy-one HAT patients were diagnosed and 50 were followed-up 6–24 months after treatment. White cell counts were used for conventional staging (stage 1, ≤5 cells/μl CSF, n = 42; stage 2, ≥20 cells/μl, n = 16) and intermediate stage (6–19 cells/μl, n = 13). Slides containing 1 μl of CSF mixed with Dynabeads® CD19 pan B were examined microscopically to detect B cell rosettes (bound to at least four beads). Results Stage 1 patients exhibited zero ( n = 37) or one CSF rosette/μl ( n = 5), contrary to most stage 2 patients (14/16: ≥2 rosettes/μl). Intermediate stage patients expressed 0 ( n = 9), 1 ( n = 3) or 2 ( n = 1) rosettes/μl of CSF. During follow-up, rosette counts correlated with white cell count staging but were much easier to read. Conclusion B cell rosettes being easily detected in the CSF in field conditions may be proposed to replace white cell count for defining HAT stages 1 and 2 and limit uncertainty in treatment decision in patients with intermediate stage. [ABSTRACT FROM AUTHOR]

Objective To determine the usefulness of IL-10 and immunoglobulin M (IgM) as biomarkers for staging HAT in vervet monkeys, a useful pathogenesis model for humans. Methods Vervet monkeys were infected with Trypanosoma brucei rhodesiense and subsequently given sub-curative and curative treatment 28 and 140 days post-infection (dpi) respectively. Matched serum and CSF samples were obtained at regular intervals and immunospecific IgM, immunoglobulin G (IgG) and IL-10 were quantified by ELISA. Results There was no detectable immunospecific IgM and IgG in the CSF before 49 dpi. CSF IgM and IgG and serum IgM were significantly elevated with peak levels coinciding with meningoencephalitis 98 dpi. The serum IL-10 was upregulated in both early and late disease stage, coinciding with primary and relapse parasitaemia respectively. CSF white cell counts (CSF WCC) were elevated progressively till curative treatment was given. After curative treatment, there was rapid and significant drop in serum IgM and IL-10 concentration as well as CSF WCC. However, the CSF IgM and IgG remained detectable to the end of the study. Conclusions Serum and CSF concentrations of immunospecific IgM and CSF IgG changes followed a pattern that mimics the progression of the disease and may present reliable and useful biomarkers of the disease stage. Due to rapid decline, serum IgM and IL-10 are, additionally, potential biomarkers of the success of chemotherapy. [ABSTRACT FROM AUTHOR]

Objective To evaluate Trypanosoma brucei gambiense infection in peri-domestic livestock from Kogo and Mbini foci (Equatorial Guinea) in order to investigate its possible implication in the sleeping sickness transmission cycle in these hypoendemic foci. Methods Samples from 698 domestic animals (goats, sheep and pigs) from trypanosomiasis-endemic localities of Kogo and Mbini foci were tested for animal trypanosomes and T. b. gambiense (group I) by species-specific polymerase chain reaction. Results Trypanosoma brucei s.l., the predominant trypanosome species, was detected in 182 (52.6%) samples from Mbini and in 127 (36.1%) samples from Kogo. T. b. gambiense was only identified in seven (2%) of the Mbini samples and one co-infection (with T. vivax) was observed. Conclusion The occurrence of T. b. gambiense in peri-domestic livestock in Mbini and its absence in Kogo could explain the epidemiological differences between the two foci and could have significant implications for sleeping sickness control in Equatorial Guinea. [ABSTRACT FROM AUTHOR]

Objective To review the geography and history of sleeping sickness (Human African trypanosomiasis; HAT) over the past 100 years in West Africa, to identify priority areas for sleeping sickness surveillance and areas where HAT no longer seems active. Method History and geography of HAT were summarized based on a review of old reports and recent publications and on recent results obtained from medical surveys conducted in West Africa up to 2006. Results/conclusions Active HAT foci seem to have moved from the North to the South. Endemic HAT presently appears to be limited to areas where annual rainfall exceeds 1200 mm, although the reasons for this remain unknown. There has also been a shift towards the south of the isohyets and of the northern distribution limit of tsetse. Currently, the most severely affected countries are Guinea and Ivory Coast, whereas the northern countries seem less affected. However, many parts of West Africa still lack information on HAT and remain to be investigated. Of particular interest are the consequences of the recent political crisis in Ivory Coast and the resulting massive population movements, given the possible consequences on HAT in neighbouring countries. [ABSTRACT FROM AUTHOR]

After the resurgence of sleeping sickness in Luba, Equatorial Guinea, a major campaign to control the disease was established in 1985. The campaign comprised no vector control, but intensive active and passive surveillance using serology for screening, and treatment of all parasitological and suspected serological cases. Total prevalence was used to classify villages as endemic, at risk, anecdotal and non-endemic which also allowed defining the geographic extent of the focus. Active case-finding was implemented from 1985 to 2004. The frequency of surveys was based on parasitological prevalence: twice a year during intensified control, once a year during ordinary control and once every 2 years during the control consolidation phase, when the parasitological prevalence in the whole focus fell to 0.1%. From 1985 to 1999, the indirect immunofluorescent antibody test (IFAT) was used as an initial screening tool, followed by parasitological confirmation of IFAT positive cases, and the Card Agglutination Trypanosomiasis Test (CATT) if necessary. In 2000, the IFAT was replaced by the CATT. Serum-positive individuals without parasitological confirmation were subsequently tested on serial dilution. All cases underwent lumbar puncture to determine the stage of the disease. First-stage cases were treated with pentamidine and second-stage cases with melarsoprol. A few relapses and very advanced cases were treated with eflornithine. The last sleeping sickness case was identified and treated in 1995. [ABSTRACT FROM AUTHOR]

Recent outbreaks of Trypanosoma brucei rhodesiense sleeping sickness in Soroti District of eastern Uganda have demonstrated the important role cattle can play as reservoirs of this parasite. To clarify the epidemiological importance of the cattle reservoir, experiments were conducted to determine the ease with which T. brucei is transmitted during the course of its development in Friesian cattle. The development of T. brucei in cattle is characterized by an acute phase with high levels of parasitaemia and a decline in PCV. The acute phase is followed by a chronic phase during which the PCV remains low but stable and the parasitaemia is low. Parasites are often difficult to detect using parasitological diagnostic tools during this chronic phase. Challenge of chronically infected cattle with T. congolense results in a sudden increase in the T. brucei parasitaemia. Despite significant differences in parasitaemia, the proportion of tsetse flies that developed metacyclic infections after a first bloodmeal on the infected cattle did not differ significantly between the acute and chronic phases or the phase of mixed T. b. brucei/ T. congolense infection. This suggests that, throughout the observation period, the parasitaemia was above the threshold above which infection rates of tsetse are independent of the parasitaemia. The repercussions of the research findings for the understanding of the epidemiology, spread and the control of T. b. rhodesiense sleeping sickness are discussed. [ABSTRACT FROM AUTHOR]

Africa is severely affected by a resurgence of human African trypanosomiasis (HAT) at epidemic proportions. We report the results of the first 5 years of a HAT control programme in northern Angola run by the non-governmental organization (NGO) ANGOTRIP. In the period between 1996 and 2001, 13 426 patients were screened for HAT. The mortality rate of patients in stage II who were treated with melarsoprol fell from 7.5% to 2.9%, possibly as a result of training and the standardization of treatment protocols. A total of 191,578 people in three provinces of Angola were screened for HAT. Vector control activities were initiated using Lancien traps. Our experiences reflect the connection between war and the increasing incidence of disease, but also demonstrate that HAT control is possible by dedicated NGOs in close cooperation with national institutions even under extremely difficult circumstances. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

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 degrees 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 >or= 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 >or= 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. [ABSTRACT FROM AUTHOR]

We tested sera from patients previously treated for human African trypanosomiasis, from patients infected with trypanosomes, and from individuals never diagnosed with African trypanosomiasis living in the Trypanosoma brucei gambiense sleeping sickness focus of Mbini in Equatorial Guinea for their trypanolytic activity against bloodstream forms of T. b. rhodesiense expressing a metacyclic and bloodstream variant surface glycoprotein (VSG). Nearly 80% of the sera from treated patients showed high trypanolytic activity against trypanosomes expressing a metacyclic VSG. The trypanolytic activity of part of these sera was mediated by IgM while that of the other part was antibody-independent. On the other hand, only 40% of the sera exhibited high trypanolytic activity against trypanosomes expressing a bloodstream VSG which also was almost completely abolished by heat-inactivation. In contrast, most sera from infected and negative individuals displayed only low to moderate trypanolytic activity against either trypanosomes expressing a metacyclic or a bloodstream VSG. These results suggest that trypanolytic activity of sera increases after African sleeping sickness and is directed against trypanosomes expressing metacyclic VSG. [ABSTRACT FROM AUTHOR]

About 160 fluoroquinolones and derivatives were tested for antitrypanosomal activity in a drug sensitivity assay followed by fluorometric evaluation. The most active quinolone compounds had IC50 values in the range from 100 to 900 ng/ml, while several derivatives were not active at a concentration of 100 microg/ml. In a structure activity relationship study, modification of the quinolones at position R1, R2, R3 and R8 did not influence trypanocidal activity. An exchange of the fluor at position 6 may contribute to an increase in activity but does not entirely control it. Pyrrolidine substituents at position R7 generally were more active than other substituents at this position. Tetracyclic quinolone derivatives were amongst the most active compounds with IC50 values in the range of 0.3-8.8 microg/ml. The in vitro cytotoxicity on HT-29 cells was determined for active compounds with IC50 values below 1 microg/ml. In addition, six drugs with an IC50 below 1 microg/ml and a selectivity index of more than 10 were chosen for in vivo experiments. Dose escalation experiments with a maximum dose of 100 mg/kg/bid were performed in a mouse model without central nervous system involvement. For unknown reasons the in vitro effect of the drugs could not be confirmed in vivo, but the class of compound remains of interest for their mode of action, the low toxicity, pharmacological properties and the availability of a large number of synthesized compounds. [ABSTRACT FROM AUTHOR]

Encephalopathies are the most feared complications of sleeping sickness treatment with melarsoprol. To investigate the existence of risk factors, the incidence of encephalopathic syndromes and the relationship between the development of different types of encephalopathies and the clinical outcome was studied in a clinical trial with 588 patients under treatment with melarsoprol. The 38 encephalopathy cases were classified into three types according to the leading clinical picture: coma type, convulsion type and psychotic reactions. Nine patients were attributed to the convulsion type, defined as a transient event of short duration with convulsions followed by a post-ictal phase, without signs of a generalized disease. None of these patients died from the reaction. Febrile reactions in the 48 h preceding the reaction were generally not observed in this group. Twenty-five patients were attributed to the coma type, which is a progredient coma lasting several days. Those patients often had signs of a generalized disease such as fever (84%), headache (72%) or bullous skin (8%) reactions. The risk of mortality was high in this group (52%). About 14/16 patients with encephalopathic syndrome of the coma type were infected with malaria. Patients with psychotic reactions or abnormal psychiatric behaviour (3/38) and one patient who died after alcohol intake were excluded from the analysis. The overall rate of encephalopathic syndromes in the cases analysed (n=34) was 5.8%, of which 38.2% died. We did not find any parameters of predictive value for the risk of developing an encephalopathic syndrome based on the symptoms and signs before treatment initiation. The appearance during treatment of febrile reactions (RR 11.5), headache (RR 2.5), bullous eruptions (RR 4.5) and systolic hypotension (RR 2.6) were associated with an increased risk for the occurrence of encephalopathic syndromes especially of the coma type. [ABSTRACT FROM AUTHOR]

Culture adapted T. b. gambiense isolated from Northwest Uganda were exposed to 0.001-0.14 microg/ml melarsoprol or 1.56-100 microg/ml DL-alpha-difluoromethylornithine (DFMO). Minimum inhibitory concentrations (MICs) of each drug were scored for each isolate after a period of 10 days drug exposure. The results indicate that T. b. gambiense isolates from Northwest Uganda had elevated MIC values for melarsoprol ranging from 0.009 to 0.072 microg/ml as compared with T. b. gambiense isolates from Cote d'Ivoire with MIC values ranging from 0.001 to 0.018 microg/ml or with T. b. rhodesiense from Southeast Uganda with MIC values from 0.001 to 0.009 microg/ml. All MIC values obtained fell below expected peak melarsoprol concentrations in serum of treated patients. However, it may not be possible to maintain constant drug concentrations in serum of patients as was the case in our in vitro experiments. Importantly, the MIC of 0.072 microg/ml exhibited by one of the isolates from Northwest Uganda was above levels attainable in CSF indicating that this isolate would probably not be eliminated from CSF of treated patients. PCR amplification of the gene encoding the P2-like adenosine transporter followed by restriction digestion with Sfa NI enzyme revealed presence of fragments previously observed in a trypanosome clone with laboratory-induced arsenic resistance. From our findings it appears that reduced drug susceptibility may be one factor for the frequent relapses of sleeping sickness after melarsoprol treatment occurring in Northwest Uganda. [ABSTRACT FROM AUTHOR]

Melarsoprol, an organo-arsenical drug, has been the drug of choice for late-stage trypanosomiasis for 50 years. Because of the lack of alternatives any abatement of this medication will have a dramatic negative impact on the perspectives for patients. As a large number of patients refractory to melarsoprol treatment was recently reported from northern Uganda and northern Angola, we investigated in northern Angola whether interpatient pharmacokinetic differences influence the outcome of melarsoprol treatment. Drug levels were determined by a biological assay in serum and cerebrospinal fluid (CSF) of 22 patients. Nine patients could be successfully treated, eight were refractory and the outcome was unclear or no adequate follow-up information was available for five patients. No differences in the pharmacokinetic parameters (maximum serum concentration Cmax, half-life t1/2 beta, total clearance CL and the volume of distribution Vss) could be detected between the groups. Serum and CSF concentrations for all patients were in the expected range. This result indicates that other underlying factors are responsible for treatment failures. [ABSTRACT FROM AUTHOR]

CGP 40 215 is an inhibitor of S-adenosylmethionine decarboxylase, a key enzyme in trypanosomal polyamine biosynthesis. It is highly active against Trypanosoma brucei rhodesiense and T. b. gambiense in vitro and in the corresponding rodent models, and therefore was a promising candidate for further development as a new drug against human African trypanosomiasis. We conducted initial pharmacokinetic and efficacy studies in African green monkeys: based on two dose-finding studies, an infection-treatment and a pharmacokinetic study in eight monkeys infected with T. b. rhodesiense in the 1st stage of infection. PK analysis revealed curative drug levels in the serum but complete absence of the drug in the cerebrospinal fluid. No adverse effects of the drug were observed, although in rats CGP 40 215 had caused hypotension. The following PK parameters were calculated using a two-compartment model: t1/2=1.8 h, VSS/f=0.4 l/kg, CL/f=3.0 ml/min x kg and AUC=21 900 ng x h/ml. Six of the eight monkeys were cured, one animal relapsed on day 222 and one animal died of unknown reasons, but was aparasitaemic. The study confirmed the curative potential of CGP 40 215 for 1st stage T. b. rhodesiense infection. Unfortunately, it was also found that the compound did not pass the blood-brain barrier, a pre-requisite for cure of 2nd stage (CNS) infection. As the majority of sleeping sickness patients seeking treatment are in the 2nd stage of the disease, further development of the compound was stopped. [ABSTRACT FROM AUTHOR]