Neopterin and CXCL-13 in Diagnosis and Follow-Up of Trypanosoma brucei gambiense Sleeping Sickness: Lessons from the Field in Angola.

Human African Trypanosomiasis may become manageable in the next decade with fexinidazole. However, currently stage diagnosis remains difficult to implement in the field and requires a lumbar puncture. Our study of an Angolan cohort of T. b. gambiense-infected patients used other staging criteria than those recommended by the WHO. We compared WHO criteria (cell count and parasite identification in the CSF) with two biomarkers (neopterin and CXCL-13) which have proven potential to diagnose disease stage or relapse. Biological, clinical, and neurological data were analysed from a cohort of 83 patients. A neopterin concentration below 15.5 nmol/L in the CSF denoted patients with stage 1 disease, and a concentration above 60.31 nmol/L characterized patients with advanced stage 2 (trypanosomes in CSF and/or cytorachia higher than 20 cells) disease. CXCL-13 levels below 91.208 pg/mL denoted patients with stage 1 disease, and levels of CXCL-13 above 395.45 pg/mL denoted patients with advanced stage 2 disease. Values between these cut-offs may represent patients with intermediate stage disease. Our work supports the existence of an intermediate stage in HAT, and CXCL-13 and neopterin levels may help to characterize it.

Stage determination in sleeping sickness: comparison of two cell counting and two parasite detection techniques.

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.

Cerebrospinal fluid B lymphocyte identification for diagnosis and follow-up in human African trypanosomiasis in the field.

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, < or =5 cells/microl CSF, n = 42; stage 2, > or =20 cells/microl, n = 16) and intermediate stage (6-19 cells/microl, n = 13). Slides containing 1 microl 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/microl (n = 5), contrary to most stage 2 patients (14/16: > or =2 rosettes/microl). Intermediate stage patients expressed 0 (n = 9), 1 (n = 3) or 2 (n = 1) rosettes/microl 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.

[Status of human African trypanosomiasis in the Nola-Salo-Bilolo in the Central African Republic in 2005]. / Situation de la trypanosomiase humaine africaine dans le foyer de Nola-Salo-Bilolo en République Centrafricaine en 2005.

In November-December 2005, the technical staff of the Organisation for Coordination of the Control of Endemic Diseases in Central Africa (OCEAC) and the National Program for Control of Human African Trypanosomiasis (PNLTHA) undertook screening for human African trypanosomiasis in the historical focus of Nola-Salo-Biolo in Central Africa. A total of 31 new cases of typanosomiasis were detected for a prevalence of 0.44%. This study also provided insight into the limits of this old focus and showed that circulation of the parasite is still heavy. The endemia of sleeping sickness has now been contained in Nola-Salo-Bilolo but maintaining control measures is essential to preventing a potentially major recrudescence.

[Diagnosis of sleeping sickness stage: towards a new approach]. / Le diagnostic du stade dans la maladie du sommeil: vers une nouvelle approche.

Diagnosis of the neurological disease stage in Trypanosoma brucei (T.b.) gambiense infection is essential to select an optimal chemotherapy. The actual parameters for stage determination, the cerebrospinal fluid (CSF) cell count, total protein concentration and trypanosome detection, are insufficiently specific and sensitive. In order to identify new parameters for stage determination, we studied the neuro-inflammatory immune response in the central nervous system, notably the intrathecal humoral immune response in sleeping sickness patients. The presence of intrathecal IgM synthesis was identified as an excellent marker of central nervous system involvement. However, intrathecal IgM detection cannot be performed under field conditions. As a consequence of the strong intrathecal IgM synthesis, extremely high concentrations of IgM are found in the CSF of sleeping sickness. We therefore developed a latex agglutination field test (LATEX/IgM) indicative for intrathecal IgM synthesis and CNS involvement in sleeping sickness. Based on our observations on the intrathecal immune response and with LATEX/IgM, we propose a new approach for stage determination in sleeping sickness.

[Human African trypanosomiasis in Ivory Coast: biological characteristics after treatment. 812 cases treated in the Daloa focus (Ivory Coast)]. / Trypanosomose humaine africaine en Côte d'Ivoire: caractéristiques biologiques après traitement. A propos de 812 cas traités dans le foyer de Daloa (Côte d'Ivoire).

The treatment and post therapeutic follow up of patients diagnosed with HAT are important for HAT control. A longitudinal survey was implemented in the focus of Daloa (Côte d'Ivoire). A total of 812 patients infected with Trypanosoma brucei gambiense in meningoencephalitic stage and treated with melarsoprol were included, this study pointed out the biological characteristics of patients after treatment. The relapse occurs between 1 and 24 months after treatment. It is essentially neurological, and characterised by the presence in the CSF of antibodies, by the increase of cell count compared with value immediately after treatment, or by the presence of trypanosomes. The cure can be confirmed from 18 months after treatment, and is characterised by the absence of antibodies and trypanosomes in the CSF, by a normal cell count and a normal proteinorachy. Biological scares were recorded on some of the patients after 18 months of follow up, but no relapse occurred among them.

Evidence for the occurrence of Trypanosoma brucei rhodesiense sleeping sickness outside the traditional focus in south-eastern Uganda.

The occurrence of Trypanosoma brucei rhodesiense west of the River Nile, in Masindi district in the mid-western part of Uganda, is confirmed. Masindi borders the traditional belt of T. b. gambiense infection in the north-west, Gulu in the north and the Democratic Republic of Congo in the west. Of the 702 persons tested for sleeping sickness in Masindi, 113 (16%) were positive by the card agglutination test for trypanosomiasis (CATT). Trypanosomes were observed in samples of cerebrospinal fluid (CSF) from two (0.3%) of the subjects: a 7-year-old girl, who had been ill for 2 weeks and yet was in good general condition, with three white blood cells (WBC)/microliter CSF; and a 47-year-old woman who had been ill for 8 months, looked sickly, had seven WBC/microliter CSF, but was still able to dig in her gardens. Rats and mice inoculated with blood from the two parasitologically confirmed cases became parasitaemic on day 3 post-inoculation, indicating that the parasites were T. b. rhodesiense. Isoenzyme analysis revealed that the parasites isolated from one of these confirmed cases belonged to a zymodeme (449) which has not been previously observed among isolates from south-eastern or north-western Uganda. Although the isolate shared PGM2 and ICD3 patterns with T. b. gambiense and T. b. rhodesiense, respectively, it did not have the SOD3:5 pattern characteristic of T. b. gambiense. The spread of T. b. rhodesiense beyond its traditional focus and the development of areas where this subspecies and T. b. gambiense are co-endemic will complicate the control of sleeping sickness in Uganda; although the CATT is very useful for the mass screening of populations for T. b. gambiense area, it is not applicable in the detection of T. b. rhodesiense.

[Human African trypanosomiasis in children. A pediatrics service experience in Libreville, Gabon]. / Trypanosomose humaine africaine chez l'enfant. Expérience d'un service de pédiatrie à Libreville, Gabon.

During a period of six years (1/1/89-12/31/94), seven children with trypanosomiasis were admitted to the Department of Pediatrics of Owendo Pediatric Hospital-Libreville, Gabon. They were 5 boys and 2 girls, aged 4-17 years, five of them under 15 years. The main reasons of hospitalization were somnolence (4 cases), psychical disorders (5 cases), neurological disorders (4 cases), asthenia (3 cases), loss of weight (3 cases) and fever (3 cases). Increased sedimentation rate (5 cases) and hypergammaglobulinemia (6 cases) were the most important biological disturbances. Serodiagnosis (CATT, indirect immunofluorescence test) was positive in all cases. The parasite was detected in blood seven times, and four times in cerebrospinal fluid (CSF). According to CSF status, six children have been classified in second stage of the disease. Six patients were treated by melarsoprol, and one by eflornithine. Tolerance and response to treatment were good in six cases. Three children presented sequels when leaving hospital. No patient was seen again after the study.

Correlation of high serum levels of tumor necrosis factor-alpha with disease severity in human African trypanosomiasis.

The levels of tumor necrosis factor-alpha (TNF-alpha) in sera from Trypanosoma brucei gambiense-infected patients from the endemic region of Boko Songho (Bouenza focus in Congo) were measured. An increase was observed in sera from patients (geometric mean = 53.75 pg/ml, n = 69) compared with control subjects from the same endemic area (6.72 pg/ml, n = 31). The patients were classified as being in the early (blood lymphatic) stage and late (meningo-encephalitic) stage of disease according to the presence of parasites and cells in cerebrospinal fluid (CSF). An increase in TNF-alpha was noted in late stage patients (68.42 pg/ml, n = 28) compared with early stage patients (43.68 pg/ml, n = 41). Those patients with fever, asthenia, and edema and those with neurologic signs had higher levels of TNF-alpha (89.36 pg/ml, n = 26) than others (38.07 pg/ml, n = 43). No differences in TNF-alpha levels were seen when trypanosomes were detected in one location (blood, lymph nodes, or CSF) or two or three locations. These data show that the levels of TNF-alpha in serum of T. b. gambiense-infected patients were correlated with disease severity (presence of signs of inflammation or presence of major neurologic signs) and indicate that TNF-alpha could be involved in some aspects of human African trypanosomiasis physiopathology.

A simple classification of different types of trypanosomiasis occurring in four camel herds in selected areas of Kenya.

Camels from herds in four different areas of Kenya were sampled at regular intervals over periods of 9-15 months and examined for the presence of trypanosomiasis. Five hundred and eight camels divided into three age categories were studied. Trypanosomiasis caused by T. evansi, T. brucei and T. congolense was shown to be present in varying degrees in all the herds. Camels were divided into 5 types according to mortality, the presence or absence of circulating trypanosomes and antibody, and the presence or absence of emaciation and anaemia. Herd profiles were constructed according to the percentage of the different types over the whole of their respective study periods. Results indicated that camels in some herds show a calf-hood resistance to trypanosomiasis in endemic areas and that the disease may be present in a relatively stable situation. Some of the implications of these results are discussed.