Longitudinal gut microbiome analyses and blooms of pathogenic strains during lupus disease flares.

OBJECTIVE: Whereas genetic susceptibility for systemic lupus erythematosus (SLE) has been well explored, the triggers for clinical disease flares remain elusive. To investigate relationships between microbiota community resilience and disease activity, we performed the first longitudinal analyses of lupus gut-microbiota communities. METHODS: In an observational study, taxononomic analyses, including multivariate analysis of ß-diversity, assessed time-dependent alterations in faecal communities from patients and healthy controls. From gut blooms, strains were isolated, with genomes and associated glycans analysed. RESULTS: Multivariate analyses documented that, unlike healthy controls, significant temporal community-wide ecological microbiota instability was common in SLE patients, and transient intestinal growth spikes of several pathogenic species were documented. Expansions of only the anaerobic commensal, Ruminococcus (blautia) gnavus (RG) occurred at times of high-disease activity, and were detected in almost half of patients during lupus nephritis (LN) disease flares. Whole genome sequence analysis of RG strains isolated during these flares documented 34 genes postulated to aid adaptation and expansion within a host with an inflammatory condition. Yet, the most specific feature of strains found during lupus flares was the common expression of a novel type of cell membrane-associated lipoglycan. These lipoglycans share conserved structural features documented by mass spectroscopy, and highly immunogenic repetitive antigenic-determinants, recognised by high-level serum IgG2 antibodies, that spontaneously arose, concurrent with RG blooms and lupus flares. CONCLUSIONS: Our findings rationalise how blooms of the RG pathobiont may be common drivers of clinical flares of often remitting-relapsing lupus disease, and highlight the potential pathogenic properties of specific strains isolated from active LN patients.

Enantiospecific reassessment of the pharmacokinetics and pharmacodynamics of oral eflornithine against late-stage Trypanosoma brucei gambiense sleeping sickness.

This study aimed to characterize the stereoselective pharmacokinetics of oral eflornithine in 25 patients with late-stage Trypanosoma brucei gambiense sleeping sickness. A secondary aim was to determine the concentrations of L- and D-eflornithine required in plasma or cerebrospinal fluid (CSF) for an efficient eradication of the T. brucei gambiense parasites. Patients were randomly allocated to receive either 100 (group I, n=12) or 125 (group II, n=13) mg/kg of body weight of drug every 6 h for 14 days. The concentrations of L- and D-eflornithine in the plasma and CSF samples were measured using a stereospecific liquid chromatographic method. Nonlinear mixed-effects modeling was used to characterize the plasma pharmacokinetics. The plasma concentrations of L-eflornithine were on average 52% (95% confidence interval [CI], 51, 54%; n=321) of the D-enantiomer concentrations. The typical oral clearances of L- and D-eflornithine were 17.4 (95% CI, 15.5, 19.3) and 8.23 (95% CI, 7.36, 9.10) liters/h, respectively. These differences were likely due to stereoselective intestinal absorption. The distributions of eflornithine enantiomers to the CSF were not stereoselective. A correlation was found between the probability of cure and plasma drug exposure, although it was not more pronounced for the L-enantiomer than for that of total eflornithine. This study may explain why oral treatment for late-stage human African trypanosomiasis (HAT) patients with racemic eflornithine has previously failed; the more potent L-enantiomer is present at much lower concentrations in both plasma and CSF than those of the D-enantiomer. Eflornithine stereoselective pharmacokinetics needs to be considered if an oral dosage regimen is to be explored further.

The gut microbiome in systemic lupus erythematosus: lessons from rheumatic fever.

For more than a century, certain bacterial infections that can breach the skin and mucosal barriers have been implicated as common triggers of autoimmune syndromes, especially post-infection autoimmune diseases that include rheumatic fever and post-streptococcal glomerulonephritis. However, only in the past few years has the importance of imbalances within our own commensal microbiota communities, and within the gut, in the absence of infection, in promoting autoimmune pathogenesis become fully appreciated. A diversity of species and mechanisms have been implicated, including disruption of the gut barrier. Emerging data suggest that expansions (or blooms) of pathobiont species are involved in autoimmune pathogenesis and stimulate clonal expansion of T cells and B cells that recognize microbial antigens. This Review discusses the relationship between the gut microbiome and the immune system, and the potential consequence of disrupting the community balance in terms of autoimmune development, focusing on systemic lupus erythematosus. Notably, inter-relationships between expansions of certain members within gut microbiota communities and concurrent autoimmune responses bear features reminiscent of classical post-infection autoimmune disease. From such insights, new therapeutic opportunities are being considered to restore the balance within microbiota communities or re-establishing the gut-barrier integrity to reinforce immune homeostasis in the host.

Sex-dependent Lupus Blautia (Ruminococcus) gnavus strain induction of zonulin-mediated intestinal permeability and autoimmunity.

Imbalances in the gut microbiome are suspected contributors to the pathogenesis of Systemic Lupus Erythematosus, and our studies and others have documented that patients with active Lupus nephritis have expansions of the obligate anaerobe, Blautia (Ruminococcus) gnavus (RG). To investigate whether the RG strains in Lupus patients have in vivo pathogenic properties in a gnotobiotic system, we colonized C57BL/6 mice with individual RG strains from healthy adults or those from Lupus patients. These strains were similar in their capacity for murine intestinal colonization of antibiotic-preconditioned specific-pathogen-free, as well as of germ-free adults and of their neonatally colonized litters. Lupus-derived RG strains induced high levels of intestinal permeability that was significantly greater in female than male mice, whereas the RG species-type strain (ATCC29149/VPI C7-1) from a healthy donor had little or no effects. These Lupus RG strain-induced functional alterations were associated with RG translocation to mesenteric lymph nodes, and raised serum levels of zonulin, a regulator of tight junction formation between cells that form the gut barrier. Notably, the level of Lupus RG-induced intestinal permeability was significantly correlated with serum IgG anti RG cell-wall lipoglycan antibodies, and with anti-native DNA autoantibodies that are a biomarker for SLE. Strikingly, gut permeability was completely reversed by oral treatment with larazotide acetate, an octapeptide that is a specific molecular antagonist of zonulin. Taken together, these studies document a pathway by which RG strains from Lupus patients contribute to a leaky gut and features of autoimmunity implicated in the pathogenesis of flares of clinical Lupus disease.

Systemic Lupus Erythematosus and dysbiosis in the microbiome: cause or effect or both?

Throughout our lives we are immersed in, and colonized by, immense and complex microbial communities. These microbiota serve as activators and early sparring partners for the progressive construction of the layers within our immune defenses and are essential to immune homeostasis. Yet, at times imbalances within the microbiota may contribute to metabolic and immune regulatory abnormalities that underlie the development of inflammatory and autoimmune diseases. Here, we review recent progress in investigations of the microbiome, with emphasis on the gut microbiota associated with systemic autoimmunity. In particular, these studies are beginning to illuminate aspects of the pathogenesis of Systemic Lupus Erythematosus, and may suggest that interconnections with specific disease-associated patterns of dysbiosis within gut communities are bidirectional and mutually reinforcing.

Mosaicism of XX and XXY cells accounts for high copy number of Toll like Receptor 7 and 8 genes in peripheral blood of men with Rheumatoid Arthritis.

The X chromosome, hemizygous in males, contains numerous genes important to immunological and hormonal function. Alterations in X-linked gene dosage are suspected to contribute to female predominance in autoimmunity. A powerful example of X-linked dosage involvement comes from the BXSB murine lupus model, where the duplication of the X-linked Toll-Like Receptor 7 (Tlr7) gene aggravates autoimmunity in male mice. Such alterations are possible in men with autoimmune diseases. Here we showed that a quarter to a third of men with rheumatoid arthritis (RA) had significantly increased copy numbers (CN) of TLR7 gene and its paralog TLR8. Patients with high CN had an upregulated pro-inflammatory JNK/p38 signaling pathway. By fluorescence in situ hybridization, we further demonstrated that the increase in X-linked genes CN was due to the presence of an extra X chromosome in some cells. Men with RA had a significant cellular mosaicism of female (46,XX) and/or Klinefelter (47,XXY) cells among male (46,XY) cells, reaching up to 1.4% in peripheral blood. Our results present a new potential trigger for RA in men and opens a new field of investigation particularly relevant for gender-biased autoimmune diseases.

Soluble HLA-G Expression Inversely Correlates With Fetal Microchimerism Levels in Peripheral Blood From Women With Scleroderma.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5'URR/3'UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p < 0.0001), independently of clinical subtypes, autoantibody profiles, disease duration, or treatments. Moreover, sHLA-G levels were inversely correlated to FMc quantities (Spearman correlation, p < 0.01). Finally, women with SSc had lower sHLA-G independently of the eight HLA-G 5'URR/3'UTR polymorphisms, although they were statistically more often homozygous than heterozygous for HLA-G polymorphism genotypes -716 (G/T), -201 (G/A), 14 bp (ins/del), and +3,142 (G/A) than healthy women. In conclusion, women with SSc display less sHLA-G expression independently of the eight HLA-G polymorphisms tested. This decreased production correlates with higher quantities of persisting FMc commonly observed in blood from SSc women. These results shed some lights on the contribution of the maternal HLA-G protein to long-term persistent fetal Mc and initiate new perspectives in this field.

Evaluation of X Chromosome Inactivation with Respect to HLA Genetic Susceptibility in Rheumatoid Arthritis and Systemic Sclerosis.

BACKGROUND: Autoimmune diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc) are characterized by a strong genetic susceptibility from the Human Leucocyte Antigen (HLA) locus. Additionally, disorders of epigenetic processes, in particular non-random X chromosome inactivation (XCI), have been reported in many female-predominant autoimmune diseases. Here we test the hypothesis that women with RA or SSc who are strongly genetically predisposed are less susceptible to XCI bias. METHODS: Using methylation sensitive genotyping of the androgen receptor (AR) gene, XCI profiles were performed in peripheral blood mononuclear cells from 161 women with RA, 96 women with SSc and 100 healthy women. HLA-DRB1 and DQB1 were genotyped. Presence of specific autoantibodies was documented for patients. XCI skewing was defined as having a ratio ≥ 80:20 of cells inactivating the same X chromosome. RESULTS: 110 women with RA, 68 women with SSc, and 69 controls were informative for the AR polymorphism. Among them 40.9% of RA patients and 36.8% of SSc patients had skewed XCI compared to 17.4% of healthy women (P = 0.002 and 0.018, respectively). Presence of RA-susceptibility alleles coding for the "shared epitope" correlated with higher skewing among RA patients (P = 0.002) and such correlation was not observed in other women, healthy or with SSc. Presence of SSc-susceptibility alleles did not correlate with XCI patterns among SSc patients. CONCLUSION: Data demonstrate XCI skewing in both RA and SSc compared to healthy women. Unexpectedly, skewed XCI occurs more often in women with RA carrying the shared epitope, which usually reflects severe disease. This reinforces the view that loss of mosaicism in peripheral blood may be a consequence of chronic autoimmunity.

Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus.

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10-4) and 60% versus 28% (P = 3.10-8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10-10) and 82% (P = 5.10-13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.

The efficacy of pentamidine in the treatment of early-late stage Trypanosoma brucei gambiense trypanosomiasis.

Fifty-eight patients in the early-late stage (early central nervous system involvement) of Trypanosoma brucei gambiense trypanosomiasis were treated with pentamidine and divided into four groups (G1, G2, G3, and G4) according to cerebrospinal fluid (CSF) indicators: white blood cell (WBC) count, protein level (CSF protein), and the presence or absence of trypanosomes. Group G1 consisted of eight patients with normal CSF WBC counts and CSF protein levels and trypanosomes in the CSF. Group G2 consisted of nine patients with elevated CSF WBC counts, normal level of CSF protein, and trypanosomes in the CSF. Group G3 consisted of 31 patients with high CSF WBC counts, normal CSF protein levels, but no trypanosomes in the CSF. Group G4 consisted of 10 patients with normal CSF WBC counts and CSF protein levels and trypanosomes demonstrated by CSF culture. Post-treatment follow-up of all patients for at least one year revealed three relapses. There were two deaths from diseases unrelated to trypanosomiasis or to the treatment protocol. Of these patients, 52 were followed for more than two years, the time necessary to confirm a complete cure, indicating a cure rate of 94%. Pentamidine is therefore effective in treating the early-late stage of T. b. gambiense trypanosomiasis, and is comparable with melarsoprol or eflornithine in terms of its tolerance and availability.

Human African trypanosomiasis: presence of antibodies to galactocerebrosides.

Improvements were made in the immunodetection of anti-galactocerebroside (anti-GalC) antibody in sera of patients with human African trypanosomiasis by thin-layer chromatography, enzyme-linked immunosorbent assay, and immunoadsorption. Rabbit anti-GalC antibodies were used to standardize these techniques and demonstrate their specificity. Anti-GalC antibodies were found in the sera of 42.8% of 63 patients with human African trypanosomiasis. Thirty-four control subjects living in the same endemic area were also tested. Anti-GalC levels were higher in human African trypanosomiasis patients with neurologic disturbances compared with patients without such disturbances. These antibodies were distributed mainly between the IgG and IgM classes, but 28% of the patients with human African trypanosomiasis had increased IgA levels without anti-GalC antibody activity.

Detection and characterization of autoantibodies directed against neurofilament proteins in human African trypanosomiasis.

In serum and in cerebrospinal fluid (CSF) from patients with human African trypanosomiasis (HAT) with central nervous system involvement, we detected autoantibodies directed to some proteins from these tissues. The characterization of antigenic proteins by Western blotting showed that the antibodies recognized the 200-kD and 160-kD proteins of neurofilament (NF). Serum anti-NF antibodies were more frequent in HAT patients than in control subjects (86% versus 24%; P < 10[-9]) and they belonged predominantly to the IgM class (anti-NF IgM = 86% versus anti-NF IgG = 4%; P < 10[-9]) in the patients with stage II (central nervous system involvement) HAT. The CSF antibodies to NF were IgM in 88% (22 of 25) of the cases and IgG in 32% (8 of 25) of the cases. Epitopes shared by NF and trypanosomes were detected by indirect immunofluorescence and this was confirmed by the disappearance of anti-NF reactivity after adsorption with trypanosome antigens (Trypanosoma brucei brucei or T. b. gambiense). Anti-NF antibodies were undetectable in the CSF from stage I HAT patients.

Twenty-four-hour plasma cortisol and prolactin in human African trypanosomiasis patients and healthy African controls.

We have previously demonstrated that human African trypanosomiasis (sleeping sickness) at the stage of meningoencephalitis results in a major disruption of the circadian rhythmicity of sleep and wakefulness that is proportional to the severity of the disease. This paper examines the corresponding 24-hourly secretion in cortisol and prolactin and compares it with the hourly distribution of sleep composition in infected patients and healthy African subjects. The secretion of cortisol in humans follows a circadian rhythm relatively independent of the sleep-wake cycle, whereas that of prolactin exhibits fluctuations over the 24-hr day that are strongly related to the sleep-wake cycle. After the clinical classification of the patients according to the severity of the disease, hourly blood samples were taken over 24 hr via an indwelling catheter. Plasma cortisol and prolactin were analyzed by radioimmunoassay, and the variations in the hourly concentrations were analyzed for the presence of a potential 24-hr rhythm (circadian). All of the healthy African subjects showed significant circadian rhythms in both cortisol and prolactin secretion, similar to data on humans from temperate regions, and a sleep-related anamnestic afternoon peak of prolactin. Major disruptions in the circadian rhythms of plasma cortisol and prolactin were found in the three patients with the most severe illness, in contrast to the four who were less severely ill and the healthy controls. Thus, it appears that as the disease progresses in severity, major disruptions begin to occur in body circadian rhythms, not only in the sleep-wake cycle as reported elsewhere, but also in cortisol and prolactin secretion, suggesting that sleeping sickness affects the circadian timing system.

Treatment of human late stage gambiense trypanosomiasis with alpha-difluoromethylornithine (eflornithine): efficacy and tolerance in 14 cases in Côte d'Ivoire.

alpha-Difluoromethylornithine (DFMO; eflornithine), an inhibitor of polyamine biosynthesis, was used to treat 14 patients with late stage gambiense sleeping sickness, 12 cases having been previously treated with and considered refractory to melarsoprol. alpha-Difluoromethylornithine was administered intravenously at a dose of 400 mg/kg/day for 14 days followed by oral treatment, 300 mg/kg/day, for 21-28 days. In all patients treatment was associated with rapid disappearance of trypanosomes from body fluids (in several cases within 24 hr) and decreased cerebrospinal fluid white blood cell counts. In all but one patient, who died of a pulmonary infection during treatment, alpha-difluoromethylornithine produced a dramatic reversal of clinical signs and symptoms of the disease. Determination of drug concentrations in serum and cerebrospinal fluid of 5 patients demonstrated that alpha-difluoromethylornithine diffuses into the central nervous system with cerebrospinal fluid levels representing up to 51% of corresponding serum concentrations. Diarrhea, abdominal pain, and anemia were the most frequent side effects associated with therapy, but were reversible and did not necessitate discontinuation of treatment. Four patients have been followed for more than 2 years post-treatment without evidence of relapse.

Diagnosis of Trypanosoma brucei gambiense sleeping sickness using an antigen detection enzyme-linked immunosorbent assay.

A monoclonal antibody-based enzyme-linked immunosorbent assay (antigen ELISA) developed for detection of trypanosome antigens in the serum and cerebrospinal fluid (CSF) of patients as a means for diagnosis of Trypanosoma brucei gambiense sleeping sickness was evaluated at the Bureau Central de la Trypanosomiase, Kinshasa, Zaire. Sixty-nine (89.6%) of 77 parasitologically confirmed cases examined at the Daloa clinic had antigens in serum; 35 (45.5%) had antigens in CSF and, in 4 of these, the antigens were detected in CSF only. Taking the serum and CSF results together, 73 (94.8%) of the 77 patients were positive in the assay. In the Kinshasa series, 168 (89.4%) of 188 parasitologically confirmed cases were positive by antigen ELISA. The controls, who included 165 blood donors and 40 patients with malaria, 2 with hydatidosis and 12 with leishmaniasis, were negative by antigen ELISA. Analysis of CSF results for 35 patients who had antigens in CSF revealed that 34 (97.1%) had elevated CSF white cell counts, 29 (82.9%) had elevated protein levels, and 23 (65.7%) had trypanosomes in their CSF. Moreover, analysis of results for 34 patients whose CSF had been shown to harbour trypanosomes by the double centrifugation technique showed that 24 (70.6%) had antigens in CSF, 28 (82.6%) had elevated protein levels, and 33 (97.1%) had elevated CSF white cell counts. Antigens were rapidly cleared from peripheral circulation following institution of treatment. Antigen clearance was accompanied by a rapid fall in CSF protein levels and white cell counts. These results demonstrate the potential of antigen ELISA, not only as a tool for diagnosis, but also for clinical staging and treatment follow-up of patients with T. b. gambiense sleeping sickness.

Long-term exposure of Trypanosoma brucei gambiense to pentamidine in vitro.

In order to study the sensitivity in vitro of Trypanosoma brucei gambiense to pentamidine, 5 x 10(4) parasites were exposed to 0, 0.1, 1.0, 2.0, 10, 100, 1000 and 10,000 micrograms/L of pentamidine isethionate for up to 10 d. The viability of parasites was determined each day by microscopy. Multiplication was retarded during continuous exposure to 2 micrograms/L. After 4 d no further multiplication took place, although the trypanosomes remained alive for another 3 d. The parasiticidal effect was more pronounced when higher concentrations were used; when exposed to 10 and 100 micrograms/L, all parasites were dead after 4 and 3 d, respectively. Despite exposure to 1000 micrograms/L, 74% of the parasites were still alive the next day. 10,000 micrograms/L killed all parasites within 24 h of exposure. Our results show that the time period of exposure to pentamidine plays a major role in determining the sensitivity in vitro of T. b. gambiense, and we suggest that prolonged exposure in vivo may be more important than attaining high but brief peak concentrations.

Pentamidine concentrations in plasma, whole blood and cerebrospinal fluid during treatment of Trypanosoma gambiense infection in Côte d'Ivoire.

Pentamidine concentrations in plasma, whole blood and cerebrospinal fluid (CSF) were determined in 11 patients with Trypanosoma gambiense infection without involvement of the central nervous system in Côte d'Ivoire. Blood samples were drawn during a 48 h period after the first and last dose of pentamidine dimesylate given as 10 intramuscular injections on alternate days. Maximum plasma concentrations were generally attained within one hour after injection but varied extensively (420-13420 nmol/litre). The median plasma concentration 48 h after the last dose was approximately 5 times higher than that after the first dose. The ratio between whole blood and plasma concentration was approximately 2. Small amounts of the drug were found in the CSF after the last dose. The findings showed inter-individual differences in the pharmacokinetics of pentamidine. The currently recommended daily dose regimen could be questioned, as drug accumulation was pronounced. All patients were cured and the concentrations attained should be considered as parasiticidal. Further studies on the kinetics and distribution after single and multiple doses of pentamidine as well as studies on the possible relationship between adverse effects and plasma concentrations are, however, needed.

A comparison of parasitological methods for the diagnosis of gambian trypanosomiasis in an area of low endemicity in Côte d'Ivoire.

The card agglutination test for trypanosomiasis (CATT) was used to examine 8974 inhabitants in 14 village areas south-west of Daloa, Côte d'Ivoire; 114 (1.3%) were CATTT or +/-, and were further examined by one or more of 6 methods for the direct detection of trypanosomes: lymphatic gland puncture, stained thick blood film (TBF), haematocrit centrifugation technique (HCT), mini-anion exchange column (MAEC), quantitative buffy coat method (QBC), and kit for in vitro isolation of trypanosomes (KIVI). Trypanosomes were seen by at least one method in 16 (14.0%) of the CATT+ group. Blood from 356 of the 8860 CATT- group was inoculated into KIVI; trypanosomes grew from the blood of 1 person. Eleven of the 17 patients with detectable trypanosomes were screened by all 6 methods: 6 were HCT+; 7 were gland+; 10 were MAEC+; 10 were KIVI+; 11 were both TBF+ and QBC+. One CATT+ patient was KIVI+ but otherwise negative, although TBF was not done. The overall prevalence of trypanosomes was 0.2% rising to 0.8% in one village area. The results support previous evidence that a reappraisal of procedures is required in the customary system of surveillance for gambian sleeping sickness.