The separation of trypanosomes from blood by anion exchange chromatography: From Sheila Lanham's discovery 50 years ago to a gold standard for sleeping sickness diagnosis

Human African trypanosomiasis (HAT), or sleeping sickness, is a neglected tropical disease that is fatal if untreated, caused by Trypanosoma brucei gambiense and T. brucei rhodesiense. In its 2012 roadmap, WHO targeted HAT for elimination as a public health problem in 2020 and for zero transmission in 2030. Diagnosis of HAT is a multistep procedure comprising of clinical suspicion, confirmation, and stage determination. Suspects are identified on clinical signs and/or on screening for specific antibodies. Parasitological confirmation of suspects remains mandatory to avoid unnecessary toxic drug administration. The positive predictive value of the antibody detection tests is low. Simple parasite detection techniques, microscopic examination of lymph node aspirate, or stained thick blood films lack sensitivity, whereas in T. brucei gambiense patients, the number of blood trypanosomes may be very low. Parasite concentration techniques are therefore indispensable. Half a century ago, Sheila Lanham discovered a technique to separate trypanosomes from the blood of infected rodents, based on anion exchange chromatography with diethyl amino ethyl (DEAE) cellulose, a weak anion exchanger. Between pH 6−9, trypanosome surface is less negatively charged than that of blood cells. When blood is poured on top of a DEAE cellulose column, blood cells are retained, whereas parasites pass the column together with the elution buffer. The result is a pure suspension of trypanosomes that retain their morphology and infectivity. Because cell surface charges vary among trypanosome and mammal species, the optimal buffer pH and ionic strength conditions for different combinations of host and trypanosome species were established. Lanham's technique revolutionized the diagnosis of HAT. It is indispensable in the production of the Card Agglutination Test for Trypanosomiasis (CATT), the most used field test for screening in T. brucei gambiense HAT foci and essential to confirm the diagnosis in suspected people. Lumsden and colleagues developed the mini anion exchange centrifugation technique (mAECT). After adaptation for field conditions, its superior diagnostic and analytical sensitivity compared to another concentration technique was demonstrated. It was recommended as the most sensitive test for demonstrating trypanosomes in human blood. At the beginning of the 21st century, the mAECT was redesigned, allowing examination of a larger volume of blood, up to 0.35 ml with whole blood and up to 10 ml with buffy coat. The plastic collector tube in the new kit is also used for detection of trypanosomes in the cerebrospinal fluid. Unfortunately, mAECT also has some disadvantages, including its price, the need to centrifuge the collector tube, and the fact that it is manufactured on a noncommercial basis at only two research institutes. In conclusion, 50 years after Sheila Lanham's discovery, CATT and mAECT have become essential elements in the elimination of HAT

Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic

Antibodies directed against L-tryptophan epitope (WE - W for tryptophan, E for epitope), a constant epitope borne by variant surface glycoproteins (VSG), have been detected in sera of all 152 Human African Trypanosomosis (HAT) patients from Angola. The WE is present in VSG hydrophobic regions of the C terminal domains. In the assay, L-tryptophan was linked to bovine serum albumin (BSA) with glutaraldehyde to synthesize W-G-BSA conjugate which was used in an enzyme-linked immunosorbent assay (ELISA) to detect the antibodies. A significant difference was found between HAT patients and controls confirming previous results obtained with a lower number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions.Key words: Tryptophan, enzyme-linked immunosorbent assay (ELISA), human African trypanosomosis, serological diagnostic

Cerebral and Peripheral Changes Occurring in Nitric Oxide (NO) Synthesis in a Rat Model of Sleeping Sickness: Identification of Brain iNOS Expressing Cells

International audienceBACKGROUND: The implication of nitric oxide (NO) in the development of human African trypanosomiasis (HAT) using an animal model, was examined. The manner by which the trypanocidal activity of NO is impaired in the periphery and in the brain of rats infected with Trypanosoma brucei brucei (T. b. brucei) was analyzed through: (i) the changes occurring in NO concentration in both peripheral (blood) and cerebral compartments; (ii) the activity of nNOS and iNOS enzymes; (iii) identification of the brain cell types in which the NO-pathways are particularly active during the time-course of the infection. METHODOLOGY/PRINCIPAL FINDINGS: NO concentration (direct measures by voltammetry) was determined in central (brain) and peripheral (blood) compartments in healthy and infected animals at various days post-infection: D5, D10, D16 and D22. Opposite changes were observed in the two compartments. NO production increased in the brain (hypothalamus) from D10 (+32%) to D16 (+71%), but decreased in the blood from D10 (-22%) to D16 (-46%) and D22 (-60%). In parallel with NO measures, cerebral iNOS activity increased and peaked significantly at D16 (up to +700%). However, nNOS activity did not vary. Immunohistochemical staining confirmed iNOS activation in several brain regions, particularly in the hypothalamus. In peritoneal macrophages, iNOS activity decreased from D10 (-83%) to D16 (-65%) and D22 (-74%) similarly to circulating NO. CONCLUSION/SIGNIFICANCE: The NO changes observed in our rat model were dependent on iNOS activity in both peripheral and central compartments. In the periphery, the NO production decrease may reflect an arginase-mediated synthesis of polyamines necessary to trypanosome growth. In the brain, the increased NO concentration may result from an enhanced activity of iNOS present in neurons and glial cells. It may be regarded as a marker of deleterious inflammatory reactions

Improvement of leucocytic Na+K+ pump activity in uremic patients on low protein diet

Improvement of leucocytic Na+ K+ pump activity in uremic patients on low protein diet. Leucocytic Na+K+ pump activity was assessed in 20 patients with advanced renal failure. Na+K+-ATPase activity was reduced when compared with the values obtained from normal subjects (101.8 ± 48.6 versus 165.13 ± 8.9 µM of Pi hr-1 · g-1 P < 0.001) and the mean 86Rb uptake by U 937 cells was depressed by 38% after the addition of patients' sera. Subsequently, patients were put on a diet providing 0.3g protein/kg body weight daily and supplemented with ketoacids. After three months of dietary treatment Na+K+-ATPase activity increased to 142 ± 48.3 (P < 0.01) and reached normal values at the sixth month (162.8 ± 54.70 µM of Pi hr-1 · g-1; P < 0.001) whereas 86Rb uptake increased by 23 percent when compared to initial values. These data suggest that among the different mechanisms which have been advanced to explain the defects in the Na+ pump observed in uremic patients, circulating inhibitors deriving from alimentary protein intake may affect cation transport