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• Filling the gap of ITS1-primers comparatively sensitivity and concordance assessment. • Ten primers targeting ITS1 of trypanosomes were assessed using single-round and nested-PCR (eight) with archived DNA samples. • Little agreement levels (k = 0.05–0.52) were noticed between ITS1-primers used to detect african trypanosome species. • Fair sensitivity (11.9 %−50.5 %) of primers in detecting trypanosomes in samples of different epidemiological settings. • Primers targeting ITS1-A sequence of trypanosomes recorded the highest sensitivity (50.5 %) and concordance with ITS1-C (k = 0.52), thus most appropriate for trypanosomes detection. Although several primers targeted to the internal transcribed-spacer 1 (ITS1) of the ribosomal DNA (rDNA) have been designed to improve the detection of African trypanosomes, no study tried to compare their agreement level and ability to amplify different trypanosome species in tsetse flies and mammals in various epidemiological settings. This study was designed to fill this gap, by targeting tsetse-infested areas of Cameroon. For this, archived DNA samples reporting at-least one trypanosome species with species-specific PCR primers were reviewed. Ten sets of primers targeting different ITS1 rDNA sequences of trypanosomes were selected for assessment using single-round and nested-PCR method. Amplification rates (sensitivity) and agreement level of different ITS1 assays were compared using Cohen's-Kappa and McNemar's x2 statistic. Little agreement level (k = 0.05–0.52) were observed between different ITS1-primers PCRs detection of African trypanosome species despite significant (X2=54.3, p = 0.0001) high amplification rate 91.6 % (339/370). This sensitivity varied from quite low for T. simiae (11.9 %) and T. vivax (27.3 %) to fairly good for T. congolence (51.9 %) , Trypanozoon (32.4 %) and T. theileri (40.3 %). Primers set targeting ITS1-A sequence of trypanosome species recorded the highest sensitivity (50.5 %) with fairly good agreement compared to 39.2 % for ITS1-C (k = 0.52), 32.4 % for ITS1-R (k = 0.47), 29.7 % for ITS1-N (k = 0.48) and 23.0 % for ITS1-KIN (k = 0.43) respectively. This study revealed a diversity in the sensitivity of different trypanosome species with different sets of ITS-primers enhancing the need to use the same sets of primers in different bio-ecological settings. The use of nested-PCR instead of single-round PCR enabled improvement of trypanosome infections detection in both tsetse and mammals. Among the sets of ITS1-primers tested, those designed by to amplify ITS1-A can be considered as the most appropriate for the detection of trypanosome infections in mammals and tsetse flies. [Display omitted] [ABSTRACT FROM AUTHOR]

• The potential new drugs for leishmaniasis and trypanosomiasis are summarized. • The antiprotozoal activities of microalgal and cyanobacterial extracts are reviewed. • The isolation, identification, and antiprotozoal activity of peptides from cyanobacteria are discussed in detail. Neglected tropical diseases (NTD) like Leishmaniasis and trypanosomiasis affect millions of people annually, while currently used antiprotozoal drugs have serious side effects. Drug research based on natural products has shown that microalgae and cyanobacteria are a promising platform of biochemically active compounds with antiprotozoal activity. These unicellular photosynthetic organisms are rich in polyunsaturated fatty acids, pigments including phycocyanin, chlorophylls and carotenoids, polyphenols, bioactive peptides, terpenes, alkaloids, which have proven antioxidant, antimicrobial, antiviral, antiplasmodial and antiprotozoal properties. This review provides up-to-date information regarding ongoing studies on substances synthesized by microalgae and cyanobacteria with notable activity against Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei , the causative agents of Leishmaniasis, Chagas disease, and human African trypanosomiasis, respectively. Extracts of several freshwater or marine microalgae have been tested on different strains of Leishmania and Trypanosoma parasites. For instance, ethanolic extract of Chlamydomonas reinhardtii and Tetraselmis suecica have biological activity against T. cruzi , due to their high content of carotenoids, chlorophylls, phenolic compounds and flavonoids that are associated with trypanocidal activity. Halophilic Dunaliella salina showed moderate antileishmanial activity that may be attributed to the high β -carotene content in this microalga. Peptides such as almiramides, dragonamides, and herbamide that are biosynthesized by marine cyanobacteria Lyngbya majuscula were found to have increased activity in micromolar scale IC 50 against L. donovani, T. Cruzi, and T. brucei parasites. The cyanobacterial peptides symplocamide and venturamide isolated from Symploca and Oscillatoria species, respectively, and the alkaloid nostocarbonile isolated from Nostoc have shown promising antiprotozoal properties and are being explored for pharmaceutical and medicinal purposes. The discovery of new molecules from microalgae and cyanobacteria with therapeutic potential against Leishmaniasis and trypanosomiasis may address an urgent medical need: effective and safe treatments of NTDs. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Trypanosoma brucei , a flagellated protozoan causing the deadly tropical disease Human African Trypanosomiasis (HAT), affects people in sub-Saharan Africa. HAT therapy relies upon drugs which use is limited by toxicity and rigorous treatment regimes, while development of vaccines remains elusive, due to the effectiveness of the parasite´s antigenic variation. Here, we evaluate a hypothetical protein Tb427.10.13790, as a potential drug target. This protein is conserved among all kinetoplastids, but lacks homologs in all other pro- and eukaryotes. Knockdown of Tb427.10.13790 resulted in appearance of monster cells containing multiple nuclei and multiple flagella, a considerable enlargement of the flagellar pocket and eventually a lethal phenotype. Furthermore, analysis of kinetoplast and nucleus division in the knockdown cell line revealed a partial cell cycle arrest and failure to initiate cytokinesis. Likewise, overexpression of the respective protein fused with enhanced green fluorescent protein was also lethal for T. brucei. In these cells, the labelled protein appeared as a single dot near kinetoplast and flagellar pocket. Our results reveal that Tb427.10.13790 is essential for the parasite´s viability and may be a suitable new anti-trypanosomatid drug target candidate. Furthermore, we suggest that it might be worthwhile to investigate also other of the many so far just annotated trypanosome genes as a considerable number of them to lack human homologs but may be of critical importance for the kinetoplastid parasites. [ABSTRACT FROM AUTHOR]

African sleeping sickness is a parasitic disease in humans and livestock caused by Trypanosoma brucei throughout sub-Saharan Africa. Absence of appropriate vaccines and prevalence of drug resistance proclaim that a new way of therapeutic interventions is essential against African trypanosomiasis. In the present study, we have looked into the effect of andrographolide (andro), a diterpenoid lactone from Andrographis paiculata on Trypanosoma brucei PRA 380. Although andro has been recognized as a promosing anti-cancer drug, its usefulness against Trypanosoma spp remained unexplored. Andro showed promising anti-trypanosomal activity with an IC 50 value of 8.3 μM assessed through SYBR Green cell viability assay and also showed no cytotoxicity towards normal murine macrophages. Cell cycle analysis revealed that andro could induce sub-G 0 /G 1 phase arrest. Flow cytometric analysis also revealed that incubation with andro caused exposure of phosphatidyl serine to the outer leaflet of plasma membrane in T. brucei PCF. This event was preceded by andro-induced depolarization of mitochondrial membrane potential (Δym) and elevation of cytosolic calcium. Andro also caused elevation of intracellular reactive oxygen species (ROS) as well as lipid peroxidation level, and depletion in reduced thiol levels. Taken together, these data indicate that andro has promising antitrypanosomal activity mediated by promoting oxidative stress and depolarizing the mitochondrial membrane potential and thereby triggering an apoptosis-like programmed cell death. Therefore, this study merits further investigation to the therapeutic possibility of using andro for the treatment of African trypanosomiasis. [ABSTRACT FROM AUTHOR]

Introduction Sleeping sickness or Human African Trypanosomiasis (HAT) is a neglected tropical disease of public health importance across much of Sub-Saharan Africa. In Uganda, chronic T. b. gambiense HAT (gHAT) and acute T. b. rhodesiense HAT (rHAT) occur in two large but discrete geographical foci. Both forms are difficult to diagnose, expensive to treat and ultimately fatal in the absence of treatment. The area affected by zoonotic rHAT has been steadily expanding, placing a high burden on local health systems. HAT is a disease of neglected populations and is notorious for being under-reported. Here we examine the factors that influence passive rHAT surveillance within the district health system in four Ugandan districts into which the disease had recently been introduced, focusing on staff knowledge, infrastructure and data management. Methods A mixed methods study was undertaken between 2011 and 2013 in Dokolo, Kaberamaido, Soroti and Serere districts to explore health facility capacity and clinical service provision, diagnostic capacity, HAT knowledge and case reporting. Structured interviews were undertaken with 86 medical personnel, including clinicians, nurses, midwives and technicians across 65 HC-II and HC-III medical facilities, where the health infrastructure was also directly observed. Eleven semi-structured interviews were undertaken with medical staff in each of the three designated HAT treatment facilities (Dokolo, Lwala and Serere HC-IV) in the area. HAT treatment centre case records, collected between 2009 and 2012, were analyzed. Results Most medical staff in HC-II and HC-III facilities had been made aware of HAT from radio broadcasts, newspapers and by word of mouth, suggestive of a lack of formal training. Key knowledge as regards the causative agent, clinical signs and that HAT drugs are provided free of charge was lower amongst HC-II than HC-III staff. Many respondents did not know whether HAT was endemic in their district. In rHAT specialist treatment centres, staff were knowledgeable of HAT and were confident in their ability to diagnose and manage cases. Between 2009–2012, 342 people were diagnosed in the area, 54% in the late stage of the disease. Over the period of this study the proportion of rHAT cases identified in early stage fell and by 2012 the majority of cases identified were diagnosed in the late stage. Conclusion This study illustrates the critical role of the district health system in HAT management. The increasing proportion of cases identified at a late stage in this study indicates a major gap in lower tier levels in patient referral, diagnosis and reporting that urgently needs to be addressed. Integrating HAT diagnosis into national primary healthcare programs and providing training to medical workers at all levels is central to the new 2030 WHO HAT elimination goal. Given the zoonotic nature of rHAT, joined up active surveillance in human and animal populations in Uganda is also needed. The role of the Coordinating Office for Control of Trypanosomiasis in Uganda in implementing a One Health approach will be key to sustainable management of zoonotic HAT. [ABSTRACT FROM AUTHOR]

Introduction Human African trypanosomiasis (HAT) comprises two fatal parasitic diseases. Uganda is home to both chronic T. b. gambiense (gHAT) and the acute zoonotic form T. b. rhodesiense (rHAT) which occur in two large but discrete geographical foci. The area affected by rHAT has been rapidly expanding due to importation of T. b. rhodesiense infected cattle into tsetse infested but previously HAT free districts. Migration of rHAT has resulted in a considerable human health burden in these newly affected districts. Here, we examined the impact of a single, district-wide, mass chemotherapeutic livestock intervention, on T. b. rhodesiense prevalence in cattle and on incidence and distribution of human rHAT cases in Kamuli and Soroti districts in eastern Uganda. Methods A single mass intervention in domestic cattle (n = 30,900) using trypanocidal drugs was undertaken in November and December 2002 under the EU funded Farming in Tsetse Controlled Areas (FITCA) programme. The intervention targeted removal of the reservoir of infection i.e. human infective T. b. rhodesiense parasites in cattle, in the absence of tsetse control. Interventions were applied in high-risk sub-counties of Kamuli district (endemic for rHAT) and Soroti district (where rHAT has been recently introduced). The prevalence of T. brucei s.l. and the human infective subspecies, T. b. rhodesiense in cattle (n = 1833) was assessed before and 3 and 12 months after intervention using PCR-based methods. A combination of descriptive statistical analysis and spatial scan statistics were applied to analyse rHAT cases reported over a 5-year period (January 2000–July 2005). Results A single intervention was highly effective at removing human infective T. b. rhodesiense parasites from the cattle reservoir and contributed to a significant decrease in human rHAT cases. Intervention coverage was higher in Kamuli (81.1%) than in Soroti (47.3%) district but despite differences in coverage both districts showed a reduction in prevalence of T. b. brucei s.l. and T. b. rhodesiense . In Kamuli, the prevalence of T. brucei s.l. decreased by 54%, from 6.75% to 3.11%, 3, months post-intervention, rising to 4.7% at 12 months. The prevalence of T. b. rhodesiense was 3% pre-intervention and no T. b. rhodesiense infections were detected 3 and 12, months post-treatment. In Soroti, the prevalence of T. brucei s.l. in cattle decreased by 38% (from 21% to 13%) 3 months after intervention decreasing to less than 10% at 12 months. The prevalence of T. b. rhodesiense was reduced by 50% at 12-months post-intervention (6%–3%). Most notably, was the impact of the intervention on the population dynamics between T. b. brucei and human infective T. b. rhodesiense . Before intervention in Kamuli district 56% of T. b. brucei s.l. circulating in cattle were T. b. rhodesiense ; at both 3 and 12 months after intervention none of the re-infecting T. b. brucei s.l. were human infective, T. rhodesiense. For human rHAT cases, there was a seven-fold decrease in rHAT incidence after intervention in Kamuli district (5.54 cases/1,000 head of population 2000–2002 to 0.76 cases/1,000, 2003–2005). Incidence data suggests that the intervention had minimal impact on the number of rHAT cases in Soroti overall, but showed a significant decrease in the seasonal peak of cases in the year following treatment. Conclusion A single intervention, targeted at cattle, introduced at district level, in the absence of tsetse control, was highly effective at removing human infective rHAT parasites from the cattle reservoir and contributed to a significant decrease in human rHAT cases. The differential impacts observed between the two districts are related to both the different stages of rHAT endemicity in the districts, and levels of intervention coverage achieved in the cattle population. Treatment of cattle to remove the reservoir of rHAT infection offers a promising and cost effective approach for the control of rHAT. It is important that cattle are treated before relocation to prevent possible merger of the two HAT foci, which would complicate diagnosis and treatment of both gHAT and rHAT. [ABSTRACT FROM AUTHOR]

• Human African trypanosomiasis (HAT) has no vaccine and is fatal without treatment. • Animal reservoirs are important for rHAT form and uncertain for gHAT form. • Insecticide treatment of livestock is better for rHAT control than trypanocides. • If gHAT has animal reservoirs, elimination goals won't be met by current strategies. . In response to large strides in the control of human African trypanosomiasis (HAT), in the early 2000s the WHO set targets for elimination of both the gambiense (gHAT) and rhodesiense (rHAT) forms as a public health (EPHP) problem by 2020, and elimination of gHAT transmisson (EOT) by 2030. While global EPHP targets have been met, and EOT appears within reach, current control strategies may fail to achieve gHAT EOT in the presence of animal reservoirs, the role of which is currently uncertain. Furthermore, rHAT is not targeted for EOT due to the known importance of animal reservoirs for this form. . To evaluate the utility of a One Health approach to gHAT and rHAT EOT, we built and parameterized a compartmental stochastic model, using the Institute for Disease Modeling's Compartmental Modeling Software, to six HAT epidemics: the national rHAT epidemics in Uganda and Malawi, the national gHAT epidemics in Uganda and South Sudan, and two separate gHAT epidemics in Democratic Republic of Congo distinguished by dominant vector species. In rHAT foci the reservoir animal sub-model was stratified on four species groups, while in gHAT foci domestic swine were assumed to be the only competent reservoir. The modeled time horizon was 2005-2045, with calibration performed using HAT surveillance data and Optuna. Interventions included insecticide and trypanocide treatment of domestic animal reservoirs at varying coverage levels. . Validation against HAT surveillance data indicates favorable performance overall, with the possible exception of DRC. EOT was not observed in any modeled scenarios for rHAT, however insecticide treatment consistently performed better than trypanocide treatment in terms of rHAT control. EOT was not observed for gHAT at 0% coverage of domestic reservoirs with trypanocides or insecticides, but was observed by 2030 in all test scenarios; again, insecticides demonstrated superior performance to trypanocides. EOT likely cannot be achieved for rHAT without control of wildlife reservoirs, however insecticide treatment of domestic animals holds promise for improved control. In the presence of domestic animal reservoirs, gHAT EOT may not be achieved under current control strategies. Our stochastic compartmental model indicates that insecticide treatment of domestic animal reservoirs provides superior control to trypanocide treatment for both forms of human African trypanosomiasis [Display omitted] [ABSTRACT FROM AUTHOR]

• Trypanosomes DNA persist after drug treatment • Trypanosome DNA was readily detected in the skin of cattle before and after treatment • No clear correlation in trypanosome DNA found in the skin and blood • There is a diagnostic potential of sampling the skin for African trypanosomiasis. African Trypanosomiasis is a debilitating disease in both humans and animals that occurs in sub-Saharan Africa and has a severe negative impact on the livelihood of people in the affected areas. The disease is caused by protozoan parasites of the genus Trypanosoma, which is often described simply as blood-borne; however, a number of studies have shown the parasite inhabits many different environments within the host. Control of the disease involves measures that include the use of trypanocidal drugs to which there are growing number of reported cases of resistance. Here, the patterns of trypanosome DNA presence during a diminazene aceturate treatment round on a cohort of cattle in Adidome, Ghana were assessed. A group of 24 cows were selected irrespective of age and sex and the infecting trypanosome species followed for 18 days before and after treatment with diminazene aceturate in the blood and skin of the animals using a diagnostic nested PCR that targeted the alpha-beta tubulin gene array. Persistence of trypanosome DNA was observed over the period and parasite DNA was readily detected in both the skin and blood, with parasite DNA disappearing and reappearing in both across the study. Moreover, there was limited correlation between the parasite DNA detected in the skin and blood. Overall, the data show the patterns of a natural trypanosome infection during drug treatment. In addition, the diagnostic potential of sampling the skin for African trypanosomiasis is highlighted. [ABSTRACT FROM AUTHOR]

Abstract: The blood meal origins of 222 tsetse flies (213 Glossina palpalis palpalis, 7 Glossina pallicera pallicera, one Glossina nigrofusca and one Glossina caliginea) caught in 2008 in two Human African trypanosomiasis foci (Bipindi and Campo) of south Cameroon were investigated. 88.7% of tsetse flies blood meals were identified using the heteroduplex method and the origin of the remaining blood meals (11.3%) was identified by sequencing the cytochrome B gene. Most of the meals were from humans (45.9%) and pigs (37.4%), 16.7% from wild animals. Interestingly, new tsetse fly hosts including turtle (Trionyx and Kinixys) and snake (Python sebae) were identified. Significant differences were recorded between Bipindi where the blood meals from pigs were predominant (66.7% vs 23.5% from humans) and Campo where blood meals from humans were predominant (62.9% vs 22.7% from pigs). Comparison with the data recorded in 2004 in the same foci (and with the same molecular approach) demonstrated significant modifications of the feeding patterns: increase in blood meals from pigs in Bipindi (66.7% in 2008 vs 44.8% in 2004) and in Campo (20.5% in 2008 vs 6.8% in 2004), decrease in that from human (significant in Bipindi only). 12.6%, 8.1% and 2.7% of the flies were, respectively, Trypanosoma congolense forest type, Trypanosoma congolense savannah type and Trypanosoma brucei gambiense infected. These results demonstrate that tsetse fly feeding patterns can be specific of a given area and can evolve rapidly with time. They show an active circulation of a variety of trypanosomes in sleeping sickness foci of southern Cameroon. [ABSTRACT FROM AUTHOR]

Abstract: To evaluate the role of wildlife in the resurgence and perenisation of human African trypanosomiasis (HAT), we investigated the influence of habitat and seasonal variations on the diversity and spatial distribution of wild mammals, with special reference to those recognised as potential host-reservoirs of Trypanosoma brucei gambiense in Bipindi (southwestern Cameroon). To achieve this, we carried out transect surveys in four habitat types over two years. A total of 31 mammal species were recorded, of which 14 occurred in the undisturbed forest, 9 in cocoa plantations, 11 in farmlands and 11 in village-adjacent gallery forests. Among them, six species (Cephalophus monticola, Cephalophus dorsalis, Atherurus africanus, Cricetomys emini, Nandinia binotata and Cercopithecus nictitans), known as reservoir hosts of T. b. gambiense, occurred in all kinds of habitats suitable or unsuited to Glossina palpalis palpalis and in all seasons. These species are the most involved in the transmission cycle (human being/tsetse flies/wild animals). Cercopithecus cephus, Miopithecus talapoin and Heliosciurus rufobrachium host Trypanosoma brucei spp.; however, only C. cephus does not occur permanently in the suitable habitat of G. palpalis palpalis. In general, some species (C. monticola, Tragelaphus spekei and C. emini) showed a slight density increase from the long dry to the heavy rainy season within the undisturbed and farmland habitats, and a slight decrease within cocoa plantations and village-adjacent forests in the same period. The density of A. africanus increased greatly from the long dry season to the heavy rainy season in the undisturbed forest while, the density of primates in this habitat decreased slightly from the long dry season to the heavy rainy season. These variations indicate a permanent movement of wild mammal reservoir or feeding hosts from one biotope to another over the seasons. Thryonomys swinderianus needs to be investigated because it occurs permanently in the suitable habitat of G. palpalis palpalis and Potamochoerus porcus for its genetic similarities to domestic pigs, favourable feeding hosts of G. palpalis palpalis. [Copyright &y& Elsevier]

Abstract: African trypanosomiasis is a severe parasitic disease affecting both man and livestock. It is crucial to expand our fundamental knowledge of the intimate interactions between trypanosomes and their vertebrate hosts in order to develop new and efficient control strategies. The mouse model of trypanosomiasis is the most popular for research purposes because of all the logistic advantages of using this species. Studies of any aspect of trypanosomiases in the mouse systematically require the quantification of some phenotypic traits which translate its degree of resistance/susceptibility to the disease, as blood cell counts. The present study presents a methodological approach combining everyday microsampling of tail blood and its analysis by flow cytometry. The technical options and conditions permitting a fast, reliable and reproducible daily quantification of erythrocyte, reticulocyte, leucocyte and trypanosome counts in the inoculated mouse were established. The protocol proposed allows the multiplication of blood samplings without being exposed to the time-consuming constraint of visual countings, without causing iatrogenic blood cell alterations in the mouse and without requiring specific anti-trypanosome antibodies. [Copyright &y& Elsevier]

Abstract: Symptoms consistent with hypothyroidism or adrenal insufficiency, such as lethargy, anorexia, cold intolerance, weakness, hypotension or paraesthesia, are frequently reported in the literature in patients with Human African Trypanosomiasis (HAT), but an endocrine origin for these symptoms has not yet been demonstrated. Thyroid and adrenocortical function were assessed in 60 patients with late-stage HAT and compared to those in 60 age- and gender-matched healthy controls. Clinical assessment and endocrine laboratory examinations were performed on admission, within 2 days after the end of treatment and at follow-up 3 months later. Signs and symptoms of hypothyroidism, such as fatigue, cold sensation, constipation, paraesthesia, peripheral oedema and dry skin, were significantly more frequent in HAT patients than in the controls. However, these signs and symptoms could not be attributed to hypothyroidism due to the lack of supporting laboratory data, and thus empirical replacement therapy for the clinically suspected hypothyroidism was not warranted. Signs and symptoms consistent with adrenal insufficiency, such as weakness, anorexia, weight loss or hypotension, were significantly more frequent in HAT patients than in controls, but they could not be associated with an insufficiency of the adrenocortical axis. Higher basal levels of cortisol were found in HAT patients than in controls, which can be viewed as a stress response to the infection. However, a transitory adrenal insufficiency was suspected in 8% of HAT patients at admission and in 9% at discharge. All values were normal at follow-up 3 months later. [Copyright &y& Elsevier]

Abstract: Background: Existing data on human African trypanosomiasis (HAT) due to Trypanosoma brucei gambiense among children are limited. Here, we described the demographic, clinical, diagnostic, treatment and outcome characteristics of HAT in pre-school children from Kajo-Keji County, South Sudan in comparison with older patients. Methods: We did a retrospective analysis of HAT patients treated at the Kiri Sleeping Sickness Treatment Centre (SSTC), Kajo-Keji County, from June 2000 to December 2002. Results: Of 1958 HAT patients, 119 (6.1%) were pre-school children (<6 years) including 56 (47%) in first-stage illness and 63 (53%) in second-stage. The proportion of children in second-stage HAT was significantly higher in very young children (<2 years). Walking and speech disturbances were more frequent in second-stage HAT but other neurological symptoms and signs were not associated with disease stage. Pentamidine treatment for first-stage illness was very safe and effective among pre-school children. In contrast, 4.9% of pre-school children in second-stage illness died during melarsoprol treatment and 46% had ≥1 severe adverse event(s). Macular rash, jaundice and skin necrosis on injection site were significantly more frequent in this age group (p <0.05). Melarsoprol-induced encephalopatic syndrome was less frequent but more severe than in older age groups. Conclusion: The clinical features of T. b. gambiense HAT among pre-school children are insufficiently stage-specific. Therefore, laboratory-based staging is mandatory to prevent unnecessary harm to HAT patients caused by the high toxicity of melarsoprol. [Copyright &y& Elsevier]

Abstract: African trypanosomiasis is a deadly disease for which few chemotherapeutic options are available. The causative agents, Trypanosoma brucei rhodesiense and T. b. gambiense, utilize a non-cytochrome, alternative oxidase (AOX) for their cellular respiration. The absence of this enzyme in mammalian cells makes it a logical target for therapeutic agents. We designed three novel compounds, ACB41, ACD15, and ACD16, and investigated their effects on trypanosome alternative oxidase (TAO) enzymatic activity, parasite respiration, and parasite growth in vitro. All three compounds contain a 2-hydroxybenzoic acid moiety, analogous to that present in SHAM, and a prenyl side chain similar to that found in ubiquinol. ACD15 and ACD16 are further differentiated by the presence of a solubility-enhancing carbohydrate moiety. Kinetic studies with purified TAO show that all three compounds competitively inhibit TAO, and two compounds, ACB41 and ACD15, have inhibition constants five- and three-fold more potent than SHAM, respectively. All three compounds inhibited the respiration and growth of continuously cultured T. b. brucei bloodstream cells in a dose-dependent manner. None of the compounds interfered with respiration of rat liver mitochondria, nor did they inhibit the growth of a continuously cultured mammalian cell line. Collectively, the results suggest we have identified a new class of compounds that are inhibitors of TAO, have trypanocidal properties in vitro, and warrant further investigation in vivo. [Copyright &y& Elsevier]

Abstract: Six sets of teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were fed on mice infected with six different isolates of Trypanosoma brucei gambiense (each mouse was infected with one of the isolates), previously isolated from patients in the sleeping sickness focus of Bonon, Côte d’Ivoire and in Makoua, Congo. All the tsetse flies were dissected 42 days post-infection and midgut and salivary glands were examined for trypanosomes by microscopical examination. No infection was observed with the reference stock whereas each of the five recently isolated trypanosome isolates was able to infect tsetse flies, with rates of infection varying between 9.7 and 18.2% depending on the isolate. Three isolates displayed only immature infections with 9.7, 17.3 and 18% of the flies showing trypanosomes in their midgut. One isolate gave both immature (12.1%) and mature infections (6.1%). Finally, the last isolate involved only mature infections in 9.7% of the Glossina species examined. These substantial differences in the cyclical transmission of T. b. gambiense in the same fly species could have important implications for the epidemiology of the transmission of Human African Trypanosomiasis. [Copyright &y& Elsevier]

Abstract: The situation of human African trypanosomiasis remains serious with one of the main threats being the increasing number of relapses or treatment failures after melarsoprol treatment. In order to investigate and to compare drug sensitivities of trypanosomes isolated at different time periods and in different locations, two sets of Trypanosoma brucei gambiense strains were used. One set was isolated in the time period 1960–1981 and the other one in 1995–2004 from different locations of West and Central Africa. These isolates were not selected based on the treatment outcome but on availability. The drug sensitivity profile for all available drugs in use and the diamidine compound DB75 was established. IC50 values were not significantly different between the “old” and “new” stocks. No indications for emerging drug resistance to any drug could be observed. The results indicate a relative stability of in vitro sensitivity of T. b. gambiense to trypanocidal drugs in space (West and Central Africa) and time (1960–2004). [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The clinical symptoms and signs of patients with second stage HAT are described for a large cohort of patients treated in a prospective multicentre, multinational study. Special emphasis is given to the influence of disease stage (duration, number of WBC in CSF) and patient age to the clinical picture. Even though the frequencies of symptoms and signs are highly variable between centres, the clinical picture of the disease is similar for all countries. Headache (78.7%), sleeping disorder (74.4%) and lymphadenopathy (56.1%) are the most frequent symptoms and signs and they are similar for all stages of the disease. Lymphadenopathy tends to be highest in the advanced second stage (59.0%). The neurological and psychiatric symptoms increase significantly with the number of WBC in the CSF indicating the stage of progression of the disease. Pruritus is observed in all stages and increases with the number of WBC in CSF from 30 to 55%. In children younger than 7 years, lymphadenopathy is less frequently reported (11.8–37.3%) than in older children or adults (56.4–61.2%). Fever is most frequently reported in children between 2 and 14 years of age (26.1–28.7%) and malnutrition is significantly more frequently observed in children of all ages (43–56%) than in adults (23.5%). [Copyright &y& Elsevier]

The current epidemic of sleeping sickness, also known as human African trypanosomiasis in sub-Saharan Africa places nearly 60 million people at risk for developing this life threatening infection. Although effective treatments for early-stage sleeping sickness exist, these drugs usually require extended dosing schedules and intravenous administration. New treatments are also needed for cerebral (late) stage trypanosomiasis. 2,5-Bis(4-amidinophenyl)furan (DB75), a pentamidine analog, has potent in vitro and in vivo anti-trypanosomal activity. However, DB75 does not exhibit significant oral bioavailability and has proved to be ineffective against mouse models of late-stage sleeping sickness regardless of administration route. To circumvent the limited oral bioavailability of DB75, an N-methoxy prodrug 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289) was designed and developed initially as a compound to treat AIDS-related Pneumocystis carinii pneumonia (PCP). Despite excellent oral activity against early-stage sleeping sickness, oral administration of DB289 exhibited limited efficacy in mouse models of late-stage disease. DB289 has recently entered Phase II(b) clinical trials to treat primary-stage sleeping sickness in Central Africa. The current study takes advantage of the innate fluorescence of DB75 and DB289 along with specific and sensitive quantitative analyses to examine plasma and brain distribution of these compounds. Animals were dosed with intravenous DB75, oral DB289, and intravenous DB289. Following intravenous administration, DB75 was readily detectable in whole brain extracts and persisted for long periods. Fluorescence microscopy revealed that DB75 did not penetrate into brain parenchyma, however, but was sequestered within cells lining the blood–brain and blood–cerebrospinal fluid barriers. In contrast, brain tissue of mice treated with oral DB289 exhibited diffuse fluorescence within the brain parenchyma, suggesting that the prodrug was not trapped within blood–brain barrier cells (BBB). However, maximal brain concentrations of the active compound DB75 were very low (13 nmol/mg of tissue at 24 h). Intravenous administration of DB289 resulted in a qualitatively similar fluorescence pattern to oral DB289, indicating again that DB289 and DB75 were present within brain parenchyma, not only in barrier regions. Furthermore, peak DB75 tissue levels were higher (61 nmol/mg of tissue at 24 h) than with oral prodrug. The near five-fold increase in brain levels of DB289 combined with parenchymal localization of compound fluorescence after intravenous administration suggest that the unaltered prodrug penetrates the blood–brain barrier, and may be subject to in situ biotransformation. Intravenous administration of DB289 should be evaluated in mouse models of late-stage sleeping sickness. [Copyright &y& Elsevier]

Phenanthridine trypanocides (isometamidium chloride hydrochloride, ISM, and Ethidium bromide, EBr) have been widely used to treat African trypanosomiasis in livestock for more than 40 years. Their main action is to inhibit nucleic acid synthesis in trypanosome parasites, by intercalation between the DNA base pairs. They can also linearise selectively kinetoplast DNA minicircles; a form of mitochondrial DNA unique to this group of parasites. However, the metabolism of these compounds by trypanosomes has not been reported. Indeed, it is not known whether or not their metabolism by the parasite contributes to their activity, selective toxicity for these parasites or to the development of chemoresistance. Therefore, we studied the metabolism of EBr and ISM, and their distribution in Trypanosoma brucei (TREU 927) using high performance liquid chromatography (HPLC), liquid chromatography combined with mass spectrometry (LC–MS) and confocal laser scanning microscopy (CLSM). Incubation of EBr with trypanosomes led to the formation of a small amount (0.606±0.191%) of one metabolite (MI). Ion chromatograms extracted from an LC–MS analysis using electrospray ionisation (ESI), showed that the difference in mass between the parent compound and its metabolite was 30. This may correspond to the addition of a hydroxyl and a methyl group. No metabolites could be detected for ISM. The distribution of the two drugs in trypanosomes was investigated by CLSM, using their intrinsic fluorescence. ISM and EBr showed differences in their distribution in trypanosomes. ISM had a greater affinity for the kinetoplast than EBr and it stained other organelles like the flagellum; in contrast the distribution of EBr was more diffuse. [Copyright &y& Elsevier]

During a medical survey the sleeping sickness focus in Bonon, Ivory Coast, PCR with Trypanosoma brucei specific primers (TBR 1–2 from Parasitology 99 (1989) 57) was tested on DNA derived from blood samples. DNA purification using a chelating resin was performed either on whole blood or on the buffy coat prepared in two different ways. The preparation based on whole blood performed better than those using the buffy-coat. Using this first method, the sensitivity was 100% on parasitologically confirmed patients, and the specificity was 92%. However, problems of reproducibility of the technique were pointed out, particularly on samples from serologically positive but apparently aparasitemic individuals. It is suggested that the PCR could help in the diagnosis of Human African Trypanosomosis, but the use of other primers should be investigated. [Copyright &y& Elsevier]

Though it has been established that domestic animals (especially the pig) are potential reservoir hosts for Trypanosoma brucei gambiense in West Africa, there is little data to this effect concerning Central Africa. Instead, some previous authors report the absence of Trypanozoon type trypanosomes in domestic animals in Cameroon. Thirty-two domestic pigs were sampled by KIVI (kit for in vitro isolation) of trypanosomes in the northern region (Bechati) of the Fontem sleeping sickness focus of Cameroon. Twenty-one of these were found positive, from 15 of which 17 isolates were successfully obtained. Isoenzyme characterization revealed that isolates from 4 of the 15 pigs belonged to zymodemes associated with T. brucei gambiense group 1. The prevalence of this disease in the local human population is, however, very low. It is evident from this study that the domestic pig may be a potential reservoir host for T. brucei gambiense in the Fontem focus. There is, however, need for an extensive study on domestic animals in Cameroon and other neighbouring countries for a better comprehension of the epidemiology of sleeping sickness within the Central African region. [Copyright &y& Elsevier]

• StS-target is more efficient in sampling Glossina fuscipes fuscipes than ES-target and biconical trap. • Cost of catching a fly using the StS-target was 3.4 times lower compared to the biconical trap. • Cost of catching a fly using StS-target was over 19 times lower compared to the ES-target. • Tsetse fly collected from StS target are suitable for PCR base molecular tests. • There is a presence of Spiroplasma in wild G. fuscipes fuscipes of western Kenya. Monitoring the effectiveness of tsetse fly control interventions that aim to reduce transmission of African trypanosomiasis requires highly efficient sampling tools that can catch flies at low densities. The sticky small target (StS-target) has previously been shown to be more effective in sampling Glossina fuscipes fuscipes compared to the biconical trap. However, its efficiency in terms of the proportion of flies it catches out of those that visit it has not been reported. Furthermore, there are no reports on whether tsetse samples caught using the StS-target can be used for subsequent processes such as molecular tests. In this study, we evaluated the efficiency of the biconical trap and targets for sampling G. f. fuscipes. All targets were tiny (0.25 × 0.50 m) but varied in their capture system. We used targets with sticky surface (StS-targets) and those with an electrified surface (ES-targets). We also assessed the suitability of flies caught on the StS-target for molecular tests by amplifying DNA of bacterial communities. Randomized block design experiments were undertaken in Mbita area and Manga Island on Lake Victoria of western Kenya. Fly catches of each sampling tool were compared to those of the sampling tool flanked by electric (E) nets and analyzed using a negative binomial regression. The total catch for each sampling tool alone was divided by the total catch of the sampling tool flanked by two E-nets to obtain its efficiency expressed as a percentage. A proportion of flies caught on the StS-target was preserved for molecular tests. Overall, the efficiencies of the biconical trap, ES-target and StS-target were 7.7%, 13.3% and 27.0%, respectively. A higher proportion of females (69 to 79%) than males approached all the sampling tools, but the trap efficiency was greater for male G. f. fuscipes than females. Furthermore, sequencing the 16S rRNA gene from fly samples caught on the StS-target revealed the presence of Spiroplasma. Our results indicate that the SS-target is the most efficient trap to monitor G. f. fuscipes population during interventions, with the biconical trap being the least efficient, and samples collected from StS-targets are suitable for molecular studies. [ABSTRACT FROM AUTHOR]

• Zebra skin-derived blend K repelled riverine tsetse flies Glossina fuscipes fuscipes. • Sex-specific variations occur in the repellent effect of blend K and WRC. • Geranyl acetone accounts for the repellent effect of blend K on G. f. fuscipes. • Olfactory cues are important in the bioecology of riverine tsetse flies. The riverine tsetse fly Glossina fuscipes fuscipes is a major vector of trypanosome pathogens causing African trypanosomiasis. This fly species uses a combination of olfactory and visual cues to locate its hosts. Previously, traps and targets baited with visual cues have been used in vector control, but the development of olfactory-based tools has been challenging. Recently, repellents have shown promise as olfactory-based tools in tsetse vector control. Here, we evaluated a three-component blend comprising 6-methyl-5-hepten-2-one, acetophenone and geranyl acetone (blend K), previously identified as a repellent for savannah tsetse flies in zebra skin odor, on G. f. fuscipes populations. Using a series of 6 × 6 randomized Latin square-designed experiments, G. f. fuscipes catches in biconical traps were monitored on four islands of Lake Victoria in western Kenya between July and September 2019, after the long rainy season. Traps were baited with blend K and individual components of this blend. The known tsetse repellent blend WRC (waterbuck repellent compounds) and trap alone were included as controls. Daily catch data in thirty-six replicate trials were analyzed using generalized linear model with negative binomial error structure using the package "MASS" in R. Treatment, day and site were set as predictor variables. Our results showed that, blend K significantly reduced G. f. fuscipes catches by 25.6% (P < 0.01) compared to the control trap alone but was not significantly different from WRC which reduced catches by 20.7% (P < 0.05). Of the individual compounds, geranyl acetone solely significantly reduced catches by 29.1% (P < 0.01) which did not differ from blend K or WRC. We conclude that geranyl acetone accounts for the repellent effect of blend K on the riverine tsetse fly, G. f. fuscipes , demonstrating the ecological importance of animal skin odors in the host-seeking behavior of medically-important tsetse fly vectors. Zebra skin-derived blend K and its component, geranyl acetone, repel Glossina fuscipes fuscipes showing their potential for use in the control of these medically-important tsetse flies. Image, graphical abstract [ABSTRACT FROM AUTHOR]