Human African Trypanosomiasis Foci Research Articles

Limited impact of vector control on the population genetic structure of Glossina fuscipes fuscipes from the sleeping sickness focus of Maro, Chad.

Tsetse flies (genus Glossina) transmit deadly trypanosomes to human populations and domestic animals in sub-Saharan Africa. Some foci of Human African Trypanosomiasis due to Trypanosoma brucei gambiense (g-HAT) persist in southern Chad, where a program of tsetse control was implemented against the local vector Glossina fuscipes fuscipes in 2018 in Maro. We analyzed the population genetics of G. f. fuscipes from the Maro focus before control (T0), one year (T1), and 18 months (T2) after the beginning of control efforts. Most flies captured displayed a local genetic profile (local survivors), but a few flies displayed outlier genotypes. Moreover, disturbance of isolation by distance signature (increase of genetic distance with geographic distance) and effective population size estimates, absence of any genetic signature of a bottleneck, and an increase of genetic diversity between T0 and T2 strongly suggest gene flows from various origins, and a limited impact of the vector control efforts on this tsetse population. Continuous control and surveillance of g-HAT transmission is thus recommended in Maro. Particular attention will need to be paid to the border with the Central African Republic, a country where the entomological and epidemiological status of g-HAT is unknown.

Population Knowledge and Practices and the Prevalence of Trypanosomes Circulating in Domestic Animals in Three Active Human African Trypanosomiasis Foci in the Republic of Congo

Human African Trypanosomiasis (HAT) is still endemic in the Republic of Congo. Although the incidence of cases has significantly decreased over years, the disease still persists in some active foci. Factors contributing to the maintenance of the disease such as the existence of an animal reservoir or population knowledge are still not well known. It is in this context that a study focusing on the knowledge and practices of the population with regard to HAT as well as on the prevalence of trypanosomes infecting animals was undertaken in three active HAT foci in the Republic of Congo. The study was performed using field surveys conducted from November 2019 to June 2021. Domestic animal blood was examined by microscopy and PCR to detect the presence of trypanosomes. A structured questionnaire was administered to the population to assess their knowledge and practices concerning HAT in these endemic foci. More than half of the animals examined were found to be infected with trypanosomes (51.22%). The main trypanosome species infecting animals were Trypanosoma congolense savannah (67.2%) and Trypanosoma brucei (s.l.) (32.8%). No trypanosomes infecting humans were detected. Concerning household surveys, more than half of the respondents (52.9%) were fully aware of the mode of transmission and symptoms of the disease. The majority of people preferred to wear clothes covering the whole body and to use locally made soap as repellents to protect themselves from tsetse fly bites. This study suggests frequent circulation of animal trypanosomes in domestic animals and the use of personal measures to protect against tsetse fly bites. Updating information on the HAT animal reservoir and population knowledge alongside regular monitoring of the tsetse fly populations and the use of traps to control tsetse flies are crucial to drive efforts towards the elimination of gHAT in the Republic of Congo.

Prevalence of blood and skin trypanosomes in domestic and wild fauna from two sleeping sickness foci in Southern Cameroon.

Although studies on African Trypanosomiases revealed a variety of trypanosome species in the blood of various animal taxa, animal reservoirs of Trypanosoma brucei gambiense and anatomical niches such as skin have been overlooked in most epidemiological settings. This study aims to update epidemiological data on trypanosome infections in animals from human African trypanosomiasis (HAT) foci of Cameroon. Blood and skin snips were collected from 291 domestic and wild animals. DNA was extracted from blood and skin snips and molecular approaches were used to identify different trypanosomes species. Immunohistochemical analyses were used to confirm trypanosome infections in skin snips. PCR revealed 137 animals (47.1%) with at least one trypanosome species in the blood and/or in the skin. Of these 137 animals, 90 (65.7%) and 32 (23.4%) had trypanosome infections respectively in the blood and skin. Fifteen (10.9%) animals had trypanosome infections in both blood and skin snip. Animals from the Campo HAT focus (55.0%) were significantly (X2 = 17.6; P< 0.0001) more infected than those (29.7%) from Bipindi. Trypanosomes of the subgenus Trypanozoon were present in 27.8% of animals while T. vivax, T. congolense forest type and savannah type were detected in 16.5%, 10.3% and 1.4% of animals respectively. Trypanosoma b. gambiense infections were detected in the blood of 7.6% (22/291) of animals. No T. b. gambiense infection was detected in skin. This study highlights the presence of several trypanosome species in the blood and skin of various wild and domestic animals. Skin appeared as an anatomical reservoir for trypanosomes in animals. Despite methodological limitations, pigs, sheep, goats and wild animals were confirmed as potential reservoirs of T. b. gambiense. These animal reservoirs must be considered for the designing of control strategies that will lead to sustainable elimination of HAT.

Tsetse flies infected with trypanosomes in three active Human african trypanosomiasis foci of the Republic of Congo

Tsetse flies infected with trypanosomes in three active Human african trypanosomiasis foci of the Republic of Congo

Population genetics of Glossina fuscipes fuscipes from southern Chad

In Subsaharan Africa, tsetse flies (genus Glossina) are vectors of trypanosomes causing Human African Trypanosomiasis (HAT) and Animal African Trypanosomosis (AAT). Some foci of HAT persist in Southern Chad, where a program of tsetse control was started against the local vector Glossina fuscipes fuscipes in the Mandoul focus in 2014, and in Maro in 2018. Flies were also sampled in 2018 in Timbéri and Dokoutou. We analyzed the population genetics of G. fuscipes fuscipes from the four tsetse-infested zones. The trapping samples were characterized by a strong female biased sex-ratio, except in Timbéri and Dokoutou that had high tsetse densities. Apparent density and effective population density appeared smaller in the main foci of Mandoul and Maro and the average dispersal distance (within the spatial scale of each zone) was as large as or larger than the total length of each respective zone. The genetic signature of a population bottleneck was found in the Mandoul and Timbéri area, suggesting a large ancient interconnected metapopulation that underwent genetic subdivision into small, isolated pockets due to adverse environmental conditions. The long-range dispersal and the existence of genetic outliers suggest a possibility of migration from remote sites such as the Central African Republic in the south (although the fly situation remains unknown there) and/or a genetic signature of recent exchanges. Due to likely isolation, an eradication strategy may be considered for sustainable HAT control in Mandoul focus. Another strategy will probably be required in Maro focus, which probably experiences much more exchanges with its neighbors.

Tsetse Flies Infected with Trypanosomes in Three Active Human African Trypanosomiasis Foci of the Republic of Congo.

Human African trypanosomiasis (HAT) is a neglected tropical disease still endemic in the Republic of Congo. Despite the continuous detection of HAT cases in the country, there is still not enough data on trypanosome infections in tsetse flies, trypanosome species and tsetse flies' species distribution in endemic foci. The present study was intended to fill this gap and improve understanding of trypanosome circulation in three active foci in the centre and south of Congo. Pyramid traps were set in various places in villages to collect tsetse flies both during the rainy and dry seasons. Once collected, tsetse flies were identified using morphological keys. DNA extracted from flies was processed by PCR for species identification and for detection of trypanosome presence. A second PCR was run for different trypanosome species identification. A total of 1291 tsetse flies were collected. The average apparent density of flies per day was 0.043 in Mpouya, 0.73 in Ngabé and 2.79 in Loudima. Glossina fuscipes quazensis was the predominant tsetse fly collected in Ngabé and Mpouya, while Glossina palpalis palpalis was the only tsetse fly found in Loudima. A total of 224 (17.7%) flies were detected infected by trypanosomes; 100 (7.91%) by Trypanosoma congolense savannah, 22 (1.74%) by Trypanosoma congolense forest, 15 (1.19%) by Trypanosoma vivax, 83 (6.56%) by Trypanosoma brucei (s.l.) and 2 (0.16%) undetermined species. No T Trypanosoma brucei gambiense was found. A total of 57 co-infections between T. brucei (s.l.) and T. congolense savannah or T. brucei (s.l.) and T. congolense forest were found only in G. p. palpalis. Loudima recorded the highest number of infected tsetse flies. The study provided updated information on the distribution of tsetse fly populations as well as on Trypanosoma species circulating in tsetse flies in the different active HAT foci in Congo. These data suggested a high risk of potential transmission of animal trypanosomes in these foci, thus stressing the need for active surveillance in this endemic area.

Human Africa Trypanosomiasis in Littoral Region of Cameroon: an Updated With First Evidence on the Circulation of Trypanosoma Brucei Gambiense in Manoka Island

Aims: A survey on Human African Trypanosomiasis (HAT) is essential for prevention and preparedness for epidemics. The objective of this study is to assess the circulation of human trypanosomes and their vectors across quiescent HAT foci of the Littoral Region of Cameroon. &#x0D; Methodology: A descriptive study on the presence of Trypanosoma brucei gambiense and its potential vectors was carried out in Youpwe, Yabassi, Sodiko, Manoka island and Cape-Cameroon Island from February to April 2022.Tsetse flies collected from the five selected locations using pyramidal traps, were first classified by species according to their morphology, then by sub-species with Polymerase Chain Reaction Diagnostic (PCR-Diag). Trypanosoma species and sub-species were subsequently identified and genotyped using a Nested PCR.&#x0D; Results: Glossina palpalis palpalis was the unique tsetse subspecies recorded across the five locations. The tsetse infection rate by Trypanosoma ssp. varied between 5.35% in Cape-island and 35.71% in Manoka island. Three Trypanosoma species were detected: T. brucei s.l. 32/500 (6.4%), T. congolense 15/500 (3.0%), and T. vivax 8/500 (1.6%). The sub-species T. b. gambiense responsible for HAT was detected in tsetse flies from Manoka (2/150: 1.33%), whereas T. congolense consisted of T. congolense “forest” and T. congolense “savannah” types.&#x0D; Conclusion: The presence of T. b. gambiense and T. congolense sub-species in Manoka and nearby suggests residual circulation of human and animal trypanosomes in quiescent HAT foci of the littoral region of Cameroon.

A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR.

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model.

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n = 10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0-15, (b) acute: 16-30, (c) sub-acute: 31-45 and (d) chronic: 46-60 dpi. Other virulence biomarkers identified included: pre-patent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B), pentamidine, diminazene aceturate and suramin, using mice groups (n = 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. A cure rate of at least 80% was achieved for all test isolates with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr clones from a single HAT focus and confirms that this variations is not a significant determinant of isolate sensitivity to anti-trypanosomal drugs.

Experiences of the one-health approach by the Uganda Trypanosomiasis Control Council and its secretariat in the control of zoonotic sleeping sickness in Uganda

Elimination of sleeping sickness from endemic countries like Uganda is key if the affected communities are to exploit the potential of the available human and livestock resources (production and productivity). Trypanosoma brucei rhodesiense, the parasite that causes acute sleeping sickness in humans, is transmitted by tsetse flies and co-exists in non-human animal reservoirs. Uganda by Act of Parliament in 1992 decided to handle the complex approach to control of sleeping sickness and animal trypanosomiasis by establishing the Uganda Trypanosomiasis Control Council (UTCC) and its secretariat the Coordinating Office for the Control of Trypanosomiasis in Uganda (COCTU). The Institutional arrangement aimed to promote engagement with key stakeholders across nine key ministries and the community, all vital for control of zoonotic sleeping sickness, creating a One Health platform, long before such practice was common. From 2006, approaches by the Public Private Partnership, Stamp Out Sleeping Sickness (SOS) have required involvement of stakeholders in the promotion of insecticide treated cattle as live tsetse baits, targeting elimination of zoonotic sleeping sickness. Experiences in promoting sustainability of these interventions have been captured in this study as part of the Tackling Infections to Benefit Africa (TIBA) partnership. Meeting transcripts, focus group discussions and questionnaires were used to collect data from the different stakeholders involved in a rapid impact live bait study over 12 months from Dec 2017. The study provides unprecedented insights into the stakeholders involved in the application of a One health approach for control of zoonotic sleeping sickness across the most important active human African trypanosomiasis focus in East Africa. This unique study is fundamental in guiding multi-stakeholder engagement if the goal to eliminate zoonotic sleeping sickness is to be realised. A major challenge is timely feedback to the community as regards human and animal disease status; rapid diagnostic services that can be delivered from facilities established in close proximity to the affected communities and well equipped in-country reference laboratories are key to delivering effective control and best One Health Approach.

Delivering \u2018tiny targets\u2019 in a remote region of southern Chad: a cost analysis of tsetse control in the Mandoul sleeping sickness focus

BackgroundSince 2012, the World Health Organisation and the countries affected by the Gambian form of human African trypanosomiasis (HAT) have been committed to eliminating the disease, primarily through active case-finding and treatment. To interrupt transmission of Trypanosoma brucei gambiense and move more rapidly towards elimination, it was decided to add vector control using ‘tiny targets’. Chad’s Mandoul HAT focus extends over 840 km2, with a human population of 39,000 as well as 14,000 cattle and 3000 pigs. Some 2700 tiny targets were deployed annually from 2014 onwards.MethodsA protocol was developed for the routine collection of tsetse control costs during all field missions. This was implemented throughout 2015 and 2016, and combined with the recorded costs of the preliminary survey and sensitisation activities. The objective was to calculate the full costs at local prices in Chad. Costs were adjusted to remove research components and to ensure that items outside the project budget lines were included, such as administrative overheads and a share of staff salaries.ResultsTargets were deployed at about 60 per linear km of riverine tsetse habitat. The average annual cost of the operation was USD 56,113, working out at USD 66.8 per km2 protected and USD 1.4 per person protected. Of this, 12.8% was an annual share of the initial tsetse survey, 40.6% for regular tsetse monitoring undertaken three times a year, 36.8% for target deployment and checking and 9.8% for sensitisation of local populations. Targets accounted for 8.3% of the cost, and the cost of delivering a target was USD 19.0 per target deployed.ConclusionsThis study has confirmed that tiny targets provide a consistently low cost option for controlling tsetse in gambiense HAT foci. Although the study area is remote with a tsetse habitat characterised by wide river marshes, the costs were similar to those of tiny target work in Uganda, with some differences, in particular a higher cost per target delivered. As was the case in Uganda, the cost was between a quarter and a third that of historical target operations using full size targets or traps.

Trypanosome infections in naturally infected horses and donkeys of three active sleeping sickness foci in the south of Chad

BackgroundEquine trypanosomiases are complex infectious diseases with overlapping clinical signs defined by their mode of transmission. Despite their economic impacts, these diseases have been neglected by the scientific community, the veterinary authorities and regulatory organizations. To fill the observed knowledge gap, we undertook the identification of different trypanosome species and subspecies naturally infecting horses and donkeys within the Chadian sleeping sickness focus. The objective of the study was to investigate the potential role of these domestic animals as reservoirs of the human-infective Trypanosoma brucei gambiense.MethodBlood samples were collected from 155 donkeys and 131 horses in three human African trypanosomiasis (HAT) foci in Chad. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) test were used to search for trypanosome infections. DNA was extracted from each blood sample and different trypanosome species and subspecies were identified with molecular tools.ResultsFrom 286 blood samples collected, 54 (18.9%) and 36 (12.6%) were positive for RDT and CTC, respectively. PCR revealed 101 (35.3%) animals with trypanosome infections. The Cohen’s kappa coefficient used to evaluate the concordance between the diagnostic methods were low; ranging from 0.09 ± 0.05 to 0.48 ± 0.07. Trypanosomes of the subgenus Trypanozoon were the most prevalent (29.4%), followed by T. congolense forest (11.5%), Trypanosoma congolense savannah (4.9%) and Trypanosoma vivax (4.5%). Two donkeys and one horse from the Maro HAT focus were found with T. b. gambiense infections. No significant differences were observed in the infection rates of different trypanosomes between animal species and HAT foci.ConclusionsThis study revealed several trypanosome species and subspecies in donkeys and horses, highlighting the existence of AAT in HAT foci in Chad. The identification of T. b. gambiense in donkeys and horses suggests considering these animals as potential reservoir for HAT in Chad. The presence of both human-infective and human non-infective trypanosomes species highlights the need for developing joint control strategies for HAT and AAT.

Molecular identification of Trypanosoma brucei gambiense in naturally infected pigs, dogs and small ruminants confirms domestic animals as potential reservoirs for sleeping sickness in Chad.

Human African trypanosomiasis (HAT) has been targeted for zero transmission to humans by 2030. Animal reservoirs of gambiense-HAT could jeopardize these elimination goals. This study was undertaken to identify potential host reservoirs for Trypanosoma brucei gambiense by detecting its natural infections in domestic animals of Chadian HAT foci. Blood samples were collected from 267 goats, 181 sheep, 154 dogs, and 67 pigs. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) were performed to search for trypanosomes. DNA was extracted from the buffy coat, and trypanosomes of the subgenus Trypanozoon as well as T. b. gambiense were identified by PCR. Of 669 blood samples, 19.4% were positive by RDT and 9.0% by CTC. PCR revealed 150 animals (22.4%) with trypanosomes belonging to Trypanozoon, including 18 (12%) T. b. gambiense. This trypanosome was found in all investigated animal species and all HAT foci. Between animal species or villages, no significant differences were observed in the number of animals harboring T. b. gambiense DNA. Pigs, dogs, sheep and goats appeared to be potential reservoir hosts for T. b. gambiense in Chad. The identification of T. b. gambiense in all animal species of all HAT foci suggests that these animals should be considered when designing new control strategies for sustainable elimination of HAT. Investigations aiming to decrypt their specific role in each epidemiological setting are important to achieve zero transmission of HAT.

Genetic diversity and population structure of Glossina morsitans morsitans in the active foci of human African trypanosomiasis in Zambia and Malawi.

The tsetse fly, Glossina morsitans morsitans, is a significant problem in Zambia and Malawi. It is the vector for the human infective parasite Trypanosoma brucei rhodesiense, which causes human African trypanosomiasis, and various Trypanosoma species, which cause African animal trypanosomiasis. Understanding the genetic diversity and population structure of G. m. morsitans is the basis of elucidating the connectivity of the tsetse fly populations, information that is essential in implementing successful tsetse fly control activities. This study conducted a population genetic study using partial mitochondrial cytochrome oxidase gene 1 (CO1) and 10 microsatellite loci to investigate the genetic diversity and population structure of G. m. morsitans captured in the major HAT foci in Zambia and Malawi. We have included 108 and 99 G. m. morsitans samples for CO1 and microsatellite analyses respectively. Our results suggest the presence of two different genetic clusters of G. m. morsitans, existing East and West of the escarpment of the Great Rift Valley. We have also revealed genetic similarity between the G. m. morsitans in Kasungu National Park and those in the Luangwa river basin in Zambia, indicating that this population should also be included in this historical tsetse belt. Although further investigation is necessary to illustrate the whole picture in East and Southern Africa, this study has extended our knowledge of the population structure of G. m. morsitans in Southern Africa.

A mathematical model for evaluating the role of trypanocide treatment of cattle in the epidemiology and control of Trypanosoma brucei rhodesiense and T. b. gambiense sleeping sickness in Uganda

BackgroundHuman and animal African trypanosomiases impose a large economic and health burden in their endemic regions. Large strides have been made in the control of human African trypanosomiasis (HAT), yet these efforts have largely focused on the non-zoonotic form of the disease. Using a mathematical model with a 10 year time horizon, we demonstrate the role of the cattle treatment with trypanocides in the epidemiology of zoonotic and non-zoonotic HAT in Uganda, and its potential implications on elimination and eradication of the disease. Methodology/principal findingsWe created two compartmental, deterministic models, each comprised of three sub-models: humans, the tsetse fly vector (Glossina fuscipes fuscipes), and cattle. We applied these models to two HAT foci in Uganda: the gambiense (chronic, non-zoonotic) form in the Northern Region, and the rhodesiense (acute, zoonotic) form in the Eastern Region. Parameters were derived from prior literature or assumed. In both foci we assumed G. fuscipes fuscipes expresses zoophilic biting behavior.With trypanocide treatment of cattle administered every 3 months, treatment in stage I (representing engagement in active or passive surveillance) had a larger impact on HAT burden than cattle treatment coverage. However increasing cattle treatment coverage allowed for further reduction in prevalence in both foci. Using these model parameters, our estimated R0 suggests humans cannot alone sustain the HAT epidemic in Uganda. Conclusions/significanceEven in the absence of zoonotic transmission, loss of a preferred tsetse host species can affect HAT risk. Thus One Health strategies which integrate HAT and animal African trypanosomiasis control may improve the timeliness and sustainability of gHAT and rHAT elimination and eradication in Uganda. Furthermore, such strategies reduce the burden of a high-morbidity livestock disease of economic importance.

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.

Variations of Glossina sp. and trypanosome species frequency within different habitats in a sleeping sickness focus, Gabon.

Knowledge of the infectious status of the Glossina is an indicator of risk of resurgence of Human African Trypanosomiasis (HAT). Environmental conditions have an impact on the density and diversity of both vector and Trypanosoma. The aim of the study was to determine the diversity and the infection rate of Glossina as well as the diversity of trypanosome species within habitats of an old HAT focus, in Gabon. Glossina were captured in September 2012 in three ecological sites. Vavoua traps were installed for twelve days. All captured flies were identified. Glossina were selected for trypanosome identification by Polymerase Chain Reaction. 1178 Glossina were captured: 55.8% in degraded forest, 28.9% in flood area and 15.4% in secondary forest. Glossina fusca congolensis (37%) and G.palpalis palpalis (36.4%) were the most abundant vector species. G. fusca congolensis was predominant in secondary forest and in flood area, while in degraded forest, it was G.palpalis palpalis. Trypanosoma infection rate was 30.7%, 42% in secondary forest, 32% in degraded forest and 18% in flood area. Trypanosoma congolense savannah was the main species detected (18.7%) followed by T.brucei brucei (10.7%) and T. brucei gambiense (4%). T. congolense savannah type was predominant in the secondary forest and in degraded forest (66.7% versus 55.5%). Glossina density and trypanosome infection rate varied according to the habitat within HAT focus. The density of tsetse was the highest in degraded forest while the infection rate was highest in secondary forest. Continuous disease surveillance and control measures are needed.

HLA-G 3’UTR 14 bp Insertion Is Associated with a Decreased Risk of Developing Human African Trypanosomiasis in the C&amp;#244;te d’Ivoire Population

Human African trypanosomiasis (HAT), or sleeping sickness, caused by Trypanosoma brucei gambiense, is associated with diverse clinical outcomes. Host’s genetic factors involved in immunity are potential factors that can regulate infection. Genetic polymorphisms within HLA-G could influence the level of HLA-G expression and therefore play a critical role in infection outcomes. The goal of our study was to investigate the association of 14 bp Indel HLA-G polymorphism with the susceptibility/resistance to HAT. DNA samples were collected from 119 cases, 221 controls and 43 seropositive individuals living in Ivorian HAT foci. The 14 bp Indel polymorphism was determined by PCR. Homozygous individuals for 14 bp insertion had a lower risk of progressing to active HAT (p = 0.012, OR = 0.27, 95% CI: 0.09 - 0.8). Moreover, the frequency of 14 bp insertion homozygous genotype was higher in the seropositive group (11%) than in the HAT cases group (3%) (p = 0.043, OR = 0.27, 95% CI: 0.07 - 0.99), which suggested a protective effect of 14 bp insertion homozygous genotype. Genetic polymorphisms in HLA-G may be associated with a variable risk to develop HAT. The 14 bp insertion appears to favour the occurrence of long-lasting T. b. gambiense latent infections.

Sleeping sickness in the historical focus of forested Guinea: update using a geographically based method.

In 2017, 1447 new cases of Human African Trypanosomiasis (HAT) were reported, which reflects considerable progress towards the World Health Organisation’s target of eliminating HAT as a public health problem by 2020. However, current epidemiological data are still lacking for a number of areas, including historical HAT foci. In order to update the HAT situation in the historical focus of forested Guinea, we implemented a geographically based methodology: Identification of Villages at Risk (IVR). The methodology is based on three sequential steps: Desk-based IVR (IVR-D), which selects villages at risk of HAT on the basis of HAT archives and geographical items; Field-based IVR (IVR-F), which consists in collecting additional epidemiological and geographical information in the field in villages at risk; and to be Medically surveyed IVR (IVR-M), a field data analysis through a Geographic Information System (GIS), to compile a list of the villages most at risk of HAT, suitable to guide active screening and passive surveillance. In an area of 2385 km2 with 1420,530 inhabitants distributed in 1884 settlements, 14 villages with a population of 11,236 inhabitants were identified as most at risk of HAT and selected for active screening. Although no HAT cases could be confirmed, subjects that had come into contact with Trypanosoma brucei gambiense were identified and two sentinel sites were chosen to implement passive surveillance. IVR, which could be applied to any gambiense areas where the situation needs to be clarified, could help to reach the objective of HAT elimination.

Detection of Wolbachia and different trypanosome species in Glossina palpalis palpalis populations from three sleeping sickness foci of southern Cameroon

BackgroundAfrican trypanosomiases are caused by trypanosomes that are cyclically transmitted by tsetse. Investigations aiming to generate knowledge on the bacterial fauna of tsetse have revealed distinct symbiotic microorganisms. Furthermore, studies addressing the tripartite association between trypanosomes-tsetse-symbionts relationship have so far been contradictory. Most studies included Sodalis glossinudius and, consequently, the association involving Wolbachia is poorly understood. Understanding the vectorial competence of tsetse requires decrypting these tripartite associations. In this study, we identified Wolbachia and trypanosomes in Glossina palpalis palpalis from three human African trypanosomiasis (HAT) foci in southern Cameroon.MethodsTsetse flies were captured with pyramidal traps in the Bipindi, Campo and Fontem HAT foci. After morphological identification, DNA was extracted from whole tsetse flies and Wolbachia and trypanosomes were identified by PCR using different trypanosome-specific primers and two Wolbachia-specific primers (Wolbachia surface protein and 16S rRNA genes). Statistical analyses were performed to compare the trypanosome and Wolbachia infection rates between villages and different foci and to look for an association between these microorganisms.ResultsFrom a total of 2122 tsetse flies, 790 G. p. palpalis were analyzed. About 25.32% of flies hosted Wolbachia and 31.84% of non-teneral flies were infected by at least one trypanosome species. There was no significant difference between the global Wolbachia prevalence revealed by the two markers while some differences were observed between HAT foci. From 248 G. p. palpalis with trypanosome infections, 62.90% were with T. vivax, 34.68% with T. congolense forest, 16.13% with T. brucei (s.l.) and 2.42% with T. congolense savannah. Of all trypanosome-infected flies, 29.84% hosted Wolbachia and no association was observed between Wolbachia and trypanosome co-infections.ConclusionsThis study revealed differences in the prevalence of Wolbachia and trypanosomes in G. p. palpalis according to HAT foci. The use of only one marker has underestimated the prevalence of Wolbachia, thus more markers in subsequent studies may improve its detection. The presence of Wolbachia seems to have no impact on the establishment of trypanosomes in G. p. palpalis. The tripartite association between tsetse, Wolbachia and trypanosomes varies according to studied areas. Studies aiming to evaluate the genetic polymorphism of Wolbachia and its density in tsetse flies could help to better understand this association.