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Background: Detection of spliced leader (SL)-RNA allows sensitive diagnosis of gambiense human African trypanosomiasis (HAT). We investigated its diagnostic performance for treatment outcome assessment., Methods: Blood and cerebrospinal fluid (CSF) from a consecutive series of 97 HAT patients, originating from the Democratic Republic of the Congo, were prospectively collected before treatment with acoziborole, and during 18 months of longitudinal follow-up after treatment. For treatment outcome assessment, SL-RNA detection was compared with microscopic trypanosome detection and CSF white blood cell count. The trial was registered under NCT03112655 in clinicaltrials.gov., Findings: Before treatment, respectively 94.9% (92/97; CI 88.5-97.8%) and 67.7% (65/96; CI 57.8-76.2%) HAT patients were SL-RNA positive in blood or CSF. During follow-up, one patient relapsed with trypanosomes observed at 18 months, and was SL-RNA positive in blood and CSF at 12 months, and CSF positive at 18 months. Among cured patients, one individual tested SL-RNA positive in blood at month 12 (Specificity 98.9%; 90/91; CI 94.0-99.8%) and 18 (Specificity 98.9%; 88/89; CI 93.9-99.8%)., Interpretation: SL-RNA detection for HAT treatment outcome assessment shows ≥98.9% specificity in blood and 100% in CSF, and may detect relapses without lumbar puncture., Funding: The DiTECT-HAT project is part of the EDCTP2 programme, supported by Horizon 2020, the European Union Funding for Research and Innovation (grant number DRIA-2014-306-DiTECT-HAT)., Competing Interests: Declaration of interests The authors have declared that no competing interests exist., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Spliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF., Methodology/principal Findings: Blood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood (78/97 positive, 80.4%, p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values., Conclusions/significance: Detection of SL-RNA in blood and CSF allows sensitive demonstration of active gambiense HAT infection, even if trypanosomes remain undetectable in blood or lymph. As this condition often occurs in treatment failures, SL-RNA detection in blood and CSF for early detection of relapses after treatment deserves further investigation., Trial Registration: This study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier: NCT03112655)., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma brucei gambiense causes human African trypanosomiasis (HAT). Between 1990 and 2015, almost 440000 cases were reported. Large-scale screening of populations at risk, drug donations, and efforts by national and international stakeholders have brought the epidemic under control with <2200 cases in 2016. The World Health Organization (WHO) has set the goals of gambiense-HAT elimination as a public health problem for 2020, and of interruption of transmission to humans for 2030. Latent human infections and possible animal reservoirs may challenge these goals. It remains largely unknown whether, and to what extend, they have an impact on gambiense-HAT transmission. We argue that a better understanding of the contribution of human and putative animal reservoirs to gambiense-HAT epidemiology is mandatory to inform elimination strategies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Trypanosoma congolense and Trypanosoma vivax are major species that infect cattle in north-eastern KwaZulu-Natal (KZN), South Africa. Of the two genetically distinct types of T. congolense, Savannah and Kilifi sub-groups, isolated from cattle and tsetse flies in KZN, the former is more prevalent and thought to be responsible for African animal trypanosomosis outbreaks in cattle. Furthermore, variation in pathogenicity within the Savannah sub-group is ascribed to strain differences and seems to be related to geographical locations. The objective of the present study was to compare the virulence of T. congolense strains isolated from African buffaloes (Syncerus caffer) inside Hluhluwe-iMfolozi Park, and from cattle on farms near wildlife parks (&lt; 5 km), to isolates from cattle kept away (&gt; 10 km) from parks. To obtain T. congolense isolates, blood of known parasitologically positive cattle or cattle symptomatically suspect with trypanosomosis, as well as isolates from buffaloes kept inside Hluhluwe-iMfolozi Park were passaged in inbred BALB/c mice. A total of 26 T. congolense isolates were obtained: 5 from buffaloes, 13 from cattle kept near parks and 8 from cattle distant from parks. Molecular characterisation revealed 80% and 20% of isolates to belong to T. congolense Savannah and Kilifi, respectively. To compare virulence, each isolate was inoculated into a group of six mice. No statistical differences were observed in the mean pre-patent period, maximum parasitaemia or drop in packed cell volume (PCV). Significant differences were found in days after infection for the drop in PCV, the patent period and the survival time. These differences were used to categorise the isolates as being of high, moderate or low virulence. Based on the virulence, 12 of 26 (46%) isolates were classified as highly virulent and 27% each as either of moderate or of low virulence. Whilst 11 of 12 high virulent strains were from buffaloes or cattle near the park, only 1 of 7 low virulent strains was from these animals. All the Kilifi T. congolense types were less virulent than the Savannah types. These results confirmed the higher virulence of T. congolense Savannah type compared to Kilifi type and indicated the prevalence of highly virulent strains to be higher in wildlife parks and in cattle near the parks than on farms further away. The geographical location of these strains in relation to the wildlife parks in the area was discussed.

Tsetse-transmitted trypanosomosis (nagana) has been the cause of stock losses in the recent past and still presents a major problem to livestock owners in certain areas of KwaZulu- Natal, South Africa. Over 10 000 cattle mortalities were reported in the 1990 nagana outbreak. Although information on the distribution and abundance of the tsetse flies Glossina brevipalpis and Glossina austeni in KwaZulu-Natal exists, data on their vector competence are lacking. This study aimed to determine the rate of natural Trypanosoma congolense infection by field-collected as well as colony-reared flies of these species. A total of 442 field-collected G. brevipalpis and 40 G. austeni flies were dissected immediately after collection to determine their infection rates, whilst 699 G. brevipalpis and 49 G. austeni flies were fed on susceptible animals in 10 and four batches, respectively, for use in xenodiagnosis experiments. Teneral colony flies were fed on infected animals and dissected 21 days post infection to confirm their infectivity testing. Glossina austeni harboured 8% immature and mature infections. In G. brevipalpis, the infection with the immature stages was lower (1%) and no mature infections were observed. Although all four batches of G. austeni transmitted T. congolense to four susceptible animals, no transmission resulted from 10 batches of G. brevipalpis fed on susceptible cattle. Colony-derived G. austeni (534) and G. brevipalpis (882) were fed on four bovines infected with different T. congolense isolates. Both G. austeni and G. brevipalpis acquired trypanosome infection from the bovines, with immature infection ranges of 20% - 33% and 1% - 4%, respectively. Parasites, however, only matured in G. austeni (average = 4%). Glossina austeni plays a larger role in the epidemiology of animal trypanosomosis in KwaZulu-Natal than G. brevipalpis and therefore more focus should be aimed at the former when control measures are implemented.

Trypanosoma congolense strains have been shown to differ in their virulence both between subgroups and within the Savannah subgroup between strains. This review revisits these findings and complements them with information on the virulence of T. congolense Savannah subgroup strains isolated from cattle (domestic transmission cycle) in different geographical areas and of strains isolated in protected areas where trypanotolerant wildlife species are the reservoir of the trypanosomes (sylvatic transmission cycle). The virulence of a total of 62 T. congolense Savannah subgroup strains (50 domestic and 12 sylvatic), determined using a standard protocol in mice, was compared. Virulence varied substantially between strains with, depending on the strain, the median survival time of infected mice varying from five to more than sixty days. The proportion of highly virulent strains (median survival time <10 days) was significantly (P = 0·005) higher in strains from the sylvatic transmission cycle. The analysis highlights repercussions of the domestication of the trypanosomiasis transmission cycle that may have to be taken in consideration in the development of trypanosomiasis control strategies., (© 2011 Blackwell Publishing Ltd.)

In order to verify whether chronic trypanosomal infections can affect the transmissibility of Trypanosoma congolense by tsetse flies, batches of Glossina morsitans morsitans were fed on mice infected with the same level of parasitemia (10(8.1)trypanosomes/ml of blood) of two cloned low virulent T. congolense strains during the acute and the chronic phases of infection. Results showed that the proportions of procyclic infections in flies that were fed during the acute phase (32.6% and 45.4% for isolates 1 and 2, respectively) were significantly higher (chi(2)=4.7, P<0.05 and chi(2)=23.7, P<0.0001, respectively) compared to the proportions of procyclic infections of flies fed during the chronic phase of infection (18.8% and 14.9% for isolates 1 and 2, respectively). Similarly the proportions of metacyclic infections in flies fed during the acute phase (32.6% and 45.4% for isolates 1 and 2, respectively) were significantly higher (chi(2)=6.3, P<0.05 and chi(2)=23.7, P<0.0001, respectively) compared to the proportions of metacyclic infections in flies fed during the chronic phase of infection (16.8% and 14.9% for isolates 1 and 2, respectively). No significant difference was found in the maturation rate of both strains during the acute phase compared to the chronic phase of infection (P>0.05). The results of this study suggest that T. congolense loses part of its transmissiblity by tsetse flies during the chronic phase of infection., (Copyright 2009. Published by Elsevier B.V.)

In order to verify whether the low impact on livestock production in endemic areas is related to a low number of trypanosome strains circulating in livestock, 37 Trypanosoma congolense isolates collected from cattle in 11 sites in an endemic trypanosomiasis area in Eastern Zambia were characterised for genotype variability using a modified amplified fragment length polymorphism technique (AFLP). Isolates were further cloned to evaluate the occurrence of mixed infections in individuals. The results obtained revealed a high genotype diversity (94.6%) among these isolates. Apart from one site, all isolates gave different AFLP profiles in each of the sites. When clones were compared, three (8%) of the 37 isolates had mixed infections. These results indicate the circulation of a high number of strains in this trypanosomiasis endemic area despite the low impact the disease has on livestock production.

The aim of this study was to assess the existence of possible cross-protection between Trypanosoma congolense strains of low and extreme virulence circulating in the same trypanosomiasis focus. Groups of six mice were infected using one of three strains of low virulence and challenged with one of three strains of extreme virulence. A group of six mice was used as control for each strain of low and extreme virulence. The results showed that mice infected with one of the strains of extreme virulence developed high parasitaemia and a significant drop of the PCV compared to mice infected with a strain of low virulence and challenged with one of the strains of extreme virulence. With an exception of one strain of extreme virulence (strain F), the survival time of mice infected with the strains of extreme virulence was shorter compared to mice infected with strains of low virulence and subsequently challenged with a strain of extreme virulence. These results suggest that in an area where trypanosomes of various virulence profiles circulate, livestock infected with T. congolense strains of low virulence can be protected against the adverse effects of extremely virulent T. congolense strains.

Two groups of Trypanosoma congolense isolates collected from cattle in 1996 (n=39) and 2003 (n=38) in the Eastern Province of Zambia were analyzed by BclI-PCR-RFLP to assess the evolution of diminazene aceturate (DA) resistance over a period of seven years. The results show a significant increase of DA resistance in this relatively short period of time. In 1996, among the 39 isolates, 61.5% were found sensitive, 12.8% resistant and 25.7% had a mixed BclI-PCR-RFLP profile. In 2004, among the 38 isolates, 10.5% were found sensitive, 63.2% were resistant and 26.3% showed a mixed BclI-PCR-RFLP profile. In vivo tests in mice showed that isolates with a sensitive or mixed RFLP profile were sensitive to DA whereas isolates with a resistant RFLP profile were resistant. Since there are no indications that the drug pressure has increased between 1996 and 2003, it is suggested that genetic exchange of resistance genes might explain the increased frequency of resistance to DA.

The virulence of 31 genetically different Trypanosoma congolense strains belonging to the Savannah subgroup and isolated from cattle at 11 sites in a trypanosomiasis endemic area of eastern Zambia was compared. Virulence testing, done in OF1 mice, revealed three virulence categories. Strains were considered extremely virulent when the median survival time ranged between 5 and 9 days. Moderately virulent strains had a median survival time between 10 and 30 days and low virulence, more than 30 days. For each strain, the prepatent period was determined and the PCV of the infected animals was measured at regular intervals. A total of six (19.4%) strains belonged to the extremely virulent category with a short prepatent period (mean 2.3+/-0.3 days), high parasitaemia, decline in PCV of 15.6+/-1.1% during the first 7 days p.i. and a short median survival time (mean 6 days). The remainder of the strains belonged to the moderate (13 strains) or low (12 strains) virulence categories with median survival times of 13 and 60 days, respectively. They had longer prepatent periods (means 3.2+/-1.6 days and 3.5+/-1.6 days for moderately virulent and strains with low virulence, respectively) and the decline in PCV was less steep (decline of 14.2+/-0.6 and 9.7+/-0.6% during the first 7 days of infection with moderately virulent strains and strains with low virulence, respectively). Extremely virulent strains were isolated from cattle at four sampling sites with 60% of the cattle from one sampling site harbouring such extremely virulent strains. Results from this study demonstrated substantial differences in the virulence of T. congolense strains of the Savannah subgroup, isolated in one geographic area from a single host species. On the assumption that information on virulence obtained from tests in mice can be extrapolated to cattle, the high proportion of strains with low to moderate virulence is thought to be attributed to the important role of susceptible cattle as reservoirs of trypanosomes in the study area and the ensuing selection against extremely virulent strains.