RESUMEN
Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.
Asunto(s)
Sistema Digestivo/parasitología , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Trypanosoma/aislamiento & purificación , Moscas Tse-Tse/parasitología , Animales , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: A critical step before treatment of human African trypanosomiasis (HAT) is the correct staging of the disease. As late stage is established when trypanosomes cross the blood-brain barrier and invade the central nervous system, we hypothesized that matrix metalloproteinases and cell adhesion molecules could indicate, alone or in combination, the disease progression from the first to the second stage of HAT. METHODS: We measured the levels of MMP-2, MMP-9, ICAM-1, VCAM-1 and E-selectin in the cerebrospinal fluid (CSF) of 63 Trypanosoma brucei gambiense-infected patients (15 stage 1 and 48 stage 2). Staging was based on counting of white blood cells (WBC) and/or parasite detection in CSF. Concentrations were obtained either by ELISA or multiplex bead suspension assays, and results were compared with three known HAT staging markers (CXCL10, CXCL8 and H-FABP). RESULTS: ICAM-1 and MMP-9 accurately discriminated between stage 1 and stage 2 patients with HAT with 95% sensitivity (SE) for 100% specificity (SP), which was better than CXCL10 (93% SE for 100% SP), one of the most promising known markers. Combination of ICAM-1 and MMP-9 with H-FABP provided a panel that resulted in 100% of SE and SP for staging HAT. CONCLUSIONS: ICAM-1 and MMP-9, alone or in combination, appeared as powerful CSF staging markers of HAT. Final validation of all newly discovered staging markers on a large multi-centric cohort including both forms of the disease as well as patients with others infections should be performed.
Asunto(s)
Molécula 1 de Adhesión Intercelular/líquido cefalorraquídeo , Metaloproteinasa 9 de la Matriz/líquido cefalorraquídeo , Tripanosomiasis Africana/diagnóstico , Adolescente , Adulto , Anciano , Biomarcadores/líquido cefalorraquídeo , Moléculas de Adhesión Celular/líquido cefalorraquídeo , Infecciones Protozoarias del Sistema Nervioso Central/líquido cefalorraquídeo , Quimiocinas/líquido cefalorraquídeo , Progresión de la Enfermedad , Métodos Epidemiológicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trypanosoma brucei gambiense/aislamiento & purificación , Tripanosomiasis Africana/líquido cefalorraquídeo , Adulto JovenRESUMEN
BACKGROUND: Glossina fuscipes fuscipes, a riverine species of tsetse, is the main vector of both human and animal trypanosomiasis in Uganda. Successful implementation of vector control will require establishing an appropriate geographical scale for these activities. Population genetics can help to resolve this issue by characterizing the extent of linkage among apparently isolated groups of tsetse. METHODOLOGY/PRINCIPAL FINDINGS: We conducted genetic analyses on mitochondrial and microsatellite data accumulated from approximately 1000 individual tsetse captured in Uganda and neighboring regions of Kenya and Sudan. Phylogeographic analyses suggested that the largest scale genetic structure in G. f. fuscipes arose from an historical event that divided two divergent mitochondrial lineages. These lineages are currently partitioned to northern and southern Uganda and co-occur only in a narrow zone of contact extending across central Uganda. Bayesian assignment tests, which provided evidence for admixture between northern and southern flies at the zone of contact and evidence for northerly gene flow across the zone of contact, indicated that this structure may be impermanent. On the other hand, microsatellite structure within the southern lineage indicated that gene flow is currently limited between populations in western and southeastern Uganda. Within regions, the average F(ST) between populations separated by less than 100 km was less than approximately 0.1. Significant tests of isolation by distance suggested that gene flow is ongoing between neighboring populations and that island populations are not uniformly more isolated than mainland populations. CONCLUSIONS/SIGNIFICANCE: Despite the presence of population structure arising from historical colonization events, our results have revealed strong signals of current gene flow within regions that should be accounted for when planning tsetse control in Uganda. Populations in southeastern Uganda appeared to receive little gene flow from populations in western or northern Uganda, supporting the feasibility of area wide control in the Lake Victoria region by the Pan African Tsetse and Trypanosomiasis Eradication Campaign.
Asunto(s)
Vectores de Enfermedades , Moscas Tse-Tse/clasificación , Moscas Tse-Tse/genética , Animales , Análisis por Conglomerados , ADN Mitocondrial/genética , Flujo Génico , Geografía , Humanos , Kenia , Repeticiones de Microsatélite , Filogenia , Sudán , UgandaRESUMEN
BACKGROUND: Glossina fuscipes fuscipes is the major vector of human African trypanosomiasis, commonly referred to as sleeping sickness, in Uganda. In western and eastern Africa, the disease has distinct clinical manifestations and is caused by two different parasites: Trypanosoma brucei rhodesiense and T. b. gambiense. Uganda is exceptional in that it harbors both parasites, which are separated by a narrow 160-km belt. This separation is puzzling considering there are no restrictions on the movement of people and animals across this region. METHODOLOGY AND RESULTS: We investigated whether genetic heterogeneity of G. f. fuscipes vector populations can provide an explanation for this disjunct distribution of the Trypanosoma parasites. Therefore, we examined genetic structuring of G. f. fuscipes populations across Uganda using newly developed microsatellite markers, as well as mtDNA. Our data show that G. f. fuscipes populations are highly structured, with two clearly defined clusters that are separated by Lake Kyoga, located in central Uganda. Interestingly, we did not find a correlation between genetic heterogeneity and the type of Trypanosoma parasite transmitted. CONCLUSIONS: The lack of a correlation between genetic structuring of G. f. fuscipes populations and the distribution of T. b. gambiense and T. b. rhodesiense indicates that it is unlikely that genetic heterogeneity of G. f. fuscipes populations explains the disjunct distribution of the parasites. These results have important epidemiological implications, suggesting that a fusion of the two disease distributions is unlikely to be prevented by an incompatibility between vector populations and parasite.
Asunto(s)
Insectos Vectores/genética , Moscas Tse-Tse/genética , Animales , ADN Mitocondrial/genética , Variación Genética/genética , Genética de Población , Humanos , Repeticiones de Microsatélite/genética , Tripanosomiasis Africana/transmisión , Uganda/epidemiologíaRESUMEN
OBJECTIVE: To assess the application of allele-specific PCR (AS-PCR) as a fast, cheap and reliable method for detecting mutant TbAT1 associated with melarsoprol relapse in Trypanosoma brucei gambiense isolates from northwest Uganda. METHODS: A total of 105 trypanosome isolates were analysed using SfaN1 restriction fragment length polymorphism (RFLP) and AS-PCR, the former used as the gold standard. Sensitivity, specificity, positive and negative predictive values of AS-PCR as well as agreement between the tests were determined. RESULTS: Eleven trypanosome isolates had mutant TbAT1 while 94 exhibited the wild-type TbAT1 genes. There was a highly significant agreement between SfaN1 RFLP and AS-PCR with kappa and intra-class correlation values of 1.0. The sensitivity and specificity of AS-PCR were both 100%, while the positive and negative predictive values were found to be equal to 1.0. Cost and time analyses were performed and AS-PCR was 4.3 times cheaper than SfaN1 RFLP, in addition to the less time required for its execution. CONCLUSION: AS-PCR should be the test of choice for screening for mutant TbAT1 in the ever-increasing numbers of field trypanosome isolates.
Asunto(s)
Resistencia a Medicamentos/genética , Melarsoprol/farmacología , Proteínas de Transporte de Nucleósidos/genética , Reacción en Cadena de la Polimerasa/métodos , Tripanocidas/farmacología , Trypanosoma brucei gambiense/genética , Alelos , Animales , Bovinos , Polimorfismo de Longitud del Fragmento de Restricción , Sensibilidad y Especificidad , Tripanosomiasis Africana/genética , UgandaRESUMEN
Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.