Reemergence of Human African Trypanosomiasis Caused by Trypanosoma brucei rhodesiense, Ethiopia.

We report 4 cases of human African trypanosomiasis that occurred in Ethiopia in 2022, thirty years after the last previously reported case in the country. Two of 4 patients died before medicine became available. We identified the infecting parasite as Trypanosoma brucei rhodesiense. Those cases imply human African trypanosomiasis has reemerged.

Target product profile: Trypanosoma brucei gambiense test to verify elimination.

Human African trypanosomiasis is a life-threatening parasitic infection transmitted by the tsetse fly in sub-Saharan Africa. The most common form is caused by Trypanosoma brucei gambiense, with humans as the main reservoir. Diagnosis in the field requires microscopic examination performed by specifically trained personnel. After over two decades of sustained efforts, the incidence of the disease is strongly declining, and some historically endemic countries are no longer detecting cases. The World Health Organization (WHO) has targeted the elimination of transmission of gambiense human African trypanosomiasis by 2030, defined as zero autochthonous cases for at least five consecutive years. Endemic countries reaching this goal must maintain dedicated surveillance to detect re-emergence or re-introduction. With this new agenda, new tools are needed for verification of the absence of transmission. WHO has therefore developed a target product profile calling for development of a method for population-level cross-cutting surveillance of T. b. gambiense transmission. The method needs to be performed in national or sub-national reference laboratories, and to test in parallel numerous samples shipped from remote rural areas. Among other characteristics the product profile specifies: (i) a simple specimen collection procedure; (ii) no cold-chain requirement to transfer specimens to reference laboratories; (iii) high sensitivity and specificity; (iv) high-throughput, substantially automatized; (v) low cost per specimen, when analysed in large batches; and (vi) applicable also in animals.

La trypanosomiase humaine africaine est une infection parasitaire potentiellement mortelle transmise par la mouche tsé-tsé en Afrique subsaharienne. La forme la plus répandue est causée par Trypanosoma brucei gambiense, les humains constituant son principal réservoir. Établir un diagnostic sur le terrain nécessite un examen microscopique réalisé par du personnel formé à cet effet. Après plus de deux décennies d'efforts soutenus, l'incidence de la maladie diminue fortement et quelques pays historiquement endémiques ne découvrent plus aucun cas. L'objectif de l'Organisation mondiale de la Santé (OMS) est d'éliminer la transmission de la trypanosomiase humaine africaine à T. b. gambiense d'ici 2030, ce qui correspond à zéro cas autochtone pendant au moins cinq années consécutives. Les pays endémiques qui atteignent cet objectif doivent maintenir une surveillance spécifique afin de détecter toute réémergence ou réintroduction. Ce nouveau programme doit s'accompagner de nouveaux outils servant à vérifier l'absence de transmission. L'OMS a donc élaboré un profil de produit cible pour le développement d'un procédé de surveillance transversale de la transmission de T. b. gambiense à l'échelle de la population. Ce procédé doit être effectué dans des laboratoires de référence nationaux ou infranationaux et tester simultanément de nombreux échantillons envoyés depuis des régions rurales isolées. Ce profil de produit comporte notamment les caractéristiques suivantes: (i) une procédure simple de collecte d'échantillons; (ii) aucune exigence concernant le respect de la chaîne du froid lors du transfert des échantillons vers les laboratoires de référence; (iii) un niveau élevé de sensibilité et de spécificité; (iv) un haut débit, en grande partie automatisé; (v) de faibles coûts par échantillon lors d'analyses en masse; et enfin, (vi) applicable aux animaux également.

La tripanosomiasis humana africana es una infección parasitaria potencialmente mortal transmitida por la mosca tsetsé en el África Subsahariana. El principal reservorio es el ser humano, y la forma más común está causada por Trypanosoma brucei gambiense. El diagnóstico práctico requiere un examen microscópico a cargo de personal con formación específica. Tras más de dos décadas de esfuerzos sostenidos, la incidencia de la enfermedad está disminuyendo considerablemente, y en algunos países históricamente endémicos ya no se detectan casos. La Organización Mundial de la Salud (OMS) se ha fijado como objetivo la eliminación de la transmisión de la tripanosomiasis africana humana gambiense para 2030, es decir, cero casos autóctonos durante al menos cinco años consecutivos. Los países endémicos que alcancen este objetivo deben mantener una vigilancia permanente para detectar la reaparición o reintroducción de la enfermedad. Con esta agenda nueva, se necesitan herramientas nuevas para verificar la ausencia de transmisión. Por consiguiente, la OMS ha elaborado un perfil de producto objetivo en el que se pide el desarrollo de un método para la vigilancia transversal a nivel de población sobre la transmisión de T. b. gambiense. El método debe realizarse en laboratorios de referencia nacionales o subnacionales y analizar en paralelo numerosas muestras enviadas desde regiones rurales remotas. Entre otras características, el perfil del producto detalla: (i) un procedimiento sencillo de recogida de muestras; (ii) ningún requisito de cadena de frío para transferir las muestras a los laboratorios de referencia; (iii) alta sensibilidad y especificidad; (iv) alto rendimiento, sustancialmente automatizado; (v) bajo coste por muestra, cuando se analizan en grandes lotes; y (vi) aplicable también en animales.

Target product profile: diagnostic test for Trypanosoma brucei rhodesiense.

Rhodesiense human African trypanosomiasis is a lethal parasitic infection caused by Trypanosoma brucei rhodesiense and transmitted by tsetse flies in eastern and southern Africa. It accounts for around 5% of all cases of human African trypanosomiasis. Currently, there is no simple serological test for rhodesiense human African trypanosomiasis and diagnosis relies on microscopic confirmation of trypanosomes in samples of blood or other tissues. The availability of a simple and accurate diagnostic test would aid the control, surveillance and treatment of the disease. A subcommittee of the World Health Organization's Neglected Tropical Diseases Diagnostics Technical Advisory Group has developed a target product profile for a diagnostic tool to identify T. b. rhodesiense infection. The optimum tool would have a sensitivity and specificity above 99% for detecting T. b. rhodesiense, but be simple enough for use by minimally trained health-care workers in unsophisticated peripheral health facilities or mobile teams in villages. The test should yield a qualitative result that can be easily observed and can be used to determine treatment. An antigen test would be preferable, with blood collected by finger-prick. Ideally, there should be no need for a cold chain, instrumentation or precision liquid handling. The test should be usable between 10 °C and 40 °C and between 10% and 88% relative humidity. Basic training should take under 2 hours and the test should involve fewer than five steps. The unit cost should be less than 1 United States dollar.

La trypanosomiase humaine africaine à T. b. rhodesiense est une infection parasitaire mortelle causée par Trypanosoma brucei rhodesiense et transmise par les mouches tsé-tsé en Afrique orientale et australe. Elle représente environ 5% de l'ensemble des cas de trypanosomiase humaine africaine. À l'heure actuelle, il n'existe aucun test sérologique simple pour l'infection à T. b. rhodesiense et le diagnostic repose sur la confirmation microscopique de la présence de trypanosomes dans des échantillons de sang ou d'autres tissus. Fournir un test de diagnostic simple et précis favoriserait la lutte, la surveillance et la prise en charge de la maladie. Un sous-comité du Groupe consultatif technique sur les produits de diagnostic des maladies tropicales négligées de l'Organisation mondiale de la Santé a donc élaboré un profil de produit cible pour un outil visant à détecter une infection par T. b. rhodesiense. L'outil le plus adapté présenterait un niveau de sensibilité et de spécificité supérieur à 99% pour la détection de T. b. rhodesiense, tout en étant à la portée de professionnels de la santé ayant reçu une formation sommaire, tant dans des structures de santé périphériques basiques qu'au sein d'équipes mobiles dans les villages. Cet outil doit fournir un résultat fiable, facile à interpréter, qui peut servir à établir un traitement. Un test antigénique serait préférable, avec prélèvement de l'échantillon sanguin par le biais d'une piqûre au bout du doigt. Idéalement, l'outil ne doit pas être thermosensible, ni nécessiter un équipement spécifique ou une manipulation de liquides délicate. Le test doit pouvoir être utilisé à une température comprise entre 10 °C et 40 °C, ainsi que dans une humidité relative de 10% à 88%. La formation requise pour son utilisation doit durer moins de deux heures et le test doit être effectué en moins de cinq étapes, Enfin, son coût unitaire doit être inférieur à un dollar américain.

La tripanosomiasis humana africana rhodesiense es una infección letal parasitaria causada por el Trypanosoma brucei rhodesiense, y es transmitida por la mosca tse-tsé en África oriental y meridional. Representa aproximadamente el 5% de todos los casos de tripanosomiasis humana africana. Actualmente, no existe ninguna prueba serológica simple para la tripanosomiasis humana africana rhodesiense, y el diagnóstico se basa en la confirmación microscópica de tripanosomas existentes en muestras de sangre u otros tejidos. Una prueba diagnóstica sencilla y precisa ayudaría a controlar, vigilar y tratar la enfermedad. Un subcomité del Grupo Asesor Técnico de Diagnóstico de Enfermedades Tropicales Desatendidas de la Organización Mundial de la Salud ha creado un perfil de producto objetivo para una herramienta de diagnóstico que permita identificar la infección T. b. rhodesiense. La herramienta óptima tendría una sensibilidad y una especificidad superiores al 99% para detectar la T. b. rhodesiense y, al ser lo suficientemente sencilla, podrían utilizarla trabajadores sanitarios mínimamente formados, en centros sanitarios periféricos no sofisticados, o bien equipos móviles. La prueba debe arrojar un resultado cualitativo de fácil lectura y que pueda utilizarse para determinar el tratamiento. Sería preferible una prueba de antígenos, con sangre extraída mediante punción digital. Idealmente, no debería ser necesaria la cadena de frío, la instrumentación ni la manipulación de líquidos de precisión. La prueba debe poder utilizarse entre 10 °C y 40 °C, con una humedad relativa de entre el 10% y el 88%. La instrucción básica debe llevar menos de 2 horas y la prueba debe incluir menos de cinco pasos. El coste de la unidad debe ser inferior a 1 dólar estadounidense.

Target product profile: Trypanosoma brucei gambiense test for low-prevalence settings.

Having caused devastating epidemics during the 20th century, the incidence of life-threatening human African trypanosomiasis has fallen to historically low levels as a result of sustained and coordinated efforts over the past 20 years. Humans are the main reservoir of one of the two pathogenic trypanosome subspecies, Trypanosoma brucei gambiense, found in western and central Africa. The expected advent of a safe and easy-to-use treatment to be given to seropositive but microscopically unconfirmed individuals would lead to further depletion; in the meantime, the presence of T. b. gambiense infection in the community must be monitored to allow the control strategy to be adapted and the elimination status to be assessed. The World Health Organization has therefore developed a target product profile that describes the optimal and minimal characteristics of an individual laboratory-based test to assess T. b. gambiense infection in low-prevalence settings. Development of the target product profile involved the formation of a Neglected Tropical Diseases Diagnostics Technical Advisory Group and a subgroup on human African trypanosomiasis diagnostic innovation needs, and an analysis of the available products and development pipeline. According to the product profile, the test should ideally: (i) require a minimally invasive or non-invasive specimen, collectable at peripheral facilities by minimally trained health workers; (ii) demonstrate good sensitivity and high specificity; (iii) have a stability of samples allowing transfer to reference laboratories preferably without cold chain; (iv) be stable over a wide range of environmental conditions for more than 2 years; and (v) after marketing, be available at low cost for at least 7 years.

Après avoir causé des épidémies dévastatrices au cours du 20e siècle, la trypanosomiase humaine africaine, potentiellement mortelle, a vu son incidence chuter à un niveau historiquement bas grâce aux efforts conjoints et soutenus déployés ces deux dernières décennies. Les humains constituent le principal réservoir de l'une des deux sous-espèces pathogéniques de trypanosome, Trypanosoma brucei gambiense, que l'on retrouve en Afrique occidentale et centrale. L'arrivée d'un traitement sûr et simple d'utilisation, qui serait administré aux individus séropositifs mais sans confirmation microscopique, devrait entraîner une nouvelle diminution; dans l'intervalle, la présence d'une infection à T. b. gambiense au sein de la communauté doit être surveillée afin de pouvoir adapter la stratégie de lutte et évaluer le statut d'élimination. Par conséquent, l'Organisation mondiale de la Santé a élaboré un profil de produit cible qui détaille les caractéristiques minimales et optimales d'un test individuel en laboratoire visant à confirmer l'infection à T. b. gambiense dans les régions à faible prévalence. La mise au point de ce profil a entraîné la formation d'un Groupe consultatif technique sur le diagnostic des maladies tropicales négligées et d'un sous-groupe consacré aux besoins en matière d'innovation diagnostique pour la trypanosomiase humaine africaine, qui a conduit une analyse des produits existants et des projets de développement. Selon le profil de produit, le test devrait idéalement: (i) nécessiter un prélèvement d'échantillon peu ou non invasif, pouvant être effectué dans des structures périphériques par des professionnels de la santé ayant reçu une formation sommaire; (ii) faire preuve d'un bon niveau de sensibilité et d'un niveau élevé de spécificité; (iii) avoir une stabilité des échantillons permettant le transfert vers des laboratoires de référence, de préférence sans chaîne de froid; (iv) rester stable dans un large éventail de conditions environnementales pendant plus de deux ans; et enfin, (v) après commercialisation, être disponible à bas coût pendant au moins sept ans.

Tras haber causado epidemias devastadoras durante el siglo XX, la incidencia de la tripanosomiasis humana africana potencialmente mortal ha descendido a niveles históricamente bajos gracias a los esfuerzos sostenidos y coordinados de los últimos 20 años. El ser humano es el principal reservorio de una de las dos subespecies patógenas del tripanosoma, Trypanosoma brucei gambiense, presente en África Occidental y Central. La prevista disponibilidad de un tratamiento seguro y fácil de administrar a personas seropositivas, pero no confirmadas al microscopio, permitiría una mayor eliminación; mientras tanto, se debe vigilar la presencia de la infección por T. b. gambiense en la comunidad para poder adaptar la estrategia de control y evaluar el estado de eliminación. Por consiguiente, la Organización Mundial de la Salud ha elaborado un perfil de producto objetivo que describe las características óptimas y mínimas de una prueba de laboratorio individual para evaluar la infección por T. b. gambiense en regiones de baja prevalencia. El desarrollo del perfil de producto objetivo implicó la formación de un Grupo de Asesoramiento Técnico sobre Diagnóstico de Enfermedades Tropicales Desatendidas y un subgrupo sobre las necesidades de innovación en el diagnóstico de la tripanosomiasis humana africana, así como un análisis de los productos disponibles y en desarrollo. Según el perfil objetivo, lo ideal sería que la prueba: (i) requiriera una muestra mínimamente invasiva o no invasiva, que pudiera ser recogida en centros periféricos por personal sanitario con una capacitación mínima; (ii) demostrara una buena sensibilidad y alta especificidad; (iii) tuviera una estabilidad de las muestras que permita su transferencia a laboratorios de referencia, preferiblemente sin cadena de frío; (iv) fuera estable en un amplio rango de condiciones ambientales durante más de 2 años; y (v) tras su comercialización, estuviera disponible a bajo coste durante al menos 7 años.

Target product profile: diagnostic test for Trypanosoma brucei gambiense.

Human African trypanosomiasis is a life-threatening parasitic infection endemic to sub-Saharan Africa. Around 95% of cases are due to Trypanosoma brucei gambiense, found in western and central Africa. Clinical signs and symptoms are nonspecific, current diagnostic tests are not sufficiently accurate, and parasitological confirmation of infection requires microscopic examination of body fluids and specialized techniques for concentrating parasites. Moreover, current treatment is not recommended on the basis of suspicion alone because it is not sufficiently safe. The availability of a simple and accurate diagnostic test to identify individuals harbouring parasites would widen treatment and help decrease disease prevalence. A subcommittee of the World Health Organization's Neglected Tropical Diseases Diagnostics Technical Advisory Group has developed a target product profile for a diagnostic tool to identify T. b. gambiense infection. This tool should have a high sensitivity for detecting T. b. gambiense but be simple enough to use in rural Africa. Ideally, the tool could be applied by any minimally trained individual in an unsophisticated peripheral health facility, or a mobile team in a village with little infrastructure. The test should be able to function under hot and humid conditions. Basic training should take under 2 hours and the test should involve fewer than five steps. There should be no need for instrumentation or precision liquid handling. The test should yield a qualitative result in under 20 minutes that can be easily observed, and one test should be sufficient for determining treatment. A unit cost below 1 United States dollar (US$) would enable mass screening.

La trypanosomiase humaine africaine est une infection parasitaire potentiellement mortelle endémique en Afrique subsaharienne. Dans près de 95% des cas, elle est causée par Trypanosoma brucei gambiense, que l'on trouve en Afrique occidentale et centrale. Les symptômes et signes cliniques sont aspécifiques, les tests de diagnostic existants ne sont pas assez précis et la confirmation parasitologique de l'infection nécessite un examen microscopique des liquides corporels ainsi que des techniques spécialisées pour concentrer les parasites. En outre, il n'est pas recommandé d'entamer le traitement actuel sur la base d'une simple suspicion car celui-ci n'est pas suffisamment sûr. Fournir un test de diagnostic simple et précis permettant d'identifier les individus porteurs de parasites contribuerait à élargir le traitement et à une diminution de la prévalence de la maladie. Un sous-comité du Groupe consultatif technique sur les produits de diagnostic des maladies tropicales négligées de l'Organisation mondiale de la Santé a élaboré un profil de produit cible pour un outil visant à détecter une infection par T. b. gambiense. Cet outil doit être suffisamment sensible pour déceler la présence de T. b. gambiense mais suffisamment simple pour être utilisé dans les régions rurales du continent. Idéalement, il doit pouvoir être employé par toute personne ayant reçu une formation sommaire, tant dans des structures de santé périphériques basiques qu'au sein d'une équipe mobile dans un village doté d'infrastructures restreintes. Par ailleurs, il doit fonctionner dans une atmosphère chaude et humide. La formation requise pour son utilisation doit durer moins de deux heures et le test doit être effectué en moins de cinq étapes, sans exiger d'équipement spécifique ni de manipulation délicate. Cet outil doit fournir un résultat fiable en moins de 20 minutes, facile à interpréter, et un seul test doit suffire à établir un traitement. Enfin, afin d'organiser un dépistage de masse, son coût unitaire ne doit pas dépasser un dollar américain.

La tripanosomiasis humana africana es una infección parasitaria potencialmente mortal endémica del África Subsahariana. Alrededor del 95% de los casos se deben al Trypanosoma brucei gambiense, presente en África Occidental y Central. Los signos y síntomas clínicos no son específicos, las pruebas diagnósticas actuales no son suficientemente precisas y la confirmación parasitológica de la infección requiere el examen microscópico de los fluidos corporales y técnicas especializadas de concentración de parásitos. Además, el tratamiento actual no se recomienda a partir de la sola sospecha porque no es suficientemente seguro. La disponibilidad de una prueba diagnóstica sencilla y precisa para identificar a las personas con parásitos ampliaría el tratamiento y ayudaría a disminuir la prevalencia de la enfermedad. Un subcomité del Grupo de Asesoramiento Técnico sobre Diagnóstico de Enfermedades Tropicales Desatendidas de la Organización Mundial de la Salud ha desarrollado un perfil de producto objetivo para una herramienta de diagnóstico destinada a identificar la infección por T. b. gambiense. Esta herramienta debe tener una alta sensibilidad para detectar T. b. gambiense, pero ser lo suficientemente sencilla para su uso en las regiones rurales de África. Lo ideal sería que la herramienta pudiera ser aplicada por cualquier persona mínimamente capacitada en un centro sanitario periférico poco sofisticado o por un equipo móvil en un pueblo con poca infraestructura. La prueba debería funcionar en condiciones de calor y humedad. La formación básica debe durar menos de 2 horas y la prueba debe constar de menos de cinco pasos. No debe necesitarse instrumentación ni manipulación precisa de líquidos. La prueba debe dar un resultado cualitativo en menos de 20 minutos que pueda observarse fácilmente y debe bastar una prueba para determinar el tratamiento. Su coste unitario, inferior a un dólar estadounidense, permitiría un cribado masivo.

Trypanosome spliced leader RNA for diagnosis of acoziborole treatment outcome in gambiense human African trypanosomiasis: A longitudinal follow-up study.

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).

Deep kinetoplast genome analyses result in a novel molecular assay for detecting Trypanosoma brucei gambiense-specific minicircles.

The World Health Organization targeted Trypanosoma brucei gambiense (Tbg) human African trypanosomiasis for elimination of transmission by 2030. Sensitive molecular markers that specifically detect Tbg type 1 (Tbg1) parasites will be important tools to assist in reaching this goal. We aim at improving molecular diagnosis of Tbg1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of these parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as Tbg1-specific, of which three were shared by the 18 studied Tbg1 strains. We developed a minicircle-based assay that is applicable on animals and as specific as the TgsGP-based assay, the current golden standard for molecular detection of Tbg1. The median copy number of the targeted minicircle was equal to eight, suggesting our minicircle-based assay may be used for the sensitive detection of Tbg1 parasites. Annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite and will thus likely be preserved in natural Tbg1 populations, the latter ensuring the reliability of our novel diagnostic assay.

Single nucleotide polymorphisms and copy-number variations in the Trypanosoma brucei repeat (TBR) sequence can be used to enhance amplification and genotyping of Trypanozoon strains.

The Trypanosoma brucei repeat (TBR) is a tandem repeat sequence present on the Trypanozoon minichromosomes. Here, we report that the TBR sequence is not as homogenous as previously believed. BLAST analysis of the available T. brucei genomes reveals various TBR sequences of 177 bp and 176 bp in length, which can be sorted into two TBR groups based on a few key single nucleotide polymorphisms. Conventional and quantitative PCR with primers matched to consensus sequences that target either TBR group show substantial copy-number variations in the TBR repertoire within a collection of 77 Trypanozoon strains. We developed the qTBR, a novel PCR consisting of three primers and two probes, to simultaneously amplify target sequences from each of the two TBR groups into one single qPCR reaction. This dual probe setup offers increased analytical sensitivity for the molecular detection of all Trypanozoon taxa, in particular for T.b. gambiense and T. evansi, when compared to existing TBR PCRs. By combining the qTBR with 18S rDNA amplification as an internal standard, the relative copy-number of each TBR target sequence can be calculated and plotted, allowing for further classification of strains into TBR genotypes associated with East, West or Central Africa. Thus, the qTBR takes advantage of the single-nucleotide polymorphisms and copy number variations in the TBR sequences to enhance amplification and genotyping of all Trypanozoon strains, making it a promising tool for prevalence studies of African trypanosomiasis in both humans and animals.

Trypanosome SL-RNA detection in blood and cerebrospinal fluid to demonstrate active gambiense human African trypanosomiasis infection.

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).

Trypanosoma brucei gambiense-iELISA: A Promising New Test for the Post-Elimination Monitoring of Human African Trypanosomiasis.

BACKGROUND: The World Health Organization targeted Trypanosoma brucei gambiense human African trypanosomiasis (gHAT) for elimination as a public health problem and for elimination of transmission. To measure gHAT elimination success with prevalences close to zero, highly specific diagnostics are necessary. Such a test exists in the form of an antibody-mediated complement lysis test, the trypanolysis test, but biosafety issues and technological requirements prevent its large-scale use. We developed an inhibition ELISA with high specificity and sensitivity that is applicable in regional laboratories in gHAT endemic countries. METHODS: The T. b. gambiense inhibition ELISA (g-iELISA) is based on the principle that binding of monoclonal antibodies to specific epitopes of T. b. gambiense surface glycoproteins can be inhibited by circulating antibodies of gHAT patients directed against the same epitopes. Using trypanolysis as reference test, the diagnostic accuracy of the g-iELISA was evaluated on plasma samples from 739 gHAT patients and 619 endemic controls and on dried blood spots prepared with plasma of 95 gHAT and 37 endemic controls. RESULTS: Overall sensitivity and specificity on plasma were, respectively, 98.0% (95% CI 96.7-98.9) and 99.5% (95% CI 98.6-99.9). With dried blood spots, sensitivity was 92.6% (95% CI 85.4-97.0), and specificity was 100% (95% CI 90.5-100.0). The g-iELISA is stable for at least 8 months when stored at 2-8°C. CONCLUSION: The g-iELISA might largely replace trypanolysis for monitoring gHAT elimination and for postelimination surveillance. The g-iELISA kit is available for evaluation in reference laboratories in endemic countries.

Assessment of Trypanosoma evansi prevalence and associated risk factors by immune trypanolysis test in camels from Ghardaïa district, southern Algeria.

Trypanosoma evansi (T. evansi) is a flagellated parasite with worldwide distribution, mainly affecting camels, horses, dogs, buffaloes and wild animals. Trypanosomosis caused by T. evansi, known as surra, is a vector borne disease that affects the health and productivity of camels. The aim of our study was to assess the prevalence of trypanosomosis due to T. evansi in camels by the immune trypanolosis test and to identify associated risk factors. Our cross-sectional study was performed on 161 camels from Ghardaïa district, southern Algeria. A structured questionnaire was used to collect data on individual characteristics (age, gender and breed) husbandry management (herd size and activity of animals) and health conditions (history of abortion and clinical symptoms). The immune trypanolysis test revealed an overall seroprevalence of 9.3% (CI 95%, 5.9-14.9). Possible factors associated with T. evansi infection were analysed by univariate and multivariate logistic regression. The results showed that risk factors for camels were history of symptoms (P = 0.002, OR = 21.91, CI95% = 3.48-169.80), racing activities (P = 0.003, OR = 0.01, CI95% = 0.001-0.18) and small herd size (P = 0.013, OR = 8.22, CI95% = 1.64-49.75). In conclusion, this study showed that T. evansi is endemic in camels of Ghardaïa district. To reduce dissemination of the disease to non-endemic areas, it is recommended to minimise risk factors associated with the infection.

Comparison of serological and molecular tests for detection of Trypanosoma evansi in domestic animals from Ghardaïa district, South Algeria.

Trypanosoma evansi (T. evansi) is a hemoflagellate parasite that affects a broad range of mammalian hosts and that causes a disease called surra. Diagnosis of surra based on clinical symptoms alone is inaccurate. Therefore, a variety of serological and molecular diagnostic tests are used to assist in the detection of T. evansi infections. The aim of this study was to compare the diagnostic performance of four serological tests (CATT/T.evansi, immune trypanolysis, ELISA with purified variant surface glycoprotein RoTat 1.2 and with whole cell lysate) and two molecular PCR tests targeting sequences within the ribosomal genes locus (ITS1 TD PCR and 18S qPCR). Tests were carried out on blood samples from 161 dromedary camels, 93 horses, 129 goats, 168 sheep, 127 bovines and 76 dogs. Latent class analysis was carried out to calculate the sensitivity and specificity of each diagnostic test. Cohen's Kappa test was used to assess the concordance between the different diagnostic tests. Overall positivity rates observed with the serological tests were as follows: 3.1 % with CATT/T.evansi, 4.9 % with ELISA/RoTat 1.2, 3.4 % with ELISA/whole lysate and 2.0 % with immune trypanolysis (TL). Among the 754 samples tested with the molecular tests, 1.7 % were positive with 18S qPCR and 1.3 % with ITS1 TD PCR. Cohen's Kappa test showed agreement ranging from fair to substantial (k = 0.2-0.8) between serological diagnostic tests. However, it showed a perfect agreement (k = 0.868) between molecular diagnostic tests. Latent class analysis showed that all serological tests were 100 % sensitive, in contrast to the molecular tests with 47 % sensitivity. All tests, though, were highly specific (≥ 97 %). Given the persistence of circulating antibodies after cure, detectable by serological tests, it is recommend combining a serological and a molecular diagnostic test for accurate diagnosis of infection with T. evansi in domestic animals.

Expression of a rK39 homologue from an Iranian Leishmania infantum isolate in Leishmania tarentolae for serodiagnosis of visceral leishmaniasis.

BACKGROUND: Kinesin-related gene diversity among strains and species of Leishmania may impact the sensitivity and specificity of serodiagnostic tests for visceral leishmaniasis (VL). METHODS: In this study, we report on the recombinant expression of this novel Iranian Leishmania infantum (MCAN14/47) homologue of rK39 (Li-rK39), in L. tarentolae. The diagnostic potential of the Li-rK39 antigen was evaluated in an ELISA, using sera from 100 VL patients, 190 healthy endemic controls, 46 non-endemic healthy controls and 47 patients with other infections. RESULTS: The results showed a sensitivity of 96% and a specificity of 93.8%. A commercial rK39 immunochromatographic test (ICT) was 90% sensitive and 100% specific on the same cohort. CONCLUSIONS: Here, we show that the K39 gene from an Iranian L. infantum isolate is heterozygous as compared to the sequence of the Brazilian L. infantum (former L. chagasi), whose antigen is incorporated in most rK39-based immunochromatographic tests. Therefore, Li-rK39 has the potential to be used as an alternative for VL diagnosis in Iran.

Equine trypanosomosis: enigmas and diagnostic challenges.

Equine trypanosomosis is a complex of infectious diseases called dourine, nagana and surra. It is caused by several species of the genus Trypanosoma that are transmitted cyclically by tsetse flies, mechanically by other haematophagous flies, or sexually. Trypanosoma congolense (subgenus Nannomonas) and T. vivax (subgenus Dutonella) are genetically and morphologically distinct from T. brucei, T. equiperdum and T. evansi (subgenus Trypanozoon). It remains controversial whether the three latter taxa should be considered distinct species. Recent outbreaks of surra and dourine in Europe illustrate the risk and consequences of importation of equine trypanosomosis with infected animals into non-endemic countries. Knowledge on the epidemiological situation is fragmentary since many endemic countries do not report the diseases to the World Organisation for Animal Health, OIE. Other major obstacles to the control of equine trypanosomosis are the lack of vaccines, the inability of drugs to cure the neurological stage of the disease, the inconsistent case definition and the limitations of current diagnostics. Especially in view of the ever-increasing movement of horses around the globe, there is not only the obvious need for reliable curative and prophylactic drugs but also for accurate diagnostic tests and algorithms. Unfortunately, clinical signs are not pathognomonic, parasitological tests are not sufficiently sensitive, serological tests miss sensitivity or specificity, and molecular tests cannot distinguish the taxa within the Trypanozoon subgenus. To address the limitations of the current diagnostics for equine trypanosomosis, we recommend studies into improved molecular and serological tests with the highest possible sensitivity and specificity. We realise that this is an ambitious goal, but it is dictated by needs at the point of care. However, depending on available treatment options, it may not always be necessary to identify which trypanosome taxon is responsible for a given infection.