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.

Specificity of SARS-CoV-2 Antibody Detection Assays against S and N Proteins among Pre-COVID-19 Sera from Patients with Protozoan and Helminth Parasitic Infections.

We aimed to assess the specificity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody detection assays among people with tissue-borne parasitic infections. We tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 neutralization antibody detection kit [cPass], Abbott SARS-CoV-2 IgG assay [Abbott Architect], and Standard Q COVID-19 IgM/IgG combo rapid diagnostic test [SD RDT IgM/SD RDT IgG]) among 559 pre-COVID-19 seropositive sera for several parasitic infections. The specificity of assays was 95 to 98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95% CI, 75 to 95]) and visceral leishmaniasis (SD RDT IgG; 80% [95% CI, 30 to 99]), and from patients with recent malaria in areas of Senegal where malaria is holoendemic (ranging from 91% for Abbott Architect and SD RDT IgM to 98 to 99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all ≥96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98 to 100%. The majority (>85%) of false-positive results were positive by only one assay. The specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.

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.

Pediatric visceral leishmaniasis: a retrospective study to propose the diagnostic tests algorithm in southern Iran.

Leishmania infantum is the most common cause of visceral leishmaniasis (VL) in Iran, where mainly the patients are children under the age of 5 years. Timely, less invasive, and accurate diagnosis and proper treatment of the disease are necessary. This retrospective study aimed to search for a less invasive but robust algorithm on VL diagnostic tests in children. Four hundred and fifteen patients with clinical suspicion of VL, 50 healthy children from VL endemic areas, 46 healthy individuals from non-endemic VL areas, and 47 non-VL diseases were tested using three diagnostic tests: indirect immunofluorescent antibody test (IFAT), rK39-rapid diagnostic test (rK39-RDT), and quantitative PCR (qPCR). One hundred and two suspected VL cases were positive in at least one test and were cured after receiving appropriate treatment. Of these 102 VL patients, 94 were positive in qPCR, 84 in IFAT, and 79 in rK39-RDT. None of the tests detected all the patients, but overall, qPCR is capable of detecting more VL patients than serological tests, i.e., 92.2%, compared to IFAT, 82.4%, and rK39, 77.5%. There was only a significant difference between the sensitivity of qPCR and rK39-RDT (p = 0.024). The specificity was 100% for qPCR and IFAT (≥128) and 98.6% for rK39-RDT. qPCR alone is capable of detecting most of the VL-suspected children. Serological tests like IFAT and rk39-RDT are recommended to increase the overall sensitivity of detection in patients with a negative molecular test. Combining qPCR with a serological test (IFAT or rK39-RDT) can help diagnose 98% of VL. In laboratories without molecular facilities, we recommend testing with the combination of rK39-RDT and IFAT yielding a combined sensitivity of 93.1% equivalent to that of qPCR in our study.

Analytical sensitivity of loopamp and quantitative real-time PCR on dried blood spots and their potential role in monitoring human African trypanosomiasis elimination.

The objective set by WHO to reach elimination of human African trypanosomiasis (HAT) as a public health problem by 2020 is being achieved. The next target is the interruption of gambiense-HAT transmission in humans by 2030. To monitor progress towards this target, in areas where specialized local HAT control capacities will disappear, is a major challenge. Test specimens should be easily collectable and safely transportable such as dried blood spots (DBS). Monitoring tests performed in regional reference centres should be reliable, cheap and allow analysis of large numbers of specimens in a high-throughput format. The aim of this study was to assess the analytical sensitivity of Loopamp, M18S quantitative real-time PCR (M18S qPCR) and TgsGP qPCR as molecular diagnostic tests for the presence of Trypanosoma brucei gambiense in DBS. The sensitivity of the Loopamp test, with a detection limit of 100 trypanosomes/mL, was in the range of parasitaemias commonly observed in HAT patients, while detection limits for M18S and TgsGP qPCR were respectively 1000 and 10,000 trypanosomes/mL. None of the tests was entirely suitable for high-throughput use and further development and implementation of sensitive high-throughput molecular tools for monitoring HAT elimination are needed.

Human African trypanosomiasis.

Human African trypanosomiasis (sleeping sickness) is a parasitic infection that almost invariably progresses to death unless treated. Human African trypanosomiasis caused devastating epidemics during the 20th century. Thanks to sustained and coordinated efforts over the past 15 years, the number of reported cases has fallen to an historically low level. Fewer than 3000 cases were reported in 2015, and the disease is targeted for elimination by WHO. Despite these recent successes, the disease is still endemic in parts of sub-Saharan Africa, where it is a considerable burden on rural communities, most notably in central Africa. Since patients are also reported from non-endemic countries, human African trypanosomiasis should be considered in differential diagnosis for travellers, tourists, migrants, and expatriates who have visited or lived in endemic areas. In the absence of a vaccine, disease control relies on case detection and treatment, and vector control. Available drugs are suboptimal, but ongoing clinical trials provide hope for safer and simpler treatments.

Case of Nigeria-Acquired Human African Trypanosomiasis in United Kingdom, 2016.

Human African trypanosomiasis has not been reported in Nigeria since 2012. Nevertheless, limitations of current surveillance programs mean that undetected infections may persist. We report a recent case of stage 2 trypanosomiasis caused by Trypanosoma brucei gambiense acquired in Nigeria and imported into the United Kingdom.

Gambiense human african trypanosomiasis and immunological memory: effect on phenotypic lymphocyte profiles and humoral immunity.

In mice, experimental infection with Trypanosoma brucei causes decreased bone marrow B-cell development, abolished splenic B-cell maturation and loss of antibody mediated protection including vaccine induced memory responses. Nothing is known about this phenomenon in human African trypanosomiasis (HAT), but if occurring, it would imply the need of revaccination of HAT patients after therapy and abolish hope for a HAT vaccine. The effect of gambiense HAT on peripheral blood memory T- and B-cells and on innate and vaccine induced antibody levels was examined. The percentage of memory B- and T-cells was quantified in peripheral blood, prospectively collected in DR Congo from 117 Trypanosoma brucei gambiense infected HAT patients before and six months after treatment and 117 controls at the same time points. Antibodies against carbohydrate antigens on red blood cells and against measles were quantified. Before treatment, significantly higher percentages of memory B-cells, mainly T-independent memory B-cells, were observed in HAT patients compared to controls (CD20+CD27+IgM+, 13.0% versus 2.0%, p<0.001). The percentage of memory T-cells, mainly early effector/memory T-cells, was higher in HAT (CD3+CD45RO+CD27+, 19.4% versus 16.7%, p = 0.003). After treatment, the percentage of memory T-cells normalized, the percentage of memory B-cells did not. The median anti-red blood cell carbohydrate IgM level was one titer lower in HAT patients than in controls (p<0.004), and partially normalized after treatment. Anti-measles antibody concentrations were lower in HAT patients than in controls (medians of 1500 versus 2250 mIU/ml, p = 0.02), and remained so after treatment, but were above the cut-off level assumed to provide protection in 94.8% of HAT patients, before and after treatment (versus 98.3% of controls, p = 0.3). Although functionality of the B-cells was not verified, the results suggest that immunity was conserved in T.b. gambiense infected HAT patients and that B-cell dysfunction might not be that severe as in mouse models.

Immune trypanolysis test with blood spotted on filter paper for epidemiological surveillance of sleeping sickness.

OBJECTIVES: The immune trypanolysis test (TL) is an accurate sero-diagnostic tool increasingly implemented for sleeping sickness medical surveillance, but it is restricted to the reference laboratories. To facilitate storage and transport of the test specimen, we developed a protocol for the examination of blood spotted on filter paper (TL-fp) that can be stored and shipped at ambient temperature. We compared its performance with the classical TL on plasma (TL-pl) that needs to be kept frozen until use. METHODS: The study was conducted in active foci of the Republic of Guinea. In total, 438 specimens from treated and untreated sleeping sickness patients and serological suspects were tested with both methods. RESULT: TL-fp gave significantly less positive results than TL-pl, but all the confirmed sleeping sickness cases were positive with the TL-fp protocol. CONCLUSION: TL-fp appears to offer a good compromise between feasibility and sensitivity to detect currently infected subjects who play a role in the transmission of Trypanosoma brucei gambiense and is useful for contributing to the elimination of gambiense sleeping sickness.

A Survey on Trypanosoma evansi (Kinetoplastida, Trypanosomatidae) Infection in Domestic Animals in a Surra Endemic Area of Southern Algeria.

Background: Trypanosoma (T.) evansi infection is endemic in dromedary camels (Camelus dromedaries) of southern Algeria. Materials and Methods: In order to assess the presence of T. evansi in other domestic animals living together with dromedary camels, a study was conducted in the wilayate of Béchar, El Bayadh, Ouargla and Tamanrasset, between 2015 and 2017. Authorisation to conduct the survey was obtained from the Direction des Services Vétérinaires (DSV, Ministry of Agriculture, Rural Development and Fisheries). A total of 190 animals were sampled, including 42 cattle (Bos taurus), 11 dogs (Canis familiaris), 44 horses (Equus caballus), 3 donkeys (Equus asinus) and 1 mule, 49 goats (Capra hircus) and 40 sheep (Ovis aries). These animals were examined by parasitological (Giemsa stained thin smear, GST), serological (card agglutination test for trypanosomosis (CATT/T. evansi), enzyme-linked immunosorbent assay/Variant Surface Glycoprotein/Rode Trypanozoon antigen type 1.2 [ELISA/VSG RoTat 1.2], immune trypanolysis [TL]) and molecular tests (T. evansi type A specific RoTat 1.2 PCR). Results and Conclusions: The CATT/T. evansi was positive in 10/42 cattle, 0/11 dogs, 2/48 equids, 27/49 goats and 15/40 sheep. On the other hand, 20/38 cattle, 1/9 dogs, 21/42 equids, 17/44 goats and 31/39 sheep were positive in ELISA/VSG RoTat 1.2. However, no single animal was positive in TL. In addition, the T. evansi parasite could not be demonstrated by either GST or RoTat 1.2 PCR in any of the examined animals. This may suggest cross-reactions of CATT/T. evansi and ELISA/VSG RoTat 1.2 with other pathogenic or commensal trypanosome species such as T. vivax or other parasites. Based on these data, in particular taking into account the high specificity of the TL for T. evansi type A, this study does not support the hypothesis that T. evansi circulates in the studied domestic animal species and that they would act as reservoirs for the parasite that causes trypanosomosis in dromedary camels.

Stage determination in sleeping sickness: comparison of two cell counting and two parasite detection techniques.

OBJECTIVES: Diagnosis of the neurological stage of human African trypanosomiasis is performed by examination of cerebrospinal fluid (CSF) for the presence of trypanosomes and numbers of white blood cells (WBC). Both CSF parameters are also used to assess treatment outcome during follow-up. In view of the importance of CSF examination, and the practical problems encountered with it, we compared the sensitivity of two trypanosome concentration techniques and the repeatability of two cell counting methods, as well as occurrence of systematic differences between them. METHODS: Patients were recruited at Dipumba hospital, in Mbuji-Mayi in the Democratic Republic of the Congo. In 94 CSF samples, trypanosome detection was performed with modified single centrifugation (MSC) and double centrifugation (DC). On 189 CSF samples with ≤30 cells/µl, cell counting was performed in duplicate in a Fuchs-Rosenthal counting chamber and in a disposable Uriglass counting chamber. RESULTS: Modified single centrifugation detected trypanosomes in significantly (P < 0.0001) more patients (85) than DC (46). Cell counts did not differ systematically in the two methods. Variability in the differences between duplicate cell counts was significantly higher (P = 0.002) in Uriglass (SD of differences 2.03) than in Fuchs-Rosenthal (SD of differences 1.62). CONCLUSIONS: For analysis of CSF in the context of sleeping sickness stage determination and follow-up after treatment, we strongly recommend the MSC for parasite detection and the application of disposable counting chambers. When the first cell count is ≤20 cells/µl, we recommend repeating the counting procedure on the same CSF specimen and taking the average of both countings.

A LiTat 1.5 variant surface glycoprotein-derived peptide with diagnostic potential for Trypanosoma brucei gambiense.

OBJECTIVE: To evaluate the accuracy of a peptide, corresponding to the variant surface glycoprotein (VSG) LiTat 1.5 amino acid (AA) sequence 268-281 and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. METHODS: A synthetic biotinylated peptide (peptide 1.5/268-281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from patients with HAT and 102 endemic HAT-negative controls. RESULTS: The area under the curve (AUC) of peptide 1.5/268-281 was 0.954 (95% confidence interval 0.918-0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982-1.000) and 0.997 (0.973-1.000), respectively, and significantly higher than the AUC of peptide 1.5/268-281. On a model of VSG LiTat 1.5, peptide 1.5/268-281 was mapped near the top of the VSG. CONCLUSIONS: A biotinylated peptide corresponding to AA 268-281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full-length native VSG LiTat 1.3 and VSG LiTat 1.5.

Performance of clinical signs and symptoms, rapid and reference laboratory diagnostic tests for diagnosis of human African trypanosomiasis by passive screening in Guinea: a prospective diagnostic accuracy study.

BACKGROUND: Passive diagnosis of human African trypanosomiasis (HAT) at the health facility level is a major component of HAT control in Guinea. We examined which clinical signs and symptoms are associated with HAT, and assessed the performance of selected clinical presentations, of rapid diagnostic tests (RDT), and of reference laboratory tests on dried blood spots (DBS) for diagnosing HAT in Guinea. METHOD: The study took place in 14 health facilities in Guinea, where 2345 clinical suspects were tested with RDTs (HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT). Seropositives underwent parasitological examination (reference test) to confirm HAT and their DBS were tested in indirect enzyme-linked immunoassay (ELISA)/Trypanosoma brucei gambiense, trypanolysis, Loopamp Trypanosoma brucei Detection kit (LAMP) and m18S quantitative PCR (qPCR). Multivariable regression analysis assessed association of clinical presentation with HAT. Sensitivity, specificity, positive and negative predictive values of key clinical presentations, of the RDTs and of the DBS tests for HAT diagnosis were determined. RESULTS: The HAT prevalence, as confirmed parasitologically, was 2.0% (48/2345, 95% CI: 1.5-2.7%). Odds ratios (OR) for HAT were increased for participants with swollen lymph nodes (OR = 96.7, 95% CI: 20.7-452.0), important weight loss (OR = 20.4, 95% CI: 7.05-58.9), severe itching (OR = 45.9, 95% CI: 7.3-288.7) or motor disorders (OR = 4.5, 95% CI: 0.89-22.5). Presence of at least one of these clinical presentations was 75.6% (95% CI: 73.8-77.4%) specific and 97.9% (95% CI: 88.9-99.9%) sensitive for HAT. HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT were respectively 97.5% (95% CI: 96.8-98.1%), 99.4% (95% CI: 99.0-99.7%) and 97.9% (95% CI: 97.2-98.4%) specific, and 100% (95% CI: 92.5-100.0%), 59.6% (95% CI: 44.3-73.3%) and 93.8% (95% CI: 82.8-98.7%) sensitive for HAT. The RDT's positive and negative predictive values ranged from 45.2-66.7% and 99.2-100% respectively. All DBS tests had specificities ≥ 92.9%. While LAMP and m18S qPCR sensitivities were below 50%, trypanolysis and ELISA/T.b. gambiense had sensitivities of 85.3% (95% CI: 68.9-95.0%) and 67.6% (95% CI: 49.5-82.6%). CONCLUSIONS: Presence of swollen lymph nodes, important weight loss, severe itching or motor disorders are simple but accurate clinical criteria for HAT referral in HAT endemic areas in Guinea. Diagnostic performances of HAT Sero-K-Set and SD Bioline HAT are sufficient for referring positives to microscopy. Trypanolysis on DBS may discriminate HAT patients from false RDT positives. Trial registration The trial was registered under NCT03356665 in clinicaltrials.gov (November 29, 2017, retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03356665 ).

Diagnostic Performance of Three rK39 Rapid Diagnostic Tests and Two Direct Agglutination Tests for the Diagnosis of Visceral Leishmaniasis in Southern Iran.

To evaluate the diagnostic performance of five alternative serodiagnostic tests, serum samples from 100 confirmed visceral leishmaniasis (VL) patients, 197 healthy endemic individuals, and 58 non-VL patients living in southern Iran were compared. The VL patients were defined as individuals with a positive result of the immunofluorescent antibody test (IFAT), having clinical signs and symptoms and appropriate response to treatment. The index tests were two direct agglutination tests, DAT-ITM (Institute of Tropical Medicine, Antwerp, Belgium) and DAT-KIT (Royal Tropical Institute, Amsterdam, The Netherlands), and three rapid diagnostic tests (RDTs), Kalazar Detect (InBios International Inc., USA), IT Leish (Bio-Rad, catalog 710124), and Leishmania test (Cypress Diagnostic Company, Belgium). Sensitivities of DAT-ITM and DAT-KIT were low, respectively, 56% and 59%, while specificities were acceptable, respectively, 98% and 93%. Observed sensitivities and specificities of RDTs were higher (71%, 81%, 70% and 99%, 99%, 98% for Kalazar Detect, IT Leish, and Leishmania test, respectively). Even with a maximum sensitivity of 81%, RDTs missed almost one-fifth of VL patients that were positive in IFAT. We conclude that RDTs in VL patients do not possess adequate performance in southern Iran and require some improvement, but they can still be helpful in the diagnosis and screening of the disease in this region due to their high specificity and speed.

Two-Year Follow-Up of Trypanosoma brucei gambiense Serology after Successful Treatment of Human African Trypanosomiasis: Results of Four Different Sero-Diagnostic Tests.

Gambiense human African trypanosomiasis (gHAT), also known as gambiense sleeping sickness, is a parasitic infection caused by Trypanosoma brucei gambiense. During the last decades, gHAT incidence has been brought to an all-time low. Newly developed serological tools and drugs for its diagnosis and treatment put the WHO goal of interruption of transmission by 2030 within reach. However, further research is needed to efficiently adapt these new advances to new control strategies. We assessed the serological evolution of cured gHAT patients over a two-year period using four different tests: the rapid diagnostic test (RDT) HAT Sero K-SeT, ELISA/T.b. gambiense, Trypanosoma brucei gambiense inhibition ELISA (iELISA), and the immune trypanolysis test. High seropositive rates were observed in all the tests, although sero-reversion rates were different in each test: ELISA/T.b. gambiense was the test most likely to become negative two years after treatment, whereas RDT HAT Sero-K-SeT was the least likely. iELISA and trypanolysis showed intermediate and comparable probabilities to become negative. Stage 1 patients were also noted to be more likely to become negative than Stage 2 patients in all four serological tests. Our results confirm previous findings that trypanosome-specific antibody concentrations in blood may persist for up to two years, implying that HAT control programs should continue to take the history of past HAT episodes into consideration.

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.