Sensitivity and specifi city of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study

Background Human African trypanosomiasis (HAT) is a life-threatening infection aff ecting rural populations in sub- Saharan Africa. Large-scale population screening by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT)/Trypanosoma brucei (T b) gambiense helped reduce the number of reported cases of gambiense HAT to fewer than 10 000 in 2011. Because low case numbers lead to decreased cost-eff ectiveness of such active screening, we aimed to assess diagnostic accuracy of a rapid serodiagnostic test (HAT Sero-K-SeT) applicable in primary health-care centres. Methods In our case-control study, we assessed participants older than 11 years who presented for HAT Sero-K-SeT and CATT/T b gambiense at primary care centres or to mobile teams (and existing patients with confi rmed disease status at these centres) in Bandundu Province, DR Congo. We defi ned cases as patients with trypanosomes that had been identifi ed in lymph node aspirate, blood, or cerebrospinal fl uid. During screening, we recruited controls without previous history of HAT or detectable trypanosomes in blood or lymph who resided in the same area as the cases. We assessed diagnostic accuracy of three antibody detection tests for gambiense HAT: HAT Sero-K-SeT and CATT/T b gambiense (done with venous blood at the primary care centres) and immune trypanolysis (done with plasma at the Institute of Tropical Medicine, Antwerp, Belgium). Findings Between June 6, 2012, and Feb 25, 2013, we included 134 cases and 356 controls. HAT Sero-K-SeT had a sensitivity of 0·985 (132 true positives, 95% CI 0·947–0·996) and a specifi city of 0·986 (351 true negatives, 0·968–0·994), which did not diff er signifi cantly from CATT/T b gambiense (sensitivity 95% CI 0·955, 95% CI 0·906–0·979 [128 true positives] and specifi city 0·972, 0·949–0·985 [346 true negatives]) or immune trypanolysis (sensitivity 0·985, 0·947–0·996 [132 true positives] and specifi city 0·980, 0·960–0·990 [349 true negatives]). Interpretation The diagnostic accuracy of HAT Sero-K-SeT is adequate for T b gambiense antibody detection in local health centres and could be used for active screening whenever a cold chain and electricity supply are unavailable and CATT/T b gambiense cannot be done

Caractérisation de Trypanosoma sp chez les animaux domestiques dans quatre foyers de la partie Ouest de la République Démocratique du Congo (RDC)

En vue d’identifier les trypanosomes circulants chez les animaux domestiques de Kinshasa, Mbanza-Ngungu, Masi-Manimba et Mushie et d’en déterminer les prévalences par PCR, une étude longitudinale a été menée. 1653 échantillons sanguins ont été prélevés chez les animaux domestiques dans les foyers de Kinshasa, Mbanza-Ngungu, Masi-Manimba et Mushie, dont 22 cas ont été positif au Trypanosoma congolense Forest, correspondant à une prévalence brute de 1,3%. Pour ce qui est des foyers, Kinshasa a eu une prévalence de 2,5%, Mbanza-Ngungu une prévalence de 2,4%, Masi-Manimba sans aucune prévalence et Mushie une prévalence de 1,3%. En rapport avec les saisons, la saison de pluie a eu une prévalence de 1,4%, alors que la saison sèche en a eu 1,3%. Concernant les espèces, les bovins ont eu une prévalence de 0,9%, les porcins une prévalence de 3,7%, les ovins une prévalence de 1,4% et les caprins une prévalence de 0,7%. Quant au sexe, les mâles ont eu une prévalence de 0,9%, alors que les femelles en ont eu 1,5%. Cette étude a montré que la Trypanosomiase Animale Africaine (TAA) serait en recul chez les animaux de ces quatre foyers. Ainsi, les cliniciens sur terrain devraient réorienter leur stratégie thérapeutique, en intégrant cette nouvelle donne. Mots clés: Trypanosoma sp, Animaux domestiques, Ouest RDCIn order to identify circulating trypanosomes in Kinshasa, Mbanza Ngungu, Masi Manimba and Mushie domestic animals and to determine their prevalence by PCR, a longitudinal study was conducted. 1653 blood samples were collected from domestic animals in the Kinshasa, Mbanza Ngungu, Masi Manimba and Mushie households, of which 22 were positive for Trypanosoma congolense Forest, corresponding to a gross prevalence of 1.3%. As for households, Kinshasa had a prevalence of 2.5%, Mbanza Ngungu a prevalence of 2.4%, Masi Manimba without any prevalence and Mushie a prevalence of 1.3%. In relation to the seasons, the rainy season had a prevalence of 1.4% while the dry season had a prevalence of 1.3%. Regarding species, cattle had a prevalence of 0.9%, pigs a prevalence of 3.7%, sheep a prevalence of 1.4% and goats a prevalence of 0.7%. As for sex, males had a prevalence of 0.9% while females had a prevalence of 1.5%. This study showed that African Animal Trypanosomiasis (AAT) is declining among animals in these four locations Thus, clinicians in the field should take into account this new situation in their therapeutic strategy. Keywords: Trypanosoma sp, Domestic animals, West DR

Diagnostic accuracy of Loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples

Background: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC). Methodology/Principal findings: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9–91.8%) in the first run and 93.0% (95% CI 87.5–96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4–96.3%) in the first run and 96.4% (95% CI 91.1–98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81. Conclusions/Significance: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions

Dog ecology, bite incidence, and disease awareness : a cross-sectional survey among a rabies-affected community in the Democratic Republic of the Congo

Despite the existence of safe and efficacious human and animal rabies vaccines, millions of people remain at risk of exposure to this deadly zoonotic disease through bites of infected dogs. Sub-Saharan African countries, such as the Democratic Republic of the Congo (DRC), bear the highest per capita death rates from rabies where dog vaccination and availability of lifesaving post-exposure prophylaxis (PEP) is scarce. Mass dog vaccination is the most cost-effective and sustainable approach to prevent human rabies deaths. We conducted a cross-sectional household survey in a rabies-affected community in Matadi, DRC, to estimate the size of the owned dog population and dog bite incidence and assess knowledge and practices regarding rabies, as preparation for future mass dog vaccination campaigns. Our study revealed that the owned dog population in Matadi was almost ten times larger than assumed by local veterinary officials, with a large proportion of free-roaming unvaccinated dogs. The annual dog bite incidence of 5.2 per 1000 person years was high, whereas community rabies knowledge was low resulting in poor practices. Given these findings, human rabies deaths are likely to occur in this community. Lack of disease awareness could negatively affect participation in future mass dog vaccination campaigns. A public sensitization campaign is needed to promote appropriate rabies prevention (washing bite wounds and PEP) and control (dog vaccination) measures in this community

Diagnostic Accuracy of Loopamp Trypanosoma brucei Detection Kit for Diagnosis of Human African Trypanosomiasis in Clinical Samples

Abstract Background: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC)

Isolation of Trypanosoma brucei gambiense from Cured and Relapsed Sleeping Sickness Patients and Adaptation to Laboratory Mice

Human African trypanosomiasis, or sleeping sickness, is still a major public health problem in central Africa. Melarsoprol is widely used for treatment of patients where the parasite has already reached the brain. In some regions in Angola, Sudan, Uganda and Democratic Republic of the Congo, up to half of the patients cannot be cured with melarsoprol. From previous investigations it is not yet clear what causes these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as relapsed patients for downstream comparative drug sensitivity profiling. From 360 sleeping sickness patients, blood and cerebrospinal fluid (CSF) was collected before treatment and along the prescribed 24 months follow-up. Blood and CSF were inoculated in thicket rats (Grammomys surdaster), Natal multimammate mice (Mastomys natalensis) and immunodeficient laboratory mice (Mus musculus). Thus, we established a unique collection of Trypanosoma brucei gambiense type I parasites, isolated in the same disease focus and within a limited period, including 12 matched strains isolated from the same patient before treatment and after relapse. This collection is now available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, Democratic Republic of the Congo

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited

A panel of trypanosoma brucei strains tagged with blue and red-shifted luciferases for bioluminescent imaging in murine infection models

BACKGROUND:Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. METHODOLOGY/PRINCIPAL FINDINGS:We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. CONCLUSIONS/SIGNIFICANCE:We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin

Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study.

BACKGROUND:The polymerase chain reaction (PCR) has been proposed for diagnosis, staging and post-treatment follow-up of sleeping sickness but no large-scale clinical evaluations of its diagnostic accuracy have taken place yet. METHODOLOGY/PRINCIPAL FINDINGS:An 18S ribosomal RNA gene targeting PCR was performed on blood and cerebrospinal fluid (CSF) of 360 T. brucei gambiense sleeping sickness patients and on blood of 129 endemic controls from the Democratic Republic of Congo. Sensitivity and specificity (with 95% confidence intervals) of PCR for diagnosis, disease staging and treatment failure over 2 years follow-up post-treatment were determined. Reference standard tests were trypanosome detection for diagnosis and trypanosome detection and/or increased white blood cell concentration in CSF for staging and detection of treatment failure. PCR on blood showed a sensitivity of 88.4% (84.4-92.5%) and a specificity of 99.2% (97.7-100%) for diagnosis, while for disease staging the sensitivity and specificity of PCR on cerebrospinal fluid were 88.4% (84.8-91.9%) and 82.9% (71.2-94.6%), respectively. During follow-up after treatment, PCR on blood had low sensitivity to detect treatment failure. In cerebrospinal fluid, PCR positivity vanished slowly and was observed until the end of the 2 year follow-up in around 20% of successfully treated patients. CONCLUSIONS/SIGNIFICANCE:For T.b. gambiense sleeping sickness diagnosis and staging, PCR performed better than, or similar to, the current parasite detection techniques but it cannot be used for post-treatment follow-up. Continued PCR positivity in one out of five cured patients points to persistence of living or dead parasites or their DNA after successful treatment and may necessitate the revision of some paradigms about the pathophysiology of sleeping sickness