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.

Dog rabies control in West and Central Africa: A review.

Rabies is a neglected but preventable zoonotic disease that predominantly affects the most vulnerable populations living in remote rural areas of resource-limited countries. To date, every country on the African mainland is considered endemic for dog-mediated rabies with an estimated 21'500 human rabies deaths occurring each year. In 2018, the United Against Rabies collaboration launched the Global Strategic Plan to end human deaths from dog-mediated rabies by 2030. The epidemiology of rabies from most Western and Central African countries remains poorly defined, making it difficult to assess the overall rabies situation and progress towards the 2030 goal. In this review, we attempt to provide an overview of the current rabies situation in 22 West and Central African countries based on published scientific literature and information obtained from rabies focal points. To this end, information was collected on i) established surveillance, ii) diagnostic capacity, iii) post-exposure prophylaxis (PEP) availability and coverage, iv) dog population estimates, v) dog vaccination campaigns, vi) animal and human health communication (One Health), vii) molecular studies, viii) Knowledge, Attitude and Practices (KAP), ix) cost estimates and x) national control strategies. Although rabies is a notifiable disease in the majority of the studied countries, national surveillance systems do not adequately capture the disease. A general lack of rabies diagnostic capacity has an additional negative impact on rabies surveillance and attempts to estimate rabies burden. Recurrent shortages of human rabies vaccine are reported by all of the countries, with vaccine availability usually limited to major urban centers but no country has yet adopted the new WHO-recommended 1-week intradermal vaccination regimen. Most countries carry out subsidized mass dog vaccination campaigns on World Rabies Day. Such activities are indispensable to keep rabies in the public consciousness but are not of the scale and intensity that is required to eliminate rabies from the dog population. Countries will need to scale up the intensity of their campaigns, if they are to progress towards the 2030 goal. But more than half of the countries do not yet have reliable figures on their dog populations. Only two countries reached stage 2 on the Stepwise Approach towards Rabies Elimination ladder - indicating that their national governments have truly prioritized rabies elimination and are thus providing the necessary support and political buy-in required to achieve success. In summary, the sub-region of West and Central Africa seems to be divided into countries which have accepted the challenge to eliminate rabies with governments committed to pushing forward rabies elimination, while other countries have achieved some progress, but elimination efforts remain stuck due to lacking government commitment and financial constraints. The possibility to meet the 2030 goal without international solidarity is low, because more than two-thirds of the countries rank in the low human development group (HDI ≤ 152). Leading countries should act as role models, sharing their experiences and capacities so that no country is left behind. Unified and with international support it is possible to reach the common goal of zero human rabies deaths by 2030.

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer's protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

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.

Melarsoprol sensitivity profile of Trypanosoma brucei gambiense isolates from cured and relapsed sleeping sickness patients from the Democratic Republic of the Congo.

BACKGROUND: Sleeping sickness caused by Trypanosoma brucei (T.b.) gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba. METHODOLOGY/PRINCIPAL FINDINGS: Forty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP) 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1. CONCLUSIONS/SIGNIFICANCE: We conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.

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.

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.