Diagnostic Tools for Human African Trypanosomiasis Elimination and Clinical Trials: Early Test-of-cure

In the last decade, the prevalence of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) has fallen and HAT has been targeted for elimination. Development of safe and efficacious drugs for HAT, applicable in an elimination context, is considered as a high priority. The drug developmental process is however slowed down by the need to follow-up treated patients for 18 months to decide on cure. For timely diagnosis of treatment failure in clinical trials, patients should have control visits with follow-up examinations at 6, 12 and 18 months after treatment. Furthermore, due to repeated lumbar punctures, treated patients refrain to present for control visits spontaneously, and tend not to comply with follow-up. Clinical trials on new drugs for HAT would therefore be accelerated by availability of an early test of cure. Trypanosomal spliced leader (SL)-RNA, neopterin \& 5-hydroxytryptophan are good candidates for accurate and shortened treatment follow-up. In particular SL-RNA detection in blood offers an opportunity for non-invasive post-treatment follow-up.

The objective of the DiTECT-HAT-WP4 study is to validate the diagnostic performance of cerebrospinal fluid neopterin \& 5-hydroxytryptophan quantification and of blood and cerebrospinal fluid trypanosomal spliced leader RNA detection for assessing treatment outcome. The DiTECT-HAT-WP4 study is embedded into an ongoing therapeutic phase II/III study (DNDi-OXA-02-HAT) testing a new oral single dose drug against HAT. Within the Framework of the therapeutical trial, patients will have post-treatment examinations, including blood and cerebrospinal fluid examination at day 11, and during follow-up at month 6, month 12 and month 18. Combination of DiTECT-HAT-WP4 with this ongoing clinical trial allows evaluation of new treatment outcome assessment markers during follow-up without the need for additional lumbar or venipunctures. The volumes of venous blood and cerebrospinal fluid taken will be increased by 2.5 mls for the DiTECT-HAT-WP4 study.

Reverse transcriptase real time PCR for spliced leader RNA detection in blood and cerebrospinal fluid and neopterin detection will be carried out in the reference laboratory in Kinshasa, (index tests). The reference laboratory will be blinded to the results of the reference standard. For evaluation of diagnostic performance of the index tests, the reference standard will consist of classification of treatment outcome according to international standards applied for the clinical trial. Receiver operator curves, sensitivity and specificity of the different index tests for treatment outcome assessment will be determined at each follow-up time point. If sufficiently accurate, trypanosomal spliced leader RNA detection in blood would allow post-treatment follow-up without the need for lumbar punctures. Improved treatment outcome assessment will not only facilitate follow-up by avoiding the feared lumbar puncture but also speed up the development and implementation of new drugs. In addition, it will also improve management of patients in routine. The proposed research will impact on clinical decision and treatment outcomes, and contribute to successful HAT elimination.

• RNA and neopterin detection (Diagnostic test), also known as Reverse transcriptase real-time PCR Trypanozoon SL-RNA, Neopterin & 5-hydroxytryptophan EIA, Mybiosource: Detection of spliced leader RNA will be performed on blood and cerebrospinal fluid taken before treatment, 11 days after treatment, 6, 12 and 18 months after treatment. Neopterin and 5-hydroxytryptophan will be quantified in cerebrospinal fluid taken at the same time points.

Primary outcomes

• Sensitivity of SL-RNA detection in blood, SL-RNA detection in cerebrospinal fluid and of neopterin & 5-hydroxytryptophan quantification in cerebrospinal fluid for relapse after human African trypanosomiasis treatment [18 months]: Index tests: qualitative detection of SL-RNA in blood, qualitative detection of SL-RNA in cerebrospinal fluid, neopterin \& 5-hydroxytryptophan quantification in cerebrospinal fluid. Reference standard: classification according to the WHO 2015 criteria as relapse or probable relapse within 18 months after treatment for human African trypanosomiasis
• Specificity of SL-RNA detection in blood, SL-RNA detection in cerebrospinal fluid and of neopterin & 5-hydroxytryptophan quantification in cerebrospinal fluid for cure after human African trypanosomiasis treatment [18 months]: Index tests: qualitative detection of SL-RNA in blood, qualitative detection of SL-RNA in cerebrospinal fluid, neopterin \& 5-hydroxytryptophan quantification in cerebrospinal fluid. Reference standard: classification according to the WHO 2015 criteria as cure or probable cure 18 months after treatment

Secondary outcomes

• Sensitivity and specificity SL-RNA detection in blood for outcome assesment after treatment for human African trypanosomiasis [post treatment day 11, month 6, month 12 and month 18]: Index tests: qualitative detection of SL-RNA in blood post treatment day 11, month 6, month 12 and month 18. Reference standard: Human African trypanosomiasis treatment outcome classification according to the WHO 2015 criteria
• Sensitivity and specificity SL-RNA detection in cerebrospinal fluid for outcome assesment after treatment for human African trypanosomiasis [post treatment day 11, month 6, month 12 and month 18]: Index tests: qualitative detection of SL-RNA in cerebrospinal fluid at post treatment day 11, month 6, month 12 and month 18. Reference standard: Human African trypanosomiasis treatment outcome classification according to the WHO 2015 criteria
• Sensitivity and specificity by ROC analysis of neopterin & 5-hydroxytryptophan quantification in cerebrospinal fluid for outcome assesment after treatment for human African trypanosomiasis [post treatment day 11, month 6, month 12 and month 18]: Index tests: quantitative detection of neopterin \& 5-hydroxytryptophan in cerebrospinal fluid at post treatment day 11, month 6, month 12 and month 18. Reference standard: Human African trypanosomiasis treatment outcome classification according to the WHO 2015 criteria

Sex: All, ages: 15 Years to any

Inclusion Criteria:

* Eligible for participation in DNDi-OXA-02-HAT clinical trial

Exclusion Criteria:

* Excluded for DNDi-OXA-02-HAT clinical trial; No informed consent for participation in the DiTECT-HAT-WP4 study

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• Veerle Lejon, PhD, Principal investigator, Institut de Recherche pour le Developpement