Diagnosis and treatment 1. Diagnosis

Parasitaemia is very different between T.b. gambiense and T.b. rhodesiense infection.

In the first case it is usually very low (<100 parasite per mL) which is very difficult to observe under microscope, whereas in the second case the amount of trypanosome is usually higher. Moreover parasitaemia usually fluctuates during the infection due to the immune response of the infected host and the ability of the parasite to evade the immune response by quickly exchanging the antigen present on the surface, rendering the detection more complicated Figure 4 [2].

The diagnosis and the treatment of the diseases caused by the two parasites form are very different. Observation of early symptoms such as swollen lymph node, repeated fever and neurological signs are not enough to start a treatment but can induce the suspicion in order to further investigate the patient. Moreover, since serological tests are also not 100% reliable, it is still important to confirm the infection by observing parasites in the body fluid.

Several serological tests are available to detect gambiense HAT. However, depending on feasibility, time consuming, material requirements and costs, they can be applied directly in the field (usually rural regions of Africa), or only in specific laboratories with specific equipment.

Most of these tests rely on the presence of two antibodies directed against two specific antigens (VSG antigen type LiTat 1.3 and 1.5) of T. b. gambiense. The card agglutination test (CATT) is a very fast, inexpensive and simple test developed in the late 1970s with a sensitivity around 87-98% and a specificity around 93-95%. It has been largely used in all control programs for serological screening of the population [69-71]. However, its use in remote areas is difficult due to the need of access to electricity and to continuously maintain the kit at cold temperatures. Several other tests relying on the same detection of specific antigens, using ELISA based immunofluorescence assays, also need expensive equipment, like fluorescent

microscopes and require electricity, high amounts of water or specific storing conditions that cannot be ensured in the field [2]. A recent test called Rapid Diagnostic test (RDT) overcomes all these problems. RDT is an immunochromatographic test that detects specific antibodies of a T. b. gambiense infection from a fresh blood drop in only 15 minutes without the need of specific equipment or specific storage conditions.

In September 2017 the second generation of RDT test (SD BIOLINE HAT 2.0) developed by FIND and supported by the Bill & Melinda Gates Foundation, UK aid from the UK government and the Swiss government was commercially launched [72].

In order to confirm the presence of the parasite, the blood or lymph fluid of a patient being positive at the serological or clinical level need to be observed under the microscope. However, these observations are very time-consuming and due to the usually low parasitemia of gambiense HAT, false-negative results occur very often. For this reason, absence of observation of trypanosoma under a microscope cannot completely exclude the possibility of an infection [2].

In some cases, molecular detection of trypanosoma DNA or RNA can be performed in order to confirm the presence of the parasite. These tests can be performed also on stored samples and are therefore a very interesting test to perform when live parasite detection fail to demonstrate the presence of parasites. Molecular detections rely on the specificity of one single copy gene, the TgsGP, which if detected on agarose gel after PCR amplification, will confirm the presence of T.b. gambiense [4, 73, 74].

In the case of rhodesiense HAT due to high antigenic variation (higher compared to the gambiense subspecies) CATT screening cannot be used as there is no specific VSG gene that can be used for diagnosis. For molecular detection the technique is based on the detection of the SRA gene which is specific to this subspecies. However, compared to gambiense HAT, rhodesiense infection usually results in a much higher parasitaemia (up to 10 000 trypanosomes/ml), so microscopic detection of parasite is much easier [2].

In order to be able to give to the patient the right treatment, it is mandatory to know the species and also of the stage of infection. In both cases, analysis of the cerebrospinal fluid (CSF) for detection of trypanosomes is necessary [75]. Since lumbar puncture is a very invasive technique it is only performed once parasitemia has already been

demonstrated or when strong clinical or serological sign are present. Before performing lumbar puncture, in particular if a T. b. rhodesiense infection is suspected, a dose of suramin is administered upfront in order to clear the blood and reduce the risk of introducing the parasite in the CSF during the manipulation. The CSF is then analysed for the presence of white blood cells and/or parasites in order to establish the progress of the disease [2].

Diagnosis outlook: Simple and rapid tests available to use in the field have been a huge progression in the control and screening of HAT. Improvement in existing test in order to render them more sensitives, less expensive and easy to use in the field are some of the concern that are taken into account for the future [76]. One important progress has been the development of Loop-mediated isothermal amplification test (LAMP) [77]. This technique relies on the detection and amplification of a specific DNA sequence by a set of specific primers used at an amplification temperature between 60-65°C. The result can be observed by simple observation in the colour change in the mixture, and the specificity and sensitivity has been proven to be very high. The adaptation to detect the specific gene marker TgsGP and SRA for gambiense and rhodosiense HAT, respectively, is in progress. Cost-efficacy compared to standard methods and sensitivity level in patient samples need to be proven before the implementation of the test in the field [2, 74].

Since 2009 a bank collecting blood, serum, CSF, saliva and urine from infected and uninfected patient from endemic areas was created by the WHO and made accessible to researchers in order to improve the diagnostic tools. [43]

1.2.3.2. Treatment

The early detection of an infection is very important to avoid disease progression to the second stage for which the therapy is risky and often unsuccessful [43]. At the present time only 5 drugs are available for fighting HAT. The currently used drugs were mostly discovered in the first half of the 20^th century. All of these drugs have serious disadvantages as they are very toxic, difficult to administer or may not cross the BBB which makes them only active against the treatment of the first stage of the disease.

For this reason, it is crucial to know the stage and the parasite subspecies to choose the right drug treatment. [2, 12].

The drugs currently used and chemotherapeutic treatment of HAT are eflornithine, melarsoprol, nifurtimox, pentamidine and suramin (Figure 8 and in Table 1)

Figure 8 Chemical structures, first year of publication of drugs use for HAT treatment and brand name.

Pentamidine: This molecule was discovered in 1940 and is used for the treatment of the first stage of T.b. gambiense infection. The mode of action of the pentamidine is unknown, however, some possible mechanisms such as binding to nucleic acids, disruption of kinetoplast DNA, inhibition of RNA-editing and inhibition of mRNA transplicing or inhibition of the plasma membrane CA²⁺-ATPase have been hypothesized [78, 79]. The treatment with pentamidine consists in one intramuscular injection per day over one week. The reported adverse reactions to the treatment are site pain, transient swelling, abdominal pain, gastrointestinal problems and hypoglycaemia (in less than 40% patients) [80-82].

Suramin: Suramin was discovered in 1920 and is used to treat first stage of T.b.

rhodesiense infection. However, the treatment is long, needing 5 slow intravenous injections (after the first test injection), one every 7 days. Nephrotoxicity, peripheral neuropathy and bone marrow toxicity accompanied by agranulocytosis and thrombocytopenia are possible rare and reversible adverse reactions to the drug [12].

As for pentamidine, the mode of action of suramin is unknown.

Melarsoprol: Melarsoprol was discovered in 1949 and is the only drug being active against second stage of T.b. rhodesiense infection, but can also be used for second stage of T.b. gambiense infection, when a different treatment regimen is considered.

For T.b. gambiense one intravenous injection per day over one week is necessary whereas for T.b. rhodesiense several protocols of injection and duration of treatment are possible [12, 83]. The melarsoprol treatment is often followed by adverse reactions that can be severe or even life-threatening. Encephalopathic syndrome, the main side-effect after drug intake, is present in 5 and 8 % of the case in T.b. gambiense and T.b.

rhodesiense infections respectively with 50% of fatality cases observed [12, 84].

Recently, about 30% of treatment failure in several foci has been reported, indicating the appearance of resistance to the drug [12, 85].

Eflornithine: Eflornithine was the only drug with antitrypanosomal activity discovered during the last 50 years, in 1981, and is used for the treatment of second stage T.b.

gambiense infection. Compared to melarsoprol it has a low mortality rate. The major problem of the drug is the accessibility to the drug and the treatment, which is very complicated. In fact, because of the very short half-life of the drug, the treatment consists in one infusion every 6 hours for 14 consecutive days. This prevent the use of eflornithine in most rural areas where the treatment is needed. The effect of the drug can induce bone marrow toxicity, leading to anemia, leucopenia and thrombocytopenia (25-50%), gastrointestinal symptoms (10-39%) and convulsions (7%) [12, 86-89]. The use of Eflornithine for T. b. rhodesiense infections is not possible due to the resistance of this subspecies to the drug.

NECT: Several combination cocktails for fighting HAT have been tested. The combination of officials drugs with other registered drugs used for related diseases were studied. Nifurtimox, a drug used to treat T. cruzi infections (Chagas disease in South America), combined with either eflornithine or melarsoprol were studied in the past. Only the combination of nifurtimox and eflornithine (NECT) showed improvement over the standard of care treatment. The NECT treatment reduced the amount of medication given and the duration of the treatment, thereby reducing side effects [12, 90, 91]. This new NECT combination was included in the first line of treatment for second stage gambiense HAT in May 2009 by WHO. [92] However, due to the complexity of the administration, some areas without trained staff still rely on melarsoprol treatment [2, 93, 94].

Table 1 Current chemotherapeutic treatment for HAT.

Treatment: Intramuscular injection of 4mg/kg daily for 7 days

ADR: Usually well tolerated. Possible observation of hypotension, dizziness and sometime collapse and shock (maximal dose of 1g per injection) [55]

ADR: Usually considered very safe. encelophathy (induce death in 5% of the patients treated), gastrointestinal and skin reactions, heart failure are common

Nufurtimox: 5mg/kg every 8h for 10 days ADR: Eflornithine: bone marrow toxicity, gastrointestinal symptoms and

New drugs: preclinical and clinical outlook

The research of new drugs for treatment of neglected tropical diseases such as Human African Trypanosomiasis is supported by Non-profit Organizations (NPO), governments and major private foundations (Wellcome Trust, Sandler Center for Basic Research and Bill & Melinda Gates Foundation). Pharmaceutical companies, which are usually at the forefront of drug discovery, are not interested in funding research with little or no commercial potential. [95, 96] In the past 30 years only 1% of the approved drugs were against neglected diseases (the major neglected disease being malaria and tuberculosis) whereas these diseases are responsible for 11.4% of the global disease burden. In order to overcome this problem new models for drugs discovery were invented. Non-governmental organizations (NGOs), such as Drugs for Neglected Disease initiative (DNDi), and Public-private partnership (PPPs), when a common interest is found, aim to bring together the research capacities of academia and Pharma in order to identify new drugs. The complexity of these collaborations results in consortia of funders that guide the work of consortia of researchers working at different stages of drug development (Figure 9). [95]

Figure 9 Outline of the drug discovery and development process and some key consortia involved at the different stages. The composition of consortia comprising researchers from academia and industry can vary over time.

Figure taken from [95]

A third model, consisting of academic, open-source networks driving drug discovery without the collaboration of an industrial partner, has risen (e.g. The Sandler Center for Basic Research in Parasitic Diseases and the tropical disease initiative at the University of Dundee).

The ideal new drug should be active when given orally, and act against both stages and infections, be safer and stable at room temperature. This would allow administration in remote areas, thus avoiding hospitalization and making lumbar puncture unnecessary [97, 98].

Fexinidazole: The new candidate for treatment of both first and second stage of gambiense HAT was first synthesized in the 1970s and proven to have trypanocidal activities, but it was not before 2006 that its development was really taken into account by DNDi. Great results were shown when tested in vitro on chronic and acute mice models of HAT diseases [99, 100]. In 2009 fexinidazole, a 5-nitroimidazole drug entered human phase-I study and in late 2012 it entered phase-II/III clinical trials with a dose of 1800 mg per day for 4 days followed by 1200 mg per day for 6 days. However, some concerns about cross-resistance between nifurtimox and fexinidazole were raised after observation of fexinidazole resistance in nifurtimox resistance T. brucei strains [101, 102]. Further investigations in order to clarify the resistance of fexinidazole are needed. Following 2016 updates on DNDi website, fexinidazole is currently in phase-IIIb clinical trials and results are to be expected soon. The treatment used is a 10 day oral treatment which is the most simple treatment for HAT disease so far [103, 104].

Benzoxaboroles: 50 compounds of the new class of boron containing molecules were analysed for their activity against HAT in a project led by Anacor, Scynexis and DNDi.

One candidate, an orally active benzoxaborole SCYX-7158, was identified as a lead candidate for first and second stage HAT. Indeed, murine studies showed activity against both gambiense and rhodesiense trypanosomes and against both stages of the disease. Moreover, in vitro absorption, distribution, metabolism, elimination and toxicology (ADMET) have been studied and confirm SCYX-7159 as a lead candidate for treating stage 2 HAT due to its stability, ability to cross the blood-brain barrier and oral availability [105]. In 2012 SCYX-7158 entered phase-I of clinical trials and is currently in phase-II/III [105, 106]. The study which is expected to be completed in 2020 consist of 3 tablet orally administrated on day 1 treatment [107].

Pafuramidine maleate (DB289) and analogs: After reaching successfully phase-I/III of clinical trials, the newly discovered oral drug for treatment of first stage HAT failed due to unexpected liver and renal toxicity. These results forced the study of pafuramidine to be stopped [108, 109]. However, studies by the consortium for parasitic drug development (CPDD), new aza-analogs of pafuramidine were performed, and compound DB829 has evolved as a promising candidate for treating both stages of HAT. Mouse experiment showed great efficacy of DB829 on both gambiense and rhodesiense infections at low doses. Second stage model of Vervet monkeys were also successfully treated with intramuscular injection of DB829 [110].

For all these reason DB829 is an interesting candidate for further development since a single dose regimen for first stage and 5 consecutives days intraperitoneal injection for second stage infections showed great results in mouse models [2, 111, 112].