Chemotherapy Strategies for Treating Human Trypanosomiasis in Africa

This report provides a detailed analysis of chemotherapy strategies employed in the treatment of Human African Trypanosomiasis (HAT), also known as sleeping sickness, a disease prevalent in sub-Saharan Africa. It begins by highlighting the challenges in disease prevention and the reliance on chemotherapy due to antigenic variation of the parasite. The report then examines the pathogenesis of HAT, differentiating between the early and late stages of infection, and explaining how the parasite evades the host's immune response. A significant portion of the report is dedicated to discussing the chemotherapeutic drugs used, including eflornithine, pentamidine, suramin, melarsoprol, and the Nifurtimox-eflornithine combination (NECT), detailing their mechanisms of action at the molecular level. The report explores assays like DNA staining and flow cytometry, to understand how these drugs impact the trypanosomes. The report concludes by summarizing the current state of chemotherapy in HAT treatment, emphasizing the importance of understanding drug mechanisms for improving therapeutic outcomes, and suggesting future research directions.

Running Head: TREATMENT OF HUMAN TRYPANOSOMIASIS IN AFRICA BY
CHEMOTHERAPY STRATEGIES
Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Name of the Student:
Name of the University:
Author’s Note:

1Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Abstract
Chemotherapy continues to majorly impact the reduction of the disease burden caused by
trypanosomatids. But it should be noted that the mode of action of the anti-trypanosomal
drugs remains completely clear and is only partially characterised. There are five current
drugs that are used for the treatment of the Human African Trypanosomiasis (HAT), they are
eflornithine, pentamidine, suramin, melarsoprol and Nifurtimox-eflornithine combination
(NECT). The assays that were exploited to detect the mechanism of action of the current
drugs included DNA staining followed by visualising the results by microscopy as well as
quantitative image analysis, flow cytometry, TUNEL for monitoring the DNA replication.
Eflornithine is an ornithine decarboxylase inhibitor. Melarsoprol inhibits mitosis. Nifurtimox
caused reduction in the abundance of the mitochondrial protein. Pentamidine causes
progressive loss of the kinetoplast DNA as well as perturbs the membrane potential and
suramin inhibits the cytokinesis. Cytology based profiling aids in effective understanding of
the mechanism of action of the drugs.

2Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Table of Contents
Introduction...........................................................................................................................3
Background...........................................................................................................................3
Aims......................................................................................................................................4
Discussion.............................................................................................................................4
Pathogenesis of Human African Trypanosomiasis (HAT)................................................4
Chemotherapeutic drugs against Human African Trypanosomiasis (HAT).....................5
NECT: Nifurtimox–Eflornithine Combination Treatment................................................8
Mode of Action of the Chemotherapeutic drugs at the Molecular Level........................10
Perspective...........................................................................................................................16
Conclusion...........................................................................................................................16
Summary.............................................................................................................................17
References...........................................................................................................................18

3Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Introduction
Human Africa trypanosomiasis (HAT) also known as sleeping sickness is a centuries-old
disease that negatively impacted the economy as well as the physical suffering in the sub-
Saharan Africa. Statistical data states that approximately 50 million people are at risk of
acquiring the disease considering an area of 10 million square kilometres (Büscher et al.,
2017). The major concern is that there is no effective vaccination for the prevention of the
disease. The creation of vaccines targeting trypanosomes is also a challenge due to the
antigenic variation observed in the species (Keating et al., 2015). This is the reason how the
parasite can evade the immune responses. There is only availability of chemotherapies that
account for the anti-trypanosomiasis measures. This article enlightens on the various
chemotherapeutic drugs exploited for effective treatment of trypanosomiasis.
Human African trypanosomiasis (HAT) also called as sleeping sickness, causes infection
by the parasite Trypanosoma that is transmitted by the vector tsetse fly. The parasite has two
subspecies, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense; and it can
lead to fatal consequences if left untreated (Wangwe et al., 2019). Of late there is an observed
reduction in the number of cases that has been reported of patients affected with African
trypanosomiasis but the treatment is a challenge for the clinicians. The treatment of the CNS
stage disease is considered to be toxic hence, diagnostic staging for differentiating the early
stage from the late stage of the disease when the CNS is invaded is indeed crucial but is
challenging. Eflornithine is combined with nifurtimox drug and acts as the first-line treatment
for late-stage Trypanosoma brucei gambiense. New drugs are in pipeline for the treatment of
CNS human African trypanosomiasis, and is giving rise to the cautious optimism (Santos et
al., 2015).

4Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Background
Human Africa trypanosomiasis (HAT) is a disease that is caused by hemoflagellates
belonging of the subgroup of Trypanosoma brusei known as Trypanosoma brusei gambiense
and Trypanosoma brusei rhodesiense which are the Gambian and the Rhodesian form
respectively (Rassi et al., 2017). Trypanosoma brusei gambiense is the first subspecies
responsible for the disease and it accounts for almost of the 98% of the reported cases of
sleeping sickness and is also responsible for cause of chronic infection (Jones and Avery,
2015). Trypanosoma brusei rhodesiense is responsible for causing acute infection and
accounts for only 2% of the cases reported (Kennedy, 2019). The tsetse fly, Glossina sp. is
the vector and aids in transmission of these parasites between the domestic as well as the wild
animals and the human beings. The other modes of infection can be accidental infection in
the laboratory or mother to child infection (Stich, 2015).
Aims
The aim of this article is to understand the action of the chemotherapeutic drugs against
the human African trypanosomiasis at the molecular level and discover the most effective
chemotherapy for the treatment of the disease.
Discussion
Pathogenesis of Human African Trypanosomiasis (HAT)
There are two stages in the clinical course of Human African Trypanosomiasis (HAT),
they are the early stage or the first stage, at this stage the parasite is present in the peripheral
circulation of the infected individual and it has not invaded the central nervous system (CNS)
(World Health Organization, 2019). In the second stage also known as the late stage in which
the parasite invades the blood-brain barrier leading to infection of the central nervous system.
The early stage causes eliciting an immune response against the pathogen (Chappuis, 2018).

5Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
At this stage there are uninterrupted waves of the parasitaemia, which leads to survival of
some parasites that evading the immune response, subsequently. The trypanosomes exploit
antigenic variation to escape the immune response (Bottieau and Clerinx, 2019). In the
process of antigenic variation, the trypanosome switches the variable surface glycoprotein
(VSG) coat to a new VSG coat which is not recognized by the immune system of the host.
This is a continual action that exhausts the defence system of the host. The process of the
evasion also involves the endocytosis of VSG-antibody complexes that allows the complexes
to escape the process of detection by antibodies that are involved in the complement-
mediated killing (Fairlamb and Horn, 2018). The mechanism of antigenic variation ensures
sufficient time for the trypanosomes to spend in the host system promoting the proliferation
and the transmission to other host system through tsetse fly that serves as the vector for
transmission of these trypanosomes. This causes infection of host systems that been already
infected with the trypanosomes and recognises the VSGs (West, 2019). The symptoms of
early stage of the disease involves fever, headaches, joint pain and irritation of the skin at the
site of infection which is a rare case though. The most important symptom is the fever which
lasts for almost about a week and may recur in a span of days or a month. The waves of
parasitaemia and the counter responses of the immune system triggers the fever (Wangwe et
al., 2019) (Santos et al., 2015).
The later stage of the trypanosomal infection is also called as the meningo-encephalitic
stage, which involves parasite invading the blood–brain barrier, entering the CNS and settling
in cerebrospinal fluid (CSF) (Winkelmann and Raether, 2016). The second stage symptoms
of infection involve confusion and poor coordination, tremors, general motor weaknesses,
irritability as well as aggressive behaviour. The most significant symptom of second stage of
the infection is disruption of the natural circadian sleep or the wake rhythm of the body, this
results in irregular and fragmented patterns of sleeping (Docampo and Moreno, 2017). Hence

6Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
the name of the disease is ‘sleeping sicknesses. If the disease is left untreated or it is treated
inadequately, the HAT infections can result in death. The subspecies of Trypanosoma brusei
determines the acute or chronic nature of trypanosomiasis infection.
Chemotherapeutic drugs against Human African Trypanosomiasis (HAT)
The effective chemotherapeutic drugs exploited to treat trypanosomiasis in the early stage
includes Pentamidine which is a water soluble aromatic diamidine which has been used since
the early times of 1930s (Steverding and Rushworth, 2017). This drug is effective against
Trypanosoma brusei gambiense but it is comparatively less effective in Trypanosoma brusei
rhodesiense. The treatment in the early stage exploits the use of 7-10 intramuscular
injections. The African trypanosomes exploits the nucleoside transporter P2 that takes up the
drug pentamidine. There are two other transporters discovered of late that uptake pentamidine
to atleast 50% (Strauss et al., 2018). Pentamidine impacts the kinetoplast that is the
mitochondrial DNA but is ineffective on the nuclear DNA. It is also observed that
pentamidine is also considered as a reversible inhibitor of the S-adenosylmethionine
(AdoMet) decarboxylase, which is an enzyme in the polyamine biosynthetic pathway but it is
not the primary mechanism of action (Santos et al., 2015). But it should be noted that this
chemotherapeutic drug does not kill the trypanosomes and even post treatment with the drug
it causes them to persist in the bloodstream forms.
Berenil is a diminazene aceturate that was discovered by Hoechst is an aromatic diamidine
exploited for treatment of bovine trypanosomiasis. This chemotherapeutic drug is effective in
the early stages of Trypanosoma brusei gambiense and Trypanosoma brusei rhodesiense
(Wenzler et al., 2016). This drug can be used in combination with melarsoprol for the late-
stage disease. This drug also acts in the kinetoplast DNA and intervenes with RNA editing
and trans-splicing (Mazzeti et al., 2018). In comparison to pentamidine, berenil is more
effective and acts as a non-competitive inhibitor of S-adenosylmethionine decarboxylase. It

7Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
results in elevation of the putrescine and elevation of the spermidine content in the parasite. It
is taken up by the P2 nucleoside transporter like pentamidine. But the use of berenil in
comparison to the pentamidine is well tolerated in humans (Rathore et al., 2016).
Suramin is a polysulphonated naphthylamine based compound which is derived from
naphthylamine dyes like trypan red and trypan blue. It is observed that the effectivity of the
drug is comparatively more when administered in the patients intravenously via injection than
by oral administration (Lombardo and Batlle, 2018). It is not absorbed by the intestines when
administered orally. This chemotherapeutic drug has several side effects as well like renal
problems, anaemia, nausea and anaphylactic shock. It is effective against the bloodstream
forms of the parasite (Fairlamb and Patterson, 2018). This chemotherapeutic drug acts on the
glycosomal enzymes that is exploited in the glycolysis pathway. This drug has been proved to
be highly effective with no resistance on Trypanosoma brusei rhodesiense. There is no
resistance against suramin is majorly because it inhibits multiple enzymes as well as
numerous metabolic pathways (Singh et al., 2016).
Melarsoprol is an organic arsenical drug was extensively exploited until the usage of the
combination treatment of nifurtimox eflornithine that got introduced in the medical world in
2009 (Leroux and Krauth-Siegel, 2016). The major cause of the replacement of this drug was
due to the elevated levels of toxicity exhibited by this chemotherapeutic agent. It is
considered to be a potent trypanocide. This drug has been proved to be effective against
Trypanosoma brusei rhodesiense and it is evident based on the literature studies that 96% of
the diseases are cured after a year of treatment with this antibiotic (Njamnshi, Gettinby and
Kennedy, 2017). This chemotherapeutic drug intervenes with the process of redox
metabolism as well as the glycolysis in the trypanosomes and causes effective lysis of the
same.

8Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Alpha difluromethylornithine, DFMO also known as eflornithine is considered as the
newest of the anti-trypanosomiasis mono therapies. It is claimed to be effective in the first
and the second stage of the disease that has been caused by Trypanosoma brusei gambiense
but it is not effective for the treatment of the Trypanosoma brusei rhodesiense (Franco,
Scarone and Comini, 2018). It is also considered as the potential chemotherapeutic agent for
the treatment of cancer. It also inhibits the enzyme ornithine decarboxylase (ODC), that are
exploited for the synthesis of polyamines (Epting et al., 2017).
Table 1: Five most recent effective chemotherapeutic drugs against Human
African Trypanosomiasis (HAT)
Drugs Mechanism Advantages Disadvantages
Pentamidine
(Pentamidine
isethionate)
This drug
accumulates in the
trypanosomes and
disrupts the
mitochondrial
process.
This drug is effective
against the stage I
Trypanosoma brucei
gambiense
It is ineffective
against stage II
Trypanosoma brucei
gambiense and both
stages of
Trypanosoma brucei
rhodesiense
Suramin
(Bayer 205,
Germanin)
This enzyme binds to
the glycosomal
enzymes and
interferes with the
glycolysis.
This drug is effective in
the stage I infection by
Trypanosoma brucei
rhodesiense
It is ineffective
against both the
strains of
Trypanosoma brucei
in the stage II of the
disease.
Melarsoprol
(Mel B)
It intervenes and
impacts the
trypanosomal redox
metabolism and the
process of glycolysis.
This drug is effective
against both the
subspecies and in either
of the stages.
This drug is toxic and
causes post treatment
reactive
encephalopathy
(PTRE). The
resistance of
trypanosome is

9Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
considered to be high
of about 30%.
Eflornithine
(difluoromethy
lornithine)
It causes inhibition of
ODC, also disrupts
the proliferation and
vulnerability to the
oxidative attack.
This drug is effective
against stage II
Trypanosoma brucei
gambiense
The treatment with
this drug is time
consuming and is
ineffective against
both the stages of
Trypanosoma brucei
rhodesiense
NECT
(Nifurtimox-
eflornithine
combination)
Eflornithine inhibits
the ODC and
Nifurtimox induces
it.
This has a high cure rate
for both the stages of
Trypanosoma brucei
This combination
drug has a potential to
be resistant to the
treatment executed in
the field.
NECT: Nifurtimox–Eflornithine Combination Treatment
NECT, Nifurtimox–Eflornithine Combination Treatment is considered as the most recent
chemotherapy against Trypanosoma brusei gambiense infection. Literature studies have
already shown that Nifurtimox has a synergism level with melarsoprol and this combination
therapy has also shown to be more effective in the treatment of trypanosomiasis, with
minimal or almost negligible relapses in comparison to the nifurtimox and the melarsoprol
monotherapies (Baker and Welburn, 2018). A major concern about utilization of nifurtimox–
melarsoprol combination therapy is that it is safe. The safety of this combination drug therapy
might be a question since there are lack of more published research data. There had been a
comparative analysis of the combination therapy of nifurtimox-melarsoprol and nifurtimox-
eflornithine was executed in a randomized clinical trial. The results from the trial exhibited
that the combination therapy of nifurtimox–eflornithine combination therapy was far better in
comparison to the combination therapy of nifurtimox–melarsoprol with respect to both

10Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
effectiveness as well as safety (Wilkowsky, 2018). The report of this clinical trial paved way
for the future tests that involved a case series that happened in Uganda, which is a phase III
randomized clinical trial in Republic of Congo and most recently, a phase III non-inferiority
trial, that compared the NECT to the standard regimen of eflornithine monotherapy
(Steverding, 2015). All the three experimental trials that were executed were a part of a wider
neglected tropical diseases control programme that was initiated and funded by the Médecins
sans Frontieres. The experiments subsequently were followed by report that stated the
addition of the NECT to the list of important medicines of the World Health Organization for
the treatment of neglected tropical diseases (Franco et al., 2017).
It is to be noted that shortly post the inclusion of NECT, it was distributed amongst the
different NSSCPs in the countries endemic for Trypanosoma brusei gambiense. The
treatment regimen of the NECT involved daily three oral doses of the drug nifurtimox for ten
days totally and fourteen infusions of the drug eflornithine for total seven days. This was
comparatively as well as significantly lesser than the fifty-six doses in a span of fourteen days
that is required for the eflornithine monotherapy treatment (Franco et al., 2017). It was also
noted that the medical care giver could efficaciously and effectively utilize the combination
therapy who previously was trained in utilization of the eflornithine drugs. Lastly, the
reduced dosages of each of the drug meant that more drug quantity could be transported at
comparatively lesser cost, when compared to only eflornithine (Aksoy et al., 2017). Hence,
all these advantages cumulatively made NECT the frontline drug for treatment of the stage II
T. b. gambiense and even accounted for almost 59% of all the considered cases in the year
2010. But the main disadvantage of the treatment with NECT is that it is comparatively
labour-intensive and implementation logistically is complicated (Mesu et al., 2018). The
treatment regimen mentioned previously exploits at least four nurses to provide the
eflornithine infusions to patients. It should also be noted that doctor should firstly prescribe

11Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
the therapy and post administration observe the adverse reaction of the patient. The medical
personnel are required to have specific training in handling eflornithine is also a factor that
needs to be considered. The drug therapy has side effects like vomiting, nausea, headaches,
abdominal pain, joint pains, insomnia and seizures. But the side effects are comparatively
lesser comparison to the other drugs. Fortunately, the side effects are less severe than the
previous drugs (MacGregor et al., 2019).
Mode of Action of the Chemotherapeutic drugs at the Molecular Level
In the combination treatment of NECT, Nifurtimox is exploited for the treatment of the
sleeping sickness in the second stage and it causes the involvement of the central nervous
system. The combination therapy with melarsoprol was initially used but due to the toxicity
caused by the later in the system the combination is replaced by nifurtimox-eflornithine
combination therapy which is safer to use and comparatively easier than the usage of
eflornithine alone. Hence, NECT is considered as the first line treatment for the second stage
of African trypanosomiasis also known as sleeping sickness (Spaulding, Gallerstein and
Ferrins, 2019).
Nifurtimox and fexinidazole both are nitro pro-drugs that gets activated by the putative
ubiquinone nitro reductase (NTR) that is located in the mitochondria of the parasite. The
chemotherapeutic drug, Nifurtimox aids in formation of a metabolite that is a nitro-anion
radical in the body of the host that reacts with the nucleic acids of the pathogen and results in
breakdown of their DNA (Njamnshi, Gettinby and Kennedy, 2017). This drug is similar to
the nitrofuran agents which are antibacterial agents possessing a furan ring with a nitro group.
The nifurtimox undergoes a reduction reaction and aids in creating an oxygen radical like the
superoxide (Mesu et al., 2018). These radicals are toxic to the pathogen. It should be noted
that the mammalian cells are protected from the superoxide due to the presence of the
catalase, glutathione, superoxide dismutase and peroxidases. The accumulation of the