assessment of treatment Efficacy: Existing biomarkers of
treatment response and new advances
The appropriate markers of CD cure (i.e. a patient being free from CD
and not at risk of developing target organ involvement such as
cardiomyopathy, cardiac failure, mega-esophagus or mega-colon, etc.)
have been subject to intense debate for decades, in part due to
prolonged persistence of T.cruzi specific antibodies, lack of
sensitivity of parasitological tests, and need for long-term follow-up
(generally years or decades) to observe negative seroconversion of
conventional serological tests, as well as a general lack of
understanding of the parasite biology in the human and the kinetics of
drug response. Serology (and, in particular, negative seroconversion)
has been heralded for many years as the gold standard for treatment
response, largely guided by the successful results observed after
treating acute infections or early chronic infections in
children8,109 (see table 1 ). However,
treatment of older patients, or even children over 7 years of age, does
not lead to negative seroconversion for decades (if ever)8,40,42,109, even if a drop in antibody titers is
observed early after pharmacological treatment. This fact is easy to
understand, if one considers that persistent immune system stimulation
(e.g. as would be the case in chronic CD due to persistent antigen
shedding by deep-tissue T cruzi nests) is bound to generate
immune responses that would last for a long time even after complete
parasite clearance by NF or BZN.
Negative seroconversion continues to be the (somehow arbitrarily) chosen
method to ascertain a treatment response, both in general practice and
research. Reported serologic response rates are as high as 96% for
congenitally infected infants 8,109–111, 76% for
acute infections112, 63% 40,113 to
90% 113 for chronically infected children, and 37%
for chronically infected adults 114. These rates have
marked variability among different published studies due to different
serologic techniques employed, with sometimes poorly evaluated,
different sensitivities and specificities, used to determine treatment
response as the primary outcome of clinical
trials115,116.
It would be reasonable to consider that more sensitive serological
techniques would under-estimate time and rates of cure (i.e. would yield
positive antibody results with lower titers), with no correlation with
clinical outcomes such as organ impact, but this still requires more
research to confirm. In order to study the correlation between serologic
response and organ damage, a recent study of a pediatric cohort
performed long-term follow up of treated children with
electrocardiograms (ECG), 24 hours ECG (Holter) and Speckle-tracking
strain echocardiography and observed no CD untoward impact on heart
function in this population years after treatment, supporting the low
correlation between serological tests and clinical
response117. Also, T. cruzi detection tests
currently in use in some countries for long term follow up of patients
such as polymerase chain reaction against T.cruzi- DNA (PCR) or
different serology techniques, were initially developed for diagnostic
purposes. Furthermore, many of the methods used have been repeatedly
changed across the years, and comparison of results from recent clinical
studies to older studies involves a degree of uncertainty even if
comparing tests that are nominally the same (e.g. RT-PCR done in recent
years would have used primers and protocols very different to those used
10 years ago) 41,46. In this context, new markers of
cure are needed. Alternative early markers of cure have been suggested,
such as decrease of total anti-T.cruzi antibody titers (i.e.
instead of negative seroconversion) or use of non-conventional
serological techniques 118,119 such as specific lytic
anti-α-Gal antibodies known as anti-F2/3
antibodies120. Other CD biomarkers suggested by
scientific literature so far have been reviewed by different authors
too, but the general impression is that they all still require more
research, and validation. Table 2 summarizes the biomarkers
studied.46,121,122
PCR has been proposed as a sensitive and specific method to detectT.cruzi parasitemia in newborns41,123,124 and
has also shown good results for the assessment of treatment failure, as
a persistently positive result after treatment clearly is evidence of
failure to eliminate the parasite125. However, while
PCR may be more sensitive than current methods in some cases, the lack
of standardization of the method across centers is a still unresolved
issue. Furthermore, actual rate of false positives is still under
debate, and may vary among testing laboratories (and different
techniques used). Other issues such as cost and instrument availability
and technical skills, conspire to limit the use of this method at the
moment, but considering its good results so far and its feasibility of
being easily applied in clinical settings, the investment in improving
PCR methodologies is worthwhile. The CD community must focus on suitable
strategies for parasite DNA extraction in lower sample volumes, the
equivalence between blood and tissue parasitemia; the reduction of false
negatives, as well as the validation and standardization of PCR assays;
and the correlation of PCR readouts with negative
seroconversion.109,126–128
Considering all available evidence, we could conclude that despite the
need of trials in this area, a negative PCR -associated to a persistent
decrease of T.cruzi antibodies titers- should be the chosen
criteria used to assess treatment response and to follow-up after
treatment in our time.
Pharmacological Treatment: New treatment strategies and
Alternative drugs
As mentioned before, there are few recent advances in BZN and NF
pharmacology, which is disappointing considering their longevity. Some
improvements in drug formulation have been proposed (e.g. application of
nanotechnologies such as nanocrystals, polymeric nanoparticles, and
lipid nanostructures) as an attractive approach to improve solubility
and dissolution of BNZ and NFX, hopefully leading to dose reductions
and, perhaps, novel treatment schemes, but virtually no clinical
research has been undertaken with this proposed formulations129,130.
New potentially effective drugs have been proposed on the basis multiple
targets in the parasite cell. Ergosterol biosynthesis enzymes in
particular have been well studied, and CYP51 (sterol 14-Demethylase) was
proposed as an interesting target, both due to its importance in
parasite survival, and the availability of multiple medications already
in the market (i.e. azole antifungal drugs) that could be easily
repositioned for clinical trials in CD 104,131–134.
This repositioning approach is advantageous in view of the cost and
time-consuming process required compared to the development of new
medicines, especially in neglected diseases, since repositioned drugs
already have their toxicological and pharmacokinetic profile assessed
when used on their previous therapeutic target 135.
Unfortunately, only allopurinol and a few azoles have been studied in
clinical trials, observational studies, and case reports - there is an
ongoing randomized double-blind, placebo controlled trial being carried
(NCT03193749) comparing Amiodarone hydrochloride with placebo but there
are no preliminary results disclosed so far. Despite allopurinol has
shown to be useful in combination with NF or Benznidazole in small
trials, evidence is still insufficient136–138. From
azoles, posaconazole was compared in high and low doses versus placebo
and research results concluded it has an acceptable antitrypanosomal
activity, but also a significant increase in treatment failure compared
with BZN group 139. Another randomized
placebo-controlled trial in adults tested E1224 (a ravuconazole pro-drug
in different dosing regimens) and BZN versus placebo, and found that
E1224 + BZN group displayed a transient, suppressive effect on parasite
clearance, whereas BZN showed early and sustained efficacy until 12
months of follow-up. This transitory effect was shown only in high dose
sub-group while parasite levels in the low-dose and short-dose E1224
groups gradually returned to placebo levels 140. In
summary, from azole‘s research, some former promising repositionable
drugs such as monotherapy with ketoconazole, ravuconazole or
posaconazole has not proven to be efficacious for the treatment of
chronic T. cruzi infection 139–141 and the
combination of posaconazole and BZN did not provide any further efficacy
or safety advantages over BZN monotherapy 142,143.
Similarly, pre-clinical studies have identified interesting targets for
drug action including cruzipain (parasite lysosomal cysteine), B
citocrome, trypanothione reductase system, cyclophilins, N-myristoylome,
carbonic anhydrases and NMDA glutamate
receptor.143,144 However, none of these targets have
drugs in clinical trials yet, and the ever-mounting costs of drug
development and human clinical trials make it difficult to believe that
many new molecules for CD would be coming into the market in the
foreseeable future.
Fexinidazole is a drug previously repositioned for Trypanosoma
brucei gambiense infection (African trypanosomiasis) after
demonstrating effectiveness in a randomized controlled trial145. Also, fexinidazole‘s safety and pharmacokinetics
had been properly studied in humans, proving that oral administration is
safe and well tolerated 132,133,146. Considering this
drug is effective in clearing T.cruzi as well in pre-clinical
studies, an ongoing randomized, double-blind, placebo controlled trial
is being carried out in Argentina, Bolivia and Spain to assess its
efficacy in CD (NCT02498782).
Interestingly, some natural compounds and dietary supplements such as
microalgae extracts147, wasp
venom148, coumarins149, South
American Vernonieae150, curcumin151and Resveratrol152 have been also studied for
anti-tripanosomal activity, but more research is required to draw
conclusions, and there is still close to no human clinical data. The use
of natural compounds to treat known diseases might lead to effective
benefit-cost resources, considering that many of these compounds are not
subject to patent restrictions and may be widely available. However,
formal clinical testing should be performed before any of these
compounds is used in patients.104
In spite of a relative abundance of preclinical molecular candidates and
potential repositionable drugs, there are currently no new classes of
drugs in the clinical development pipeline for CD and BZN and NF remain
the only two available drugs for treatment with relatively solid
clinical data to support their use.