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.