3.3. Natural course of CD infection
First, the course of infection was monitored during the acute-phase (Figure 4A ). Parasitaemia was detected from the 9th dpi, it showed the highest levels between the 22nd and 24th dpi, reaching more than 5 million BTs per mL of blood, and it was undetectable from the 45th dpi in all infected mice. Therefore, it can be established that the acute-phase ends on this day (Rassi Jr et al. , 2010; Pérez-molina and Molina, 2018).
However, parasites were detected at 20st dpi in 6 out of the 14 analysed organs/tissues, and 9 out of them were infected at the end of the acute-phase (40th dpi). The same organs/tissues were infected at 60th dpi.Figure 4B shows the PCR analysis of 14 organs/tissues for each experimental time. Therefore, this assay reveals the tropism of T. cruzi Arequipa for 9 target organs/tissues: adipose tissue, bone marrow, brain, oesophagus, heart, lung, muscle, spleen and stomach. Analysis of murine model has identified the gastrointestinal tract as a primary reservoir using different T. cruzi strains (Lewiset al. , 2014, 2016; Lewis and Kelly, 2016). Hence, the intestine may also be parasitized in some areas along its length using T. cruzi Arequipa, but we did not observe parasitization in the selected fragments for PCR. Other reason would be that the parasite load to be below the PCR limit detection (~1 parasite per 10 mg) (Francisco et al. , 2015), although three rounds of PCR were performed for all tissues/organs.
It should be noted that the chronic-phase begins when the amastigote forms are nested inside target organs/tissues (Guarner et al. , 2001; Rassi Jr et al. , 2010; Pérez-molina and Molina, 2018). Here, amastigote forms were already found nesting in 6 out of the 9 target organs/tissues at 20th dpi; hence, the chronic-phase begins when parasitaemia has not yet peaked. It should be highlighted that the chronic-phase is characterized by a low and intermittent parasitaemia (Bilate and Cunha-Neto, 2008; Rassi Jret al. , 2010; Pérez-molina and Molina, 2018), but not detectable by counting BTs. In addition, sophisticated parasite-detection bioluminiscence technologies are leading to a better appreciation of the spatiotemporal and quantitative dynamics of chronic infections (Lewiset al. , 2014, 2016, 2018), limited using the PCR method. In summary, acute-phase treatment should be performed once the parasitaemia is confirmed, that is, ~9th dpi; and parasitaemia should be monitored until ~55th dpi, when parasitaemia is not detected. For chronic-phase treatment, it should be performed from 70th dpi, when this phase is established. Thereafter, assessment of cure should be confirmed by PCR of the 9 target organs/tissues in late chronic-phase.