The master mix (one per molecular target) was performed at a final volume of 15 μL which contained 1.5 μL of reaction buffer 10X (Invitrogen), 0.125 μL of dNTPs (10 mM), 0.365 μL of MgCl2 (25 mM), 0.5 μL of each primer (HSP70f and HSP70r; KDNAf and kDNAr, LITS and L5.8S and R223 and R333) (10 μM), 0.05 μL of Taq platinum DNA polymerase (Invitrogen) (0.1U) and 5 μL of DNA. PCR was performed on T100^TM Thermal Cycler (Bio-Rad) using thermal cycle conditions as follows: An initial denaturation of 95°C for 5 min followed by 40 cycles at 95°C for 1 min, 1 min at 60°C and 1 min at 72°C, with a final extension at 72°C per 10 min (For the LITS and L5.8S primers, the annealing temperature was 56°C). To determine the band size, the amplification products were run on 2% agarose gels and stained with Sybr Safe.

The master mix (one per molecular target) was performed at a final volume of 12 μL which contained 5.0 μL of Fast SYBR Green (Applied Biosystems Ref. 4385370), 0.6 μL of each of the same sets of primers used in PCR (shown above) and 2 μL of DNA. The thermal profile consisted of the first stage of 50°C for 2 min followed by 40 cycles at 95°C for 30 s and 15 s at 60°C. The qPCR was executed with a 7500 Fast Real-Time PCR System (Applied Biosystems). After the qPCR assay, a melting curve analysis was performed to detect any primer dimerization that could affect the efficiency of the assays.

Describes the ability of the tests to detect the existing diversity of blank DNA (Leishmania DNA). Therefore, we performed standard PCR and qPCR with the four molecular markers (HSP70, ITS-1, 18S and kDNA) with the conditions described above to all the DNAs of the six Leishmania reference strains within a single day.

Determines the non-response of DNA tests from closely related but not considered target sample sources. In this case, we selected microorganisms phylogenetically related to Leishmania and also those associated with differential diagnosis of CL. DNAs of parasites belonging to the order Kinetoplastida and obtained from a biological supply vendor (ATCC: The Global Bioresource Center) (ATCC PRA-330 Trypanosoma cruzi and ATCC 30032 Trypanosoma rangeli) and 8 microorganisms of differential diagnosis of CL (ATCC 25923 Staphylococcus aureus, ATCC 12344 Streptococcus pyogenes, ATCC 26033 Histoplasma capsulatum, ATCC 27294 Mycobacterium tuberculosis, ATCC 26329 Sporothrix schenckii and ATCC 18827 Fonsecaea pedrosoi) were subjected to conventional PCR and qPCR within a single day.

It refers to a range of concentrations in which the analyte can be determined with an adequate level of confidence and accuracy. To achieve this, seven serial dilutions (1 × 10⁴ to 1 × 10^-2 parasites equivalents/mL) of each DNA of the Leishmania species (six species) were taken and subjected to the two molecular platforms (PCR and qPCR) with each of the four genetic targets (HSP70, kDNA, 18S and ITS-1). Each dilution was amplified in triplicate within a single day. For the case of conventional PCR, the minimum dilution to which a positive result (present of amplification band in electrophoresis) was consistently generated was identified. For qPCR, the reaction efficiency was evaluated through linear regression analysis, by calculating: (i) the slope of the linear logarithmic phase of the reaction, representing the accuracy and reproducibility of the results (values between -3.0 and -3,6, with -3.32 considered the expected value, corresponding to 100% efficiency), (ii) Y-intercept, corresponding to the theoretical detection limit of the reaction and (iii) correlation coefficient (R²), as a measure of linearity of the obtained curves and reflection of the reproducibility. Amplification efficiencies for the qPCR were graphically represented using the program GraphPad Prism 7.

The LoD was calculated as the lowest dilution providing 95% positive results, as established by NCCLS standards (NCCLS, 2004). Five serial dilutions of each DNA of the six Leishmania species were used and subjected to both molecular platforms with each of the four genetic targets. The amplification of each dilution was performed with 8 replicates and during 5 consecutive days. The LoD was determined by Probit Regression (Probit Minitab 15 software, United States).

Intra-assay reproducibility was assessed in terms of accuracy for each test. A dilution above and below the LoD of each DNA of the 6 Leishmania species were evaluated in triplicate for 10 days (one run per day) under the same conditions. For qPCR: Mean, Standard Deviation (SD) and Coefficient of Variation (CV) were estimated. For PCR, the presence/absence of a band was considered as a result for the subsequent analyses.

We determined the analytical sensitivity for PCR in terms of ARR, LoD and accuracy (Figure 1 and Table 1). Santamaria et al. (2005) determined the LoD in 1 equivalent parasite in the PCR using kDNA (Santamaria et al., 2005), unlike the findings of this study, the LoD of this technique with the same marker was 1 × 10^-1 parasites equivalents/mL (Figure 1). These results are consistent with those described by other authors where is reported that kDNA presents high copy number and can detect up to less than one parasite in conventional PCR (de Bruijn and Barker, 1992; Mary et al., 2004; Francino et al., 2006; Oliveira et al., 2011; Gualda et al., 2015). The LoD for the assays directed to the 18S and ITS-1 markers was 1 × 10⁰ parasites equivalents/mL, similar results reported by Deborggraeve et al. (2008) where the technique was able to detect up to 1 parasite/180 μL. On the other hand, the LoD with the HSP70 marker was 1 × 10¹ parasites equivalents/mL. These findings support the reported in previous studies where the LoD of HSP70 is lower than other molecular markers. This can be explained by the low number of HSP70 copies across the Leishmania genome compared to kDNA and ribosomal markers (Hernández et al., 2014; Hitakarun et al., 2014). Also, this might be explained by the variation across the HSP70 gene in several Leishmania species from the old world and new world (Hernández et al., 2014).

When the same parameters were evaluated in qPCR, it was observed that the technique was more sensitive and reproducible than conventional PCR (Figures 1, 2). Previous studies have described these same findings not only for Leishmania (Pourmohammadi et al., 2010; Sterkers et al., 2010; Eroglu et al., 2014) but for other microorganisms such as Helicobacter pylori (de Bruijn and Barker, 1992), Plasmodium spp (Gama et al., 2007), Salmonella enterica (Parker et al., 2011) and viruses such as ZIKV (Francino et al., 2006) and DENV (Bai et al., 2008; Faye et al., 2013). For the ARR, we observed that when kDNA is used a Ct between 5 and 27 is observed, a much lower range than the one reported for the other molecular markers (Figure 2). According to data reported by Jara et al. (2013), where its amplification range was between a Ct of 7 and 27 (Jara et al., 2013). For the other markers, there was greater variability of the range among strains such as L. panamensis and L. braziliensis in HSP70 and a low efficiency in L. amazonensis and L. guyanensis species in ITS-1 (Figure 2). In general, good reproducibility and efficiency were presented with kDNA and 18S genetic targets (R²) between replicates in accordance with Bezerra-Vasconcelos et al. (2011). However, kDNA is not exclusive and the best analytical performance is finally observed for 18S.

The qPCR LoD was 1 × 10^-2 parasites equivalents/mL for the kDNA and 18S markers, and 1 × 10^-1 parasites equivalents/mL for HSP70 and ITS-1 (Figure 3). These results are consistent with those found by Mary et al. (2004) and Bezerra-Vasconcelos et al. (2011) when using the kDNA, a LoD of 0.0125 parasites/mL was reported but contrasts with Nicolas et al. (2002) where they only report a limit of 0.1 parasites. For the ITS-1 marker, a number of copies from 20 to 200 have been estimated in the Leishmania genome and might explain its good performance in low concentrations of parasites (Schonian et al., 2001a,b; Odiwuor et al., 2011). Nevertheless, our results contrast with the reported limit, since the LoD was lower than that found with the 18S gene. For the HSP70, LoD was lower in comparison to kDNA and 18S, but higher than that reported by Hernández et al. (2014), where they report a LoD of 10 parasites/mL (Hernández et al., 2014). For this gene, the number of copies present in the different species, although variable, are few, fluctuating between 1 and 15 copies (MacFarlane et al., 1990; Bock and Langer, 1993; Zurita et al., 2003), which could, theoretically, explain the lowest LoD obtained. It is quite unlikely to estimate the LoD of HSP70 as 1 × 10^-1 equivalent parasites/mL due to previous reports of 10–15 copies across the genome. However, herein we used a smaller fragment of HSP70 designed exclusively for the New World species. This might have improved the efficiency of the test that allowed us to reach that LoD. This set of primers has never been applied in the old world Leishmania species. Also, it is currently unknown the precise organization of the HSP70 gene in the New World species. The advent of Genomic studies will provide further insights about the true number of copies and genomic organizations of HSP70 cluster in new world species. Nevertheless, this is not sufficient to explain the obtained LoD for HSP70. The accuracy results shows that at this dilution (1 × 10^-1 parasites equivalent/mL), the percentage of variation was 21–40% (Figure 4) which was not the case for the other markers demonstrating that there is massive variation at the LoD dilution. This could envisage that small DNA fragments of the gene are subject of amplification via the qPCR but no stable equivalent parasites are truly amplified. Therefore, a plausible explanation might be that the LoD is not stable due to massive variation in terms of gene arrangements or intra-specific variation or enough template availability and plausibly suggesting that in future studies this LoD has to be subject of investigation.

The literature reports that each parasite species contains large numbers of copies of the 18S ribosomal gene (∼ 160). Therefore, several studies choose this marker as the ideal target for molecular studies. In 2011, Bezerra et al., established a LoD of 40 parasites/mL when using 18S. However, Schulz et al. (2003) reported a LoD of 100 parasites/mL. Our results differ from these two studies in determining a lower LoD for 18S (1 × 10^-2 parasites equivalents/mL). There was no variation in LoD between strains when using this molecular marker (Table 1). The data analyzed by ANOVA with three parameters (Variable result: CT, Fixed factors: concentration, species, marker and Covariable: days) had no effect of the covariate nor of the repetition through the days. The same was found when analyzing the difference between the Ct means obtained (P-value: 0.86) (Supplementary Figure S2). It was also observed that the means comparison tests allowed to identify that the tendency to increase Ct, is dependent on the increase of the dilution, showing a significant difference for all the dilutions and in all the markers (P-value < 0.05), When comparing the Ct means of each molecular marker used in each dilution evaluated, it was found that most have a different behavior (statistically significant differences P-value < 0.05).

For the accuracy, we did not observe variation between the replicates of the tests, independent of the day of execution of the technique (average coefficient of variation of 0.98), but when handling lower dilutions the coefficient of variation increases in the days of repetition of the technique. This has been demonstrated in studies evaluating the performance of qPCR in T. cruzi and in Leishmania (Duffy et al., 2013; Jara et al., 2013). The highest coefficient of variation was obtained with the HSP70 marker.

In general, the comparison herein conducted showed the limitations of HSP70 in the detection of Leishmania in terms of sensitivity by PCR and qPCR. Also, the fact that kDNA is not an exclusive marker due to the amplification for T. cruzi and M. tuberculosis DNA (Supplementary Figure S1). Also, the low efficiency of the qPCR using the ITS-1 with L. amazonensis and L. guyanensis species (Figure 2). However, we report the good analytical performance (in terms of accuracy among the species) that the 18S marker exhibits for molecular diagnostics. As mentioned initially, studies on the comprehensive evaluation of the analytical performance of molecular methods for the diagnosis of CL are scarce and are affected by the difficulty of not having a consensus of established diagnostic tests. Implementing a molecular technique such as qPCR for field work in endemic areas for leishmaniasis leads to having a special infrastructure as well as the acquisition of costly equipment and reagents (its cost is reported to be up to three times higher than that of conventional PCR) (Bock and Langer, 1993). This reflects the need to develop new technologies more sensitive but easy to acquire and to be implemented in this type of regions, such as LAMP (Notomi et al., 2000; Tomita et al., 2008; Nzelu et al., 2014, 2016; Abbasi et al., 2016) and nanoparticles (Andreadou et al., 2014). Although microscopy remains the gold standard for routine diagnosis, the high incidence of CL in different regions of South America highlights the need to rethink the implementation of specific strategies for the correct and timely diagnosis of this disease. The results herein obtained provide the basis for the subsequent evaluation of the diagnostic performance with a panel of varied samples and its interlaboratory comparison of real-time PCR with the 18S. We also suggest the inclusion of more species from the Americas. We employed only six species that are the most frequent in CL cases in Colombia but further studies should be considered.