Methods,and,parameters,of,melting,curve,analysis,for,identification,of,Leishmania,species:,A,scoping,review

Juliana J G Ferreira, Fernanda S Nascimento, Gláucia E B Marcon, Eros A de Almeida, Sandra C B Costa

1Laboratório de Diagnóstico de Doenças Infecciosas por Técnicas de Biologia Molecular, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, 13083-887, SP, Brazil

2Fundação Oswaldo Cruz Mato Grosso do Sul (FIOCRUZ MS), Rua Gabriel Abrão, 92, Jardim das Nações, Campo Grande, 79081-746 MS, Brazil

3Grupo de Estudos em Doença de Chagas, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas(UNICAMP), Campinas, 13083-887, SP, Brazil

ABSTRACT

Leishmaniasis is a set of diseases with a worldwide distribution that affects mainly economically underprivileged populations in developing countries. It has a major impact on public health, with a global cost of billions of dollars per year. The treatment and control of leishmaniasis vary according to the Leishmania species involved,which require reliable methods for species identification. Since most of the currently used methods have limitations, there is a need for assays that allow rapid, precise identification of the offending species. Real-time polymerase chain reactions in conjunction with dissociation curve analysis have been used to detect differences in the DNA composition of selected genes of Leishmania spp. Kinetoplast DNA is the main molecular target used because of its high copy number per parasite, but other targets have also been studied. As part of an effort to establish melting temperature standards for each target gene, we have reviewed the pertinent literature available in public databases, including PubMed, Web of Science, SciELO and LILACS, using the keywords “Leishmania”, “leishmaniasis”, “realtime PCR”, “melting temperature”, and “melting curve”, alone or in combination. After applying eligibility criteria, 27 articles were selected for analysis. A considerable variation in the methodologies analyzed was found regarding molecular targets, standardization of the methods, reproducibility and specificity. Because of this,statistical analysis was not performed. In most cases, the methods were able to differentiate the parasite at the subgenus level or few species regardless of the target chosen.

KEYWORDS: Leishmaniasis; Melting temperature; Real-time PCR

Leishmaniasis is a vector-borne disease caused by parasites of the genus Leishmania that are transmitted by phlebotomine insects to a variety of mammals, mainly humans and domestic animals such as dogs. Leishmania parasites are classified into two subgenera, Leishmania (Leishmania) and Leishmania (Viannia),with the clinical symptoms of the disease varying according to the offending species. The main forms of the disease are visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucosal leishmaniosis (ML). Globally, leishmaniasis is associated mainly with poverty and affects more than 12 million people, with 0.9 to 1.6 million new cases per year, estimated yearly deaths between 20 000 and 30 000, approximately 350 million people are at risk of infection[1]. In the Americas, CL and ML are endemic in 18 countries, 17 of which reported 892 846 new cases of CL from 2001 to 2016; VL occurs in 76 countries (12 of which are located in the Americas) and, in the same 15-year period indicated above,55 530 cases of this form of leishmaniasis were reported (mean of 3 457 cases/year)[2].

The wide variety of species involved in leishmaniasis, together with the large spectrum of clinical manifestation, makes the diagnosis of this disease complex, particularly since the various clinical symptoms require different therapeutic approaches.Visceral leishmaniasis is the systemic and the most severe form of the disease, leading to death if untreated. The most common features include fever, splenomegaly, hepatomegaly, pancytopenia,anemia, and weight loss, but VL may also be asymptomatic.In addition, the clinical symptoms and manifestations may vary among geographic regions, as may the treatment used,primarily because of different patterns of parasite resistance to medicines[3]. The drugs currently recommended by the World Health Organization (WHO) includes amphotericin B, miltefosine,paromomycin, and pentavalent antimonials (or combination of these), depending on the case or region[4].

The initial symptom of CL is a papule or nodule at the site of the infection that subsequently progresses to ulcerated nodules. CL has some rare variations, such as diffuse CL, disseminated CL and CL relapse, that are associated with the immune responses[5]. The type and severity of lesions and their spontaneous healing also depend on the immune response, in addition to the Leishmania species responsible for the infection[4-6]. The choice of treatment will depend on the species causing Old World or New World CL infection, as well as on the availability of medicines, thermotherapy and cryotherapy[4].

ML, which is characterized by destructive lesions of the nasal septum, lips, and palate, occurs mainly in immunocompromised subjects and requires rapid treatment to avoid permanent disfigurement[5]. ML is generally treated with pentavalent antimonials, amphotericin B or miltefosine, with the latter being used only in certain countries[4].

The standard visualization of amastigote forms of Leishmania in diverse biological samples shows high specificity, but is unable to distinguish the species[4]. Serological techniques, although useful for screening in endemic areas, do not distinguish the species or the status of infection. Detection of the parasite DNA by polymerase chain reaction (PCR) has high sensitivity and specificity, and certain variations of this method, such as restriction fragment length polymorphism (RFLP-PCR), allow identification of the species[7,8]. The gold standard for typing Leishmania species and strains is multilocus enzyme electrophoresis (MLEE), introduced in 1990 by Rioux et al., for classification of the genus Leishmania[9].This method identifies the parasites by their enzymatic profile(known as zymodemes) based on their electrophoretic mobility,and several enzyme panels can be analyzed simultaneously. The use of MLEE is limited because of the large amount of proteins required that can only be obtained by culturing Leishmania[10].

Real-time PCR (qPCR) with molecular probes or an intercalating dye can be used to detect the parasite and provide quantitative results. SYBR Greentechnology, which uses an intercalating dye,allows melting curve analysis (MCA) of the dissociation profile of double-stranded DNA[11]. High-resolution melting analysis(HRMA) fulfills the same function but provides better performance with regard instrumentation and the fluorescence of DNA-binding dyes[12,13]. Both qPCR and HRMA are used for various applications in microbiological and parasitological analysis, as well as to discriminate Leishmania subgenera and/or species[14-20].

The choice of the molecular target is an important issue for accurate parasite identification by PCR because of the wide variety of targets available. The kinetoplast DNA (kDNA) is one of the most studied targets among species of the class Kinetoplastida because of its peculiar organization in maxicircles and minicircles,with the latter being highly conserved. Since minicircles are very abundant, specific and repetitive, they have been used as a molecular target to detect and identify Leishmania species by different PCR strategies.

Targets other than kDNA have also been used. For example, the ribosomal internal transcribed spacer 1 (ITS1) is a region with a high number of copies of the rRNA gene and shows interspecific variations and single nucleotide polymorphisms (SNPs)[16,21].HSP70, a heat-shock protein with an important role in cellular and protein interactions, has been used for the molecular identification of Leishmania and for phylogenetic studies because of its conserved nature[22,23]. The tryparedoxin peroxidase gene has antioxidant activity that contributes to parasite viability. This gene is located on chromosome 15 and has three copies in the form of a tandem repeat that allow detection of even low levels of expression[24].Cathepsin L-like cysteine proteinase B is encoded by the gene for Cpb, a major antigen of Leishmania parasites. This gene, which is highly conserved, occurs in multiple copies among species, is polymorphic and is a sensitive target for parasite detection[25,26].Concomitant sequencing of mannose phosphate isomerase (MPI)and 6-phosphogluconate dehydrogenase (6PGD) genes was able to differentiate seven species of New World Leishmania because the genetic polymorphisms involved were species-specific[27,28].Other genes that have been used include that of the single-copy,housekeeping Leishmania RNA-binding protein (DRBD3), which is considered ideal to this purpose[29].

Together, these studies indicate that a wide variety of targets has been used for species identification in human and animal samples,with marked inter-specific differences being observed in melting temperature (Tm) analysis, even when using the same target.The large variety of biological samples that can be used for DNA extraction may also influence the assay sensitivity.

Based on the foregoing studies, the objectives of this investigation were to: 1) review the use of Tm for Leishmania identification in humans, dogs, mice and/or cultured parasites; 2) analyze the Tm of each Leishmania species, according to the gene/target chosen; 3)analyze the criteria used to assess method quality; and 4) analyze the procedures for method standardization.

Literature searches of four public databases (PubMed, Web of Science, Scielo and LILACS) were done in May, 2020. The keywords used for search were “Leishmania”, “leishmaniasis”,“real-time PCR”, “melting temperature” and “melting curve”, alone or in combination, using the Boolean operators “AND” and/or“OR”. Search was performed in duplicate and papers were selected after reading the title and/or abstract and assessing whether they met the inclusion criteria. In addition, the reference lists of studies included in the review were carefully scanned for possible articles not retrieved from the databases.

The eligibility criteria used were: 1) articles written in English,Spanish or Portuguese idiom; 2) publication within the period from January, 2000 until April, 2020; 3) studies that involved humans,dogs, mice and/or cultured parasites; 4) studies done in any country; and 5) studies that used real-time PCR to describe Tm or HRMA for different Leishmania subgenera or species. Studies that exclusively analyzed phlebotomine insects and other mammals,review articles, and articles not containing a precise description of the method and the Tm found were excluded.

The data obtained were classified into different variables and organized in tables to support the interpretation of data analysis(Tables 1-5). In addition, the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR)[30] was applied to identify and report the data.

Table 1. Distribution of species in the subgenera Leishmania (Leishmania)and Leishmania (Viannia).

This study involved a descriptive analysis of the Tm for each Leishmania species, in addition to each target/gene analyzed. The wide variation among techniques meant that the application of meta-analyses was impracticable.

The literature search identified 1 312 references, of which 1 285 were excluded because they did not meet the inclusion criteria orwere duplicates. A total of 27 references that used of MCA and or HRMA for identification of the Leishmania species or subgenus were selected (Figure 1).

Figure 1. Flowchart of the inclusion criteria used in this study.

The two methods most widely used in the studies were MCA and HRMA. Of those that used MCA, five employed integrated fluorescent probes in conjunction with fluorescence-resonance energy transfer (FRET; referred to hereafter as FRET/MCA).

These methods were applied for different subjects: cultured parasites, humans, mice, dogs, cats and insects; however, we only included the analysis samples from parasites, humans, mice and dogs. Samples were also diverse: blood, buffy coat, biopsy,cutaneous lesions, dermal scrapings, bone marrow aspirate, urine and conjunctival swab.

To assess the quality of qPCR validation, we identified researches that performed tests of sensitivity (18/27, 66.6%), specificity(17/27, 62.9%) and reproducibility (12/27, 44.4%). In addition,it was analyzed which studies estimated DNA concentration(14/27, 51.8%), which used replicates (17/27, 62.7%), which ran independent reactions (17/27, 62.7%), which used serial dilutions(21/27, 77.7%), which used intern control (6/27, 22.2%) and which verified cross-reactions with other microorganisms (14/27, 51.8%)(Table 2). The confirmation of Leishmania species found through sequencing or cloning was performed in 59.2% (16/27) and 25.9%(7/27), respectively. Incomplete analysis of all method validations represent the study limitation which hinders a comparative analysis among them.

A detailed description of primer sequences and Tm for each Leishmania specie according to molecular target are discriminated in Tables 3 and 4. The main characteristics of selected studies as first author, year of publication, country, population, molecular target, methodology, main objective and outcome are available in Table 5.

Few papers reported study limitations or risk of bias for the sample included in its analysis. The main reported issues were the sample size, absence of multicentric studies, absence of negative samples resulting in imprecise negative predictive value and the impossibility to apply all tests to all study participants.

Identification of the species causing the disease is a crucial step for better management and treatment, as well as for possible control measures. In addition, adequate methods for species identification are essential for eco-epidemiological studies since they allow determination of the species circulating among phlebotomies in domestic and wild reservoirs[4].

Reference strains or cultured isolates, previously identified by MLEE and/or other methods such as PCR-RFLP or single target sequencing, were listed in 59.3% of the reports. Although most of the studies mentioned the use of species in culture, in several cases the method used for species identification was unclear. In addition to the use of cultured promastigotes, specimens obtained from a variety of sources (peripheral blood, bone marrow, tissue biopsy,skin lesion fluid, skin lesion scrap, urine) from humans and/or animals (dogs, cats, mice) were also tested. Four studies used filter paper to improve sample collection and preservation[15,31-33]. However,this procedure was less invasive and proved useful for field studies,two reports found it to be less sensitive when compared with the use of directly collected specimens[15,32]. In 51.8% of the reports,the concentration of DNA extracted from different samples were analyzed (Table 2). Several authors analyzed the interference of template DNA concentration[18,33-39] and host DNA[34,36,37,39]on the variation in Tm, but only a few observed any shift in the melting curve in relation to the template[18,36] and host DNA concentration[18,39]. Although these variables do not appear to affect all Leishmania spp., they should nevertheless be carefully evaluated since they could lead to species misidentification,especially when using HRMA that allows the use of a smaller Tm difference to distinguish among species.

Although quantitative PCR has been widely used as a diagnostic tool, the nomenclature and concepts used have not always been applied appropriately, including in some of the articles examined here. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines[40],the correct abbreviation for quantitative PCR is qPCR, while RTPCR refers to quantitative reverse transcription PCR. Two reports used the abbreviation RT-PCR to refer to real-time PCR[24,41].

Analytical sensitivity is the “minimum number of copies that can be measured accurately with an assay, generally known as the limit of detection (LOD)”, while clinical sensitivity is “the percentage of individuals with a given disorder whom the assay identifies as positive for that condition”. Analytical specificity is “the detection of the appropriate target sequence rather than other, nonspecific targets also present in a sample” and diagnostic specificity is “the percentage of individuals without a given condition whom the assay identifies as negative for the condition”. Repeatability is the intraassay variation, or the precision of the same samples evaluated in one assay, while reproducibility is the inter-assay variance, or the variation in results among different runs or laboratories[40]. These definitions are very important for guaranteeing the homogeneity of analyses. However, given the variation in the methods and analyses reported in the different studies, it was not possible to compare several of these aspects.

The test sensitivity was calculated in 66.6% of the articles (Table 2),and in 77.7% of these was tested by serial dilutions, with a minimum of 0.001 parasite/reaction being detected by Ceccarelli et al. using kDNA[42]. By using plasmids in a duplex method that targeted MPI and 6PGD genes, Tsukayama et al. were able to increase the assay sensitivity to detect as little as 10-100 copies of parasite DNA[27].

Test specificity was calculated in 62.9% of the articles, although only 51.8% of the studies assessed cross-reactivity. More than 23 species were screened, with Trypanosoma (T.) cruzi and T.brucei being the most used trypanosomatids. In one study, T. cruzi DNA was detected by kDNA amplification (Tm: 81.5 ℃), but this Tm did not overlap with Leishmania species. This interesting finding could be used to differentiate leishmaniasis from Chagas disease in areas where both diseases are endemic since the current serological tests used to distinguish between them may show crossreactivity[43,44]. The use of an internal control to detect inhibitors in DNA samples was reported in only six studies (Table 2). These controls were used to confirm DNA quality and ensure that false negative results were not attributable to the low quality of the samples, and. When these controls are not used, negative results are unreliable and may affect the clinical sensitivity.

Although 17 studies used replicates to calculate the standard deviation for each Tm, most of them did not specify how many replicates were run. Fifteen reports calculated the reproducibility by performing more than one run for the same samples or control and the remaining did not calculate this parameter, did not specify how they calculated it or calculated it using a positive control in the same run (Table 2).

A range of Tm values was reported for the same species of Leishmania and even for the same targeted sequence. Table 4 lists all the Leishmania species, targets and Tm, as well as the PCRequipment used in these references. The most explored target was kDNA that was used in 13 reports, with the primers pair JW13/JW14 being used in four of them[31,35,41,45].

Table 2. Assay parameters in studies of melting point analysis.

Table 3. Primer and nucleotide sequences of the target genes analyzed.

Table 4. Comparative analysis of the Tm for each Leishmania spp. according to the target selected by different authors.

Table 4. Continued.

Table 5. Characteristics of studies included in the analysis.

Table 5. Continued.

Eleven references used only MCA for the diagnosis and/or identification of Leishmania spp. The first report to use MCA was published in 2002[45] and was able to distinguish among cultured strains based on their Tm peaks: L. major (83.18 ℃), L. donovani(87.12 ℃), L. tropica (88.89 ℃) and L. infantum (89.21 ℃). These latter two species were difficult to differentiate because the Tm difference between them was less than 1 ℃. The MCA targeted a fragment of the kDNA minicircle using the primers JW13/JW14, which were also employed in the next three references. De Mombrison et al.[31] correctly identified the parasite in 55% of the samples from human cutaneous lesions in Algeria based on preestablished Tm values for reference strains. The species identified were L. major [(87.5 ± 0.3) ℃], L. donovani [(88.5 ± 0.4) ℃], L.tropica (89.3 ± 0.2) ℃] and L. infantum [(90.3 ± 0.2) ℃]. A certain degree of overlapping (≤1 ℃) among the strains and a distinct peak [(84.4 ± 0.4) ℃] were detected in 24% of the samples in which the species could not be determined. Abda et al.[41] used dermal scrapings collected from lesion borders of 27 patients in Tunisia and reported positive results/species identification in 81.5% of cases. Again, the Tm provided better discrimination between L. major [(87 ± 0.5) ℃] and L. infantum [(89.3 ± 0.3) ℃],although overlap with L. tropica [(88.5 ± 0.2) ℃] led to a couple of misidentifications, later resolved by MLEE as L. tropica killicki. As previously reported, a distinct Tm of (84.3 ± 0.3) ℃ was detected in four samples that were associated with L. infantum. In 2011,Khademvatan et al., reported the analyses of 102 stained samples from CL patients in Iran, 94% of whom were positive by qPCR,with MCA identifying three species: L. infantum [(89.4 ± 0.3) ℃],L. tropica [(88.3 ± 0.2) ℃] and L. major [(86.5 ± 0.2) ℃], the Tm of which were compared to those of reference strains; the amplicons were also sequenced to confirm the species identification[35].

By targeting another fragment of kDNA minicircle (120 bp), Pita-Pereira et al. used MCA to identify the species that caused VL and CL in 30 Brazilian patients. The initial analysis of reference strains allowed classification of the species to the subgenera Viannia(77.34 ℃) and Leishmania (78.95 ℃), with the biopsies from CL patients being placed in the Viannia group and the peripheral blood and bone marrow aspirates from VL patients in the Leishmania group. The specimens had been previously diagnosed by microscopy, culture and/or PCR-RFLP[46]. Similarly, another study using kDNA analyzed blood samples from humans, cats, and dogs(n=300). A total of 223 samples were characterized into two ranges based on the Tm average obtained from the different strains of species tested. Thus, range 1 (78-79.99 ℃) contained 58 specimens that included species from the subgenus Viannia: L. braziliensis, L.panamensis, L. guyanensis, L. lainsoni and L. shawi, and range 2 (80-82.2 ℃) contained 165 specimens from the subgenus Leishmania:L. mexicana and L. amazonensis, but also included L. naiffi from the subgenus Viannia. There was however, some disagreement between the results with MCA and other techniques such as MLEE (20%),PCR-RFLP (72.2%) and sequencing (56.4%), this discrepancy indicated the incorrect classification of some samples in the two established ranges[47]. MCA has also been used to target kDNA in urine from patients suspected of having VL, in which L. infantum was detected, thus confirming the results found in blood[48].

MCA has been used to target several other genes. In 2008,a study used the highly conserved GP63 gene that encodes a surface metalloprotease, implicated in Leishmania virulence. The authors designed a pair of primers able to detect several species of reference strains by qPCR, but the MCA indicated similar Tm among them, L. major [(89.4 ± 0.1) ℃], L. donovani [(89.5 ± 0.2) ℃],L. chagasi [(89.5 ± 0.3) ℃], L. mexicana [(89.4 ± 0.1) ℃], and L.braziliensis [(88.6 ± 0.1) ℃] with the latter species differing from the others by a Tm difference ≥1 ℃ from the others[49]. Khosravi et al., analyzed 100 biopsies from CL patients in Iran. These authors targeted the tryparedoxin peroxidase gene (three copies)and performed a qPCR with MCA that yelded two peaks with the reference strains used being L. major [(91.3 ± 0.7) ℃] and L. tropica[(89.5 ± 0.2) ℃]. Seventy-five samples had L. major and five had L.tropica[24].

Another study based on MCA that targeted a 220-275 bp fragment of the ITS1 rDNA used 11 different species from 33 World Health Organization and Centers for Disease Control and Prevention reference strains, all of which were classified on MLEE and ITS2 sequencing. The species were classified in groups, as follows: G1 that included species from the subgenus Viannia: L. braziliensis, L.guyanensis, L. panamensis, G2A that included L. donovani and L.infantum/L. chagasi, G2B that consisted only L. tropica, and G3 that included L. aethiopica, L. amazonensis, L. mexicana and L. major. A total of 1 051 specimens (biopsies, peripheral blood, bone marrow and stained slides) were tested, with 45% being positive. The human internal control used to monitor inhibition yielded positive results in all the samples tested. The use of two different realtime PCR platforms demonstrated a variation of 1.0-2.1 ℃ among the established groups[50]. De Paiva-Cavalcante et al. used about six pairs of primers from conventional PCR and evaluated them in several qPCR assays that targeted kDNA, ITS1 and SSU rDNA to preferentially detect L. braziliensis relative to other species in specimens from dogs, cats and skin scrapings from humans with CL. The results showed that kDNA was the best for detecting L.braziliensis (80 ℃) in 90% of the species, followed by SSU rDNA(85 ℃) in 82%[51], in contrast, the discriminatory power among the other species tested was low.

Several studies have used qPCR in conjunction with specific fluorescent probes for MCA based on probe-template Tm. Here we describe five reports that used a FRET/MCA combination to detect Leishmania species. The first one was published in 2003,just a year after the first report of MCA in leishmaniasis. The authors targeted the 18S rDNA gene to classify the species into three clinically relevant groups. They ran a qPCR with one pair of primers and two probes (L. braziliensis and L. donovani complexes)and evaluated the usefulness of the assay against 17 reference strains of several species and 12 human specimens (blood, bone marrow and biopsies). The FRET/MCA results showed no overlapping Tm among the three groups established (A, B, C) and all the samples were positive. Group A included L. aethiopica, L.tropica, L. major and L. mexicana (64.9-65.1 ℃), group B included L. donovani complex (71.5-71.8 ℃) and group C the L. braziliensis complex (66.5-67.6 ℃)[52]. Not long after this report, Quispe-Quintaya et al. described the use of a hemi-nested PCR followed by FRET/MCA, with the addition of three probes at the end of each of the nested reactions. The targets were two cysteine protein B (CPB) isogenes used to distinguish the species within the L.donovani complex (L. donovani, L. infantum and L. archibaldi). The authors tested reference strains of several species from different geographical locations, including L. tropica, L. major, L. aethiopica and clinical samples from humans and dogs diagnosed with VL. The combination of peaks detected in both reactions would reflect the species’ geographic origin. Although the analyses were useful in excluding non-L. donovani complex species, the Tm was apparently unable to distinguish among species of the complex since different species were classified in the same group and the same species were classified in different groups[53].

The ITS1 was also used as a target by Toz et al.[54], who designed two probes to detect the L. donovani complex (L. donovani and L.infantum), L. major and L. tropica. The analyses involved cultured isolates previously identified by MLEE (n=51), reference strains(n=4) and specimens from humans and dogs with CL or VL(n=315). The TM obtained in the pilot study varied among the species: L. major (53 ℃), L. tropica (62 ℃) and L. donovani complex(68 ℃). Among the specimens tested, L. infantum (60.6%) and L. tropica (21%) were identified, in addition to a two-peak (Tm)profile detected in 18.5% of the samples. Sequencing results corroborated 98% of the 102 sequenced samples, while MLEE matched 94.1% of the 51 isolates tested.

Tsukayama et al. targeted two loci, with the single copy genes MPI and 6PGD being able to discriminate among six New World species in 79.1% of the clinical samples tested[27]. Although the technique could distinguish L. amazonensis and L. infantum from others species, these two were indistinguishable from each other because of their identical Tm. A similar approach, in which the polymorphic and multi-copy CPB gene was targeted using a single reaction, was able to distinguish three Old World species of Leishmania based in comparison with reference strains and cultured promastigotes from previously diagnosed specimens; tests on seven human biopsies detected L. major and L. tropica[55]. The use of FRET/MCA probes increased the sensitivity in detecting differences in Tm among species or groups when compared with MCA alone, in which overlapping curves hampered the identification and distinction of a higher number of species.

The use of HRMA to diagnose leishmaniasis was first reported in 2010. As with MCA, the technique measures changes in the fluorescence intensity of the dsDNA intercalating dye (SYBR Green) during dissociation, enabling the detection of SNPs in the targeted sequence. Advances in software/analysis features have increased the sensitivity in distinguishing among very similar Tm.Nasereddin and Jaffe[33] targeted 7SL RNA and used reference strains to identify L. tropica [(89.49 ± 0.09) ℃], L. major [(89.27± 0.06) ℃] and the L. donovani [(90.8 ± 0.02) ℃] complex. The same gene and apparently the same sequence were later targeted by Mohammadi et al.[56], who concluded that HRMA was cheaper and less time-consuming compared with other techniques, such as sequencing (7SL RNA) and PCR-RFLP (ITS), and showed equal sensitivity and specificity for L. major [(87.03 ± 0.03) ℃], L. tropica[(88.09 ± 0.35) ℃] and L. infantum [(88.86 ± 0.02) ℃].

The use of kDNA in HRMA was first explored by Ceccarelli et al., who tested two reactions, only one of which, using primers MLF-MLR, was able to distinguish between the subgenera Viannia and Leishmania in reference strains and dog specimens[36]. The same group later reported several cases of L. infantum that showed two peaks, with others having an undetermined Tm because of the lack of reproducibility among replicates, as also seen in L.amazonensis[37]. To validate their previous work, Diottalevi et al.[15]used the same approach and applied two qPCR to Brazilian strains and specimens (n=47) from humans and dogs with VL or CL; the Leishmania spp. was identified in 78.7% of the cases. Two human samples had L. infantum and the subgenus Viannia; the others were positive for L. infantum (n=36) and L. amazonensis (n=1). In Turkey,Eroglu et al.[32] compared the sensitivity of different diagnostic methods for chronic CL. The use HRMA to target the ITS1 region yielded positive results in 63.4% of the cases with confirmed leishmaniasis; the species detected were L. tropica (54.5%), L. major(24.3%) and L. infantum (21.2%).

The first report to describe the use of the HSP70 gene in combination with ITS1 as a potential genotyping tool for Leishmania spp. used reference strains characterized by PCR-RFLP/MLEE. The strains were isolated from humans, sand flies, and marsupials (n=39) from different geographical areas of Colombia.The established algorithm was able to distinguish among six New World species[17]. Later, Zampieri et al. amplified two targets within the HSP70 gene and, among the reference strains tested,the first reaction (amplicon 1) was able to distinguish L. tropica,from other groups based on a Tm difference of 0.3 ℃, while the second reaction (amplicon 2) placed five species in two different groups: L. braziliensis/L. guyanensis and L. lainsoni/L. amazonensis/L.mexicana[18]. In Colombia, Marin et al., searched the HSP70 gene of L. panamensis, L. braziliensis and L. guyanensis for targets that could be validated by HRMA using reference strains. Three pairs of primers were selected based on their specificity for each of the species of interest. The runs were done separately and resulted in distinct peaks. Thirty-three isolates from biopsies previously tested by PCR-RFLP were blindly tested by HRMA; 87.8% of these were positive, with agreement of 90.6%[57]. However, the reproducibility of this assay was compromised because the authors did not report the sequences of the primers used.

Kuang et al.[38] explored the lack gene as a target for HRMA since it encodes an antigenic protein expressed in amastigotes and promastigote stages. Six reference strains from the WHO isolated from different countries and ten isolates from China (human, sand fly, rodent and canine) were tested. Two distinct reactions were run, the first of which detected and distinguished among four species: L. donovani [(91.31± 0.02) ℃], L. tropica [(88.75±0.01)℃], L.major [(88.30±0.03) ℃] and L. braziliensis [(90.85±0.02) ℃], while the second distinguished between L. turanica [(90.75±0.02) ℃] and L. braziliensis [(91.60±0.03) ℃].

Miller et al. targeted a coding sequence of the two-copy gene for amino acid permease 3 (AAP3), which has been less explored in studies of leishmaniasis. Reference strains from the WHO and additional ones from cultured isolates were tested. Three pair of primers were designed and tested in distinct reactions. Based on the authors’ analyses, who considered a Tm difference of ±0.25 ℃as distinct, the first reaction classified strains of the subgenus Leishmania into three clusters and those of Viannia also into three clusters, with L. lainsoni and L. braziliensis being distinct from one another and from the other clusters. The second reaction yielded a similar profile for the subgenus Leishmania, but now distinguished L. major from L. tropica and these from the other two clusters. The third reaction was able to distinguish among some species that had been clustered together by the previous reaction, e.g. L. amazonensis from L. mexinana, L. donovani from L. infantum and, as in the second reaction, L. major from L. tropica[39].

In summary, kDNA has been the main target used for Leishmania spp. identification, followed by ITS1. Most investigations have explored a single target, using only pair of primers, although 2-3 pairs have also been used to amplify different regions in the same target/gene. In addition, 2-3 targets/genes have been used in combination to resolve species grouped by Tm (Table 3). All the strategies used so far have been able to identify Leishmania spp. by their Tm, with the number of the species ranging from one to 11 species.

To overcome the difficulty of differentiating among species with highly similar Tm, several reports have used Tm ranges that grouped species within subgenera or grouped species from distinct subgenera based on a pre-established algorithm. Reference strains and/or cultured parasites were used to establish “reference Tm”that were later applied to the classification of results from a wide variety of specimens. Although the use of different reactions has not always resulted in the detection of more species, Müller et al.created an algorithm using three qPCR for the (AAP3 gene) that was able to identify 11 species of Leishmania by HRMA, while Tsukayama et al. were able to distinguish six species using two reactions (FRET/MCA)[27,39].

The use of appropriate equipment to perform HRMA allows the detection of small differences in the melting curve, but distinct enough to separate species. The use of probes (FRET/MCA)provides greater sensitivity in distinguish among the Tm of species or groups when compared with MCA alone since the occurrence of overlapping curves in the latter assay can hamper the identification of a large number of species.

Many studies have sought to identify Leishmania spp. based on their geographical incidence and the clinical symptoms caused by certain species. Indeed, geographical variation in the clinical manifestations of leishmaniasis is an important epidemiological issue. For example, genetic variability has been reported for the L. donovani complex[58], for L. infantum and L. tropica in southern Turkey[59], and for L. infantum from humans and dogs in different regions of Brazil[60,61].

Although many investigators have described algorithms based on MCA/HRMA for the simple, rapid detection/discrimination of Leishmania spp., such algorithms are not that simple or reproducible since variation in the discriminatory power of the assay has been noted even when using the same target/primers and the same species. In addition, as shown by Almeida et al, the use of different qPCR platforms can also introduce variation in the melting curve temperatures[16]. Indeed, MCA/HRMA alone has often been unable to identify the parasites, with a need for confirmation by sequencing (59.2%) or cloning (25.9%) (Table 2).In conclusion, although the use of MCA/HRM has limitations in reproducibility and specificity, it has been considered a good tool to distinguish among Leishmania species and assist in choosing the appropriate therapeutic approach. The reports discussed here show that this method can distinguish Leishmania at the subgenus level and possibly identify some species within each subgenus.

The wide range of methodological variations to each of the assays discussed here limited the use of comparative analyses tools in this analysis. These limitations could be overcome by a multicentric study, with participants from different countries using strains from distinct geographical areas to standardize the methods and ensure the quality and robustness of Leishmania identification.Such a coordinated effort would also be helpful in optimizing the techniques and algorithms involved.

Conflict of interest statement

The authors have no conflicts of interest with this work.

Authors’ contributions

JJGF designed the study, performed the data acquisition. JJGF and FSN defined the paper’s content and wrote the manuscript.JJGF, FSN and GEBM analyzed the data, assisted in content definition and manuscript review. GEBM approved the grand,EAA and SCBC approved the project and lead the research and infrastructure needed. All the authors analyzed the data, reviewed,edited and approved the manuscript draft.