Immunological tests may represent valuable tools for the diagnosis of human tegumentary leishmaniasis (TL) due to their simple execution, less invasive nature and potential use as a point-of-care test. Indeed, several antigenic targets have been used with the aim of improving the restricted scenario for TL-diagnosis. We performed a worldwide systematic review to identify antigenic targets that have been evaluated for the main clinical forms of TL, such as cutaneous (CL) and mucosal (ML) leishmaniasis. Included were original studies evaluating the sensitivity and specificity of immunological tests for human-TL, CL and/or ML diagnosis using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies to detect Leishmania-specific antibodies or antigens. The review methodology followed PRISMA guidelines and all selected studies were evaluated in accordance with QUADAS-2. Thirty-eight original studies from four databases fulfilled the selection criteria. A total of 79 antigens were evaluated for the detection of antibodies as a diagnostic for TL, CL and/or ML by ELISA. Furthermore, three antibodies were evaluated for the detection of antigen by immunochromatographic test (ICT) and immunohistochemistry (IHC) for CL-diagnosis. Several antigenic targets showed 100% of sensitivity and specificity, suggesting potential use for TL-diagnosis in its different clinical manifestations. However, a high number of proof-of-concept studies reinforce the need for further analysis aimed at verifying true diagnostic accuracy in clinical practice.
Citation: Freire ML, Rêgo FD, Cota G, Pascoal-Xavier MA, Oliveira E (2021) Potential antigenic targets used in immunological tests for diagnosis of tegumentary leishmaniasis: A systematic review. PLoS ONE 16(5): e0251956. https://doi.org/10.1371/journal.pone.0251956
Editor: Albert Schriefer, Universidade Federal da Bahia, BRAZIL
Received: January 6, 2021; Accepted: May 6, 2021; Published: May 27, 2021
Copyright: © 2021 Freire et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico, grant number 301159/2016-5 to Edward Oliveira Conselho. Nacional de Desenvolvimento Científico e Tecnológico, grant number 301384/2019-3 to Gláucia Cota. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, grant number Finance Code 001 to Mariana Lourenço Freire. Conselho Nacional de Desenvolvimento Científico e Tecnológico, grant number 155839/2018-7 to Felipe Dutra Rêgo.
Competing interests: The authors have declared that no competing interests exist.
Tegumentary Leishmaniasis (TL) is a neglected tropical disease caused by different species of the genus Leishmania (Kinetoplastea: Trypanosomatidae), transmitted to vertebrate hosts by sand flies (Diptera: Psychodidae) . TL is considered an emergent and re-emergent disease, since a worrisome increase in its incidence has been reported . On a the global scale, the number of new autochthonous TL cases reported annually to the World Health Organization (WHO) increased from 71,486 to 251,553 during 1998 to 2018 . Several factors are involved with the spread of TL, such as human migration from rural to urban areas, conflicts and wars, disturbances in microenvironments due to climate change and human intervention and deterioration of socioeconomic conditions in endemic countries .
TL comprises a broad spectrum of clinical manifestations ranging from single or multiple ulcerative skin lesions (cutaneous leishmaniasis—CL), to diffuse (diffuse leishmaniasis-DL) and mucosal (mucosal leishmaniasis—ML) lesions, with the last two being typical in the Americas. TL is associated with physical deformities and psychological alterations, affecting the health and wellness of the patient [4, 5].
The range of clinical manifestations can hinder rapid and accurate diagnoses, a key step to initiate treatment promptly and control the disease. Although several advances, TL-diagnosis remains based on the triad of epidemiological background, clinical signs and laboratory diagnosis, including direct and histopathological examination of skin biopsy and molecular detection of Leishmania DNA. Despite high specificity, low sensitivities have been described for direct and histopathological examination, especially in New World countries, where chronic cases and ML are frequent [6–9]. Molecular techniques are complex, expensive, still without a standardized protocol for routine use and are restricted to reference and research centers. Therefore, these limitations make the TL-diagnosis scenario restricted, particularly in resource limited settings [10–12].
In this sense, immunological tests may present remarkable advantages for TL-diagnosis, due to the use of less invasive sampling compared to skin biopsy and their potential to be automated, quantitative and used as point-of-care tests. The anti-Leishmania delayed-type hypersensitivity reaction, known as the Montenegro skin test (MST), has been the most used immunological test for CL-diagnosis in Brazil, even though it presents significant limitations such as positive results associated with previous leishmaniasis or asymptomatic infections [13, 14]. Nonetheless, the production of the MST antigen was discontinued in Brazil, hampering even more CL-diagnosis in the country . Other immunological tests, mainly Enzyme-Linked Immunosorbent Assay (ELISA), have presenting promising results in the Americas and beyond .
Several studies using soluble Leishmania antigen (SLA) in ELISA for TL-diagnosis, have presented variable sensitivity especially due to antigen preparation and antigenic differences among Leishmania isolates and species. Moreover, reduced specificity due to the cross-reactivity with other infectious diseases has been frequently reported [16–18]. Since CL-patients commonly produce low levels of anti-Leishmania antibodies, there is growing interest in high sensitivity antigens for immunological tests. Different methodologies have been employed, such as bioinformatics tools [19–23], cDNA expression library , phage display [25, 26], immunoproteomic approach [18, 27–32] and isolation and purification of glycoconjugates [33, 34] to identify potential antigens. Furthermore, immunological tools have already been used to detect Leishmania antigens using monoclonal and polyclonal antibodies by immunochromatographic test (ICT) or immunohistochemistry (IHC), such as the CL Detect Rapid Test (InBios International Inc., Seattle, WA, USA), which detects peroxidoxin from Leishmania and has been used especially in Old World countries, with limited sensitivity [35, 36].
In this sense, we consider immunnodiagosis as potential tools to increase the access and improve TL-diagnosis. Although systematic reviews have been conducted on some aspects of this form of diagnosis, it is essential to identify potential antigenic targets that have been evaluated as TL-immunodiagnostic, point out knowledge gaps that still remain and encourage other studies to allow its application in clinical practice [37, 38]. In this way, we performed a worldwide systematic review to identify potential antigenic targets, with reported sensitivity and specificity, used as TL-immunodiagnostic.
Material and methods
Protocol and registration
The review protocol was registered in the International Prospective Record of Systematic Reviews (PROSPERO: CRD42020213311) and was developed based on the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy . This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (S1 Table) .
Information sources and study selection
Structured searches were conducted in the following databases: MEDLINE, Virtual Health Library, Embase and Cochrane. A comprehensive list of key terms including tegumentary leishmaniasis and its different clinical forms AND immunological diagnosis or targets (antigens and antibodies) AND techniques or outcomes (sensitivity and specificity), was constructed in MEDLINE (S1 Fig). Similar searches were adapted to each database. Complementary searches were performed by analysis of reference lists of selected articles. Searches were performed on 23rd March 2020, without restriction of publication date.
Inclusion and exclusion criteria
Original research articles reporting on the performance (sensitivity and specificity) of immunological tests based on the detection of antibodies or antigens using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies for diagnosis of human-TL, CL or ML were included. Exclusion criteria were: evaluation of serological tests based on SLA; only non-human samples were tested (e.g. canine samples); both sensitivity and specificity of the immunological tests were not presented or were impossible to be calculated; less than five samples were tested; the absence of information about the reference test and a non-specific Leishmania antigen was used.
For each database, all publications were retrieved and duplicate citations were excluded by EndNote software . Based on the inclusion and exclusion criteria, two independent reviewers analyzed each publication by title and abstract using Rayyan software . Articles with no reason for rejection were included for full text reading. All discrepancies were solved by consensus after discussion. Selected studies were read in full to confirm their eligibility, to extract data or to exclude if exclusion criteria were identified during this step.
Data were independently extracted by two researchers (MLF and FDR) directly from full-length articles and were checked by a third researcher (EO). In case of disagreements, the final decision was reached by consensus. In this study, data were extracted and a 2x2 contingency table set up for immunological tests, containing the true positives, false positives, true negatives and false negatives. Furthermore, the following items were extracted: origin of the participants; the immunological test used; antigen or antibody types; Leishmania species and reference standard test used for disease confirmation. The phase of development of each study was classified according to Leeflang & Allerberger (2019) .
Study quality assessment
The quality of the studies was assessed using the second version of Quality Assessment of Studies of Diagnostic Accuracy Approach (QUADAS-2) . This tool allows a more transparent rating of risk of bias for studies included in systematic reviews on diagnostic accuracy.
The performance of antigenic targets was presented in four groups according immunological tests and clinical form: 1) ELISA for TL; 2) ELISA for CL; 3) Other immunological tests for CL and 4) ELISA for ML. The performance outcomes for each antigen or antibody were sensitivity (probability of a positive test among cases or disease confirmed individuals) and specificity (probability of a negative test among controls or individuals without disease). Forest plots showing sensitivity and specificity values of all antigens, including 95% confidence intervals (CI) and Summary Receiver Operating Characteristic (SROC) curves were created using RevMan 5.3.
Several studies considered a set of results for the same antigen (e.g. different cut-off points were available or different non-case groups were used in the analysis, such as healthy patients and those with other diseases). If possible, these results were grouped and only one sensitivity rate and one specificity rate including all evaluated patients. When impossible, we chose to present data that reflect the best field conditions (e.g. non-case group of patients with other diseases) or the better performance (e.g. cut-off point with best performance).
A total of 1642 articles from four databases were initially identified. Of this total, 261 were excluded due to duplicity (the same study was found in different databases). The title and abstract of each of the 1381 articles were checked and 139 were selected for full text reading. Finally, 98 articles presented exclusion criteria and so 38 were included (Fig 1).
Descriptive analysis of included studies
The characteristics of all included studies are presented in Table 1. In several studies, test performance was analyzed according to the clinical form (CL and ML) or globally (TL). In 19 studies, the antigenic targets were evaluated for TL-diagnosis, in 21 for CL and in 9 for ML. Sample size ranged from 26 to 500 patients. A total of three different immunological tests using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies were reported: ELISA, ICT and IHC. Different reference standard tests were used to confirm leishmaniasis cases. Thirty-one studies (81.6%) considered at least one parasitological method as a reference standard test, such as microscopy examination or in vitro culture for isolation of the parasite. On the other hand, seven studies (18.4%) considered some immunological or molecular tests as a reference standard. A total of 89.5% (34 out of 38) of the studies was classified as phase I (proof-of-concept), and the remaining 10.5% (4 out of 38) was classified as phase III.
ELISA for TL diagnosis
Nineteen studies used ELISA to evaluate the performance of a total of 56 antigens for TL-diagnosis, without specification of the clinical form (CL or ML). These studies evaluated 38 recombinant proteins, 14 synthetic peptides and 4 purified proteins. Forty-seven antigens were evaluated in studies that considered at least one parasitological method, such as microscopy examination or in vitro culture isolation of the parasite, as a reference standard test. The number of TL-patients ranged from 20 to 219 and the number of non-TL patients ranged from 8 to 281. The highest performance (100% of sensitivity and specificity) was reported for four recombinant proteins (cytochrome c oxidase; hypothetical protein XP_003886492.1; putative IgE histamine releasing factor; tryparedoxin peroxidase) and four synthetic peptides (A10, B7, C12 and H7) selected by the phage display technique [18, 25, 27, 30]. Nine other antigens were evaluated in studies that considered at least one immunological method as a reference standard test. For these antigens the sensitivity ranged from 39.8% to 76.9% and the specificity from 53.4% to 97%. The forest plots for sensitivity and specificity of ELISA considering parasitological methods and other tests as reference standard tests for TL-diagnosis are presented in Fig 2; more details about each evaluated antigen are available in S2 Table.
ELISA for CL diagnosis
Seventeen studies used ELISA to evaluate the performance of 44 antigens for CL-diagnosis, which comprised 20 recombinant proteins, 13 synthetic peptides and 11 purified proteins. The performance of 35 antigens was evaluated considering at least one parasitological method as a reference standard test. Among these, the sample size for studies of CL-patients ranged from 12 to 74 and for non-CL-patients from 10 to 177. Peroxidoxin was the only antigen presenting 100% sensitivity and specificity . Nine antigens were evaluated considering at least one immunological test as a reference standard. Overall, HSP83 presented the highest performance (100% sensitivity and specificity)  (Fig 3, S3 Table).
Other immunological tests for CL diagnosis
The performance of ICT and IHC using different monoclonal and/or polyclonal antibodies is presented in Fig 4 and detailed in S4 Table. Four studies evaluated the CL Detect Rapid Test (InBios International Inc., Seattle, WA, USA) in different countries. The sensitivity ranged from 35.6 to 67.6 and the specificity was higher than 80%. For IHC, two monoclonal antibodies were employed to detect antigens in fixed skin fragments. The highest performance was reported for IS22B4/XLVI5B8 mAbs, with 96% and 100% sensitivity and specificity, respectively .
ELISA for ML results
Nine studies used ELISA to evaluate the performance of 23 antigens for ML-diagnosis, which comprised 19 recombinant proteins, three synthetic peptides and one purified protein. The sample size from ML-patients in these studies ranged from 14 to 53 and from non-ML-patients from 20 to 92. At least one parasitological method was used as a reference standard test for the evaluation of sixteen antigens. The highest performance was obtained for Hypothetical protein XP_001467126.1, with 100% sensitivity and 98% specificity . Seven antigens were evaluated in studies considering at least one immunological test as a reference standard. As noted for CL-diagnosis, 100% sensitivity and specificity were reported for HSP83 . The performance of these antigens is presented in Fig 5 and more details are available in S5 Table.
The SROC curves with the antigen performances for the diagnostic of different clinical forms, using parasitological or other tests (such as ELISA and MST) as a reference standard, are presented in Fig 6. The antigens tended to have greater accuracy in studies that have used the parasitological methods as reference standard tests, regardless of TL-clinical manifestation.
Quadas-2 based quality assessment
Quality assessment of the study according to risk of bias and concern with applicability (low, high and unclear) is shown in Fig 7. Of the 38 studies assessed, 21 had high risk of bias in patient selection. The risk was unclear for the index test in 34 studies and for flow and timing in 30 studies. Nineteen studies had high concerns regarding applicability of patient selection criteria.
TL is considered a multifactorial disease, responsible for psychological and social impacts due to scars and mutilating lesions generating stigma and self-deprecation in affected patients . Improvements in healthcare access and laboratory diagnosis are needed to overcome the impacts of this disease and should be encouraged . According to WHO’s Special Programme for Research and Training in Tropical Diseases (TDR), the ideal test must be affordable, sensitive, specific, user-friendly, rapid, equipment-free and delivered to end-users (ASSURED) . Immunological tests may fill these criteria since they are usually easy to perform, accessible and require minimally invasive sample collection. Therefore, the identification of sensitive and specific antigenic targets seems to be a promising step toward the improvement of TL-diagnosis.
The studies analyzed here were conducted from 1996 to 2019, however, almost 50% of them were conducted in the last five years, mostly in Brazil or another country in the Americas. The increase in the number of studies is coincident with the interruption of the production of MST antigen in Brazil in 2015, which extinguished the simple and rapid immunodiagnostic for TL . This fact may have boosted research aimed at finding new diagnostic tools.
Parasitological diagnosis was considered a reference standard test in 89.5% of the studies. Despite this technique being highly specific for TL-diagnosis, its sensitivity is limited and inversely correlated with disease duration [9, 70]. However, no test seems to present sufficiently high sensitivity and specificity to be used as a gold standard test. We observed a tendency for index tests to be more accurate if parasitological tests were used as a reference standard than other reference test such as MST and histopathology (Fig 6). Polymerase chain reaction (PCR) was used as a reference standard test in 18 studies, generally in association with parasitological diagnosis. Overall, PCR appears to be a more suitable reference test, however, a standard protocol is urgently needed and encouraged, since distinct extraction methods, protocols and molecular targets have been used overtime .
The ability to accurately identify TL-patients is essential for a diagnostic test, in view of the range of clinical forms, disease severity and treatment toxicity. Several studies have included patients with Chagas disease as non-TL cases, however, despite phylogenetic proximity, the inclusion of patients with clinical signs that do not resemble TL is at least questionable. For tests with diagnostic purposes, a better sample panel needs to be encouraged, including diseases such as sporotrichosis, paracoccidioidomycosis, hanseniasis, vasculitis, syphilis and other dermal or mucosal diseases, that represent confounding factors in clinical practice.
Despite the distinct profiles in immune response usually reported for each clinical form of TL, some antigens presented high values of sensitivity, even for CL-patients. In general, higher levels of antibodies have been reported for ML-patients compared to CL-patients, the latter being characterized by a moderate Th1 immune response [72, 73]. In this way, it seems that problems related to antibody detection in CL-patients may be reduced by using sensitive targets and well-standardized procedures . Some antigenic targets were evaluated for TL-diagnosis without distinction of clinical form and, consequently, immune response profile. We believe that the accuracy of these antigenic targets may be improperly estimated in these specific cases.
This systematic literature review found 79 different antigens, comprising 40 recombinant proteins, 24 synthetic peptides and 15 purified proteins. The identification and more refined selection of protein targets using recombinant proteins or synthetic peptides allows the development of more standardized techniques due to the possibility of generating the purest inputs. Some protein-families have been widely evaluated as antigenic targets for TL-immunodiagnosis, such as heat shock proteins (HSPs), histones and peroxiredoxins, with promising results.
HSPs represent a highly conserved family of intracellular proteins of varying molecular weights in prokaryotic and eukaryotic cells, including cytosolic, mitochondrial, nuclear and endoplasmic reticulum resident proteins. They act as a chaperon in peptide folding and in the translocation of proteins to organelles, the prevention of protein aggregation, and the stabilization and degradation of proteins [74, 75]. HSPs have usually been identified by amino acid sequence homology and molecular weight, with HSP70 and HSP83 being the most abundant [76, 77]. These proteins are constitutively expressed throughout the life cycle of Leishmania, increasing expression in the vertebrate host due to variation in temperature and pH . The recombinant proteins HSP70 and HSP83, and the synthetic peptides extracted from those proteins, have been widely evaluated for TL-diagnosis [20, 24, 46, 47, 62, 66]. The performance of these targets seems to be promising, with HSP83 presenting sensitivity of over 90% and high specificity with few cross reactions [20, 46, 47].
Histones are conserved proteins bound to DNA establishing chromatin structure in eukaryotes. Several biological functions have been described for histones during Leishmania infection in susceptible hosts. Core nucleosomal Leishmania histones have been proposed as prominent intracellular pathoantigens, since immunological responses against histones seem to be involved in the pathological mechanisms of visceral leishmaniasis (VL) [79, 80]. In this way, this protein family has been extensively employed in ELISA for both human and canine VL [81–84]. The presence of antibodies against rH2B of L. peruviana , rH1 of L. braziliensis  and rH2A, rH2B, rH3 and rH4 of L. infantum have been detected in sera from CL or ML patients. CARMELO et al. (2002) demonstrated that the antibody against histone H1 was specific for the parasite without cross reaction with human histones. However, moderate cross reactivity has been observed in a sample panel composed of Systemic Lupus Erythematosus (SLE) and Chagas disease [45, 55, 62].
Peroxidoxin, also known as thiol-specific antioxidant protein, as well as tryparedoxin peroxidase, are peroxiredoxins, an antioxidant enzyme family [85–87]. This protein family has been described in a wide variety of organisms and several biological functions have been reported for Leishmania parasites, such as virulence factor and protection against reactive oxygen and nitrogen species . In this manner, they are directly associated with cell proliferation, senescence, apoptosis, and circadian rhythms . These proteins have been described in the secretome of L. braziliensis and the antigenicity of tryparedoxin peroxidase has also been evaluated for both human and canine VL-diagnosis [90–92]. Peroxidoxin is the protein target identified by the CL Detect Rapid Test (InBios International Inc.) for CL-diagnosis. Variable performance has been reported for this ICT, according to endemic region and, consequently, the Leishmania species involved, with better results for infections caused by L. tropica, with sensitivity ranging 65.4–73% and specificity 92–100% [35, 36]. This test, however, has not been evaluated in Brazil.
Other recombinant proteins, such as cytochrome c oxidase, putative IgE histamine releasing factor, prohibitin, eukaryotic initiation factor 5a, cathepsin L-like peptide and small myristoylated protein-3, as well as hypothetical proteins, were evaluated in preliminary studies demonstrating potential as candidates for TL-immunodiagnosis, and so more studies are desirable [27, 29, 30, 93].
Some promising synthetic peptides have been identified and employed in ELISA. The use of small fragments containing potent antigenic determinants is able to minimize non-specific reactions. LINK et al. (2017) identified three peptides by phage display, probably from GP63 glycoprotein, and presented 79% sensitivity in ELISA . COSTA et al. (2016) found high performance for three clones (A10, C12 and H7) in discriminating TL-patients from patients with other diseases and healthy individuals (100% sensitivity and specificity) . However, these short linear peptides may have some drawbacks, such as limited passive adsorption on polystyrene titration plates (ELISA-standard procedure), inability to identify serum antibodies that recognize conformational epitopes and problems considering reproducibility due to variation in inter-assay reactivity producing different batches .
Despite the advantages, the absence of post-translational modifications of bacterially-expressed and chemically synthesized proteins comprises an important limitation for the employment of this biotechnology for immunodiagnosis. In this way, purified proteins can represent significant advantages, especially regarding immunoreactivity. This review found iron-superoxide dismutase to be a purified protein with interesting results, with more than 80% sensitivity for CL or ML diagnosis. However, being purified proteins, sensitivity and specificity may vary according to the type, source, and purity of the antigen used [53, 54].
Three polyclonal and monoclonal antibodies were evaluated for detecting Leishmania antigen by ICT and IHC [35, 36, 48, 51, 58, 59]. The phase III studies included were ICT tests, that is, prospective studies in which the index and reference test were performed simultaneously in patients with clinical suspicion [35, 36, 48, 58]. This is a commercial test that, despite its low sensitivity, has been useful in some localities due to the simple realization and high specificity, reducing the number of CL patients referred for diagnosis confirmation. High performance was observed in phase I studies for species-specific monoclonal antibody (IS2-2B4—A11/ XLVI-5B8-B3) employed in IHC, with 96% sensitivity and 100% specificity . More robust studies using monoclonal or polyclonal antibodies for TL-diagnosis need to be encouraged evaluating the performance in clinical practice.
The strength of the present literature review is that it employed a comprehensive search strategy with four databases. One of the meaningful limitations may be the limited number of studies evaluating the same protein target, and so results need to be interpreted with caution. For this reason, a meta-analysis was not performed here. Additionally, it is important to consider that the risk of bias for many of the included studies was unclear and/or was high for some of the evaluated parameters: “Patient selection”, “Flow and Timing” and "Index test". Here, we identified a large number of antigenic targets that could help clinical diagnosis. However, the high number of proof-of-concept and phase I studies highlights the need to move forward with more refined and mainly prospective studies including patients with clinical suspicion of TL from different endemic regions and the most sensitive reference standard tests, to evaluate the diagnostic accuracy of antigenic targets reported in clinical practice.
S2 Table. Antigenic targets used in ELISA for diagnosis of tegumentary leishmaniasis.
S3 Table. Antigenic targets used in ELISA for diagnosis of cutaneous leishmaniasis.
S4 Table. Antigenic targets for diagnosis of cutaneous leishmaniasis by other tests.
We are grateful for support from the Programa de Pós Graduação em Ciências da Saúde of the Instituto René Rachou and Coordination for the Improvement of Higher Education Personnel (CAPES).
- 1. PAHO/WHO. Leishmaniases. Epidemiological Report of the Americas. 2019.: https://iris.paho.org/handle/10665.2/51734
- 2. WHO. Global leishmaniasis surveillance, 2017–2018, and first report on 5 additional indicators. Wkly Epidemiol Rec. 2020;95: 265–280. https://www.who.int/publications/i/item/who-wer9525
- 3. Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, Brooker S. Cutaneous leishmaniasis. Lancet Infect Dis. 2007;7: 581–596. pmid:17714672
- 4. d. C Toledo AC, da Silva RE, Carmo RF, Amaral TA, Luz ZMP, Rabello A. Assessment of the quality of life of patients with cutaneous leishmaniasis in Belo Horizonte, Brazil, 2009–2010. A pilot study. Trans R Soc Trop Med Hyg. 2013;107: 335–336. pmid:23474473
- 5. Chahed MK, Bellali H, Ben Jemaa S, Bellaj T. Psychological and Psychosocial Consequences of zoonotic cutaneous leishmaniasis among women in Tunisia: Preliminary findings from an exploratory study. PLoS Negl Trop Dis. 2016;10: e0005090. pmid:27788184
- 6. Al-Hucheimi SN, Sultan BA, Al-Dhalimi MA. A comparative study of the diagnosis of Old World cutaneous leishmaniasis in Iraq by polymerase chain reaction and microbiologic and histopathologic methods. Int J Dermatol. 2009;48: 404–408. pmid:19335428
- 7. Goto H, Lindoso JAL. Current diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis. Expert Rev Anti Infect Ther. 2010;8: 419–433. pmid:20377337
- 8. Sotto MN, Yamashiro-Kanashiro EH, da Matta VLR, de Brito T. Cutaneous leishmaniasis of the New World: diagnostic immunopathology and antigen pathways in skin and mucosa. Acta Trop. 1989;46: 121–130. pmid:2565073
- 9. Weigle KA, de Davalos M, Heredia P, Molineros R, Saravia NG, D’Alessandro A. Diagnosis of cutaneous and mucocutaneous leishmaniasis in Colombia: a comparison of seven methods. Am J Trop Med Hyg. 1987;36: 489–496. pmid:2437815
- 10. Kar K. Serodiagnosis of leishmaniasis. Crit Rev Microbiol. 1995;21: 123–152. pmid:7639932
- 11. Reithinger R, Coleman PG. Treating cutaneous leishmaniasis patients in Kabul, Afghanistan: cost-effectiveness of an operational program in a complex emergency setting. BMC Infect Dis. 2007;7: 3. pmid:17263879
- 12. de Paiva-Cavalcanti M, de Morais RCS, Pessoa-e-Silva R, Trajano-Silva LAM, Gonçalves-de-Albuquerque S da C, Tavares D de HC, et al. Leishmaniases diagnosis: An update on the use of immunological and molecular tools. Cell and Bioscience. BioMed Central Ltd.; 2015. pp. 1–10. pmid:26097678
- 13. Guarín N, Palma G, Pirmez C, Valderrama L, Tovar R, Saravia N. Comparative immunohistological analysis of the Montenegro skin test reaction in asymptomatic infection and in acute and chronic cutaneous leishmaniasis. Biomédica. 2006. http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-41572006000500006. Accessed 29 Mar 2021. pmid:17361840
- 14. Sassi A, Louzir H, Ben-Salah A, Mokni M, Ben-Osman A, Dellagi K. Leishmanin skin test lymphoproliferative responses and cytokine production after symptomatic or asymptomatic Leishmania major infection in Tunisia. Clin Exp Immunol. 1999;116: 127–132. pmid:10209516
- 15. Braz LMA. Tegumentary leishmaniasis diagnosis: What happened with MST (Montenegro skin test) in Brazil? Rev Inst Med Trop Sao Paulo. 2019;61: 1–3. pmid:30864622
- 16. Romero GAS, Orge M de la GO, de F Guerra MV, Paes MG, de O Macêdo V, de Carvalho EM. Antibody response in patients with cutaneous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil. Acta Trop. 2005;93: 49–56. pmid:15589797
- 17. Caballero ZC, Sousa OE, Marques WP, Saez-Alquezar A, Umezawa ES. Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and Leishmania spp. Clin Vaccine Immunol. 2007;14: 1045–1049. pmid:17522327
- 18. Lage DP, Machado AS, Ramos FF, Silveira PC, Dias DS, Ribeiro PAF, et al. A biomarker for tegumentary and visceral leishmaniasis based on a recombinant Leishmania hypothetical protein. Immunobiology. 2019;224: 477–484. pmid:31164242
- 19. Menezes-Souza D, De Oliveira Mendes TA, Pinto Nagem RA, De Oliveira Santos TT, Teixeira Silva AL, Santoro MM, et al. Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis. PLoS One. 2014;9. pmid:24921246
- 20. Menezes-Souza D, De Mendes TAO, De Gomes MS, Reis-Cunha JL, Nagem RAP, Carneiro CM, et al. Epitope mapping of the HSP83.1 protein of Leishmania braziliensis discloses novel targets for immunodiagnosis of tegumentary and visceral clinical forms of leishmaniasis. Clin Vaccine Immunol. 2014;21: 949–959. pmid:24807053
- 21. Menezes-Souza D, de O Mendes TA, de S Gomes M, Bartholomeu DC, Fujiwara RT. Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis Cathepsin L-like protein and a synthetic peptide containing its linear B-cell epitope. PLoS Negl Trop Dis. 2015;9: e3426. pmid:25569432
- 22. Menezes-Souza D, de Oliveira Mendes TA, de Araújo Leão AC, de Souza Gomes M, Fujiwara RT, Bartholomeu DC. Linear B-cell epitope mapping of MAPK3 and MAPK4 from Leishmania braziliensis: implications for the serodiagnosis of human and canine leishmaniasis. Appl Microbiol Biotechnol. 2015;99: 1323–1336. pmid:25359475
- 23. Florez MM, de Oliveira CI, Puerta C, Guzman F, Ayala M, Montoya G, et al. Synthetic peptides derived from ribosomal proteins of Leishmania spp. in mucocutaneous leishmaniasis: Diagnostic usefulness. Protein Pept Lett. 2017;24: 982–988. pmid:28758598
- 24. Amorim AG, Carrington M, Miles MA, Barker DC, Almeida LC, de Almeida ML. Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response. Cell Stress Chaperones. 1996;1: 177–187. pmid:9222603
- 25. Costa LE, Salles BCS, Alves PT, Dias ACS, Vaz ER, Ramos FF, et al. New serological tools for improved diagnosis of human tegumentary leishmaniasis. J Immunol Methods. 2016;434: 39–45. pmid:27090730
- 26. Link JS, Alban SM, Soccol CR, Pereira GVM, Thomaz Soccol V. Synthetic peptides as potential antigens for cutaneous leishmaniosis diagnosis. J Immunol Res. 2017;2017: 5871043. pmid:28367456
- 27. Duarte MC, Pimenta DC, Menezes-Souza D, Magalhães RDM, Diniz JLCP, Costa LE, et al. Proteins selected in Leishmania (Viannia) braziliensis by an immunoproteomic approach with potential serodiagnosis applications for tegumentary leishmaniasis. Clin Vaccine Immunol. 2015;22: 1187–1196. pmid:26376929
- 28. Lima BSS, Pires SFF, Fialho LCCJ, Oliveira EJJ, Machado-de-Avila RAA, Chavez-Olortegui C, et al. A proteomic road to acquire an accurate serological diagnosis for human tegumentary leishmaniasis. J Proteomics. 2017;151: 174–181. pmid:27262223
- 29. Carvalho AMRSS, Costa LE, Salles BCSS, Santos TTO, Ramos FF, Lima MP, et al. An ELISA immunoassay employing a conserved Leishmania hypothetical protein for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. Cell Immunol. 2017;318: 42–48. pmid:28602279
- 30. Coelho EAF, Costa LE, Lage DP, Martins VT, Garde E, de Jesus Pereira NC, et al. Evaluation of two recombinant Leishmania proteins identified by an immunoproteomic approach as tools for the serodiagnosis of canine visceral and human tegumentary leishmaniasis. Vet Parasitol. 2016;215: 63–71. pmid:26790739
- 31. Lima MP, Costa LE, Lage DP, Dias DS, Ribeiro PAF, Machado AS, et al. Diagnostic application of recombinant Leishmania proteins and evaluation of their in vitro immunogenicity after stimulation of immune cells collected from tegumentary leishmaniasis patients and healthy individuals. Cell Immunol. 2018;334: 61–69. pmid:30287082
- 32. Salles BCS, Dias DS, Steiner BT, Lage DP, Ramos FF, Ribeiro PAF, et al. Potential application of small myristoylated protein-3 evaluated as recombinant antigen and a synthetic peptide containing its linear B-cell epitope for the serodiagnosis of canine visceral and human tegumentary leishmaniasis. Immunobiology. 2019;224: 163–171. pmid:30266201
- 33. Gomes-Silva A, Souza MA, Afonso-Cardoso SR, Andrade LR, Dietze R, Lemos E, et al. Serological reactivity of different antigenic preparations of Leishmania (Leishmania) amazonensis and the Leishmania braziliensis complex. Rev Soc Bras Med Trop. 2008;41: 135–141. pmid:18545832
- 34. De Souza LMB, Thomaz Soccol V, Petterle RR, Bates MD, Bates PA, et al. Analysis of Leishmania mimetic neoglycoproteins for the cutaneous leishmaniasis diagnosis. Parasitology. 2018;145: 1938–1948. pmid:29806570
- 35. Bennis I, Verdonck K, el Khalfaoui N, Riyad M, Fellah H, Dujardin JC, et al. Accuracy of a rapid diagnostic test based on antigen detection for the diagnosis of cutaneous leishmaniasis in patients with suggestive skin lesions in Morocco. Am J Trop Med Hyg. 2018;99: 716–722. pmid:29988004
- 36. Vink MMT, Nahzat SM, Rahimi H, Buhler C, Ahmadi BA, Nader M, et al. Evaluation of point-of-care tests for cutaneous leishmaniasis diagnosis in Kabul, Afghanistan. EBioMedicine. 2018;37: 453–460. pmid:30396855
- 37. Zanetti ADS, Sato CM, Longhi FG, Ferreira SMB, Espinosa OA. Diagnostic accuracy of enzyme-linked immunosorbent assays to detect anti-leishmania antibodies in patients with American tegumentary leishmaniasis: A systematic review. Rev Inst Med Trop Sao Paulo. 2019;61: e42. pmid:31432991
- 38. Pena HP, Belo VS, Xavier-Junior JCC, Teixeira-Neto RG, Melo SN, Pereira DA, et al. Accuracy of diagnostic tests for American tegumentary leishmaniasis: a systematic literature review with meta-analyses. Trop Med Int Heal. 2020;25: 1168–1181. pmid:32677284
- 39. Higgins J, Thomas J. Cochrane Handbook for Systematic Reviews of Interventions | Cochrane Training. 2020. https://training.cochrane.org/handbook/current
- 40. McInnes MDF, Moher D, Thombs BD, McGrath TA, Bossuyt PM, Clifford T, et al. Preferred reporting items for a systematic review and meta-analysis of diagnostic test accuracy studies the PRISMA-DTA Statement. JAMA—J Am Med Assoc. 2018;319: 388–396. pmid:29362800
- 41. Lorenzetti DL, Ghali WA. Reference management software for systematic reviews and meta-analyses: An exploration of usage and usability. BMC Med Res Methodol. 2013;13: 141. pmid:24237877
- 42. Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan-a web and mobile app for systematic reviews. Syst Rev. 2016;5: 1–10.
- 43. Leeflang MMG, Allerberger F. How to: evaluate a diagnostic test. Clin Microbiol Infect. 2019;25: 54–59. pmid:29906592
- 44. Whiting PF, Rutjes AWS, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, et al. Quadas-2: A revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med. 2011;155: 529–536. pmid:22007046
- 45. Carmelo E, Martinez E, Gonzalez AC, Pinero JE, Patarroyo ME, Del Castillo A, et al. Antigenicity of Leishmania braziliensis histone H1 during cutaneous leishmaniasis: localization of antigenic determinants. Clin Diagn Lab Immunol. 2002;9: 808–811. pmid:12093677
- 46. Celeste BJ, Angel SO, Castro LGM, Gidlund M, Goto H. Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis. Brazilian J Med Biol Res. 2004;37: 1591–1593. pmid:15517072
- 47. Celeste BJ, Sanchez MCA, Ramos-Sanchez EM, Castro LGM, Costa FAL, Goto H. Recombinant Leishmania infantum heat shock protein 83 for the serodiagnosis of cutaneous, mucosal, and visceral leishmaniases. Am J Trop Med Hyg. 2014;90: 860–865. pmid:24615136
- 48. De Silva G, Somaratne V, Senaratne S, Vipuladasa M, Wickremasinghe R, et al. Efficacy of a new rapid diagnostic test kit to diagnose Sri Lankan cutaneous leishmaniasis caused by Leishmania donovani. Gannavaram S, editor. PLoS One. 2017;12: 1–14. pmid:29135995
- 49. González AC, Martínez E, Carmelo E, Piñero JE, Alonso V, Del Castillo A, et al. Analysis of NLS and rRNA binding motifs in the L25 ribosomal protein from Leishmania (Viannia) braziliensis: Investigation of its diagnostic capabilities. Parasitology. 2002;125: 51–57. pmid:12166520
- 50. Jensen AT, Gaafar A, Ismail A, Christensen CB, Kemp M, Hassan AM, et al. Serodiagnosis of cutaneous leishmaniasis: Assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein. Am J Trop Med Hyg. 1996;55: 490–495. pmid:8940979
- 51. Kenner JR, Aronson NE, Bratthauer GL, Turnicky RP, Jackson JE, Tang DB, et al. Immunohistochemistry to identify Leishmania parasites in fixed tissues. Journal of Cutaneous Pathology 1999 pp. 130–136. pmid:10235378
- 52. Lima MP, Costa LE, Duarte MC, Menezes-Souza D, Salles BCS, de Oliveira Santos TT, et al. Evaluation of a hypothetical protein for serodiagnosis and as a potential marker for post-treatment serological evaluation of tegumentary leishmaniasis patients. Parasitol Res. 2017;116: 1197–1206. pmid:28150041
- 53. Longoni SS, Marín C, Sánchez-Moreno M, Longoni SS, Marin C, Sanchez-Moreno M, et al. Excreted Leishmania peruviana and Leishmania amazonensis iron-superoxide dismutase purification: Specific antibody detection in Colombian patients with cutaneous leishmaniasis. Free Radic Biol Med. 2014;69: 26–34. pmid:24440468
- 54. Marin C, Longoni SS, Urbano JJ, Minaya G, Mateo H, De Diego JA, et al. Enzyme-linked immunosorbent assay for superoxide dismutase-excreted antigen in diagnosis of sylvatic and andean cutaneous leishmaniasis of Peru. Am J Trop Med Hyg. 2009;80: 55–60. pmid:19141840
- 55. Montoya Y, Leon C, Talledo M, Nolasco O, Padilla C, Muñoz-Najar U, et al. Recombinant antigens for specific and sensitive serodiagnosis of Latin American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg. 1997;91: 674–676. pmid:9580116
- 56. Padilla R. C, Montoya P. Y. Characterization and immunoreactivity of Leishmania braziliensis P2ß acidic ribosomal protein. Rev Peru Med Exp Salud Publica. 2003;20: 92–96.
- 57. Sato CM, Sanchez MCA, Celeste BJ, Duthie MS, Guderian J, Reed SG, et al. Use of recombinant antigens for sensitive serodiagnosis of American tegumentary leishmaniasis caused by different Leishmania species. J Clin Microbiol. 2017;55: 495–503. pmid:27927927
- 58. Schallig H, Hu RVPF, Kent AD, van Loenen M, Menting S, Picado A, et al. Evaluation of point of care tests for the diagnosis of cutaneous leishmaniasis in Suriname. BMC Infect Dis. 2019;19: 25. pmid:30616544
- 59. Shirian S, Oryan A, Hatam G-RR, Panahi S, Daneshbod Y, et al. Comparison of conventional, molecular, and immunohistochemical methods in diagnosis of typical and atypical cutaneous leishmaniasis. Arch Pathol Lab Med. 2014;138: 235–240. pmid:24476521
- 60. Skraba CM, Pedroso RB, Fiorini A, Rosado FR, Aristides SMA, Lonardoni MVC, et al. Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis. Diagn Microbiol Infect Dis. 2014;78: 411–417. pmid:24485589
- 61. Soto M, Requena JM, Quijada L, Alonso C. Specific serodiagnosis of human leishmaniasis with recombinant Leishmania P2 acidic ribosomal proteins. Clin Diagn Lab Immunol. 1996;3: 387–391. pmid:8807201
- 62. Souza AP, Soto M, Costa JMLL, Boaventura VS, de Oliveira CI, Cristal JR, et al. Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins. PLoS One. 2013;8: e66110. pmid:23776617
- 63. de Souza LMB, Carvalho J, Bates MD, Petterle RR, Thomaz-Soccol V, Bates PA, et al. Production of a kinesin-related recombinant protein (Lbk39) from Leishmania braziliensis by Leishmania tarentolae promastigotes and its application in the serodiagnosis of leishmaniasis. One Heal. 2019;8: 100111. pmid:31788531
- 64. Vidigal CP, Marcussi VM, Marcussi LM, Mikcha JMG, Arraes SMAA, Lonardoni MVC, et al. Enzyme immunoassay using Leishmania (Viannia) braziliensis antigens for laboratorial diagnosis of American cutaneous leishmaniasis. Acta Trop. 2008;107: 208–212. pmid:18561892
- 65. Yeganeh F, Barkhordari F, Omidi M, Samiei A, Adeli A, Mahboudi F, et al. Cloning and expression of Leishmania major superoxide dismutase b1: A potential target antigen for serodiagnosis of leishmaniasis. Iran J Immunol. 2009;6: 130–140. pmid:19801786
- 66. Zurita AI, Rodriguez J, Pinero JE, Pacheco R, Carmelo E, del Castllo A, et al. Cloning and characterization of the Leishmania (Viannia) braziliensis HSP70 gene. Diagnostic use of the C-terminal fragment rlb70(513–663). J Parasitol. 2003;89: 372–378. pmid:12760657
- 67. Bennis I, Belaid L, De Brouwere V, Filali H, Sahibi H, Boelaert M. The mosquitoes that destroy your face. Social impact of cutaneous leishmaniasis in south-eastern Morocco, A qualitative study. Fortin A, editor. PLoS One. 2017;12: e0189906. pmid:29261762
- 68. WHO. Accelerating work to overcome the global impact of neglected tropical diseases. 2012. https://www.who.int/neglected_diseases/NTD_RoadMap_2012_Fullversion.pdf
- 69. Land KJ, Boeras DI, Chen XS, Ramsay AR, Peeling RW. REASSURED diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes. Nat Microbiol. 2019;4: 46–54. pmid:30546093
- 70. Jara M, Adaui V, Valencia BM, Martinez D, Alba M, Castrillon C, et al. Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: Exploratory study of parasite load and clinical parameters. J Clin Microbiol. 2013;51: 1826–1833. pmid:23554201
- 71. De Oliveira DM, Valdrinez M, Lonardoni C, Teodoro U, Gomes T, Silveira V, et al. Comparison of different primes for PCR-based diagnosis of cutaneous leishmaniasis. Brazilian J Infect Dis. 2011;15: 204–210.
- 72. Cuba CC, Llanos-Cuentas EA, Barreto AC, Magalhães A V., Lago EL, Reed SG, et al. Human mucocutaneous leishmaniasis in Três Braços, Bahia—Brazil: an area of Leishmania braziliensis braziliensis transmission. I. Laboratory diagnosis. Rev Soc Bras Med Trop. 1984;17: 161–167.
- 73. Pedrosa Valli LC, Azeredo Passos VM, Dietze R, Lee Callahan H, Berman JD, Grogl M. Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis. J Parasitol. 1999;85: 1076–1083. pmid:10647040
- 74. Stewart GR, Young DB. Heat-shock proteins and the host-pathogen interaction during bacterial infection. Curr Opin Immunol. 2004;16: 506–510. pmid:15245747
- 75. Priya S, Sharma SK, Goloubinoff P. Molecular chaperones as enzymes that catalytically unfold misfolded polypeptides. FEBS Lett. 2013;587: 1981–1987. pmid:23684649
- 76. Clare DK, Saibil HR. ATP-driven molecular chaperone machines. Wittinghofer A, editor. Biopolymers. 2013;99: 846–859. pmid:23877967
- 77. Wiesgigl M, Clos J. Heat shock protein 90 homeostasis controls stage differentiation in Leishmania donovani. Mol Biol Cell. 2001;12: 3307–3316. pmid:11694568
- 78. Zilka A, Garlapati S, Dahan E, Yaolsky V, Shapira M. Developmental regulation of heat shock protein 83 in Leishmania: 3′ processing and mRNA stability control transcript abundance, and translation is directed by a determinant in the 3′-untranslated region. J Biol Chem. 2001;276: 47922–47929. pmid:11598129
- 79. Chang KP, McGwire BS. Molecular determinants and regulation of Leishmania virulence. Kinetoplastid Biol Dis. 2002;1: 1. pmid:12234388
- 80. Chang KP, Reed SG, McGwire BS, Soong L. Leishmania model for microbial virulence: The relevance of parasite multiplication and pathoantigenicity. Acta Tropica. Acta Trop; 2003. pp. 375–390. pmid:12659975
- 81. Maalej IA, Chenik M, Louzir H, Ben Salah A, Bahloul C, Amri F, et al. Comparative evaluation of ELISAs based on ten recombinant or purified Leishmania antigens for the serodiagnosis of Mediterranean visceral leishmaniasis. Am J Trop Med Hyg. 2003;68: 312–320. pmid:12685637
- 82. Soto M, Requena JM, Quijada L, García M, Guzman F, Patarroyo ME, et al. Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with leishmaniasis. Immunol Lett. 1995;48: 209–214. pmid:8867853
- 83. Soto M, Requena JM, Quijada L, Gomez LC, Guzman F, Patarroyo ME, et al. Characterization of the antigenic determinants of the Leishmania infantum histone H3 recognized by antibodies elicited during canine visceral leishmaniasis. Clin Exp Immunol. 1996;106: 454–461. pmid:8973612
- 84. Soto M, Requena JM, Quijada L, Perez MJ, Nieto CG, Guzman F, et al. Antigenicity of the Leishmania infantum histones H2B and H4 during canine viscerocutaneous leishmaniasis. Clin Exp Immunol. 1999;115: 342–349. pmid:9933463
- 85. Jirata D, Kuru T, Genetu A, Barr S, Hailu A, Aseffa A, et al. Identification, sequencing and expression of peroxidoxin genes from Leishmania aethiopica. Acta Trop. 2006;99: 88–96. pmid:16962062
- 86. Levick MP, Tetaud E, Fairlamb AH, Blackwell JM. Identification and characterisation of a functional peroxidoxin from Leishmania major. Mol Biochem Parasitol. 1998;96: 125–137. pmid:9851612
- 87. Webb JR, Campos-Neto A, Ovendale PJ, Martin TI, Stromberg EJ, Badaro R, et al. Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect Immun. 1998;66: 3279–89. http://www.ncbi.nlm.nih.gov/pubmed/9632596
- 88. Barr SD, Gedamu L. Role of peroxidoxins in Leishmania chagasi survival. Evidence of an enzymatic defense against nitrosative stress. J Biol Chem. 2003;278: 10816–10823. pmid:12529367
- 89. Castro H, Rocha MI, Silva R, Oliveira F, Gomes-Alves AG, Cruz T, et al. Functional insight into the glycosomal peroxiredoxin of Leishmania. Acta Trop. 2020;201: 105217. pmid:31605692
- 90. Cuervo P, De Jesus JB, Saboia-Vahia L, Mendonça-Lima L, Domont GB, Cupolillo E. Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes. J Proteomics. 2009;73: 79–92. pmid:19703603
- 91. Santarém N, Silvestre R, Cardoso L, Schallig H, Reed SG, Cordeiro-da-Silva A. Application of an improved enzyme-linked immunosorbent assay method for serological diagnosis of canine leishmaniasis. J Clin Microbiol. 2010;48: 1866–74. pmid:20164286
- 92. Todolí F, Pérez-Filgueira M, Galindo I, Gómez-Sebastián S, Escribano JM, Rodríguez-Cortés A, et al. Seroreactivity against raw insect-derived recombinant KMPII, TRYP, and LACK Leishmania infantum proteins in infected dogs. Vet Parasitol. 2009;164: 154–161. pmid:19570612
- 93. Rodrigues MR, Santos LMO, Miyazaki CK, Martins VT, Ludolf FR, Kursancew AC, et al. Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope. J Immunol Methods. 2019;474: 112641. pmid:31400411
- 94. Gomara M, Haro I. Synthetic peptides for the immunodiagnosis of human diseases. Curr Med Chem. 2007;14: 531–546. pmid:17346145