HRM Accuracy and Limitations As a Species Typing Tool for Parasites

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2023 Oct 14

PMID 37834232

Authors

Camila Patricio Braga Filgueira

Daniela Pitta-Pereira

Lilian Motta Cantanhede

Gabriel Eduardo Melim Ferreira

Sayonara Dos Reis

Elisa Cupolillo

Otacilio C Moreira

Constanca Britto

Mariana Cortes Boite

Affiliations

Soon will be listed here.

Abstract

High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published quantifying approach as combined tools for clinicians, surveillance, and research.

References

Torrico M, Fernandez-Arevalo A, Ballart C, Solano M, Rojas E, Ariza E . Tegumentary leishmaniasis by Leishmania braziliensis complex in Cochabamba, Bolivia including the presence of L. braziliensis outlier: Tegumentary leishmaniasis in Cochabamba, Bolivia: Tegumentary leishmaniasis in Cochabamba, Bolivia. Transbound Emerg Dis. 2021; 69(4):2242-2255. DOI: 10.1111/tbed.14228. View

Hernandez C, Alvarez C, Gonzalez C, Ayala M, Leon C, Ramirez J . Identification of six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay. Parasit Vectors. 2014; 7:501. PMC: 4239372. DOI: 10.1186/s13071-014-0501-y. View

Davila M, Pineda V, Calzada J, Saldana A, Samudio F . Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama. Mem Inst Oswaldo Cruz. 2021; 116:e200572. PMC: 8061342. DOI: 10.1590/0074-02760200572. View

Alaeenovin E, Parvizi P, Ghafari S . Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR. Rev Soc Bras Med Trop. 2022; 55. PMC: 9757716. DOI: 10.1590/0037-8682-0186-2022. View

Motta Cantanhede L, Mattos C, de Souza Ronconi C, Braga Filgueira C, Silva Junior C, Limeira C . First report of Leishmania (Viannia) lindenbergi causing tegumentary leishmaniasis in the Brazilian western Amazon region. Parasite. 2019; 26:30. PMC: 6532396. DOI: 10.1051/parasite/2019030. View

Graca G, Volpini A, Romero G, de Oliveira Neto M, Hueb M, Porrozzi R . Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species. Mem Inst Oswaldo Cruz. 2012; 107(5):664-74. DOI: 10.1590/s0074-02762012000500014. View

Talmi-Frank D, Nasereddin A, Schnur L, Schonian G, Ozensoy Toz S, Jaffe C . Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis. 2010; 4(1):e581. PMC: 2797090. DOI: 10.1371/journal.pntd.0000581. View

Brito M, Andrade M, Mendonca M, Silva C, Almeida E, Lima B . Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil. Trop Med Int Health. 2009; 14(10):1278-86. DOI: 10.1111/j.1365-3156.2009.02361.x. View

Kubista M, Andrade J, Bengtsson M, Forootan A, Jonak J, Lind K . The real-time polymerase chain reaction. Mol Aspects Med. 2006; 27(2-3):95-125. DOI: 10.1016/j.mam.2005.12.007. View

10.

de Almeida J, Souza C, Fuzari A, Joya C, Valdivia H, Bartholomeu D . Diagnosis and identification of Leishmania species in patients with cutaneous leishmaniasis in the state of Roraima, Brazil's Amazon Region. Parasit Vectors. 2021; 14(1):32. PMC: 7791761. DOI: 10.1186/s13071-020-04539-8. View

11.

Leon C, Munoz M, Hernandez C, Ayala M, Florez C, Teheran A . Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Species DNA in Colombia. Front Microbiol. 2017; 8:1907. PMC: 5632848. DOI: 10.3389/fmicb.2017.01907. View

12.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

13.

Garay A, Fraenkel S, Diaz J, Salvioni Recalde O, Vega Gomez M, Riquelme J . Sensitivity comparison for the Leishmania spp. detection in different canine tissues using PCR-HRM. Rev Soc Bras Med Trop. 2022; 55. PMC: 9757722. DOI: 10.1590/0037-8682-0069-2022. View

14.

Slomka M, Sobalska-Kwapis M, Wachulec M, Bartosz G, Strapagiel D . High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies. Int J Mol Sci. 2017; 18(11). PMC: 5713285. DOI: 10.3390/ijms18112316. View

15.

Romero G, Ishikawa E, Cupolillo E, Toaldo C, Guerra M, Paes M . The rarity of infection with Leishmania (Viannia) braziliensis among patients from the Manaus region of Amazonas state, Brazil, who have cutaneous leishmaniasis. Ann Trop Med Parasitol. 2002; 96(2):131-6. DOI: 10.1179/000349802125000745. View

16.

Andrade Zampieri R, Laranjeira-Silva M, Muxel S, de Lima A, Shaw J, Floeter-Winter L . High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species. PLoS Negl Trop Dis. 2016; 10(2):e0004485. PMC: 4771719. DOI: 10.1371/journal.pntd.0004485. View

17.

da Silva L, de Sousa C, Graca G, Porrozzi R, Cupolillo E . Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil. Infect Genet Evol. 2009; 10(1):77-83. DOI: 10.1016/j.meegid.2009.11.001. View

18.

Espada C, Ortiz P, Shaw J, Barral A, Costa J, Uliana S . Identification of Leishmania (Viannia) species and clinical isolates of Leishmania (Leishmania) amazonensis from Brazil using PCR-RFLP of the heat-shock protein 70 gene reveals some unexpected observations. Diagn Microbiol Infect Dis. 2018; 91(4):312-318. DOI: 10.1016/j.diagmicrobio.2018.03.004. View

19.

Boite M, L Mauricio I, Miles M, Cupolillo E . New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis. 2012; 6(11):e1888. PMC: 3486886. DOI: 10.1371/journal.pntd.0001888. View

20.

Kuhls K, Cupolillo E, Silva S, Schweynoch C, Cortes Boite M, Mello M . Population structure and evidence for both clonality and recombination among Brazilian strains of the subgenus Leishmania (Viannia). PLoS Negl Trop Dis. 2013; 7(10):e2490. PMC: 3814519. DOI: 10.1371/journal.pntd.0002490. View