Llanos-Cuentas A et al., 2008. Clinical and parasite species risk factors for pentavalent antimonial treatment failure in cutaneous leishmaniasis in Peru. Clin Infect Dis 46: 223–231.
Arevalo J et al., 2007. Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Infect Dis 195: 1846–1851.
Aronson N et al., 2016. Diagnosis and treatment of leishmaniasis: clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH).
Reithinger R , Dujardin J-C , Louzir H , Pirmez C , Alexander B , Brooker S , 2007. Cutaneous leishmaniasis. Lancet Infect Dis 7: 581–596.
Boggild AK et al., 2010. Clinical and demographic stratification of test performance: a pooled analysis of five laboratory diagnostic methods for American cutaneous leishmaniasis. Am J Trop Med Hyg 83: 345–350.
Boggild AK et al., 2011. Diagnostic performance of filter paper lesion impression PCR for secondarily infected ulcers and nonulcerative lesions caused by cutaneous leishmaniasis. J Clin Microbiol 49: 1097–1100.
Boggild AK et al., 2010. Detection and species identification of Leishmania DNA from filter paper lesion impressions for patients with American cutaneous leishmaniasis. Clin Infect Dis 50: e1–e6.
Boggild AK et al., 2011. Non-invasive cytology brush PCR diagnostic testing in mucosal leishmaniasis: superior performance to conventional biopsy with histopathology. PLoS One 6: e26395.
Akhoundi M et al., 2017. Leishmania infections: molecular targets and diagnosis. Mol Aspects Med 57: 1–29.
Valencia BM et al., 2016. Quantitative kinetoplast DNA assessment during treatment of mucosal leishmaniasis as a potential biomarker of outcome: a pilot study. Am J Trop Med Hyg 94: 107–113.
Kotb Elmahallawy E et al., 2014. Diagnosis of leishmaniasis. J Infect Dev Ctries 8: 961–972.
World Health Organization , 2010. Expert Committee on the Control of the Leishmaniases & World Health Organization. Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22–26 March 2010. Available at: https://apps.who.int/iris/handle/10665/44412.
Valencia BM et al., 2012. Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru. PLoS One 7: e49738.
Van der Auwera G , Dujardin J-C , 2015. Species typing in dermal leishmaniasis. Clin Microbiol Rev 28: 265–294.
Montalvo AM , Fraga J , Maes I , Dujardin J-C , Van der Auwera G , 2012. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol Infect Dis 31: 1453–1461.
Veland N et al., 2013. Simultaneous infection with Leishmania (Viannia) braziliensis and L. (V.) lainsoni in a Peruvian patient with cutaneous leishmaniasis. Am J Trop Med Hyg 88: 774–777.
Fraga J et al., 2012. Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World. Diagn Microbiol Infect Dis 74: 142–150.
de Almeida ME , Steurer FJ , Koru O , Herwaldt BL , Pieniazek NJ , da Silva AJ , 2011. Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA Internal Transcribed Spacer 2. J Clin Microbiol 49: 3143–3149.
Mahdy MAK et al., 2010. Molecular characterization of Leishmania species isolated from cutaneous leishmaniasis in Yemen. PLoS One 5: e12879.
Rotureau B et al., 2006. Use of PCR-restriction fragment length polymorphism analysis to identify the main New World Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples. J Clin Microbiol 44: 459–467.
Schönian G et al., 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47: 349–358.
Victoir K et al., 2003. Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg 97: 80–87.
Zhang W-W et al., 2006. Development of a genetic assay to distinguish between Leishmania viannia species on the basis of isoenzyme differences. Clin Infect Dis 42: 801–809.
Garcia AL et al., 2005. American tegumentary leishmaniasis: antigen-gene polymorphism, taxonomy and clinical pleomorphism. Infect Genet Evol 5: 109–116.
Garcia L et al., 2004. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol 42: 2294–2297.
Tsokana CN , Athanasiou LV , Valiakos G , Spyrou V , Manolakou K , Billinis C , 2014. Molecular Diagnosis of Leishmaniasis, Species Identification and Phylogenetic Analysis. Leishmaniasis—Trends in Epidemiology, Diagnosis and Treatment. InTech.
Van der Auwera G et al., 2016. Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014. Euro Surveill 21: 30418.
Reithinger R , Dujardin J-C , 2007. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45: 21–25.
Cassagne C et al., 2014. Identification of Leishmania at the species level with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Microbiol Infect 20: 551–557.
Ubeda J-M et al., 2014, Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol. 12: e1001868.
Quispe Tintaya KW , Ying X , Dedet J , Rijal S , De Bolle X , Dujardin J , 2004. Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex. J Infect Dis 189: 1035–1043.
NCBI GenBank xm_001562090.1. Available at: https://www.ncbi.nlm.nih.gov/search/all/?term=xm_001562090.1. Accessed July 3, 2019.
NCBI GenBank GQ180933 , 1. Available at: https://www.ncbi.nlm.nih.gov/search/all/?term=GQ180933.1. Accessed July 3, 2019.
NCBI GenBank EU289030 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/EU289030.1. Accessed July 3, 2019.
NCBI GenBank FN395040 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/FN395040.1. Accessed July 3, 2019.
NCBI GenBank FN395052 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/FN395052.1. Accessed July 3, 2019.
NCBI GenBank FN395055 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/FN395055.1. Accessed July 3, 2019.
Leishmania peruviana (ID 40809) - Genome - NCBI. Available at: https://www.ncbi.nlm.nih.gov/genome/?term=L.+peruviana. Accessed July 3, 2019.
Leishmania panamensis (ID 13991) - Genome - NCBI. Available at: https://www.ncbi.nlm.nih.gov/genome/?term=MHOM%2FPA%2F94%2FPSC-1. Accessed July 3, 2019.
Leishmania braziliensis (ID 718) - Genome - NCBI. Available at: https://www.ncbi.nlm.nih.gov/genome/?term=L.+braziliensis+MHOM%2FBR%2F75%2FM2904.
Kuhls K et al., 2013. Population structure and evidence for both clonality and recombination among Brazilian strains of the subgenus Leishmania (Viannia). PLoS Negl Trop Dis 7: e2490.
Montalvo AM , Fraga J , Monzote L , Montano I , De Doncker S , Dujardin JC , Van der Auwera G , 2010. Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World. Parasitology 137: 1159–1168.
Cruz-Barrera ML , Ovalle-Bracho C , Ortegon-Vergara V , Pérez-Franco JE , Echeverry MC , 2015. Improving Leishmania species identification in different types of samples from cutaneous lesions. J Clin Microbiol 53: 1339–1341.
Coelho AC , Boisvert S , Mukherjee A , Leprohon P , Corbeil J , Ouellette M , 2012. Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing. PLoS Negl Trop Dis 6: e1512.
Teixeira DG et al., 2017. Comparative analyses of whole genome sequences of Leishmania infantum isolates from humans and dogs in northeastern Brazil. Int J Parasitol 47: 655–665.
Downing T et al., 2011. Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res 21: 2143–2156.
Rogers MB et al., 2014. Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population. PLoS Genet. 10: e1004092.
Gannavaram S et al., 2016. Modulation of innate immune mechanisms to enhance Leishmania vaccine-induced immunity: role of coinhibitory molecules. Front Immunol 7: 187.
Raja HA , Miller AN , Pearce CJ , Oberlies NH , 2017. Fungal identification using molecular tools: a primer for the natural products research community. J Nat Prod 80: 756–770.
Veland N et al., 2012. Leishmania (Viannia) species identification on clinical samples from cutaneous leishmaniasis patients in Peru: assessment of a molecular stepwise approach. J Clin Microbiol 50: 495–498.
Cupolillo E , Grimaldi Júnior G , Momen H , Beverley SM , 1995. Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol 73: 145–155.
Montalvo AM et al., 2008. PCR-RFLP and RAPD for typing neotropical Leishmania. Biomedica 28: 597–606. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19462565. Accessed July 3, 2019.
Past two years | Past Year | Past 30 Days | |
---|---|---|---|
Abstract Views | 1073 | 340 | 14 |
Full Text Views | 429 | 24 | 0 |
PDF Downloads | 321 | 15 | 0 |
The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.
Disclosure: This study was approved by the Ethics Research Board of Public Health Ontario.
Financial support: This work was funded by Public Health Ontario via a Project Initiation Fund.
Authors’ addresses: Rachel Lau, Public Health Ontario Laboratory, Toronto, Ontario, Canada, E-mail: rachel.lau@oahpp.ca. Avinash N. Mukkala, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada, E-mail: avinash.mukkala@mail.utoronto.ca. Ruwandi Kariyawasam, University of Alberta, Edmonton, Alberta, Canada, E-mail: ruwandi@ualberta.ca. Shareese Clarke, Faculty of Nursing, University of Toronto, Toronto, Ontario, Canada, E-mail: shareese.clarke@mail.utoronto.ca. Braulio M. Valencia, Kirby Institute, New South Wales, Australia, E-mail: braulio.valencia@upch.pe. Alejandro Llanos-Cuentas, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mail: alejandro.llanos.c@upch.pe. Andrea K. Boggild, Institute of Medical Science and the Department of Medicine, University of Toronto, Toronto, Ontario, Canada, E-mail: andrea.boggild@utoronto.ca,
Llanos-Cuentas A et al., 2008. Clinical and parasite species risk factors for pentavalent antimonial treatment failure in cutaneous leishmaniasis in Peru. Clin Infect Dis 46: 223–231.
Arevalo J et al., 2007. Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Infect Dis 195: 1846–1851.
Aronson N et al., 2016. Diagnosis and treatment of leishmaniasis: clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH).
Reithinger R , Dujardin J-C , Louzir H , Pirmez C , Alexander B , Brooker S , 2007. Cutaneous leishmaniasis. Lancet Infect Dis 7: 581–596.
Boggild AK et al., 2010. Clinical and demographic stratification of test performance: a pooled analysis of five laboratory diagnostic methods for American cutaneous leishmaniasis. Am J Trop Med Hyg 83: 345–350.
Boggild AK et al., 2011. Diagnostic performance of filter paper lesion impression PCR for secondarily infected ulcers and nonulcerative lesions caused by cutaneous leishmaniasis. J Clin Microbiol 49: 1097–1100.
Boggild AK et al., 2010. Detection and species identification of Leishmania DNA from filter paper lesion impressions for patients with American cutaneous leishmaniasis. Clin Infect Dis 50: e1–e6.
Boggild AK et al., 2011. Non-invasive cytology brush PCR diagnostic testing in mucosal leishmaniasis: superior performance to conventional biopsy with histopathology. PLoS One 6: e26395.
Akhoundi M et al., 2017. Leishmania infections: molecular targets and diagnosis. Mol Aspects Med 57: 1–29.
Valencia BM et al., 2016. Quantitative kinetoplast DNA assessment during treatment of mucosal leishmaniasis as a potential biomarker of outcome: a pilot study. Am J Trop Med Hyg 94: 107–113.
Kotb Elmahallawy E et al., 2014. Diagnosis of leishmaniasis. J Infect Dev Ctries 8: 961–972.
World Health Organization , 2010. Expert Committee on the Control of the Leishmaniases & World Health Organization. Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22–26 March 2010. Available at: https://apps.who.int/iris/handle/10665/44412.
Valencia BM et al., 2012. Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru. PLoS One 7: e49738.
Van der Auwera G , Dujardin J-C , 2015. Species typing in dermal leishmaniasis. Clin Microbiol Rev 28: 265–294.
Montalvo AM , Fraga J , Maes I , Dujardin J-C , Van der Auwera G , 2012. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol Infect Dis 31: 1453–1461.
Veland N et al., 2013. Simultaneous infection with Leishmania (Viannia) braziliensis and L. (V.) lainsoni in a Peruvian patient with cutaneous leishmaniasis. Am J Trop Med Hyg 88: 774–777.
Fraga J et al., 2012. Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World. Diagn Microbiol Infect Dis 74: 142–150.
de Almeida ME , Steurer FJ , Koru O , Herwaldt BL , Pieniazek NJ , da Silva AJ , 2011. Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA Internal Transcribed Spacer 2. J Clin Microbiol 49: 3143–3149.
Mahdy MAK et al., 2010. Molecular characterization of Leishmania species isolated from cutaneous leishmaniasis in Yemen. PLoS One 5: e12879.
Rotureau B et al., 2006. Use of PCR-restriction fragment length polymorphism analysis to identify the main New World Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples. J Clin Microbiol 44: 459–467.
Schönian G et al., 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47: 349–358.
Victoir K et al., 2003. Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg 97: 80–87.
Zhang W-W et al., 2006. Development of a genetic assay to distinguish between Leishmania viannia species on the basis of isoenzyme differences. Clin Infect Dis 42: 801–809.
Garcia AL et al., 2005. American tegumentary leishmaniasis: antigen-gene polymorphism, taxonomy and clinical pleomorphism. Infect Genet Evol 5: 109–116.
Garcia L et al., 2004. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol 42: 2294–2297.
Tsokana CN , Athanasiou LV , Valiakos G , Spyrou V , Manolakou K , Billinis C , 2014. Molecular Diagnosis of Leishmaniasis, Species Identification and Phylogenetic Analysis. Leishmaniasis—Trends in Epidemiology, Diagnosis and Treatment. InTech.
Van der Auwera G et al., 2016. Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014. Euro Surveill 21: 30418.
Reithinger R , Dujardin J-C , 2007. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45: 21–25.
Cassagne C et al., 2014. Identification of Leishmania at the species level with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Microbiol Infect 20: 551–557.
Ubeda J-M et al., 2014, Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol. 12: e1001868.
Quispe Tintaya KW , Ying X , Dedet J , Rijal S , De Bolle X , Dujardin J , 2004. Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex. J Infect Dis 189: 1035–1043.
NCBI GenBank xm_001562090.1. Available at: https://www.ncbi.nlm.nih.gov/search/all/?term=xm_001562090.1. Accessed July 3, 2019.
NCBI GenBank GQ180933 , 1. Available at: https://www.ncbi.nlm.nih.gov/search/all/?term=GQ180933.1. Accessed July 3, 2019.
NCBI GenBank EU289030 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/EU289030.1. Accessed July 3, 2019.
NCBI GenBank FN395040 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/FN395040.1. Accessed July 3, 2019.
NCBI GenBank FN395052 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/FN395052.1. Accessed July 3, 2019.
NCBI GenBank FN395055 , 1. Available at: https://www.ncbi.nlm.nih.gov/nuccore/FN395055.1. Accessed July 3, 2019.
Leishmania peruviana (ID 40809) - Genome - NCBI. Available at: https://www.ncbi.nlm.nih.gov/genome/?term=L.+peruviana. Accessed July 3, 2019.
Leishmania panamensis (ID 13991) - Genome - NCBI. Available at: https://www.ncbi.nlm.nih.gov/genome/?term=MHOM%2FPA%2F94%2FPSC-1. Accessed July 3, 2019.
Leishmania braziliensis (ID 718) - Genome - NCBI. Available at: https://www.ncbi.nlm.nih.gov/genome/?term=L.+braziliensis+MHOM%2FBR%2F75%2FM2904.
Kuhls K et al., 2013. Population structure and evidence for both clonality and recombination among Brazilian strains of the subgenus Leishmania (Viannia). PLoS Negl Trop Dis 7: e2490.
Montalvo AM , Fraga J , Monzote L , Montano I , De Doncker S , Dujardin JC , Van der Auwera G , 2010. Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World. Parasitology 137: 1159–1168.
Cruz-Barrera ML , Ovalle-Bracho C , Ortegon-Vergara V , Pérez-Franco JE , Echeverry MC , 2015. Improving Leishmania species identification in different types of samples from cutaneous lesions. J Clin Microbiol 53: 1339–1341.
Coelho AC , Boisvert S , Mukherjee A , Leprohon P , Corbeil J , Ouellette M , 2012. Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing. PLoS Negl Trop Dis 6: e1512.
Teixeira DG et al., 2017. Comparative analyses of whole genome sequences of Leishmania infantum isolates from humans and dogs in northeastern Brazil. Int J Parasitol 47: 655–665.
Downing T et al., 2011. Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res 21: 2143–2156.
Rogers MB et al., 2014. Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population. PLoS Genet. 10: e1004092.
Gannavaram S et al., 2016. Modulation of innate immune mechanisms to enhance Leishmania vaccine-induced immunity: role of coinhibitory molecules. Front Immunol 7: 187.
Raja HA , Miller AN , Pearce CJ , Oberlies NH , 2017. Fungal identification using molecular tools: a primer for the natural products research community. J Nat Prod 80: 756–770.
Veland N et al., 2012. Leishmania (Viannia) species identification on clinical samples from cutaneous leishmaniasis patients in Peru: assessment of a molecular stepwise approach. J Clin Microbiol 50: 495–498.
Cupolillo E , Grimaldi Júnior G , Momen H , Beverley SM , 1995. Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol 73: 145–155.
Montalvo AM et al., 2008. PCR-RFLP and RAPD for typing neotropical Leishmania. Biomedica 28: 597–606. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19462565. Accessed July 3, 2019.
Past two years | Past Year | Past 30 Days | |
---|---|---|---|
Abstract Views | 1073 | 340 | 14 |
Full Text Views | 429 | 24 | 0 |
PDF Downloads | 321 | 15 | 0 |