- INTRODUCTION
- METHODS
- Ethical statement.
- Reference strains.
- DNA extraction and serial dilutions.
- Loop-mediated isothermal amplification assays.
- Loop-mediated isothermal amplification analytical specificity.
- Loop-mediated isothermal amplification analytical sensitivity.
- Evaluation of LAMP in biological samples (sandflies and direct smears from skin lesions of patients with CL).
- RESULTS
- DISCUSSION
- Supplementary Material
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Akhoundi M et al. 2017. Leishmania infections: molecular targets and diagnosis. Mol Aspects Med 57: 1–29.
-
2.
Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M; WHO Leishmaniasis Control Team, 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671.
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3.
World Health Organization (WHO)/Department of Control of Neglected Tropical Diseases, 2017. Global leishmaniasis update, 2006–2015: a turning point in leishmaniasis surveillance. Weekly Epidemiological Record 38: 557–565.
-
4.
Montalvo AM, Fraga J, Tirado D, Blandón G, Alba A, Van der Auwera G, Vélez ID, Muskus C, 2017. Detection and identification of Leishmania spp.: application of two Hsp70-based PCR-RFLP protocols to clinical samples from the New World. Parasitol Res 116: 1843–1848.
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5.
Ramirez JD, Hernandez C, Leon CM, Ayala MS, Florez C, González C, 2016. Taxonomy, diversity, temporal and geographical distribution of cutaneous leishmaniasis in Colombia: a retrospective study. Sci Rep 6: 28266.
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Mohammadiha A, Mohebali M, Haghighi A, Mahdian R, Abadi AR, Zarei Z, Yeganeh F, Kazemi B, Taghipour N, Akhoundi B, 2013. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol 133: 89–94.
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7.
Munoz EB, Santander S, Rojas-Silva P, Cardenas PA, Fornasini M, Cifuentes SC, Salvador D, Baldeón ME, 2016. Diagnostic efficacy of molecular techniques for detection and identification of Leishmania species in human whole blood and skin samples from Ecuador. Am J Trop Med Hyg 95: 803–805.
-
8.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T, 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63.
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9.
Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877–882.
-
10.
Adams ER, Gomez MA, Scheske L, Rios R, Marquez R, Cossio A, Albertini A, Schallig H, Saravia NG, 2014. Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR. Parasitology 141: 1891–1897.
-
11.
Mikita K, Maeda T, Yoshikawa S, Ono T, Miyahira Y, Kawana A, 2014. The Direct Boil-LAMP method: a simple and rapid diagnostic method for cutaneous leishmaniasis. Parasitol Int 63: 785–789.
-
12.
Abbasi I, Kirstein OD, Hailu A, Warburg A, 2016. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples. Acta Trop 162: 20–26.
-
13.
Ghasemian M, Gharavi MJ, Akhlaghi L, Mohebali M, Meamar AR, Aryan E, Oormazdi H, 2014. Development and assessment of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of human visceral leishmaniasis in Iran. Iran J Parasitol 9: 50–59.
-
14.
Sriworarat C, Phumee A, Mungthin M, Leelayoova S, Siriyasatien P, 2015. Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection. Parasit Vectors 8: 591.
-
15.
Qiao YM, Guo YC, Zhang XE, Zhou YF, Zhang ZP, Wei HP, Yang RF, Wang DB, 2007. Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol Lett 29: 1939–1946.
-
16.
Villari C, Tomlinson JA, Battisti A, Boonham N, Capretti P, Faccoli M, 2013. Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus. Appl Environ Microbiol 79: 2527–2533.
-
17.
Inacio J, Flores O, Spencer-Martins I, 2008. Efficient identification of clinically relevant Candida yeast species by use of an assay combining panfungal loop-mediated isothermal DNA amplification with hybridization to species-specific oligonucleotide probes. J Clin Microbiol 46: 713–720.
-
18.
Wozniakowski G, Samorek-Salamonowicz E, Kozdrun W, 2013. Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek’s disease virus serotypes 1, 2, and 3. Avian Dis 57: 539–543.
-
19.
Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, Hasebe F, Morita K, 2004. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 42: 1956–1961.
-
20.
Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I, 2003. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41: 5517–5524.
-
21.
Poon LL et al. 2006. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52: 303–306.
-
22.
Besuschio SA et al. 2017. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples. PLoS Negl Trop Dis 11: e0005779.
-
23.
Nzelu CO, Gomez EA, Caceres AG, Sakurai T, Martini-Robles L, Uezato H, Mimori T, Katakura K, Hashiguchi Y, Kato H, 2014. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Trop 132: 1–6.
-
24.
Khan MG, Bhaskar KR, Salam MA, Akther T, Pluschke G, Mondal D, 2012. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors 5: 280.
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Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis
Cielo M. León
Cielo M. León
Grupo de Investigaciones Microbiológicas-UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia;
Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia;
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Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia;
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Marina Muñoz
Marina Muñoz
Grupo de Investigaciones Microbiológicas-UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia;
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Juan H. Tabares
Juan H. Tabares
Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia;
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Carolina Hernandez
Carolina Hernandez
Grupo de Investigaciones Microbiológicas-UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia;
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Carolina Florez
Carolina Florez
Grupo de Parasitología, Instituto Nacional de Salud, Bogotá, Colombia
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Martha S. Ayala
Martha S. Ayala
Grupo de Parasitología, Instituto Nacional de Salud, Bogotá, Colombia
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Juan David Ramírez
Juan David Ramírez
Grupo de Investigaciones Microbiológicas-UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia;
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Loop-mediated isothermal amplification (LAMP) is ideal for the detection of Leishmania DNA as it is a quick and easy-to-perform test that does not require complex or sophisticated equipment or infrastructure. However, the application of this technique in the detection of Leishmania DNA has not been comprehensively analyzed to date (analytical validation). Our objective was to evaluate the sensitivity and analytical specificity (anticipated reportable range [ARR], the limit of detection [LoD], and accuracy) of LAMP targeting the 18S rRNA gene in the diagnosis of six New World Leishmania species. We then applied the validated LAMP assay across 50 samples of sandflies and 50 direct smears from a recent outbreak of cutaneous leishmaniasis in Colombia to determine its diagnostic performance. The LAMP assay exclusively amplified the DNA of Leishmania spp., and an ARR of between 1 × 104 and 1 × 10−2 equivalent parasites/mL was determined. An LoD of 1 × 10−2 equivalent parasites/mL was established and there was no statistically significant variation in terms of accuracy. Finally, a sensitivity of 100% in direct smears and sandflies samples was calculated and a specificity of 90.9% for direct smears using microscopy as reference and 96.8% for sandflies using real-time polymerase chain reaction as reference were determined. To our knowledge, this is the first attempt to analytically validate a LAMP test to detect Leishmania DNA, which showed good diagnostic potential from sandflies and direct smear samples.
- Supplemental Materials (PDF 26 KB)
Author Notes
Financial support: We thank the Departamento Administrativo de Ciencia, Tecnología e Innovación ‘COLCIENCIAS’ for funding the Project “Fortalecimiento de la capacidad diagnóstica, de investigación y de vigilancia de enfermedades transmisibles emergentes y reemergentes en Colombia” grant number 757-13.
Authors’ addresses: Cielo M. León, Marina Muñoz, Carolina Hernandez, and Juan David Ramírez, Facultad de Ciencias Naturales y Matemáticas, Grupo de Investigaciones Microbiológicas, Universidad del Rosario, Bogotá, Colombia, E-mails: cmlr8527@gmail.com, claudiamarina23@gmail.com, dcahernandez@gmail.com or dcahernandezc@gmail.com, and juand.ramirez@urosario.edu.co. Juan H. Tabares, Departamento de Salud Publica, Universidad Nacional de Colombia, Bogotá, Colombia, E-mail: jhtabaresg@unal.edu.co. Carolina Florez and Martha S. Ayala, Grupo de Parasitología, Instituto Nacional de Salud, Bogotá, Colombia, E-mails: aflorez@ins.gov.co and mayalas@ins.gov.co.
- INTRODUCTION
- METHODS
- Ethical statement.
- Reference strains.
- DNA extraction and serial dilutions.
- Loop-mediated isothermal amplification assays.
- Loop-mediated isothermal amplification analytical specificity.
- Loop-mediated isothermal amplification analytical sensitivity.
- Evaluation of LAMP in biological samples (sandflies and direct smears from skin lesions of patients with CL).
- RESULTS
- DISCUSSION
- Supplementary Material
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Akhoundi M et al. 2017. Leishmania infections: molecular targets and diagnosis. Mol Aspects Med 57: 1–29.
-
2.
Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M; WHO Leishmaniasis Control Team, 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671.
-
3.
World Health Organization (WHO)/Department of Control of Neglected Tropical Diseases, 2017. Global leishmaniasis update, 2006–2015: a turning point in leishmaniasis surveillance. Weekly Epidemiological Record 38: 557–565.
-
4.
Montalvo AM, Fraga J, Tirado D, Blandón G, Alba A, Van der Auwera G, Vélez ID, Muskus C, 2017. Detection and identification of Leishmania spp.: application of two Hsp70-based PCR-RFLP protocols to clinical samples from the New World. Parasitol Res 116: 1843–1848.
-
5.
Ramirez JD, Hernandez C, Leon CM, Ayala MS, Florez C, González C, 2016. Taxonomy, diversity, temporal and geographical distribution of cutaneous leishmaniasis in Colombia: a retrospective study. Sci Rep 6: 28266.
-
6.
Mohammadiha A, Mohebali M, Haghighi A, Mahdian R, Abadi AR, Zarei Z, Yeganeh F, Kazemi B, Taghipour N, Akhoundi B, 2013. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol 133: 89–94.
-
7.
Munoz EB, Santander S, Rojas-Silva P, Cardenas PA, Fornasini M, Cifuentes SC, Salvador D, Baldeón ME, 2016. Diagnostic efficacy of molecular techniques for detection and identification of Leishmania species in human whole blood and skin samples from Ecuador. Am J Trop Med Hyg 95: 803–805.
-
8.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T, 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63.
-
9.
Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877–882.
-
10.
Adams ER, Gomez MA, Scheske L, Rios R, Marquez R, Cossio A, Albertini A, Schallig H, Saravia NG, 2014. Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR. Parasitology 141: 1891–1897.
-
11.
Mikita K, Maeda T, Yoshikawa S, Ono T, Miyahira Y, Kawana A, 2014. The Direct Boil-LAMP method: a simple and rapid diagnostic method for cutaneous leishmaniasis. Parasitol Int 63: 785–789.
-
12.
Abbasi I, Kirstein OD, Hailu A, Warburg A, 2016. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples. Acta Trop 162: 20–26.
-
13.
Ghasemian M, Gharavi MJ, Akhlaghi L, Mohebali M, Meamar AR, Aryan E, Oormazdi H, 2014. Development and assessment of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of human visceral leishmaniasis in Iran. Iran J Parasitol 9: 50–59.
-
14.
Sriworarat C, Phumee A, Mungthin M, Leelayoova S, Siriyasatien P, 2015. Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection. Parasit Vectors 8: 591.
-
15.
Qiao YM, Guo YC, Zhang XE, Zhou YF, Zhang ZP, Wei HP, Yang RF, Wang DB, 2007. Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol Lett 29: 1939–1946.
-
16.
Villari C, Tomlinson JA, Battisti A, Boonham N, Capretti P, Faccoli M, 2013. Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus. Appl Environ Microbiol 79: 2527–2533.
-
17.
Inacio J, Flores O, Spencer-Martins I, 2008. Efficient identification of clinically relevant Candida yeast species by use of an assay combining panfungal loop-mediated isothermal DNA amplification with hybridization to species-specific oligonucleotide probes. J Clin Microbiol 46: 713–720.
-
18.
Wozniakowski G, Samorek-Salamonowicz E, Kozdrun W, 2013. Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek’s disease virus serotypes 1, 2, and 3. Avian Dis 57: 539–543.
-
19.
Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, Hasebe F, Morita K, 2004. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 42: 1956–1961.
-
20.
Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I, 2003. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41: 5517–5524.
-
21.
Poon LL et al. 2006. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52: 303–306.
-
22.
Besuschio SA et al. 2017. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples. PLoS Negl Trop Dis 11: e0005779.
-
23.
Nzelu CO, Gomez EA, Caceres AG, Sakurai T, Martini-Robles L, Uezato H, Mimori T, Katakura K, Hashiguchi Y, Kato H, 2014. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Trop 132: 1–6.
-
24.
Khan MG, Bhaskar KR, Salam MA, Akther T, Pluschke G, Mondal D, 2012. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors 5: 280.
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