Diagnostic Accuracy of Loop-mediated Isothermal Amplifica-tion Assay as a Field Molecular Tool for Rapid Mass Screening of Old World Leishmania Infections in Sand Flies and In Vitro Culture
AbstractBackground: We employed a highly sensitive loop-mediated isothermal amplification (LAMP) by targeting 18S rRNA gene to identify the rapid mass screening of Leishmania infections in captured sand flies of southwest Iran and In vitro culture. Methods: One hundred fifty sand flies were collected from 11 sites adjacent to Iraqi’s borders in southern parts of Khuzestan Province by using sticky sheets of paper and CDC miniature light traps during late May 2014 to Nov 2015. Following morphological identification of sand flies species, the DNA of infected samples was extracted and amplified by PCR and LAMP assays by targeting ITS-rDNA and 18S rRNA genes. The PCR amplicons were directly sequenced to conduct the phylogenetic analysis Results: Ten (6.6%) Leishmania infections were identified by LAMP assay (detection limit 0.01 parasites DNA) among infected Sergentomyia baghdadis, S. sintoni and Phlebotomus papatasi sand flies that was more sensitive than PCR (n=6.4%; (detection limit 101parasites DNA). LAMP can identify 101-106promastigotes/100 µl RPMI 1640 while PCR recognized104-106 promastigotes. The majority infection rate of sand flies was confirmed to L. major inferred by phylogenetic analysis. Conclusion: This is the first exploration characterized the Old World Leishmania infections by LAMP technique in both infected sand flies and In vitro conditions. The LAMP method because of its shorter reaction time, robustness, more sensitivity, lack of requirement of complicated equipment and visual discriminatory of positivity can be appeared a promising tool instead of PCR to identify low Leishmania loads and entomological monitoring of leishmaniasis in resource-limited endemic of the world.
Alvar J, Yactayo S, Bern C. Leishmaniasis and poverty. Trends Parasitol. 2006; 22(12):552-7.
Rouhani S, Mirzaei A, Spotin A et al. Novel identification of Leishmania major in Hemiechinus auritus and molecular detection of this parasite in Meriones libycus from an important foci of zoonotic cutaneous leishmaniasis in Iran. J Infect Public Health. 2014;7(3):210-7.
Spotin A, Rouhani S, Ghaemmaghami P et al. Different morphologies of Leishmania major amastigotes with no molecular diversity in a neglected endemic area of zoonotic cutaneous leishmaniasis in Iran. Iran Biomed J. 2015;19(3):149-59.
Spotin A, Rouhani S, Parvizi P. The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan province, Iran. Biomed Res Int. 2014;2014:913510.
Shirzadi MR, Esfahania SB, Mohebalia M et al. Epidemiological status of leishmaniasis in the Islamic Republic of Iran, 1983–2012. East Mediterr Health J. 2015;21(10):736-42.
Holakouie-Naieni K, Mostafavi E, Boloorani AD et al. Spatial modeling of cutaneous leishmaniasis in Iran from 1983 to 2013. Acta Trop. 2017;166:67-73.
Heydarpour F, Sari AA, Mohebali M et al. Incidence and Disability-Adjusted Life Years (Dalys) Attributable to Leishmaniasis In Iran, 2013. Ethiop J Health Sci. 2016;26(4):381-8.
Ready PD. Biology of phlebotomine sand flies as vectors of disease agents. Annu Rev Entomol. 2013;58:227-50.
Parvizi P, Taherkhani H, Ready PD. Phlebotomus caucasicus and Phlebotomus mongolensis (Diptera: Psychodidae): indistinguishable by the mitochondrial cytochrome b gene in Iran. Bull Entomol Res. 2010;100(4):415-20.
Ashford RW, Desjeux P, Deraadt P. Estimation of population at risk of infection and number of cases of leishmaniasis. Parasitol Today. 1992;8(3):104-5.
Spotin A, Parvizi P. Comparative study of viscerotropic pathogenicity of Leishmania major amastigotes and promastigotes based on identification of mitochondrial and nucleus sequences. Parasitol Res. 2016;115(3):1221-8.
Jorquera A, González R, Marchán-Marcano E et al. Multiplex-PCR for detection of natural Leishmania infection in Lutzomyia spp. captured in an endemic region for cutaneous leishmaniasis in state of Sucre, Venezuela. Mem Inst Oswaldo Cruz. 2005;100(1):45-8.
Garcia AL, Tellez T, Parrado R et al. Epidemiological monitoring of American tegumentary leishmaniasis: molecular characterization of a peridomestic transmission cycle in the Amazonian lowlands of Bolivia. Trans R Soc Trop Med Hyg. 2007;101(12):1208-13.
Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS‐rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health. 2008;13(9):1159-71.
Ranasinghe S, Rogers ME, Hamilton JG et al. A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis. Trans R Soc Trop Med Hyg. 2008;102(9):875-82.
Sharbatkhori M, Spotin A, Taherkhani H et al. Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran. J Vector Borne Dis. 2014;51(1):16-21.
Akane A, Matsubara K, Nakamura H et al. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci. 1994;39(2):362-72.
Bélec L, Authier J, Eliezer-Vanerot MC et al. Myoglobin as a polymerase chain reaction (PCR) inhibitor: a limitation for PCR from skeletal muscle tissue avoided by the use of Thermus thermophilus polymerase. Muscle Nerve. 1998;21(8):1064-7.
Al-Soud WA, Jönsson LJ, Râdström P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000;38(1):345-50.
Ikadai H, Tanaka H, Shibahara N et al. Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol. 2004;42(6):2465-9.
Poon LL, Wong BW, Ma EH et al. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem. 2006;52(2):303-6.
Njiru ZK, Traub R, Ouma JO et al. Detection of Group 1 Trypanosoma brucei gambiense by loop-mediated isothermal amplification. J Clin Microbiol. 2011;49(4):1530-6.
Ghasemian M, Gharavi MJ, Akhlaghi L et al. Development and Assessment of Loop-Mediated Isothermal Am-plification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran. Iran J Parasitol. 2014; 9: 50-59.
Nzelu CO, Gomez EA, Cáceres AG et al. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Trop. 2014;132:1-6.
Ebrahimi S, Bordbar A, Rastaghi AR et al. Spatial distribution of sand fly species (Psychodidae: Phlebtominae), ecological niche, and climatic regionalization in zoonotic foci of cutaneous leishmaniasis, southwest of Iran. J Vector Ecol. 2016;41(1):103-9.
Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006;23(2):254-67.
Khan MG, Bhaskar KR, Salam MA et al. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors. 2012;5:280.
Adams ER, Schoone GJ, Ageed AF et al. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples. Am J Trop Med Hyg. 2010;82(4):591-6.
Nzelu CO, Cáceres AG, Guerrero-Quincho S et al. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool. Acta Trop. 2016;153:116-9.
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