Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients

Particle and Fibre Toxicology

Dinesh Mondal¹  Gerd Pluschke²  Tania Akther¹  Md Abdus Salam³  Khondaker Rifat Hasan Bhaskar¹  Md Gulam Musawwir Khan¹ 

[1] International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka, 1212, Bangladesh;Head of Department, Medical Parasitology & Infection Biology, Molecular Immunology, Basel, Switzerland;Rajshahi Medical College, Rajshahi, 6000, Bangladesh

关键词: Bangladesh;    PCR;    Diagnosis;    Visceral leishmaniasis;    LAMP;   

Others  :  1228820
DOI  :  10.1186/1756-3305-5-280

 received in 2012-08-02, accepted in 2012-11-21,  发布年份 2012

【 摘 要 】

Background

Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world’s total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise required in the classical parasitological diagnosis, the goal of the VL experts has been to develop noninvasive procedure(s) applicable in the field settings. Several serological and molecular biological approaches have been developed over the last decades, but only a few are applicable in field settings that can be performed with relative ease. Recently, loop-mediated isothermal amplification (LAMP) has emerged as a novel nucleic acid amplification method for diagnosis of VL. In this study, we have evaluated the LAMP assay using buffy coat DNA samples from VL patients in Bangladesh and compared its performance with leishmania nested PCR (Ln-PCR), an established molecular method with very high diagnostic indices.

Methods

Seventy five (75) parasitologically confirmed VL patients by spleen smear microcopy and 101 controls (endemic healthy controls −25, non-endemic healthy control-26, Tuberculosis-25 and other diseases-25) were enrolled in this study. LAMP assay was carried out using a set of four primers targeting L. donovani kinetoplast minicircle DNA under isothermal (62 °C) conditions in a heat block. For Ln-PCR, we used primers targeting the parasite’s small-subunit rRNA region.

Results

LAMP assay was found to be positive in 68 of 75 confirmed VL cases, and revealed its diagnostic sensitivity of 90.7% (95.84-81.14, 95% CI), whereas all controls were negative by LAMP assay, indicating a specificity of 100% (100–95.43, 95% CI). The Ln-PCR yielded a sensitivity of 96% (98.96-87.97, 95% CI) and a specificity of 100% (100–95.43, 95% CI).

Conclusion

High diagnostic sensitivity and excellent specificity were observed in this first report of LAMP diagnostic evaluation from Bangladesh. Considering its many fold advantages over conventional PCR and potential to be used as a simple and rapid test in the VL endemic areas of the Indian subcontinent, our findings are encouraging, but further evaluation of LAMP is needed.

【 授权许可】

   
2012 Khan et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M: Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLoS One 2012, 7(5):e35671.
• [2]WHO: Regional Technical Advisory Group on Kala-azar Elimination. Report of the first meeting, Manesar, Haryana, 20–23 December 2004. New Delhi: WHO Regional Office for South-East Asia; 2005.
• [3]Badaro R, Benson D, Eulalio MC, Freire M, Cunha S, Netto EM, Pedral-Sampaio D, Madureira C, Burns JM, Houghton RL, et al.: rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis 1996, 173(3):758-761.
• [4]Khan MG, Alam MS, Podder MP, Itoh M, Jamil KM, Haque R, Wagatsuma Y: Evaluation of rK-39 strip test using urine for diagnosis of visceral leishmaniasis in an endemic area in Bangladesh. Parasit Vectors 2010, 3:114. BioMed Central Full Text
• [5]Khan MG, Alam MS, Bhuiyan AT, Jamil MA, Saha B, Islam M, Haque R, Hossain M, Jamil KM: Short communication: evaluation of a new rapid diagnostic test for quality assurance by kala azar elimination programme in Bangladesh. J Parasitol Res 2011, 2011:862475.
• [6]Deniau M, Canavate C, Faraut-Gambarelli F, Marty P: The biological diagnosis of leishmaniasis in HIV-infected patients. Ann Trop Med Parasitol 2003, 97(Suppl 1):115-133.
• [7]Rijal S, Boelaert M, Regmi S, Karki BM, Jacquet D, Singh R, Chance ML, Chappuis F, Hommel M, Desjeux P, et al.: Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal. Trop Med Int Health 2004, 9(6):724-729.
• [8]Singh DP, Goyal RK, Singh RK, Sundar S, Mohapatra TM: In search of an ideal test for diagnosis and prognosis of kala-azar. J Health Popul Nutr 2010, 28(3):281-285.
• [9]Salam MA, Khan MG, Mondal D: Urine antigen detection by latex agglutination test for diagnosis and assessment of initial cure of visceral leishmaniasis. Trans R Soc Trop Med Hyg 2011, 105(5):269-272.
• [10]Lachaud L, Chabbert E, Dubessay P, Reynes J, Lamothe J, Bastien P: Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood. J Clin Microbiol 2001, 39(2):613-617.
• [11]Cruz I, Canavate C, Rubio JM, Morales MA, Chicharro C, Laguna F, Jimenez-Mejias M, Sirera G, Videla S, Alvar J: A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg 2002, 96(Suppl 1):S185-189.
• [12]Salam MA, Mondal D, Kabir M, Ekram ARMS, Haque R: PCR for diagnosis and assessment of cure in kala-azar patients in Bangladesh. Acta Trop 2010, 113(1):52-55.
• [13]Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T: Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000, 28(12):E63.
• [14]Takagi H, Itoh M, Islam MZ, Razzaque A, Ekram ARMS, Hashighuchi Y, Noiri E, Kimura E: Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification. Am J Trop Med Hyg 2009, 81(4):578-582.
• [15]Chulay JD, Bryceson AD: Quantitation of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis. Am J Trop Med Hyg 1983, 32(3):475-479.
• [16]Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, Kibona SN, Thompson RC, Ndung'u JM: African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 2008, 38(5):589-599.
• [17]Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I: Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 2003, 41(12):5517-5524.
• [18]Thekisoe OM, Kuboki N, Nambota A, Fujisaki K, Sugimoto C, Igarashi I, Yasuda J, Inoue N: Species-specific loop-mediated isothermal amplification (LAMP) for diagnosis of trypanosomosis. Acta Trop 2007, 102(3):182-189.
• [19]Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, 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):303-306.
• [20]Lau YL, Fong MY, Mahmud R, Chang PY, Palaeya V, Cheong FW, Chin LC, Anthony CN, Al-Mekhlafi AM, Chen Y: Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP) in blood samples. Malar J 2011, 10:197. BioMed Central Full Text
• [21]Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M, Sanga E, Hoelscher M, Notomi T, Hase T, et al.: Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin Microbiol 2007, 45(6):1936-1940.
• [22]Tao ZY, Zhou HY, Xia H, Xu S, Zhu HW, Culleton RL, Han ET, Lu F, Fang Q, Gu YP, Liu YB, Zhu GD, Wang WM, Li JL, Cao J, Gao Q: Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection. Parasit Vectors 2011, 115(4):1-8.
• [23]Aonuma H, Yoshimura A, Perera N, Shinzawa N, Bando H, Oshiro S, Nelson B, Fukumoto S, Kanuka H: Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model. Parasit Vectors 2009, (1):15. BioMed Central Full Text
• [24]Adams ER, Schoone GJ, Ageed AF, Safi SE, Schallig HD: 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):591-596.
• [25]Alam MZ, Shamsuzzaman AK, Kuhls K, Schonian G: PCR diagnosis of visceral leishmaniasis in an endemic region, Mymensingh district, Bangladesh. Trop Med Int Health 2009, 14(5):499-503.
• [26]Roelfsema JH, Nozari N, Herremans T, Kortbeek LM, Pinelli E: Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients. Exp Parasitol 2011, 127(1):36-41.
• [27]Marfurt J, Niederwieser I, Makia ND, Beck HP, Felger I: Diagnostic genotyping of Old and New World Leishmania species by PCR-RFLP. Diagn Microbiol Infect Dis 2003, 46(2):115-124.
• [28]Enomoto Y, Yoshikawa T, Ihira M, Akimoto S, Miyake F, Usui C, Suga S, Suzuki K, Kawana T, Nishiyama Y, et al.: Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method. J Clin Microbiol 2005, 43(2):951-955.
• [29]Nagamine K, Hase T, Notomi T: Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 2002, 16(3):223-229.
• [30]Hill J, Beriwal S, Chandra I, Paul VK, Kapil A, Singh T, Wadowsky RM, Singh V, Goyal A, Jahnukainen T, et al.: Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. J Clin Microbiol 2008, 46(8):2800-2804.
• [31]Njiru ZK, Ouma JO, Bateta R, Njeru SE, Ndungu K, Gitonga PK, Guya S, Traub R: Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA. Vet Parasitol 2011, 180(3–4):358-362.
• [32]Mori Y, Nagamine K, Tomita N, Notomi T: Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 2001, 289(1):150-154.