Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application.
We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL).
The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse.
The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.
World Health Organization. Leishmaniasis in high-burden countries: an epidemiological update based on data reported in 2014. World Health Organ, Week Epidemiol Rec. 2016.
Desjeux P, Ghosh R, Dhalaria P, Strub-Wourgaft N, Zijlstra E. Report of the Post Kala-Azar Dermal Leishmaniasis (PKDL) consortium meeting, New Delhi, India, 27–29 June 2012. Parasit & Vectors. 2013;6:196. CrossRef
Kumar R, Bumb RA, Ansari NA, Mehta RD, Salotra P. Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: parasite identification and characterization using molecular and immunologic tools. Am J Trop Med Hyg. 2007;76(5):896–901. PubMed
Salotra P, Singh R. Challenges in the diagnosis of post kala-azar dermal leishmaniasis. Indian J Med Res. 2006;123(3):295–310. PubMed
Salotra P, Sreenivas G, Pogue G, Lee N, Nakhasi H, Ramesh V, et al. Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol. 2001;39(3):849–54. CrossRefPubMedPubMedCentral
Antinori S, Calattini S, Longhi E, Bestetti G, Piolini R, Magni C, et al. Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in hiv-infected and hiv-uninfected patients: a single-center, 8-year experience in Italy and review of the literature. Clin Infect Dis. 2007;44(12):1602–10. CrossRefPubMed
Gonçalves-de-Albuquerque S, Pessoa e Silva R, De Morais R, Trajano-Silva L, Régis-da-Silva C, Brandão-Filho S, et al. Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis. J Venom Anim Toxins Incl Trop Dis. 2014;20:16. doi: 10.1186/1678-9199-20-16. CrossRefPubMedCentral
Rodriguez-Gonzalez I, MarÃn C, Longoni S, Mateo H, Alunda J, Minaya G, et al. Identification of New World Leishmania species from Peru by biochemical techniques and multiplex PCR assay. FEMS Microbiol Lett. 2007;267(1):9–16. http://dx.doi.org/ 10.1111/j.1574-6968.2006.00574.x. CrossRefPubMed
Verma S, Kumar R, Katara GK, Singh LC, Negi NS, Ramesh V, et al. Quantification of parasite load in clinical samples of leishmaniasis patients: IL-10 level correlates with parasite load in visceral leishmaniasis. PLoS One. 2010;5:e10107. doi: 10.1371/journal.pone.0010107. CrossRefPubMedPubMedCentral
World Health Organization. Control of the leishmaniases. World Health Organ, Tech Rep Ser. 2010;949:22–6.
Bakheit M, Torra D, Palomino L, Thekisoe O, Mbati P, Ongerth J, et al. Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet Parasitol. 2008;158(1-2):11–22. CrossRefPubMed
Verma S, Bhandari V, Avishek K, Ramesh V, Salotra P. Reliable diagnosis of post-kala-azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures. Trop Med Int Health. 2012;18(3):268–75. PubMed
- Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection
Ram Avtar Bumb
Narendra Singh Negi
- BioMed Central
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