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. 2019 Jul 30;2019:9392414.
doi: 10.1155/2019/9392414. eCollection 2019.

Application of kDNA Minicircle PCR-RFLP to Characterize Leishmania donovani Clinical Isolates Obtained From Post-Kala-Azar Dermal Leishmaniasis in Eastern Nepal

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Free PMC article

Application of kDNA Minicircle PCR-RFLP to Characterize Leishmania donovani Clinical Isolates Obtained From Post-Kala-Azar Dermal Leishmaniasis in Eastern Nepal

Ojesh Pokhrel et al. Can J Infect Dis Med Microbiol. .
Free PMC article

Abstract

Post-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation of visceral leishmaniasis (VL) which develops after apparent cure in some patients. PKDL is considered as the potential reservoir for the VL infection. Molecular epidemiological characterization of L. donovani isolates obtained from VL and PKDL isolates is essentially required in order to understand the transmission dynamics of the VL infection. To date, genetic variation among the VL and PKDL L. donovani isolates was not fully elucidated. Therefore, 14 clinical isolates from VL and 4 clinical isolates from PKDL were speciated by hsp70 and rDNA genes. Further characterization of L. donovani by haspB PCR demonstrates two different genotypes. All PKDL isolates have the same genetic structure. kDNA PCR-RFLP assay revealed 18 different genotypes; however, structural analysis showed the two distinct kDNA genotype population (k = 2). The kDNA fingerprint patterns of parasites from hilly districts were clustered separately from low-land districts. Therefore, further study with a large number of samples is urgently required for systematic characterization of the clinical isolates to track the molecular epidemiology of the Leishmania donovani causing VL and the role of PKDL as a reservoir.

Conflict of interest statement

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
Gel picture of hsp70 restriction digested products showing the isolates of L. donovani species.
Figure 2
Figure 2
Gel picture of haspB PCR showing PCR products at 320 bp and 640 bp. Note: M: DNA ladder, 1: BPK806/0, 2: BPK811/0, 3: BPK814/0, 4: BPK813/0, 5: 63232-Fx, 6: 63233-Fx, 7: 63238-Fx, 8: 63241-Fx, 9: 63245-Fx, 10: 63256-Fx, 11: 63269-Fx, 12: BPK-PKN435, 13: 63277-Fx, 14: BPK-PKN467, 15: BPK-PKN468, 16: negative control N1, and 17: negative control N2.
Figure 3
Figure 3
Spatial distribution of different genotypes and disease in Eastern Nepal.
Figure 4
Figure 4
UPGMA dendrogram of all the 18 isolates obtained from kDNA PCR-RFLP (HaeIII) fingerprint data considering the densitometric curve. #: duplicate samples; s2–s4: four consecutive subcultures; ‡ control samples. A and B: groups separated according to haspB PCR product size.
Figure 5
Figure 5
Gel picture of kDNA PCR-RFLP using HaeIII restriction digested products. Note: M: DNA ladder, Lane 1: BPK806, Lane 2: BPK806 (duplicate sample), Lane 3: BPK813, Lane 4: BPK814, Lane 5: 63233-Fx, Lane 6: 63241-Fx, Lane 7: 63245-Fx, Lane 8: 63256-Fx, Lane 9: 63269-Fx, Lane 10: 63277-Fx, Lane 11: 63291-Fx, Lane 12: 63294-Fx, Lane 13: BPK-PKN435, Lane 14: BPK-PKN467, Lane 15: BPK-PKN468, Lane 16: BPK811, and Lane 17: BPK-PKN466.
Figure 6
Figure 6
Gel picture of kDNA PCR-RFLP using HaeIII restriction digested products. Note: M: DNA ladder, Lane 1: BPK806, Lane 2: BPK813, Lane 3: BPK814, Lane 4: 63232-Fx, Lane 5: 63233-Fx, Lane 6: 63238-Fx, Lane 7: 63241-Fx, Lane 8: 63245-Fx, Lane 9: 63256-Fx, Lane 10: 63269-Fx, Lane 11: 63277-Fx, Lane 12: 63294-Fx, Lane 13: BPK-PKN435, Lane 14: BPK-PKN467, Lane 15: BPK-PKN468, Lane 16: BPK811, and Lane 17: BPK-PKN466.
Figure 7
Figure 7
L. donovani kDNA genotypes determined by Structure v2.3.4. (a) Structure Harvester output for the determination of K (http://taylor0.biology.ucla.edu/structureHarvester/). Two distinct populations of kDNA genotypes are determined (k = 2). (b) Summary plot of estimates of Q value. Each individual data is represented by a single vertical line broken into K coloured segments, with length proportional to each of the K inferred clusters.

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References

    1. Ganguly S., Das N. K., Barbhuiya J. N., Chatterjee M. Post-kala-azar dermal leishmaniasis—an overview. International Journal of Dermatology. 2010;49(8):921–931. doi: 10.1111/j.1365-4632.2010.04558.x. - DOI - PubMed
    1. Zijlstra E. E., Musa A. M., Khalil E. A., El Hassan I. M., El-Hassan A. M. Post-kala-azar dermal leishmaniasis. The Lancet Infectious Diseases. 2003;3(2):87–98. doi: 10.1016/s1473-3099(03)00517-6. - DOI - PubMed
    1. Uranw S., Ostyn B., Rijal A., et al. Post-kala-azar dermal leishmaniasis in Nepal: a retrospective cohort study (2000–2010) PLoS Neglected Tropical Diseases. 2011;5(12) doi: 10.1371/journal.pntd.0001433.e1433 - DOI - PMC - PubMed
    1. Singh S., Sharma U., Mishra J. Post-kala-azar dermal leishmaniasis: recent developments. International Journal of Dermatology. 2011;50(9):1099–1108. doi: 10.1111/j.1365-4632.2011.04925.x. - DOI - PubMed
    1. Banjara M. R., Das M. L., Gurung C. K., et al. Integrating case detection of visceral leishmaniasis and other febrile illness with vector control in the post-elimination phase in Nepal. The American Journal of Tropical Medicine and Hygiene. 2019;100(1):108–114. doi: 10.4269/ajtmh.18-0307. - DOI - PMC - PubMed

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