Efficacy of a new rapid diagnostic test kit to diagnose Sri Lankan cutaneous leishmaniasis caused by Leishmania donovani

Gayani De Silva, Vijani Somaratne, Sujai Senaratne, Manuja Vipuladasa, Rajitha Wickremasinghe, Renu Wickremasinghe, Shalindra Ranasinghe, Gayani De Silva, Vijani Somaratne, Sujai Senaratne, Manuja Vipuladasa, Rajitha Wickremasinghe, Renu Wickremasinghe, Shalindra Ranasinghe

Abstract

Background: Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani. This study assessed the diagnostic value of a new rapid diagnostic immunochromatographic strip (CL-Detect™ IC-RDT), that captures the peroxidoxin antigen of Leishmania amastigotes.

Methodology/principal findings: We sampled 74 clinically suspected CL lesions, of which 59 (79.7%) were positive by PCR, 43 (58.1%) by Giemsa stained slit skin smear (SSS) and 21 (28.4%) by the new IC-RDT. All samples which were positive either by SSS or IC-RDT or both were positive by PCR. The sensitivities of the IC-RDT and SSS compared to PCR were 36% and 73%, respectively. Fifteen patients from this endemic region were negative by all three tests. Twenty two clinically non-CL skin lesions from a CL non-endemic region were also negative by all three methods. Specificity and PPV of both IC-RDT and SSS compared to PCR were 100%; the NPVs of IC-RDT and SSS were 37% and 58%, respectively. The median parasite grading of the 59 PCR positive samples was 2+ (1-10 parasites/100 HPFs) and IC-RDT positive lesions was 3+ (1-10 parasites /10HPFs). The duration of the lesion was not associated with IC-RDT positivity.

Conclusions/significance: The median parasite grade of Sri Lankan CL lesions is low. The low sensitivities of SSS and CL Detect™ IC-RDT may be due to low parasite counts or low expression of peroxidoxin antigen in amastigotes of the Sri Lankan L. donovani strain. Our results indicate that negative SSS has to be combined with PCR for confirmation of CL in Sri Lanka. The current commercially available IC-RDT is not suitable to diagnose CL in Sri Lanka; an IC-RDT with improved sensitivity to detect L. donovani would be a valuable addition in the diagnostic tool kit for Sri Lanka.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Venn diagram showing the distribution…
Fig 1. Venn diagram showing the distribution of positive laboratory results.
Samples were taken from a total of 74 suspected skin lesions. Out of them only 59 became positive by at least one laboratory test. All three tests were positive only in 21 patients.
Fig 2. Comparison of laboratory results.
Fig 2. Comparison of laboratory results.
(2A) ITS1 PCR result of n = 6/59 samples. Lane 1:100bp DNA ladder, Lane 2: negative control, Lane 3: sample 67, Lane 4: sample 65, Lane 5: sample 63, Lane 6: sample 64, Lane 7: sample 74, Lane 8: sample 73, Lane 9: positive control. (2B) Slit skin smear image. Characteristic amastigotes (sample 64; *grade 4+) x1000 magnification. (2C) Tested IC-RDT strips. Left to right; Strip 1: sample 30 (*grade 2+), Strip 2: sample 9 (*grade 4+), Strip 3 = sample 46 (*grade 3+), Strip 4 = sample 64 (*grade 4+), Strip 5 = sample 67 (*grade 2+), Strip 6 = sample 74 (*grade 2+), Strip 7 = sample 63 (*grade 2+), Strip 8 = sample 65 (*grade 3+), Strip 9 = sample 69 (*grade 3+). *grade = parasite grade [33].

References

    1. Kassi M, Kassi M, Afghan AK, Rehman R, Kasi PM. Marring leishmaniasis: the stigmatization and the impact of cutaneous leishmaniasis in Pakistan and Afghanistan. PLoS Negl Trop Dis. 2008;2(10):e259 doi:
    1. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS one. 2012;7(5):e35671 doi:
    1. Kolivand M, Fallah M, Salehzadeh A, Davari B, Poormohammadi A, Pazoki Ghohe H, et al. An epidemiological study of cutaneous leishmaniasis using active case finding among elementary school students in Pakdasht, southeast of Tehran, Iran 2013–2014. J Res Health Sci. 2015;15(2): 104–8
    1. Siriwardana HV, Thalagala N, Karunaweera ND. Clinical and epidemiological studies on the cutaneous leishmaniasis caused by Leishmania (Leishmania) donovani in Sri Lanka. Ann Trop Med Parasitol. 2010;104(3): 213–23. doi:
    1. Semage SN, Pathirana KP, Agampodi SB. Cutaneous leishmaniasis in Mullaitivu, Sri Lanka: a missing endemic district in the leishmaniasis surveillance system. Int J Infect Dis. 2014;25:53–5. doi:
    1. Athukorale DN, Seneviratne JK, Ihalamulla RL, Premaratne UN. Locally acquired cutaneous leishmaniasis in Sri Lanka. J Trop Med Hyg.1992;95(6): 432–3
    1. Epidemiology Unit. Weekly Epidemiology Reports. Ministry of Health Sri Lanka. 2009–2016.
    1. Ranasinghe S, Wickremasinghe R, Munasinghe A, Hulangamuwa S, Sivanantharajah S, Seneviratne K, et al. Cross-sectional study to assess risk factors for leishmaniasis in an endemic region in Sri Lanka. The American journal of tropical medicine and hygiene. 2013;89(4):742–9. Epub 2013/08/07. doi:
    1. Kariyawasam KK, Edirisuriya CS, Senerath U, Hensmen D, Siriwardana HV, Karunaweera ND. Characterisation of cutaneous leishmaniasis in Matara district, southern Sri Lanka: evidence for case clustering. Pathogens and global health. 2015;109(7):336–43. Epub 2015/09/09. doi:
    1. Districts of Sri lanka.
    1. Karunaweera ND, Pratlong F, Siriwardane HV, Ihalamulla RL, Dedet JP. Sri Lankan cutaneous leishmaniasis is caused by Leishmania donovani zymodeme MON-37. Trans R Soc Trop Med Hyg. 2003;97(4): 380–1
    1. Nawaratna SS, Weilgama DJ, Wijekoon CJ, Dissanayake M, Rajapaksha K. Cutaneous leishmaniasis in Sri Lanka. Emerg Infect Dis. 2007. July;13(7):1068–70.
    1. Zhang WW, Ramasamy G, McCall LI, Haydock A, Ranasinghe S, Abeygunasekara P, et al. Genetic analysis of Leishmania donovani tropism using a naturally attenuated cutaneous strain. PLoS Pathog. 2014;10(7):e1004244 doi:
    1. Ranasinghe S, Wickremasinghe R, Hulangamuwa S, Sirimanna G, Opathella N, Maingon RD, et al. Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani. Mem Inst Oswaldo Cruz. 2015;110(8): 1017–23. doi:
    1. McCall LI, Zhang WW, Ranasinghe S, Matlashewski G. Leishmanization revisited: immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis. Vaccine. 2013;31(10): 1420–5. doi:
    1. Rosypal AC, Tripp S, Kinlaw C, Hailemariam S, Tidwell RR, Lindsay DS, et al. Surveillance for antibodies to Leishmania spp. in dogs from Sri Lanka. J Parasitol. 2010;96(1): 230–1. doi:
    1. Nawaratna SS, Weilgama DJ, Rajapaksha K. Cutaneous leishmaniasis in Sri Lanka: a study of possible animal reservoirs. Int J Infect Dis. 2009;13(4): 513–7. doi:
    1. Ranawaka RR, Abeygunasekara PH, Weerakoon HS. Correlation of clinical, parasitological and histopathological diagnosis of cutaneous leishmaniasis in an endemic region in Sri Lanka. Ceylon Med J. 2012;57(4):149–52. doi:
    1. El Tai NO, Osman OF, el Fari M, Presber W, Schonian G. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg. 2000;94(5):575–9
    1. Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Experimental parasitology. 1990;71(3):267–75
    1. Ronet C, Beverley SM, Fasel N. Muco-cutaneous leishmaniasis in the New World: the ultimate subversion. Virulence. 2011;2(6):547–52. doi:
    1. Medley GF, Hollingsworth TD, Olliaro PL, Adams ER. Health-seeking behaviour, diagnostics and transmission dynamics in the control of visceral leishmaniasis in the Indian subcontinent. Nature. 2015;528(7580):S102–8. doi:
    1. Inbios International, Inc. CL DetectTM Rapid test FDA cleared.
    1. Inbios International, Inc. CL DetectTM Rapid Test for Cutaneous Leishmaniasis, Package insert.
    1. Webb JR, Campos-Neto A, Ovendale PJ, Martin TI, Stromberg EJ, Badaro R, et al. Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect Immun. 1998;66(7): 3279–89
    1. Bayih AG, Daifalla NS, Gedamu L. DNA-protein immunization using Leishmania peroxidoxin-1 induces a strong CD4+ T cell response and partially protects mice from cutaneous leishmaniasis: role of fusion murine granulocyte-macrophage colony-stimulating factor DNA adjuvant. PLoS Negl Trop Dis. 2014;8(12):e3391 doi:
    1. Harder S, Bente M, Isermann K, Bruchhaus I. Expression of a mitochondrial peroxiredoxin prevents programmed cell death in Leishmania donovani. Eukaryotic cell. 2006;5(5):861–70. doi:
    1. Castro H, Teixeira F, Romao S, Santos M, Cruz T, Flórido M,et al. Leishmania mitochondrial peroxiredoxin plays a crucial peroxidase-unrelated role during infection: insight into its novel chaperone activity. PLoS Pathog. 2011;7(10): e1002325 doi:
    1. Teixeira F, Castro H, Cruz T, Tse E, Koldewey P, Southworth DR,et al. Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum. Proc Natl Acad Sci U S A. 2015; 17;112(7): E616–24. doi:
    1. Ben Salah A, N B, Zaatour A, Gharbi A, Chen H, Morkowski S, Needham J, et al. CL Detect: selection of an optimal sampling device and evaluation of assay performance againt microscopy and Leishmania real time PCR. ASTMH; Atlanta, GA: 2012.
    1. Ben Salah A, N B, Zaatour A, Gharbi A,Bettaieb J, Ghawar W, Khedher A, et al. Clinical evaluation of CL detect TM rapid test for cutaneous leishmaniasis: performance characteristics when compared to smear microscopy at multiple test sites. ASTMH; New Orleans, LA USA: 2014.
    1. RJG Rycroft and SJ Robertson (Editors) 2002. A colour handbook of dermatology. Mansons Publishing Limited, 73 Corringngham Road,London Nw11 7 DL, UK
    1. World Health Organization. Control of the leishmaniases. technical report series. 2010 (949).
    1. El Tai NO, El Fari M, Mauricio I, Miles MA, Oskam L, El Safi SH, et al. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001;97(1): 35–44. doi:
    1. Sirimanna GMP, Seneviratne JKK, Samaraweera ESN, Ranawaka RR, Hulangamuwa CS, de Silva VNH, et al. Guidelines on management of leishmaniasis. Sri Lanka College of Dermatologists; 2013:7.
    1. Campbell M J and Swinscow T D V. Statistics at square one. 11th edition ISBN-978-1-4051-9100-5. Wiley-Blckwell, John Wiley & Sons Ltd; October 2009.
    1. Gajapathy K, Peiris LB, Goodacre SL, Silva A, Jude PJ, Surendran SN. Molecular identification of potential leishmaniasis vector species within the Phlebotomus (Euphlebotomus) argentipes species complex in Sri Lanka. Parasites & vectors. 2013;6(1):302 Epub 2014/02/07. doi:
    1. 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 research international. 2014;2014:913510 Epub 2014/10/16. doi:
    1. Jing BQ, Deng SS, Zhang RG, Zhang J. Comparative proteomic analysis of the promastigotes and amastigotes of Leishmania donovani. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2009;27(2):102–6.
    1. Harder S, Bente M, Isermann K, Bruchhaus I. Expression of a mitochondrial peroxiredoxin prevents programmed cell death in Leishmania donovani. Eukaryot Cell. 2006;5(5):861–70. doi:
    1. McCall LI, Zhang WW, Dejgaard K, Atayde VD, Mazur A, Ranasinghe S, et al. Adaptation of Leishmania donovani to cutaneous and visceral environments: in vivo selection and proteomic analysis. Journal of proteome research. 2015;14(2):1033–59. Epub 2014/12/24. doi:
    1. Barr SD, Gedamu L. Cloning and characterization of three differentially expressed peroxidoxin genes from Leishmania chagasi. Evidence for an enzymatic detoxification of hydroxyl radicals. J Biol Chem. 2001;276(36):34279–87. Epub 2001 Jul 3. doi:

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi