Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka

Overview

Journal Microorganisms

Specialty Microbiology

Date 2022 May 28

PMID 35630433

Authors

Nirmitha Lalindi De Silva

Viraji Nefertiti Hiromel De Silva

Arachchige Theja Hemapala Deerasinghe

Upeksha Lakmini Rathnapala

Makoto Itoh

Hidekazu Takagi

Mirani Vasanthamala Weerasooriya

Hirotomo Kato

Thishan Channa Yahathugoda

Affiliations

Soon will be listed here.

Abstract

The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by Leishmania donovani) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (103 to 10−2) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy.

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References

Karunaweera N, Ginige S, Senanayake S, Silva H, Manamperi N, Samaranayake N . Spatial Epidemiologic Trends and Hotspots of Leishmaniasis, Sri Lanka, 2001-2018. Emerg Infect Dis. 2019; 26(1):1-10. PMC: 6924882. DOI: 10.3201/eid2601.190971. View

Naotunne T, Rajakulendran S, Abeywickreme W, Kulasiri C, Perera J, Premaratne U . Cutaneous leishmaniasis in Sri Lanka. An imported disease linked to the Middle East and African employment boom. Trop Geogr Med. 1990; 42(1):72-4. View

Lypaczewski P, Matlashewski G . Leishmania donovani hybridisation and introgression in nature: a comparative genomic investigation. Lancet Microbe. 2022; 2(6):e250-e258. DOI: 10.1016/S2666-5247(21)00028-8. View

Kothalawala H, Karunaweera N . Loop-mediated isothermal amplification assay as a sensitive diagnostic tool for Leishmania donovani infections in Sri Lanka. Ceylon Med J. 2016; 61(2):68-70. PMC: 6206497. DOI: 10.4038/cmj.v61i2.8286. View

Silva H, Liyanage A, Deerasinghe T, Chandrasekara V, Chellappan K, Karunaweera N . Treatment failure to sodium stibogluconate in cutaneous leishmaniasis: A challenge to infection control and disease elimination. PLoS One. 2021; 16(10):e0259009. PMC: 8535432. DOI: 10.1371/journal.pone.0259009. View

Cupolillo E, Grimaldi Junior G, Momen H, Beverley S . Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol. 1995; 73(1-2):145-55. DOI: 10.1016/0166-6851(95)00108-d. View

Giantsis I, Chaskopoulou A, Bon M . Direct Multiplex PCR (dmPCR) for the Identification of Six Phlebotomine Sand Fly Species (Diptera: Psychodidae), Including Major Leishmania Vectors of the Mediterranean. J Econ Entomol. 2016; 110(1):245-249. DOI: 10.1093/jee/tow269. View

El Tai N, Osman O, 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. 2001; 94(5):575-9. DOI: 10.1016/s0035-9203(00)90093-2. View

Silva M, Soares C, Medeiros R, Medeiros Z, Melo F . Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol. 2013; 134(2):206-10. DOI: 10.1016/j.exppara.2013.03.003. View

10.

Karunaweera N, Pratlong F, Siriwardane H, Ihalamulla R, Dedet J . Sri Lankan cutaneous leishmaniasis is caused by Leishmania donovani zymodeme MON-37. Trans R Soc Trop Med Hyg. 2004; 97(4):380-1. DOI: 10.1016/s0035-9203(03)90061-7. View

11.

Akhoundi M, Downing T, Votypka J, Kuhls K, Lukes J, Cannet A . Leishmania infections: Molecular targets and diagnosis. Mol Aspects Med. 2017; 57:1-29. DOI: 10.1016/j.mam.2016.11.012. View

12.

Sharquie K, Hassen A, Hassan S . Evaluation of diagnosis of cutaneous leishmaniasis by direct smear, culture and histopathology. Saudi Med J. 2002; 23(8):925-8. View

13.

Iddawela D, Vithana S, Atapattu D, Wijekoon L . Clinical and epidemiological characteristics of cutaneous leishmaniasis in Sri Lanka. BMC Infect Dis. 2018; 18(1):108. PMC: 5838877. DOI: 10.1186/s12879-018-2999-7. View

14.

Ranasinghe S, Wickremasinghe R, Hulangamuwa S, Sirimanna G, Opathella N, Maingon R . 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. PMC: 4708022. DOI: 10.1590/0074-02760150286. View

15.

Koarashi Y, Caceres A, Saca F, Flores E, Trujillo A, Alvares J . Identification of causative Leishmania species in Giemsa-stained smears prepared from patients with cutaneous leishmaniasis in Peru using PCR-RFLP. Acta Trop. 2016; 158:83-87. DOI: 10.1016/j.actatropica.2016.02.024. View

16.

Rajapaksa U, Ihalamulla R, Karunaweera N . First report of mucosal tissue localisation of leishmaniasis in Sri Lanka. Ceylon Med J. 2005; 50(2):90-1. View

17.

Franssen S, Durrant C, Stark O, Moser B, Downing T, Imamura H . Global genome diversity of the complex. Elife. 2020; 9. PMC: 7105377. DOI: 10.7554/eLife.51243. View

18.

Deepachandi B, Weerasinghe S, Soysa P, Karunaweera N, Siriwardana Y . A highly sensitive modified nested PCR to enhance case detection in leishmaniasis. BMC Infect Dis. 2019; 19(1):623. PMC: 6631494. DOI: 10.1186/s12879-019-4180-3. View

19.

McGwire B, Satoskar A . Leishmaniasis: clinical syndromes and treatment. QJM. 2013; 107(1):7-14. PMC: 3869292. DOI: 10.1093/qjmed/hct116. View

20.

Fotakis E, Giantsis I, Avgerinou A, Kourtidis S, Agathaggelidou E, Kapoula C . Identification of Leishmania Species in Naturally Infected Sand Flies from Refugee Camps, Greece. Emerg Infect Dis. 2018; 25(2):361-364. PMC: 6346468. DOI: 10.3201/eid2502.181359. View