Leishmania (Cutaneous, Mucosal, or Visceral Leishmaniasis) - Microscopy and PCR

Testing Indications

CL and ML: PCR is the main diagnostic test method and should be requested whenever either disease is suspected. Microscopic examination is helpful when positive but has poor sensitivity in CL and ML. Serology has limited value in CL and ML and should not be routinely requested.¹

VL: Microscopy and PCR are the main diagnostic test methods and should be requested whenever VL is suspected. Serology is an adjunct but not a replacement for microscopy and PCR in VL.¹

Acceptance/Rejection Criteria

Specimens submitted without relevant clinical or exposure information (including travel history) may be delayed or rejected.

Storage and Transport

Place specimen in biohazard bag and seal. Specimens should be stored at room temperature following collection and shipped to PHO’s laboratory as soon as possible. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

CL or ML: To increase the likelihood of detection, collect multiple specimens from the most active areas (e.g. oozing ulcer base and advancing inflammatory margins) of each lesion using different techniques as below. Avoid healed areas or areas with secondary bacterial/fungal infection. Clean affected areas with 70% alcohol (not iodine) and remove overlying necrotic debris/scabs. If needed, a low concentration of local anesthetics (e.g. 1% lidocaine) can be used to reduce discomfort.

1. If using lesion scraping technique: scrape most active areas (without making incision; bleeding should be minimal but oozing may occur) and transfer scraped material into a sterile container with minimal amount of sterile saline to keep material moistened during transit.
2. If using lesion brushing/swabbing technique: brush most active areas multiple times until pink-tinged tissue fluids are wicked onto brush/swab and transfer brush/swab into a sterile container with minimal amount of sterile saline keep material moistened during transit.
3. If using lesion aspirate technique: using 0.1-0.2 ml of sterile saline and a 23- to 27-gauge needle, gently insert and rotate the needle back and forth tangentially in the most active area while aspirating pink-tinged tissue fluids up to the needle hub (note: if insufficient fluid is obtained, inject 0.05-0.1 ml of sterile saline in the area and resume suction) and transfer aspirated fluid into a sterile container.
4. If using filter paper lesion impression technique: using sterile coarse-porosity 7-cm filter paper, gently press paper onto ulcer base multiple times to cover ulcer surface until pink-tinged tissue fluids are wicked onto the paper and transfer paper into a sterile container.
5. If using lesion biopsy technique: obtain a 2-4 mm punch biopsy of the most active area (down to the level of the dermis) and transfer biopsied material into a sterile container with minimal amount of sterile saline to keep material moistened during transit.

Note that PCR sensitivity varies by cutaneous or mucosal lesion type, lesion severity, collection technique, lesion area sampled, and Leishmania species. Boggild et al.^2,3 reported comparable sensitivity between filter paper impressions (86.6-92.3%) and aspirates or scrapings (92.5-94.2%). Valencia et al.⁴ similarly reported comparable sensitivity between filter paper impressions (99.1%), foam swabs (99.1%), and scrapings (96.6%). Boggild et al.⁵ reported comparable sensitivity between foam swabs (95.7%) and biopsy specimens (95.7%). Adams et al.⁶ reported superior sensitivity of foam swabs (97.5%) versus aspirates (80%). Blaizot et al.⁷ reported superior sensitivity of post-biopsy cotton swabs (98%) versus biopsy specimens (89%). Suarez et al.⁸ reported superior parasite loads at the ulcer base compared to the raised border, as well as superior parasite loads using foam swabs or scrapings versus biopsies.

VL: PCR sensitivity also varies by specimen type and Leishmania species. Although splenic aspirates are usually considered of highest yield, their collection is a high-risk procedure, and blood and bone marrow specimens alternatively also have high accuracy (93.1-95.3% sensitivity and 92.6-95.6% specificity per de Ruiter et al.⁹).

Test Frequency and Turnaround Time (TAT)

Leishmania PCR and microscopy are performed Monday to Friday at PHO’s Toronto laboratory site.

Turnaround time is up to 3 calendar days for PCR and 5 calendar days for microscopy from receipt at PHO.

If strongly positive by PCR, samples are forwarded to the National Reference Centre for Parasitology (NRCP) in Montreal for species identification. Turnaround time for species identification is up to 42 calendar days.

Leishmania PCR detection is performed at PHO using three different laboratory-developed assays performed in parallel: a genus-level 18s rRNA gene real-time PCR (Wortmann et al.⁹), a Leishmania Leishmania subgenus kDNA real-time PCR (Mary et al.¹⁰), and a Leishmania Viannia subgenus kDNA real-time PCR(Lopez et al.¹¹).

Leishmania microscopy is performed at PHO using Giemsa staining. Whole blood specimens undergo buffy coat concentration.

Leishmania species identification is performed at the NRCP

Performance and Limitations
At PHO, microscopy has an overall sensitivity of 27% and specificity of 100% (Lau et al.¹⁴). The 18s rRNA gene real-time PCR has 95% sensitivity for the Leishmania genus but does not differentiate between Leishmania (L.) and Leishmania (V.) subgenera. The Leishmania (L.) subgenus kDNA real-time PCR has > 95% sensitivity for the Leishmania (L.) subgenus with around 25% cross-reactivity with Leishmania (V.) species at higher cycle threshold values. For L. (L.) amazonensis and L. (L.) tropica however, the Leishmania (L.) kDNA target sensitivity may be inferior compared to 18s rRNA gene target sensitivity due to sequence mismatches. The Leishmania (V.) kDNA real-time PCR has > 95% sensitivity for the Viannia subgenus with no cross-reactivity with the Leishmania subgenus. Both kDNA assays may cross-react with other organisms including Trypanosoma species. The performance of either 18s rRNA gene or kDNA PCR targets for rarer Leishmania species, including Leishmania Mundinia sugenus, has not yet been established.

The NRCP species identification assays may be unable to provide identification at low parasite loads.

Algorithm

PCR is performed on all specimens received. If PCR is positive with a strong enough signal (18s rRNA cycle threshold value < 30), the specimen will be automatically forwarded to the NRCP for further Leishmania species identification, otherwise species identification will not be performed.

Microscopy is only performed if specifically requested on the requisition.

Interpretation

PCR detection results:

Microscopy results (if requested):

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

References

1. Aronson N, Herwaldt BL, Libman M, Pearson R, Lopez-Velez R, Weina P, Carvalho EM, Ephros M, Jeronimo S, Magill A. Diagnosis and Treatment of Leishmaniasis: Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH). Clin Infect Dis. 2016 Dec 15;63(12):1539-1557. doi: 10.1093/cid/ciw742. PMID: 27941143.
2. Boggild AK, Valencia BM, Espinosa D, Veland N, Ramos AP, Arevalo J, et al. Detection and species identification of Leishmania DNA from filter paper lesion impressions for patients with American cutaneous leishmaniasis. Clin Infect Dis. 2010;50(1):e1-6. Available from: https://doi.org/10.1086/648730
3. Boggild AK, Ramos AP, Valencia BM, Veland N, Calderon F, Arevalo J, et al. Diagnostic performance of filter paper lesion impression PCR for secondarily infected ulcers and nonulcerative lesions caused by cutaneous leishmaniasis. J Clin Microbiol. 2011;49(3):1097-100. Available from: https://doi.org/10.1128/JCM.02457-10
4. Valencia BM, Veland N, Alba M, Adaui V, Arevalo J, Low DE, et al. Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru. PLoS One. 2012;7(11):e49738. Available from: https://doi.org/10.1371/journal.pone.0049738
5. Boggild AK, Valencia BM, Veland N, Pilar Ramos A, Calderon F, Arevalo J, et al. Non-invasive cytology brush PCR diagnostic testing in mucosal leishmaniasis: superior performance to conventional biopsy with histopathology. PLoS One. 2011;6(10):e26395. Available from: https://doi.org/10.1371/journal.pone.0026395
6. Adams ER, Gomez MA, Scheske L, Rios R, Marquez R, Cossio A, et al. Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR. Parasitology. 2014;141(14):1891-7. Available from: https://doi.org/10.1017/S0031182014001280
7. Blaizot R, Simon S, Ginouves M, Prévot G, Blanchet D, Ravel C, et al. Validation of swab sampling and SYBR green-based real-time PCR for the diagnosis of cutaneous leishmaniasis in French Guiana. J Clin Microbiol. 2021;59(2):e02218-20. Available from: https://doi.org/10.1128/JCM.02218-20
8. Suárez M, Valencia BM, Jara M, Alba M, Boggild AK, Dujardin JC, et al. Quantification of Leishmania (Viannia) kinetoplast DNA in ulcers of cutaneous leishmaniasis reveals inter-site and inter-sampling variability in parasite load. PLoS Negl Trop Dis. 2015;9(7):e0003936. Available from: https://doi.org/10.1371/journal.pntd.0003936
9. de Ruiter CM, van der Veer C, Leeflang MM, Deborggraeve S, Lucas C, Adams ER. Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy. J Clin Microbiol. 2014;52(9):3147-55. Available from: https://doi.org/10.1128/JCM.00372-14
10. Wortmann G, Sweeney C, Houng HS, Aronson N, Stiteler J, Jackson J, et al. Rapid diagnosis of leishmaniasis by fluorogenic polymerase chain reaction. Am J Trop Med Hyg. 2001;65(5):583-7. Available from: https://doi.org/10.4269/ajtmh.2001.65.583
11. Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol. 2004;42(11):5249-55. Available from: https://doi.org/10.1128/JCM.42.11.5249-5255.2004
12. Lopez M, Inga R, Cangalaya M, Echevarria J, Llanos-Cuentas A, Orrego C, et al. Diagnosis of Leishmania using the polymerase chain reaction: a simplified procedure for field work. Am J Trop Med Hyg. 1993;49(3):348-56. Available from: https://doi.org/10.4269/ajtmh.1993.49.348
13. Lau R, Valencia B, Ralevski F, Llanos-Cuentas A, Boggild AK. Validation of a molecular diagnostic algorithm for leishmania detection in a non-endemic setting. Int J Infect Dis. 2014;21(1):162-3. Available from: https://doi.org/10.1016/j.ijid.2014.03.761