Sampling is decisive to determination of Leishmania (Viannia) species

PLoS Negl Trop Dis. 2024 Apr 25;18(4):e0012113. doi: 10.1371/journal.pntd.0012113. eCollection 2024 Apr.

Abstract

Background: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays.

Methodology: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample.

Principal findings: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples.

Conclusions: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopsy
  • DNA, Protozoan / genetics
  • Humans
  • Leishmania* / classification
  • Leishmania* / genetics
  • Leishmania* / isolation & purification
  • Leishmaniasis, Cutaneous* / diagnosis
  • Leishmaniasis, Cutaneous* / parasitology
  • Molecular Diagnostic Techniques / methods
  • Peru
  • Polymerase Chain Reaction / methods
  • Sensitivity and Specificity*
  • Specimen Handling / methods

Substances

  • DNA, Protozoan

Grants and funding

This work was supported by an award from the Armed Forces Health Surveillance Division, Global Emerging Infection Surveillance Branch (AFHSD/GEIS), ProMIS ID 17_N6_1.1.3, 2017-2018, to HOV. The funders had no role in data collection and analysis of study design, decision to publish, or preparation of the manuscript.