Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study

Abstract

Introduction: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. Objective: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. Materials and methods: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. Results: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Conclusion: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

“…Differences in enzyme mobilities are due to differences in their protein structures, which comprise different amino acids, and lead to the creation of banding patterns from which zymodemes (populations with similar isoenzyme patterns) can be assigned [ 24 ]. MLEE is laborious as it requires a large volume of cell culture and can take 1–2 months to produce results, which cannot be compared, with confidence, between laboratories; e.g., different enzyme panels are used in Europe and South America [ 204 , 205 ]. Furthermore, one zymodeme, MON-1, for L. infantum , the causative agent of most cases of VL in the Mediterranean Basin and South America, was shown to be heterogeneous and polymorphic [ 129 ].…”

Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods

“…This method allows identification of microorganisms by electrophoretic mobility of various intracellular enzymes (Ovalle-Bracho et al 2018). Isoenzymes play a significant role in metabolic processes in different cellular components and are produced by multiple genes.…”

Molecular Typing of Two Suspected Cutaneous Leishmaniasis Isolates in Baghdad

“…This method has previously been reported for species-specific identification of Old World species and New World leishmaniasis (23,34,36 be accomplished by digesting the PCR product with just one restriction enzyme and this one-step typing can be used to detect the sub-genus of medically important isolates (36). Moreover, recent laboratory assessment proved that PCR-RFLP is more applicable with less complexity than multi-locus enzyme electrophoresis and less expensive from the time consuming Isoenzyme technique; it is also more accurate and sensitive than routine microscopic or Giemsa staining diagnostic methods (37,38). Similar applications of RFLP-PCR assay is recommended for other Leishmania isolates from all parts of Iraqi provinces, for the detection of another local Leishmania species.…”