Polymerase chain reaction–<i>miniexon</i>: a promising diagnostic method for mucocutaneous leishmaniasis

Ovalle-Bracho, Clemencia; Díaz-Toro, Yira Rosalba; Muvdi-Arenas, Sandra

doi:10.1111/ijd.12910

Cited by 8 publications

(8 citation statements)

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“…The isolates were collected between 2000 and 2015 and were selected randomly from the center’s biological bank. The identification of species was performed by using PCR-RFLP profiling of the hsp70 and miniexon gene according to the reported methodology [ 31 , 32 ].…”

Section: Methodsmentioning

confidence: 99%

Intraspecies differences in natural susceptibility to amphotericine B of clinical isolates of Leishmania subgenus Viannia

2018

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Amphotericin B (AmB) is a recommended medication for the treatment of cutaneous and mucosal leishmaniasis in cases of therapeutic failure with first-line medications; however, little is known about the in vitro susceptibility to AmB of clinical isolates of the subgenus Viannia, which is most prevalent in South America. This work aimed to determine the in vitro susceptibility profiles to AmB of clinical isolates of the species L. (V.) panamensis, L. (V.) guyanensis and L. (V.) braziliensis. In vitro susceptibility to AmB was evaluated for 65 isolates. Macrophages derived from the U937 cell line were infected with promastigotes and exposed to different AmB concentrations. After 96 hours, the number of intracellular amastigotes was quantified by qPCR, and median effective concentration (EC50) was determined using the PROBIT model. The controls included sensitive strains and experimentally derived less sensitive strains generated in vitro, which presented EC50 values up to 7.57-fold higher than the values of the sensitive strains. The isolates were classified into groups according to their in vitro susceptibility profiles using Ward’s hierarchical method. The susceptibility to AmB differed in an intraspecies-specific manner as follows: 28.21% (11/39) of L. (V.) panamensis strains, 50% (3/6) of L. (V.) guyanensis strains and 34.61% (9/26) of L. (V.) braziliensis strains were classified as less sensitive. The latter subset featured three susceptibility groups. We identified Colombian isolates with different AmB susceptibility profiles. In addition, the capacity of species of subgenus Viannia to develop lower susceptibility to AmB was demonstrated in vitro. These new findings should be considered in the pharmacovigilance of AmB in Colombia and South America.

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Section: Methodsmentioning

confidence: 99%

Intraspecies differences in natural susceptibility to amphotericine B of clinical isolates of Leishmania subgenus Viannia

2018

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“…This claim is supported by experimental studies of the miniexon gene performed by Marfur, et al, as well as studies performed by Serin, et al, who evaluated the functionality of the miniexon gene as a phylogenetic marker of 17 species of the New and the Old World (22)(23)(24). The hsp70 gene has been evaluated for almost all circulating species in the world, with results that demonstrate its discriminatory capacity (28,32,44).…”

Section: Discussionmentioning

confidence: 85%

“…Previous studies from our group have focused on identifying circulating species of Leishmania in Colombia by using MLEE, monoclonal antibodies and PCR with kDNA, ITS rDNA, miniexon and hsp70 as targets, with the last two being those that allow differentiation of all subgenus Leishmania and Viannia species circulating in Colombia (18,(26)(27)(28)(29).…”

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confidence: 99%

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

et al. 2018

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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.

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“…Unlike in L. major , overexpression of the miniexon region in L. braziliensis led to complete virulence attenuation in a hamster model (de Toledo et al, 2009 ), suggesting species-specific functions of this array. This is further supported by the genetic divergence of this array between new world and old world dermotropic species (Fernandes et al, 1994 ), which is used as a diagnostic signal for parasite genotyping (Serin et al, 2005 ; Ovalle-Bracho et al, 2016 ).…”

Section: Cosmid-based Functional Genetic Screening In Leishmentioning

confidence: 98%

Reverse Epidemiology: An Experimental Framework to Drive Leishmania Biomarker Discovery in situ by Functional Genetic Screening Using Relevant Animal Models

Piel

Pescher

Späth

2018

Front. Cell. Infect. Microbiol.

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Leishmania biomarker discovery remains an important challenge that needs to be revisited in light of our increasing knowledge on parasite-specific biology, notably its genome instability. In the absence of classical transcriptional regulation in these early-branching eukaryotes, fluctuations in transcript abundance can be generated by gene and chromosome amplifications, which have been linked to parasite phenotypic variability with respect to virulence, tissue tropism, and drug resistance. Conducting in vitro evolutionary experiments to study mechanisms of Leishmania environmental adaptation, we recently validated the link between parasite genetic amplification and fitness gain, thus defining gene and chromosome copy number variations (CNVs) as important Leishmania biomarkers. These experiments also demonstrated that long-term Leishmania culture adaptation can strongly interfere with epidemiologically relevant, genetic signals, which challenges current protocols for biomarker discovery, all of which rely on in vitro expansion of clinical isolates. Here we propose an experimental framework independent of long-term culture termed “reverse” epidemiology, which applies established protocols for functional genetic screening of cosmid-transfected parasites in animal models for the identification of clinically relevant genetic loci that then inform targeted field studies for their validation as Leishmania biomarkers.

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Polymerase chain reaction–miniexon: a promising diagnostic method for mucocutaneous leishmaniasis

Abstract: We consider PCR-miniexon as a diagnostic method of first choice for mucocutaneous leishmaniasis due to its excellent diagnostic performance and its ability to discriminate between Leishmania and Viannia subgenera as well as between species belonging to Leishmania subgenus.

Cited by 8 publications

References 57 publications

Intraspecies differences in natural susceptibility to amphotericine B of clinical isolates of Leishmania subgenus Viannia

Intraspecies differences in natural susceptibility to amphotericine B of clinical isolates of Leishmania subgenus Viannia

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

Reverse Epidemiology: An Experimental Framework to Drive Leishmania Biomarker Discovery in situ by Functional Genetic Screening Using Relevant Animal Models

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