Improving Leishmania species identification in different types of samples from cutaneous lesions.

The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification.

Functional evaluation of gene silencing on macrophages derived from U937 cells using interference RNA (shRNA) in a model of macrophages infected with Leishmania (Viannia) braziliensis.

Leishmaniasis development is multifactorial; nonetheless, the establishment of the infection, which occurs by the survival and replication of the parasite inside its main host cell, the macrophage, is mandatory. Thus, the importance of studying the molecular mechanisms involved in the Leishmania-macrophage interaction is highlighted. The aim of this study was to characterize a cellular model of macrophages derived from U937 cells that would allow for the identification of infection phenotypes induced by genetic silencing with interference RNA in the context of macrophages infected with Leishmania (Viannia) braziliensis. The model was standardized by silencing an exogenous gene (gfp), an endogenous gene (lmna) and a differentially expressed gene between infected and non-infected macrophages (gro-ß). The silencing process was successful for the three genes studied, obtaining reductions of 88·9% in the GFP levels, 87·5% in LMNA levels and 74·4% for Gro-ß with respect to the corresponding control cell lines. The cell model revealed changes in the infection phenotype of the macrophages in terms of number of amastigotes per infected macrophage, number of amastigotes per sampled macrophage and percentage of infected macrophages as a result of gene silencing. Thus, this cell model constitutes a research platform for the study of parasite-host interactions and for the identification of potentially therapeutic targets.

Detection of Leishmania RNA Virus in Clinical Samples from Cutaneous Leishmaniasis Patients Varies according to the Type of Sample.

Leishmania RNA virus (LRV) is a double-stranded RNA virus belonging to the Totiviridae family detected as cytoplasmic inclusions in some strains of the human parasite Leishmania spp. Experimental evidence supports the hypothesis that human coinfection with Leishmania spp.-LRV triggers an exacerbated immune response in the host that can be responsible for the observed complicated outcomes in cutaneous leishmaniasis (CL), such as mucosal leishmaniasis (ML) and treatment failure of CL. However, the reported frequencies of LRV associated with complicated outcomes in patient's series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. To assess whether or not the inconsistent information about the frequency of LRV associated with CL complicated outcomes could be related to the virus detection approach, the present study evaluated the LRV presence in clinical samples using a diagnostic algorithm according to the type of the sample. In 36 samples with diagnosis of complicated forms of CL (15 of ML and 21 of CL antimony treatment failure) and six samples with non-Leishmania spp. infection, the LRV presence was assessed by RT-PCR, RT-qPCR, and nested RT-PCR. Viral load was estimated in parasite clinical isolates. By combining the methods, LRV1 presence was confirmed in 45% (9/20) of isolates and 37.5% (6/16) of the incisional biopsies. Remarkably, in some cases (4/8), LRV1 was undetectable in the isolates but present in their respective biopsies, and less frequently, the opposite was observed (1/8), suggesting the possibility of loss of parasites harboring LRV1 during the in vitro growth.

Mucosal leishmaniasis: A forgotten disease, description and identification of species in 50 Colombian cases / Mucosal leishmaniasis: A forgotten disease, description and identification of species in 50 Colombian cases

INTRODUCTION: Mucosal leishmaniasis has a progressive course and can cause deformity and even mutilation in the affected areas. It is endemic in the American continent and it is mainly caused by Leishmania (Viannia) braziliensis. OBJECTIVE: To describe a series of mucosal leishmaniasis cases and the infectious Leishmania species. MATERIALS AND METHODS: We included 50 patients with a clinical diagnosis of mucosal leishmaniasis and parasitological confirmation, and we described their clinical and laboratory results. We performed species typing by PCR-RFLP using the miniexon sequence and hsp70 genes; confirmation was done by sequencing. RESULTS: The median time of disease evolution was 2.9 years (range: 1 month to 16 years). The relevant clinical findings included mucosal infiltration (94%), cutaneous leishmaniasis scar (74%), total loss of the nasal septum (24%), nasal deformity (22%), and mucosal ulceration (38%). The symptoms reported included nasal obstruction (90%), epistaxis (72%), rhinorrhea (72%), dysphonia (28%), dysphagia (18%), and nasal pruritus (34%). The histopathological study revealed a pattern compatible with leishmaniasis in 86% of the biopsies, and amastigotes were identified in 14% of them. The Montenegro skin test was positive in 86% of patients, immunofluorescence in 84%, and culture in 8%. Leishmania (V.) braziliensis was identified in 88% of the samples, L. (V) panamensis in 8%, and L. (V.) guyanensis and L. (L.) amazonensis in 2% respectively. CONCLUSION: In this study, we found a severe nasal disease with destruction and deformity of the nasal septum in 25% of the cases, probably associated with late diagnosis. Leishmania (V.) braziliensis was the predominant species. We described a case of mucosal leishmaniasis in Colombia caused by L. (L.) amazonensis for the first time.

Introducción. La leishmaniasis mucosa tiene un curso progresivo y puede causar deformidad e incluso mutilación de las zonas afectadas. Es endémica en el continente americano y es causada principalmente por Leishmania (Viannia) brasiliensis. Objetivo. Describir una serie de casos de leishmaniasis mucosa y las especies de Leishmania infecciosas. Materiales y métodos. Se estudiaron 50 pacientes con diagnóstico clínico de leishmaniasis mucosa y confirmación parasitológica. Se describieron sus características clínicas y los resultados de laboratorio. La tipificación de especies se hizo mediante reacción en cadena de la polimerasa de los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism Polymerase Chain Reaction, PCR-RFLP) en la secuencia del miniexon y el gen hsp70 y se confirmó por secuenciación. Resultados. La evolución de la enfermedad fue de un mes a dieciséis años (mediana de 2,8 años). Los hallazgos clínicos fueron los siguientes: infiltración mucosa (94 %), cicatriz de leishmaniasis cutánea (74 %), pérdida total del tabique nasal (24 %), deformidad nasal (22 %) y ulceración (38 %). Los síntomas reportados fueron: obstrucción nasal (90 %), epistaxis (72 %), rinorrea (72 %), disfonía (28 %), disfagia (18 %) y prurito nasal (34 %). La histopatología mostró un patrón compatible con leishmaniasis en 86 % de las biopsias y se identificaron amastigotes en 14 % de ellas. La prueba de Montenegro fue positiva en 86 % de los pacientes, la inmunofluorescencia en 84 %, y el cultivo en 8 %. Leishmania (V.) brasiliensis se identificó en 88 % de las muestras, L. (V) panamensis en 8 %, y L. (V.) guyanensis y L. (L.) amazonensis en 2 %, respectivamente. Conclusión. Se encontró enfermedad nasal grave con destrucción y deformidad del tabique nasal en una cuarta parte de los casos, probablemente debido a un diagnóstico tardío. Leishmania (V.) brasiliensis fue la especie predominante. Se describe por primera vez un caso de leishmaniasis mucosa causado por L. (L.) amazonensis en Colombia.

Evaluating the spatial distribution of Leishmania parasites in Colombia from clinical samples and human isolates (1999 to 2016).

In Colombia, nine species of parasites of the genus Leishmania circulate in more than 20 sand fly species, putting at risk of contracting the disease approximately 60% of the population. The Federico Lleras Acosta Dermatological Center, a reference center in Colombia, has been treating patients with cutaneous and mucosal leishmaniasis for more than 15 years, identifying the infecting Leishmania species from different clinical samples, and recording systematically all the epidemiological and geographic information related to each diagnosed patient. With this valuable information, the objective of this work was to perform a long term and large-scale study, aiming to identify the Leishmania species circulating in Colombia from clinical samples from 1999 to 2016, and to assess their current and potential spatial distribution. In all, four Leishmania species were identified in 688 samples from 183 municipalities distributed in 28 of the 32 departments of the country, and 387 records were georeferenced, from 20 departments. The most widespread species was L. (V.) braziliensis, showing new collection records, and the species related to areas with highest leishmaniasis transmission was L. (V.) panamensis. Ecological niche models were built for the three species that had more than 20 georeferenced records, showing a potential distribution for L. (V.) braziliensis on 42% of the national territory mainly in the interandean valleys, and the Orinoquia and Amazon regions. Leishmania (V.) guyanensis potential distribution covers 36% of Colombia continental territory with a spatial distribution similar to that of L. (V.) braziliensis. There was a marked tendency of L. (V.) panamensis to be distributed in the northwest of the country occupying 35% of the national area and mainly in areas of transformed ecosystems. Species were identified in patients from areas where the occurrence of cases was unprecedented, which suggests that the distribution of Leishmania may be greater than currently known. To improve the predictive capacity of the models, we suggest incorporating, in future studies, Leishmania samples from vectors and reservoirs that have a greater dependence on environmental variables. Our results are an important tool for health systems because they allow potential areas of transmission and information gaps to be identified.

Cost-effectiveness analysis of Mucosal Leishmaniasis diagnosis with PCR-based vs parasitological tests in Colombia.

To estimate the cost-effectiveness of available diagnosis alternatives for Mucosal Leishmaniasis (ML) in Colombian suspected patients. A simulation model of the disease's natural history was built with a decision tree and Markov models. The model´s parameters were identified by systematic review and validated by expert consensus. A bottom-up cost analysis to estimate the costs of diagnostic strategies and treatment per case was performed by reviewing 48 clinical records of patients diagnosed with ML. The diagnostic strategies compared were as follows: 1) no diagnosis; 2) parasite culture, biopsy, indirect immunofluorescence assay (IFA), and Montenegro skin test (MST) combined ; 3) parasite culture, biopsy, and IFA combined; 4) PCR-miniexon; and 5) PCR-kDNA. Three scenarios were modeled in patients with ML clinical suspicion, according to ML prevalence scenarios: high, medium and low. Adjusted sensitivity and specificity parameters of a combination of diagnostic tests were estimated with a discrete event simulation (DES) model. For each alternative, the costs and health outcomes were estimated. The time horizon was life expectancy, considering the average age at diagnosis of 31 years. Incremental cost-effectiveness ratios (ICERs) were calculated per Disability Life Year (DALY) avoided, and deterministic and probabilistic sensitivity analyses were performed. A threshold of willingness to pay (WTP) of three-time gross domestic product per capita (GDPpc) (US$ 15,795) and a discount rate of 3% was considered. The analysis perspective was the third payer (Health System). All costs were reported in American dollars as of 2015. PCR- kDNA was the cost-effective alternative in clinical suspicion levels: low, medium and high with ICERs of US$ 7,909.39, US$ 5,559.33 and US$ 4,458.92 per DALY avoided, respectively. ML diagnostic tests based on PCR are cost-effective strategies, regardless of the level of clinical suspicion. PCR-kDNA was the most cost-effective strategy in the competitive scenario with the parameters included in the present model.

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

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.

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

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.

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

BACKGROUND: Mucocutaneous leishmaniasis is a chronic disease caused mainly by Leishmania species that belong to Viannia subgenus. It affects upper respiratory airways and may lead to deformity, dysphagia, and even death in severe cases. Diagnosis is a challenge because clinical and histopathologic changes are easily confused with other diseases, and conventional methods for parasite identification and culture have a low sensitivity. Molecular methods have been used in the last two decades. In 2007, we published a validation study using internal transcript spacers and kinetoplast DNA as molecular targets with satisfactory results. In this research, we tested miniexon gene as the target. METHODS: Mucosal tissue samples from 60 Colombian patients with clinical signs of mucocutaneous leishmaniasis were included. A composite reference standard defined 30 cases and 30 controls. Two blind observers performed patient classification and test application independently. Miniexon gene amplification generated: 226-230 bp fragment for subgenus Viannia; 308 bp fragment for L. amazonensis; 340 bp fragment for L. mexicana; and 418 bp fragment for L. infantum-chagasi. RESULTS: Polymerase chain reaction (PCR) sensitivity for fresh samples was 87.5% (95% confidence interval [CI] 72.2-100), specificity, 95% (95% CI 83.0-100), and positive likelihood ratio was 17.5 (95% CI 2.58-118.93), similar to results obtained with paraffin-embedded samples. Agreement between observers was 96% (kappa = 0.912; 95% CI 0.815-1.000) for both subgenus Viannia and Leishmania. CONCLUSIONS: 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.

Changes in Macrophage Gene Expression Associated with Leishmania (Viannia) braziliensis Infection.

Different Leishmania species cause distinct clinical manifestations of the infectious disease leishmaniasis. It is fundamentally important to understand the mechanisms governing the interaction between Leishmania and its host cell. Little is known about this interaction between Leishmania (Viannia) braziliensis and human macrophages. In this study, we aimed to identify differential gene expression between non-infected and L. (V) braziliensis-infected U937-derived macrophages. We deployed a whole human transcriptome microarray analysis using 72 hours post-infection samples and compared those samples with their non-infected counterparts. We found that 218 genes were differentially expressed between infected and non-infected macrophages. A total of 71.6% of these genes were down-regulated in the infected macrophages. Functional enrichment analyses identified the steroid and sterol/cholesterol biosynthetic processes between regulatory networks down-regulated in infected macrophages. RT-qPCR further confirmed this down-regulation in genes belonging to these pathways. These findings contrast with those from studies involving other Leishmania species at earlier infection stages, where gene up-regulation for this metabolic pathway has been reported. Sterol biosynthesis could be an important biological process associated with the expression profile of macrophages infected by L. (V.) braziliensis. Differential transcriptional results suggest a negative regulation of the genetic regulatory network involved in cholesterol biosynthesis.

Mucosal leishmaniasis: A forgotten disease, description and identification of species in 50 Colombian cases / Leishmaniasis mucosa: una enfermedad olvidada, descripción e identificación de especies en 50 casos colombianos

Abstract Introduction: Mucosal leishmaniasis has a progressive course and can cause deformity and even mutilation in the affected areas. It is endemic in the American continent and it is mainly caused by Leishmania (Viannia) braziliensis. Objective: To describe a series of mucosal leishmaniasis cases and the infectious Leishmania species. Materials and methods: We included 50 patients with a clinical diagnosis of mucosal leishmaniasis and parasitological confirmation, and we described their clinical and laboratory results. We performed species typing by PCR-RFLP using the miniexon sequence and hsp70 genes; confirmation was done by sequencing. Results: The median time of disease evolution was 2.9 years (range: 1 month to 16 years). The relevant clinical findings included mucosal infiltration (94%), cutaneous leishmaniasis scar (74%), total loss of the nasal septum (24%), nasal deformity (22%), and mucosal ulceration (38%). The symptoms reported included nasal obstruction (90%), epistaxis (72%), rhinorrhea (72%), dysphonia (28%), dysphagia (18%), and nasal pruritus (34%). The histopathological study revealed a pattern compatible with leishmaniasis in 86% of the biopsies, and amastigotes were identified in 14% of them. The Montenegro skin test was positive in 86% of patients, immunofluorescence in 84%, and culture in 8%. Leishmania (V.) braziliensis was identified in 88% of the samples, L. (V) panamensis in 8%, and L. (V.) guyanensis and L. (L.) amazonensis in 2% respectively. Conclusion: In this study, we found a severe nasal disease with destruction and deformity of the nasal septum in 25% of the cases, probably associated with late diagnosis. Leishmania (V.) braziliensis was the predominant species. We described a case of mucosal leishmaniasis in Colombia caused by L. (L.) amazonensis for the first time.

Resumen Introducción. La leishmaniasis mucosa tiene un curso progresivo y puede causar deformidad e incluso mutilación de las zonas afectadas. Es endémica en el continente americano y es causada principalmente por Leishmania (Viannia) brasiliensis. Objetivo. Describir una serie de casos de leishmaniasis mucosa y las especies de Leishmania infecciosas. Materiales y métodos. Se estudiaron 50 pacientes con diagnóstico clínico de leishmaniasis mucosa y confirmación parasitológica. Se describieron sus características clínicas y los resultados de laboratorio. La tipificación de especies se hizo mediante reacción en cadena de la polimerasa de los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism Polymerase Chain Reaction, PCR-RFLP) en la secuencia del miniexon y el gen hsp70 y se confirmó por secuenciación. Resultados. La evolución de la enfermedad fue de un mes a dieciséis años (mediana de 2,8 años). Los hallazgos clínicos fueron los siguientes: infiltración mucosa (94 %), cicatriz de leishmaniasis cutánea (74 %), pérdida total del tabique nasal (24 %), deformidad nasal (22 %) y ulceración (38 %). Los síntomas reportados fueron: obstrucción nasal (90 %), epistaxis (72 %), rinorrea (72 %), disfonía (28 %), disfagia (18 %) y prurito nasal (34 %). La histopatología mostró un patrón compatible con leishmaniasis en 86 % de las biopsias y se identificaron amastigotes en 14 % de ellas. La prueba de Montenegro fue positiva en 86 % de los pacientes, la inmunofluorescencia en 84 %, y el cultivo en 8 %. Leishmania (V.) brasiliensis se identificó en 88 % de las muestras, L. (V) panamensis en 8 %, y L. (V.) guyanensis y L. (L.) amazonensis en 2 %, respectivamente. Conclusión. Se encontró enfermedad nasal grave con destrucción y deformidad del tabique nasal en una cuarta parte de los casos, probablemente debido a un diagnóstico tardío. Leishmania (V.) brasiliensis fue la especie predominante. Se describe por primera vez un caso de leishmaniasis mucosa causado por L. (L.) amazonensis en Colombia.

Geographical distribution of Leishmania species in Colombia, 1985-2017 / Distribución geográfica de las especies de Leishmania en Colombia, 1985-2017

Abstract Introduction: Knowledge of the geographical distribution of Leishmania species allows guiding the sampling to little-studied areas and implementing strategies to define risk zones and priority areas for control. Objective: Given that there is no publication that collects this information, the search, review, and compilation of the available scientific literature that has identified species in Colombia is presented in this paper. Materials and methods: A bibliographic search was performed in PubMed, Web of Knowledge, Google Scholar, SciELO and LILACS with the terms "(Leishmania OR Leishmaniasis) AND species AND Colombia", without restrictions on publication year, language or infected organism; records of national scientific events and repositories of theses from Colombian universities were also included. Results: Eighty-six scientific documents published between 1985 and 2017 were found in which the species of Leishmania and their geographical origin were indicated. The species reported, in descending order of frequency, were: Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) infantum, L. (L.) amazonensis, L. (L.) mexicana, L. (V.) colombiensis, L. (V.) lainsoni and L. (V.) equatorensis; the last three were found with the same frequency. Leishmania species were reported from 29 departments. Conclusion: Information on the distribution of Leishmania species in Colombia is limited; therefore, it is necessary to gather existing data and propose studies that consolidate the distribution maps of Leishmania species in Colombia. This would allow the detection of areas where species have not been identified as well as the comparison of existing parasite and vector distributions.

Resumen Introducción. El conocimiento de la distribución geográfica de las especies de Leishmania permite orientar el muestreo hacia áreas poco estudiadas e implementar estrategias para detectar zonas de riesgo y áreas prioritarias de control. Objetivo. Dado que no existe una publicación que reúna esta información, se planteó la revisión y compilación de la literatura científica disponible de estudios de identificación de especies del país. Materiales y métodos. Se llevó a cabo una búsqueda bibliográfica en PubMed, Web of Knowledge, Google Académico, SciELO y Lilacs con los términos "(Leishmania OR Leishmaniasis) AND especie AND Colombia", así como en memorias de eventos científicos nacionales y repositorios de tesis y trabajos de grado de universidades del país. Resultados. Se encontraron 86 documentos científicos publicados entre 1985 y 2017, en los cuales se informaron la especie de Leishmania y el origen geográfico. Las especies circulantes reportadas, en su orden de frecuencia, fueron: Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) infantum, L. (L.) amazonensis, L. (L.) mexicana, L. (V.) colombiensis, L. (V.) lainsoni y L. (V.) equatorensis, las últimas tres, con igual frecuencia. Los reportes proceden de 29 departamentos. Conclusión. La información de la distribución de las especies de Leishmania en Colombia es limitada. Por lo tanto, se necesita reunir los datos existentes y plantear trabajos que permitan consolidar el mapa de distribución de las especies en el país, lo cual permitiría detectar las zonas sin información de las especies circulantes y establecer la concordancia entre su distribución y la de los vectores.

Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study / Tipificación molecular de Leishmania (Leishmania) amazonensis y especies del subgénero Viannia asociadas a la leishmaniasis cutánea y la mucosa en Colombia: un estudio de concordancia

Resumen 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.

Abstract Introducción. La electroforesis de enzimas multilocus (Multilocus Enzyme Electrophoresis, MLEE) es el estándar de referencia para la tipificación de las especies de Leishmania. La prueba está restringida a laboratorios especializados por su complejidad técnica, sus costos y el tiempo necesario para obtener resultados. La PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) se utiliza para tipificar especies de Leishmania. Objetivo. Establecer la concordancia entre las dos pruebas como métodos de tipificación de las especies circulantes en Colombia. Materiales y métodos. Se seleccionaron 96 aislamientos de pacientes con leishmaniasis cutánea o mucocutánea y se tipificaron mediante MLEE y PCR-RFLP con los blancos moleculares miniexon y hsp70 usados en serie. Las enzimas de restricción aplicadas fueron la HaeIII y la BccI, respectivamente. Se calculó el coeficiente kappa y un intervalo de confianza (IC) de 95 %. Resultados. Se determinó que la concordancia fue "muy buena" al obtener un coeficiente de 0,98 (IC95%: 0,98-1,00). Las especies identificadas fueron: Leishmania Viannia braziliensis, L. (V.) panamensis, L. (V.) guyanensis y L. (L,) amazonensis. De los 96 aislamientos, 80 se enviaron a secuenciación y se confirmaron los resultados obtenidos mediante PCR-RFLP. Conclusión. Dada la concordancia obtenida con la PCR-RFLP amplificando los genes miniexon y hsp70, se propone esta prueba como alternativa para la tipificación de especies de Leishmania circulantes en Colombia.

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study.

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6% (95% CI 59.2-72.6) sensitivity and 92% (95% CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3) and negative likelihood ratio: 0.3 (95% CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3) sensitivity and 96% (95% CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8) and negative likelihood ratio: 0.6 (95% CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6 percent (95 percent CI 59.2-72.6) sensitivity and 92 percent (95 percent CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95 percent CI 2.8-31.3) and negative likelihood ratio: 0.3 (95 percent CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40 percent (95 percent CI 31.5-42.3) sensitivity and 96 percent (95 percent CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95 percent CI 2.0-58.8) and negative likelihood ratio: 0.6 (95 percent CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.