Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques

Morais, Rayana Carla Silva de; Oliveira, Cíntia Nascimento da Costa; Albuquerque, Suênia da Cunha Gonçalves de; Trajano-Silva, Lays Adrianne Mendonça; Silva, Rômulo Pessoa e; Cruz, Heidi Lacerda Alves da; Brito, Maria Edileuza Felinto de; Paiva-Cavalcanti, Milena de

doi:10.1016/j.exppara.2016.03.005

Cited by 19 publications

(16 citation statements)

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“…In another study, the Tm analysis of amplicons generated from the conserved region of minicircle kDNA allowed the differentiation of the subgenus L. ( Leishmania ) from L. ( Viannia ), but species identification was not possible [ 98 ]. Successively, de Morais et al [ 99 ] identified two Tm ranges using previously developed primers, designed on minicircle kDNA [ 59 ], to differentiate two groups of species causing CL in Brazil. The group 1 included L. ( V. ) braziliensis , L. ( V. ) panamensis , L. ( V. ) lainsoni , L. ( V. ) guyanensis , L. ( V. ) shawi (Tm = 78–79.99 °C), and the group 2 included L. ( V. ) naiffi , L. ( L. ) amazonensis and L. ( L. ) mexicana (Tm = 80–82.2 °C).…”

Section: Qpcr Assays For Leishmania Genotypingmentioning

confidence: 99%

Real-time PCR applications for diagnosis of leishmaniasis

et al. 2018

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Leishmaniasis is a vector-borne disease caused by many Leishmania species, which can infect both humans and other mammals. Leishmaniasis is a complex disease, with heterogeneous clinical manifestations ranging from asymptomatic infections to lesions at cutaneous sites (cutaneous leishmaniasis), mucosal sites (mucocutaneous leishmaniasis) or in visceral organs (visceral leishmaniasis), depending on the species and host characteristics. Often, symptoms are inconclusive and leishmaniasis can be confused with other co-endemic diseases. Moreover, co-infections (mainly with HIV in humans) can produce atypical clinical presentations. A correct diagnosis is crucial to apply the appropriate treatment and the use of molecular techniques in diagnosis of leishmaniasis has become increasingly relevant due to their remarkable sensitivity, specificity and possible application to a variety of clinical samples. Among them, real-time PCR (qPCR)-based approaches have become increasingly popular in the last years not only for detection and quantification of Leishmania species but also for species identification. However, despite qPCR-based methods having proven to be very effective in the diagnosis of leishmaniasis, a standardized method does not exist. This review summarizes the qPCR-based methods in the diagnosis of leishmaniasis focusing on the recent developments and applications in this field.

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Section: Qpcr Assays For Leishmania Genotypingmentioning

confidence: 99%

Real-time PCR applications for diagnosis of leishmaniasis

et al. 2018

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“…PCR can be used for detection of Leishmania in naturally infected dog samples, and PCR-RFLP (restriction fragment length polymorphism) is sensitive for identification of Leishmania species [28]. In addition, qPCR is effective in quantifying Leishmania DNA loading in clinical samples [29]. The blood sample from dogs infected by L. infantum was found by real-time PCR to have a sensitivity of 100% and specificity of 96.4% [30].…”

Section: Discussionmentioning

confidence: 99%

Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis

Silva¹,

Sousa²,

Almeida³

et al. 2020

Parasitology and Microbiology Research

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Visceral leishmaniasis (VL) is a zoonotic parasitic disease caused by Leishmania infantum (L. chagasi) that infects cells of the monocyte-phagocyte system. This work aims to describe the bone marrow parasitism in dogs naturally infected by L. chagasi, and to correlate with serum concentrations of cytokines and antibody level. It evaluated 42 dogs, 21 uninfected and 21 infected by L. infantum, of both sexes and of different ages; dogs were classified into three clinical stages: stage I, mild disease; stage II, moderate disease; and stage III, severe disease. Parasitic index was determined by real-time polymerase chain reaction (PCR) and cytokine serum concentration by flow cytometry. The average parasitic index of infected dogs was 4.59 × 1010 copies/μl. IL-4 and TNF-α concentrations were higher in infected dogs than in the control group. Antibody levels were positively correlated with IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. Antibody levels were positively correlated with IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. However, this cytokine can be used as a marker to distinguish between different clinical stages.

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“…However, this methodology does not permit the direct identification of clinical specimens, the culture is required and it has low sensibility for samples from patients with mucosal and chronic cutaneous leishmaniasis, which prevents obtaining large amounts of parasites. Besides, MLEE is technically demanding and requires prolonged times for obtaining results (10)(11)(12)41). An alternative to discriminate between Leishmania species is PCR-RFLP, a widely used test that is performed with different molecular targets to determine the ability to differentiate species (42).…”

Section: Discussionmentioning

confidence: 99%

“…Worldwide, nine reference centers provide species identification by MLEE, and only three of them are in the Americas (1). This, added to the complexity of the technique, limits the frequent application of this methodology to characterize Leishmania species in Colombia and the world (10)(11)(12).…”

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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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Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques

Cited by 19 publications

References 19 publications

Real-time PCR applications for diagnosis of leishmaniasis

Real-time PCR applications for diagnosis of leishmaniasis

Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis

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

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