Real-time PCR applications for diagnosis of leishmaniasis

Abstract

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.

Cutaneous and mucocutaneous leishmaniasis: experience of a Mediterranean hospital

“…On the other hand, in our experience, the usefulness of Giemsa stained preparation from the lesion smear is acceptable, and should be considered as the first diagnostic step, especially in the paediatric population. Therefore, an appropriate diagnosis would be performed in the first instance based on examination of the smear of the lesion, and for Giemsa-negative cases, based on biopsy and NAAT [33].…”

Introductory Chapter: Leishmaniasis: An Emerging Clinical Syndrome

“…This underscores the need for highly sensitive and specific diagnostic modalities. In this regard, molecular techniques such as real-time polymerase chain reaction (qPCR)-based methods are gaining ground for detection and quantification of Leishmania as well as for species identification [57]. However, to rule out false negatives, combination of two PCR techniques is advisable in patients with cutaneous lesions [58].…”

Evaluation of a pan-Leishmania SL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

“…The golden standard for parasite detection in sand flies and animal tissues is microscopic examination. This method allows to confirm the presence of viable parasites, but is time consuming and requires a substantial level of expertise [7]. These drawbacks resulted in a shift towards sample screening with molecular assays.…”

“…The most commonly used PCR assay for Leishmania detection in sandflies [12,13] and small mammals [14–16] is targeting the minicircle kinetoplast DNA (kDNA). Because of the high kDNA copy number (10 4 minicircles per parasite), very low numbers of parasites can be detected [7]. However, the nucleotide sequence and copy number sometimes differ among Leishmania species, impeding consistent quantification [17,18].…”