Detection of DNA from Leishmania (Viannia): Accuracy of Polymerase Chain Reaction for the Diagnosis of Cutaneous Leishmaniasis

Abstract

Cutaneous leishmaniasis (CL) can occur in skin and mucosa, causing disfiguring lesions. The laboratory diagnosis of CL involves immunological methods and optical detection of the parasite, al of which have limitations. There is a need for more effective diagnostic methods for CL which wil allow treatment to be initiated more promptly in order to help prevent the development of severe forms of mucosal disease, and to estimate the prognosis of the infection. The polymerase chain reaction (PCR) has been widely used to diagnose CL, because of its higher sensitivity. This study estimated the accuracy and compared PCRs of samples from lesion scarification (PCR-L) and blood sample-enriched leukocytes (PCR-B) with three conventional diagnostic techniques: parasite direct search (DS), Montenegro skin test (MST), and indirect immunofluorescence reaction (IIF). The study included 276 patients under suspicion of CL. We conducted a cross-sectional study, in which patients were selected by convenience sampling. We used MP3H/MP1L primers to generate a Leishmania (Viannia) (minicircle kDNA) fragment of 70-bp. Of 106 patients with CL, 83.87%, 51.67%, 64.52%, 85.71%, or 96.10% tested positive by PCR-L, PCR-B, DS, IIF, or MST, respectively. Five patients tested positive only by PCR-L, and two other patients only by PCR-B. PCR-L is indicated for use in patients with chronic lesions or Leishmania reinfection, which may progress to mucosal lesion. PCR-B is indicated for use in patients with negative results in conventional tests or for patients with no apparent lesion. PCR is not only useful in diagnosing CL but also helps to identify the infecting species.

Serological and Molecular Investigation of Cutaneous Leishmaniasis in Healthy Individuals from an American Cutaneous Leishmaniasis-Endemic Region

“…The samples were amplified and subsequently subjected to agarose gel electrophoresis according to the protocol provided by Nietzke-Abreu et al (2013). The reaction mixture (25 µL) contained 1 µM of each primer (Invitrogen), 1.5 mM MgCl 2 , 1X enzyme buffer, 0.2 mM dNTP (Invitrogen), 1 U Taq DNA polymerase (Invitrogen) and 2 µL of the sample DNA.…”

Diagnostic Efficacy of Molecular Techniques for Detection and Identification of Leishmania Species in Human Whole Blood and Skin Samples from Ecuador

“…Vergel and others 11 detected the presence of Leishmania DNA in 7.5% of skin lesions and in 19.2% in peripheral-blood monocytes isolated from 59 patients with confirmed diagnosis of CL. In a recent study, Neitzke-Abreu and others 21 reported the identification of Leishmania DNA in 83.87% of skin lesions and 51.67% of peripheral-blood monocytes isolated from 106 patients with CL. The percentages of Leishmania DNA detected in the studies described above are lower than the percentage of Leishmania DNA detected in the skin and whole blood of the present study.…”

“…11,12,21 Venazzi and others, 12 studying 68 patients with CL, detected parasite DNA in 3.4% of prepared blood samples. In that study, blood samples were treated with a solution containing 25 mM citric acid, 50 mM sodium citrate, and 81 mM glucose before DNA isolation.…”

Detection of Leishmania (Viannia) DNA in leucocytes from the blood of patients with cutaneous leishmaniasis

“…The mucosal form of CL, which manifests as destructive and mutilating injuries in the mucous membranes of the upper airways, is estimated to occur in 3-5% of cases (2) . In State of Paraná, the mucosal form was identifi ed in 23.1% of cases in 2010, indicating a higher prevalence of this form of the disease compared to other Brazil States (4) .…”

“…All samples were tested for the presence of possible inhibitors of PCR by using specifi c primers for the human β-globin gene (GH20: 5'-GAA GAG CCA AGG ACA GGT AC-3' and PC04: 5'-CAA CTT CAT CCA CGT TCA CC-3') (9) , intended to amplify a fragment of 268bp (4) .…”