A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis

Abstract

Background: Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling.

Methods: We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan.

Results: The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 microL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%-99.7%) and 95.6% (95% CI, 85.2%-98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%-97.7%), 91.7% (95% CI, 64.6%-98.5%), and 86% (95% CI, 72.7%-93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively.

Conclusions: The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.

Figure 1

Alignment of the internal control (IC) DNA sequence and the Leishmania OligoC-TesT DNA target sequence within the 18S rRNA gene of the Trypanosomatidae parasites Leishmania donovani (L.d.; GenBank accession no. X07773), Trypanosoma brucei gambiense (T.b.; GenBank accession no. AJ009141), and Trypanosoma cruzi (T.c.; GenBank accession no. AF303660). Gaps in the sequences are represented by dots. Black, dark gray, and light gray shading indicate consensus among all 4, 3, or 2 sequences, respectively. The forward (18S-L-F) and reverse (18S-L-R) primers (arrows), detection probe, and biotinylated (B) IC and Leishmania (L) capture probes are shown. Lowercase letters indicate nucleotides that were added to avoid steric hindrance by the biotin-avidin binding during hybridization.

Figure 2

Schematic overview of the Leishmania OligoC-TesT. Polymerase chain reaction (PCR) amplification of a sample containing Leishmania DNA (1) is performed using a PCR mix containing single-stranded internal control (IC) template (2). This IC template is amplified with the same primers as the Leishmania DNA target but contains a specific internal sequence (dotted line). When the PCR and subsequent denaturation is completed, the PCR product solution contains single-stranded Leishmania and IC DNA (3) and is mixed with an equal volume of migration buffer preheated at 55°C. The Leishmania Oligo-Strip is dipped into the solution, and test results are read after a 10-min migration at 55°C (4). During migration, the solution takes up the impregnated probes: the gold-labeled detection probe (5) hybridizes with both types of amplicons, while the biotinylated Leishmania capture probe on the test side (6) and the biotinylated IC capture probe on the control side (7) hybridize with their respective amplicons. Gold and biotin labeling is indicated by white circles and black circles, respectively. The biotinylated capture probes accumulate the hybridized complex on the Neutralite avidin lines on both sides of the strip, resulting in visible red lines (8). For a negative sample, only the IC amplicon is present and is captured on the control side (9). The excess detection probes migrate further and hybridize on the complementary probes coated on both sides of the dipstick as a control for migration (10). The IC line and the migration control lines determine whether the test is valid or invalid. An invisible migration control line indicates an invalid detection step, while an invalid PCR is indicated by a negative IC line in combination with a negative test line. The latter is possibly due to inhibitory factors in the extracted DNA. When a sample contains high concentrations of Leishmania DNA, competition between the Leishmania DNA and the IC template DNA during PCR can result in an invisible IC control line combined with a strongly visible Leishmania test line. In this case, the test is considered valid.

Figure 3

Analytical sensitivity and specificity of the Leishmania OligoC-TesT. The upper line is the migration control, and the lower line is the Leishmania test line. A, Test results for a serial dilution of Leishmania donovani DNA in water containing 0.1 mg/mL acetylated bovine serum albumin. Dipsticks 1–9 are for 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, and 0 fg per assay, respectively. B, Test results for a serial dilution of L. donovani promastigotes in naive human blood. Dipsticks 1–6 are for 10,000, 1000, 100, 10, 1, and 0 parasites in 180 µL of blood. C, Test results for different Leishmania species (1 ng of DNA per assay) and nontarget pathogens (50 ng of DNA per assay). Dipsticks 1–20 are for L. amazonensis, L. donovani, L. infantum, L. braziliensis, L. peruviana, L. guyanensis, L. panamensis, L. mexicana, L. major, L. lainsoni, L. tropica, L. aethiopica, Sporothrix schenckii, Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, Mycobacterium tuberculosis, Schistosoma mansoni, a negative control for polymerase chain reaction, and a negative control for DNA extraction.

Table 1

Diagnostic accuracy of the Leishmania OligoC-TesT on clinical samples from nonendemic control subjects, endemic control subjects, and patients with cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), or visceral leishmaniasis (VL).