Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

Abstract

The precise diagnosis of American tegumentary leishmaniasis (ATL) is an essential task due to the disease's associated morbidity. A noninvasive, extremely sensitive, and highly specific exam is critical, particularly for mucosal leishmaniasis (ML), in which a low parasite quantity is expected. We aimed to compare the diagnostic accuracy of swab and biopsy sample analysis using SYBR Green-and TaqManbased real-time PCR (qPCR) assays with that of a composite reference standard consisting of the Montenegro skin test, serology, histopathology, smears, culture, and conventional PCR. In total, 55 patients with ATL (ML, 18 patients; cutaneous leishmaniasis [CL], 37 patients) and 36 patients without ATL were studied. qPCR analysis of swabs was more accurate when using SYBR Green (87.88%; 95% confidence interval [CI], 77.86 to 93.73 patients) than when using TaqMan (78.79%; 95% CI, 67.49 to 86.92%) (P ϭ 0.031). SYBR Green (84.72%; 95% CI, 74.68 to 91.25%) was also more accurate than TaqMan (73.61%; 95% CI, 62.42 to 82.41%) for biopsy samples (P ϭ 0.008). All qPCR methods were 100% specific. Swabs and biopsy specimens had similar sensitivity when using the same chemistry (P ϭ 0.125 for SYBR Green and P ϭ 0.625 for TaqMan). Moreover, qPCR achieved better performance than most existing techniques used for the diagnosis of ATL and also detected the Leishmania parasite in a greater proportion of patients than the associated histopathology, smear, culture, and conventional PCR techniques did. Swabs therefore represent a useful diagnostic tool because they not only are noninvasive but also can achieve an accuracy similar to that of biopsy samples. The high accuracy of SYBR Green-based qPCR may also reduce the requirement for associated parasitological tests for ATL diagnosis.

Molecular Diagnosis of Visceral Leishmaniasis

“…However, quantitative PCR (qPCR) does not directly measure the number of viable parasites circulating in the blood, but rather the amount of circulating parasite DNA. Therefore, sensitivity of qPCR depends on assay design (primer and target region), chemistry used (SYBER Green or TaqMan), nature of clinical samples (blood, skin, bone marrow or splenic fluids) and the DNA extraction methods (manual vs commercial kits) [126] (Table-2). Using this technique, it was earlier demonstrated that the simultaneous quantitative evaluation of Leishmania DNA and cytokine by Real Time PCR assay allows prediction of the development of disease in asymptomatic infected dogs [127].…”

Accuracy of a TaqMan‐based real‐time polymerase chain reaction combined to a Novy‐MacNeal‐Nicolle medium culture for the diagnosis of American tegumentary leishmaniasis

“…1,2 Real-time PCR (qPCR) is considered a very efficient technique, and it adds quantitative results to clinical analyses. [3][4][5] We aimed to test the accuracy of the combination of a Novy-MacNeal-Nicolle (NNN) medium culture and TaqManbased qPCR analysis for the diagnosis of TL. Sample size was calculated 6 considering a 70% prevalence of ATL in this population 5,7 with a null hypothesis for sensitivity set at 70%, an alternative hypothesis for sensitivity set at 90%, power set at ≥80% and the P-value set to be <0.05.…”

“…Case and control definition was performed according to a composite reference standard as described elsewhere. 4 For the index test, the preparation of the culture media was adapted according to the methodology described by Romero et al 8 Culture material was withdrawn from a lesion edge aspirate 8 (Fig. 2).…”

“…Quantitative and qualitative TaqMan-based qPCR was performed with primers targeting sequences of the kDNA of Leishmania. The primer pair selected was 5 0 -TGC TAT AAA ATC GTA CCA CCC GACA-3 0 and 5 0 -GAA CGG GGT TTC TGT ATG CCA TTT-3 0 with the following specific hydrolysis probe for Leishmania (Viannia) braziliensis: FAM-TTG CAG AAC GCC CCT ACC CAG AGGC-TAMRA (FAM, 6-carboxyfluorescein, TAMRA, 6-carboxyethionyl triamine) (Applied Biosystems â , Foster City, CA, USA) as described elsewhere 4,5,9 (R 2 = 0.949, efficiency = 99.722, slope = À3.329). The limit of quantification of the present standard curve was 1 parasite.…”

“…The positivity of the microscopic analysis in culture Letters to the Editor e189 tubes with 1000 or more parasites was 100% (11/11). In 12 patients, the subgenera Viannia was identified by a previously described restriction fragment length polymorphism PCR, 4 and in only one patient, the subgenera Leishmania was identified. The present result is probably explained by the fact that samples extracted from active ATL lesions will present a significant quantity of Leishmania amastigotes even in the absence of differentiation to promastigotes in culture.…”