Filter paper performance in PCR for cutaneous leishmaniasis diagnosis

Abstract INTRODUCTION: The objective of this study was to evaluate the performance of filter paper (FP) for lesion scraping collection in a polymerase chain reaction (PCR) for cutaneous leishmaniasis (CL) diagnosis. METHODS: Lesion scrapings from 48 patients were collected and analyzed for PCR. RESULTS: PCR with FP detected up to three Leishmania braziliensis promastigotes. Considering the direct search by microscopy or PCR of samples collected in STE buffer as standards, the sensitivity of PCR with FP was 100%. CONCLUSIONS: FP can be useful for CL diagnosis in remote regions, allowing high sensitivity in the detection of the parasite by PCR.

Cutaneous leishmaniasis (CL) is a neglected disease and significant public health problem. In the last five years, one million cases of CL were reported worldwide, and more than 90% of them occurred in Afghanistan, Algeria, Brazil, Colombia, Iran, Pakistan, Peru, Saudi Arabia, and Syria 1 .
Collecting material from CL lesions by biopsy is invasive and demands extra care when it comes to sample preservation 2 . A precise molecular diagnosis depends on obtaining adequate samples and also on DNA integrity 3 . Filter paper (FP) is an important tool for collecting, storing and transporting samples for the diagnosis of neglected diseases 4 .
Considering the importance of accurate CL diagnosis, especially for populations living in regions which are difficult to access, the objective of this study was to evaluate the performance of FP for lesion scraping collection, including the sensitivity of the test and the number of Leishmania parasites that can be detected on the FP with the purified material used directly in PCR.
To determine the sensitivity of PCR with FP, lesion scrapings were collected from CL-suspected patients referred to the Laboratório de Ensino e Pesquisa em Análises Clínicas (LEPAC) of the State University of Maringá (UEM) for CL diagnosis. These patients lived in municipalities belonging to the North-Central Paraná Mesoregion, Brazil, which is endemic for CL (Supplementary Figure 1) Samples were collected by scraping of the lesion's inner edge. For DS, lesion scrapings were placed on glass slides, stained with Giemsa, and examined by microscopy for Leishmania sp. amastigotes. The lesion scrapings were also distributed into microtubes containing 50 μL of sodium chloride-Tris-EDTA (STE) buffer (10 mM Tris, 1mM Na 2 EDTA•H 2 O; 0.1 M NaCl, pH 8.0) and stored at -20 ºC for further PCR testing. The DNA was obtained by incubation at 95 ºC for 30 min in a Veriti ® Thermocycler (Applied Biosystems ® , USA) followed by centrifugation at 13.000 g for 1 min. The supernatant containing the DNA was precipitated and stored at 4 ºC.
Lesion scrapings were also placed on FP (Whatman ® FTA TM Classic card, GE Healthcare, UK) and stored in plastic bags at room temperature. A disk (d = 2 mm) was cut out of the FP using an FTA TM Harris Micro-Punch instrument and transferred to a PCR microtube. The total DNA of the lesion scraping contained in the disk was purified with 200 μL of FTA TM Purification Reagent (Amersham Buckinghamshire, UK) according to the manufacturer's instructions (Figure 1). For performance assays, a serial dilution of L. (V.) braziliensis promastigotes was used to measure FP's capacity to retain and preserve Leishmania DNA and to determine the minimal number of Leishmania parasites per punch that are required for successful PCR amplification. Assay 1 was performed with L. (V.) braziliensis promastigotes (3x10 5 to 3x10 -3 /μL) suspended in PBS and spotted on FP to determine the detection limit of the test for the parasite alone. Assay 2 was a mixture of lesion scrapings from patients with negative CL diagnosis plus 3x10 5      In this study, we assessed FP performance, encompassing its capacity to preserve DNA and to be used directly in PCR for CL diagnosis 6 . According to our search of the scientific literature, some studies have already evaluated the sensitivity of PCR with samples collected on FP for CL diagnosis 6-8 , but no work has shown the detection limit of PCR with the direct use of FP containing the Leishmania DNA as we did in our research, i.e., in a serial dilution placed on the paper. Also, the primer chosen has already been applied in several studies involving DNA detection of Leishmania (Viannia) 9,10 . Assay 2 was done to evaluate the interference of non-target DNA in the detection of Leishmania from lesion samples collected on FP and was constructed in a way that imitates real biological conditions to the maximum extent. In this assay, it was possible to detect up to three promastigotes on a single punched disk, equivalent to 0.25 pg of DNA, considering the sequenced genome of L. (V.) braziliensis (MHOM/BR/75/M2904) 9 . The intensity of the amplicon band of assay 2 corresponding to three Leishmania parasites, was weak compared to the band of assay 1 that amplified the same quantity of parasites. When comparing assay 2 with the control dilution, a 10-fold difference in the number of detected parasites was observed. These differences are related to the interference of non-target DNA, carried with the patient's cells. For this reason, high sensitivity primers should be chosen to perform PCRs with FP. Santos (2012) shows that the direct use of the FTA TM elute card in the mix was more effective in identifying HPV-DNA (92% of positive samples) than DNA extracted from the card by elution (54%) 11 . Studies involving Leishmania that used FP for the collection of lesion scrapings, biopsy, or lesion imprint had greater sensitivity (92% to 100%) compared to DS or PCR with DNA obtained from lesion aspirate or biopsy. These results were obtained either by extracting the DNA from the card or by using it directly in the PCR mixture 6,12 . FP does not appear to interfere with DNA amplification. The increase in temperature of the PCR mix was probably enough for the DNA to escape the FP, thus allowing an efficient reaction 11 .
When tested with clinical specimens from CL-suspected patients, the PCR with FP was positive for two patients with negative CL diagnosis by DS, which was fortunate for these people. Still, most likely due to the number of patients analyzed, this difference in positivity was not statistically significant. There is a reduction in the number of parasites in the lesion with the progression of the disease, and DS (by microscopy) sensitivity can be limited by the expertise of the laboratory technician who performs the test 13 . Using microscopy, culture, and PCR methods, Eroglu (2014) evaluated the sensitivities of skin samples taken in smear, aspiration fluid, and FP for CL diagnosis in the Old World 14 .
Using similar techniques to those in the previously cited study, Al-Jawabreh (2018) compared unstained smears, smears stained with Giemsa stain, and FP, using microscope, culture, and PCR for CL diagnosis. In both of these studies, the lesion samples collected on FP revealed the most CL cases 8 .
The sensitivity of PCR using FP was 100% compared to both DS and PCR with lesion scrapings collected in STE, and specificity was 92% and 96%, respectively. This small decrease in specificity can be explained by the test results of one patient who was DS negative but positive for both types of PCR (STE and FP), and another who was showed negative using DS and PCR with STE, but positive with PCR using FP. The method of specimen collection affects DNA yield and, consequently, the test's sensitivity 7,8 . Samples collected on FP may have a higher capacity to preserve the DNA 7,13 .
This study had a limitation due to the lack of a control group of healthy patients, due to the impossibility of collecting biological material from these patients. For this reason, lesions from patients with other dermatologic diseases who had a negative diagnosis for CL were considered as a control group.
We also suggest the use of a less invasive and painful collection method, such as scraping instead of a biopsy, for the search of Leishmania in patients with cutaneous lesions 2 . FP preserves the biological material, when it is properly collected, and its association with a sensitive technique such as PCR can contribute significantly to disease diagnosis and appropriate treatment. In particular, it can be applied in cases of individuals who live in difficult-to-access areas, where preservation of the collected material becomes precarious. FP is a safe and easy collecting tool that avoids contamination risks. It allows preservation of material obtained by a less invasive method, such as lesion scraping or imprinting, and allows quick and effective DNA collection 6,7 . It also provides a functional way to transport the samples, and can be stored at room temperature for extended periods 15 . This study showed that it is possible to detect up to three Leishmania parasites in lesion scrapings spotted on FP with a single punched disk, directly used in the PCR amplification mix. FP may be an alternative tool, with high performance, to preserve samples for field collection, especially in difficult-to-access regions, where, until recently, CL has caused stigma and affected the most impoverished populations.