Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

Acurácia da reação em cadeia da polimerase quantitativa em amostras de pele íntegra congelada e parafinada no diagnóstico da leishmaniose visceral canina

ABSTRACT

The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

Keywords:
diagnosis; qPCR; canine visceral leishmaniasis

RESUMO

O objetivo deste trabalho foi avaliar a acurácia da PCR quantitativa (qPCR) realizada em amostras de pele íntegra congelada (FT) e parafinada (FFPE). Trata-se de um estudo de validação, com amostras provenientes de 46 cães de uma área endêmica no Brasil. Após as coletas de amostras, as extrações de DNA foram realizadas utilizando-se kits comerciais, e a qPCR foi orientada para alvos do kDNA da espécie Leishmania infantum. Os resultados obtidos para as amostras FFPE foram 63,6% de sensibilidade e 77,1% de especificidade; para as amostras FT, 100% e 48,6%, respectivamente. A concordância dos resultados da técnica de qPCR com amostras FT e FFPE foi pobre. Os resultados sugerem que o congelamento é o método mais adequado de conservação para banco de amostras para recuperação de DNA.

Palavras-chave:
diagnóstico; qPCR; leishmaniose visceral canina

INTRODUCTION

Visceral leishmaniasis is a chronic infectious disease of public health importance worldwide, affecting both human beings and other species of wild and domestic mammals (Control…, 2010).

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania (Leishmania) infantum (sin. L. chagasi) (Kuls et al., 2011), and Lutzomyia longipalpis is its main vector (Brasil, 2006). Domestic dogs (Canis familiaris) have been considered its main reservoir and source of infection in urban areas; therefore, as a control measure, the euthanasia of dogs with positive diagnosis for CVL (Brasil, 2006) has been advocated.

Currently, the diagnosis of leishmaniasis is accomplished through the combination of clinical, laboratorial and epidemiological data, because the disease presents a wide spectrum of clinical manifestations, which complicates its identification (Gontijo and Melo, 2004).

Polymerase chain reaction (PCR) -based techniques show high specificity and sensitivity, besides the ability to exponentially amplify DNA copies from a small sample amount (Mesquita et al., 2001) and different types of clinical materials (Almeida et al., 2013). Moreover, the immune status of dogs does not interfere with the test results, which allows the use of PCR in cases with inconclusive outcome by other techniques, as well as in cases of clinical (or immune) anergy or cross-reactivity in serological tests (Moreira et al., 2007; Lachaud et al., 2002). In addition, the PCR technique can be used in studies on the development of potential vaccines and monitoring of CVL treated animals (Fiuza et al., 2015; Nunes et al., 2016).

The quality of the DNA used in PCR is an important factor to obtain reliable results. Although DNA extraction requires a differentiated protocol, its extraction for PCR can be performed from fresh frozen tissues (FT) (Oliveira et al., 2005) and formalin-fixed, paraffin-embedded (FFPE) tissues (Roura et al., 1999). FT samples result in better quality DNA, while FFPE samples can result in partially degraded or impure DNA (Ferrer et al., 2007).

However, FFPE samples can be stored without cooling, which facilitates their transportation and storage in locations with poor infrastructure (Laskay et al., 1995). In this study, we evaluated the accuracy of quantitative polymerase chain reaction (qPCR) conducted with FT and FFPE samples of healthy skin, collected from the scapular region of dogs, for L. infantum DNA detection.

MATERIALS AND METHODS

The sample bank comprised dogs from a CVL endemic area in Belém, Para state, Brazil. The animals were submitted to biopsy of healthy skin in order to perform reference techniques for CVL diagnosis. Dogs which tested positive in at least one of the following tests were considered infected with Leishmania: parasitological culture (Silva et al., 2011) followed by isoenzymatic characterization (Cupolillo et al., 1994); immunohistochemistry (IHC) (Quintella et al., 2009); and microscopy identification after hematoxylin and eosin staining (HE) (Carson and Hladick, 2009).

This is a diagnostic validation study using a convenience sample. We used two fragments - one prepared with FFPE and another with FT - of healthy skin collected from the scapular region, with a three-millimeter (mm) punch, of each of the 46 dogs surveyed. The protocol for obtaining the clinical samples was approved by the Ethics Committee on Animal Use - CEUA/Fiocruz (N. L-038/08).

We used the guideline of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) (Bustin et al., 2009). All phases of qPCR were performed in a reference center for CVL.

Five 5μm-thick histological sections were obtained from each block of FFPE samples using a microtome (Leica Microsystems RM 2025). To prevent contamination between samples, the microtome was cleaned with 70% ethanol and had its disposable blade replaced at the start of every new sample processing.

DNA extraction was performed for each tissue using a specific kit, in accordance with the manufacturers’ protocols, aiming to achieve the best DNA yield results.

Deparaffinization and DNA extraction were conducted using a QIAamp^® DNA FFPE Tissue kit (Qiagen, California, USA), whereas an Illustra^TM Tissue & Cells Genomicprep Mini spin kit (GE Healthcare, UK) was used for the extraction and purification of DNA from FT samples. The manufacturers’ recommendations were followed for both processes.

DNA quantification was conducted by fluorimetry using a Qubit® 2.0 Fluorometer (Invitrogen®, California, USA) platform together with a Qubit® dsDNA HS Assay (Invitrogen®, California, USA) reagent specific for double-stranded DNA, following the manufacturers’ recommendations.

Standardized tests using culture of promastigote forms of L. infantum and sample of negative dogs were performed to determine reaction quality. The standard curve was made with a DNA sample obtained from the culture of promastigote forms of L. infantum (MHOM/BR/1974/PP75) in exponential growth phase. The DNA of 1 x 10⁶ parasites was extracted using a DNeasy® Blood & Tissue kit (Qiagen® California, USA), following the manufacturer’s recommendations.

Considering that the DNA extraction method showed 100% yield, the resulting DNA sample from 1x10⁶ parasites eluted in 200(L buffer solution presented a concentration of 5,000 genome equivalents (gEq) of L. infantum/(L. Serial decimal dilutions of this DNA sample were performed upon assembly of the reaction plate. Each point of the curve was built in triplicate, using 5(L of each dilution. We used dilutions relating to 500, 50, 5, 0.5 and 0.05gEq of L. infantum/(L, which represent the points 2,500, 250, 25, 2.5, 0.25gEq of L. infantum/qPCR reaction. Standard curves were established for each reaction plate to determine the number of genome equivalents (gEq) of L. infantum in the DNA samples assessed in the study.

After extraction, the DNA samples in triplicate were submitted to amplification by qPCR using a StepOne™ (Applied Biosystems® CA, USA) platform and a TaqMan® (TaqMan® MGB Applied Biosystems®, CA, USA) hydrolysis probe. The probe (FAM-5’-AAAAATGGGTGCAGAAAT-3’-NFQ-MGB) and the indicators LEISH-1 (5’-AACTTTTCTGGTCCTCCGGGTAG-3’) and LEISH-2 (5’-ACCCCCAGTTTCCCGCC-3’) were designed to recognize conserved regions of kDNA of L. Infantum (Francino et al., 2006).

The reactions were performed with Universal Mastermix (Applied Biosystems, CA, USA) reagent, according to the manufacturer's instructions, in a final volume of 25(L, containing 5(L of DNA sample, 1.35(ML of the indicators LEISH-1 and LEISH-2, 0.5(M of the probe, and 2(L of bovine albumin serum (BSA) (Sigma-Aldrich®) at 5(g/(L concentration.

The qPCR methodology is reported in the paper by Solcà et al., (2014), briefly, the reactions were conducted in a 48-well plate (Applied Biosystems), sealed with adhesive film (Applied Biosystems), with the following DNA amplification protocol: 1 denaturation cycle at 50(C for 2 minutes, 1 denaturation cycle at 95(C for 10 minutes, and 40 cycles of denaturation at 95(C for 15 seconds and of annealing/extension at 60(C for 1 minute.

In each amplification plate, we used positive (standard curve of L. infantum culture) and negative (ultrapure water) controls of reaction, and threshold set at 0.1, to determine the quantification cycle (Cq). The cutoff point and the detection limit were determined after qPCR reaction standardization under laboratory conditions. Samples with Cq lower than 37 cycles were considered positive for L. infantum DNA, whereas samples with Cq greater than 37 cycles were considered undetectable. The detection limit was 0.0025 GEQ of L. infantum/reaction.

The results were then normalized using the concentration of DNA present in the 5(L of sample as the denominator.

In order to identify possible false-negative results caused by the presence of inhibitors in the reaction or by degraded DNA sample, a test was conducted on all samples undetected by qPCR. We used the Taqman® Gene Expression Assay system (Applied Biosystems, CA, USA), which contains a primer pair and a probe preset for the realization of qPCR, for the amplification of a segment of the canine constitutive gene that encodes the β-actin protein subunit (REF: Cf03023880_ g1, Applied Biosystems, CA, USA), in a final total reaction volume of 25(L, containing 2(L of DNA and 23(l of reaction mixture composed of 12.5(L of Universal Mastermix, 1.25(L of the primers and probe solution, and 9.25(L of ultrapure water. In this phase, the result was qualitative, considered to be detectable or undetectable. DNA samples that showed amplification were considered positive, thus containing intact and inhibitor-free DNA.

The results obtained were organized into a database and processed by the software program Statistical Package for the Social Science (SPSS), version 16.0. The median, minimum and maximum of DNA quantification were obtained and compared according to the method of conservation (FT and FFPE) by the Wilcoxon test. The results of the qPCR were compared, and the degree of agreement between the paired tissue samples preserved in FT or FFPE was determined by the Kappa coefficient. The degree of agreement was classified according Landis and Koch (1977). The values for sensitivity, specificity and confidence intervals of the tests were calculated using the reference standard (positive in at least one of the diagnostic tests: parasitological culture, IHC, or HE staining). The Mann-Whitney nonparametric test was used for comparison of genomic equivalents (parasitic load) in detectable FT and FFPE samples. Results were considered statistically significant at P<0.05.

RESULTS

Of the 46 dogs included in the study, 23.9% tested positive in at least one of the three techniques used as standard diagnostic reference of CVL. Regarding the results of DNA quantification by fluorimetry, we observed that the median of DNA concentration in the FFPE skin samples (0.1540ng/µL, min: 0.0005 and max: 1.5400) was significantly lower (P<0.001) than that observed in the FT samples (6.9600ng/µL, min: 0.0005 and max: 77.7000); we also observed a large variation between the minimum and maximum DNA concentration values, mainly in the FT specimens.

Sensitivity, specificity, accuracy, negative and positive predictive values of qPCR were calculated using the results obtained with FT and FFPE samples of healthy skin. The results are shown in Table 1.

Table 1
Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of Quantitative Polymerase Chain Reaction (qPCR) using DNA samples of FT and FFPE skin

Healthy skin samples	Sensitivity	Specificity	PPV	NPV	Accuracy
FFPE	63.6% (43-90%)	77.1% (90-100%)	46.7%	87.1%	73.9%
FT	100% (62-98%)	48.6% (75-96%)	37.9%	100%	60.9%

• Note: FFPE: formalin-fixed, paraffin-embedded sample. FT: frozen tissue. PPV: positive predictive value. NPP: negative predictive value. REFERENCE STANDARD: parasitological culture, IHC, HE.

When comparing the 46FT and FFPE samples by qPCR, we observed that 24 of them showed concordant results (11 detectable and 13 undetectable) and 22 presented discordant results (18 detectable on FT and undetectable on FFPE; and 4 detectable on FFPE and undetectable on FT).

The complete agreement of the results of the samples for qPCR (detectable/undetectable) was 52.2%. The Kappa index presented a degree of 0.12, which is a poor degree of agreement.

The result of the medians of genome equivalents (gEq) of L. infantum (parasitic load) in the qPCR was 5.463gEq (min: 0.431 max: 1687515.00) for the 29 FT detectable samples and 1.421 gEq (min: 0.616 max: 29.030) for the 14 FFPE detectable samples (P=0.001) (Table 2).

Table 2
Medians of gEq of L. infantum (parasitic load) obtained by Quantitative Polymerase Chain Reaction (qPCR) and the results of reference standard in FFPE and FT skin samples

Healthy skin samples	(+) qPCR (-) reference standard (Discordant)	(+) qPCR (+) reference standard (Concordant)	Total
FFPEa	0.933gEq (n=7)	3.141gEq (n=7)	1.421gEq (n=14)
FTb	3.366gEq (N=18)	517.967gEq (n=11)	5.463gEq (n=29)

• Note: FFPE: formalin-fixed, paraffin-embedded sample. FT: frozen tissue. REFERENCE STANDARD: parasitological culture, IHC, HE. +: positive, -: negative. (a) discordant FFPE versus concordant FFPE skin samples, Mann-Whitney test, P=0.209; (b) discordant FT versus concordant FT skin samples, Mann-Whitney test, P=0.084.

The medians of genome equivalents (gEq) of L. infantum (parasitic load) in concordant and discordant samples between the reference standard and qPCR are also shown in Table 2. However, no statistically significant differences were observed.

All 17 FT negative samples showed amplification of the gene for (-actin, and six FFPE negative samples (12%, n=31) showed no amplification.

DISCUSSION

In this study, fragments of healthy skin collected from the scapular region of dogs were used as biological samples for the diagnosis of CVL by specific qPCR targeting L. infantum kDNA sequence. The skin has proved to be a good site for parasitological confirmation (Madeira et al., 2009), and it is an important indicator, considering that the transmission of infection occurs during the blood feeding of the insect vector. Furthermore, the material collected from the same place on the skin can be used to perform other diagnostic tests, as the three used in this study as reference standard (parasitological culture, HE, and IHC), with no need to increase the number of biological specimens collected from the animal (Xavier et al., 2006).

With the extraction of DNA from FT samples, it was possible to obtain samples with a higher mean concentration of DNA compared with those prepared with FFPE tissue (Isola et al., 1994).

This outcome contradicts authors who have found good results with human FFPE samples (Vilanova-costa et al., 2008). One of the limiting factors for DNA recovery from FFPE tissues is the incubation time for fixation for later paraffinization (Ferrer et al., 2007). In our study, the residence time in formalin was 2-10 days. In the specific literature, studies show that acid fixatives interfere with the quality of nucleic acids, contributing to DNA degradation (Ben-ezra et al., 1991; Greer et al., 1991), and that samples fixed and stored for more than six months present undefined or poor results in PCR owing to DNA degradation (Ferrer et al., 2007).

The deparaffinization process and the different phases of extraction can also cause fragmentation, loss of DNA, and inhibition of the reaction (Dietrich et al., 2013). The efficiency of the DNA extraction technique is also related to the method used (Fernandes et al., 2004). In our study, we did not assess the efficiency of the techniques used for DNA extraction, but we tried to use appropriate methodologies and reagents for each type of biological sample. However, because of the low concentration of DNA in some samples, it was not possible to repeat some analyses, particularly those that showed high standard deviation in triplicate, probably due to the same fact.

The low DNA concentration extracted from FFPE samples has prevented the use of a single, predefined concentration in qPCR assays for the different samples. Therefore, we used a fixed volume of DNA sample per reaction, irrespective of its concentration. For this reason, it is possible that the validity of qPCR may have been compromised, because the lower the concentration of DNA in this reaction, the lower the chance of detection of the target DNA sequence.

To overcome such limitations and increase the sensitivity of the PCR technique for detection of the parasite genome in FFPE samples, the use of a larger number of sections of FFPE tissue for DNA extraction has been suggested, considering that the number of parasites per section is small (Roura et al., 1999). In the present study, we used five 5mm-thick slices of FFPE healthy skin samples for DNA extraction, whereas FT samples were prepared with 3mm punches on the skin. Because the obtained DNA samples were eluted in 50uL buffer solution, in both cases, the differences between the mass of FT and FFPE tissue subjected to DNA extraction may have contributed to the large difference in the mean final concentration values of DNA obtained from these two sources of material.

The difficulties found in DNA amplification by PCR may also be related to the presence of inhibitors of DNA polymerase and to the small initial number of molecules containing target sequences with sufficient integrity for amplification. However, the process of PCR inhibition can be reversed with adaptations of the technique, such as the use of a higher concentration of polymerase and dNTP, and increased extension time (Dietrich et al., 2013).

The qPCR for detection of L. infantum using FT samples presented high sensitivity but low specificity. Other authors have found positivity of approximately 100% with PCR (Queiroz et al., 2010).

The low specificity of qPCR found in the present study may have resulted from the low sensitivity of the techniques used as standard diagnostic reference (Schubach et al., 2014; Solcá et al., 2014). In our study, qPCR was able to detect Leishmania DNA in samples with parasite load below the limit required to obtain positive results in parasitological culture, HE, and IHC. The median for gEq/ng of DNA in the FT samples was significantly higher compared with that of the FFPE samples. Four samples of FFPE tissue showed undetectable results in qPCR and were positive in the reference standard and FT qPCR. In addition, low parasite load may be a predictive factor for false-negative results on FFPE tissue (Frickmann et al., 2013).

In this study, six FFPE samples showed undetectable results in the DNA quality test (qPCR for (-actin). However, there was not sufficient sample volume for subsequent analysis of DNA degradation in any of the cases.

Analysis of the medians of genome equivalents (gEq) of L. infantum (Table 2) showed that samples that are negative in the reference standard but positive in qPCR tend to present lower parasitic load (median of gEq/ng of DNA), although not statistically significant due to the wide dispersion of data. This fact suggests that positive results in the reference standard require levels of parasitic load higher than the minimum necessary for detection by qPCR.

The qPCR technique presented 100% sensitivity on FT samples, but low specificity was observed for this technique owing to the low parasitic detection by reference standards; these findings suggest that qPCR on FT samples should be used as the reference standard for CVL. Thus, the use of qPCR would allow better monitoring of the parasitic load of treated dogs (Manna et al., 2008), considering that the immune status of dogs does not interfere with the result (Moreira et al., 2007).

CONCLUSION

Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery.

ACKNOWLEDGMENTS

The authors are grateful to the Coordination for the Improvement of Higher Education Personnel - CAPES for the financial support (scholarship) and to the graduate program. This study was supported by the Rio de Janeiro Research Foundation - FAPERJ, Project “Jovem Cientista do Nosso Estado” and the Brazilian Council for Scientific and Technological Development - CNPq. FBF and MFM hold grants from CNPq for productivity in research.

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Publication Dates

• Publication in this collection
  Nov-Dec 2017

History

• Received
  30 Mar 2016
• Accepted
  23 Feb 2017