Comparison of Six PCR Methods Using Peripheral Blood for Detection of Canine Visceral Leishmaniasis

Animals.Thirty-seven dogs of different breeds and ages living in outdoor conditions in the endemic focus of the Cévennes (southern France) were recruited for the study. The CVL diagnosis was classically established from clinical signs, specific serology, and/or direct examination of lymph node or bone marrow aspirates. For serology, two techniques were used: immunofluorescence (cutoff value, 1/40) and counterimmunoelectrophoresis (cutoff value, one line). Peripheral blood for PCR was collected by jugular venipuncture in an EDTA-coated tube. A control group was made up of 10 dogs living in an area where the disease is not endemic in the north of Corrèze (France); the dogs were born there and had never left the area.

Leishmania serial dilution assay.Promastigotes from a 4-day-old culture of a reference strain of L. infantum (MHOM/FR/78/LEM75) were washed twice in 1× phosphate-buffered saline and precisely counted on a Thoma hemacytometer (mean of 10 counts). The pellet was lysed with proteinase K at 320 μg/ml in 2 volumes of TNNT buffer (0.5% Tween 20, 0.5% Nonidet P-40, 10 mM NaOH, 10 mM Tris, pH 7.2). The DNA was extracted by a simplified phenol-chloroform method and ethanol precipitated. A series of dilutions was performed, yielding DNA solutions corresponding to decreasing concentrations from 20 to 0.00001 parasite/μl.

Preparation of seeded blood samples.All samples consisted of 4.5 ml of peripheral blood collected in EDTA-coated tubes. The blood was collected from five healthy dogs living in an area where the disease is not endemic and was then pooled before being aliquoted again. Seeded samples were made by adding live L. infantum promastigotes to the buffy coat (BC) collected as described below. The concentrations of parasites tested were 1,000, 100, 10, 5, 2, and 1 per ml of whole blood, corresponding to 10, 1, 0.1, 0.05, 0.02, and 0.01 parasite per PCR. Concentrations of <1 parasite/ml of blood (i.e., <0.01 parasite/PCR) were obtained by serial dilutions of the lowest concentration given above in a solution of negative control dog blood DNA.

Sample and DNA preparation.For both seeded samples and dog samples, the BC was isolated from 4.5 ml of peripheral blood after a simple centrifugation step for 10 min at 1,600 × g. The BC (300 μl) was lysed in 2 volumes of TNNT buffer and 960 μl of proteinase K/ml, and the tube was incubated for between 2 and 24 h at 56°C, boiled for 10 min, and then stored at 4°C (10). Extraction lysates were processed as follows: 500 μl of lysate was subjected to phenol-chloroform extraction (i.e., phenol and phenol-chloroform followed by chloroform), the DNA was precipitated with ethanol and resuspended in 150 μl of sterile distilled water. The DNA extraction was controlled by measurement of the optical density at 260 nm in a GeneQuantII spectrophotometer (Pharmacia).

PCR amplification.Six primer pairs were compared. Their characteristics are presented in Table 1. A thorough optimization of the PCR conditions was carried out as described previously (11) for each of the PCR methods, using seeded dog blood samples instead of purified cultivated promastigotes. The optimized conditions for each method are summarized in Table 1. For all reactions, 5 μl of 10× buffer, 0.6 mg of bovine serum albumin/ml, 200 μM (each) deoxynucleoside triphosphate, and 50 pmol of primers were used in a total reaction volume of 50 μl, including 10 μl of sample DNA. The variable factors included the MgCl₂ concentration, the amount of Taq DNA polymerase (Eurogentec), and the primer annealing temperature. The “hot-start” technique (Dynawax; Eurogentec) was used routinely in all experiments to increase the technical specificity. The reactions were cycled in a Biometra thermal cycler for K13A-K13B and RV1-RV2 and in an MJResearch thermal cycler for the remaining primer pairs. The following conditions were used: initial annealing at 94°C for 4 min, 40 cycles of 94°C for 30 s, variable annealing temperature according to the primer set (Table 1) for 30 s, and 72°C for a variable duration according to the primer set, followed by a final elongation at 72°C for 10 min. Each concentration for the sensitivity assays (both with purified culture parasites and seeded blood samples) was tested at least twice in quadruplicate. The dog samples were tested twice in duplicate. Additionally, in each test, one internal positive control tube for the detection of PCR inhibition was included for each sample (11). In cases of reaction inhibition, a second DNA extraction was performed, and both DNA preparations were tested as is and diluted to 1/5 or 1/10. Finally, three to five negative control tubes that each received 10 μl of H₂O instead of DNA were included in each test to detect any carryover contamination. Uracil DNA N-glycosylase (UNG) was used for primers K13A-K13B and RV1-RV2: 1 U of the enzyme was added to the PCR mixture, and dTTP was replaced by dUTP (400 μM); the tube was then incubated for 10 min at 50°C prior to the first denaturation.

PCR product analysis and DNA hybridization.The reaction products were visualized under UV light after electrophoresis of 20 μl of the reaction solution in a 2 or 3% agarose gel. All gels were then Southern blotted and hybridized with the corresponding ³²P-labeled PCR product. The filters were washed twice at high stringency (0.1× SSPE [1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA, pH 7.7], 1% sodium dodecyl sulfate) before the autoradiogram was made. Hybridization was systematically carried out for all methods. It was found necessary for methods Deb8-Ajs3 and Pia1-Pia2.

Decontamination procedures.Complete physical separation (of rooms, materials, and personnel) as well as decontamination (e.g., UV exposure of rooms, consumables, and materials and bleaching of all materials and surfaces) procedures were used routinely to avoid carryover contamination from previously amplified DNA (9, 11). Additional separation measures were taken for two primer pairs, K13A-K13B and RV1-RV2, for which the PCR and analysis of PCR products were performed in distinct hospital buildings. Enzymatic decontamination using uracil DNA N-glycosylase was also used with both of these primer pairs.

Comparison of six PCR methods with artificial Leishmania samples.The comparisons were based on examination of the specificity, sensitivity, ease of interpretation of results, and reproducibility of each method. The specificities of the six methods against other microorganisms had previously been tested by different authors and were not tested again here. The technical specificity was assessed from the presence of spurious amplification products revealed by artifactual bands on the electrophoresis gels. One of our positive criteria was to avoid specific DNA hybridization, which is cumbersome in routine practice. Using purified cultivated parasites, no artifactual bands were observed, except for some with Deb8-Ajs3. By contrast, the presence of dog blood DNA tended to yield spurious amplification products that hampered the interpretation of the electrophoresis gels and often made a hybridization step necessary. Great differences in this respect were observed among the different methods tested (Table 2).

Sensitivity was assessed on one hand by a Leishmania serial dilution assay (SDA) using cultivated parasites and on the other hand with seeded dog blood samples containing known parasite concentrations (see Materials and Methods). Using purified cultivated parasites, the sensitivities varied from 0.05 to 10⁻⁴ parasite per reaction tube between the least (R221-R332) and the most (K13A-K13B and RV1-RV2) sensitive methods (Table 2). The presence of blood led to a decrease in sensitivity of 10- to 100-fold with the least sensitive methods, which then detected 0.2 to 1 parasite per reaction tube. By contrast, it did not affect the sensitivities of the most sensitive methods, which detected 10⁻⁴ parasite per reaction tube. More pragmatically, the sensitivity thresholds varied from 10 to 10⁻³ parasite per ml of blood according to the PCR method used (Table 2). In our hands, whichever method was used, the DNA hybridization step did not improve sensitivity compared with direct visualization of the gels under UV light. Finally, for all primer pairs, the intramethod reproducibility was high with both the Leishmania SDA and seeded dog blood samples, including with low parasite DNA concentrations, and even for artifactual bands.

On the whole, using these criteria, method Deb8-Ajs3 was thought not to be adequate for further evaluation. The five remaining primers were assessed for the diagnosis of CVL using dog peripheral blood.

Diagnosis of CVL by different PCR assays.Peripheral blood was drawn from 24 dogs living in an area of endemicity and was grouped according to clinical status and specific serology. Two groups were considered for the study: 17 dogs presenting typical CVL, i.e., with clinical signs and positive serology and a positive direct examination in the six cases where it was performed (group 1) and 7 asymptomatic dogs with positive serology (group 2). The results obtained with the different PCR methods for Leishmania detection are shown in Tables 3 and 4. The sensitivities of the different methods with dog clinical samples correlated well with the assessment previously made with artificial samples. Unexpectedly, however, primers Pia3-Pia4 yielded very poor results even in group 1, partly due to a high rate of PCR inhibitions which could not be reversed by any means (Table 3). For this reason, they were not tested further in group 2. Methods K13A-K13B and RV1-RV2 were the most sensitive in both groups of dogs. Again, whichever primer pair was used, systematic Southern blot analysis did not improve the sensitivities obtained after simple electrophoresis. The PCR results, as well as the specific serological tests, were all negative in the control group of 10 dogs living in a area where the disease was not endemic.