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This study was conducted according to the principles specified in the Declaration of Helsinki and under local ethical guidelines (Universidad Peruana Cayetano Heredia Institutional Review Board). The study protocol, informed consent and sampling procedures were approved by the Institutional Review Boards of the Hospital Nacional Cayetano Heredia and Universidad Peruana Cayetano Heredia (Lima, Peru) for studies involving human subjects. Written informed consent was obtained from all participants prior to enrolment.
The sample size was calculated using the G*Power 3.1 software (release 3.1.9.2; available from: http://www.gpower.hhu.de/) [27] to assess the null hypothesis of no difference in parasite load levels between different sampling sites within the CL ulcer. Assuming a medium effect size of 0.5, a significance level of 5%, and a power of 80%, 35 matched pairs of lesion samples were required to be examined (two-sided, Wilcoxon signed-rank test for matched pairs). For significant results, the effect size was assumed to be ‘medium’, which means an effect visible to the naked eye. Non-significant results were assumed to have a ‘small’ effect size. We managed to study 31 paired lesion samples from patients presenting with acute CL.
Patients that attended the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Hospital Nacional Cayetano Heredia, in Lima, Peru, between January and June 2013 for the examination of skin lesions were invited to participate in the study and evaluated for possible eligibility. Patients were considered for enrolment if they presented with ulcerative skin lesions of recent onset (≤3 months of evolution), with elevated and infiltrative borders and a lesion size over 1 cm in diameter; and were able to give written informed consent for the sampling procedures. We included adult patients with laboratory confirmed diagnosis of CL, as defined by a positive result on at least 1 of these 3 tests: direct microscopy on Giemsa-stained lesion smears [7], lesion aspirate microculture [28], and qualitative PCR targeting kDNA minicircles [29] on a biopsy specimen obtained from the ulcer border. This diagnostic PCR includes internal control primers for amplifying the human beta-globin gene as previously described [13]. The intradermal leishmanin skin test (LST), used to assess exposure to Leishmania infection, was performed on CL patients before treatment, as described elsewhere [30,31]. We excluded patients allergic to local anesthetics, with clinical evidence of bacterial or fungal superinfection of the ulcer (when possible), with any contraindication to skin biopsy and those undergoing active treatment for CL. In three cases with secondarily infected ulcers, patients were treated with a 5-day course of antibiotics before sample collection.
In order to analyze the distribution and load of Leishmania amastigotes within the cutaneous lesion, samples were collected from 3 different sites, in the following order: the center of the ulcer, the base (inner border) of the ulcer, and the raised border of the ulcer (Fig 1); using a randomly chosen coordinate defined as North, South, East or West, taking as reference the lateral and longitudinal axes of the human body. If the patient had more than one lesion, the most active and typical indurated ulcer was selected. Eight specimens were collected from a single lesion per patient: a punch biopsy and a dermal scraping from each of the 3 lesion sites, and a cytology brush from each the center and base of the ulcer. The order of sampling was: biopsy, scraping, and cytology brush. All samples were taken by the same physician in order to avoid inter-individual variation.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pntd.0003936.g001 Sites of sample collection within the cutaneous ulcer. (A) Macroscopic aspect of an ulcerated lesion. (B) Schematic representation of a typical CL ulcer. The sites where samples were collected are indicated: border (1), base (2), and center (3) of the ulcer. Figure adapted from: Zvietcovich et al. [32].
Prior to sampling, lesions were cleansed with topical antiseptics, removed from any overlying scab or crust with saline solution and anesthetized with 1 cc of lidocaine 1%. A small tissue fragment of 1.5 mm in diameter was obtained from the ulcer center, base and raised border, using a sterile disposable punch (Miltex), at a randomly chosen coordinate within the cutaneous lesion.
Lesion material was scraped from the ulcer center, base and from an incision made at the raised border, using a sterile lancet; this was done in the same coordinate but adjacent to the point from where biopsy samples were obtained.
A sterile cervical cytology brush (Cervisoft, Puritan Medical Products) was rolled clockwise at a single point of the ulcer center and base 5 times each in sequence in order to collect lesion cellular and exudative material, as described by Valencia et al. [15]; this was done in the same coordinate but adjacent to the point from where scrapings were obtained. Clinical specimens were stored at −20°C in a 1.5 mL Eppendorf tube containing 700 μL 100% ethanol for subsequent molecular analysis.
Isolation of DNA from biopsies, lancets and cytology brushes: Prior to DNA extraction, samples were centrifuged at 8000 g for 2 min and ethanol was discarded. Biopsied tissue was disaggregated with a sterile scalpel. Disaggregated tissue, lancets and cytology brushes were subjected to overnight lysis with Proteinase K and processed for DNA isolation using a column-based method (High Pure PCR template preparation kit, Roche), according to the manufacturer’s instructions. The isolated DNA was then quantified by fluorometry using the Quant–iT Broad Range dsDNA Assay kit (for biopsies) and the Quant-iT High Sensitivity dsDNA Assay kit (for scrapings and cytology brushes) on the Qubit fluorometer (Invitrogen). DNA samples were diluted to 5 ng/μL; those samples below this concentration were added directly into the PCR reaction.
Parasites were typed using the heat-shock protein 70 gene (hsp70) PCR-N variant followed by restriction fragment length polymorphism (RFLP) analysis using the restriction enzymes BsaJI and RsaI as in Montalvo et al. [33].
Detection and quantification of Leishmania (Viannia) spp. We applied a SYBR Green-based qPCR assay targeting kDNA minicircles to detect and quantify Leishmania (Viannia) parasites in clinical samples, as previously described [26]. Each kDNA-qPCR run included a standard curve of L. (V.) braziliensis (MHOM/BR/75/M2904) DNA ranging from 5 × 104 to 5 × 10−3 parasite DNA equivalents/reaction (run in duplicate); a positive control with known amount of Leishmania parasites, which consisted of a mix of Leishmania DNA and human genomic DNA in order to mimic clinical specimens (run in triplicate); a negative control (human genomic DNA from peripheral blood mononuclear cells of a healthy donor; run in triplicate); and a blank (no-template control; run in triplicate). The standard curves (inter-assay reproducibility, n = 11) showed a mean square error (MSE) of ≤0.111, correlation coefficient (r2) of ≥0.998 and slopes of 3.28 (mean) ± 0.05 (standard deviation), indicating a high amplification efficiency (≥1.99) (2 would indicate 100% PCR efficiency). The positive control showed a mean of 7,640 parasites and an inter-assay coefficient of variation of 7.8% (n = 11 independent runs). All clinical samples were run in duplicate; if replicates differed by a standard deviation of >0.35 in Cq (quantification cycle) values (>0.5 cycles), they were retested. A sample was quantified when it had a Cq value falling within the range of the standard curve. The highest dilution of template of the standard curve was defined as the lower limit of quantification (LOQ). Samples with Leishmania DNA levels below the LOQ could be detected; they were considered positive (qualitative detection) only if their melting curves had the same profile as those of the standards included in the same experiment. The Leishmania parasite load was calculated as follows: [parasite DNA equivalents per reaction/amount of tissue DNA per reaction] × 103, expressed as the number of Leishmania parasites per μg of tissue DNA.
Frequencies and proportions were used to describe categorical variables while median and interquartile range or mean and standard deviation were used for numeric continuous variables. To assess whether the median parasite load in clinical specimens differed significantly according to the skin lesion site or the sampling method, analyses for paired samples using Friedman (with Dunn’s post-hoc test) and Wilcoxon signed rank tests were performed. The correlation degree between the parasite load measurements in scraping and cytology brush specimens with respect to those in biopsy specimens was calculated using the Spearman’s rank correlation test. The association between the Leishmania load and the parasite species was evaluated using the Mann-Whitney U test. Statistical analyses were performed under a 5% significance level, using the GraphPad Prism v5.02 software.
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